**Part 3**

**Microbial Biotechnology** 

208 Innovations in Biotechnology

Yang, I. H., Co, C. C. & Ho, C. C. (2005). *Alteration of human neuroblastoma cell morphology and neurite extension with micropatterns*, Biomaterials, Vol. 26, No. 33, pp. 6599-6609 Yin, C., Liao, K., Mao, H. Q., Leong, K. W., Zhuo, R. X. & Chan, V. (2003). *Adhesion contact* 

Zheng, J. Q. & Poo, M. M. (2007). *Calcium signaling in neuronal motility*, Annu Rev Cell Dev

5, pp. 837-850

Biol, Vol. 23, pp. 375-404

*dynamics of HepG2 cells on galactose-immobilized substrates*, Biomaterials, Vol. 24, No.

## **Microbial Expression Systems and Manufacturing from a Market and Economic Perspective**

Hans-Peter Meyer and Diego R. Schmidhalter *Lonza AG Switzerland* 

#### **1. Introduction**

Biotechnology generates global sales in the order of well over 200 billion US\$ in all markets and has thus become an important economic factor in manufacturing. The buzz word 'biotechnology' carries expectations that it can provide sustainable solutions for greenhouse gas reduction in manufacturing industries, trigger a 'clean tech' boom and create new jobs. It is no wonder that biotechnology has gained significant attention even in high level politics as it can give a 'green' touch to administrations. Many consumers are not even aware of the surprising array of products and services which biotechnology can or could provide today; these range from high-tech pharmaceutical applications to snow making. Table 1 lists some new or unusual applications of biotechnology products which include, for example, skin protection compounds from the oceans or biopolymers for drag reduction in transport pipelines.


Table 1. A few examples of new and unusual applications of biotechnology and its products in different fields.

These high expectations are merited due for 4 reasons:


Consequently, biotechnology will have an especially high impact in the production of complex chemicals used for pharmaceuticals, fine chemicals and specialities (Meyer, 2011). Other promising areas are biopolymers and protein-based novel biomaterials for consumer goods, car parts, medical devices or as support for the 2D and 3D cultivation of tissue and organ replacements.

It is industrial or white biotechnology which is of growing academic and private interest, as it represents an equal or even bigger business potential than red biotechnology in the long term. But how can application fields and markets of biotechnology be classified? One way to describe the different markets of biotechnology is the colour code (red, white, green, blue and grey).

#### **2. The markets**

A useful way to classify the applications of biotechnology is the colour code of biotechnology shown in Table 2.

Estimates and definitions may vary, but there is one common denominator in various assessments, namely that the proportion of products manufactured using biotechnology will increase significantly. While the development and the market introduction of new biopharmaceuticals such as monoclonal antibodies will continue at its present rate, it is especially industrial biotechnology which is expected to realise high growth rates.

The different "biotechnologies" do overlap and especially the boundaries especially between red and white biotechnologies for pharmaceutical applications can be confusing. There is one important additional difference between the red biotechnology of therapeutic proteins and monoclonal antibodies and the white biotechnology pharmaceuticals which includes a large variety of products: red biotechnology is characterised more by its products whereas white biotechnology is defined more by its technology platform.

#### **2.1 The chemical market**

The sales of global chemical markets are expected to grow from 2292 billion Euros in 2007 to 3235 billion Euros in 2015 and to 4012 billion Euros in 2020 (Perlitz, 2008). It is estimated that only about 3-4% of all chemical sales have been generated with some help from biotechnology (Nieuwenhuizen et al., 2009), but this figure is anticipated to grow faster than the average market rate. It is speculated that at least 20% of the global chemicals will be derived using

1. The unmatched precision in the production and assembly of small and large molecules. This precision of the natural biosynthetic machinery cannot be reached using chemical

2. The fantastic speed, at which these production systems can reproduce themselves. The reason for this is that bacteria have by far the largest surface-to-volume ratio in the living world, leading to maximal metabolic rates. A single bacterium, weighing about 10-12 grams, grows so fast that its biomass would theoretically reach the mass of the

3. The inherent safety of biological systems as metabolic heat makes run-away reactions

Consequently, biotechnology will have an especially high impact in the production of complex chemicals used for pharmaceuticals, fine chemicals and specialities (Meyer, 2011). Other promising areas are biopolymers and protein-based novel biomaterials for consumer goods, car parts, medical devices or as support for the 2D and 3D cultivation of tissue and

It is industrial or white biotechnology which is of growing academic and private interest, as it represents an equal or even bigger business potential than red biotechnology in the long term. But how can application fields and markets of biotechnology be classified? One way to describe the different markets of biotechnology is the colour code (red, white,

A useful way to classify the applications of biotechnology is the colour code of

Estimates and definitions may vary, but there is one common denominator in various assessments, namely that the proportion of products manufactured using biotechnology will increase significantly. While the development and the market introduction of new biopharmaceuticals such as monoclonal antibodies will continue at its present rate, it is

The different "biotechnologies" do overlap and especially the boundaries especially between red and white biotechnologies for pharmaceutical applications can be confusing. There is one important additional difference between the red biotechnology of therapeutic proteins and monoclonal antibodies and the white biotechnology pharmaceuticals which includes a large variety of products: red biotechnology is characterised more by its products

The sales of global chemical markets are expected to grow from 2292 billion Euros in 2007 to 3235 billion Euros in 2015 and to 4012 billion Euros in 2020 (Perlitz, 2008). It is estimated that only about 3-4% of all chemical sales have been generated with some help from biotechnology (Nieuwenhuizen et al., 2009), but this figure is anticipated to grow faster than the average market rate. It is speculated that at least 20% of the global chemicals will be derived using

especially industrial biotechnology which is expected to realise high growth rates.

whereas white biotechnology is defined more by its technology platform.

approaches.

organ replacements.

green, blue and grey).

biotechnology shown in Table 2.

**2.1 The chemical market** 

**2. The markets** 

earth in only a few days!

impossible, when compared to organic chemistry. 4. The biocatalyst and biomass are fully recyclable.

industrial biotechnology in 2020, which translates into almost 1000 billion Euros. This means that the sales generated by industrial biotechnology will increase by an order of magnitude as the recent estimates of the global sales of industrial biotechnology products vary between 50 and 150 billon Euros, depending on whether biofuels are included or not. There is a consensus that biotechnology will play a much greater role in future manufacturing as it can deliver complex products using economically and ecologically sustainable processes.


Table 2. Classification of the applications of biotechnology with respect to markets; for each class sales volumes, compound annual growth rate and number of companies globally active in the field are noted; there is of course an overlap between industrial and pharmaceuticals biotechnology, some sectors of industrial biotechnology are dependent on cheap and reliable sources from agro biotechnology (Clive, 2010). The market size of >11 billion US\$ for agro biotechnology refers to "seed biotech". The global value of marketed harvested goods resulting from these seeds would be much larger, by one or two orders of magnitude.

The products potentially produced by biotechnology range from commodities (e.g. succinic acid), biopolymers (e.g. polyhydroxybutyric acid), flavour & fragrance products (e.g. vanillin), agroproducts (*Bacillus thuringiensis*) to small molecule pharmaceuticals and more.

Unfortunately there are no shortcuts in biotechnology, and in order to meet the anticipated 1000 billion Euros derived from white or industrial biotechnology and to keep red biotechnology humming we need to further develop appropriate tools while keeping in mind that it took 200 years to complete today's chemical toolbox (Ghisalba et al., 2010).

#### **2.2 The feed, food and dietary supplement markets**

Biotechnology plays an important role in the kitchen through the food and beverage industry. Enzymes are used in large scale food production of glucose from starch (hydrolytic enzymes), high fructose syrup (isomerase), conversion of lactose to galactose and glucose (hydrolase), cheese production (proteases), meat processing (proteases) and many more. Phytase (phosphohydrolase) is an example of an enzyme used in the feed industry. However, only about 25% to 30% of all industrial enzymes are used for food and feed purposes. It is also worthwhile to note that over 90% of industrial enzymes sales come from less than 30 enzymes, most of them hydrolytic enzymes. As these enzymes are used as an auxiliary material in the food manufacturing process, most of them can be produced using recombinant technology. Thus microbial expression systems play a crucial role for enzyme production.

The market of functional foods more than doubled between 2001 and 2010 and is estimated at 5 billion € (Welck & Ohlig, 2011). Other important biotechnology products are amino acids, vitamins or PUFAs (polyunsaturated fatty acids).

Flavours and fragrances are also biotechnology-relevant markets with a volume of over 22 billion US\$ (Leffingwell & Associates, 2011). More than 10% of the supply is derived from bioprocesses, with more than 100 commercial aroma chemicals derived via different biotechnological methods (Berger, 2009). In order to relieve the pressure on natural resources, companies are increasingly turning towards novel biotechnological sources and methods including genetic engineering approaches for the production of these raw materials.

This is also true for a part of the pharma market described below. To supply the anticancer secondary metabolite taxol (paxclitaxel) from its original natural source (the bark of the Pacific yew tree *Taxus brevifolia*) would be impossible. Taxol is produced in minute amounts (0.4 g/kg of bark) and synthetic alternatives including biotechnological steps had to be developed for this blockbuster drug with sales of about 3.5 billion US\$. It is difficult to estimate the global market value of botanical or plant derived drugs (BCC Research, 2009), but they seem to grow at a CAGR of 11% and alternative commercially viable expression and production systems will be needed for their production to replace the extraction from endangered or slow growing plants.

#### **2.3 The pharmaceutical market**

According to the IMS Health Forecasts, the global pharmaceutical market is expected to grow at an overall rate of 5-7% per annum, to reach \$880 billion US\$ in 2011 (Gatyas, 2011). Of this total pharmaceutical market, monoclonal antibodies alone represented 43 billion US\$ and therapeutic proteins 66 billion US\$ (2009 figures, Market Research News, 2011). The numbers for small molecule pharmaceuticals, which represent a very fragmented market, are even more difficult to estimate. Franssen et al. estimated the market segment in 2010 for pharmaceutical ingredients using industrial biotechnology at about 20 bn US\$ (Franssen et al, 2010). This could be conservative, as the number of small molecule APIs (Active Pharmaceutical Ingredients) using biotechnology in their (chemical) synthesis is rapidly increasing. Blockbuster such as Merck's Sitagliptin or Pfizer's Atorvastatin now include biotechnological steps in their manufacturing. Atorvastatin (Lipitor®), a cholesterollowering small molecule drug is the largest selling drug in history, which peaked at 12.7 billion US\$ annual sales. The chemical synthesis includes a biocatalysis step using a ketoreductase. The same is true for MERCK's Sitagliptin, the small molecule compound used in the antidiabetes drug Januvia®, which includes a transaminase step in its chemical synthesis. Taking such products into consideration, the number will increase well above 30 billion US\$ in 2011. Many classical biotechnological products like antibiotics (market of 42 billion US\$ in 2009) or steroids are even not included in these 30 billion US\$. What is important is that the small molecule market remains by far the most important driver for innovation in industrial biotechnology (Meyer et al., 2009).

It is especially red biotechnology with therapeutic proteins and monoclonal antibodies which is thriving and driving growth with established technologies. Remicade was the top selling mAbs brand followed by Avastin®, Rituxan®, Humira® and Herceptin®. The top 5 brands had sales of over \$ 5 billion each. However, besides that they often serve narrower disease phenotypes due to their specificity. The downsides of therapeutic proteins and monoclonal antibodies are that

1. they are not consumable as pills

214 Innovations in Biotechnology

many more. Phytase (phosphohydrolase) is an example of an enzyme used in the feed industry. However, only about 25% to 30% of all industrial enzymes are used for food and feed purposes. It is also worthwhile to note that over 90% of industrial enzymes sales come from less than 30 enzymes, most of them hydrolytic enzymes. As these enzymes are used as an auxiliary material in the food manufacturing process, most of them can be produced using recombinant technology. Thus microbial expression systems play a crucial role for

The market of functional foods more than doubled between 2001 and 2010 and is estimated at 5 billion € (Welck & Ohlig, 2011). Other important biotechnology products are amino

Flavours and fragrances are also biotechnology-relevant markets with a volume of over 22 billion US\$ (Leffingwell & Associates, 2011). More than 10% of the supply is derived from bioprocesses, with more than 100 commercial aroma chemicals derived via different biotechnological methods (Berger, 2009). In order to relieve the pressure on natural resources, companies are increasingly turning towards novel biotechnological sources and methods

This is also true for a part of the pharma market described below. To supply the anticancer secondary metabolite taxol (paxclitaxel) from its original natural source (the bark of the Pacific yew tree *Taxus brevifolia*) would be impossible. Taxol is produced in minute amounts (0.4 g/kg of bark) and synthetic alternatives including biotechnological steps had to be developed for this blockbuster drug with sales of about 3.5 billion US\$. It is difficult to estimate the global market value of botanical or plant derived drugs (BCC Research, 2009), but they seem to grow at a CAGR of 11% and alternative commercially viable expression and production systems will be needed for their production to replace the extraction from

According to the IMS Health Forecasts, the global pharmaceutical market is expected to grow at an overall rate of 5-7% per annum, to reach \$880 billion US\$ in 2011 (Gatyas, 2011). Of this total pharmaceutical market, monoclonal antibodies alone represented 43 billion US\$ and therapeutic proteins 66 billion US\$ (2009 figures, Market Research News, 2011). The numbers for small molecule pharmaceuticals, which represent a very fragmented market, are even more difficult to estimate. Franssen et al. estimated the market segment in 2010 for pharmaceutical ingredients using industrial biotechnology at about 20 bn US\$ (Franssen et al, 2010). This could be conservative, as the number of small molecule APIs (Active Pharmaceutical Ingredients) using biotechnology in their (chemical) synthesis is rapidly increasing. Blockbuster such as Merck's Sitagliptin or Pfizer's Atorvastatin now include biotechnological steps in their manufacturing. Atorvastatin (Lipitor®), a cholesterollowering small molecule drug is the largest selling drug in history, which peaked at 12.7 billion US\$ annual sales. The chemical synthesis includes a biocatalysis step using a ketoreductase. The same is true for MERCK's Sitagliptin, the small molecule compound used in the antidiabetes drug Januvia®, which includes a transaminase step in its chemical synthesis. Taking such products into consideration, the number will increase well above 30 billion US\$ in 2011. Many classical biotechnological products like antibiotics (market of 42 billion US\$ in 2009) or steroids are even not included in these 30 billion US\$. What is

including genetic engineering approaches for the production of these raw materials.

enzyme production.

acids, vitamins or PUFAs (polyunsaturated fatty acids).

endangered or slow growing plants.

**2.3 The pharmaceutical market** 


Although the market value of small molecule drugs which use one or more biocatalysis steps in their synthesis amounts to well over 30 billion US\$ and is growing, the share of biotechnology is well below what it could be. The pharmaceutical industry is under great pressure due to exploding R&D costs, politically due to the health care cost burden and from the market side due to an expected reduction of growth from 7% to 3%.

Both large and small molecules could face a technology gap and manufacturing bottlenecks to meet these production challenges. In both cases we need innovative, sustainable and costeffective production methods.

The vaccine market is a special subset of the pharmaceutical market, in which innovative solutions are sought for cancer, infectious diseases (e.g. malaria), pandemics (influenza) or bioterrorism. The vaccine market of 20 billion US\$/year is expected to grow to 35 billion US\$ in five years. Unusual new solutions such as production in transgenic plants may be required to achieve the scale and price targets (Langer, 2011).

The number of market introductions of small molecule pharmaceuticals or NCEs (New Chemical Entities) has been steadily decreasing since the late 1980s, whereas new therapeutic proteins and monoclonal antibodies have increased. To make things worse, small molecule drugs are positioned in markets that are becoming increasingly generic, thereby adding further pressure. The only small molecule drugs with an annual growth of over 10% were high potency drugs and to a lesser extent peptides. However, keeping healthcare costs under control will require efficient and affordable drugs, which are generally smaller entities without complicated and folded structures such as large proteins which need expensive production and logistics and can only be administered by injection.

However, solutions of the manufacturing challenges in the pharmaceutical industry will also provide solutions for other markets.

We need new cost-effective production for large and small molecule drugs (Meyer & Turner, 2009) with more chirality, more complex functionalities, and composed of various chemical structures. Examples are the glycosylation of proteins, aryl- or alkyl-organics drugs for modification of their biological efficacy and the functionalisation of novel biomaterials for medical devices and scaffolds for tissue generation using stem cells. The current biotechnological and chemical toolbox is reaching its technical limits and needs expansion to meet the economic and ecological manufacturing standards of the future (Meyer & Werbitzky, 2011).


1 Chinese hamster ovary cells, baby hamster kidney cells

Table 3. This table gives an overview of all biological methods and frequently used genus which can be used for the production of small and large molecules. The microbial pro- and eukaryotes are further tabulated in Table 6.

#### **3. The biotechnology toolbox**

216 Innovations in Biotechnology

*Generalists, well established technology, cost efficient, remains the standard method* 

The standard microbial workhorse

*Mostly specialist, niche applications with* 

Uniform human like N-linked glycans,

PUFAs, pigments, in discussion for

Potential host for human antibodies,

Expression system for ciliates for the production of therapeutic proteins

Veterinary products, easier cultivation

than mammalian cells

Mouse cell lines Human cell line

pluripotent cells

Several pharma blockbusters, established technology

Tissue and organ repair, mass production in development

Human embryonic stem cells, induced

Host for secreted proteins

Source of biologically active compounds but tricky to manufacture

*increasing importance*  Will lose its importance

host for industrial enzymes Nutritional flexibility, efficient secreters, industrially widely used

expression in 3 genomes Cost advantage, vaccines, "plantibodies" (antibodies)

with

biofuels

*Small and large molecules* 

Secondary metabolites

*Large and small molecules* 

Proteins and enzymes

Secondary metobolites Citric acid, enzymes, proteins

Mainly small molecules

Small and large molecules

Bovine trypsin Different products

Large molecules

Large molecules

Cells, tissues, organs

Table 3. This table gives an overview of all biological methods and frequently used genus which can be used for the production of small and large molecules. The microbial pro- and

Large molecules and enzymes Secondary metabolites Small and large molecules

**Organism Genus, species Products Comment** 

*Prokaryotes* 

*Bacillus Actinomycetes Escherichia coli Pseudomonas Gluconobacter Myxobacterium*

*Saccharomyces Pichia* 

*Penicillium Aspergillus* 

*Chlamidomonas* 

*Zea mais Nicotiana tabacum Pisum sativum* 

*Tetrahymena Trochoplusia Spodoptera Trochoplusia* 

CHO , BHK1 C127, NSO PerC6 Stem cells hESC, iPS

1 Chinese hamster ovary cells, baby hamster kidney cells

eukaryotes are further tabulated in Table 6.

**Gram+** 

**Gram-**

*Eukaryotes* 

**Fungi** 

**Algae** 

**Plants** 

**Protozoa** 

**Insect cells** 

**Mammalian cells** 

Table 3 gives an overview of the production systems available today for the production of small and large molecules. Old technologies still dominate the industry, especially for pharmaceuticals. Other than pharmaceuticals still extracted from plants, animals (mostly now forbidden), only a few biotechnological methods are used. For example of the 130 recombinant protein products in the US and European markets, 48 are expressed in microbes (34% *E. coli* specifically, 1% other bacteria, yeast 13%, see also Figure 6). Another 43% of products are produced in mammalian cells, primarily in CHO cells. *E. coli* and CHO cells have the longest history of use since the commercialisation of the first recombinant proteins in the 1980s. CHO and *E. coli* account for 64% of the expression systems used in manufacturing of currently marketed recombinant therapeutics (US and Europe). Novel expression systems in evaluation for biopharmaceuticals include e.g. *Pseudomonas fluorescens, Staphylococcus carnosus, Bacillus subtilis, Caulobacter crescentus, Chrysosporium lucknowense, Arxula sp*. as we will see later.

#### **3.1 Cultivation options**

While Table 3 lists the organism which can be used, it does not include the cultivation methods which are listed in Table 4 below.

	-
	-
	-
	-
	-

Table 4. Overview of the principal biotechnological production methods. Fermentation in sterile containments is by far the most important and suitable for bacteria, fungi, protozoa, algae, plants cells, insect cells, mammalian cells. Stem cells are preferentially produced adherent in biofilms, but the mass production for therapeutic purposes will probably also use suspension culture.

The standard manufacturing procedure in Table 4 is the submersed production of organisms in sterile containments as shown in Figure 1. This method allows the controlled growth of one organisms in a closed tank (fermenter). In over 15 years Armin Fiechter of the Swiss Federal Institute of Technology in Zürich developed a standardised biological test system and systematically tested many different bioreactor designs at the m3 scale. Although many new and interesting bioreactor designs exist, nothing has really changed since the 1980s and the numbers prove that the classic stirred tank with Rushton impellers remains the most versatile fermenter design (Meyer, 1987). It can be used for unicellular and filamentous cells, and is even used in large scale fermentation for mammalian and plant cell cultures. Moreover, the design gives good performance over a wide range of viscosities. It is a truly multipurpose equipment, with decades of depreciation times for the invested capital for the tanks themselves. The average cost for a large scale sterile fermentation plant (without down stream processing) ranges between 130000 €/m3 for an ISO plant to 300000 €/m3 nominal volume for a cGMP plant for injectable products.

The technology transfer and engineering challenges of fermentation, which include control of the physicochemical environment, mass and gas transfer and other items are discussed elsewhere (Sharma et al., 2011; Meyer & Klein, 2006; Meyer & Rohner, 1995; Meyer & Birch, 1999; Hoeks et al., 1997). Table 4 is a very general overview and many variants of suspension cultures cannot be discussed here. For example the immobilisation of cells by filtration in combination with continuous culture is a powerful tool to reach outstanding productivities with mammalian and microbial cells if sterile operation can be achieved and the cell type allows extended number of cell divisions and generations Hoeks et al., 1992).

Fig. 1. Left side: photograph of a 15m3 state of the art sterile containment for high cell density fermentations. LONZA has built and operates such high performance equipment up to the 75m3 for a range of organisms including methylotrophic yeasts such as *Pichia pastoris.* The flow breakers are designed as cooling elements needed to remove metabolic heat. The stirrers are concave Rushton impellers which, in combination with specially designed dip pipes (not shown), allow optimal mixing. In most cases the stirrer configuration is completed by the use of a top radially downward pumping marine impeller. Right: Two chromatography columns for the purification of injectable therapeutic proteins with a diameter of 2 meters to demonstrate size and complexity of down stream processing (Copyright@Lonza).

#### **3.2 The production organisms**

*The production strain is not everything, but everything is nothing without a good production strain*. Irrespective of the organism used in suspension culture for biotechnological manufacturing, there are numerous common problems, but also some typical differences, which will be discussed. Strain development is, however, the key issue for a commercially viable bioprocess. We have seen above, that companies have invested in expensive multipurpose fermentation equipment. Thus, the biology must be adapted to existing equipment and not

remains the most versatile fermenter design (Meyer, 1987). It can be used for unicellular and filamentous cells, and is even used in large scale fermentation for mammalian and plant cell cultures. Moreover, the design gives good performance over a wide range of viscosities. It is a truly multipurpose equipment, with decades of depreciation times for the invested capital for the tanks themselves. The average cost for a large scale sterile fermentation plant (without down stream processing) ranges between 130000 €/m3 for an ISO plant to 300000

The technology transfer and engineering challenges of fermentation, which include control of the physicochemical environment, mass and gas transfer and other items are discussed elsewhere (Sharma et al., 2011; Meyer & Klein, 2006; Meyer & Rohner, 1995; Meyer & Birch, 1999; Hoeks et al., 1997). Table 4 is a very general overview and many variants of suspension cultures cannot be discussed here. For example the immobilisation of cells by filtration in combination with continuous culture is a powerful tool to reach outstanding productivities with mammalian and microbial cells if sterile operation can be achieved and the cell type allows extended number of cell divisions and generations Hoeks et al., 1992).

Fig. 1. Left side: photograph of a 15m3 state of the art sterile containment for high cell density fermentations. LONZA has built and operates such high performance equipment up to the 75m3 for a range of organisms including methylotrophic yeasts such as *Pichia pastoris.* The flow breakers are designed as cooling elements needed to remove metabolic heat. The stirrers are concave Rushton impellers which, in combination with specially designed dip pipes (not shown), allow optimal mixing. In most cases the stirrer configuration is completed by the use of a top radially downward pumping marine impeller. Right: Two chromatography columns

*The production strain is not everything, but everything is nothing without a good production strain*. Irrespective of the organism used in suspension culture for biotechnological manufacturing, there are numerous common problems, but also some typical differences, which will be discussed. Strain development is, however, the key issue for a commercially viable bioprocess. We have seen above, that companies have invested in expensive multipurpose fermentation equipment. Thus, the biology must be adapted to existing equipment and not

for the purification of injectable therapeutic proteins with a diameter of 2 meters to demonstrate size and complexity of down stream processing (Copyright@Lonza).

**3.2 The production organisms** 

€/m3 nominal volume for a cGMP plant for injectable products.

the other way round. Table 5 summarises our experience with regard to the different factors influencing manufacturing costs with mammalian and bacterial cell cultures.

Generally, the strain and its growth characteristics define medium composition, cycle time and final product concentrations. Consequently, the number of steps needed in downstream processing (DSP) and volumetric sterile productivity (the two key cost drivers) are directly related to the choice of strain. The ideal strain is genetically stable, has a high specific (*qp*) and volumetric productivity (*Qp*), forms no by-products, and uses a well-defined medium resulting in a DSP with a limited number of steps. Fermentation is where value is created, downstream processing needs to conserve that created value.

A key issue in achieving a high specific and volumetric production rate is the choice of a highly efficient expression system and finally choosing the right recombinant strain for production.


Table 5. Effect of strain, process and plant on the overall process outcome. A highly productive strain is the most important factor in a bioprocess. I & D = Interest & depreciation. Y = Yield.

#### **3.2.1 Prokaryotes and lower eukaryotes**

#### **3.2.1.1 Bacteria, yeast and fungi**

Prokaryotes such as bacteria and lower eukaryotes such as yeasts or fungi are by far the most productive organisms in biotechnology. We will call them collectively microorganisms in this paper. Because of their small size (Figure 2), microorganisms have by far the largest surface to volume ratio in the living world which allows them to maximize their metabolic rates because of a high rate of exchange of molecules through their surface. With the right cultivation conditions, microorganisms grow exponentially according to the equation

$$\chi\_t = \chi\_o \ast \operatorname{\mathbf{e}}^{\left(\mu \ast t\right)}$$

Xo is the biomass concentration at time zero, or the start of cultivation. Xt is the biomass concentration at the time of harvest. μ is a strain specific growth rate. Some of the fastest growing bacteria weighing maybe 10-12 g are theoretically able to duplicate and grow so fast that their biomass would reach the mass of the earth (9 \*1054 tons) in less than a week. This means, that if a bacterial strain produces a protein or another product which can be industrially applied, large amounts can theoretically be produced economically.

Not only are microorganisms able to increase biomass at breathtaking rates, many are also able to grow under different conditions and on a great variety of substrates. This metabolic flexibility requires the ability to produce thousands of different enzymes and other proteins for all sorts of reactions and purposes. With the advent of genetic engineering, these enzymes and proteins can be overproduced in great quantities. Today entire pathways are modified and completed with heterologous genes, which allows the expression and production of molecules foreign to the species.

Fig. 2. Left a budding cell of the yeast *Saccharomyces cerevisiae*. During the reproductive phase a cell multiplies by forming buds. After the buds enlarge, nuclear division occurs and a cross wall is formed between the two cells. *S. cerevisiae* is easy to cultivate at large scale and it serves as host in many biotransformation processes, mainly after genetic recombination. The photograph in the center shows a few cells of the workhorse par excellence in biotechnology: *Escherichia coli*. The diameter of the *E. coli* cells varies between 0.5 and 1.5 micrometer. The bacterium to the right belongs to the genus *Rhizobium*, of which the strain HK1349 is used at large scale for the production of L-carnitine (Copyright@Lonza).

However, it does not end with the ability of microorganisms to produce large amounts of biomass and a great variety of different enzymes and proteins in a short time. There is an even more important reason why one wants to use microorganisms, namely the chemo-, regio- and enantio-selectivity of the microbial toolbox, and this is true for all living matter. No chemical manufacturing technology can match the precision of natural systems for the production of chiral and complex small and large molecules.

While this article focuses on the production of recombinant proteins and small molecules with *Escherichia coli*, it is important to understand the advantages of the different expression and production systems and compare them with the work-horse *Escherichia coli*.

There are now such a multitude of microbial host and expression systems theoretically available (Meyer et al., 2008) that it becomes difficult to choose the right combination. Emerging alternative production systems including those for glycosylated biopharmaceutical proteins for therapeutic use also are numerous (Jostock, 2007). *Escherichia coli* is currently the almost exclusively used prokaryotic production system but alternatives in discussion and developed on top of the commercially available expression systems are listed in Table 6. Examples are *Caulobacter crescentus*, *Proteus mirabilis*, *Pseudomonas* strains, *Staphylococcus carnosus*, *Streptomyces,* as well as fungi and yeasts such as *Pichia, Hansenula*, *Arxula*, *Yarrowia, Aspergillus* or *Trichoderma*.


Table 6. Commercialised microbial expression platforms and hosts; B, bacterial; F, fungal; Y, yeast.

220 Innovations in Biotechnology

Not only are microorganisms able to increase biomass at breathtaking rates, many are also able to grow under different conditions and on a great variety of substrates. This metabolic flexibility requires the ability to produce thousands of different enzymes and other proteins for all sorts of reactions and purposes. With the advent of genetic engineering, these enzymes and proteins can be overproduced in great quantities. Today entire pathways are modified and completed with heterologous genes, which allows the expression and

Fig. 2. Left a budding cell of the yeast *Saccharomyces cerevisiae*. During the reproductive phase a cell multiplies by forming buds. After the buds enlarge, nuclear division occurs and a cross wall is formed between the two cells. *S. cerevisiae* is easy to cultivate at large scale and it serves

However, it does not end with the ability of microorganisms to produce large amounts of biomass and a great variety of different enzymes and proteins in a short time. There is an even more important reason why one wants to use microorganisms, namely the chemo-, regio- and enantio-selectivity of the microbial toolbox, and this is true for all living matter. No chemical manufacturing technology can match the precision of natural systems for the

While this article focuses on the production of recombinant proteins and small molecules with *Escherichia coli*, it is important to understand the advantages of the different expression

There are now such a multitude of microbial host and expression systems theoretically available (Meyer et al., 2008) that it becomes difficult to choose the right combination. Emerging alternative production systems including those for glycosylated biopharmaceutical proteins for therapeutic use also are numerous (Jostock, 2007). *Escherichia coli* is currently the almost exclusively used prokaryotic production system but alternatives in discussion and developed on top of the commercially available expression systems are listed in Table 6. Examples are *Caulobacter crescentus*, *Proteus mirabilis*, *Pseudomonas* strains, *Staphylococcus carnosus*, *Streptomyces,* as well as fungi and yeasts such as *Pichia, Hansenula*,

and production systems and compare them with the work-horse *Escherichia coli*.

as host in many biotransformation processes, mainly after genetic recombination. The photograph in the center shows a few cells of the workhorse par excellence in biotechnology: *Escherichia coli*. The diameter of the *E. coli* cells varies between 0.5 and 1.5 micrometer. The bacterium to the right belongs to the genus *Rhizobium*, of which the strain HK1349 is used at

large scale for the production of L-carnitine (Copyright@Lonza).

production of chiral and complex small and large molecules.

*Arxula*, *Yarrowia, Aspergillus* or *Trichoderma*.

production of molecules foreign to the species.

Fig. 3. The fungus *Aspergillus niger* is a "generally recognised as safe" (GRAS) organism used industrially for the production of many substances such as citric acid or gluconic acid, enzymes (e.g. glucose oxidase, glucoamylase, alpha-galactosidase) and other compounds. It is also a versatile host for the heterologius expression of proteins (Copyright@Lonza).

*Aspergilli* (Figure 3) are ideally suited for recombinant protein expression as they have an enormous nutritional flexibility combined with a particularly efficient secretion system and secretion capacity (Fleissner & Dersch, 2010). They are amongst preferred organisms for the production of commercial food enzymes. Genetic engineering of different *Aspergillus* host strains has also allowed the synthesis of industrially relevant amounts of various heterologous proteins (such as human lactoferrin, calf chymosin or the plant-derived sweeteners thaumatin or neoculin peptide sweeteners, Nakajiama et al., 2008). Proteins are also efficiently glycosylated in *Aspergilli*, while undesired hyperglycosylation is usually not observed. Whole genome sequences of several *Aspergillus* species are now available.

#### **3.2.1.2 Algae**

Recently, *Chlamydomonas reinhardtii*, a unicellular eukaryotic green algae has been proposed as a host to produce several forms of a human IgA antibody directed against herpes simplex virus (Franklin & Mayfield, 2005; Specht et al., 2010). The main reason for turning to algae is the claimed cost advantage and the absence of viral or prion contaminations that can harm humans. One can frequently read that microalgae grow faster (by an order or two of magnitude!) than terrestrial plants and biomass titers of 600-1000 mg/l dry weight are reached. However, one has to be careful. Microalgae can grow very fast when grown heterotrophically on glucose for example. In this case the performance of high cell density fermentations are almost as productive as those reported for bacteria and yeasts (Xiong et al., 2008). But things look different when microalgae are mass-produced phototrophically! For a number of technical and biological reasons not discussed here, doubling time of around 10 hours are probably a realistic assumption, leading to growth rates of μ= 0.07 per hour. This is very low when compared to the bacterium *Escherichia coli* where growth rates of 1 and higher are quite common in large scale. But growth rates are perceptibly higher than with all other eukaryotes except the yeasts and fungi mentioned above. The advantage

Fig. 3. The fungus *Aspergillus niger* is a "generally recognised as safe" (GRAS) organism used industrially for the production of many substances such as citric acid or gluconic acid, enzymes (e.g. glucose oxidase, glucoamylase, alpha-galactosidase) and other compounds. It is also a versatile host for the heterologius expression of proteins (Copyright@Lonza).

*Aspergilli* (Figure 3) are ideally suited for recombinant protein expression as they have an enormous nutritional flexibility combined with a particularly efficient secretion system and secretion capacity (Fleissner & Dersch, 2010). They are amongst preferred organisms for the production of commercial food enzymes. Genetic engineering of different *Aspergillus* host strains has also allowed the synthesis of industrially relevant amounts of various heterologous proteins (such as human lactoferrin, calf chymosin or the plant-derived sweeteners thaumatin or neoculin peptide sweeteners, Nakajiama et al., 2008). Proteins are also efficiently glycosylated in *Aspergilli*, while undesired hyperglycosylation is usually not

observed. Whole genome sequences of several *Aspergillus* species are now available.

Recently, *Chlamydomonas reinhardtii*, a unicellular eukaryotic green algae has been proposed as a host to produce several forms of a human IgA antibody directed against herpes simplex virus (Franklin & Mayfield, 2005; Specht et al., 2010). The main reason for turning to algae is the claimed cost advantage and the absence of viral or prion contaminations that can harm humans. One can frequently read that microalgae grow faster (by an order or two of magnitude!) than terrestrial plants and biomass titers of 600-1000 mg/l dry weight are reached. However, one has to be careful. Microalgae can grow very fast when grown heterotrophically on glucose for example. In this case the performance of high cell density fermentations are almost as productive as those reported for bacteria and yeasts (Xiong et al., 2008). But things look different when microalgae are mass-produced phototrophically! For a number of technical and biological reasons not discussed here, doubling time of around 10 hours are probably a realistic assumption, leading to growth rates of μ= 0.07 per hour. This is very low when compared to the bacterium *Escherichia coli* where growth rates of 1 and higher are quite common in large scale. But growth rates are perceptibly higher than with all other eukaryotes except the yeasts and fungi mentioned above. The advantage

**3.2.1.2 Algae** 

over terrestrial plants is the smaller amount of soil used which leads to much better productivities per hectare for algae.

Algae might also have some other distinctive features, which could give them an advantage over other expression system for selected products. For example all three genomes (chloroplast, mitochondrial, nuclear) have been sequenced and can be transformed and each has distinct transcriptional, translational and post-translational properties. Proteins can accumulate at particularly high levels in the chloroplasts because of the absence of the silencing mechanisms. However, proteins in the chloroplasts are not glycosylated. Another feature of algae is, that they can be grown using sunlight or heterotrophically, or using a combination of both.

Fig. 4. Polyunsaturated fatty acids (PUFAs) such as docosohexanoic (DHA) acid are important building blocks of human brain tissue or the retina of the eye. The molecule can be produced by fermentation of marine algae. The picture shows the algae *Ulkenia sp*. which is used for the industrial production of DHA. They grow to form "footballs" consisting of single cells – 5 in this case with the product accumulated in the cells as oil drops. The size of the "football" is about 2 μm. Picture by Stefan Geimer, University of Bayreuth, Germany for Lonza (Copyright@Lonza).

The reality is that algae are routinely used for the production of polyunsaturated fatty acids only, and that other small molecule products such as carotenoids (Fernández-Sevilla et al., 2010 ) are merely in discussion. Although their growth is slow when compared to microbes, algae grow faster than terrestrial plants especially when grown heterotrophically with sugar as carbon and energy source. However, large-scale mass cultivation of algae using sunlight is far from being solved, and the calculations are sobering. Algae using CO2 as a carbon source are a theoretically ideal solution but are a long way from being cost-competitive in practice (Van Beilen, 2010).

Whether algae will ever play a role for recombinant protein production and especially for recombinant monoclonal antibodies is very doubtful for two reasons. Firstly, the industry is very risk averse, conservative and one does not want to change well established production systems. Secondly, there are microbial systems being successfully developed which are also able to provide human-like post translational modifications as we will see later.

#### **3.2.2. Higher eukaryonts**

#### **3.2.2.1 Plants**

**Whole plants**. Pharming or molecular farming describes the use of transgenic plants (potato, tobacco, banana, tomato, maize, rice, lettuce) and animals for the production of recombinant therapeutic proteins or other recombinant drugs. As with algae, the driver to use plants for recombinant proteins and monoclonal antibodies is the lower unit cost of agricultural production combined with easy scalability while post-translational modifications are possible (Ahmad et al., 2010).

Starting in the late 1980s human interferon, mouse immunoglobulins and human serum albumin were the first recombinant proteins to be tested for transgenic plant production. The first molecular farming project was in 1999 to trigger an immune response in humans with a safe and cost-effective edible vaccine (Langer, 2011). By 2007, about 370 plant made pharmaceuticals (PMPs) were undergoing field trials and about 16 of them were reported to be in clinical trials (Spök, 2007). Recombinant plants are an interesting form of production, and we see important differences to algae.


Because of the cost advantage, it is claimed that insulin could be a candidate for transgenic plant production as diabetes becomes a globally widespread disease, where affordable insulin is an absolute necessity. Vaccines in particular can be interesting products for plant made therapeutic proteins and other pharmaceuticals. MEDICAGO has developed a plantbased manufacturing platform (Proficia vaccine and antibody production system) with PHILIP MORRIS that produces (proficia) vaccine doses (H5N1 flu) in *Nicotiana benthamiana* leaf cells, a wild Australian relative of cultivated tobacco (Vécina et al., 2011). It is a transient expression system based on *Agrobacterium tumefaciens*, infecting plant cells and transferring genetic information to leaf cells. 600 million US\$ have been invested in NOVARTIS plant R&D facilities with 167 acres of land. SIGMA ALDRICH uses molecular farming to make avidin, aprotinin, lysozyme, lactoferrin and trypsin at small scale for use as chemicals. TrypZeanTM of SIGMA ALDRICH FINE CHEMICALS is a commercial recombinant bovine trypsin from transgenic corn.

Plant seeds are also being investigated as bioreactors for recombinant protein production (Lau & Sun, 2009), because they naturally contain large amounts of proteins but have low protease activities and low water content. Antibodies, vaccine antigens and other recombinant proteins have been shown to accumulate at high levels in seeds and remain stable and functional for years at ambient temperatures. As seeds can be eaten they allow oral delivery of vaccine antigens or pharmaceutical proteins for immunisation where oral delivery is an option. Unfortunately, most protein based therapeutics are usually not biologically active after oral consumption.

However, recombinant production of, for example, proteins in genetically modified plants has also several drawbacks.


**Whole plants**. Pharming or molecular farming describes the use of transgenic plants (potato, tobacco, banana, tomato, maize, rice, lettuce) and animals for the production of recombinant therapeutic proteins or other recombinant drugs. As with algae, the driver to use plants for recombinant proteins and monoclonal antibodies is the lower unit cost of agricultural production combined with easy scalability while post-translational

Starting in the late 1980s human interferon, mouse immunoglobulins and human serum albumin were the first recombinant proteins to be tested for transgenic plant production. The first molecular farming project was in 1999 to trigger an immune response in humans with a safe and cost-effective edible vaccine (Langer, 2011). By 2007, about 370 plant made pharmaceuticals (PMPs) were undergoing field trials and about 16 of them were reported to be in clinical trials (Spök, 2007). Recombinant plants are an interesting form of production,

1. If the product is in an edible form (for example in seeds or fruits) formulation may not

Because of the cost advantage, it is claimed that insulin could be a candidate for transgenic plant production as diabetes becomes a globally widespread disease, where affordable insulin is an absolute necessity. Vaccines in particular can be interesting products for plant made therapeutic proteins and other pharmaceuticals. MEDICAGO has developed a plantbased manufacturing platform (Proficia vaccine and antibody production system) with PHILIP MORRIS that produces (proficia) vaccine doses (H5N1 flu) in *Nicotiana benthamiana* leaf cells, a wild Australian relative of cultivated tobacco (Vécina et al., 2011). It is a transient expression system based on *Agrobacterium tumefaciens*, infecting plant cells and transferring genetic information to leaf cells. 600 million US\$ have been invested in NOVARTIS plant R&D facilities with 167 acres of land. SIGMA ALDRICH uses molecular farming to make avidin, aprotinin, lysozyme, lactoferrin and trypsin at small scale for use as chemicals. TrypZeanTM of SIGMA ALDRICH FINE CHEMICALS is a commercial recombinant bovine

Plant seeds are also being investigated as bioreactors for recombinant protein production (Lau & Sun, 2009), because they naturally contain large amounts of proteins but have low protease activities and low water content. Antibodies, vaccine antigens and other recombinant proteins have been shown to accumulate at high levels in seeds and remain stable and functional for years at ambient temperatures. As seeds can be eaten they allow oral delivery of vaccine antigens or pharmaceutical proteins for immunisation where oral delivery is an option. Unfortunately, most protein based therapeutics are usually not

However, recombinant production of, for example, proteins in genetically modified plants

3. logistics can be easier as cooling may not be needed for storage and transport.

2. the product in plant material will be easily and stably stored and

**3.2.2. Higher eukaryonts** 

modifications are possible (Ahmad et al., 2010).

and we see important differences to algae.

be necessary

trypsin from transgenic corn.

has also several drawbacks.

biologically active after oral consumption.

**3.2.2.1 Plants** 


Plant transformation using physical methods is rather inefficient (gene gun). Transient expression is less time consuming but limited in scale. Recombinant *Agrobacterium tumefaciens* or plant viruses like the tobacco mosaic virus are used for transient expression.

Let us consider growth rates and use rapeseed as an example as it is one of the industrially used genetically modified crops. At the end of August one hectare is inoculated with 3-4 kg of rapeseeds. At the end of July in the following year (the plant needs a cold period to thrive – the so called vernalisation) the same hectare yields on average 4000 kg of seeds, from which 1700 l of rapeseed oil can be extracted. The biomass has increased by a factor of 1000 in 11 months. This represents a growth rate of μ = 0,05 h-1 when calculated for the seeds growing on one hectare. This is relatively high, but it should be considered that one uses 1 hectare during one full year to produce 1'700 l of oil. Using heterotrophic algal fermentation probably 2000 times more oil could probably be produced on the same hectare. But then again, one still needs hectares to produce the necessary carbon and energy source for the fermentation, and we are thus back to square one with algae.

Coming back to transgenic plants and recombinant proteins: because of strong negative pressure from non-governmental environmental organisations and consumer organisations, transgenic plants in general will be slow to be used, also for the manufacturing of therapeutic proteins, unless they offer some of the advantages mentioned, such as edible and cheap recombinant drugs and vaccines for example.

**Plant cell culture**. An alternative to grow whole plants is to grow plant cells in suspension cultures. GREENVAX produces influenza vaccines by growing tobacco using XCELLEREX XDR single use bioreactors. GREENOVATION (Greenovation 2011) proposes bryotechnology (bryophytes = mosses) for recombinant protein production. PROTALIX's BIOTHERAPEUTICS Inc produces the enzyme replacement drug taliglucerase alfa for the treatment of Gauchers disease in disposable 800 litre bioreactors with carrot cells. Despite the fact that large companies such as PFIZER also stake a claim in plant cell-made biopharmaceuticals (taligurase alfa in carrot cells against Gaucher's disease of PROTALIX, Ratner, 2010), we conclude that commercial heterologous protein production in transgenic plants will not be economically relevant in the foreseeable future.

Whole plants and plant cell suspension culture, both will not play a relevant role in the production of therapeutic proteins and monoclonal antibodies. However, as with whole plants the situation is a different one for the recombinant production of other products and molecules such as secondary metabolites. The shift of wealth from west to east and north to south will fortunately eliminate economic inequalities. But an increasing global prosperity also increases pressure on naturally sourced raw materials especially from plants. Plant cell culture, recombinant or not, is a great alternative and will help to relieve pressure on partly even endangered species.

#### **3.2.2.2 Protozoa**

Heterologous expression of proteins or protein fragments is also possible in protozoa. *Dictyostelium discoideum* (Han et al., 2004), *Leishmania tarentolae*, *Perkinsus marinus* and especially *Tetrahymena thermophila* are organisms used, for which recombinant protozoa techniques exist. The ciliate-based expression system (CIPEX) of CILIAN AG (Cilian, 2011) is a proven tool which makes protozoa a potential competitor of mammalian cells such as CHO cells. For example viral influenza haemaglutinin, parasite surface proteins and a human intestinal alkaline phosphatase and a human DNase I were expressed and secreted in the unicellular non-pathogenic protozoan ciliate *Tetrahymena thermophila* (Hartmann et al., 2010; Aldag et al., 2011). The genome of *Tetrahymena thermophila* is entirely sequenced, and it is one of the best-characterized unicellular eukaryotes as it has served for long time as a laboratory model in biology.

Protozoa are naturally mostly feeding on bacteria. However, they can also easily be grown by pinocytosis in a culture medium containing only soluble components. Growth rates of protozoa in non-optimized media and culture conditions reach values of μ = 0.02 h-1 and viable cell densities of 1 x 107 ml-1. Protozoa can also be cultivated in normal continuously stirred tank bioreactors and possess the sub-cellular machinery to perform eukaryotic posttranslational protein modifications. However, protozoan-based expression systems have not yet made the transition from a laboratory model to an established recombinant protein platform at large scale. The main advantage of protozoa is that they are free of endogenous infectious agents as their genetics and phylogenetic distance to higher animals make viral infection unlikely. As with other expression systems high gene doses allow relatively high volumetric productivities [Qp] and scale-up has been proven up to the 1.5 m3 scale. Protozoa have a consistent oligo-mannose N-glycosylation albeit not of mammalian nature.

Fig. 5. Picture of *Tetrahymena thermopile*, one of the best characterized unicellular nonpathogenic protozoa, expressing a recombinant green fluorescent protein in phagosomes; protozoa can also secrete recombinant products. The ciliates, distributed on the cell surface, cannot been seen because the photograph was taken with a light microscope. The cell measures about 50 μm in diameter (Copyright@Cilian).

In the case of *Tetrahymena thermophila* (Figure 5) and with optimized media and culture conditions, cell densities of over 2 x 107 cells per milliliter and dry biomass titers of 8 g/l can be reached in high cell density protozoa fermentations and 50 g/l in continuous fermentations with cell retention. Generation times become very short and values between 1.4 and 3 hours can be obtained.

In conclusion, protozoa such as *Tetrahymena thermophila* have a certain potential for the recombinant production of selected recombinant proteins. To some extent they combine the advantages of the microbial and the mammalian world as they can grow rapidly to relatively high cell densities and have a posttranslational modification apparatus. If the necessary tools for the manufacturing with stable expression, conserved sequences and target glycosylation can be established, we may see cultivation of protozoa in large scale fermenters in the future for human enzymes, monoclonal antibodies and, in particular protein vaccines. Because of their faster growth, the first vaccine targets in preclinical phase are already in development using protozoan recombinant expression.

#### **3.2.2.3 Insect cells**

226 Innovations in Biotechnology

Heterologous expression of proteins or protein fragments is also possible in protozoa. *Dictyostelium discoideum* (Han et al., 2004), *Leishmania tarentolae*, *Perkinsus marinus* and especially *Tetrahymena thermophila* are organisms used, for which recombinant protozoa techniques exist. The ciliate-based expression system (CIPEX) of CILIAN AG (Cilian, 2011) is a proven tool which makes protozoa a potential competitor of mammalian cells such as CHO cells. For example viral influenza haemaglutinin, parasite surface proteins and a human intestinal alkaline phosphatase and a human DNase I were expressed and secreted in the unicellular non-pathogenic protozoan ciliate *Tetrahymena thermophila* (Hartmann et al., 2010; Aldag et al., 2011). The genome of *Tetrahymena thermophila* is entirely sequenced, and it is one of the best-characterized unicellular eukaryotes as it has served for long time as a

Protozoa are naturally mostly feeding on bacteria. However, they can also easily be grown by pinocytosis in a culture medium containing only soluble components. Growth rates of protozoa in non-optimized media and culture conditions reach values of μ = 0.02 h-1 and viable cell densities of 1 x 107 ml-1. Protozoa can also be cultivated in normal continuously stirred tank bioreactors and possess the sub-cellular machinery to perform eukaryotic posttranslational protein modifications. However, protozoan-based expression systems have not yet made the transition from a laboratory model to an established recombinant protein platform at large scale. The main advantage of protozoa is that they are free of endogenous infectious agents as their genetics and phylogenetic distance to higher animals make viral infection unlikely. As with other expression systems high gene doses allow relatively high volumetric productivities [Qp] and scale-up has been proven up to the 1.5 m3 scale. Protozoa

have a consistent oligo-mannose N-glycosylation albeit not of mammalian nature.

Fig. 5. Picture of *Tetrahymena thermopile*, one of the best characterized unicellular nonpathogenic protozoa, expressing a recombinant green fluorescent protein in phagosomes; protozoa can also secrete recombinant products. The ciliates, distributed on the cell surface, cannot been seen because the photograph was taken with a light microscope. The cell

In the case of *Tetrahymena thermophila* (Figure 5) and with optimized media and culture conditions, cell densities of over 2 x 107 cells per milliliter and dry biomass titers of 8 g/l can

measures about 50 μm in diameter (Copyright@Cilian).

**3.2.2.2 Protozoa** 

laboratory model in biology.

**Insect cells culture**. A few recombinant proteins made using insect cell lines have already been approved for veterinary use. Only one vaccine, Cervarix® of GLAXO SMITH KLINE, has been approved for human use within the EU (in 2007) and in the US in 2009. Production with insect cells has the following advantages:


The *Spodoptera frugiperda* cell lines (Sf-9 and Sf-21, Figure 6) and *Trichoplusia ni* are frequently used host cell lines for recombinant protein expression and production via infection with a genetically modified baculovirus expression vector system, BEVS (Invitrogen, 2011; Quiagen, 2011; Tiwary et al., 2010). As for other cell lines used for production, insect cell lines need to be "immortalized" or rendered permanent.

Fig. 6. Cells from the ovarium of this moth *Spodopera frugiperda* are used in combination with baculoviruses for the recombinant production of proteins in suspension culture in fermenters. The method was first introduced in 1982 and in 1985 the first recombinant protein interleukin-2 was expressed in moth cells (Copyright@Canadian Biodiversity Information Facility, 2011).

Taticek et al. (2001) compared the growth and recombinant protein expression in suspension culture and attached cells culture of *Spodoptera frugiperda* and *Trichoplusia ni* (Taticek et al., 2001) expressing *Escherichia coli* beta-galactosidase or human secreted alkaline phosphatase. The production of both enzymes varied as a function of inoculum size, media, culture conditions etc. Cell densities of 5 x 106 viable cells/ml and doubling of 20 hours were reached, which corresponds to a growth rate of about μ = 0.04 h-1. It is not clear whether a typical large scale fed batch cycle for insect cells would also be around 4 to 5 days which would about 2 to 3 times faster than a batch with mammalian cell culture. However, generally insect cells have two major disadvantages:


Vermasvori et al. compared the production of a model protein (Negative factor or Nef) in *Escherichia coli*, *Pichia pastoris* and the *Drosophila* S2 cell line (Vermasvuori et al., 2009). When studying the systems purely economically the microbial system had significantly lower manufacturing costs than the insect cell lines. The most significant difference between the manufacturing costs of the two microbial systems was due to the much longer strain construction time with the *Pichia pastoris*. The manufacturing costs for the production of 100 mg Nef protein were


3. *Drosophila* S2 21111 €

Omitting the strain construction costs, the microbial systems were cost-wise fairly comparable. These numbers from small scale experiments insinuate that insect cells could be theoretically a threat for mammalian cell culture. However, there are no compelling data showing an advantage by switching to insect cells for large scale production.

**Transgenic insects**. The production of recombinant proteins in live animals is an option, as production of therapeutic proteins and monoclonal antibodies is an option in living plants. The most frequently proposed method is that of producing and extracting recombinant proteins from the milk of transgenic livestock. However, insects such as silkworms (Fraser & Jarvis, 2010), can theoretically also be used for recombinant protein production. However, we believe that transgenic insects will not play a role in the production of pharmaceuticals rather than in the control of insect populations and prevention and control of parasites and disease transmission in man, animals and plants.

#### **3.2.2.4 Mammalian cells**

**Mammalian cells in culture**. The clinical and commercial success of mainly monoclonal antibodies has led to the rapid development of mammalian cell culture within a short time after the landmark findings of Cesar Milstein and Georges Köhler in the mid 70s. Mammalian cell culture is a well established production method for the production of therapeutic proteins and monoclonal antibodies. One of the most frequent mammalian hosts used is the CHO cell line (Chinese hamster ovary) in combination with the GS expression system or the DHFR (dihydroolate reductase) expression system (Birch & Racher, 2006). The mouse NS0 cells cell line is another option which is less frequently chosen today.

Taticek et al. (2001) compared the growth and recombinant protein expression in suspension culture and attached cells culture of *Spodoptera frugiperda* and *Trichoplusia ni* (Taticek et al., 2001) expressing *Escherichia coli* beta-galactosidase or human secreted alkaline phosphatase. The production of both enzymes varied as a function of inoculum size, media, culture conditions etc. Cell densities of 5 x 106 viable cells/ml and doubling of 20 hours were reached, which corresponds to a growth rate of about μ = 0.04 h-1. It is not clear whether a typical large scale fed batch cycle for insect cells would also be around 4 to 5 days which would about 2 to 3 times faster than a batch with mammalian cell culture. However,

1. the baculovirus system results in cell death and lysis of the host insect cells and the

Vermasvori et al. compared the production of a model protein (Negative factor or Nef) in *Escherichia coli*, *Pichia pastoris* and the *Drosophila* S2 cell line (Vermasvuori et al., 2009). When studying the systems purely economically the microbial system had significantly lower manufacturing costs than the insect cell lines. The most significant difference between the manufacturing costs of the two microbial systems was due to the much longer strain construction time with the *Pichia pastoris*. The manufacturing costs for the production of 100

Omitting the strain construction costs, the microbial systems were cost-wise fairly comparable. These numbers from small scale experiments insinuate that insect cells could be theoretically a threat for mammalian cell culture. However, there are no compelling data

**Transgenic insects**. The production of recombinant proteins in live animals is an option, as production of therapeutic proteins and monoclonal antibodies is an option in living plants. The most frequently proposed method is that of producing and extracting recombinant proteins from the milk of transgenic livestock. However, insects such as silkworms (Fraser & Jarvis, 2010), can theoretically also be used for recombinant protein production. However, we believe that transgenic insects will not play a role in the production of pharmaceuticals rather than in the control of insect populations and prevention and control of parasites and

**Mammalian cells in culture**. The clinical and commercial success of mainly monoclonal antibodies has led to the rapid development of mammalian cell culture within a short time after the landmark findings of Cesar Milstein and Georges Köhler in the mid 70s. Mammalian cell culture is a well established production method for the production of therapeutic proteins and monoclonal antibodies. One of the most frequent mammalian hosts used is the CHO cell line (Chinese hamster ovary) in combination with the GS expression system or the DHFR (dihydroolate reductase) expression system (Birch & Racher, 2006). The mouse NS0 cells cell line is another option which is less frequently chosen today.

showing an advantage by switching to insect cells for large scale production.

release of cell proteins, which offsets again the high productivity of the cells

2. N-glycosylation of insect cells is different from mammalian cells.

generally insect cells have two major disadvantages:

mg Nef protein were

**3.2.2.4 Mammalian cells** 

1. Escherichia coli 6456 € 2. *Pichia pastoris* 13382 € 3. *Drosophila* S2 21111 €

disease transmission in man, animals and plants.

Continuously stirred tank fermenters are operated (Varley & Birch, 1999) with volumes of up to 20 m3. An average batch lasts about two weeks with specific productivities up to 100 pg/cell x day. Product titers for antibodies of several grams per litre are almost routine today, making product recovery and purification the greater challenge from a manufacturing perspective. Defined media are available for high growth rates, stable expression, low lactate and byproduct formation to facilitate fermentation, isolation and purification. Lonza (Lonza, 2011) recently introduced a novel medium and feeding platform for its GS-expression systemTM, which leads to product titers of up to 10 g per litre!

Rapid and efficient development of stable production cell lines is a critical step and transfection alternatives for the quick preparation of stable mammalian production cell lines for recombinant proteins are needed (Wurm, 2004; Birch et al., 2005). The PiggyBac transposon is an example of a novel delivery vehicle for rapid and efficient recombinant cell line generation (Matasci et al., 2011).

Unfortunately, the generation of a stable production cell line remains a time consuming endeavour. However, for early feasibility tests with a protein, transient gene expression and protein production may be sufficient (Ye et al., 2009). EXCELLGENE SA (Excellgene, 2011) has successfully established transient gene expression for fast protein delivery. Production and recovery of several grams of purified protein can be done within 3-4 weeks. However, transient recombination is limited by two factors:


Nevertheless, transient technology allows a reduction of cost and time for biopharmaceuticals with mammalian cells. It is not yet a valid method for regular production or even material for clinical material testing of e.g. drug candidates. Cell lines may not be stable but at least production can start within a matter of weeks. The combination of transient expression with disposable reactors, which are now available up to the m3 scale (Eibl et al., 2010), makes the production of several 100 g of new proteins possible in a short time.

**Transgenic animals**. Clones and transgenics of livestock and companion animals have become a reality in animal farming, meat production and even pharmaceutical production since the first transgenic animals were created in the laboratory. Mice were the first mammals to be genetically modified in the early 1980s and in 1987 the breeding of a tPA (human tissue plasminogen activator) producing transgenic mouse was reported by Simons *et al.* (1987). As with any other organism mentioned above, it is also possible to introduce or delete existing genes in an animal cell and have the modification passed on to the next generation resulting in a new phenotype of the living animal.

In 2004 about a dozen companies had products in development in transgenic animals (Keefer, 2004) but many of them have been abandoned. Human antithrombin III (hAT) of GTC BIOTHERAPETICS, a glycoprotein controlling blood clotting, was the first approved biopharmaceutical from transgenic animals. NEXIA BIOPHARMACEUTICALS published a patent in 2002 for the production of spider silk biofilaments in transgenic animals using milk or urine-specific promoters (Karazas & Turcotte, 2003) but the process is not competitive with microbial production. The Dutch company GEN PHARMING was a pioneer in the field of production of human lactoferrin in ruminants. Alpha 1-antitrypsin, fibrinogen, tissue plasminogen activator, vaccines, human monoclonal and polyclonal antibodies are other example of products.

This technology has, however, been the subject of controversial discussions (for example "cloned meat") as these biotechnology applications have been judged with evident hierarchies of acceptability (Einsiedel, 2005). Transgenic animals are particularly sensitive but they nevertheless offer options to produce a therapeutic protein in a fluid of the animal (milk, blood, urine) of mice, goats or cows. Coupling the target protein to a signal one can direct the expression into the mammary glands and milk - the optimal choice.

Nonmammalian animals such as birds or insects (see above) can also be used for the production of glycosylated therapeutic proteins and monoclonal antibodies, especially in the egg white or egg yolk of transgenic chicken.

Patel et al., 2007 claim, that animals are more cost effective bioreactors, with 16 therapeutic proteins in development in transgenic animals (sheep 5, pig 2, goat 4, cow 5) plus several monoclonal antibodies in cows, goats and chicken. Productivities in terms of milk containing recombinant protein given are ~8000 litres containing a total 40 – 80 kg of recombinant protein. However we disagree, and think that transgenic animals will only be used in a very few and exceptional cases for the following reasons:


Take productivity as one example, a single cow will produce ~60 kg of a therapeutic protein in one full year which has still to be isolated and purified. In the case of a goat or a sheep that number drops to 4 kg and 2.5 kg per year, respectively. This should be compared with the productivity of a 20m3 bioreactor with mammalian cells which produces on average 125 kg in a single month! That means one bioreactor replaces 25 cows, 375 goats or 600 sheep.

How do depreciations between 25 cows and a 20m3 plant differ? The cost of one transgenic animal is high, 500000 US\$ for one calf according to Keefer (2004). This is due to inefficiencies in this technique as over 1000 bovine, 300 sheep and 200 goat oocytes must be injected. Goats and sheep with their shorter generation interval are less costly, and the figures may look somewhat better today. On the other hand and based on numbers given by Patel et al (2007), we calculated an average of about 50000 US\$ in value created per animal and per year. It now all depends how fast a productive herd can be created by cloning and how long the productive period of a cloned transgenic animal is? Over how many productive years do we have to depreciate the animals? One important disadvantage of a capital investment in a transgenic cow is that a cow is a dedicated "plant" while a bioreactor is a multipurpose and multiproduct installation.

We do not believe that transgenic animals are an attractive alternative except for a few very special exceptions. However, the one argument for transgenic animals (as for transgenic plants and plant seeds) is the case in which the product can be consumed directly with the milk. It would make therapy affordable, and one can imagine that an oral protein-based vaccine would be an ideal candidate for a transgenic animal.

tissue plasminogen activator, vaccines, human monoclonal and polyclonal antibodies are

This technology has, however, been the subject of controversial discussions (for example "cloned meat") as these biotechnology applications have been judged with evident hierarchies of acceptability (Einsiedel, 2005). Transgenic animals are particularly sensitive but they nevertheless offer options to produce a therapeutic protein in a fluid of the animal (milk, blood, urine) of mice, goats or cows. Coupling the target protein to a signal one can

Nonmammalian animals such as birds or insects (see above) can also be used for the production of glycosylated therapeutic proteins and monoclonal antibodies, especially in the

Patel et al., 2007 claim, that animals are more cost effective bioreactors, with 16 therapeutic proteins in development in transgenic animals (sheep 5, pig 2, goat 4, cow 5) plus several monoclonal antibodies in cows, goats and chicken. Productivities in terms of milk containing recombinant protein given are ~8000 litres containing a total 40 – 80 kg of recombinant protein. However we disagree, and think that transgenic animals will only be

Take productivity as one example, a single cow will produce ~60 kg of a therapeutic protein in one full year which has still to be isolated and purified. In the case of a goat or a sheep that number drops to 4 kg and 2.5 kg per year, respectively. This should be compared with the productivity of a 20m3 bioreactor with mammalian cells which produces on average 125 kg in a single month! That means one bioreactor replaces 25 cows, 375 goats or 600 sheep. How do depreciations between 25 cows and a 20m3 plant differ? The cost of one transgenic animal is high, 500000 US\$ for one calf according to Keefer (2004). This is due to inefficiencies in this technique as over 1000 bovine, 300 sheep and 200 goat oocytes must be injected. Goats and sheep with their shorter generation interval are less costly, and the figures may look somewhat better today. On the other hand and based on numbers given by Patel et al (2007), we calculated an average of about 50000 US\$ in value created per animal and per year. It now all depends how fast a productive herd can be created by cloning and how long the productive period of a cloned transgenic animal is? Over how many productive years do we have to depreciate the animals? One important disadvantage of a capital investment in a transgenic cow is that a cow is a dedicated "plant" while a bioreactor

We do not believe that transgenic animals are an attractive alternative except for a few very special exceptions. However, the one argument for transgenic animals (as for transgenic plants and plant seeds) is the case in which the product can be consumed directly with the milk. It would make therapy affordable, and one can imagine that an oral protein-based

direct the expression into the mammary glands and milk - the optimal choice.

used in a very few and exceptional cases for the following reasons:

1. low success rate of gene transfer (e.g. 0,1% with cows)

4. low consumer acceptance in view of better alternatives

2. cost and time to produce transgenic animals 3. possible infectious agents from mammals

is a multipurpose and multiproduct installation.

vaccine would be an ideal candidate for a transgenic animal.

5. many alternatives exist

other example of products.

egg white or egg yolk of transgenic chicken.

Fig. 7. Microbial and mammalian cell culture are used in 93% of all cases for the production of therapeutic proteins. See also Figure 8 with the spread of the individual expression systems.

In summary, microbial fermentation and mammalian cell culture will continue to carry the main burden for the production of recombinant proteins as it is already the case today (Figure 7). Other expression systems, especially plant-based and algae, will have potential for recombinant protein niche applications. The situation is different for small molecule pharmaceuticals, neutraceuticals and fine chemicals, where a more varied host-expression system combination will be needed. However, even in the latter case one will first fall back on proven methods. We will now describe in more details the beacon in recombinant microbial expression – *Escherichia coli*.

#### **4.** *Escherichia coli* **as work horse**

In the year 1885 the German paediatrician Theodor Escherich (1857-1911) described a bacterium, which he called "*Bacterium coli comunale*". At that time nobody could anticipate that this bacterium, which later on was named after him *Escherichia coli*, would become world famous as a model organism in the field of molecular biology and as "the" minifactory for recombinant protein manufacturing (Piechocki, 1989).

This is best demonstrated by statistical figures related to expression platforms in use (Figure 8). In the reported year 34% of all recombinant therapeutic proteins registered in the US and EU were produced by means of *Escherichia coli* based expression technology. The second and third most successful expression platforms were Chinese Hamster Ovary cells with a 30% and yeast systems, mostly *Sacharomyces cerevisiae*, with a 12% shares respectively (Rader, 2008).

#### **4.1 Why is** *Escherichia coli* **such a popular expression host?**

Although there is no gold standard platform in microbial expression, expression systems based on *Escherichia coli* have dominated microbial expression for more than 30 years. One can only speculate on the reasons for this domination. *Escherichia coli* and its phages were early objects and models for studying molecular biology topics, especially aspects related to the understanding of gene functions and regulation. More than 10 scientists received the Nobel prize for exciting discoveries connected to research on *Escherichia coli* (Piechocki, 1989). Worth mentioning is the isolation and purification of a restriction enzyme for the first time by Werner Arber in 1968. These enzymes are enabling tools in the area of rDNA technology. The rapid pace in the development of expression technology and of genetic engineering tools is best reflected by the quite early launch of a first biopharma product, expressed in *Escherichia coli*, recombinant human insulin in 1982 (Humulin®, licensed by GENENTECH to ELI LILLY). This is even more remarkable if one considers the lengthy approval procedure for therapeutics. *Escherichia coli* based biotechnology profited directly from the multitude of fundamental discoveries made on this model organism, giving this species a timely technical advantage in use as expression host.

Fig. 8. Percentage of expression platforms used for the manufacture of bio-therapeutics in the US and the EU. The figure is based on numbers published by Rader (2008).

Other explanations for the success of this microorganism are low genome complexity and the extra-chromosomal genetic elements, plasmids, which ease both (a) in-vitro manipulation of genetic elements and (b) insertion of homologous and foreign genes into the organism.

Besides low safety concerns and high regulatory acceptance, ease of use and familiarity with the organism was in favour of *Escherichia coli*. There is hardly a student in biology who has not run at least one cloning experiment in one of the *Escherichia coli* expression systems used in academia. Since its first industrial applications, *Escherichia coli* expression technology has been continuously improved with the aims of gaining control of the quality of the recombinant products and increasing the product titre in fermentation, the latter obviously being crucial to process economy.

#### **4.2 What are the characteristics of an industrial** *Escherichia coli* **expression platform?**

Incremental improvements led to the development of *Escherichia coli* based expression platforms that are suitable for industrial use. More precisely these systems allow for robust, reliable and scalable processes and economical manufacturing. High performance expression technology is characterized by two properties: (a) high volumetric productivity Qp, preferentially due to a high specific product production rate qp and (b) high control on product quality, meaning that no or only a negligible amount of product variants are produced.

Industrial expression systems distinguish themselves from academic systems by an optimized combination of the various components of which an expression system is made. Basically, a bacterial expression system is composed of a host and a vector which contains the product coding DNA, a selection marker and various regulatory elements. Regulatory elements are promoters, signal sequences, ribosome binding sites, transcription terminators and vector replication or integration regions.

**Host**. The host organism provides specific features to an expression system as a result of its genetic background; these features include:


232 Innovations in Biotechnology

engineering tools is best reflected by the quite early launch of a first biopharma product, expressed in *Escherichia coli*, recombinant human insulin in 1982 (Humulin®, licensed by GENENTECH to ELI LILLY). This is even more remarkable if one considers the lengthy approval procedure for therapeutics. *Escherichia coli* based biotechnology profited directly from the multitude of fundamental discoveries made on this model organism, giving this

**7%** E. coli

Streptococci S. cerevisiae P. pastoris Insect cells

CHO Murine cells Monkey cells Human cells

Chicken embryos (eggs) Chicken embryo cells

Transgenic mammals Viruses as life vaccines

**34%**

Fig. 8. Percentage of expression platforms used for the manufacture of bio-therapeutics in

Other explanations for the success of this microorganism are low genome complexity and the extra-chromosomal genetic elements, plasmids, which ease both (a) in-vitro manipulation of genetic elements and (b) insertion of homologous and foreign genes into

Besides low safety concerns and high regulatory acceptance, ease of use and familiarity with the organism was in favour of *Escherichia coli*. There is hardly a student in biology who has not run at least one cloning experiment in one of the *Escherichia coli* expression systems used in academia. Since its first industrial applications, *Escherichia coli* expression technology has been continuously improved with the aims of gaining control of the quality of the recombinant products and increasing the product titre in fermentation, the latter obviously

**4.2 What are the characteristics of an industrial** *Escherichia coli* **expression platform?**  Incremental improvements led to the development of *Escherichia coli* based expression platforms that are suitable for industrial use. More precisely these systems allow for robust, reliable and scalable processes and economical manufacturing. High performance expression technology is characterized by two properties: (a) high volumetric productivity Qp, preferentially due to a high specific product production rate qp and (b) high control on product quality, meaning that no or only a negligible amount of product

the US and the EU. The figure is based on numbers published by Rader (2008).

species a timely technical advantage in use as expression host.

**12%**

**30%**

the organism.

being crucial to process economy.

variants are produced.


High cell densities are most desirable since a positive correlation exists between the amount of biomass (X) and the product production rate (rp). The corresponding equation is

rp = qp \* X(product production rate = specific product production rate x biomass).

The relationship above should not be confused with growth rate dependency on the product production rate, which can be optimal at high or low growth rates. It is possible that in the worst case maximal specific production rates rp correlate with very low growth rate close to maintenance (Meyer & Fiechter,1985). In that case production requires two separate phases, growth and production phase.

Commonly used *Escherichia coli* host strains are listed in Table 7. BL21 is the most frequently used *Escherichia coli host*. BL21 popularity is based on


The organism is not sensitive to high glucose concentration due to its active glyoxylate shunt, gluconeogenesis and anaplerotic pathways and a more active TCA cycle, which leads to better glucose utilisation and lower acetate production (Phue et al., 2008). However, when used in combination with the T7 expression system and when exposed to stress, this host is at risk of bacteriophage DE3 excision. For this reason laboratories started to promote the use of BLR, a recA¯ mutant of BL21. In our experience an increased use of W3110 is taking place in the industry. This can be attributed to the excellent production capabilities of this host. Orgami strains may allow for better formation of disulfide bonds in the cytoplasm due to lower reducing power in the cytoplasm (Novagen, 2011). The endA¯ and recA¯ hosts DH5 and JM109 are the organisms of choice for the manufacture of pDNA. The lack of endonuclease 1 which degrades double stranded DNA positively affects stability of pDNA (Phue et al., 2008). In conclusion, product nature and product characteristics determine the selection of the most optimal host.


Table 7. Frequently used *Escherichia coli* host strains and related specific characteristics.

**Promoters**. Promoters control the expression to the extent of how much and at which point in time mRNa is synthesized. As a consequence they control production of product. A large number of promoters that allow modulation of the mode of induction in a desired way are used in the industry. Lactose or lactose-analogue IPTG induced T7 promoterbased expression systems currently dominate the market. Apart from T5, araB and phoA, other classical promoters such as lambda, lac, trp, PL, PR, tetA and trc/tac are rather seldom used.

Novel promoters are under development and continuously make their way into industrial applications. New disaccharide inducible promoters, which induce protein production during the stationary growth phase, have recently been successfully applied in *Escherichia coli* based biopharmaceutical processes. Some of these are part of Lonza's XS TechnologiesTM *Escherichia coli* platform, which has been chosen as an example to discuss performance of current leading industrial *Escherichia coli* expression platforms (Lonza). Depending on the promoter the induction signal is of a chemical or physical nature. Some of the above mentioned *Escherichia coli* promoters have been successfully used in other bacterial systems such as *Bacillus subtilis* (Alexander et al., 2007).

State of the art industrial expression platforms allow for product specific modulation of the rate of protein synthesis. Proteins of high complexity, having disulphide bonds are typically best produced at a lower production rate. In contrast proteins of low complexity are often produced at a high production rate, thus achieving high concentrations after a short time of fermentation. Productivity is often affected by interaction between specific promoters and recombinant target proteins. Therefore, in general, it makes sense to screen for the performance of different promoters.

**Signal Sequences**. Signal sequences determine whether a product is directed through the cellular membrane and out of the cytoplasma; the signal sequence is cleaved during the secretion step. Secretion is desirable in many cases, since a large proportion of target proteins do not fold correctly in the reducing cytoplasmic environment. Folding requires oxidative conditions which are provided outside the cytoplasm. Secretion sequences

B strain, recA¯ mutant of BL21 with decreased likelihood of excision of DE3

K-12 or B strains which supply tRNAs for codons that are rare in E. coli

K-12 strain, recA¯, endA¯, often used for pDNA manufacture

**Host Strains Strain Characteristics**

K-12 strain, recA¯

K-12 strain K-12 strain K-12 strain

such as *Bacillus subtilis* (Alexander et al., 2007).

performance of different promoters.

B strain, *lon* and *ompT* protease deficiencies

K-12 or B strains with mutations in *trxB* and *gor* 

Table 7. Frequently used *Escherichia coli* host strains and related specific characteristics.

**Promoters**. Promoters control the expression to the extent of how much and at which point in time mRNa is synthesized. As a consequence they control production of product. A large number of promoters that allow modulation of the mode of induction in a desired way are used in the industry. Lactose or lactose-analogue IPTG induced T7 promoterbased expression systems currently dominate the market. Apart from T5, araB and phoA, other classical promoters such as lambda, lac, trp, PL, PR, tetA and trc/tac are rather

Novel promoters are under development and continuously make their way into industrial applications. New disaccharide inducible promoters, which induce protein production during the stationary growth phase, have recently been successfully applied in *Escherichia coli* based biopharmaceutical processes. Some of these are part of Lonza's XS TechnologiesTM *Escherichia coli* platform, which has been chosen as an example to discuss performance of current leading industrial *Escherichia coli* expression platforms (Lonza). Depending on the promoter the induction signal is of a chemical or physical nature. Some of the above mentioned *Escherichia coli* promoters have been successfully used in other bacterial systems

State of the art industrial expression platforms allow for product specific modulation of the rate of protein synthesis. Proteins of high complexity, having disulphide bonds are typically best produced at a lower production rate. In contrast proteins of low complexity are often produced at a high production rate, thus achieving high concentrations after a short time of fermentation. Productivity is often affected by interaction between specific promoters and recombinant target proteins. Therefore, in general, it makes sense to screen for the

**Signal Sequences**. Signal sequences determine whether a product is directed through the cellular membrane and out of the cytoplasma; the signal sequence is cleaved during the secretion step. Secretion is desirable in many cases, since a large proportion of target proteins do not fold correctly in the reducing cytoplasmic environment. Folding requires oxidative conditions which are provided outside the cytoplasm. Secretion sequences

**Escherichia coli** 

Orgami strains Rosetta strains

seldom used.

BL21 HMS174 BLR

W3110 MG1655 RV308 DH5

frequently used in *Escherichia coli* are MalE, OmpA and PelB. Yeast organisms such as *Saccharomyces*, *Pichia*, *Hansenula*, *Yarrowia* and Gram-positive bacteria such as *Bacillus* and *Corynebacterium* secrete proteins which carry a secretion signal into the medium, whereas Gram-negative genera such as *Escherichia*, *Pseudomonas* and *Ralstonia* direct the product through the inner membrane into the periplasmic space. This is what the theory says. According to the authors' experience, the *Escherichia coli* outer membrane is leaky for a large proportion of secreted proteins which are supposed to accumulate in the periplasmic space. The observed partitioning of the secreted protein between fermentation medium and periplasmic space can be influenced to some extent by modifying the fermentation conditions. The latter behaviour is product dependent and for the time being not predictable.

**Selection markers**. Selection markers are necessary for the cloning process and crucial for controlling plasmid stability. Typical microbial selection markers are antibiotic resistance genes. However, the prevalence of β-lactam allergies strongly suggests avoidance of the use of ampicillin and other β-lactam derivatives for the purpose of selective pressure in the manufacture of clinical products. Optional stabilization systems used in *Escherichia coli* are based on antidote and poison gene systems with the poison gene being integrated into the bacterial chromosome and the antidote gene located on the plasmid, respectively (Peubez et al., 2010). Constitutive expression of the antidote gene stabilizes plasmid-containing cells. A system based on the mode of action described above is marketed by DELPHI GENETICS Inc (Delphigenetics, 2011).

Besides the above mentioned regulatory aspect, Rozkov et al. (2004) note another one that should be taken into consideration when selecting the plasmid stabilizing system. According to these authors, the presence of an antibiotic selection marker imposes a huge metabolic burden on an expression system. They found that the product of the selection marker gene accounted for up to 18% of the cell protein. A negative effect on the recombinant expression of the genes of interest is highly likely. Due to constitutive expression this is the case even in the absence of the corresponding antibiotic in the medium. One way to circumvent this problem is to use complementation markers, i.e. marker genes that complement an auxotrophic chromosomal mutation.

A majority of successful technologies, genetic elements and related know-how, are subject to patent protection or trade secrets, as shown also in Table 6. In particular, multiple license requirements for the use of a specific production technology can lead to an unfavourable economic situation. On the other hand, off-patent expression systems and elements thereof are usually not state of the art. Since process economy depends to a large extent on productive and robust strains, outsourcing strain development to a specialised laboratory is often justified, given that licensing cost remain reasonable. The resulting economic benefits on the process side typically offset the costs related to accessing a productive and robust state of the art industrial strain platform.

#### **4.3 A more critical view on** *Escherichia coli* **expression platforms**

Despite their dominant position within microbial expression *Escherichia coli* based expression platforms also exhibit weaknesses which should not be ignored. These drawbacks are shared with other commercialised Gram-negative expression platforms as *Pseudomonas* and *Ralstonia*. Among these disadvantages are


WACKER Chemie has commercialised a K-12 derivative that exhibits higher secretion ability than other K-12 and B strains (Mücke et al., 2009). Other expression system aspects such as:


are often referred to as disadvantages. These apparent drawbacks can, however, be turned to advantages depending on the target protein's specifics.

Table 8 compares the suitability of the 3 leading expression platforms related to characteristics of the expression candidate protein. Apart from the two characteristics (a) requirement for human-like glycosylation, which includes monoclonal antibodies whose efficacy depends on Fc effector functions and (b) peptide nature of the recombinant target, most of the aspects captured in the table, do not give a clear indication regarding choice of the ideal expression platform. There is a large grey zone which typically needs to be explored empirically.

Active enzymes up to a size of 220 kDa and 250 kDa recombinant spider silk protein have been successfully expressed in *Escherichia coli* at high concentrations, questioning the dogma that bacterial systems are not suitable for the expression of large proteins. This thesis is further supported by successful expression of complex heterodimers, such as aglycosylated functional antibodies, in bacterial systems. For an in-depth analyis of expression of complex heterodimers in *Escherichia coli* we recommend the paper of Jeong et al. (2011). We also question the criticism towards inclusion body formation that often is cited as a disadvantage. Rather than a drawback we consider this as a capability that adds flexibility to the use of *Escherichia coli* based platforms. Industrial expression platforms allow for inclusion body concentrations as high as 10 g/l culture broth and above. This consideration combined with an efficient refolding process provides high potential for a competitive process from a cost point of view.

Some therapeutic protein candidates are not glycosylated, such as a non-glycosylated version of an antibody. In particular, recombinant proteins produced by yeast expression systems may carry undesired O-glycans. In these cases a lack of glycosylation capability can be considered as advantage rather than a system weakness. Intracellular expression in Escherichia coli may lead to product variants (a) with N-terminal formyl-methionine and (b) without formyl-methionine at the N-terminus. Methionine cleavage by the methionylaminopeptidase depends on the characteristics of the adjacent amino acid, which consequently determines the ratio of the 2 product fractions.

Earlier on endotoxin formation and low control of secretion into the medium were mentioned as problematic aspects for expression systems which are based on Gram-negative bacteria such as *Escherichia*, *Pseudomonas* and *Ralstonia*. On the other hand Table 8 also

1. the presence of high levels of endotoxins that need to be removed from therapeutic

WACKER Chemie has commercialised a K-12 derivative that exhibits higher secretion ability than other K-12 and B strains (Mücke et al., 2009). Other expression system aspects

1. the lack of posttranslational modification capability including a lack of glycosylation

3. the difficulty of expressing complex, multimeric proteins with a high number of

are often referred to as disadvantages. These apparent drawbacks can, however, be turned

Table 8 compares the suitability of the 3 leading expression platforms related to characteristics of the expression candidate protein. Apart from the two characteristics (a) requirement for human-like glycosylation, which includes monoclonal antibodies whose efficacy depends on Fc effector functions and (b) peptide nature of the recombinant target, most of the aspects captured in the table, do not give a clear indication regarding choice of the ideal expression platform. There is a large grey zone which typically needs to be

Active enzymes up to a size of 220 kDa and 250 kDa recombinant spider silk protein have been successfully expressed in *Escherichia coli* at high concentrations, questioning the dogma that bacterial systems are not suitable for the expression of large proteins. This thesis is further supported by successful expression of complex heterodimers, such as aglycosylated functional antibodies, in bacterial systems. For an in-depth analyis of expression of complex heterodimers in *Escherichia coli* we recommend the paper of Jeong et al. (2011). We also question the criticism towards inclusion body formation that often is cited as a disadvantage. Rather than a drawback we consider this as a capability that adds flexibility to the use of *Escherichia coli* based platforms. Industrial expression platforms allow for inclusion body concentrations as high as 10 g/l culture broth and above. This consideration combined with an efficient refolding process provides high potential for a competitive

Some therapeutic protein candidates are not glycosylated, such as a non-glycosylated version of an antibody. In particular, recombinant proteins produced by yeast expression systems may carry undesired O-glycans. In these cases a lack of glycosylation capability can be considered as advantage rather than a system weakness. Intracellular expression in Escherichia coli may lead to product variants (a) with N-terminal formyl-methionine and (b) without formyl-methionine at the N-terminus. Methionine cleavage by the methionylaminopeptidase depends on the characteristics of the adjacent amino acid, which

Earlier on endotoxin formation and low control of secretion into the medium were mentioned as problematic aspects for expression systems which are based on Gram-negative bacteria such as *Escherichia*, *Pseudomonas* and *Ralstonia*. On the other hand Table 8 also

2. the difficulty of controlling full secretion into the medium.

products

machinery

disulfide bonds

explored empirically.

process from a cost point of view.

2. the capability of intracellular expression

to advantages depending on the target protein's specifics.

consequently determines the ratio of the 2 product fractions.

such as:

indicates some weaknesses of yeast platforms. On the one hand yeast N- and Oglycosylation capability can negatively impact product quality so that adverse immunogenic reactions in the clinic are the result. Another problem often observed with *Pichia* and *Hansenula* are product variants produced through incomplete N-terminal processing and proteolytic degradation (Meyer et al., 2008). This, together with an on average lower observed productivity, negatively affects broad usage of yeast systems, despite their advantageous secretion capability.


Table 8. Criteria that drive selection of an expression platform. Legend: –, not suitable; • low, •• medium, ••• high suitability; 1) mostly cited as limiting criterion, nevertheless, up to 220 kDa proteins have been expressed in Lonza's *E. coli* XS TechnologiesTM platform with a very high titre, 2) Unigene and Lonza developed *E. coli* based peptide platforms, 3) secretion required for most recombinant proteins, 4) proteins exhibiting low solubility or a high aggregation propensity are often expressed at high titres as inclusion bodies, 5) yeast type glycosylation, mainly mannose comprising oligosaccharides, is highly immunogenic, 6) Nterminal product variants are frequently observed with *Pichia pastoris* and *Hansenula polymorpha* as a result of incomplete N-terminal processing.

Table 9 compares bacterial Gram-negative and yeast platforms to selected bacterial Grampositive expression platforms, i.e. to *Bacillus* and *Corynebacterium* platforms (White, 2011). The comparison suggests that the disadvantages of the existing bacterial Gram-negative platforms and yeast platforms can be overcome by moving into bacterial Gram-positive platforms. Gram-positive bacteria, in contrast to Gram-negative bacteria do not produce endotoxins and they naturally secrete proteins. Comparing them to yeast, they do not glycosylate proteins and there are no N-terminal processing problems. Both *Bacillus* and *Corynebacterium* hosts need to be engineered to resolve the problematic aspects of the corresponding wildtype strains such as low plasmid stability and secretion of undesired proteases.


Table 9. Suitability of yeast, Gram-negative and Gram-positive expression platforms related to classical microbial platform weaknesses.

#### **4.4 Production performance of relevant industrial** *Escherichia coli* **expression platforms**

In contrast to the expression of antibodies in CHO cells, expression success cannot be predicted in microbial expression systems. What is good for a specific recombinant protein A does not necessarily work for protein B, even if B is a protein variant of A. An integral part of the various strain platforms are generic high cell density fermentations. When considering industrialisation, strains and fermentation procedures should be looked at as single entities rather than separate process aspects. This is the main reason for the difficulty in judging the performance of expression platforms in general. Data from one single product are not sufficient, since the performance of one expression platform can differ greatly from product to product for as yet unknown reasons. One platform typically shows exceptional productivity only for a small number of products and rather low productivity for the majority of desired expression targets.

Expression titres of commercial products are typically handled as trade secrets. The authors have access to an informative set of expression titre data of leading *Escherichia coli* expression systems which are part of Lonza's XS TechnologiesTM platform (Figure 9). This platform is a broad one which in itself encompasses various *Escherichia coli*, *Pichia pastoris* and *Bacillus subtilis* platforms. In our experience, heterogeneity of the recombinant protein pipeline demands access to a variety of powerful expression tools in order to cope with specific expression challenges. On a few occasions the platform performance could be directly benchmarked against competitive CMO and other commercialised platforms based on *Escherichia coli* and *Pseudomonas*. On these occasions XS TechnologiesTM showed superior or equal performance. Therefore we consider the performance data shown in Figure 10 as representative for leading bacterial Gram-negative expression platforms.

glycosylate proteins and there are no N-terminal processing problems. Both *Bacillus* and *Corynebacterium* hosts need to be engineered to resolve the problematic aspects of the corresponding wildtype strains such as low plasmid stability and secretion of undesired

> **Gram+ Platforms**  *Bacillus Corynebacterium*

> > suitable

suitable

suitable

suitable

**Gram-Platforms**  *Escherichia Pseudomonas*

not suitable

not suitable

suitable

suitable

**Yeast Platforms**  *Pichia Hansenula*

suitable

suitable

not suitable

not suitable

**4.4 Production performance of relevant industrial** *Escherichia coli* **expression** 

Table 9. Suitability of yeast, Gram-negative and Gram-positive expression platforms related

In contrast to the expression of antibodies in CHO cells, expression success cannot be predicted in microbial expression systems. What is good for a specific recombinant protein A does not necessarily work for protein B, even if B is a protein variant of A. An integral part of the various strain platforms are generic high cell density fermentations. When considering industrialisation, strains and fermentation procedures should be looked at as single entities rather than separate process aspects. This is the main reason for the difficulty in judging the performance of expression platforms in general. Data from one single product are not sufficient, since the performance of one expression platform can differ greatly from product to product for as yet unknown reasons. One platform typically shows exceptional productivity only for a small number of products and rather low productivity for the

Expression titres of commercial products are typically handled as trade secrets. The authors have access to an informative set of expression titre data of leading *Escherichia coli* expression systems which are part of Lonza's XS TechnologiesTM platform (Figure 9). This platform is a broad one which in itself encompasses various *Escherichia coli*, *Pichia pastoris* and *Bacillus subtilis* platforms. In our experience, heterogeneity of the recombinant protein pipeline demands access to a variety of powerful expression tools in order to cope with specific expression challenges. On a few occasions the platform performance could be directly benchmarked against competitive CMO and other commercialised platforms based on *Escherichia coli* and *Pseudomonas*. On these occasions XS TechnologiesTM showed superior or equal performance. Therefore we consider the performance data shown in Figure 10 as

representative for leading bacterial Gram-negative expression platforms.

proteases.

**Problematic Characteristics** 

Endotoxins

**platforms** 

Control of secretion

N-terminal product variants

to classical microbial platform weaknesses.

majority of desired expression targets.

Undesired glycosylation

Fig. 9. Example of an industrial expression platform, XS TechnologiesTM (Lonza). The platform comprises a number of powerful expression technologies for expressing recombinant proteins in *Escherichia*, *Pichia* and *Bacillus* in order to cope with the expression challenges related to the heterogeneity of the recombinant proteins pipeline, including recombinant peptides and pDNA.

With Gram-negative organisms such as *Escherichia coli* and *Pseudomonas,* the recombinant product can be localized in different spaces, either intracellular (cytoplasmic) or extracellular. We define the latter as proteins expressed with a secretion sequence, and thus directed through the inner membrane, which means that the recombinant protein can be localized either in the periplasm or in the cell free medium. As a second aspect to consider, product is formed in either a soluble form or as insoluble aggregates. Apart from intentional inclusion body formation, production in a soluble, functional form is preferred. Therefore 4 effective expression modes are to be distinguished. Recombinant protein can be localised (C1) in the cytoplasm, insoluble as inclusion bodies, (C2) in the cytoplasm in a soluble form, (C3) in the cell-free medium in a soluble form and (C4) in the periplasm in a soluble form. Periplasmic insoluble material is typically not accessed and therefore ignored in the productivity figures.

Figure 10 shows expression levels of 24 recombinant proteins, mostly biopharmaceuticals that are expressed in *Escherichia coli* platforms. Induction is platform-dependent either by the addition of a corresponding sugar or by entering the stationary phase.

Fig. 10. Expression titres obtained for 24 different recombinant proteins mostly biopharmaceuticals. The proteins were expressed in either one of the sugar inducible or one of the stationary phase inducible *Escherichia coli* systems belonging to Lonza XS TechnologiesTM platform. Among the 24 recombinant products were fragment antibodies, Fab-fusions, singlechain antibodies, virus-like particles, novel non-antibody type binders, growth factors, recombinant enzymes, amphipathic proteins, recombinant vaccines, peptides (hormones and others), affinity ligands and monomers of biopolymers; size of the proteins varied between 2 and 220 kDa. Legend: C1, insoluble as inclusion bodies in cytoplasm; C2, soluble in cytoplasm; C3, soluble in cell-free medium; C4, soluble in periplasm.

**Cytoplasmic expression (categories C1 and C2)**. Among the products expressed in the cytoplasm, either soluble or insoluble as inclusion bodies, were highly soluble recombinant proteins as well as proteins prone to high aggregation propensity belonging to product classes such as recombinant vaccines, novel non-antibody based binders, recombinant therapeutic and non-therapeutic enzymes, virus like particles (VLPs), peptides (hormones and others), monomers of biopolymers, affinity ligands and others. The proteins were mostly monomeric with the size ranging from 2 to 40 kDa. Highest expression titres are obtained in the case of cytoplasmic soluble expression (C2 in Figure 10) with a median titre of 11 g/l culture broth and a range of 3 – 20 g/l. Intentional intracellular expression of recombinant protein in an insoluble state as inclusion bodies (C1 in Figure 10) resulted in a median titre of about 9 g/l, with a range of 3 - 15 g/l dependent on the target protein.

**Extracellular expression, periplasmic and into cell free medium (categories C3 and C4)**. Products that were expressed with a signal sequence were fragment antibodies (Fab), Fab fusion proteins, single chain antibodies (scFv), growth factors, enzymes and various formats of amphipathic proteins. The size of the corresponding products varied between 20 and 220

20

11.0

16

14

12

10

8

8.8

C3, soluble in cell-free medium; C4, soluble in periplasm.

dependent on the target protein.

**Fermentation Titre [g/l]**

6

4

2

0

C1 C2 C3 C4

biopharmaceuticals. The proteins were expressed in either one of the sugar inducible or one of the stationary phase inducible *Escherichia coli* systems belonging to Lonza XS TechnologiesTM platform. Among the 24 recombinant products were fragment antibodies, Fab-fusions, singlechain antibodies, virus-like particles, novel non-antibody type binders, growth factors, recombinant enzymes, amphipathic proteins, recombinant vaccines, peptides (hormones and others), affinity ligands and monomers of biopolymers; size of the proteins varied between 2 and 220 kDa. Legend: C1, insoluble as inclusion bodies in cytoplasm; C2, soluble in cytoplasm;

**Cytoplasmic expression (categories C1 and C2)**. Among the products expressed in the cytoplasm, either soluble or insoluble as inclusion bodies, were highly soluble recombinant proteins as well as proteins prone to high aggregation propensity belonging to product classes such as recombinant vaccines, novel non-antibody based binders, recombinant therapeutic and non-therapeutic enzymes, virus like particles (VLPs), peptides (hormones and others), monomers of biopolymers, affinity ligands and others. The proteins were mostly monomeric with the size ranging from 2 to 40 kDa. Highest expression titres are obtained in the case of cytoplasmic soluble expression (C2 in Figure 10) with a median titre of 11 g/l culture broth and a range of 3 – 20 g/l. Intentional intracellular expression of recombinant protein in an insoluble state as inclusion bodies (C1 in Figure 10) resulted in a median titre of about 9 g/l, with a range of 3 - 15 g/l

**Extracellular expression, periplasmic and into cell free medium (categories C3 and C4)**. Products that were expressed with a signal sequence were fragment antibodies (Fab), Fab fusion proteins, single chain antibodies (scFv), growth factors, enzymes and various formats of amphipathic proteins. The size of the corresponding products varied between 20 and 220

Fig. 10. Expression titres obtained for 24 different recombinant proteins mostly

1.5 2.0

kDa. Among them were both soluble and fairly soluble monomers and multimers, homoand heteromers. Extracellular product (C3 in Figure 10) reached concentrations in the range of 0.5 to 8.5 g/l in the cell free medium with a median of 1.5 g/l. Proteins which accumulated in the periplasm (C4 in Figure 10) reached titres of functional product between 0.5 and 10 g/l with a median titre of 2.0 g/l. Dependent on the product-specific aggregation propensity sometimes significant amounts of precipitated recombinant protein were observed in the periplasm. This fraction has been ignored, since it does not contribute to functional product. The extent of product precipitation can be influenced by the choice of the promoter system, the related induction mode and fermentation conditions. Similarly, the distribution of product between the periplasm and the cell-free medium can be partly controlled by changes in physical and chemical environmental conditions. However, ideal conditions need to be identified empirically.

The above mentioned product titres have been typically obtained within 36 to 72 hours of fermentation.

#### **4.5 Posttranslational modification in** *Escherichia coli*

Proteins often require posttranslational modification in order to gain full biological activity. Therefore, missing posttranslational protein modification capabilities such as glycosylation, formation of pyroglutamic acid at the N-terminus, N-terminal acylation and C-terminal amidation are frequently cited as a disadvantage of bacterial expression.

However, over the last decade big advances have been made in understanding glycosylation mechanisms and in glycoengineering of microbial organisms. Gerngross and coworkers (Choi et al., 2003) and Contreras and coworkers (Vervecken et al., 2004) were among the first to succeed in glycoengineering yeast more precisely, *Pichia pastoris*, towards the formation of defined glycoforms. The yeast related work culminated in successful expression of humanlike glycosylated antibody in a *Pichia pastoris* host, that enables specific human Nglycosylation with high fidelity (Potgieter et al., 2009).

In parallel it became evident that protein glycosylation is also abundant in prokaryotes. Whereas N-linked protein glycosylation is the most abundant posttranslational modification in eukaryotes, within prokaryotes it seems to be restricted to the domain of the Archea where S-layer proteins show N-linked glycosylation. Already in 2002 Aebi and coworkers (Wacker et al., 2002) demonstrated successful transfer of the *Campylobacter jejuni* protein N-glycosylation machinery into *Escherichia coli*. This opened up an exciting opportunity to produce N-glycoproteins within bacterial expression platforms. Nevertheless, two features were inhibitory to a broad application of the new system. (a) The *Campylobacter jejuni* glycan is immunogenic for humans. (b) The glycan is linked to asparagine through an unusual deoxysugar, bacillosamin. Recently the system has been further developed towards formation of the required N-acetylglucosamin-asparagine linkage that is commonly found in glycoproteins of eukaryotic origine (Schwarz et al., 2010). The same paper proposes a semi-synthetic approach towards human glycosylation based on the new developed technology.

The goal of any microbial glycoengineered system must be to overcome the weaknesses of the existing mammalian platforms that are, (a) mammalian glycosylation is characterized by naturally occurring heterogeneity in the glycan structure and (b) by limited possibilities to tailor glycosylation towards improved therapeutic performance. Consequently microbial glycoengineered expression platforms should allow for tailored, homogenous and humanlike glycosylation. However the challenges on the way to the development of a well performing microbial glycoengineered platform are manifold. The following lists the technical obstacles that need to be addressed:


As mentioned before, existing mammalian expression technology is not fulfilling all of the desirable requirements and there is an even longer way to go for the existing yeast systems in order to compete with mammalian systems. Not all of the above mentioned technical challenges have been successfully addressed in yeast. Even further away from technical maturity are bacterial glycoengineered systems. Nevertheless technical advances are achieved at high pace. The authors would not be surprised if bacterial expression technology would one day be a viable solution for large scale manufacturing of glycoproteins.

#### **4.6 Cost considerations**

From a commercial point of view, bacterial and yeast systems share many advantages over mammalian systems such as high growth rate, the potential to reach high biomass concentration, structural and segregational robustness and a higher product production rate rP, resulting in significantly shorter fermentation times. While mammalian cells such as CHO cells are characterized by a high specific product production rate qP, volumetric productivity QP is typically negatively affected by a relatively low growth rate and more importantly by the lower achievable biomass concentration as compared to *Pichia pastoris* (Kunert et al., 2008). The same is true, when comparing CHO cells to *Escherichia coli*. Other aspects such as time required for the development of a stable CHO cell line and media costs should be considered as well. All these aspects add to the attractiveness of microbial and yeast systems when the manufacture of aglycosylated non-antibody type of recombinant proteins is considered. Table 10 shows cost drivers in fermentation of the current key biopharmaceuticals production platforms.

naturally occurring heterogeneity in the glycan structure and (b) by limited possibilities to tailor glycosylation towards improved therapeutic performance. Consequently microbial glycoengineered expression platforms should allow for tailored, homogenous and humanlike glycosylation. However the challenges on the way to the development of a well performing microbial glycoengineered platform are manifold. The following lists the

2. tailoring, access to a number of specific glycoforms through defined host backgrounds 3. productivity, volumetric productivity should not be below the productivity of existing

4. O-glycosylation, existing yeast O-glycosylation causes immunogenic reactions with

8. plug and play, access to stable glycoengineered hosts, such that only the target gene

10. good growth characteristics, system viability is affected by the amount of genetic

As mentioned before, existing mammalian expression technology is not fulfilling all of the desirable requirements and there is an even longer way to go for the existing yeast systems in order to compete with mammalian systems. Not all of the above mentioned technical challenges have been successfully addressed in yeast. Even further away from technical maturity are bacterial glycoengineered systems. Nevertheless technical advances are achieved at high pace. The authors would not be surprised if bacterial expression technology would one day be a viable solution for large scale manufacturing of

From a commercial point of view, bacterial and yeast systems share many advantages over mammalian systems such as high growth rate, the potential to reach high biomass concentration, structural and segregational robustness and a higher product production rate rP, resulting in significantly shorter fermentation times. While mammalian cells such as CHO cells are characterized by a high specific product production rate qP, volumetric productivity QP is typically negatively affected by a relatively low growth rate and more importantly by the lower achievable biomass concentration as compared to *Pichia pastoris* (Kunert et al., 2008). The same is true, when comparing CHO cells to *Escherichia coli*. Other aspects such as time required for the development of a stable CHO cell line and media costs should be considered as well. All these aspects add to the attractiveness of microbial and yeast systems when the manufacture of aglycosylated non-antibody type of recombinant proteins is considered. Table 10 shows cost drivers in fermentation of the current key

11. N-terminal variability, often seen in yeast systems, needs to be under control

5. glycosylation efficiency, the whole of the target protein is expected to be glycosylated 6. secretion efficiency, needs to be high, since glycosylation is connected to secretion 7. expression of complex proteins such as antibodies, capability to produce hetero-

technical obstacles that need to be addressed:

multimers (disulphide bridges)

mammalian systems

needs to be inserted

humans

changes

glycoproteins.

**4.6 Cost considerations** 

biopharmaceuticals production platforms.

1. glyoform homogeneity, ideally one glycoform should be formed

9. proteases, deletion of all undesirable proteolytic activity


Table 10. Comparison of bacterial, yeast and mammalian system characteristics which drive cost of goods in fermentation; 1) the figures have been modelled based on typical production key figures and assuming an equal product titre of 5 g/l at the end of the fermentation; USP, upstream processing.

Methylotrophic yeast fermentation can be very demanding on equipment performance as a result of the high oxygen demand, high cooling requirements and explosion-proof design because of methanol feeding. Corresponding bioreactor layout requirements are described by Hoeks et al. (2005).

Figure 7 shows that about 9% of all recombinant DNA products are supposedly manufactured with transgenic animals, avian cells, insect cells and viral platforms. On top of these, there are early projects of recombinant expression in plants, filamentous fungi, plants and protozoa. The decision to opt for one of these systems is mostly driven by specific product aspects, cost or IP reasons in order to gain freedom to operate. A cost advantage through higher productivity or lower depreciation compared to more conventional systems is not obvious. Cost allocated to fermentation is typically in the range of 30% to 50% of overall manufacturing costs. Irrespective of the recombinant biosynthesis method used, the DSP costs remain. Therefore the sometimes cited 10X overall cost improvement through the use of one specific expression system and the related USP production platform is difficult to understand if not unrealistic.

The cost of downstream processing (DSP) is more or less independent of the chosen system, if we assume product localization in the cell-free medium. When using Gram-negative expression technology special attention needs to be paid to endotoxin removal. On the other hand a mammalian system makes viral clearance mandatory.

Intracellular production obviously requires cell disruption or product release from the cells followed by a usually more complex biomass removal step. The latter is more or less standardized for conventional expression technologies. Other operations such as inclusion body isolation and purification followed by protein refolding typically drive DSP costs up. Theses higher costs for DSP can only be justified through higher upstream productivity as shown in Figure 10 or a lack of production alternatives. It is also obvious that no significant cost advantage is to be expected on the DSP side, if product needs to be extracted and purified out of whole plants. However, in the latter case a significant cost advantage arises if for example, a therapeutic or a vaccine is administered through oral consumption of the whole plant or a non-purified low-cost plant extract.

Please note that other costs for so called secondary manufacturing (e.g fill and finish, formulation) accrue for the finished product, which we can not discuss here.

#### **5. Conclusions**

The industry has become very conservative, risk averse and reluctant to change established and successful manufacturing platforms because of a very strict interpretation of regulatory guidelines. This is also the main reason why the authors think that the main load of biotechnological manufacturing production has remained with the already industrially established microbial (*E. coli*, yeast) and mammalian production systems and will continue to do so. Nevertheless, regulatory government bodies do welcome novel manufacturing methods for the production of affordable pharmaceuticals because of ever increasing health care costs. Indeed, it cannot be denied that cost pressure and novel applications will help to disturb the established situation. We consider two alternative expression systems to have some potential.


These two options, however, will again be hampered by another expected or even partly realised breakthrough: the successful targeted humanised glycosylation in yeast and later in bacteria. On top of that, we will sooner or later experience the realisation of extensive pathway engineering and synthetic biology principles, where production organisms will be designed using engineering principles as in the automotive or aerospace industry. It is even harder to imagine how and where alternatives such as plants or protozoa can beat such advanced microbial or mammalian options.

#### **6. Acknowledgments**

We thank our former and actual Lonza colleagues John R Birch, Gareth Griffiths, Christoph Kiziak and Joachim Klein for their critical lecture and valuable comments on the manuscripts. The remarks of Professor Florian Wurm of the Swiss Federal Institute in Lausanne on the section "Mammalian cells" were very much appreciated.

#### **7. References**

Ahmad, A., Pereira, E.O., Conley, A.J., Richman, A.S., Menassa, R. (2010). Green Biofactories: Recombinant Protein Production in Plants. *Recent Patents on Biotechnology,* 4, pp. 242-259.

purified out of whole plants. However, in the latter case a significant cost advantage arises if for example, a therapeutic or a vaccine is administered through oral consumption of the

Please note that other costs for so called secondary manufacturing (e.g fill and finish,

The industry has become very conservative, risk averse and reluctant to change established and successful manufacturing platforms because of a very strict interpretation of regulatory guidelines. This is also the main reason why the authors think that the main load of biotechnological manufacturing production has remained with the already industrially established microbial (*E. coli*, yeast) and mammalian production systems and will continue to do so. Nevertheless, regulatory government bodies do welcome novel manufacturing methods for the production of affordable pharmaceuticals because of ever increasing health care costs. Indeed, it cannot be denied that cost pressure and novel applications will help to disturb the established situation. We consider two alternative expression systems to have

1. Transgenic plants have the possibility to combine therapeutic with nutrition needs. The production of edible vaccines for human or veterinary applications for example appear to be an attractive option especially as the active crop can be phototrophically and

2. Due to their short doubling times and easier cultivation, protozoa offer themselves as a possibility between microbial and mammalian cell culture. Insect cell culture seem to be not as attractive as protozoa as they do not grow as fast and the frequently used BEVS

These two options, however, will again be hampered by another expected or even partly realised breakthrough: the successful targeted humanised glycosylation in yeast and later in bacteria. On top of that, we will sooner or later experience the realisation of extensive pathway engineering and synthetic biology principles, where production organisms will be designed using engineering principles as in the automotive or aerospace industry. It is even harder to imagine how and where alternatives such as plants or protozoa can beat such

We thank our former and actual Lonza colleagues John R Birch, Gareth Griffiths, Christoph Kiziak and Joachim Klein for their critical lecture and valuable comments on the manuscripts. The remarks of Professor Florian Wurm of the Swiss Federal Institute in

Ahmad, A., Pereira, E.O., Conley, A.J., Richman, A.S., Menassa, R. (2010). Green

Biofactories: Recombinant Protein Production in Plants. *Recent Patents on* 

results in more complex isolation and purification procedures.

Lausanne on the section "Mammalian cells" were very much appreciated.

formulation) accrue for the finished product, which we can not discuss here.

whole plant or a non-purified low-cost plant extract.

**5. Conclusions** 

some potential.

cheaply grown locally.

advanced microbial or mammalian options.

*Biotechnology,* 4, pp. 242-259.

**6. Acknowledgments** 

**7. References** 


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### **Exogenous Catalase Gene Expression as a Tool for Enhancing Metabolic Activity and Production of Biomaterials in Host Microorganisms**

Ahmad Iskandar Bin Haji Mohd Taha1, Hidetoshi Okuyama1,2, Takuji Ohwada3, Isao Yumoto4 and Yoshitake Orikasa3 *1Graduate School of Environmental Science 2Faculty of Environmental Earth Science, Hokkaido University 3Department of Food Science, Obihiro University of Agriculture and Veterinary Medicine 4National Institute of Advanced Industrial Science and Technology (AIST) Japan* 

#### **1. Introduction**

250 Innovations in Biotechnology

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Heterologous gene expression is a widely used and vital biotechnology in basic and applied biology research fields. In particular, this technology (bioengineering ) is emerging as a useful tool in fields of applied biology, such as medical, pharmaceutical, and agricultural sciences including food science. With this technology, animals, fishes, plants, and eukaryotic and prokaryotic microorganisms can be used as host organisms for transformation, and various types of vectors have been developed and have become commercially available. It is only about 40 years since a heterologous cloned DNA was expressed in *Escherichia coli* cells (Annie et al., 1974; Old & Primrose, 1989). Development of various types of gene transfer systems has assisted the widespread use of gene engineering technologies in all research fields.

The production of high-value compounds is a requisite purpose of gene engineering for all the fields of applied biology. Such production can occur in two ways: host cells are used to produce a product that is new to them by expressing a foreign gene(s); or host cells are enhanced to produce higher levels of the target product(s), which can be also inherently synthesized at normal levels by host organisms. The examples of the former are the production of insulin (Goeddel et al., 1979) or α-fetprotein (Nishi et al., 1988) by *E. coli* cells that were transformed with genes encoding these proteins. The literature on the latter means of production has accumulated rapidly. In such cases, two or more kinds of foreign gene carried in one or more vectors was used to transform the host organisms, by which a highvalue compound(s) can be generated as the new product in the transformed host organisms or can enhance the metabolic activities of the host organisms.

Yumoto et al. (1998) isolated a bacterium with a remarkably high catalase activity from a waste pool at a fish-processing factory in Hokkaido, Japan. This bacterium was identified as *Vibrio rumoiensis* strain S-1T (strain S-1 hereafter; Yumoto et al., 1999). Details of this bacterium and its catalase protein (VktA**)** and gene (*vktA*) are described in the following section (**Section 2, VktA catalase and its gene)**. The VktA catalase had a significantly high specific activity after being purified (Yumoto et al., 2000). The *vktA* gene encoding VktA was cloned and expressed in various strains of *E. coli* (Ichise et al., 1999; Orikasa, 2002). Cell-free extracts prepared from *vktA*-transformed *E. coli* cells exhibited almost the same specific activity of catalase as those prepared from the parent strain S-1. Biochemical and molecular studies on VktA prepared from the parent and *vktA*-transformed *E. coli* transformants showed that strain S-1 could accumulate VktA protein at a level as high as a concentration of 2% of total soluble proteins and that the high catalytic activity of purified VktA enzyme is 4 times greater than that of bovine liver catalase (Yumoto et al., 2000).

In this chapter, we describe two biotechnological uses of the *vktA* gene as a foreign gene. First it was used to enhance the nitrogen-fixing activity in a root nodule bacterium (**Section 3, Enhancement of nitrogen fixation by** *vktA* **in root nodule bacteria**), which has its own catalase. Second, it was used to enhance eicosapentaenoic acid (EPA) biosynthesis in *E. coli* that had already been transformed with the EPA biosynthesis gene cluster (*pfa* genes) cloned from a marine bacterium (**Section 4, Enhancement of eicosapentaenoic acid production in**  *E. coli* **through expression of** *vktA*). The host *E. coli* cell has its own catalase but no ability to synthesize EPA. In both cases, the catalase activity of *vktA*-transformed host cells increased remarkably. Furthermore, the nitrogen-fixing ability in *R. leguminosarum* was definitely enhanced, and the EPA contents in *E. coli* transformed with *pfa* and *vktA* became greater than the EPA content in *E. coli* transformed with *pfa* genes only. The physiological and molecular roles of the increased catalase activity in catalase gene (*vktA*)-engineered bacterial cells are discussed in each section. **Section 5** contains concluding remarks and discusses possibilities for further use of VktA. Catalase comparable with VktA or those with much higher performance than VktA are described as a tool for producing biomaterials by biotechnologies in various research fields.

#### **2. VktA catalase and its gene**

The VktA catalase is the sole catalase protein detected in strain S-1 (Yumoto et al., 1999). This enzyme is characterized by its significantly high catalytic activity when compared with other known catalases. General information on catalases and detailed characteristics of VktA and its gene (*vktA)* are provided in this section.

#### **2.1 Catalases and their genes**

Aerobic organisms metabolize oxygen through respiration for production of energy to sustain their life. During aerobic respiration, organisms generate toxic reactive oxygen species (ROSs), such as superoxide anion radicals (O2•–), hydrogen peroxide (H2O2), and hydroxyl radicals (OH•), as by-products. The presence of H2O2 in cells has a possibility to generate a more toxic ROS, OH•, by a Fenton-type reaction (Halliwell & Gutteridge, 1999). Excessive amounts of H2O2 and OH• are harmful to cell components. Therefore, aerobic organisms eliminate H2O2 with scavenging enzymes, such as catalase, peroxidase, and glutathione peroxidase. On the other hand, elimination of extracellular H2O2 by catalase is also important for either aerobic or anaerobic organisms to distribute their niche among several microorganisms. For example, parasitic and symbiotic microorganisms are attacked

bacterium and its catalase protein (VktA**)** and gene (*vktA*) are described in the following section (**Section 2, VktA catalase and its gene)**. The VktA catalase had a significantly high specific activity after being purified (Yumoto et al., 2000). The *vktA* gene encoding VktA was cloned and expressed in various strains of *E. coli* (Ichise et al., 1999; Orikasa, 2002). Cell-free extracts prepared from *vktA*-transformed *E. coli* cells exhibited almost the same specific activity of catalase as those prepared from the parent strain S-1. Biochemical and molecular studies on VktA prepared from the parent and *vktA*-transformed *E. coli* transformants showed that strain S-1 could accumulate VktA protein at a level as high as a concentration of 2% of total soluble proteins and that the high catalytic activity of purified VktA enzyme is

In this chapter, we describe two biotechnological uses of the *vktA* gene as a foreign gene. First it was used to enhance the nitrogen-fixing activity in a root nodule bacterium (**Section 3, Enhancement of nitrogen fixation by** *vktA* **in root nodule bacteria**), which has its own catalase. Second, it was used to enhance eicosapentaenoic acid (EPA) biosynthesis in *E. coli* that had already been transformed with the EPA biosynthesis gene cluster (*pfa* genes) cloned from a marine bacterium (**Section 4, Enhancement of eicosapentaenoic acid production in**  *E. coli* **through expression of** *vktA*). The host *E. coli* cell has its own catalase but no ability to synthesize EPA. In both cases, the catalase activity of *vktA*-transformed host cells increased remarkably. Furthermore, the nitrogen-fixing ability in *R. leguminosarum* was definitely enhanced, and the EPA contents in *E. coli* transformed with *pfa* and *vktA* became greater than the EPA content in *E. coli* transformed with *pfa* genes only. The physiological and molecular roles of the increased catalase activity in catalase gene (*vktA*)-engineered bacterial cells are discussed in each section. **Section 5** contains concluding remarks and discusses possibilities for further use of VktA. Catalase comparable with VktA or those with much higher performance than VktA are described as a tool for producing biomaterials by

The VktA catalase is the sole catalase protein detected in strain S-1 (Yumoto et al., 1999). This enzyme is characterized by its significantly high catalytic activity when compared with other known catalases. General information on catalases and detailed characteristics of VktA

Aerobic organisms metabolize oxygen through respiration for production of energy to sustain their life. During aerobic respiration, organisms generate toxic reactive oxygen species (ROSs), such as superoxide anion radicals (O2•–), hydrogen peroxide (H2O2), and hydroxyl radicals (OH•), as by-products. The presence of H2O2 in cells has a possibility to generate a more toxic ROS, OH•, by a Fenton-type reaction (Halliwell & Gutteridge, 1999). Excessive amounts of H2O2 and OH• are harmful to cell components. Therefore, aerobic organisms eliminate H2O2 with scavenging enzymes, such as catalase, peroxidase, and glutathione peroxidase. On the other hand, elimination of extracellular H2O2 by catalase is also important for either aerobic or anaerobic organisms to distribute their niche among several microorganisms. For example, parasitic and symbiotic microorganisms are attacked

4 times greater than that of bovine liver catalase (Yumoto et al., 2000).

biotechnologies in various research fields.

and its gene (*vktA)* are provided in this section.

**2. VktA catalase and its gene** 

**2.1 Catalases and their genes** 

by ROSs which are produced by the external defense system of their host organisms (Katsuwon & Anderson, 1992; Rocha et al., 1996; Visick & Ruby, 1998).

The dismutation reaction of H2O2 has evolved into three unrelated groups in the category of catalase (EC 1.11.1.6). The first group consists of so-called typical catalase. These catalases consist of four identical subunits each equipped with protoheme IX (heme *b*) or heme *d* in the active site as the prosthetic group, and their subunit molecular masses are 55-84 kDa. These catalases can be subdivided into small subunits (subunit molecular mass: 55-69 kDa) possessing heme *b* as the prosthetic group and large subunits (75-84 kDa) possessing mainly heme *d* as the prosthetic group. This group of catalases is the most widespread in nature and exhibits efficient catalytic reactions. These enzymes have a broad pH optimum range, are specifically inhibited by 3-amino-1,2,4-triazole, which reacts with a catalytic intermediate state, compound I (see below), and are resistant to reduction by dithionite (Kim et al., 1994; Nadler et al., 1986). The second group of catalases is catalase-peroxidase. Catalaseperoxidases exhibit a bifunctional character: catalase and peroxidase activities. The maximal catalatic activities of catalase-peroxidases are two or three orders of magnitude lower than those of typical catalases. These enzymes typically have a dimeric or tetrameric structure with a subunit with a molecular mass of approximately 80 kDa, containing only one or two hemes *b* per molecule. Catalase-peroxidases have been detected in Bacteria, Archaea and Eukarya domains (although only in fungi in Eukarya). In addition, these enzymes have a sharp pH optimum, are not inhibited by 3-amino-1,2,4-triazole, and are readily reduced by dithionite (Hochman & Shemesh, 1987; Kengen et al., 2001). The deduced primary structures of these enzymes are closely related to each other and their three-dimentional structures are similar to those of plant peroxidase. The third group of catalases consists of manganese catalase (Mn-catalase). Mn-catalases, in contrast with the other two catalase groups, are not equipped with heme as the prosthetic group; rather these enzymes use manganese ions. Therefore, activities of these enzymes are not inhibited by cyanide or azide, which are inhibitors of catalases in the other groups. Mn-catalases are mostly hexameric and the molecular size of their subunit ranges 28 kDa to 35 kDa (Kono & Fridovich, 1983; Allgood & Perry, 1986). These catalases, which are sometimes referred to as pseudocatalases, are distributed in lactic acid bacteria and thermophilic bacteria.

All catalases possessing heme as the prosthetic group commonly exhibit a two-step mechanism for the degradation of H2O2. In the first step, one H2O2 molecule oxidizes the enzyme in the resting state (Fe3+ Por) to ferryl porphyrin with a porphyrin π-cation radical (Fe4+=O Por+•), so-called compound I. In the second step, compound I oxidizes a second H2O2 molecule to molecular oxygen and water (eqs. 1 and 2; Deisseroth & Dounce, 1970; Schonbaum & Chance, 1976).

Enzyme (Fe3+ Por) + H2O2 → Compound I (Fe4+=O Por+•) + H2O (Reaction 1)

$$\text{Compound I (Fe}^{4+} \text{O } \text{Por}^{\star}\text{) + H}\_{2}\text{O}\_{2} \rightarrow \text{Enzyme (Fe}^{3+} \text{ Por}) + \text{H}\_{2}\text{O} + \text{O}\_{2} \text{ (Reaction 2)}$$

Phylogenic analysis based on the amino-acid sequence deduced from the gene sequence of typical catalases has revealed their subdivision into three distinct clades (Klotz et al., 1997). Clade 1 catalases are small-subunit catalases and are mainly of plant origin, but also includes one algal representative and a subgroup of bacterial origin. Clade 2 catalases are all large-subunit catalases of bacterial, archaeal, and fungal origins. The one archaeal enzyme belonging to clade 2 catalase is postulated to have arisen in a horizontal transfer event from *Bacillus* species. This clade of catalases exhibits a strong resistance to denaturation by heat and proteolysis. Clade 3 catalases are small-subunit catalases and their origins are bacteria, archaea, fungi, and other eukaryotes. There are no pronounced functional difference between clade 3 and clade 1 catalase. Bacteria harboring clade 3 catalase as the single catalase isozyme are distributed to a restricted environment.

Most aerobic bacteria contain one or more catalases, which are produced in response to oxidative stress or depending on the growth phase. *Escherichia coli* possess two types of catalase gene, *katG* and *katE*. These encode periplasmic HPI (catalase-peroxidase) and cytoplasmic catalase HPII (typical catalase, clade 2), respectively. *KatG* is induced by H2O2, while *katE* is induced by the entry into the stationary phase of growth (Storz & Zheng, 2000). Both of the genes are regulated by the alternative sigma factor, σs, which is produced by the *rpoS* gene. The expression of *katG* is regulated by OxyR, a transcriptional regulator that senses H2O2 (Ivanova et al., 1994; Storz & Zheng, 2000). OxyR can switch rapidly between reduced and oxidized states, and only the oxidized form acts as a transcriptional activator for target genes (Aslund et al., 1999; Christman et al., 1985).

#### **2.2 Characteristics of VktA catalase and its gene**

Even though there have been many reports of bacterial oxidative stress responses, there had been few reports on the microorganisms that are able to survive in highly oxidative environments. Therefore, studies were conducted in order to understand how a bacterium adapts to an oxidative environment and what kind of H2O2 eliminating system it possesses. A facultatively psychrophilic bacterium exhibiting high catalase activity was isolated from a drain pool of fish egg processing factory that uses H2O2 as a bleaching agent (Yumoto et al., 1998, 1999). The isolate, strain S-1, was identified as a new species, *Vibrio rumoiensis*. The catalase activity in cell extract of strain S-1 was 2 orders higher than those of *E*. *coli* and *Bacillus subtilis*. Although S-1 cells exhibit high catalase activity, individual cells do not exhibit strong resistance to H2O2. It is probably due to the fragility of the cell structure (Ichise et al., 1999).

Catalase (VktA) from strain S-1 has been purified and characterized (Yumoto et al., 2000). Molecular mass of the subunit of the catalase is 57.3 kDa and the enzyme consists of four identical subunits. The enzyme was not apparently reduced by dithionite. The activity showed a broad optimum pH range (pH 6–10) and was inhibited by 3-amino-1,2,4-triazole.

Therefore, the enzyme belongs to the typical small subunit catalase. The catalase activity of VktA was 1.5 and 4.3 times faster than *Micrococcus luteus* and bovine catalases, respectively, under 30 mM H2O2 in 50 mM potassium phosphate buffer (pH 7) at 20°C. Therefore, VktA is considered to be a catalase with very high activity and one with the highest turnover numbers in all known catalases. The thermoinstability of VktA was significantly higher than that of *M*. *luteus* and bovine catalases. It is suggested that the unique properties of VktA may reflect protective strategies for the survival of strain S-1 under oxidative environmental conditions, where this bacterium was inhabited.

The gene of VktA, *vktA*, has been isolated and sequenced. The *vktA* consisted of an open reading frame of 1530 bp encoding a 508 amino-acid protein with a calculated molecular mass of 57657.79 Da (Ichise et al., 2000). A putative ribosome binding site (AGGAGA) was

belonging to clade 2 catalase is postulated to have arisen in a horizontal transfer event from *Bacillus* species. This clade of catalases exhibits a strong resistance to denaturation by heat and proteolysis. Clade 3 catalases are small-subunit catalases and their origins are bacteria, archaea, fungi, and other eukaryotes. There are no pronounced functional difference between clade 3 and clade 1 catalase. Bacteria harboring clade 3 catalase as the single

Most aerobic bacteria contain one or more catalases, which are produced in response to oxidative stress or depending on the growth phase. *Escherichia coli* possess two types of catalase gene, *katG* and *katE*. These encode periplasmic HPI (catalase-peroxidase) and cytoplasmic catalase HPII (typical catalase, clade 2), respectively. *KatG* is induced by H2O2, while *katE* is induced by the entry into the stationary phase of growth (Storz & Zheng, 2000). Both of the genes are regulated by the alternative sigma factor, σs, which is produced by the *rpoS* gene. The expression of *katG* is regulated by OxyR, a transcriptional regulator that senses H2O2 (Ivanova et al., 1994; Storz & Zheng, 2000). OxyR can switch rapidly between reduced and oxidized states, and only the oxidized form acts as a transcriptional activator

Even though there have been many reports of bacterial oxidative stress responses, there had been few reports on the microorganisms that are able to survive in highly oxidative environments. Therefore, studies were conducted in order to understand how a bacterium adapts to an oxidative environment and what kind of H2O2 eliminating system it possesses. A facultatively psychrophilic bacterium exhibiting high catalase activity was isolated from a drain pool of fish egg processing factory that uses H2O2 as a bleaching agent (Yumoto et al., 1998, 1999). The isolate, strain S-1, was identified as a new species, *Vibrio rumoiensis*. The catalase activity in cell extract of strain S-1 was 2 orders higher than those of *E*. *coli* and *Bacillus subtilis*. Although S-1 cells exhibit high catalase activity, individual cells do not exhibit strong resistance to H2O2. It is probably due to the fragility of the cell structure

Catalase (VktA) from strain S-1 has been purified and characterized (Yumoto et al., 2000). Molecular mass of the subunit of the catalase is 57.3 kDa and the enzyme consists of four identical subunits. The enzyme was not apparently reduced by dithionite. The activity showed a broad optimum pH range (pH 6–10) and was inhibited by 3-amino-1,2,4-triazole. Therefore, the enzyme belongs to the typical small subunit catalase. The catalase activity of VktA was 1.5 and 4.3 times faster than *Micrococcus luteus* and bovine catalases, respectively, under 30 mM H2O2 in 50 mM potassium phosphate buffer (pH 7) at 20°C. Therefore, VktA is considered to be a catalase with very high activity and one with the highest turnover numbers in all known catalases. The thermoinstability of VktA was significantly higher than that of *M*. *luteus* and bovine catalases. It is suggested that the unique properties of VktA may reflect protective strategies for the survival of strain S-1 under oxidative environmental

The gene of VktA, *vktA*, has been isolated and sequenced. The *vktA* consisted of an open reading frame of 1530 bp encoding a 508 amino-acid protein with a calculated molecular mass of 57657.79 Da (Ichise et al., 2000). A putative ribosome binding site (AGGAGA) was

catalase isozyme are distributed to a restricted environment.

for target genes (Aslund et al., 1999; Christman et al., 1985).

**2.2 Characteristics of VktA catalase and its gene** 

conditions, where this bacterium was inhabited.

(Ichise et al., 1999).

found 5 bases upstream of the start codon (Fig. 1). In further upstream, putative promoter sequences, (TATAAT) and (TTGGCT), corresponding to -10 and -35, respectively were also found. The promoter sequences were probably recognized by RNA polymerase carrying the housekeeping sigma subunit σ70 (Hawley & McClure, 1983). Another putative promoter binding site for OxyR was in further upstream (Fig. 1).

Fig. 1. Nucleotide sequence of the *vktA* promoter region. Nucleotides in the putative ribosome binding site, Shine-Dalgarno ribosome-binding site (SD) is in the boxed. Arrow indicates the putative transcription initiation site. Potential promoter sites of -10 and -35 are indicated as underlined bold letters. An alignment with OxyR consensus sequences of OxyR consensus (Tartaglia et al., 1989; Toledano et al., 1994), *Haemophilus influenza* (Bishai et al., 1994); and *Salmonella typhimurium* (Tartaglia et al., 1989) is shown upstream of the putative -35 region.

The deduced amino acid sequence of VktA shows high similarity with that of typical small subunit catalases belonging to clade 3 (Fig. 2). Many calatases possessing as the sole isozyme in parasitic or symbiotic microorganisms belong to clade 3. Therefore, there is a possibility that strain S-1 inherently be a symbiont of fish, which is supported by the fact that the strain was originally obtained from the fish egg processing factory. It is also pointed that bacteria possessing clade 3 catalases as the sole isozyme like strain S-1 was naturally selected in the environment that is frequently exposed to H2O2.

Localization of the VktA catalase in the cytoplasmic and periplasmic spaces has been demonstrated by the enzymatic activity of fractionated cells and immunological detection methods (Ichise et al., 1999; Yumoto et al., 2000). In addition, it has been shown that cell density and release of VktA from disrupted cells play an important role in the survival of strain S-1 cells when they reacted with H2O2 (Ichise et al., 1999). However, almost no growth hindrance is observed when 100 mM H2O2 is introduced into the culture. Accounts of strain S-1's strong tolerance to H2O2 in high concentrations of H2O2 during cultivation, concomitant with very rapid elimination of H2O2 due to the strong catalase activity of the cells themselves, have remained unresolved. The contribution of catalase to H2O2 tolerance and the presence of cell surface catalase have been demonstrated by H2O2 tolerance of catalase-deficient *E. coli* strain UM2 carrying *vktA* and immunoelectron microscopic observation on strain S-1 and catalase-deficient mutant, strain S-4, derived from strain S-1 using an antibody for the intracellular catalase of strain S-1, respectively (Ichise et al., 2008). Cell surface catalase is considered to contribute to the elimination of extracellular H2O2.

To further characterize the VktA catalase protein, it was analyzed by polyacrylamide gel electrophoresis (PAGE) using partially purified bovine liver catalase (Sigma, St Louis, USA), two types of *Aspergillus niger* catalase (one from Sigma and the other from NAGASE Co., Ltd, Tokyo, Japan), and *Corynebacterium glutamicum* catalase (Sigma) as references. All samples except for strain S-4 exhibited one activity band detected by the method by Uriel (1958) in native gel (data not shown). Fig. 3 shows protein band profiles and indications of the activity band position of all the samples in SDS-PAGE under different conditions. In the presence of SDS with neither 2-mercaptoethanol (2-ME) nor heating, all samples except for strain S-4 showed one activity band, whereas no activity band was observed in the presence of SDS, 2- ME and heating. The bands with activity are considered to be proteins with a dimeric form. It is interesting that two *A*. *niger* and C. *glutamicum* catalases were stained positive for activity in the presence of SDS, 2-ME, but no heating. The dimeric form of these catalases was resistant to reduction by 2-ME, at room temperature. By contrast, VktA was sensitive to 2-ME treatment at room temperature. A very faint oxygen bubble formation was observed for the position of the dimeric form of bovine liver catalase in the presence of SDS, suggesting that the tetrameric form of bovine liver catalase is apt to be easily dissociated into monomers in the presence of SDS only. These results suggest that VktA has an intermediate characteristic of bovine liver catalase and other microbial catalases against treatment with 2-ME at room temperature. The VktA catalase, which is tetrameric in its native form (Yumoto et al. 2000), can be dissociated into active dimers in the presence of SDS; however, the structure and activity of the dimers are sensitive to treatment with 2-ME at room temperature, suggesting that VktA could be a structurally and actively flexible enzyme.

Fig. 2. Phylogenic tree of catalases. The tree was constructed by the CLUSTAL W program (Thompson et al., 1994) with multiple alignment using neighbor-joining method (Saitou & Nei, 1987). Numbers at the branches are bootstrap percentages based on 1000 replicates. Bar, 0.1 changes per amino acid position.

band position of all the samples in SDS-PAGE under different conditions. In the presence of SDS with neither 2-mercaptoethanol (2-ME) nor heating, all samples except for strain S-4 showed one activity band, whereas no activity band was observed in the presence of SDS, 2- ME and heating. The bands with activity are considered to be proteins with a dimeric form. It is interesting that two *A*. *niger* and C. *glutamicum* catalases were stained positive for activity in the presence of SDS, 2-ME, but no heating. The dimeric form of these catalases was resistant to reduction by 2-ME, at room temperature. By contrast, VktA was sensitive to 2-ME treatment at room temperature. A very faint oxygen bubble formation was observed for the position of the dimeric form of bovine liver catalase in the presence of SDS, suggesting that the tetrameric form of bovine liver catalase is apt to be easily dissociated into monomers in the presence of SDS only. These results suggest that VktA has an intermediate characteristic of bovine liver catalase and other microbial catalases against treatment with 2-ME at room temperature. The VktA catalase, which is tetrameric in its native form (Yumoto et al. 2000), can be dissociated into active dimers in the presence of SDS; however, the structure and activity of the dimers are sensitive to treatment with 2-ME at room temperature, suggesting that VktA could be a

Fig. 2. Phylogenic tree of catalases. The tree was constructed by the CLUSTAL W program (Thompson et al., 1994) with multiple alignment using neighbor-joining method (Saitou & Nei, 1987). Numbers at the branches are bootstrap percentages based on 1000 replicates. Bar,

structurally and actively flexible enzyme.

0.1 changes per amino acid position.

Fig. 3. SDS-PAGE profiles of cell-free extracts from *Vibrio rumoiensis* strain S-1 (S1) and its catalase-deficient mutant strain S-4 (S4), and commercially available catalases: B from bovine liver; C from *Corynebacterium glutamicum;* A from *Aspergillus niger* (Sigma); R from *A. niger* (Ryonet F Plus; Nagase Co., Ltd). +, catalase activity positive (oxygen bubble formation) at the band shown by solid arrows; –, no catalase activity band; superscripts, m and mh, SDS-PAGE conditions in the presence of 2-mercaptoethanol (2-ME) at room temperature for 5 min and in the presence of 2-ME with heating at 100ºC for 5 min, respectively. No superscript means SDS-PAGE conditions with SDS but no 2-ME. The open arrows indicate the position of monomeric subunits of catalases. VktA, bovine liver (Sigma) and two *A. niger* catalases from Sigma and Nagase have a tetrameric native structure with a molecular mass of 230 kDa, 240 kDa, 250 kDa and approximately 350 kDa, respectively. Information on the native form of the *C. glutamicum* catalase is not available. Gels (e-Pagel, 10%; Atto, Tokyo) were stained with Coomassie Brilliant Blue. Lane M, molecular marker standard (kDa).

#### **3. Enhancement of nitrogen fixation by** *vktA* **in root nodule bacteria**

Root nodule bacteria are characterized by their ability to fix dinitrogen in root nodules, where microaerobic conditions are maintained. However, the nitrogen-fixing process requires a large amount of ATP, suggesting that molecular oxygen is requisite for oxidative phosphorylation. This section describes the use of *vktA* to modulate oxygen metabolism in root nodule bacteria. The *vktA* gene was introduced into *Rhizobium leguminosarum* cells and the strain with a remarkably high catalase activity was constructed. Results show that the increase of catalase activity in rhizobia could be a valuable way to improve the nodulation and nitrogen-fixing ability of nodules (Orikasa et al., 2010).

#### **3.1 Nitrogen fixation and catalases in rhizobia: General information**

Rhizobia infect the roots of leguminous plants and form nodules with elaborate control by exchanging molecular signals between two partners (Wei et al., 2008, 2010). The rhizobia are present as bacteroids such as microsymbiotic organelles in the nodules and convert atmospheric dinitrogen into biologically available ammonia (nitrogen fixation). Nitrogen fixation is an energy-requiring process and needs large amounts of ATP, which is supplied by oxidative phosphorylation. This process is extremely oxygen-sensitive and the partial pressure of oxygen inside the nodules is maintained at very low levels, resulting in strongly reduced conditions with the production of ROSs such as H2O2 (Tjepkema & Yocum, 1974). Leghemoglobins present in nodules play a role in the effective diffusion of oxygen and their autoxidation results in the production of O2・- and H2O2 (Appleby, 1984; Puppo et al., 1981). However, as the host plant can control the nodulation in such a way that the infection during symbiotic interaction is aborted due to a hypersensitive reaction, it has been suggested that the release of ROS such as H2O2, termed the oxidative burst, may occur in the early stage of the infection thread formation (Vasse et al., 1993). It was reported that a striking release of ROS occurred under the conditions of plant defenses against pathogens (Mehdy, 1994). Since it is believed that ROSs such as H2O2, O2•– and OH• are also generated naturally during the metabolism of cells growing aerobically and that these ROS could damage the protein, lipids, and DNA components in organisms (Sangpen et al., 1995), these reports suggest that the response of rhizobia to ROSs such as H2O2 would exert a serious influence on both the nodulation process and nitrogen-fixing abilities.

Catalase in rhizobia cells was not previously considered to play a crucial role in the nitrogen-fixing abilities because *Sinorhizobium meliloti* has three kinds of catalase (KatA, KatB, KatC) and the *katA*-minus mutant did not impair both nodulation and nitrogen fixing abilities (Herouart et al., 1996). Rhizobia are symbiotic microorganisms. They were previously considered to be able to rely on their host plant for defense against toxic forms of oxygen such as H2O2; that is, plant-derived catalases, as well as defense systems against H2O2 toxicity such as the ascorbate-glutathione reaction system, were considered to be able to contribute to the removal of H2O2 toxicity. It was reported that the production of oxidative protection enzymes by plant cells in nodules was positively correlated with the increase in nitrogenase activity and leghemoglobin content (Dalton et al., 1986). In addition, parasitic bacteria were reported not to need their own catalase because they could depend upon their host catalase (Steiner et al., 1984). However, subsequent experiments showed that the double-catalase mutants (∆*katA*∆*katC* or ∆*katB*∆*katC*) had considerably lower capability for both nodulation and nitrogen fixation (Jamet et al., 2003; Sigaud et al., 1999), and their activities decreased during nodule senescence (Hernandez-Jimenez et al., 2002). These results showed that the catalase in rhizobia could be important for the efficient function of both nodulation and nitrogen fixation. Another rhizobium strain, *Bradyrhizobium*, showed the important role of catalase for H2O2 decomposition, and the *katG*-minus mutant resulted in the loss of both catalase activity and exogenous H2O2 consumption (Loewen et al., 1985b; Panek & Obrian, 2004).

Unexpectedly, rhizobia had a tendency for less catalase activity than other genera of aerobic and facultative anaerobic bacteria, resulting in higher susceptibility to H2O2 (Ohwada et al., 1999). We compared catalase and peroxidase activities in rhizobia (11 strains) with those in other genera of bacteria (six strains). Catalase activities (units per mg protein) of the rhizobia strains tested were in the range of 0.9-5.8, although the addition of H2O2 increased activities in all strains to the range of 2.5-11.3. By contrast, the activities in the other genera strains tested ranged 12.3 to 893.3, and, in the presence of

are present as bacteroids such as microsymbiotic organelles in the nodules and convert atmospheric dinitrogen into biologically available ammonia (nitrogen fixation). Nitrogen fixation is an energy-requiring process and needs large amounts of ATP, which is supplied by oxidative phosphorylation. This process is extremely oxygen-sensitive and the partial pressure of oxygen inside the nodules is maintained at very low levels, resulting in strongly reduced conditions with the production of ROSs such as H2O2 (Tjepkema & Yocum, 1974). Leghemoglobins present in nodules play a role in the effective diffusion of oxygen and their autoxidation results in the production of O2・- and H2O2 (Appleby, 1984; Puppo et al., 1981). However, as the host plant can control the nodulation in such a way that the infection during symbiotic interaction is aborted due to a hypersensitive reaction, it has been suggested that the release of ROS such as H2O2, termed the oxidative burst, may occur in the early stage of the infection thread formation (Vasse et al., 1993). It was reported that a striking release of ROS occurred under the conditions of plant defenses against pathogens (Mehdy, 1994). Since it is believed that ROSs such as H2O2, O2•– and OH• are also generated naturally during the metabolism of cells growing aerobically and that these ROS could damage the protein, lipids, and DNA components in organisms (Sangpen et al., 1995), these reports suggest that the response of rhizobia to ROSs such as H2O2 would exert a serious influence on both the nodulation

Catalase in rhizobia cells was not previously considered to play a crucial role in the nitrogen-fixing abilities because *Sinorhizobium meliloti* has three kinds of catalase (KatA, KatB, KatC) and the *katA*-minus mutant did not impair both nodulation and nitrogen fixing abilities (Herouart et al., 1996). Rhizobia are symbiotic microorganisms. They were previously considered to be able to rely on their host plant for defense against toxic forms of oxygen such as H2O2; that is, plant-derived catalases, as well as defense systems against H2O2 toxicity such as the ascorbate-glutathione reaction system, were considered to be able to contribute to the removal of H2O2 toxicity. It was reported that the production of oxidative protection enzymes by plant cells in nodules was positively correlated with the increase in nitrogenase activity and leghemoglobin content (Dalton et al., 1986). In addition, parasitic bacteria were reported not to need their own catalase because they could depend upon their host catalase (Steiner et al., 1984). However, subsequent experiments showed that the double-catalase mutants (∆*katA*∆*katC* or ∆*katB*∆*katC*) had considerably lower capability for both nodulation and nitrogen fixation (Jamet et al., 2003; Sigaud et al., 1999), and their activities decreased during nodule senescence (Hernandez-Jimenez et al., 2002). These results showed that the catalase in rhizobia could be important for the efficient function of both nodulation and nitrogen fixation. Another rhizobium strain, *Bradyrhizobium*, showed the important role of catalase for H2O2 decomposition, and the *katG*-minus mutant resulted in the loss of both catalase activity and exogenous H2O2

Unexpectedly, rhizobia had a tendency for less catalase activity than other genera of aerobic and facultative anaerobic bacteria, resulting in higher susceptibility to H2O2 (Ohwada et al., 1999). We compared catalase and peroxidase activities in rhizobia (11 strains) with those in other genera of bacteria (six strains). Catalase activities (units per mg protein) of the rhizobia strains tested were in the range of 0.9-5.8, although the addition of H2O2 increased activities in all strains to the range of 2.5-11.3. By contrast, the activities in the other genera strains tested ranged 12.3 to 893.3, and, in the presence of

process and nitrogen-fixing abilities.

consumption (Loewen et al., 1985b; Panek & Obrian, 2004).

H2O2, the levels of all strains were increased to the range of 16.1-1,460.2, indicating that the catalase activity of the tested rhizobia tended to be considerably lower than those of the other genera tested (Fig. 4). Results of Southern analyses imply that rhizobia have no DNA region similar to *katE* of *E. coli*, which is inducible tenfold during the stationary phase of growth (Loewen et al., 1985a), and the kinetics of catalase induction in *R. leguminosarum* bv. *phaseoli* was different from that in *E. coli* (Crockford et al., 1995; Hassan & Fridovich, 1978). Crockford et al. (1995) mentioned that, during growth, unidentified compounds accumulate in the cells and repress catalase activity, although the significance of growth-phase-dependent regulation of catalase activity remains obscure.

Fig. 4. Correlation between catalase activities and H2O2 tolerance. Cells were incubated with H2O2 (0.6, 1.5, 5.9, 14.7, 29.4, 58.8, and 147 mM) and the maximum concentration of H2O2 (mM) in which the cells could grow within 24 h (96 h for *Bradyrhizobium japonicum*) was plotted as ordinate. Catalase activities in the cells in the early stationary phase with (panel B) or without (panel A) H2O2 (0.6 mM) were plotted as the abscissa. *Rhizobium, Sinorhizobium* and *Bradyrhizobium* strains tested are enclosed within a circle. Data points are means of results from at least three trials. *Rhizobium, Sinorhizobium* & *Bradyrhizobium* strains used: *Rhizobium leguminosarum* bv. *phaseoli* USDA2667, 2676; *R. leguminosarum* bv. *trifolii* USDA2053, 2145; *R. leguminosarum* bv. *viciae* USDA2370, 2443; *Sinorhizobium fredii* USDA191, 206; *S. meliloti* USDA1021, 1025; *Bradyrhizobium japonicum* S32. Other strains used: *Bacillus subtilis* AHU1390; *Escherichia coli* JM109; *Micrococcus luteus* AHU1427; *Proteus vulgaris* AHU1144; *Pseudomonas fluorescens* AHU1719; *Serratia marcescens* AHU1488. Adapted with permission from Ohwada et al. (1999).

The relationships between catalase activities and H2O2 tolerance in all bacteria tested are shown in Figure 4. The results indicate that a tendency for a positive and mutual correlation between them for all strains tested. Particularly, both the catalase activities and the H2O2 tolerance of the rhizobia tested were lower than those of the others. On the other hand, peroxidase activities in cells with different electron donors (NADH, *o*-dianisidine, and *p*phenylenediamine) were considerably lower than the catalase activities and there was no significant difference between rhizobia and the others. In addition, the growth repression caused by the addition of catalase inhibitor in the presence of H2O2 was observed in rhizobia such as *R. leguminosarum* bv. *phaseoli*, bv. *trifolii* and *S. meliloti*. These results indicate that catalase could be mainly responsible for the defense mechanism against H2O2 toxicity. For *E. coli*, it was reported that catalase-deficient and catalase-overproductive mutants were more sensitive and resistant to H2O2, respectively (Greenberg & Demple, 1988; Loewen, 1984). The peroxisome-targeting signal (SKL sequence) of catalase (KatA) in *S. meliloti*, which is supposed to be connected with the export into the periplasmic region, suggests that the *S. meliloti* catalase is located in this region (Herouart et al, 1996). The location of protecting enzymes such as catalase against H2O2 seems to be important because periplasmic enzymes could be advantageous to the defense against exogenous H2O2. Therefore, the location of catalase in rhizobia would have an effect on the mutual correlation between catalase activities and H2O2 tolerance.

Since rhizobia are symbiotic microorganisms, they might be able to rely on their host plant in nodules to some extent for defense against toxic forms of oxygen such as H2O2. However, the catalase activity of *Bradyrhizobium* strain from effective nodules was reported to be higher than that in a strain from ineffective nodules (Francis & Alexander, 1972). These results prompted the construction of rhizobia with higher catalase activity to improve the nitrogen-fixing ability of nodules.

#### **3.2 Enhancement of nitrogen fixation through expression of** *vktA* **in** *Rhizobium leguminosarum*

The DNA fragment (4.9 kbp) including the catalase gene (*vktA*), which is controlled by its own promoter activity, was ligated into pBluescriptII SK+ to construct pBSsa1 (Ichise et al., 2000). The *Bam*HI-*Xho*I fragment including a coding region of *vktA* (4.9 kbp) was isolated from the pBSsa1 and ligated into a broad host range vector, pBBR1MCS-2 (Kovach et al., 1995) at the site of *Bam*HI-*Xho*I. Then, the recombinant plasmid was introduced into *R. leguminosarum* bv. *phaseoli* USDA2676 cells to construct the *vktA*-transformant by triparental mating (Simon, 1984). The result showed that the *vktA*-transformed *R. leguminosarum*  exhibited a remarkably high catalase activity of up to around 10,000 units per mg protein. This activity was three orders of magnitude greater than that of the parent strain and comparable to that of strain S-1 (Yumoto et al., 1999). To confirm the production of VktA catalase, activity staining of the catalase and immunoblot analysis with anti-VktA antiserum were conducted. The activity staining was carried out according to Clare *et al*. (1984). Results showed an obvious band of VktA catalase in both logarithmic and stationary phases.

Although the parent strain of *R. leguminosarum* originally had two bands of different mobility from the VktA catalase, their intensities were considerably weak compared with that of the VktA catalase and were not major in the *vktA*-transformant. Additionally, the immunoblot analysis was conducted using polyclonal anti-VktA and a goat anti-rabbit IgGhorseradish peroxidase conjugate as primary and secondary antibodies, respectively. The results indicated that a positive antigen-antibody reaction occurred as a single band between the anti-VktA antiserum and the VktA catalase. These results clearly showed that the efficient production of VktA catalase was responsible for the high catalase activity in the *vktA*-transformant. As described above, since catalase could be mainly responsible for the H2O2 tolerance in bacteria and the catalase activities in the rhizobia tested were considerably lower compared with the other genera tested, the growth of almost all of rhizobia tested was severely repressed even in the presence of 1.5 mM H2O2. However, the *vktA*-transformant showed almost the same cell density as the parent in the presence of 10 mM H2O2; even with 50 mM H2O2, the *vktA*-transformant could grow and the cell density reached levels half that of the parent at 24 h, then almost the same as the parent at 30 h after incubation. These results indicate that the *vktA*-transformant acquired resistance against H2O2 through the enhancement of catalase production in the cells.


1) Acetylene reduction activity (μmol of C2H2/h).

260 Innovations in Biotechnology

significant difference between rhizobia and the others. In addition, the growth repression caused by the addition of catalase inhibitor in the presence of H2O2 was observed in rhizobia such as *R. leguminosarum* bv. *phaseoli*, bv. *trifolii* and *S. meliloti*. These results indicate that catalase could be mainly responsible for the defense mechanism against H2O2 toxicity. For *E. coli*, it was reported that catalase-deficient and catalase-overproductive mutants were more sensitive and resistant to H2O2, respectively (Greenberg & Demple, 1988; Loewen, 1984). The peroxisome-targeting signal (SKL sequence) of catalase (KatA) in *S. meliloti*, which is supposed to be connected with the export into the periplasmic region, suggests that the *S. meliloti* catalase is located in this region (Herouart et al, 1996). The location of protecting enzymes such as catalase against H2O2 seems to be important because periplasmic enzymes could be advantageous to the defense against exogenous H2O2. Therefore, the location of catalase in rhizobia would have an effect on the mutual correlation between catalase

Since rhizobia are symbiotic microorganisms, they might be able to rely on their host plant in nodules to some extent for defense against toxic forms of oxygen such as H2O2. However, the catalase activity of *Bradyrhizobium* strain from effective nodules was reported to be higher than that in a strain from ineffective nodules (Francis & Alexander, 1972). These results prompted the construction of rhizobia with higher catalase activity to improve the

**3.2 Enhancement of nitrogen fixation through expression of** *vktA* **in** *Rhizobium* 

showed an obvious band of VktA catalase in both logarithmic and stationary phases.

Although the parent strain of *R. leguminosarum* originally had two bands of different mobility from the VktA catalase, their intensities were considerably weak compared with that of the VktA catalase and were not major in the *vktA*-transformant. Additionally, the immunoblot analysis was conducted using polyclonal anti-VktA and a goat anti-rabbit IgGhorseradish peroxidase conjugate as primary and secondary antibodies, respectively. The results indicated that a positive antigen-antibody reaction occurred as a single band between the anti-VktA antiserum and the VktA catalase. These results clearly showed that the efficient production of VktA catalase was responsible for the high catalase activity in the *vktA*-transformant. As described above, since catalase could be mainly responsible for the H2O2 tolerance in bacteria and the catalase activities in the rhizobia tested were considerably

The DNA fragment (4.9 kbp) including the catalase gene (*vktA*), which is controlled by its own promoter activity, was ligated into pBluescriptII SK+ to construct pBSsa1 (Ichise et al., 2000). The *Bam*HI-*Xho*I fragment including a coding region of *vktA* (4.9 kbp) was isolated from the pBSsa1 and ligated into a broad host range vector, pBBR1MCS-2 (Kovach et al., 1995) at the site of *Bam*HI-*Xho*I. Then, the recombinant plasmid was introduced into *R. leguminosarum* bv. *phaseoli* USDA2676 cells to construct the *vktA*-transformant by triparental mating (Simon, 1984). The result showed that the *vktA*-transformed *R. leguminosarum*  exhibited a remarkably high catalase activity of up to around 10,000 units per mg protein. This activity was three orders of magnitude greater than that of the parent strain and comparable to that of strain S-1 (Yumoto et al., 1999). To confirm the production of VktA catalase, activity staining of the catalase and immunoblot analysis with anti-VktA antiserum were conducted. The activity staining was carried out according to Clare *et al*. (1984). Results

activities and H2O2 tolerance.

nitrogen-fixing ability of nodules.

*leguminosarum*

2) Significant differences were evaluated by Student's *t* test (*P* < 0.01).

Table 1. Number, weight and acetylene reduction activity (ARA) of nodules formed in the combination of *Phaseolus vulgaris* (L.) cv. Yukitebou and *vktA*-transformed *R. leguminosarum*. Values were obtained from 20 determinants of at least two independent experiments. The values given are the means ± S. D. of 20 different tests. Adapted with permission from Orikasa et al. (2010).

The host plant, *Phaseolus vulgaris* (L.), was inoculated with *vktA*-transformed *R. leguminosarum* cells (106 cells per seed) and, after cultivation in a seed bag with Norris and Date medium (Dye, 1980) or in a pot filled with vermiculite, the number, weight, and nitrogenase activity (acetylene reduction activity, ARA) of the nodules were measured (Table 1). For the seed bag, the number and weight of nodules did not show a significant difference between *vktA*transformant and the parent cells. However, the acetylene reduction activity (ARA) of nodules formed with *vktA*-transformed cells was significantly higher than that formed with the parent cells, and around 1.7 times as many nodules were formed as with the parent cells (around 1.4 times per plant). For the pot, the number and weight of the nodules formed with *vktA*transformant were larger than those of the parent cells, with around 1.2 and 1.3 times those of the parent, respectively, although these levels tended to be lower than those for seed bag cultivation. Higher levels of ARA in the nodules formed with *vktA*-transformant were also observed and the levels reached around 2.3 times those of the parent (around 3.0 times per plant). Another set of experiments with the combination of *vktA*-transformed *S. fredii* and *Glycine max* (L.) also showed that the production of VktA significantly increased the ARA per nodule or plant weight. These results indicate that enhancing the catalase activity in *Rhizobium* cells significantly increased the nodules' nitrogen-fixing ability.

Next, catalase production in bacteroids of *vktA*-transformed *R. leguminosarum* was measured. Bacteroids were separated immediately after the nodules were detached from the plant roots (Kouchi & Fukai, 1989). The result showed that the *vktA*-transformant maintained an even higher catalase activity compared with the parent (around 150 units per mg protein). Results of western blot analysis using the anti-VktA antiserum showed a single band for VktA catalase, indicating that higher production of VktA catalase resulted in a high catalase activity even in bacteroids. However, the catalase activity in bacteroids was considerably low as compared with free-living cells. Given that a decrease in the relative amounts of DNA, as well as the dynamic conversion of cellular metabolism such as the repression of sugar degradation, was reported during the differentiation process of bacteroids (Bergersen & Turner, 1967; Verma et al., 1986; Vierny & Laccarino, 1989), the loss of a certain number of the *vktA*-recombinant plasmids and/or the repressive production of VktA catalase might occur through the differentiation to bacteroids in the absence of antibiotics. The localization of the VktA catalase in free-living cells and bacteroids of *vktA*-transformant was studied by immunoelectron microscopy using the polyclonal antiserum against VktA with a secondary anti-rabbit antibody, which was coupled with gold particles. The number of gold particles at the periphery of the freeliving cells including periplasm accounted for about 57.4 % of the sum total. For bacteroids, a relatively large number of gold particles (about 52.3% of the sum total), were observed at the periphery of the bacteroids including the symbiosome. These results indicate that the VktA catalase was preferentially distributed at the peripheral part of the cells for both free-living cells and bacteroids. H2O2 and leghemoglobin contents in the nodule formed with *vktA*-transformant were also measured. Nodules were detached 35 days after planting and H2O2 was extracted by grinding in 1 M HClO4 (Ohwada & Sagisaka, 1987). The H2O2 content in the extracts was measured by Quantitative Hydrogen Peroxide Assay (OXIS International, Portland, USA). The extraction and quantification of leghemoglobin components using capillary electrophoresis were carried out according to Sato et al. (1998). The results showed that the H2O2 content (nmol/g fresh wt of nodule) in the nodules formed with the parent cells was around 21.0, but this level was decreased to around 15.4 by the production of VktA catalase in the cells. By contrast, the VktA production increased the content of the leghemoglobins (Lba and Lbb) and the levels in the nodules formed with *vktA*-transformant were around 1.2 (Lba) and 2.1 (Lbb) times higher than those with the parent cells.

Considering that ROSs such as H2O2 are released from the plant root not only under pathogenic conditions but also during the infection process (Mehdy, 1994; Vasse et al., 1993), it is possible that *Rhizobium* cells with a higher catalase activity are advantageous to the infection process because they decrease the amounts of H2O2 around them. This supports the possibility that the VktA catalase is preferentially located near the surface area of the cells, suggesting that they could be effective in decomposing H2O2. The peripheral distribution of VktA was also observed in strain S-1 (Ichise et al., 2000). In nodules, lack of the ability to remove H2O2 caused the reduction of both nodulation and nitrogen-fixing ability (Bergersen et al., 1973). Given that electron microscopic observation did not seem to reveal any difference in the density of bacteroids inside the nodules between *vktA*-transformant and the parent, it is thought that the enhancement of the ability to decrease H2O2 by higher catalase activity is responsible for the increased levels of nitrogen-fixing activity. On the other hand, it was reported that leghemoglobins accumulated in the infected plant cells before nitrogen fixation in order to decrease the partial pressure of oxygen inside the nodule and protect nitrogenase from inactivation by oxygen (Appleby, 1984). Adding leghemoglobin to bacteroid suspensions enhanced the nitrogenase-mediated reactions, and the nitrogenase activity of bacteroids was dependent on the concentration of leghemoglobin (Bergersen et al., 1973). Furthermore, the deficiency of leghemoglobin synthesis in nodules of *Lotus japonicus* using RNAi led to the

in a high catalase activity even in bacteroids. However, the catalase activity in bacteroids was considerably low as compared with free-living cells. Given that a decrease in the relative amounts of DNA, as well as the dynamic conversion of cellular metabolism such as the repression of sugar degradation, was reported during the differentiation process of bacteroids (Bergersen & Turner, 1967; Verma et al., 1986; Vierny & Laccarino, 1989), the loss of a certain number of the *vktA*-recombinant plasmids and/or the repressive production of VktA catalase might occur through the differentiation to bacteroids in the absence of antibiotics. The localization of the VktA catalase in free-living cells and bacteroids of *vktA*-transformant was studied by immunoelectron microscopy using the polyclonal antiserum against VktA with a secondary anti-rabbit antibody, which was coupled with gold particles. The number of gold particles at the periphery of the freeliving cells including periplasm accounted for about 57.4 % of the sum total. For bacteroids, a relatively large number of gold particles (about 52.3% of the sum total), were observed at the periphery of the bacteroids including the symbiosome. These results indicate that the VktA catalase was preferentially distributed at the peripheral part of the cells for both free-living cells and bacteroids. H2O2 and leghemoglobin contents in the nodule formed with *vktA*-transformant were also measured. Nodules were detached 35 days after planting and H2O2 was extracted by grinding in 1 M HClO4 (Ohwada & Sagisaka, 1987). The H2O2 content in the extracts was measured by Quantitative Hydrogen Peroxide Assay (OXIS International, Portland, USA). The extraction and quantification of leghemoglobin components using capillary electrophoresis were carried out according to Sato et al. (1998). The results showed that the H2O2 content (nmol/g fresh wt of nodule) in the nodules formed with the parent cells was around 21.0, but this level was decreased to around 15.4 by the production of VktA catalase in the cells. By contrast, the VktA production increased the content of the leghemoglobins (Lba and Lbb) and the levels in the nodules formed with *vktA*-transformant were around 1.2 (Lba) and 2.1 (Lbb) times

Considering that ROSs such as H2O2 are released from the plant root not only under pathogenic conditions but also during the infection process (Mehdy, 1994; Vasse et al., 1993), it is possible that *Rhizobium* cells with a higher catalase activity are advantageous to the infection process because they decrease the amounts of H2O2 around them. This supports the possibility that the VktA catalase is preferentially located near the surface area of the cells, suggesting that they could be effective in decomposing H2O2. The peripheral distribution of VktA was also observed in strain S-1 (Ichise et al., 2000). In nodules, lack of the ability to remove H2O2 caused the reduction of both nodulation and nitrogen-fixing ability (Bergersen et al., 1973). Given that electron microscopic observation did not seem to reveal any difference in the density of bacteroids inside the nodules between *vktA*-transformant and the parent, it is thought that the enhancement of the ability to decrease H2O2 by higher catalase activity is responsible for the increased levels of nitrogen-fixing activity. On the other hand, it was reported that leghemoglobins accumulated in the infected plant cells before nitrogen fixation in order to decrease the partial pressure of oxygen inside the nodule and protect nitrogenase from inactivation by oxygen (Appleby, 1984). Adding leghemoglobin to bacteroid suspensions enhanced the nitrogenase-mediated reactions, and the nitrogenase activity of bacteroids was dependent on the concentration of leghemoglobin (Bergersen et al., 1973). Furthermore, the deficiency of leghemoglobin synthesis in nodules of *Lotus japonicus* using RNAi led to the

higher than those with the parent cells.

absence of symbiotic nitrogen fixation (Ott et al., 2005). Therefore, it is considered that the increase of leghemoglobin content also contributed toward the improvement of nitrogenfixing ability, although the accelerated mechanism of the leghemoglobin production is still under investigation. It was reported that the effective nodules of white clover and soybean contained higher activity of catalase compared with the ineffective nodules (Francis & Alexander, 1972). It seems that catalase is disadvantageous to protect nitrogenase from the cytotoxic effect of H2O2 because oxygen, which represses nitrogenase activity, is generated through the decomposition of H2O2. However, considering that a large amount of ATP, which could be supplied by bacteroidal oxidative phosphorylation, is required for the nitrogen-fixing reaction and that the leghemoglobins maintain a high oxygen flux for respiration through the facilitated oxygen diffusion (Ott et al., 2005; Tajima et al., 1986; Wittenberg et al., 1975), it might be possible that the oxygen generated by the catalase reaction could also be useful for energy production. The results here show that an increase in catalase activity reduced H2O2 levels in the nodules concomitantly with the enhancement of leghemoglobin contents, followed by improvement of the nitrogen-fixing ability in the nodules. The enhanced nitrogen fixation from the expression of *vktA* in rhizobia would lead to the growth of the host plant with reduced use of chemical nitrogen fertilizer.

#### **4. Enhancement of eicosapentaenoic acid production in** *E. coli* **through expressing** *vktA*

Eicosapentaenoic acid (EPA) is an essential nutrient for humans and animals. Its derivatives, such as eicosanoids, are known as signal compounds in blood and nervous systems. Therefore, the ethyl ester of EPA is used a medicine. Fish oils, which have been the most widely used source of EPA to date, have been recognized as unsuitable because of their low EPA content and their unavoidable contamination with heavy metals from seawater; therefore new sources of EPA have been sought. Bacteria or fungi, which inherently produce EPA, constitute one of such possible source. Another possibility is the heterologous expression of EPA biosynthesis genes or chain elongation/desaturase genes of fatty acids in various types of host organism. This section describes the EPA biosynthesis in *E. coli* transformed only with EPA biosynthesis genes and the enhancement of EPA biosynthesis by coexpression of the *vktA* gene.

Fig. 5. Domain structure of *pfa* genes responsible for the biosynthesis of EPA from *Shewanella pneumatophori* SCRC-2738.

#### **4.1 Bacterial biosynthesis of EPA**

Bacterial species belonging to *Shewanella*, *Vibrio*, *Flexibacter*, and *Halomonas* (Salunkhe et al., 2011) are known to produce EPA as a major long-chain polyunsaturated fatty acid. EPA is synthesized de novo in a polyketide biosynthesis mode by the enzyme complex consisting of PfaA, PfaB, PfaC, PfaD, and PfaE, which are encoded by *pfaA*, *pfaB*, *pfaC*, *pfaD*, and *pfaE*, respectively. These five genes (designated as an EPA biosynthesis gene cluster) generally locate in proximity on the chromosome (Fig. 5). PfaA and PfaC are multifunctional proteins and have some functional domains (Fig. 5). Only one functional domain for each of acyltransferase, enoyl reductase, and phosphopantetheinyl transferase is found, in PfaB, PfaD, and PfaE, respectively. Since the EPA gene cluster was first cloned from *Shewanella* sp. SCRC 2738 (*S. pneumatophori* SCRC-2738; Hirota et al., 2005) in 1996 (Yazawa, 1996), much attention has been paid to increasing the content of EPA in *E. coli* host cells and to its heterologous expression of these genes in various organisms, such as bacteria, yeast, and plants (Yazawa, 1996). The EPA gene clusters have been successfully expressed in various types of *E. coli* strains (Orikasa et al., 2004). Furthermore, numerous attempts have been made to express bacterial EPA biosynthesis genes in bacteria other than *E. coli* and in eukaryotic cells. However, to our knowledge, the report by Yu et. al. (2000) is the only one, in which a marine cyanobacterium is used as a host organism to express the EPA gene cluster.

#### **4.2 Enhanced production of EPA by expression of** *vktA* **in** *E. coli* **carrying** *pfa* **genes**

The enhanced production of EPA was observed in recombinant systems of *E. coli* that carried both EPA biosynthesis genes (*pfa*) and a *vktA* catalase gene. Although no molecular mechanism has been determined for this enhanced production of EPA, this technique may become another useful method to increase the productivity of EPA using recombinant systems. Docosahexaenoic acid (DHA) can be synthesized also in bacteria using DHA biosynthesis *pfa* genes, because the two *pfa* genes have a very similar structure (Okuyama et al., 2007).

*E. coli* DH5α transformants carrying pEPAΔ1 that included *pfaA-E* genes to the host cell led to the production of EPA (approximately 3% of total fatty acids; Table 2). The production of EPA in host organisms carrying pEPAΔ1 was increased to 12% of total fatty acids by the introduction of a *vktA* insert in pGBM3 [strain DH5α (pEPAΔ1) (pGBM3::*vktA*)]. The empty pGBM3 had no effect on EPA production. In strain DH5α carrying (pEPAΔ1) and partially deleted *vktA* in pGBM3(pGBM3::Δ*vktA*), EPA made up 6% of total fatty acids. The increase in EPA production in strain DH5α (pEPAΔ1)(pGBM3::*vktA*) was accompanied by a decrease in the proportions of palmitoleic acid [16:1(9)] (Table 2). When pGBM3 and pGBM3::*vktA* were replaced in the *E. coli* transformants with pKT230 and pKT230::*vktA*, respectively, similar trends were observed (data not shown). The yield of EPA per culture was approximately 1.5 μg/ml for DH5α(pEPAΔ1) and DH5α(pEPAΔ1)(pGBM3). It increased to 7.3 μg/ml for DH5α(pEPAΔ1) (pGBM3::*vktA*). The yield of EPA from DH5α (pEPAΔ1) pGBM3::Δ*vktA*) was 3.3 μg/ml (Table 2). *E. coli* DH5α has an inherent catalase activity of 2–3 U/mg protein (Nishida et al., 2006). The plasmid pEPAΔ1 had no effect on the catalase activity of the host cells. Catalase activity was increased to 535 U/mg protein for DH5α(pEPAΔ1)(pGBM3::*vktA*). However, there was no enhancement of catalase activity in DH5α(pEPAΔ1)(pGBM3::Δ*vktA*)

Bacterial species belonging to *Shewanella*, *Vibrio*, *Flexibacter*, and *Halomonas* (Salunkhe et al., 2011) are known to produce EPA as a major long-chain polyunsaturated fatty acid. EPA is synthesized de novo in a polyketide biosynthesis mode by the enzyme complex consisting of PfaA, PfaB, PfaC, PfaD, and PfaE, which are encoded by *pfaA*, *pfaB*, *pfaC*, *pfaD*, and *pfaE*, respectively. These five genes (designated as an EPA biosynthesis gene cluster) generally locate in proximity on the chromosome (Fig. 5). PfaA and PfaC are multifunctional proteins and have some functional domains (Fig. 5). Only one functional domain for each of acyltransferase, enoyl reductase, and phosphopantetheinyl transferase is found, in PfaB, PfaD, and PfaE, respectively. Since the EPA gene cluster was first cloned from *Shewanella* sp. SCRC 2738 (*S. pneumatophori* SCRC-2738; Hirota et al., 2005) in 1996 (Yazawa, 1996), much attention has been paid to increasing the content of EPA in *E. coli* host cells and to its heterologous expression of these genes in various organisms, such as bacteria, yeast, and plants (Yazawa, 1996). The EPA gene clusters have been successfully expressed in various types of *E. coli* strains (Orikasa et al., 2004). Furthermore, numerous attempts have been made to express bacterial EPA biosynthesis genes in bacteria other than *E. coli* and in eukaryotic cells. However, to our knowledge, the report by Yu et. al. (2000) is the only one, in which a marine cyanobacterium is used as a host organism to

**4.2 Enhanced production of EPA by expression of** *vktA* **in** *E. coli* **carrying** *pfa* **genes**  The enhanced production of EPA was observed in recombinant systems of *E. coli* that carried both EPA biosynthesis genes (*pfa*) and a *vktA* catalase gene. Although no molecular mechanism has been determined for this enhanced production of EPA, this technique may become another useful method to increase the productivity of EPA using recombinant systems. Docosahexaenoic acid (DHA) can be synthesized also in bacteria using DHA biosynthesis *pfa* genes, because the two *pfa* genes have a very similar structure

*E. coli* DH5α transformants carrying pEPAΔ1 that included *pfaA-E* genes to the host cell led to the production of EPA (approximately 3% of total fatty acids; Table 2). The production of EPA in host organisms carrying pEPAΔ1 was increased to 12% of total fatty acids by the introduction of a *vktA* insert in pGBM3 [strain DH5α (pEPAΔ1) (pGBM3::*vktA*)]. The empty pGBM3 had no effect on EPA production. In strain DH5α carrying (pEPAΔ1) and partially deleted *vktA* in pGBM3(pGBM3::Δ*vktA*), EPA made up 6% of total fatty acids. The increase in EPA production in strain DH5α (pEPAΔ1)(pGBM3::*vktA*) was accompanied by a decrease in the proportions of palmitoleic acid [16:1(9)] (Table 2). When pGBM3 and pGBM3::*vktA* were replaced in the *E. coli* transformants with pKT230 and pKT230::*vktA*, respectively, similar trends were observed (data not shown). The yield of EPA per culture was approximately 1.5 μg/ml for DH5α(pEPAΔ1) and DH5α(pEPAΔ1)(pGBM3). It increased to 7.3 μg/ml for DH5α(pEPAΔ1) (pGBM3::*vktA*). The yield of EPA from DH5α (pEPAΔ1) pGBM3::Δ*vktA*) was 3.3 μg/ml (Table 2). *E. coli* DH5α has an inherent catalase activity of 2–3 U/mg protein (Nishida et al., 2006). The plasmid pEPAΔ1 had no effect on the catalase activity of the host cells. Catalase activity was increased to 535 U/mg protein for DH5α(pEPAΔ1)(pGBM3::*vktA*). However, there was no enhancement of catalase activity in DH5α(pEPAΔ1)(pGBM3::Δ*vktA*)

**4.1 Bacterial biosynthesis of EPA** 

express the EPA gene cluster.

(Okuyama et al., 2007).

(Table 2). Figure 6 shows the profiles of proteins prepared from various *E. coli* DH5α transformants using SDS-PAGE. A significant amount of protein in the VktA band of 57 kDa, was detected only for DH5α(pEPAΔ1)(pGBM3::*vktA*). No notable novel band was observed in DH5α (pEPAΔ1)(pGBM3::Δ*vktA*) or in any of the other transformants.

Fig. 6. SDS-PAGE profiles of cell-free extracts from various *Escherichia coli* DH5α transformants. Lane 1, *E. coli* DH5α carrying pEPAΔ1; lane 2, *E. coli* DH5α carrying pEPAΔ1 plus empty pGBM3; lane 3, *E. coli* DH5α carrying pEPAΔ1 plus pGBM3::*vktA*; lane 4, *E. coli* DH5α carrying pEPAΔ1 plus pGBM3::Δ*vktA*. Lane M, molecular marker standard (kDa). Arrow indicates the position of running dye. Adapted with permission from Orikasa et al. (2007).

It is evident that bacterial EPA (and DHA) is synthesized by the polyketide biosynthesis pathway, and that this process operates independently of the de novo biosynthesis of fatty acids up to C16 or C18 (Metz et al., 2001; Morita et al., 2000). However, it is likely that acetyl-CoA would be commonly used as a priming substrate in both processes, as specific inhibition of the de novo synthesis of fatty acids up to C18 by cerulenin enhanced the production of EPA and DHA in bacteria and probably also in *Schizochytrium* (Hauvermale et al., 2006). This is analogous to the situation in the unsaturated fatty acid auxotroph *E. coli fabB*– that was transformed with bacterial *pfa* genes, where EPA accounted for more than 30% of total fatty acids (Metz et al., 2001; R.C. Valentine & D. L Valentine, 2004). All of these findings suggest that the metabolic regulation of host organisms carrying *pfa* genes responsible for EPA biosynthesis could potentially be used commercially to enhance the production of EPA. *V. rumoiensis* S-1 accumulates high levels of VktA protein, the amount of which is calculated approximately 2% of total soluble proteins (Yumoto et al., 2000). A significant accumulation of VktA was observed in DH5α(pEPAΔ1)(pGBM3::*vktA*) (Fig. 6). However, the fact that a slight increase in EPA production was also observed in DH5α(pEPAΔ1)(pGBM3::Δ*vktA*) excludes the possibility that the catalytic activity of VktA protein per se was involved in this increased EPA production.


1) The cells were grown at 20C until the culture had an OD660 of 1.0

2) Fatty acids are denoted as number of carbon atoms:number of double bond. The Δ-position of double bond is presented in parenthesis

3) Others incude 12:0, 14:0, 18:0 and 3-hydroxyl 14:0.

Table 2. Fatty acid composition of *E. coli* DH5α and its various transformants and recovered amount of EPA from cultures. Adapted with permission from Orikasa et al. (2007).

At present, the mechanism for the enhanced production of EPA in *E. coli* recombinant systems carrying DH5α(pEPAΔ1)(pGBM3::*vktA*) is unknown. One possibility is that the increase in production of EPA is a response against intracellular stress. DH5α(pEPAΔ1)(pGBM3::*vktA*) accumulated a large amount of VktA protein, which may have increased the stress for the host cells. This would have delayed their growth. Nishida et al. (2006) provided evidence that cellular EPA has an antioxidative function against extracellular H2O2 in bacterial recombinant systems expressing EPA biosynthesis (*pfa*) genes. Interestingly, levels of protein carbonyls were much lower in *E. coli* carrying *pfa* genes (with EPA) than in *E. coli* carrying no vector (without EPA), even if they had not been treated with H2O2. That is, cellular EPA may exert an antioxidative effect on ROS produced intracellularly (Nishida et al., 2006). A variety of stressful conditions, such as heat shock, osmotic shock, nutrient deprivation, and oxidative stress, are known to induce the synthesis of specific proteins. In *E. coli*, the induction of a protein was elicited in response to the overexpression of foreign proteins (Arora et al., 1995). However, to our knowledge, instances where the expression of one foreign gene (DNA) induces the expression of another foreign gene(s) have not been reported.

Clarification of the mechanism of increased EPA (and probably DHA) biosynthesis and the combined use of this technique with the others described above would create the possibility of greater production of these useful polyunsaturated fatty acids.

#### **5. Conclusions**

The VktA catalase is characterized by its high specific activity (Yumoto et al., 1998; 1999; 2000). However, the molecular mechanism of this notable future has not been clarified by its primary structure of protein. VktA accumulate predominantly in the periplasmic space at a level of approximately 2% of total soluble proteins of strain S-1 cells (Yumoto et al., 2000) and part of it is localized at the surface of cells. Such specific distribution of VktA may protect it from attack by protein-degrading enzymes. We are not able to conclude whether the high specific activity of VktA and/or VktA accumulation in the cell are involved in enhancing the nitrogen-fixing activity in *R. leguminosarum* and the increased production of EPA (and probably DHA) in *E. coli* cells. If the high accumulation of catalase with a significantly high specific activity is essential for metabolic modifications (discussed above) in *vktA*-transformed host cells, it is desirable to use other kinds of catalase that accumulate in the cells and have a high specific activity. Such catalases are the *Exiguobacterium oxidotolerans* catalase *(*EKTA catalase; Hara et al., 2007) and the *Psychrobacter piscatorii catalase (*Kimoto et al., 2008)*,* whose specific activity is comparable to that of VktA, indicating that these could be used instead of VktA.

#### **6. Acknowledgements**

266 Innovations in Biotechnology

commercially to enhance the production of EPA. *V. rumoiensis* S-1 accumulates high levels of VktA protein, the amount of which is calculated approximately 2% of total soluble proteins (Yumoto et al., 2000). A significant accumulation of VktA was observed in DH5α(pEPAΔ1)(pGBM3::*vktA*) (Fig. 6). However, the fact that a slight increase in EPA production was also observed in DH5α(pEPAΔ1)(pGBM3::Δ*vktA*) excludes the possibility that the catalytic activity of VktA protein per se was involved in this increased EPA

2) Fatty acids are denoted as number of carbon atoms:number of double bond. The Δ-position of double

Table 2. Fatty acid composition of *E. coli* DH5α and its various transformants and recovered

At present, the mechanism for the enhanced production of EPA in *E. coli* recombinant systems carrying DH5α(pEPAΔ1)(pGBM3::*vktA*) is unknown. One possibility is that the increase in production of EPA is a response against intracellular stress. DH5α(pEPAΔ1)(pGBM3::*vktA*) accumulated a large amount of VktA protein, which may have increased the stress for the host cells. This would have delayed their growth. Nishida et al. (2006) provided evidence that cellular EPA has an antioxidative function against extracellular H2O2 in bacterial recombinant systems expressing EPA biosynthesis (*pfa*) genes. Interestingly, levels of protein carbonyls were much lower in *E. coli* carrying *pfa* genes (with EPA) than in *E. coli* carrying no vector (without EPA), even if they had not been treated with H2O2. That is, cellular EPA may exert an antioxidative effect on ROS produced intracellularly (Nishida et al., 2006). A variety of stressful conditions, such as heat shock, osmotic shock, nutrient deprivation, and oxidative stress, are known to induce the synthesis of specific proteins. In *E. coli*, the induction of a protein was elicited in response to the overexpression of foreign proteins (Arora et al., 1995). However, to our knowledge, instances where the expression of one foreign gene (DNA) induces the expression of another

Clarification of the mechanism of increased EPA (and probably DHA) biosynthesis and the combined use of this technique with the others described above would create the possibility

The VktA catalase is characterized by its high specific activity (Yumoto et al., 1998; 1999; 2000). However, the molecular mechanism of this notable future has not been clarified by its

of greater production of these useful polyunsaturated fatty acids.

amount of EPA from cultures. Adapted with permission from Orikasa et al. (2007).

1) The cells were grown at 20C until the culture had an OD660 of 1.0

3) Others incude 12:0, 14:0, 18:0 and 3-hydroxyl 14:0.

foreign gene(s) have not been reported.

**5. Conclusions** 

production.

bond is presented in parenthesis

Plasmid vector of pEPAΔ1 and *A. niger* catalase (Ryonet F Plus) were kindly provided by Sagami Chemical Research Institute and Nagase Co. Ltd, respectively. A. I. B. H. M. T. is a recipient of the scholarship of the Ministry of Education, Science, Sports, and Culture of Japan (MEXT). This work was partly supported by Grant-in-Aid for Scientific Research ((C) no. 22570130) from MEXT and a grant from Nationa Institute of Polar Research, Japan.

#### **7. References**


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## **Part 4**

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### **Acupuncture for the Treatment of Simple Obesity: Basic and Clinical Aspects**

Wei Shougang and Xie Xincai *Capital Medical University China* 

#### **1. Introduction**

Obesity is a serious, prevalent, and refractory health problem. Individuals who are overweight or obese are at greater risk for a variety of medical conditions including diabetes, hypertension, dyslipidemia, fatty liver, cardiovascular disease, and polycystic ovary syndrome.

Typical therapy for obesity includes: diet restriction, regulation of physical activity, behavior treatment, pharmacotherapy, operation, or the use of any of these methods in combination. The cost of treatment of obesity and obesity-related diseases is significant in general health expenditures of various countries. Moreover, pharmacotherapy and surgical operations have side effects and may be unsafe for some people. Even the behavioral treatments, including diet restriction and regulation of physical activity, seem to produce unfavorable psychological changes. The introduction of new therapies for obesity is in demand.

Acupuncture, practiced for several thousand years in China as monotherapy or complementary therapy that іѕ safe and inexpensive, is increasingly used worldwide in the treatment of a wide spectrum of clinic symptoms and diseases. Acupuncture has been found effective in weight control since 1980s'. An increasing body of evidence demonstrates that acupuncture has good effects for weight loss without adverse reactions. Although relatively new, acupuncture therapy for obesity is increasingly accepted by more and more people around the world. In addition, acupuncture can help with the treatment of obesity-related diseases. Extensive research on acupuncture weight loss has been conducted in both basic and clinic areas in recent years. In this chapter we try to provide a comprehensive review of the most recent basic and clinical advances relating to acupuncture in the treatment of obesity.

#### **2. Traditional chinese medicine view of obesity**

Obesity is a medical condition in which excess body fat has accumulated to the extent that it may have an adverse effect on health. The major symptoms of obesity are excessive weight gain and the presence of large amounts of fatty tissue. People are considered obese if they weigh 20 percent or more above average for their height and build. A widely applied crude population measure of obesity is the body mass index (BMI), a person's weight (in kilograms) divided by the square of his or her height (in metres). A person with a BMI of 30 or more is generally considered obese. As Asian populations develop negative health consequences at a lower BMI than Caucasians, some nations have redefined obesity; the Japanese have defined obesity as any BMI greater than 25 (Kanazawa et al., 2002) while China uses a BMI of greater than 28 (Beifan et al., 2002).

In the view of Western medicine, obesity is primarily caused by a combination of excessive food energy intake, lack of physical activity, and genetic susceptibility. Although excess food (calories) in any form can be converted into fat and stored, the amount of fat in a person's diet may have a greater impact on weight than the number of calories it contains. A sedentary lifestyle can contribute to positive energy balance and the spared calories are stored as fat (adipose) tissue. Eating habits and patterns of physical activity also play a significant role in the amount of weight a person gains. Genetic factors can influence how the body regulates the appetite and the rate at which it turns food into energy (metabolic rate). Psychological factors such as depression and low self-esteem may, in some cases, also play a role in weight gain.

Not surprisingly, the molecular biology of obesity is only partially understood. This is likely due to the heterogeneity of "garden variety" obesity and the fact that it is caused, like other complex diseases, not by a single genetic mutation but by multiple allelic defects, which determine susceptibility to environmental factors . Individuals who carry only one or some of these alleles may still not develop the disease, because they either lack another allele (gene-gene interaction) or are not exposed to the precipitating environment (geneenvironment interaction). Furthermore, there is controversial evidence for a direct association between genotypes and lifestyle or anatomical phenotypes of obesity.

Obesity can be classified in several different ways: for example, by BMI intervals, by anatomic phenotypes or by the stage of life a person becomes obese. Most common form of obesity classification is done using BMI values. According to the World Health Organization (WHO), obesity is classified into 3 classes as listed in the below table.


Table 1. Obesity classification using BMI values

The most common anatomical characterization refers to a prevalently visceral or a prevalently subcutaneous deposition of fat. The ratio of waist circumference to hip circumference (WHR) has served the purpose of defining the degree of central (ie visceral) vs. peripheral (ie subcutaneous) obesity. It is known that visceral adiposity is a major risk factor for metabolic complications of obesity, while subcutaneous fat seems to be much more benign, and in some cases even protective against the development of metabolic complications.

At what stage of life a person becomes obese can affect his or her ability to lose weight. In childhood, excess calories are converted into new fat cells (hyperplastic obesity), while excess calories consumed in adulthood only serve to expand existing fat cells (hypertrophic obesity). Since dieting and exercise can only reduce the size of fat cells, not eliminate them, persons who were obese as children can have great difficulty losing weight, since they may have up to five times as many fat cells as someone who became overweight as an adult.

#### **2.1 Causes of obesity from a TCM perspective**

278 Innovations in Biotechnology

consequences at a lower BMI than Caucasians, some nations have redefined obesity; the Japanese have defined obesity as any BMI greater than 25 (Kanazawa et al., 2002) while

In the view of Western medicine, obesity is primarily caused by a combination of excessive food energy intake, lack of physical activity, and genetic susceptibility. Although excess food (calories) in any form can be converted into fat and stored, the amount of fat in a person's diet may have a greater impact on weight than the number of calories it contains. A sedentary lifestyle can contribute to positive energy balance and the spared calories are stored as fat (adipose) tissue. Eating habits and patterns of physical activity also play a significant role in the amount of weight a person gains. Genetic factors can influence how the body regulates the appetite and the rate at which it turns food into energy (metabolic rate). Psychological factors such as depression and low self-esteem may, in some cases, also

Not surprisingly, the molecular biology of obesity is only partially understood. This is likely due to the heterogeneity of "garden variety" obesity and the fact that it is caused, like other complex diseases, not by a single genetic mutation but by multiple allelic defects, which determine susceptibility to environmental factors . Individuals who carry only one or some of these alleles may still not develop the disease, because they either lack another allele (gene-gene interaction) or are not exposed to the precipitating environment (geneenvironment interaction). Furthermore, there is controversial evidence for a direct

Obesity can be classified in several different ways: for example, by BMI intervals, by anatomic phenotypes or by the stage of life a person becomes obese. Most common form of obesity classification is done using BMI values. According to the World Health Organization

**BMI Classification BMI(for Asian)** 

< 18.5 underweight < 18.5 18.5–24.9 normal weight 18.5–22.9 25.0–29.9 overweight 23.0–24.9 30.0–34.9 class I obesity 25.0–29.9 35.0–39.9 class II obesity ≥ 30.0

The most common anatomical characterization refers to a prevalently visceral or a prevalently subcutaneous deposition of fat. The ratio of waist circumference to hip circumference (WHR) has served the purpose of defining the degree of central (ie visceral) vs. peripheral (ie subcutaneous) obesity. It is known that visceral adiposity is a major risk factor for metabolic complications of obesity, while subcutaneous fat seems to be much more benign, and in some cases even protective against the development of metabolic

At what stage of life a person becomes obese can affect his or her ability to lose weight. In childhood, excess calories are converted into new fat cells (hyperplastic obesity), while

association between genotypes and lifestyle or anatomical phenotypes of obesity.

(WHO), obesity is classified into 3 classes as listed in the below table.

≥ 40.0 class III obesity

Table 1. Obesity classification using BMI values

complications.

China uses a BMI of greater than 28 (Beifan et al., 2002).

play a role in weight gain.

In Traditional Chinese Medicine (TCM), good health consists of the body's systems acting in harmony according to the individual's constitution. If all is working well, there will not be any weight problem. TCM takes a holistic approach to obesity by focussing on the underlying changes in the body. According to TCM principles, development of obesity is due to the following pathological changes (Integrated Chinese Medicine Holdings LTD. [ICMHL], Shen-Nong Info. a).

#### **2.1.1 Dyspepsia causes stomach heat and poor spleen functioning**

Over consumption of heavy, greasy and spicy foods or alcohol facilitate production of heat evils in the stomach. Meanwhile, inadequate exercise after eating these types of foods damages the spleen function. The over-heated stomach will ripen an excessive amount of food.Therefore, the stomach will digest food easier and make an individual feel hungry, but the spleen cannot handle an excessive food load causing it to under function and be unable to carry out its transformation and transportation functions properly. As a result, the spare metabolic products turn into turbid fluid and phlegm which intermix with blood and qi (vital energy) filling up the organs, bones and muscles.

#### **2.1.2 Exogenous evils giving rise to obesity**

Invasion of exogenous evils or over consumption of greasy foods leads to poor transformation and transportation functions of the spleen. Dampness evils then begin to accumulate in the middle burner, which is part of the triple burner (the passage through which water, food and fluid are transported). When dampness and turbid fat enter these passages, they are further distributed by the lungs, allowing penetration into all the organs internally. Additionally, exogenous evils can also penetrate the skin, subcutaneous tissue and muscles through the body's surface giving rise to obesity.

#### **2.1.3 Qi (vital energy) stagnation causes turbid phlegm accumulation**

For those who are emotionally disturbed, experience trauma, have menstruation problems or are elderly, the liver can fail to regulate qi flow which in turn affects digestion and blood flow. The resulting sluggish qi and blood flow tend to block the meridians. Therefore, in these people, dampness is likely to endure in the body. Over time, this will congeal into phlegm and result in obesity.

#### **2.1.4 Kidney essence exhaustion leads to disharmony**

Lifestyles, which consume kidney essence, such as being sexually over active can lead to the excitation of the internal ministerial fire. The excessive ministerial fire is a kind of "evil fire" which makes the body produce an over abundance of heat. This "evil fire" affects the middle burner, leading to a malfunction of the stomach and spleen. When this persists over a long period, the vaporization processes in the bladder and triple burner are impaired causing more evils to accumulate and worsen the obesity condition.

From TCM experience, the above causes of obesity can appear together or separately. In short, the fundamental causes of obesity are spleen and kidney deficiencies, which manifest as an overflow of body fluids, accumulation of dampness and phlegm evils and stagnation in blood flow. Sometimes stomach heat and qi stagnation are associated. Moreover, improper vaporization of body fluids by the triple burner may also appear. All of these factors play an important role in the development of obesity.

#### **2.2 Types of obesity from a TCM perspective**

Syndrome identification is the premise and foundation of TCM treatment. Currently there are still no standardized obesity patterns, ranging from 3 to 12 patterns by clinical reports. Most often, simple obesity was classified into deficiency syndrome and excess syndrome by syndrome differentiation of TCM, which further classified into four types (ICMHL, Shen-Nong Info.b).

#### **2.2.1 Excessive internal phlegm and dampness due to spleen deficiency**

Phlegm is an important concept in TCM. TCM holds that fat or adipose tissue is mostly due to phlegm and dampness evils. The spleen is regarded to be at the root of all phlegm production. When the spleen becomes damaged, such as eating too many sweet foods and getting too little exercise, it will fail in its duty to move and transform waste fluids and foods. Instead these metabolic wastes will gather, collect and transform into evil dampness. If dampness evils endure, over time they will congeal into phlegm, and become fat tissue. The excessive internal phlegm manifests itself as excess weight, accompanied by tiredness, body heaviness, chest and/or stomach distension, and in some cases poor appetite. The tongue has a slimy covering of fur, while there is a rolling, taut pulse. This type of obesity is generally due to an eating disorder, or secondarily by some other illness.

Treatment revolves around sweeping away phlegm and removing stagnation. Once the phlegm is swept away, the qi can move smoothly and easily. This promotes the movement of phlegm and reduction of fat with the ultimate result of decreasing obesity.

#### **2.2.2 Stagnation of qi and blood**

Patients exhibiting this condition may suffer from irritability or low motivation, chest or breast fullness, insomnia, a dreamy state, menstrual disorder or amenorrhea (absence of menstruation), and infertility. Some patients may complain of headaches. There may be dizziness and numbness of the four extremities; and the tongue is dark red with a white thin fur or a thin and greasy fur. The pulse is thready and rolling. This is because the movement of blood is not smooth or easily flowing. Stasis obstructs the vessels and inhibits the qi mechanism. Therefore, fat and dampness collect and accumulate within the vessels, making the blood more viscous. If this continues over time, obesity and arteriosclerosis (thickening and hardening of the arteries) will result.

The principle treatment is to speed up the blood flow and remove stagnation.

#### **2.2.3 Yang deficiency of spleen and kidney**

In the case of yang deficiency of spleen and kidney, there is not enough qi to transform or melt the phlegm. People in this category often feel exhausted or fatigued. They may experience lower back and knee weakness, shortness of breath, impotence or low libido. The pulse is deep and fine, the tongue is pale and without any fur covering. Genetic factors can play a part in this condition. It may also be the result of other illnesses, stress or an unhealthy lifestyle.

Treatment involves fortifying the spleen and rectifying the kidney deficiency.

#### **2.2.4 Liver stagnation**

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burner, leading to a malfunction of the stomach and spleen. When this persists over a long period, the vaporization processes in the bladder and triple burner are impaired causing

From TCM experience, the above causes of obesity can appear together or separately. In short, the fundamental causes of obesity are spleen and kidney deficiencies, which manifest as an overflow of body fluids, accumulation of dampness and phlegm evils and stagnation in blood flow. Sometimes stomach heat and qi stagnation are associated. Moreover, improper vaporization of body fluids by the triple burner may also appear. All of these

Syndrome identification is the premise and foundation of TCM treatment. Currently there are still no standardized obesity patterns, ranging from 3 to 12 patterns by clinical reports. Most often, simple obesity was classified into deficiency syndrome and excess syndrome by syndrome differentiation of TCM, which further classified into four types (ICMHL,

Phlegm is an important concept in TCM. TCM holds that fat or adipose tissue is mostly due to phlegm and dampness evils. The spleen is regarded to be at the root of all phlegm production. When the spleen becomes damaged, such as eating too many sweet foods and getting too little exercise, it will fail in its duty to move and transform waste fluids and foods. Instead these metabolic wastes will gather, collect and transform into evil dampness. If dampness evils endure, over time they will congeal into phlegm, and become fat tissue. The excessive internal phlegm manifests itself as excess weight, accompanied by tiredness, body heaviness, chest and/or stomach distension, and in some cases poor appetite. The tongue has a slimy covering of fur, while there is a rolling, taut pulse. This type of obesity is

Treatment revolves around sweeping away phlegm and removing stagnation. Once the phlegm is swept away, the qi can move smoothly and easily. This promotes the movement

Patients exhibiting this condition may suffer from irritability or low motivation, chest or breast fullness, insomnia, a dreamy state, menstrual disorder or amenorrhea (absence of menstruation), and infertility. Some patients may complain of headaches. There may be dizziness and numbness of the four extremities; and the tongue is dark red with a white thin fur or a thin and greasy fur. The pulse is thready and rolling. This is because the movement of blood is not smooth or easily flowing. Stasis obstructs the vessels and inhibits the qi mechanism. Therefore, fat and dampness collect and accumulate within the vessels, making the blood more viscous. If this continues over time, obesity and arteriosclerosis (thickening

**2.2.1 Excessive internal phlegm and dampness due to spleen deficiency** 

generally due to an eating disorder, or secondarily by some other illness.

of phlegm and reduction of fat with the ultimate result of decreasing obesity.

The principle treatment is to speed up the blood flow and remove stagnation.

more evils to accumulate and worsen the obesity condition.

factors play an important role in the development of obesity.

**2.2 Types of obesity from a TCM perspective** 

Shen-Nong Info.b).

**2.2.2 Stagnation of qi and blood** 

and hardening of the arteries) will result.

Liver stagnation caused by prolonged strong emotions or depression leads to disharmony between the spleen and the liver and gives rise to fluid retention. Due to the liver being depressed, the gall bladder is also depressed and exhausted; the ebb and flow of these organs become unbalanced, and the qi mechanism does not flow freely. Hence fat turbidity is difficult to be transformed and over time it leads to obesity.

Fig. 1. Changes of tongue coating in different types of fat person(ICMHL, Shen-Nong Info.b)

People in this category tend to have excessive fatty material deposited in the abdomen. The physique is bloated and individuals feel drained of energy. Individuals may also experience excess sputum secretion, dizziness, vertigo, retching, a dry mouth, lack of desire for food or drink and discomfort in chest and abdomen. A white glossy or greasy coating usually covers their tongues. The pulse is rolling.

Treatment involves improving liver functioning, unblocking the gallbladder and moving stagnation.

#### **3. Individualized acupuncture for the treatment of obesity: Effects and methods**

In terms of more healthy and holistic methods of weight loss in Chinese medicine, the fundamental prescriptive methodology is to "bian zheng lun zhi"—base treatment on the patient's personal pattern discrimination. TCM doctors diagnose the name of a disease, followed by the differentiation diagnosis of syndromes, for prescribing a treatment. Acupuncture treatment based on a patient's syndrome differentiation is both safe and effective because it addresses that person's own metabolic reasons for being overweight or obese. Using this method, each obese patient can receive his or her own individually tailored acupuncture treatment plan.

Very basically, acupuncture is the insertion of stainless steel filiform needles into precisely specified acupoints on the body's surface , in order to influence physiological functioning of the body.

Fig. 2. Acupuncture being applied to the abdomen

According to TCM, the meridian system provides the transportation channel for the fundamental substances of **qi**, **blood**, and **body fluids**, and along the fourteen **main meridians** there are a total of 365 acupoints have been identified, each point belongs to a particular meridian and connects to a corresponding organ that make it exert particular therapeutic properties.

TCM holds that obesity is caused by anomaly transportation and transformation of the body fluid, accumulation of water-dampness and phlegm turbidity, which are the result of disorders of zang-fu organs, stagnation of qi and blood, disharmony of the Thoroughfare and Conception vessels. Therefore the weight loss can be achieved by needling meridian points to balance yin-yang, regulate zang-fu organs, promote flow of qi and blood of the meridians, and eliminate the inner pathogenic factors by means of dredging meridian and collateral. Accordingly, different set of points would be used, depending on which organ(s) needed to be energized or inhibited. Furthermore, acupuncture needles can be twirled, electrically stimulated, penetrated to different depths and left in place for variable lengths of time.

#### **3.1 Individualized acupuoints selection**

Acupuncture for weight loss refers to the therapeutic approach applying acupuncture or moxibustion on some special points under the guidance of meridian theory of the TCM. For point selection, the acupoints zhong-wan, xia-wan, liang-men and tai-yi were offen used to regulate stomach qi, remove dampness to restore normal function of the spleen. Acupoints tian-shu and da-heng were selected to promote qi circulation and remove obstruction in the collaterals. Acupoints qi-hai and guan-yuan were used to reinforce the kidney. Acupoints wailing, shui-dao, qu-chi, zhi-gou and nei-ting were selected to eliminate the dampness and heat, induce diuresis to alleviate edema, and promote qi flow to relax the bowels.

followed by the differentiation diagnosis of syndromes, for prescribing a treatment. Acupuncture treatment based on a patient's syndrome differentiation is both safe and effective because it addresses that person's own metabolic reasons for being overweight or obese. Using this method, each obese patient can receive his or her own individually

Very basically, acupuncture is the insertion of stainless steel filiform needles into precisely specified acupoints on the body's surface , in order to influence physiological functioning of

According to TCM, the meridian system provides the transportation channel for the fundamental substances of **qi**, **blood**, and **body fluids**, and along the fourteen **main meridians** there are a total of 365 acupoints have been identified, each point belongs to a particular meridian and connects to a corresponding organ that make it exert particular

TCM holds that obesity is caused by anomaly transportation and transformation of the body fluid, accumulation of water-dampness and phlegm turbidity, which are the result of disorders of zang-fu organs, stagnation of qi and blood, disharmony of the Thoroughfare and Conception vessels. Therefore the weight loss can be achieved by needling meridian points to balance yin-yang, regulate zang-fu organs, promote flow of qi and blood of the meridians, and eliminate the inner pathogenic factors by means of dredging meridian and collateral. Accordingly, different set of points would be used, depending on which organ(s) needed to be energized or inhibited. Furthermore, acupuncture needles can be twirled, electrically

stimulated, penetrated to different depths and left in place for variable lengths of time.

induce diuresis to alleviate edema, and promote qi flow to relax the bowels.

Acupuncture for weight loss refers to the therapeutic approach applying acupuncture or moxibustion on some special points under the guidance of meridian theory of the TCM. For point selection, the acupoints zhong-wan, xia-wan, liang-men and tai-yi were offen used to regulate stomach qi, remove dampness to restore normal function of the spleen. Acupoints tian-shu and da-heng were selected to promote qi circulation and remove obstruction in the collaterals. Acupoints qi-hai and guan-yuan were used to reinforce the kidney. Acupoints wailing, shui-dao, qu-chi, zhi-gou and nei-ting were selected to eliminate the dampness and heat,

tailored acupuncture treatment plan.

Fig. 2. Acupuncture being applied to the abdomen

**3.1 Individualized acupuoints selection** 

therapeutic properties.

the body.

Fig. 3. Some examples of acupoints used for promoting weight loss (ICMHL, Shen-Nong Info. c)

Besides these routine points (mainly the points of Conception Vessel, Spleen, Stomach, Kidney, and Bladder Meridians) which were often selected as chief acupoints in the acupuncture treatment of obesity, different adjunct acupoints were added depending on the types of disharmony pattern.

Liu et al. (2004) used the following therapeutic principle and acupoints selection in their clinical practice:


In case of heart palpitations and shortness of breath add shen-men and nei-guan; for scanty urine add shui-fen and yin-ling-quan; for qi depression, nei-guan and tai-chong need to be added; for yin deficiency and heat, tai-xi and zhao-hai need to be added; for menopausal obesity, qi-hai, guan-yuan, pi-shu, shen-shu, tai-xi, or ming-men need to be added; for complications of high blood sugar (e.g., deficiency of both qi and yin), yang-chi, wan-gu, ran-gu, san-yin-jiao, yi-shu and shen-shu need to be added; for excessive appetite (e.g., excessive heat of spleen and stomach), liang-men, liang-qiu, nei-ting, gong-sun, fei-shu and wei-shu need to be added.

#### **3.2 Selection of acupuncture weight loss methods**

Currently there are a variety of acupuncture weight loss methods being used in clinic practice, including body acupuncture, electric acupuncture, magnetic acupuncture, laser acupuncture, warming acupuncture, acupressure, auricular acupuncture, integrative acupuncture and herbal prescription, integrative acupuncture and tuina, integrative warming acupuncture and herbal moxibustion, elongated-needle therapy, catgut embedment, gua-sha(scraping), etc.

#### **3.2.1 Classical body acupuncture**

The filiform needles with diameter of 0.25-0.30 mm and length of 40-75 mm were typically selected based on the case's obesity degree. In applying simple body acupuncture, needling maneuvers were stressed. The reducing method with twirling-rotating techniques was used for excessive syndrome, and the reinforcing method with twirling-rotating techniques was used for deficient syndrome.

For assessment of therapeutic effects of simple obesity, most acupuncturists and Traditional Chinese Medicine practitioners in China use the criteria stipulated at 3rd national conference on obesity by integrated Chinese and western medicine in 1991(Wei, 1992).

Recent clinical cure: Body weight decreases to the standard level; BMI is near to 23.0 kg/m2.

Remarkable effect: Body weight decreases>5.0 kg, and BMI decreases ≥ 4.0 kg/m2.

Effect: Body weight decreases>2.0 kg but<5.0 kg, and BMI decreases ≥ 2.0 kg/m2 but<4.0 kg/m2.

Failure: Body weight decreases<2.0 kg, and BMI decreases<2.0 kg/m2.

The treatment was designed to nourish the liver and kidney. The auricular points selected were liver, kidney and endocrine, and the body acupoints selected were gan-

The treatment was designed to tonify and benefit the lung and spleen. The auricular points selected were lung, spleen and san-jiao, and the body acupoints selected were

The treatment was designed to tonify and benefit the heart and lung. The auricular points selected were heart, lung and ear shen-men, and the body acupoints selected

In case of heart palpitations and shortness of breath add shen-men and nei-guan; for scanty urine add shui-fen and yin-ling-quan; for qi depression, nei-guan and tai-chong need to be added; for yin deficiency and heat, tai-xi and zhao-hai need to be added; for menopausal obesity, qi-hai, guan-yuan, pi-shu, shen-shu, tai-xi, or ming-men need to be added; for complications of high blood sugar (e.g., deficiency of both qi and yin), yang-chi, wan-gu, ran-gu, san-yin-jiao, yi-shu and shen-shu need to be added; for excessive appetite (e.g., excessive heat of spleen and stomach), liang-men, liang-qiu, nei-ting, gong-sun, fei-shu and

Currently there are a variety of acupuncture weight loss methods being used in clinic practice, including body acupuncture, electric acupuncture, magnetic acupuncture, laser acupuncture, warming acupuncture, acupressure, auricular acupuncture, integrative acupuncture and herbal prescription, integrative acupuncture and tuina, integrative warming acupuncture and herbal moxibustion, elongated-needle therapy, catgut embedment, gua-sha(scraping), etc.

The filiform needles with diameter of 0.25-0.30 mm and length of 40-75 mm were typically selected based on the case's obesity degree. In applying simple body acupuncture, needling maneuvers were stressed. The reducing method with twirling-rotating techniques was used for excessive syndrome, and the reinforcing method with twirling-rotating techniques was

For assessment of therapeutic effects of simple obesity, most acupuncturists and Traditional Chinese Medicine practitioners in China use the criteria stipulated at 3rd national conference

Recent clinical cure: Body weight decreases to the standard level; BMI is near to 23.0 kg/m2.

Effect: Body weight decreases>2.0 kg but<5.0 kg, and BMI decreases ≥ 2.0 kg/m2 but<4.0

on obesity by integrated Chinese and western medicine in 1991(Wei, 1992).

Failure: Body weight decreases<2.0 kg, and BMI decreases<2.0 kg/m2.

Remarkable effect: Body weight decreases>5.0 kg, and BMI decreases ≥ 4.0 kg/m2.

• Pattem of yin deficiency in the liver and kidney:

shu, shen-shu, guan-yuan and san-yin-jiao. • Pattem of deficiency in both lung and spleen:

were xin-shu, pi-shu, fei-shu, nei-guan and dan-zhong.

**3.2 Selection of acupuncture weight loss methods** 

fei-shu, pi-shu, zu-san-li and lie-que. • Pattern of qi deficiency in the heart and lung:

wei-shu need to be added.

**3.2.1 Classical body acupuncture** 

used for deficient syndrome.

kg/m2.


Table 2. Needle techniques in acupuncture treatment (ICMHL, Shen-Nong Info. d)

Zhang (2008) reported a 72 cases of obesity treated by body acupuncture which were divided into two groups: heng-gu, da-he, qi-xue, si-man, zhong-zhu and zhi-gou were used in the first group, and da-chang-shu, guan-yuan-shu, xiao-chang-shu, pang-guang-shu, baihuan-shu and tai-xi were selected in the second group, among which bilateral points were alternately used. A lifting-thrusting and twirling reduction method was applied in the first two weeks, which was followed by a lifting-thrusting and twirling uniform reinforcingreducing method with the intensity tolerable to patients. Needles were retained for 30 minutes, during which needles were manipulated twice. The treatment was given 5 times weekly in the first two weeks and then followed by 3 times a week, and 3 months of treatments constituted a therapeutic course. Short-term results showed that 16 cases were clinically cured, 18 cases markedly effective, 34 cases effective and 4 cases failed.

Sun (2008) treated 31 cases of abdominal obesity and 52 cases of symmetrical obesity with the same acupuncture methods for 3 months: zhong-wan, cheng-man, tian-shu, shui-dao, qihai, zu-san-li and san-yin-jiao were used as main points. A reinforcing maneuver was used for qi-hai, zu-san-li and san-yin-jiao, and an even maneuver for others. Liang-men, dai-mai, feng-long, ji-men, yin-bao, nao-hui and zhi-gou were selected as subordinate points and punctured with an even maneuver. The symptomatic points: liang-qiu and nei-ting for stomach heat, reducing; tai-chong for liver depression, reducing; yin-ling-quan for deficiency of spleen, reinforcing; and guan-yuan for deficiency of qi, reinforcing. Needles were remained for 20 min, the treatment was given 3 times weekly, and one month constituted a course. In abdominal obesity group, body mass index (BMI), waist circumference (WC) and skin fat thickness (SFT) in the upper limbs, trunk and abdomen were very significantly reduced (*P*<0.01) after the 1st and the 2nd course of treatment respectively, but no significant difference was found in all indices after the 3rd course of treatment (*P*>0.05). In symmetrical obesity group, all the indices of BMI, WC and SFT were reduced in the successive 3 months of treatment (*P*<0.01).

A majority of obesity patients have substantial accumulation of fat in abdomen and waist, and the more severe the accumulation, the higher relative risk of obesity-related diseases. Mu et al. (Mu &Yuan, 2008) applied abdomen acupuncture therapy to 30 obese patients with elongated needles (75 mm in length). After routine disinfection in the acupoints areas, needles were inserted into the abdominal points of zhong-wan, xia-wan, qi-hai and guanyuan to reinforce the spleen and kidney; of bilateral hua-rou-men and wai-ling to regulate qi and blood; bilateral da-heng to reinforce spleen and dispel dampness; bilateral zhi-gan to regulate the body's qi movement; and of shui-dao to clear heat in triple energizer and downbear the urine and stool. After insertion, the needles were perpendicularly punctured into the earth level (deep level) and retained for 3-5 min to await qi, then the needles were manipulated with twirling-rotating techniques to produce needling sensation. The needles were retained for 30 mm and manipulated once every 5 min to strengthen the needling sensation. The treatment was given once every day in the first 5 times, and once every other day in the latter l 0 times. Fifteen treatments constituted a therapeutic course. Another 30 comparable obese patients receiving the same acupuncture treatment at the points of zhongwan, tian-shu, da-heng, shui-dao, qu-chi, zhi-gou, yin-ling-quan, shang-ju-xu, feng-long and nei-ting were taken as body acupuncture control group in this randomized controlled trial. Before treatment, after l treatment course and after 2 treatment courses were selected as observation time point. The results showed that there was no significant difference in BMI between the two groups after 1-course treatment, but the BMI of abdomen acupuncture group was significant lower than body acupuncture group after 2-course treatment (P<0.05). The cure and total effective rates in abdomen acupuncture group were also higher than those in body acupuncture group after 2-course treatment (see table 3), indicating the curative effect is better in the abdomen acupuncture group.

Abdomen acupuncture divides the insertion depth into three levels of heaven, earth and human. Heaven level (shallow needling) is for those with shorter duration or the pathogen in exterior. Human level (middle needling) is for those have long duration but the pathogen doesn't affect zang-fu organs or pathogen in interstices. For those have long duration and the pathogen involve zang-fu organs or the pathogen in interior, earth level (deep needling) is used.

De qi is viewed as essential to acupuncture's therapeutic effectiveness. The therapeutic depth is the depth to which a needle can be manipulated to achieve the characteristic de qi reaction. Although many studies have used computed tomography (CT) to measure the safe depths (the distance from the surface of the skin of the acupoint to the transverse fascia of the abdominal cavity) of acupoints, few studies have reported on the relative ratio between the therapeutic depth and the safe depth. This ratio may be of clinical importance because it may have an impact on the safety and the therapeutic effectiveness of acupuncture. Chen et al. (2009) analyzed the ratio between the therapeutic depth and safe depth of 12 abdominal

were remained for 20 min, the treatment was given 3 times weekly, and one month constituted a course. In abdominal obesity group, body mass index (BMI), waist circumference (WC) and skin fat thickness (SFT) in the upper limbs, trunk and abdomen were very significantly reduced (*P*<0.01) after the 1st and the 2nd course of treatment respectively, but no significant difference was found in all indices after the 3rd course of treatment (*P*>0.05). In symmetrical obesity group, all the indices of BMI, WC and SFT were

A majority of obesity patients have substantial accumulation of fat in abdomen and waist, and the more severe the accumulation, the higher relative risk of obesity-related diseases. Mu et al. (Mu &Yuan, 2008) applied abdomen acupuncture therapy to 30 obese patients with elongated needles (75 mm in length). After routine disinfection in the acupoints areas, needles were inserted into the abdominal points of zhong-wan, xia-wan, qi-hai and guanyuan to reinforce the spleen and kidney; of bilateral hua-rou-men and wai-ling to regulate qi and blood; bilateral da-heng to reinforce spleen and dispel dampness; bilateral zhi-gan to regulate the body's qi movement; and of shui-dao to clear heat in triple energizer and downbear the urine and stool. After insertion, the needles were perpendicularly punctured into the earth level (deep level) and retained for 3-5 min to await qi, then the needles were manipulated with twirling-rotating techniques to produce needling sensation. The needles were retained for 30 mm and manipulated once every 5 min to strengthen the needling sensation. The treatment was given once every day in the first 5 times, and once every other day in the latter l 0 times. Fifteen treatments constituted a therapeutic course. Another 30 comparable obese patients receiving the same acupuncture treatment at the points of zhongwan, tian-shu, da-heng, shui-dao, qu-chi, zhi-gou, yin-ling-quan, shang-ju-xu, feng-long and nei-ting were taken as body acupuncture control group in this randomized controlled trial. Before treatment, after l treatment course and after 2 treatment courses were selected as observation time point. The results showed that there was no significant difference in BMI between the two groups after 1-course treatment, but the BMI of abdomen acupuncture group was significant lower than body acupuncture group after 2-course treatment (P<0.05). The cure and total effective rates in abdomen acupuncture group were also higher than those in body acupuncture group after 2-course treatment (see table 3), indicating the

Abdomen acupuncture divides the insertion depth into three levels of heaven, earth and human. Heaven level (shallow needling) is for those with shorter duration or the pathogen in exterior. Human level (middle needling) is for those have long duration but the pathogen doesn't affect zang-fu organs or pathogen in interstices. For those have long duration and the pathogen involve zang-fu organs or the pathogen in interior, earth level

De qi is viewed as essential to acupuncture's therapeutic effectiveness. The therapeutic depth is the depth to which a needle can be manipulated to achieve the characteristic de qi reaction. Although many studies have used computed tomography (CT) to measure the safe depths (the distance from the surface of the skin of the acupoint to the transverse fascia of the abdominal cavity) of acupoints, few studies have reported on the relative ratio between the therapeutic depth and the safe depth. This ratio may be of clinical importance because it may have an impact on the safety and the therapeutic effectiveness of acupuncture. Chen et al. (2009) analyzed the ratio between the therapeutic depth and safe depth of 12 abdominal

reduced in the successive 3 months of treatment (*P*<0.01).

curative effect is better in the abdomen acupuncture group.

(deep needling) is used.

acupoints with factors sex, body weight, age, and waist girths by one-way analysis of variance and multiple linear regression analysis to show that the therapeutic depth of abdominal acupoints is closer to the safe depth in overweight and in older children aged 7 to 15 years old, ranged from 0.67 to 0.88 and increased significantly with body weight, age, and waist circumference, but there was no significant difference between genders.


Table 3. Comparison of the therapeutic effects between two groups (Cases)

#### **3.2.2 Classical auricular acupuncture**

From a TCM viewpoint, the ears are an important pivot point for the meridians to communicate with each other. When the organs are in disharmony, it will be reflected on the auricle of the ear. Some of the common auricle acupoints selected for needling in weight loss are: large intestine,small intestine, lung, triple burner, endocrine, subcortex, hunger center, thirst center, constipation center, sympathetic, stomach, esophagus, mouth, adrenal gland and spleen (See Fig.4 for reference). Obese patient with spleen and kidney disharmonies, for instance, can be treated by stimulating corresponding acupoints on the ears to regulate these organs' functions.

Special stainless steel thumbtack form ear needles were used for auricular acupuncture. After sterilizing the acupoints with 75% alcohol, the ear needles were inserted into the auricular acupoints using forceps or fingers. In each treatment, three to five acupoints were needled to induce soreness, numbness, or heat sensation. The bilateral points can be simultaneously used and the needles retained for 30 minutes, once a day, or be alternately used and the needles be embedded with 3M ventilation tape and kept on the ear for several days. Moreover, Cowherb seeds instead of ear needles can be fixated on the auricular acupoints (acupoint embedding) and the points were pressed several times a day or press when hungry by the patients themselves to cause pain sensation.

Although both body acupuncture and auricular acupuncture were effective for weight reduction in obese subjects, combining the application of both body acupuncture and otopuncture has a better result in reducing body weight with its reliable short-term and stable long-term effect.

Fig. 4. Selected aural acupoints in weight loss (ICMHL, Shen-Nong Info. e)

#### **3.2.3 Electroacupuncture**

Meng et al. (Zhang, 2008, as cited in Meng et al, 2002) treated 180 cases of female simple obesity by using electroacupuncture (EA) and 60 cases by manual acupuncture as control. Zhong-wan, tian-shu, guan-yuan and zu-san-li were selected as main points in both groups. In the EA treatment group, bilateral tian-shu were stimulated by a G6805 electric apparatus with disperse and dense wave and the intensity tolerable to patients. Needles were retained for 40 minutes, and the treatment was given 5 times a week followed by a 2 day-interval in both groups, and 20 sessions made up of a therapeutic course. The total effective rate of 97.8% and 88.0% was achieved in the EA treatment and control group respectively. Yin (Zhang, 2008, as cited in Yin, 2000) selected zhong-wan, da-heng, guanyuan and san-yin-jiao as main points, and added secondary points according to differentiation of symptoms and signs. After the arrival of qi by lifting and thrusting for reinforcing and reducing, a G6805 electric apparatus was applied to the main points with continuous waves and 20/sec in frequency and intensity tolerable to patients. The treatment was given once every other day, and 10 treatments constituted a therapeutic course with an interval of 3 days between two courses. The total effective rate after two courses of treatments was 87.5%.

As the parameters of the EA can be precisely characterized and the results are more or less reproducible, an attempt was made by Han Jisheng's research team to clarify whether EA of strictly identified parameters is effective to suppress the simple obesity induced by high energy diet in a rat model. In the diet-induced obese rats, EA was applied at the hind leg acupoints three times per week for 4 weeks with high energy diet and water provided ad libitum. A significant reduction of the body weight accompanied by a reduction in food intake was observed. 2 Hz EA was more effective than 100 Hz EA (Tian et al., 2005).

Fig. 5. An electric apparatus for electroacupuncture stimulation

As was known to all, diet-induced obese rats showed an increased level of plasma cholesterol and triglyceride. EA stimulation produced a reduction of plasma level of total cholesterol and triglyceride. In this respect, 100 Hz EA was more effective than 2 Hz EA. If it is verified that 2 Hz EA is more effective in body weight loss and 100 Hz EA more effective in decreaing plasma lipid content, it may be worthwhile to try the 2/100 Hz alternative mode of stimulation to cover both sides of the disorder.

#### **3.2.4 Laser acupuncture**

288 Innovations in Biotechnology

Fig. 4. Selected aural acupoints in weight loss (ICMHL, Shen-Nong Info. e)

Meng et al. (Zhang, 2008, as cited in Meng et al, 2002) treated 180 cases of female simple obesity by using electroacupuncture (EA) and 60 cases by manual acupuncture as control. Zhong-wan, tian-shu, guan-yuan and zu-san-li were selected as main points in both groups. In the EA treatment group, bilateral tian-shu were stimulated by a G6805 electric apparatus with disperse and dense wave and the intensity tolerable to patients. Needles were retained for 40 minutes, and the treatment was given 5 times a week followed by a 2 day-interval in both groups, and 20 sessions made up of a therapeutic course. The total effective rate of 97.8% and 88.0% was achieved in the EA treatment and control group respectively. Yin (Zhang, 2008, as cited in Yin, 2000) selected zhong-wan, da-heng, guanyuan and san-yin-jiao as main points, and added secondary points according to differentiation of symptoms and signs. After the arrival of qi by lifting and thrusting for reinforcing and reducing, a G6805 electric apparatus was applied to the main points with continuous waves and 20/sec in frequency and intensity tolerable to patients. The treatment was given once every other day, and 10 treatments constituted a therapeutic course with an interval of 3 days between two courses. The total effective rate after two

As the parameters of the EA can be precisely characterized and the results are more or less reproducible, an attempt was made by Han Jisheng's research team to clarify whether EA of strictly identified parameters is effective to suppress the simple obesity induced by high energy diet in a rat model. In the diet-induced obese rats, EA was applied at the hind leg acupoints three times per week for 4 weeks with high energy diet and water provided ad

**3.2.3 Electroacupuncture** 

courses of treatments was 87.5%.

37 adrenal gland 45 sub-cortex 47 lung 49 spleen 50 endocrine 51 triple burners 52 mouth 53 esophagus 55 stomach 57 small intestine 59 large intestine 64 sympathetic 86 constipation center 87 thirst center 88 hunger center

> One of the latest developments in acupuncture stimulation methods are laser needles, which are applied to the surface of the skin but are not inserted into the skin. This non-invasive, painless laser stimulation can induce reproducible peripheral and specific cerebral changes that can be measured in different ways, for example, cerebral blood flow velocity. Several studies show that the cerebral effects induced by laser needles are similar to those evoked by manual needle acupuncture. The "low-power" segment of the beam was postulated to be responsible for the clinical therapeutic effects. Laser devices were manufactured in which power densities and energy densities of laser were lowered to a point where no photothermal effects occurred; but the photoosmotic, photoionic, and photoenzymatic effects were still operative. The latest new laser devices are designed at infrared wavelength combined with highfrequency pulses that allow the photons to penetrate deep into tissue without heat effect.

> It has been observed that laser acupuncture application to obese people increases excitability of the satiety center in the ventromedial nuclei of the hypothalamus, thus suppressing appetite. John et al (2008) made a randomized controlled pilot study on the effects of laser acupuncture on body weight with subjects divided into control and experimental groups. The experimental group was treated with an activated laser and received 16 J of laser energy output to the he-gu and qu-chi. The control group was given a sham low-level laser therapy

treatment with no power output. During the treatment period, each subject received 2 treatments per week for 12 weeks with 4 minutes of active laser or sham treatment to the acupoints in each treatment. Perhaps due to the subjects recruited were not overt obese and the limited laser acupoint stimulation time in this study, no significant weight reduction was observed after the laser acupoint treatment. More studies are needed to investigate the effect of laser acupuncture therapy on body weight.

For the first time, laser needle acupuncture allows simultaneous optical stimulation of individual point combinations. Systematic, double-blind studies of acupuncture can also be performed using optical stimulation because the patient does not notice the activation or deactivation of red or infrared laser needles.

#### **3.2.5 Warming acupuncture**

Yang (Zhang, 2008, as cited in Yang, 2002) used moxibustion with warming needle to treat 32 cases of simple obesity of deficiency type by selecting qi-hai, guan-yuan, zu-san-li, tianshu, yin-ling-quan, and san-yin-jiao as the main points and secondary points according to differentiation of symptoms and signs. Following the arrival of qi, 1-2 lighted moxa sticks about 2 cm in length were consecutively put on the handles of the needles of the 2-3 main points, and the other needles were retained as usual. The treatment was given 6 times weekly, and 30 sessions constituted a therapeutic course. A total effective rate of 90.6% was achieved after one course of treatments.

Fig. 6. A pattern of warming needle moxibusiton

Shi et al. (2008) investigated the clinical effect of acupuncture-moxibustion therapy on simple obesity due to spleen deficiency. Sixty-eight cases of simple obesity of deficiency syndrome types, including internal dampness due to spleen deficiency, qi deficiency of lung-spleen, yang deficiency of spleen-kidney, were randomly allocated into two groups, treatment group (36 cases) and control group (32 cases). The former group was treated with warm needling moxibustion method, and the latter was treated with electroacupuncture. Zhong-wan, shui-fen, qi-hai, zhong-ji and bilateral tian-shu, shui-dao, nei-guan, he-gu, xuehai, zu-san-li, feng-long, san-yin-jiao were selected as main points in both groups, Biliteral

treatment with no power output. During the treatment period, each subject received 2 treatments per week for 12 weeks with 4 minutes of active laser or sham treatment to the acupoints in each treatment. Perhaps due to the subjects recruited were not overt obese and the limited laser acupoint stimulation time in this study, no significant weight reduction was observed after the laser acupoint treatment. More studies are needed to investigate the

For the first time, laser needle acupuncture allows simultaneous optical stimulation of individual point combinations. Systematic, double-blind studies of acupuncture can also be performed using optical stimulation because the patient does not notice the activation or

Yang (Zhang, 2008, as cited in Yang, 2002) used moxibustion with warming needle to treat 32 cases of simple obesity of deficiency type by selecting qi-hai, guan-yuan, zu-san-li, tianshu, yin-ling-quan, and san-yin-jiao as the main points and secondary points according to differentiation of symptoms and signs. Following the arrival of qi, 1-2 lighted moxa sticks about 2 cm in length were consecutively put on the handles of the needles of the 2-3 main points, and the other needles were retained as usual. The treatment was given 6 times weekly, and 30 sessions constituted a therapeutic course. A total effective rate of 90.6% was

Shi et al. (2008) investigated the clinical effect of acupuncture-moxibustion therapy on simple obesity due to spleen deficiency. Sixty-eight cases of simple obesity of deficiency syndrome types, including internal dampness due to spleen deficiency, qi deficiency of lung-spleen, yang deficiency of spleen-kidney, were randomly allocated into two groups, treatment group (36 cases) and control group (32 cases). The former group was treated with warm needling moxibustion method, and the latter was treated with electroacupuncture. Zhong-wan, shui-fen, qi-hai, zhong-ji and bilateral tian-shu, shui-dao, nei-guan, he-gu, xuehai, zu-san-li, feng-long, san-yin-jiao were selected as main points in both groups, Biliteral

effect of laser acupuncture therapy on body weight.

deactivation of red or infrared laser needles.

achieved after one course of treatments.

Fig. 6. A pattern of warming needle moxibusiton

**3.2.5 Warming acupuncture** 

da-heng, fu-jie, yin-ling-quan, gong-sun, pi-shu, wei-shu and qi-hai-shu were added as adjunct acupoints for pattern of internal dampness due to spleen deficiency; dan-zhong and biliteral chi-ze, lie-que, yin-ling-quan, fei-shu, pi-shu, gao-huang-shu were added for pattern of qi deficiency of lung-spleen; guan-yuan, ming-men and bilateral gui-lai, shousan-li, tai-xi, fu-liu, pi-shu, shen-shu were added for pattern of yang deficiency of spleenkidney. After the needling sensation was obtained by routine acupuncture, the main acupoints were connected to the electroacupuncture apparatus in the control group, with continuous wave and at frequency of 2 Hz, by the intensity of stimulation within the patients's tolerance. For the treatment group, warming needle moxibustion was done on 3-4 pairs acupoints for each pattern [qi-hai and biliteral shui-dao, yin-ling-quan, san-yin-jiao were selected for pattern of internal dampness due to spleen deficiency; shui-fen and biliteral chi-ze, zu-san-li, san-yin-jiao for pattern of deficiency of lung-spleen qi; shui-fen, guan-yuan and biliteral tai-xi, zu-san-li for pattern of yang deficiency of spleen-kidney]. Two cones of moxa roll with length of 1.5-2.0 cm were inserted into the needle handle and light it. The needles were retained for 30 min. The treatment was done every other day and 15 times made up of a course. After one course of treatment, the therapeutic efficacy was analyzed and indicated that the weight losing value of treatment group was obviously higher than that of control group. It indicated that for treating simple obesity due to spleen deficiency, warming needle moxibustion method has more advantage than electroacupuncture method. Some cases in two groups were followed up to 6 months after the treatment ended, no obvious rebound phenomenon of body weight were found in two groups, moreover, for some cases treated by warming needle moxibusiton, their body weight continued to decrease in different degrees.

Warm acupuncture is the combination of neediing and moxibustion. Acupuncture has better effect in dredging the channels and collaterals, qi and blood; moxibustion has the double function of warm-dredge and warm-tonify. The combination of acupuncture and moxibustion can give both reinforcing and reducing, address both the symptoms and root causes.

Usually acupuncture weight loss consists of such three phases as fast, stable, and slow weigh-loss phases (Xu et al., 2004). Clinically most people showed marked effect in the first several times of treatment, followed by a stable phase, which may last different time period for different individuals, as the new metabolic balance was being reconstructed by the acupuncture stimulation. Some TCM doctors consider that formula of the points as well as the reinforcing or reducing manipulations need to be regulated during this phase according to the syndrome differentiation, and then the third phase may come smoothly.

#### **4. Mechanisms of acupuncture on weight loss**

The mechanisms of acupuncture's therapeutic effects for simple obesity are not completely understood. TCM holds that obesity belongs to the mixture of root-deficiency (mainly qi deficiency) and symptoms excess (excess of phlegm-dampness). Acupuncture acts to strengthen the function of spleen, stomach, 1iver and kidney, supplement antipathogenic qi and remove pathogenic qi by stimulating points and regulating meridians. Syndrome differentiation is especially important for the treatment. According to the syndrome differentiation of TCM theory, combination of both chief and supplementory points can regulate qi and blood of meridians, correct yin-yang disorder of zang-fu organs and make lasting force with marked effect.

Although the exact mechanism by which acupuncture works is often unknown, the mechanism that helps to suppress appetite in patients who use acupuncture has been of interest to researchers. Ear has close relation with both meridians and zang-fu organs. There have been reports of reduced appetite or craving for food from subjects wearing auricular acupuncture devices (Dung,1986; Richards & Marley, 1998). The conclusion was usually obtained according to subjective reports rather than quantitative analysis of the food intake. Wang et al (2008), in their animal study, measured daily food intake of the rats, and found a reduction of food consumption in the electroacupuncture (EA) treatment group compared to control group subject to restraint only (P<0.001). This reduction was positively correlated with weight loss. It was noticeable that when rats were administered with 2 Hz EA every other day, a reduction of food consumption was observed only on the day of EA administration, suggesting that the effect of EA on appetite suppression lasted for <24 h. Considering the fact that most of the food consumption occurred in the dark phase, the researchers compared the effect of EA treatment delivered in the dark versus light. 2 Hz EA delivered just before the dark phase was more effective than that at the end of the dark phase, suggesting once again that the effect of EA in reducing food intake was immediate and short lasting.

Shiraishi et al. (1995) reported that auricular acupuncture applied to rats produced a reduction of the neural activity of lateral hypothalamus (LH, considered as the ''feeding center'') and an increase of the neural activity of ventral medial hypothalamus (VMH, considered as the ''satiety center''). Because of this, it could help to control the sense of hunger. Furthermore, it has been determined that auricular acupuncture suppresses the appetite by stimulation of the auricular branch of nervous vagus, which has been shown to increase tone in the smooth muscle of the stomach, thus suppressing appetite (Richards & Marley, 1998).

Steyer and Ables(2009)reported that acupuncture affected the ventromedial nucleus of the hypothalamus. Rats that were stimulated with acupuncture needles demonstrated decreased levels of tyrosine and dopamine and increased levels of 5-hydroxytryptamine and 5-hydroxyindoline in this area of the brain. In a study by Wei and Liu in 2003, levels of tryptophan and 5-hydroxyindoleacetic acid were increased, and 5- hydroxytryptamine were decreased in the raphe nuclei of acupuncture treated rats. Thus, acupuncture appears to work on neurotransmitters within the brain to suppress appetite levels and thus help with weight loss.

Electroacupuncture stimulation of the somatic acupoints Zu-san-li and Nei-ting can also increase excitability of the satiety center in the ventral medial nucleus of the hypothalamus (Zhao et al., 2000). The arcuate nucleus of hypothalamus (ARH) is a crucial integrative center for modulation of food intake (Niswender & Schwartz, 2003; Cowley, et al. 2001). The ARH contains at least two populations of neurons that have opposite influence on food intake. One population expresses the anorexigenic peptide "alphamelanocytestimulating hormone" (a-MSH). The other population expresses the orexigenic peptide ''neuropeptide Y (NPY)''. It was demonstrated that in obese rats with

regulate qi and blood of meridians, correct yin-yang disorder of zang-fu organs and make

Although the exact mechanism by which acupuncture works is often unknown, the mechanism that helps to suppress appetite in patients who use acupuncture has been of interest to researchers. Ear has close relation with both meridians and zang-fu organs. There have been reports of reduced appetite or craving for food from subjects wearing auricular acupuncture devices (Dung,1986; Richards & Marley, 1998). The conclusion was usually obtained according to subjective reports rather than quantitative analysis of the food intake. Wang et al (2008), in their animal study, measured daily food intake of the rats, and found a reduction of food consumption in the electroacupuncture (EA) treatment group compared to control group subject to restraint only (P<0.001). This reduction was positively correlated with weight loss. It was noticeable that when rats were administered with 2 Hz EA every other day, a reduction of food consumption was observed only on the day of EA administration, suggesting that the effect of EA on appetite suppression lasted for <24 h. Considering the fact that most of the food consumption occurred in the dark phase, the researchers compared the effect of EA treatment delivered in the dark versus light. 2 Hz EA delivered just before the dark phase was more effective than that at the end of the dark phase, suggesting once again that the effect of EA in reducing food intake was

Shiraishi et al. (1995) reported that auricular acupuncture applied to rats produced a reduction of the neural activity of lateral hypothalamus (LH, considered as the ''feeding center'') and an increase of the neural activity of ventral medial hypothalamus (VMH, considered as the ''satiety center''). Because of this, it could help to control the sense of hunger. Furthermore, it has been determined that auricular acupuncture suppresses the appetite by stimulation of the auricular branch of nervous vagus, which has been shown to increase tone in the smooth muscle of the stomach, thus suppressing appetite (Richards

Steyer and Ables(2009)reported that acupuncture affected the ventromedial nucleus of the hypothalamus. Rats that were stimulated with acupuncture needles demonstrated decreased levels of tyrosine and dopamine and increased levels of 5-hydroxytryptamine and 5-hydroxyindoline in this area of the brain. In a study by Wei and Liu in 2003, levels of tryptophan and 5-hydroxyindoleacetic acid were increased, and 5- hydroxytryptamine were decreased in the raphe nuclei of acupuncture treated rats. Thus, acupuncture appears to work on neurotransmitters within the brain to suppress appetite levels and

Electroacupuncture stimulation of the somatic acupoints Zu-san-li and Nei-ting can also increase excitability of the satiety center in the ventral medial nucleus of the hypothalamus (Zhao et al., 2000). The arcuate nucleus of hypothalamus (ARH) is a crucial integrative center for modulation of food intake (Niswender & Schwartz, 2003; Cowley, et al. 2001). The ARH contains at least two populations of neurons that have opposite influence on food intake. One population expresses the anorexigenic peptide "alphamelanocytestimulating hormone" (a-MSH). The other population expresses the orexigenic peptide ''neuropeptide Y (NPY)''. It was demonstrated that in obese rats with

lasting force with marked effect.

immediate and short lasting.

& Marley, 1998).

thus help with weight loss.

hyperphagia, the expression of a-MSH in ARH was significantly decreased (Lin et al, 2000; Tian et al., 2004). 2 Hz EA treatment produced an increase in the expression of mRNA encoding a-MSH as well as an increase of the peptide level of a-MSH (Tian et al., 2003). In the meantime, there was a downregulation of NPY expression (Tian et al., 2006). Thus, an increase of a-MSH expression and a reduction of NPY expression in the hypothalamic arcuate nuclei may constitute at least part of the mechanisms underlying the effect of 2 Hz EA for decrease of appetite and reduction of body weight.

Wenhe and Yucun (1981) observed that the level of serotonin (5-HT) in the central nervous system increased with acupuncture application. Serotonin has been implicated in the control of eating behavior and body weight. It is thought that an increase in the level of serotonin in the central nervous system with acupuncture application can provide weight loss, as it has a role in both reducing food intake and arranging the psychomotor balance.

It has been shown that uncoupling protein 3(UCP3) in the muscle accelerates the utilization of fatty acids as energy substrate and UCP3 mRNA expression is positively associated with energy expenditure (Costford et al., 2007). The expression of UCP3 could be up-regulated by the activation of 5'-AMP-activated protein kinase (AMPK) (Suwa et al., 2003), and a deletion of AMPKa resulted in a decrease of UCP3 expression in muscle [Jorgensen et al., 2005]. Several studies had implied that UCP3 may serve as a new target in reducing body weight by up-regulating energy expenditure (Schrauwen et al.,1999; Tiraby et al.,2007; Yoon et al.,2007). Wang et al. (2008) reported no significant change in the content of UCP3 protein in the muscle of obese rats was observed after 2 Hz EA treatment, and, in consistent with this, neither phosphorylated nor total protein level of AMPKa were changed. Therefore, the effects of 2 Hz EA in reducing body weight seem to be a result of decrease of food intake rather than an increase of energy expenditure through AMPK–UCP3 pathway.

In many studies it has been observed that electroacupuncture application caused an increase in the levels of beta endorphin both in serum and in the central nervous system (Jin et al., 1996; Takeshige et al., 1992, 1993; Fu, 2000; Petti et al., 1998). It also has been observed that low current frequency (2 Hz) electroacupuncture application increases the concentration of endomorphins, enkephalins, and beta endorphin but high current frequency (100 Hz) electroacupuncture application increased the concentration of dynorphin in the central nervous system (Han et al., 1999). Richter et al. (1983) investigated the lipolitic activity of beta endorphin in the isolated fat cells of rabbits in vivo. It was determined that as a result of the effect of beta endorphin on fat cells, the levels of free fatty acid and glycerol increased in the rabbit plasma. Vettor et al. (1993) studied the lipolitic activity of beta endorphin in isolated human fat tissue to observed that beta endorphin application caused the increase of glycerol secretion from isolated fat cells. According to the results obtained from these studies, it is thought that electroacupuncture, which increases the plasma beta endorphin levels, can contribute to the weight loss by increasing the lipolitic activity (Cabýoglu et al., 2006a).

As was reported before, diet-induced obese rats showed an increased level of plasma cholesterol and triglyceride. Sun (2005) applied ear- and body-acupuncture to obese subjects, and found a decrease in plasma levels of triglyceride, total cholesterol and LDL cholesterol as well as an increase in the HDL level. Since the manipulation of the needle according to traditional Chinese medicine is difficult to characterize, Wang et al. (2008) took advantage of using precisely identified frequency and intensity of the electrical stimulation applied on needles inserted into the acupoints. EA produced a reduction of plasma level of total cholesterol and triglyceride. In this respect, 100 Hz EA was more effective than 2 Hz EA. If it is verified that 2 Hz EA is more effective in body weight loss and 100 Hz EA more effective in decreasing plasma lipid content, it may be worthwhile to try the 2/100 Hz alternative mode of stimulation to cover both sides of the disorder.

Leptin is a peptide known to decrease the body weight and appetite. Kim et al. (2006) applied 100 Hz EA to ad libitum fed normal rats and revealed a significant increase of plasma leptin level. You et al. (2005) found that 100 Hz EA produced a significant decrease of plasma leptin in obese rats. These results suggest that the effect of EA in modulating plasma level of leptin depends on the energy balance state of the animal. On the other hand, the subject's sensitivity to leptin should be regarded as a more important factor in determining the occurrence of obesity than the plasma level of leptin (Kim et al., 2006; You et al. 2005). In other words, resistance to leptin is more important for the induction of obesity than the insufficient supply of leptin. Therefore, study should be proceeded to characterize whether the sensitivity of leptin can be improved by EA treatment.

In sum, the weight loss function of acupuncture might work through the following three means: 1) Regulating nerve system. It is believed that when needling certain acupoints, peripheral nerves were stimulated to regulate the autonomic nerve of the internal organs and make the intercoordination between sympathetic and parasympathetic nerve, which can inhibit gastric emptying and correct abnormal appetite on one side, and promote intestinal peristalsis and reduce the food absorption on the other side; It has been observed that acupuncture application causes changes in the concentrations of K+, Na+, and Ca+ in the neurons (Deng, 1995), and the amount of neuropeptides like beta endorphin, leucine, encephalin, and neurotransmitters like aspartate in the central nervous system (Fu, 2000). Researchers strongly support the opinions that the effect of acupuncture is arranged by the brain (Futaesaku et al., 1995) and that EA application causes a great change in the action potential of nerve cells (Fu, 2000). 2) Regulating endocrine system. Actually endocrine disorder is both the cause and result of obesity. Acupuncture can restore normal endocrine by regulating the two systems of "hypothalamus-pituitary-adrenal cortex" and "sympathetic adrenal cortex" (Shi & Zhang, 2005). It has been determined that endomorphin-1, beta endorphin, encephalin, and serotonin levels increase in plasma and the central nervous system through acupuncture application. Encephalins as well as serotonin has an effect on feeling well, producing happiness, being pleased, producing a normal level of appetite, and achieving psychomotor balance. These effects play a role in the arrangement of psychological behaviors, including dietary behavior (Cabýoglu et al., 2006b). 3) Regulating lipid metabolism. It has been observed that the increases of endomorphin-1, beta endorphin, encephalin, serotonin, and dopamine cause lipolitic effects on metabolism. Needling certain points can reduce the content of lipid peroxide in the blood and accelerate the fat decomposition. In addition, acupuncture can regulate water and salt metabolism and thus correct the condition of water-salt retention (Sun et al., 1996).

cholesterol as well as an increase in the HDL level. Since the manipulation of the needle according to traditional Chinese medicine is difficult to characterize, Wang et al. (2008) took advantage of using precisely identified frequency and intensity of the electrical stimulation applied on needles inserted into the acupoints. EA produced a reduction of plasma level of total cholesterol and triglyceride. In this respect, 100 Hz EA was more effective than 2 Hz EA. If it is verified that 2 Hz EA is more effective in body weight loss and 100 Hz EA more effective in decreasing plasma lipid content, it may be worthwhile to try the 2/100 Hz

Leptin is a peptide known to decrease the body weight and appetite. Kim et al. (2006) applied 100 Hz EA to ad libitum fed normal rats and revealed a significant increase of plasma leptin level. You et al. (2005) found that 100 Hz EA produced a significant decrease of plasma leptin in obese rats. These results suggest that the effect of EA in modulating plasma level of leptin depends on the energy balance state of the animal. On the other hand, the subject's sensitivity to leptin should be regarded as a more important factor in determining the occurrence of obesity than the plasma level of leptin (Kim et al., 2006; You et al. 2005). In other words, resistance to leptin is more important for the induction of obesity than the insufficient supply of leptin. Therefore, study should be proceeded to characterize whether the sensitivity of leptin can be improved by EA

In sum, the weight loss function of acupuncture might work through the following three means: 1) Regulating nerve system. It is believed that when needling certain acupoints, peripheral nerves were stimulated to regulate the autonomic nerve of the internal organs and make the intercoordination between sympathetic and parasympathetic nerve, which can inhibit gastric emptying and correct abnormal appetite on one side, and promote intestinal peristalsis and reduce the food absorption on the other side; It has been observed that acupuncture application causes changes in the concentrations of K+, Na+, and Ca+ in the neurons (Deng, 1995), and the amount of neuropeptides like beta endorphin, leucine, encephalin, and neurotransmitters like aspartate in the central nervous system (Fu, 2000). Researchers strongly support the opinions that the effect of acupuncture is arranged by the brain (Futaesaku et al., 1995) and that EA application causes a great change in the action potential of nerve cells (Fu, 2000). 2) Regulating endocrine system. Actually endocrine disorder is both the cause and result of obesity. Acupuncture can restore normal endocrine by regulating the two systems of "hypothalamus-pituitary-adrenal cortex" and "sympathetic adrenal cortex" (Shi & Zhang, 2005). It has been determined that endomorphin-1, beta endorphin, encephalin, and serotonin levels increase in plasma and the central nervous system through acupuncture application. Encephalins as well as serotonin has an effect on feeling well, producing happiness, being pleased, producing a normal level of appetite, and achieving psychomotor balance. These effects play a role in the arrangement of psychological behaviors, including dietary behavior (Cabýoglu et al., 2006b). 3) Regulating lipid metabolism. It has been observed that the increases of endomorphin-1, beta endorphin, encephalin, serotonin, and dopamine cause lipolitic effects on metabolism. Needling certain points can reduce the content of lipid peroxide in the blood and accelerate the fat decomposition. In addition, acupuncture can regulate water and salt metabolism and

alternative mode of stimulation to cover both sides of the disorder.

thus correct the condition of water-salt retention (Sun et al., 1996).

treatment.

#### **5. Reflection about acupuncture weight loss research approach**

Most of the literature on acupuncture for the treatment of obesity is based on uncontrolled trials. Among the controlled trials with positive results, the interpretation of these results is limited by such methodological problems as short duration, inadequate placebo controls, and nonstandard treatment protocols. A recently conducted systematic review and metaanalysis of acupuncture for obesity by Lee et al. (2009), which included a total of 31 randomized controlled trials (RCTs) and 3013 individual cases,reported that: Compared to control of lifestyle, such as diet, exercise or *qigong,*acupuncture was associated with a significant reduction of average body weight (95% confidence interval, CI) of 1.72 kg (0.50– 2.93 kg) and associated with an improvement in obesity (relative risk =2.57; 95% CI, 1.98– 3.34). Acupuncture significantly reduced a body weight of 1.56 kg (0.74–2.38 kg), on average, compared to placebo or sham treatments. Acupuncture also showed more improved outcomes for body weight (mean difference =1.90 kg; 1.66–2.13 kg), as well as for obesity (relative risk =1.13; 1.04–1.22), than conventional medication.

However, most studies have been of short duration, varying from 4 to 12 weeks. Noting that obesity is a chronic condition, it is likely to require longer periods of acupuncture treatment. Moreover, obesity may wax and wane with or without treatment, and thus a longer followup period with serial measurements of outcomes is suggested to determine the genuine effect of acupuncture as well as its long-term efficacy. Acupuncture may also be considered during the maintenance phase of weight loss programs to prevent relapse. In addition, attrition data at each phase of treatment would provide a more thorough evaluation of this alternative treatment (Lacey et al., 2003).

Concealing allocation of treatment vs control is uniquely challenging. When a nonacupuncture point ('sham') is used, it is important for the treatment to be blinded by all except the acupuncturist, since a needle is applied to the same depth and for the same duration as the treatment group but in a location that has no known effect. Although blinding of the therapist who applies acupuncture would be difficult, blinding of patients and other care providers, as well as outcome assessors should be attempted to minimize the performance and assessment bias of trials. Standardized controls should be used in all future clinical trials of acupuncture treatment in obesity. Nevertheless, there have been studies showing that up to 50% of individuals treated by 'sham acupuncture' processes show some physiological effect (Liang & Koya, 2010). The more recently developed placebo needle may be a more appropriate method to ensure validity in assessing the effectiveness of acupuncture. This placebo needle, with a nonpenetrating, blunt tip, held in place by a bandaid and plastic ring, was shown to be perceived by volunteers as similar to the true penetrating acupuncture needle (also held in place via bandaid and ring), and was significantly less effective (Liang & Koya, 2010). With or without appropriate blinding and placebo controls, expectations about the credibility (usefulness and efficacy) of unconventional methods such as acupuncture may influence outcome. Such moderating variables may obscure real differences between groups especially in small samples. One suggested mechanism to control for patients' expectations is the treatment credibility assessment, adapted from Borkovec and Nau (Lacey et al., 2003, as cited in Borkovec & Nau, 1982), which is a simple series of four questions designed to measure the individual's belief in the efficacy of treatment. Ideally, the mean scale scores should be equivalent for both treatment and control groups to ensure that the groups are comparable.

Laser acupuncture is currently being used in double-blind studies (Liang & Koya, 2010). In this technique, a laser needle is rather fixed onto the skin than pricked into the skin, to deliver the laser power to the acupoints, and the precise power intensity can determined by using the intensity curve (Litscher & Schikora, 2002). In this way, the patient can hardly feel the stimulation and the operator may also be unaware of whether the laser needle system is active, and therefore true doubleblind studies in acupuncture research can be performed. In this regard, previous studies indicated that laser acupuncture applied to the placebo points did not produce marked cerebral changes compared with that applied to the acupoints (Litscher et al.,2004).

To assess accurately any potential benefits for treating obesity, the art of acupuncture must be effectively bridged with the science of evaluation. Standard algorithms need to be developed, based on principles used by practitioners, for example, criteria for selecting and changing point locations, and spacing of treatments (Lacey et al., 2003).

Several randomized controlled trials have suggested that acupuncture has a positive impact on short-term weight loss. These positive effects are typically not observed when acupuncture is used in the absence of dietary and/or behavioral interventions. Therefore, future studies should include a behavioral component across conditions in order to maximize success, provide an active treatment for the controls, and decrease attrition in the comparison groups (Lacey et al., 2003).

The 31 RCTs results in Lee's systematic review (Lee et al., 2009) do not make any consistent suggestions about which form of acupuncture may be the most effective for various types of obesity. So, in light of the evidence, it is a fair summary that acupuncture is an effective treatment for obesity, and further studies, especially rigorous long-term RCTs, are justified to overcome a number of challenges such as effective evaluation of acupuncture while meeting research standards required for evidence-based medicine, to provide conclusive evidence as to the efficacy of acupuncture for weight loss and which particular type of acupuncture should be offered in accordance with the syndrome differentiation of obesity.

Animal research studies are of great importance to identify the underlying mechanism of acupuncture in treatment of obesity. Originating in China centuries ago, acupoints were described in human body rather than in animals. Animal research of acupuncture was initiated in China in the early 1950s and various mammals such as monkey, horse, dog, mouse, rabbit and rat have been applied in acupuncture studies. Although there have been standardized acupoints in human body (WHO Regional Office for the Western Pacific,2008), no such acupoints have been defined in animals. Most animal studies applied acupoints corresponding anatomically to their original locations in humans. A recent report on transpositional acupoint location in mice and rats may be supplied as a reference (Yin et al., 2008).

Mapping the precise location of needles at specific acupoints, including insertion points, depth, direction and angle will definitely have impact on the effect of acupuncture. Further studies are needed to clearly map the site and depth of needle prick/insertion at acupoints (Liang & Koya, 2010).

Since the manipulation of the needle according to TCM is difficult to characterize, a wide spectrum of high-tech methods including Laser Doppler flowmetry and imaging, multidirectional transcranial ultrasonography, cerebral near infrared spectroscopy as well as functional magnetic imaging and a range of bioelectrical methods have been utilized for research in the field of modernization of acupuncture (Liang & Koya, 2010; Litscher et al.,2004; Litscher & Schikora, 2002). Today it is possible to perform transcontinental studies, for example, using teleacupuncture (Litscher, 2009). Slowly but surely the secrets of acupuncture will be demystified.

#### **6. Acknowledgements**

This work was supported by Beijing Natural Science Foundation (No. 7112014).

#### **7. References**

296 Innovations in Biotechnology

in the efficacy of treatment. Ideally, the mean scale scores should be equivalent for both

Laser acupuncture is currently being used in double-blind studies (Liang & Koya, 2010). In this technique, a laser needle is rather fixed onto the skin than pricked into the skin, to deliver the laser power to the acupoints, and the precise power intensity can determined by using the intensity curve (Litscher & Schikora, 2002). In this way, the patient can hardly feel the stimulation and the operator may also be unaware of whether the laser needle system is active, and therefore true doubleblind studies in acupuncture research can be performed. In this regard, previous studies indicated that laser acupuncture applied to the placebo points did not produce marked cerebral changes compared with that applied to the acupoints

To assess accurately any potential benefits for treating obesity, the art of acupuncture must be effectively bridged with the science of evaluation. Standard algorithms need to be developed, based on principles used by practitioners, for example, criteria for selecting and

Several randomized controlled trials have suggested that acupuncture has a positive impact on short-term weight loss. These positive effects are typically not observed when acupuncture is used in the absence of dietary and/or behavioral interventions. Therefore, future studies should include a behavioral component across conditions in order to maximize success, provide an active treatment for the controls, and decrease attrition in the

The 31 RCTs results in Lee's systematic review (Lee et al., 2009) do not make any consistent suggestions about which form of acupuncture may be the most effective for various types of obesity. So, in light of the evidence, it is a fair summary that acupuncture is an effective treatment for obesity, and further studies, especially rigorous long-term RCTs, are justified to overcome a number of challenges such as effective evaluation of acupuncture while meeting research standards required for evidence-based medicine, to provide conclusive evidence as to the efficacy of acupuncture for weight loss and which particular type of acupuncture should be offered in accordance with the syndrome differentiation of obesity. Animal research studies are of great importance to identify the underlying mechanism of acupuncture in treatment of obesity. Originating in China centuries ago, acupoints were described in human body rather than in animals. Animal research of acupuncture was initiated in China in the early 1950s and various mammals such as monkey, horse, dog, mouse, rabbit and rat have been applied in acupuncture studies. Although there have been standardized acupoints in human body (WHO Regional Office for the Western Pacific,2008), no such acupoints have been defined in animals. Most animal studies applied acupoints corresponding anatomically to their original locations in humans. A recent report on transpositional acupoint location in mice and rats may be supplied as a

Mapping the precise location of needles at specific acupoints, including insertion points, depth, direction and angle will definitely have impact on the effect of acupuncture. Further studies are needed to clearly map the site and depth of needle prick/insertion at acupoints

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### **Spermatogonial Stem Cells and Animal Transgenesis**

Flavia Regina Oliveira de Barros, Mariana Ianello Giassetti and José Antônio Visintin *School of Veterinary Medicine and Animal Sciences – University of Sao Paulo Brazil* 

#### **1. Introduction**

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Spermatogonial stem cells (SSCs) are unipotent adult stem cells responsible for the maintenance of the spermatogenesis throughout the entire life of the male. We could say that the mammalian spermatogenesis is a classic adult stem cell-dependent process, sustained by self renewal and differentiation of SSCs. They are the only germline stem cells in adults. These cells can be found in the seminiferous tubule, lying near to the basement membrane. The SSC may choose to self-renewal or generate a daughter cell committed to differentiation. Studying SSCs provides a model to better understand adult stem cell biology and decipher the mechanisms that control SSC functions. It was reported that these cells hold the ability to colonize the seminiferous tubules after transplantation, restoring spermatogenesis. Besides the biomedical potential to perform studies of infertility in many species, SSCs present a promising application in biotechnology in the production of transgenic animals. This alternative route for transgenesis is of interest because a single male will generate by regular mate a variety of transgenic progenies. The production of a transgenic gonad can overcome the obstacles faced with the sperm-mediated gene transfer (SMGT) due to the high specialization of sperm. The use of SSC for transgenesis relies on targeting a much more undifferentiated germ cell and the potential permanent modification of the germ line. In this manner, this chapter aims to review the following topics regarding SSCs: (1) Mammalian spermatogenesis and SSCs; (2) Characterization of SSCs; (3) Isolation and *in vitro* culture of SSCs; (4) Transplantation of SSCs and animal transgenesis.

#### **2. Spermatogonial stem cells and spermatogenesis**

Spermatogenesis is a highly organized and complex process that is responsible for sperm production in male individuals (Russell et al., 1990). Besides providing continuous source of spermatozoa, it is responsible for maintenance of its stem cell population by constant replication of SSCs. In mammals, millions of sperm cells are produced everyday from SSC (Meistrich & van Beek, 1993). In the testis, only SSCs hold the self-renewal ability, i.e. the ability to undergo a series of mitotic cycles without differentiating. In this manner, we can say that spermatogonial stem cells are at the foundation of spermatogenesis. They are the adult stem cell population of the testis, which is responsible for the maintenance of spermatogenesis throughout the entire life of the male. As observed in other tissue-specific stem cells, SSCs are rare, being only 0.03 percent present in an adult mouse testis (Tagelenbosch & de Rooij, 1993). They are present in the testis in such a small number due to the high density of differentiated germ cells, as differentiating spermatogonia, spermatocytes, spermatids and sperm, all originated from SSCs. We define SSCs as stem cells based on their ability to balance self-renewal and differentiation. The self-renewal sustains the stem cell pool at the testis because SSC undergo multiple mitosis producing new SSCs. These new SSCs hold the same self-renewal and differentiation potential as their precursors. Upon demand, SSCs start the cell division in order to produce a differentiated daughter cell. The balance of these two cell divisions maintains spermatogenesis, which can produce millions of sperm each day without causing depletion of cell source.

SSCs originate from primordial germ cells (PGC), which migrate from the embryonic ectoderm in the epiblast, through the allantois and hindgut until reaching genital ridges (Lawson & Pedersen, 1992; Clark & Eddy, 1975). Once PGCs colonize the genital ridge, they are enclosed by differentiating Sertoli cells, starting the formation of seminiferous cords, that will eventually give rise to seminiferous tubules (Byskov & Hø´yer, 1994). From this stage on, the germ cells are called gonocytes because they differ morphologic from PGCs (Clermont & Perey 1957; Huckins & Clermont 1968). In the late stage of gestation, the gonocytes undergo proliferation and become quiescent, i.e., arrested in the G0/G1 phase of the cell cycle (Clermont & Perrey, 1957). These cells remain in mitotic arrest until the peripubertal period, when they start proliferating again, this time to produce type A SSCs. Increasing levels of gonadotrophic hormones concentration triggers this massive proliferation of type A SSCs, marking the onset of spermatogenesis (Huckins & Clermont, 1968; Belveé et al., 1977).

It is known that in rhesus monkey (de Rooij et al., 2002) and human (Clermont, 1966), two subtypes of type A spermatogonia are morphologic distinguishable: Adark and Apale. The Adark spermatogonium act as a true SSC, forming the testis regenerative reserve while Apale has a progenitor role, constituting the functional reserve (Ehmcke et al., 2006). In rhesus monkey, these spermatogonia are followed by four generations of spermatogonia in different stages of differentiation (B1,B2, B3 and B4; de Rooij, 1986). In human, only one generation of type B spermatogonium can be observed before formation of spermatocytes (Clermont, 1966). In rodents, seven subtypes of type A spermatogonia have been reported: A simple (As), A pared (Apr), A aligned (Aal), A1, A2, A3 and A4 (Huckins, 1971a,b; Huckins & Oakberg, 1978). As spermatogonia are considered the SSCs. Although still undifferentiated, Apr and Aal produce expanded colonies of SSCs because they have already undergone mitosis. However, due to a formation of an intercellular bridge that connects their daughter cells, their division is considered incomplete (Zamboni & Merchant 1973). As a result, these cells no longer possess the self-renewal ability as subtype As spermatogonia. Subtypes A1-A4 spermatogonia also constitute expanded SSCs colonies, but differently from Aal and Apr, they are already synchronized with the cycle of the seminiferous epithelium. In this manner, it is possible to say that A1-A4 spermatogonia are already committed to differentiation into future spermatozoa. We refer as the cycle of seminiferous epithelium the synchronic evolution of germ cells from one stage of spermatogenesis to the next. In other words, the cycle of seminiferous epithelium is the completion of ordered events of cell association, divisions and stages in the seminiferous epithelium over time (Russell et al., 1990). In this cycle, the succession of spermatogonia, spermatocytes and spermatids from basement

stem cells, SSCs are rare, being only 0.03 percent present in an adult mouse testis (Tagelenbosch & de Rooij, 1993). They are present in the testis in such a small number due to the high density of differentiated germ cells, as differentiating spermatogonia, spermatocytes, spermatids and sperm, all originated from SSCs. We define SSCs as stem cells based on their ability to balance self-renewal and differentiation. The self-renewal sustains the stem cell pool at the testis because SSC undergo multiple mitosis producing new SSCs. These new SSCs hold the same self-renewal and differentiation potential as their precursors. Upon demand, SSCs start the cell division in order to produce a differentiated daughter cell. The balance of these two cell divisions maintains spermatogenesis, which can

SSCs originate from primordial germ cells (PGC), which migrate from the embryonic ectoderm in the epiblast, through the allantois and hindgut until reaching genital ridges (Lawson & Pedersen, 1992; Clark & Eddy, 1975). Once PGCs colonize the genital ridge, they are enclosed by differentiating Sertoli cells, starting the formation of seminiferous cords, that will eventually give rise to seminiferous tubules (Byskov & Hø´yer, 1994). From this stage on, the germ cells are called gonocytes because they differ morphologic from PGCs (Clermont & Perey 1957; Huckins & Clermont 1968). In the late stage of gestation, the gonocytes undergo proliferation and become quiescent, i.e., arrested in the G0/G1 phase of the cell cycle (Clermont & Perrey, 1957). These cells remain in mitotic arrest until the peripubertal period, when they start proliferating again, this time to produce type A SSCs. Increasing levels of gonadotrophic hormones concentration triggers this massive proliferation of type A SSCs, marking the onset of spermatogenesis (Huckins & Clermont,

It is known that in rhesus monkey (de Rooij et al., 2002) and human (Clermont, 1966), two subtypes of type A spermatogonia are morphologic distinguishable: Adark and Apale. The Adark spermatogonium act as a true SSC, forming the testis regenerative reserve while Apale has a progenitor role, constituting the functional reserve (Ehmcke et al., 2006). In rhesus monkey, these spermatogonia are followed by four generations of spermatogonia in different stages of differentiation (B1,B2, B3 and B4; de Rooij, 1986). In human, only one generation of type B spermatogonium can be observed before formation of spermatocytes (Clermont, 1966). In rodents, seven subtypes of type A spermatogonia have been reported: A simple (As), A pared (Apr), A aligned (Aal), A1, A2, A3 and A4 (Huckins, 1971a,b; Huckins & Oakberg, 1978). As spermatogonia are considered the SSCs. Although still undifferentiated, Apr and Aal produce expanded colonies of SSCs because they have already undergone mitosis. However, due to a formation of an intercellular bridge that connects their daughter cells, their division is considered incomplete (Zamboni & Merchant 1973). As a result, these cells no longer possess the self-renewal ability as subtype As spermatogonia. Subtypes A1-A4 spermatogonia also constitute expanded SSCs colonies, but differently from Aal and Apr, they are already synchronized with the cycle of the seminiferous epithelium. In this manner, it is possible to say that A1-A4 spermatogonia are already committed to differentiation into future spermatozoa. We refer as the cycle of seminiferous epithelium the synchronic evolution of germ cells from one stage of spermatogenesis to the next. In other words, the cycle of seminiferous epithelium is the completion of ordered events of cell association, divisions and stages in the seminiferous epithelium over time (Russell et al., 1990). In this cycle, the succession of spermatogonia, spermatocytes and spermatids from basement

produce millions of sperm each day without causing depletion of cell source.

1968; Belveé et al., 1977).

membrane toward the lumen of seminiferous tubule is established in a stepwise manner during postnatal development.

In bovine, a livestock species, a similar classification was proposed in 1995 by Wrobel et al. According to this classification, there are basal stem cells, corresponding to type As and Apr spermatogonia in rodents, aggregated spermatogonial precursor cells, equivalent to Aal spermatogonia and finally committed spermatogonial precursor cells, equivalent to A1-A4 spermatogonia. It was suggested that type Apr spermatogonia also hold stem cell properties in bulls.

#### **3. Characterization of spermatogonial stem cells**

SSC are the foundation of the productive spermatogenesis that results in the continuous production of spermatozoa in the postnatal life, but studies with SSCs are complicated because these cells are few in number and no unique identifying characteristics have been reported to date. Thus, little is known of their morphology, functional assay or biochemical characteristics and those evaluations become harder in postnatal tissue. Togelenbosch and de Rooij (1993) performed a quantitative study with spermatogonial cells in mouse testis comprising 1 in 3333 cells from adult mouse testis.

In the spermatogenic cycle with each division the number of cells theoretically double, but is important to remember that generally there is no divisions between Aal to A1 cells. Although morphological changes occur and A1 cells slightly resemble Aal cells. Only some spermatogonial cell types can be distinguished by morphologic characteristics and this may actually cause many disturbances in spermatogonial kinetics studies. In almost all species the type A have very similar morphologic characteristics when these cells are analyzed in whole seminiferous tubules. On the other hand, the type A (as a class), Intermediate and type B spermatogonia can be distinguished by minor morphological changes, using either light or electron microscopy (Russell et al., 1990).

The type A spermatogonia have two different surfaces: one flattened and another rounded. The first surface acquires this format because of its direct contact with basal lamina and the second surface is surrounded by Sertoli cells. In the nucleus is observed little presence of heterochromatin and the nucleolus is visible. The Intermediate spermatogonia typically show an ovoid nucleus, present more heterochromatin located close to the nuclear envelop compared to type A and have also a rounded and a flattened surface. Finally, type B spermatogonia has a rounded nucleus with a moderated quantity of heterochromatin allocated around nuclear edge. A smaller part of the cellular membrane is in contact with basal lamina then the most part of surface is rounded. Thus, main morphological aspects that are analyzed to distinguish the types of spermatogonial cells are, first, the amount of heterochromatin in the nucleus and its relation to nuclear membrane. The type A basically has no heterocromatin, Intermediate displays a moderate quantity and type B an abundant amount. The second important aspect is that spermatogonial cells are part of seminiferous epithelium and always have a flattened surface in contact with basal lamina, and rounded, in contact with Sertoli Cells (Russell et al., 1990). Approximately 300,000 cell (types As, Apr, Aal and Intermediated) were counted and characterized from mice seminiferous tubules (Togelenbosch & de Rooij, 1993) being identified approximately 35,000 type As cells from each testis (Meistrich & van Beek, 1993). The morphological evaluation is an important tool for permatogonial studies but it provides many disturbance in the analysis of data, mainly in cells that is analyzed outside of seminiferous tubules environment. Nevertheless, espermatogonial cells can be identified with functional assays or molecular techniques besides the morphological characterization.

In the functional assay, the presence of SSC, for example: from a new purification protocol, was checked by the transfer of progenitor germ cell to the testis of a recipient animal. Spermatogenesis of the recipient testis was previously depleted by the treatment with an alkylating agent, Busulfan or fractionated X-irradiation (local testicular doses of 1.5 and 12 Gy, 24 h apart; Aponte et al., 2005). After transplantation, SSC repopulate the recipient animal seminiferous tubules, that produces a spermatic cycle with donor progenitor cells from the same specie or not.

Whilst in the undifferentiated stage, SSCs express different proteins and genes. In both cases, they are not produced in greater differentiation (Type A1-A4, Intermediated, spermatocytes and spermatids; Caires et al., 2010). A key step in studying the biology of SSCs is to determine their gene expression profile. However, a scarce knowledge of molecular markers has been accumulated in recent years (Kokkinaki et al., 2010). Some research groups have demonstrated that glial cell-.derived neurotrophic factor (GDNF) is the most essential factor for SSC self-renewal and *in vitro* maintenance in rodents (Caires et al., 2010; Ryu et al., 2005; Meng et al., 2000; Kubota et al., 2004a,b; Kanatsu-Shinohara et al., 2005; Kanatsu-Shinohara et al., 2008; and Braydish-Stalle et al., 2005) and that GDNF receptor (GFRA1) is expressed by SSC/progenitor cells (Naughton et al., 2006; Hofmann et al., 2005 and He et al., 2007). The activation of GDNF pathway probably is related with other pathways that promote the proliferation and the self-renewal of SSCs (Caires et al., 2010; Jijiwa et al., 2004; Braydich-Stolle et al., 2007; Oatley et al., 2007 and Lee et al., 2006). Thereby, many molecular markers for SSC are associated with GDNF pathway.

#### **4. SSC markers and differences among species**

The establishment of molecular signatures for SSCs are a complex and difficult process but some molecular markers have been defined for SSCs and undifferentiated spermatogonia (Caires et al., 2010). It is important to know that all these markers (Table 1) were established for different species using a pool containing undifferentiated germ cells.

One of the most important molecular marker for progenitor germ cells is the GRFA1 that is a co-receptor of RET for the GDNF (He et al., 2007). GDNF is related to neural development (He et al., 2007 and Garces et al., 2000) and renal morphogenesis (He et al., 2007 and Vega et al., 1996). In spermatogonial cells, this factor plays an important role in the regulation of proliferation and differentiation or undifferentiated (He et al., 2007, Naughton et al., 2006; Takakoro et al., 2002; Meng et al., 2000 and Hofmann et al., 2005). Others important markers are Nanog and Pou5f1 (Oct3/4). They are essential transcription factors for the maintenance of pluripotency (Goel et al., 2008). THY-1, a member of the Ig super family is highly expressed in rat stem cells and in SSCs from pre-pubertal bulls (Aponte et al., 2005, Ryu et al., 2004 and Reding et al., 2010), However, the role of THY-1 in the male fertility is still unknown (Aponte et al., 2005 and Barlow et al., 2002). PLZF (*Zfp145*) is a molecular marker for As, Apr and Aal spermatogonia and as GRFA1, it is related with self-renewal of SSC (Aponte et al., 2005; Buaas et al., 2004 and Costoya et al., 2004). NGN3 also is expressed in

for permatogonial studies but it provides many disturbance in the analysis of data, mainly in cells that is analyzed outside of seminiferous tubules environment. Nevertheless, espermatogonial cells can be identified with functional assays or molecular techniques

In the functional assay, the presence of SSC, for example: from a new purification protocol, was checked by the transfer of progenitor germ cell to the testis of a recipient animal. Spermatogenesis of the recipient testis was previously depleted by the treatment with an alkylating agent, Busulfan or fractionated X-irradiation (local testicular doses of 1.5 and 12 Gy, 24 h apart; Aponte et al., 2005). After transplantation, SSC repopulate the recipient animal seminiferous tubules, that produces a spermatic cycle with donor progenitor cells

Whilst in the undifferentiated stage, SSCs express different proteins and genes. In both cases, they are not produced in greater differentiation (Type A1-A4, Intermediated, spermatocytes and spermatids; Caires et al., 2010). A key step in studying the biology of SSCs is to determine their gene expression profile. However, a scarce knowledge of molecular markers has been accumulated in recent years (Kokkinaki et al., 2010). Some research groups have demonstrated that glial cell-.derived neurotrophic factor (GDNF) is the most essential factor for SSC self-renewal and *in vitro* maintenance in rodents (Caires et al., 2010; Ryu et al., 2005; Meng et al., 2000; Kubota et al., 2004a,b; Kanatsu-Shinohara et al., 2005; Kanatsu-Shinohara et al., 2008; and Braydish-Stalle et al., 2005) and that GDNF receptor (GFRA1) is expressed by SSC/progenitor cells (Naughton et al., 2006; Hofmann et al., 2005 and He et al., 2007). The activation of GDNF pathway probably is related with other pathways that promote the proliferation and the self-renewal of SSCs (Caires et al., 2010; Jijiwa et al., 2004; Braydich-Stolle et al., 2007; Oatley et al., 2007 and Lee et al., 2006).

Thereby, many molecular markers for SSC are associated with GDNF pathway.

for different species using a pool containing undifferentiated germ cells.

The establishment of molecular signatures for SSCs are a complex and difficult process but some molecular markers have been defined for SSCs and undifferentiated spermatogonia (Caires et al., 2010). It is important to know that all these markers (Table 1) were established

One of the most important molecular marker for progenitor germ cells is the GRFA1 that is a co-receptor of RET for the GDNF (He et al., 2007). GDNF is related to neural development (He et al., 2007 and Garces et al., 2000) and renal morphogenesis (He et al., 2007 and Vega et al., 1996). In spermatogonial cells, this factor plays an important role in the regulation of proliferation and differentiation or undifferentiated (He et al., 2007, Naughton et al., 2006; Takakoro et al., 2002; Meng et al., 2000 and Hofmann et al., 2005). Others important markers are Nanog and Pou5f1 (Oct3/4). They are essential transcription factors for the maintenance of pluripotency (Goel et al., 2008). THY-1, a member of the Ig super family is highly expressed in rat stem cells and in SSCs from pre-pubertal bulls (Aponte et al., 2005, Ryu et al., 2004 and Reding et al., 2010), However, the role of THY-1 in the male fertility is still unknown (Aponte et al., 2005 and Barlow et al., 2002). PLZF (*Zfp145*) is a molecular marker for As, Apr and Aal spermatogonia and as GRFA1, it is related with self-renewal of SSC (Aponte et al., 2005; Buaas et al., 2004 and Costoya et al., 2004). NGN3 also is expressed in

**4. SSC markers and differences among species** 

besides the morphological characterization.

from the same specie or not.

the same cell types as PLZF and acts in the differentiation of spermatogonia (Aponte et al., 2005 and Yoshida et al., 2004), but also is present in spermatocytes (Aponte et al., 2005 and Reverot et al., 2005).


Table 1. Spermatogonial cells molecular markers in different species

#### **5. Isolation and** *in vitro* **culture of spermatogonial stem cells**

#### **5.1 Isolation techniques**

As discussed earlier, SSCs are found close to the basement membrane and their presence in the adult testis is restricted to less than 0.1 percent of all germ cells (Togelenbosch & De Rooij, 1993). In this manner, the election of the most suitable technique to isolate them is very important to establish an *in vitro* culture of SSC. Nowadays, the two step enzymatic digestion is the most popular technique used to isolate SSCs. This technique is based on two incubations of testicular tissue fragments in the presence of enzymes to digest it. It was first proposed by Davis and Schuetz, (1975) in rats and Bellvé et al., (1977) in mice. The enzymatic digestion have been adapted and applied to many other species since then. The isolation of SSCs is often followed by a purification or enrichment step, in order to increase the amount of SSCs in the cell culture. For that, many approaches have been reported, including the discontinuous Percoll density gradient (Van Pelt et al., 1996), differential plating (Izadyar et al., 2002), flow cytometry cell sorting and magnetic cell sorting using SSCs specific antibodies. We can highlight the Percoll gradient as the most popular enrichment protocol for SSCs. However, many modifications have been proposed to this technique regarding the Percoll density adopted and number of layers used to prepare the gradient. The differential plating consists on the overnight *in vitro* culture of freshly isolated SSCs followed by subculture of only non adherent cells. Germ cells tend to remain in suspension, while supporting cells and other testicular cells adhere to the culture dish. These two techniques are usually combined in order to enrich the population of SSCs.

Other important factor in SSCs isolation if the age of donor individuals at the moment of germ cell isolation. Kanatsu-Shinohara et al. (2004) isolated SSCs from newborn mice (0-2 days of age) because at this age, the most primitive types of spermatogonia are predominant in the testis. When Guan et al. (2006) isolated SSCs from mice with 4-6 weeks of age, they obtained a less pure population of SSCs. Similar results were obtained by Seandel at el. (2007) with 3-5 weeks mice. We believe the same happens in livestock species, as bovine. Izadyar et al. (2002) isolated 65-87% type A SSC population from 5-7 months calves.

#### **5.2** *In vitro* **culture of SSCs**

*In vitro* culture of SSCs faces similar hurdles to those commonly observed in *in vitro* culture of adult stem cells. However, many advances have been achieved in this area, and we can find in the literature protocols with satisfactory outcomes.

It was first reported that SSCs can be *in vitro* cultured for months by Nagano et al. (1998). The same group later suggested the addition of GDNF is important to short-term *in vitro* maintenance of SSCs (Nagano et al., 2003). Aponte et al. (2006) demonstrated the importance of this growth factor in the *in vitro* culture of bovine SSCs. Kanatsu-Shinohara et al. (2003a,b) studied the dynamics of gonocytes throughout *in vitro* culture, assessing the cell number increasing. In this study, it was possible to observe a 1014-fold increase in cell number. In 2005, Kanatsu-Shinohara et al. developed a serum-free culture condition, when germ stem cells were cultured *in vitro* over 6 months. Serum-free conditions to culture SSCs have been optimized in rodents (Kubota et al., 2004a,b and Ryu et al., 2005) in order to support longterm maintenance.

The co-culture of SSCs with monolayers of other cell types as mitotically inactivated murine embryonic fibroblasts (MEF) or STO feeder cells (Nagano et al., 1998) are still discussible, presenting different results so far. MEF is widely used in *in vitro* culture of embryonic stem cells (Evans & Kaufman, 1981) and has been applied as feeder cells to murine SSCs cultures (Kanatsu-Shinohara et al., 2004a). Oatley et al., (2002) cultured bovine SSCs over a monolayer of STO feeder cells and in 2004, the same group developed a lineage of bovine embryonic cells (BEF), which was shown to be effective in the *in vitro* maintenance of bovine SSCs. However, Lee et al. (2001) and Aponte et al. (2006) successfully cultured bovine SSCs under feeder-free conditions. Izadyar et al. (2003a) adopted a lamimin based extracelular matrix to support bovine SSCs *in vitro*.

#### **5.3 Cryopreservation of SSCs**

308 Innovations in Biotechnology

The enzymatic digestion have been adapted and applied to many other species since then. The isolation of SSCs is often followed by a purification or enrichment step, in order to increase the amount of SSCs in the cell culture. For that, many approaches have been reported, including the discontinuous Percoll density gradient (Van Pelt et al., 1996), differential plating (Izadyar et al., 2002), flow cytometry cell sorting and magnetic cell sorting using SSCs specific antibodies. We can highlight the Percoll gradient as the most popular enrichment protocol for SSCs. However, many modifications have been proposed to this technique regarding the Percoll density adopted and number of layers used to prepare the gradient. The differential plating consists on the overnight *in vitro* culture of freshly isolated SSCs followed by subculture of only non adherent cells. Germ cells tend to remain in suspension, while supporting cells and other testicular cells adhere to the culture dish. These two techniques are usually combined in order to enrich

Other important factor in SSCs isolation if the age of donor individuals at the moment of germ cell isolation. Kanatsu-Shinohara et al. (2004) isolated SSCs from newborn mice (0-2 days of age) because at this age, the most primitive types of spermatogonia are predominant in the testis. When Guan et al. (2006) isolated SSCs from mice with 4-6 weeks of age, they obtained a less pure population of SSCs. Similar results were obtained by Seandel at el. (2007) with 3-5 weeks mice. We believe the same happens in livestock species, as bovine. Izadyar et al. (2002) isolated 65-87% type A SSC population from 5-7

*In vitro* culture of SSCs faces similar hurdles to those commonly observed in *in vitro* culture of adult stem cells. However, many advances have been achieved in this area, and we can

It was first reported that SSCs can be *in vitro* cultured for months by Nagano et al. (1998). The same group later suggested the addition of GDNF is important to short-term *in vitro* maintenance of SSCs (Nagano et al., 2003). Aponte et al. (2006) demonstrated the importance of this growth factor in the *in vitro* culture of bovine SSCs. Kanatsu-Shinohara et al. (2003a,b) studied the dynamics of gonocytes throughout *in vitro* culture, assessing the cell number increasing. In this study, it was possible to observe a 1014-fold increase in cell number. In 2005, Kanatsu-Shinohara et al. developed a serum-free culture condition, when germ stem cells were cultured *in vitro* over 6 months. Serum-free conditions to culture SSCs have been optimized in rodents (Kubota et al., 2004a,b and Ryu et al., 2005) in order to support long-

The co-culture of SSCs with monolayers of other cell types as mitotically inactivated murine embryonic fibroblasts (MEF) or STO feeder cells (Nagano et al., 1998) are still discussible, presenting different results so far. MEF is widely used in *in vitro* culture of embryonic stem cells (Evans & Kaufman, 1981) and has been applied as feeder cells to murine SSCs cultures (Kanatsu-Shinohara et al., 2004a). Oatley et al., (2002) cultured bovine SSCs over a monolayer of STO feeder cells and in 2004, the same group developed a lineage of bovine embryonic cells (BEF), which was shown to be effective in the *in vitro* maintenance of bovine SSCs. However, Lee et al. (2001) and Aponte et al. (2006) successfully cultured bovine SSCs

the population of SSCs.

months calves.

term maintenance.

**5.2** *In vitro* **culture of SSCs** 

find in the literature protocols with satisfactory outcomes.

There are two ways to preserve SSCs, long-term *in vitro* culture and cryopreservation. Culture and cryopreservation in combination could be used to immortalize a male's genetic line through the germ cells due to the spermatogonial stem cells ability to self-replicate (Oatley et al., 2004). Since it is still hard to maintain pure populations of SSCs for long periods under in vitro condition, the development of effective cryopreservation protocols have been considered of high interest. Izadyar et al., (2002) cryopreserved type A bovine SSCs using DMSO obtaining a survival rate of 50% after thawing cells. Kaul et al., (2010) also adopted DMSO as SSC cryoprotectant in caprine, also observing a 50% survival rate after cryopreservation. Due to the fact enrichment protocols for type A SSCs are still being improved, many groups adopted the cryopreservation of testicular tissue instead of isolated cells. It appears to provide good results in preserving germ cells (Sato et al., 2011; Kaul et al., 2010).

#### **6. SSCs transplantation and transgenesis**

The development of male germ cells transplantation methods provided a powerful means to study the biology of SSCs and its role in spermatogenesis and opened a door to a new potential tool for transgenesis. In addition, the testis cells transplantation is considered the unique *in vivo* functional assay for SSCs. This technique was first used to verify the function of *in vitro* cultured SSCs in mice (Brinster & Zimmermann, 1994; Brinster & Avarbock, 1994). Testicular cells are isolated from a fertile donor and microinjected into the seminuferous tubule of an infertile recipient. It is expected to observe the resumption of spermatogenesis from a donor SSC-derived colonies in the recipient testes. These cell colonies rise from a single transplanted SSC, what allows the quantification of these clonal events (Brinster, 2002). Besides the possibility to study male infertility, the transplantation of SSCs also provides another way to conserve reproductive potential of genetically valuable individuals within or between species and, finally, can be used to produce transgenic animals after generation of transgenic sperm cells. This last application is especially important for species in which embryonic stem cell lines have not been established and other transgenic techniques present limited efficiency. Transgenic animals have huge applications from basic science such as the creation of animal models for human diseases, like Parkinson´s (Crabtree & Zhang, 2011) to production of recombinant pharmaceutic proteins in the animal's fluid: blood, milk (Houdebine, 2000a,b and Houdebine, 2002), egg white (Zhu et al., 2005; van de Lavoir et al., 2006 and Lillico et al., 2007) and seminal plasma (Dyck et al., 2003). Ever since the generation of the first transgenic animal, in 1980, through pronuclei microinjection in an embryo's pronuclei (Houdebine, 2009), this method has been used in other prolific species as rat, rabbit and pig (Houdebine, 2000a,b). However, along the years, many disadvantages of pronuclei microinjection were reported. One of the most important is the misplaced injection of DNA in the cell cytoplasm and not in its pronucleus. Additionally, the exogenous DNA interaction with the host cells genome is quite variable (Houdebine, 2009). Alternatively, other techniques were developed, such as: gene transfer with transposons, lentiviral vectors, sperm, pluripotent, stem and somatic cells. In 2002, Lavitrano et al. obtained a large number of transgenic pigs using sperm-mediated gene transfer (SMGT). The authors reported that up to 80% of the animal had the exogenous gene integrated in the genome, thus SMGT was more efficient than other techniques previously described. SSCs of all mammalian species examined, including human, can replicate in mouse seminiferous tubules following transplantation, the growth factors required for SSCs self-renewal are probably conserved among mammalian species (Kubota et al., 2006).

Although most studies have been performed in rodents, germ cell transplantation has also been applied to non-rodent species as pigs, goats, cattle, monkeys and recently fish and chickens (Honaramooz et al., 2002a,b, 2003; Schlatt et al., 2002; Izadyar et al., 2003b; Takeuchi et al., 2003; Yoshizaki et al., 2005; Lee et al., 2006; Mikkola et al., 2006; Okutsu et al. 2006; Trefil et al. 2006), as reviewed by Dobrinski, (2008). The first hurdle found when germ cell transplantation was applied to livestock species was the differences in testicular anatomy and physiology. While in rodents it is possible to microinject germ cells directly into the seminiferous tubule via efferent ducts, the same is not feasible in larger animals. Thus, the alternative use of ultrasoung to guide the needle during the injection of cells was successfully reported by Kaul et al. (2010) in goats. In bovine, this ultrasound guided needle technique was successfully applied when an autologous transplantation (Izadyar et al., 2002) as well as a heterologous transplantation (Herrid et al., 2006) was performed. It has been demonstrated that germ cell transplantation can be performed more efficiently after suppression of spermatogenesis in recipient animals. The most popular chemical treatment consists of administering a DNA alkylating agent, Busulfan, that destroys proliferating cells. Busulfan is commonly used to suppress spermatogenesis in rodents. An alternative to Busulfan, irradiation of the testis (Creemers et al., 2002; Schlatt et al., 2002), being frequently adopted in studies with large animals as bulls (Izadyar et al., 2003). Despite all the described potential of SSCs to produce transgenic animals, until now, few groups have genetically modified these cells, being the efforts more restricted to laboratory species.

#### **7. Conclusions and perspectives**

As discussed in this chapter, SSCs, like every other adult stem cell in mammals, retains the ability of either self-renewal or differentiation. In this case, the differentiation process is known as spermatogenesis. Interest in spermatogonia has grown in recent years as a result of exciting developments in stem cell research in general and the development of new research tools allowing the isolation, culture and transplantation of these cells.

Because there are a low concentration of SSCs in mammal testis and isolation processes are difficult (Meachem et al., 2001), the assessment of biological activity and cell viability are essentials for the maintenance of the SSC (Potten & Loeffler, 1990; van der Kooy & Weiss, 200; Watt & Hogan, 2000). Brinster & Avarbock (1994) performed the first SSC transplantation and reported this technique to be a good functional assay. In this way, the progenitor germ cells would be in the correct environment having direct contact with somatic niches (Brinster, 2002). Because SSCs are capable of restoring spermatogenesis after transplantation into testes which spermatogenesis had been suppressed, their transplantation opened the door to many possibilities of usage. In this context, we can include the preservation and reestablishment of the reproductive potential of the animals. For example, and animal with desirable genetic traits which can no longer mate, can continue to spread its genetics through germ cell transplantation. In humans, cancer patients now have the opportunity to cryopreserve their SSCs during chemotherapy or radiation treatments. However, there is so much to study and to understand regarding the biology of SSCs before their transplantation in human becomes a routine procedure. When SSC culture becomes available for clinical use, efficient protocols for cryopreservation of these cells and testicular tissue will be of great value.

Finally, the most exciting potential usage of SSCs relies on their capability to transfer genetic modifications to the next generation in a fast manner. The potential use of SSCs in animal transgenesis has attracted the attention of many research groups all over the globe. As discussed, it is almost impossible to describe the value of a transgenic animal, since they can be used in basic science as animal models to human diseases. In addition, transgenic animals can serve as bioreactors, producing proteins of high interest in the human pharmaceutical industry.

In conclusion, SSCs has many future research perspectives, such as: infertility treatment, contraceptive strategy*, in vitro* spermatogenesis, the development of markers for identification of spermatogonial subtypes, innovative research using germ cell transplantation, preservation of fertility for cancer patients, generation of transgenic animals and preservation of valuable animals (Meachem et al., 2001).

#### **8. References**

310 Innovations in Biotechnology

The authors reported that up to 80% of the animal had the exogenous gene integrated in the genome, thus SMGT was more efficient than other techniques previously described. SSCs of all mammalian species examined, including human, can replicate in mouse seminiferous tubules following transplantation, the growth factors required for SSCs self-renewal are

Although most studies have been performed in rodents, germ cell transplantation has also been applied to non-rodent species as pigs, goats, cattle, monkeys and recently fish and chickens (Honaramooz et al., 2002a,b, 2003; Schlatt et al., 2002; Izadyar et al., 2003b; Takeuchi et al., 2003; Yoshizaki et al., 2005; Lee et al., 2006; Mikkola et al., 2006; Okutsu et al. 2006; Trefil et al. 2006), as reviewed by Dobrinski, (2008). The first hurdle found when germ cell transplantation was applied to livestock species was the differences in testicular anatomy and physiology. While in rodents it is possible to microinject germ cells directly into the seminiferous tubule via efferent ducts, the same is not feasible in larger animals. Thus, the alternative use of ultrasoung to guide the needle during the injection of cells was successfully reported by Kaul et al. (2010) in goats. In bovine, this ultrasound guided needle technique was successfully applied when an autologous transplantation (Izadyar et al., 2002) as well as a heterologous transplantation (Herrid et al., 2006) was performed. It has been demonstrated that germ cell transplantation can be performed more efficiently after suppression of spermatogenesis in recipient animals. The most popular chemical treatment consists of administering a DNA alkylating agent, Busulfan, that destroys proliferating cells. Busulfan is commonly used to suppress spermatogenesis in rodents. An alternative to Busulfan, irradiation of the testis (Creemers et al., 2002; Schlatt et al., 2002), being frequently adopted in studies with large animals as bulls (Izadyar et al., 2003). Despite all the described potential of SSCs to produce transgenic animals, until now, few groups have genetically modified these cells, being the efforts more restricted to

As discussed in this chapter, SSCs, like every other adult stem cell in mammals, retains the ability of either self-renewal or differentiation. In this case, the differentiation process is known as spermatogenesis. Interest in spermatogonia has grown in recent years as a result of exciting developments in stem cell research in general and the development of new

Because there are a low concentration of SSCs in mammal testis and isolation processes are difficult (Meachem et al., 2001), the assessment of biological activity and cell viability are essentials for the maintenance of the SSC (Potten & Loeffler, 1990; van der Kooy & Weiss, 200; Watt & Hogan, 2000). Brinster & Avarbock (1994) performed the first SSC transplantation and reported this technique to be a good functional assay. In this way, the progenitor germ cells would be in the correct environment having direct contact with somatic niches (Brinster, 2002). Because SSCs are capable of restoring spermatogenesis after transplantation into testes which spermatogenesis had been suppressed, their transplantation opened the door to many possibilities of usage. In this context, we can include the preservation and reestablishment of the reproductive potential of the animals. For example, and animal with desirable genetic traits which can no longer mate, can

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**7. Conclusions and perspectives** 


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### **Gene Expression Microarrays in Microgravity Research: Toward the Identification of Major Space Genes**

Jade Q. Clement *Department of Chemistry, Texas Southern University, Houston, Texas USA* 

#### **1. Introduction**

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Crewmembers of space flights commonly experience certain health condition changes such as immune system dysregulation, musculoskeletal changes (e.g., significant bone and muscle loss), and neurological alterations. Since the space exploration of the 1960s and 1970s, physiological changes in several organ systems due to weightlessness have been identified. Some of the adverse effects are a decline in cellular immune responses (Leach et al, 1990; Cogoli 1993; Pippia et al; 1996; Borchers et al, 2002;), cardiovascular deconditioning (Fritsch-Yelle, et al 1996), bone deterioration (Mack et al 1967; Vose et al 1974; Atkov 1992; Schneider et al, 1995; Collet et al, 1997) and muscular atrophy (Thomason and Booth 1990; Aubers et al, 2005; Trappe et al, 2009). Human exposure to microgravity has been demonstrated to be a major environmental factor during space flight (Cogoli et al 1993; Ullrich et al, 2008). Many of the adverse effects of microgravity have much in common with earthbound health problems related to low physical activity or less mechanical loading. For example, bone and muscle loss as well as immune system dysfunction are some of the main consequences common to both extended spaceflight and physical inactivity such as that associated with the aging population and people suffering from degenerative disorders. In a recent review article, mechanotransduction is attributed as the possible convergence point for all the "abnormalities" associated with aging and microgravity because human adaptation to microgravity has all the features of accelerated aging (reviewed by Vernikos & Schneider 2010). More recently, similarities between the clinical presentation (such as atrophy in muscle and bone, cardiovascular disturbances, and alterations in renal, immune and sensory motor systems) of individuals living with spinal cord injury (SCI) and those who experience prolonged gravity unloading (especially astronauts) are reviewed (Scott et al, 2011). It is evident that continued effort in microgravity research will deeper our understanding of space adaptation response and improve many of our health-related problems on earth. Thus, microgravity based research can further our understanding of human diseases such as SCI, diabetes, osteoporosis and premature aging that are related to physical inactivity. Most effective counter measures can then be formulated to ensure safe experience in microgravity and promote healthy beings especially at the senior level.

Despite over 50 years of manned space flight, there is still much to be learned about the consequences of living in space for extended periods of time. Microgravity exposure from spaceflight has global effects on cells in virtually all organ systems in the body. Most cell types, ranging from bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting that microgravity affects fundamental cellular activities. Studies at the cellular and molecular levels have been reported from both space flight and groundbased microgravity simulations. Ground-based gravity-simulation experiments at the cellular and molecular levels have gained much insight into the underlying molecular and cellular alterations induced by microgravity stress as well as the mechanisms of the microgravity effects (reviewed in Cogoli, 1993, 1996; Sonnenfeld & Shearer 2002; Sonnenfeld 2005). Environmental change as drastic as sudden gravity change is likely to alter the function and transcriptional activities of groups of genes. This is because any change in the physiological activity of a cell or an organism is most likely the result of changes in certain genes' expressions. Through the 1990s a number of gene expression studies were carried out to determine microgravity effect on organisms and these studies tended to focus on a few genes at a time. It was only with the advent of high-throughput genomic technology such as microarrays that large scale genome-wide studies have been performed. For most genes (especially structural genes), gene expression in response to an environmental change is mainly controlled at the level of transcription, which provides the base for the successful development of mRNA-based high-throughput assays such as DNA microarray technology. The integrated application of biotechnologies in microgravity research with high throughput microarrays for gene expression analysis and various ground-based simulated microgravity models makes it possible for well controlled experimental studies (Hammond & Hammond, 2001). In this chapter, I will first give a brief overview of the ground based simulated microgravity technology and microarray technology for space lifescience research. Then I will review the combined use of these biotechnologies in the study of microgravity effect on gene expression of mammalian cells with specific focus on the areas where most studies tend to focus on such as cells in the immune system, bone, and muscles. In addition, I will make an attempt toward the identification of major space genes by combing data from all the retrievable microarray-based microgravity studies for each of the specific areas as well as an overall combination of these areas with other less studied areas through bioinformatics analysis. Furthermore, I will discuss the initial list of candidate major space genes that are most frequently altered by microgravity environments through microarray based assays and cross-platform, cross-species bioinformatics analysis.

#### **1.1 An overview of two relevant biotechnologies in space bioscience research: Simulated microgravity and gene expression microarrays**

As we depend on the unit gravity (1g) on earth for our daily lives, most of the cells in our body depend on the gravity for proper growth and function. When the cells are placed in reduced gravity environment, many of their functions are affected at various degrees. A clear understanding of microgravity effects on our genes of cells in our organ systems is essential for extraterrestrial health of space travelers. Because of the high-cost and low efficiency of space flown experiments, ground-based methods for simulating the microgravity environment have been developed. These simulated microgravity studies include head down bed rest for humans (LeBlanc et al, 2007), tail suspension for rodents (Sonnenfeld & Shearer 2002; Sonnenfeld 2005), cell and microorganism cultures with high aspect ratio wall vessel bioreactors (RWV) (Schwarz et al, 1992; Tsao et al 1992; Hammond & Hammond 2001; Nickerson et al, 2003) and random positioning machines (RPM) (Hoson et al 1993; Walther et

spaceflight has global effects on cells in virtually all organ systems in the body. Most cell types, ranging from bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting that microgravity affects fundamental cellular activities. Studies at the cellular and molecular levels have been reported from both space flight and groundbased microgravity simulations. Ground-based gravity-simulation experiments at the cellular and molecular levels have gained much insight into the underlying molecular and cellular alterations induced by microgravity stress as well as the mechanisms of the microgravity effects (reviewed in Cogoli, 1993, 1996; Sonnenfeld & Shearer 2002; Sonnenfeld 2005). Environmental change as drastic as sudden gravity change is likely to alter the function and transcriptional activities of groups of genes. This is because any change in the physiological activity of a cell or an organism is most likely the result of changes in certain genes' expressions. Through the 1990s a number of gene expression studies were carried out to determine microgravity effect on organisms and these studies tended to focus on a few genes at a time. It was only with the advent of high-throughput genomic technology such as microarrays that large scale genome-wide studies have been performed. For most genes (especially structural genes), gene expression in response to an environmental change is mainly controlled at the level of transcription, which provides the base for the successful development of mRNA-based high-throughput assays such as DNA microarray technology. The integrated application of biotechnologies in microgravity research with high throughput microarrays for gene expression analysis and various ground-based simulated microgravity models makes it possible for well controlled experimental studies (Hammond & Hammond, 2001). In this chapter, I will first give a brief overview of the ground based simulated microgravity technology and microarray technology for space lifescience research. Then I will review the combined use of these biotechnologies in the study of microgravity effect on gene expression of mammalian cells with specific focus on the areas where most studies tend to focus on such as cells in the immune system, bone, and muscles. In addition, I will make an attempt toward the identification of major space genes by combing data from all the retrievable microarray-based microgravity studies for each of the specific areas as well as an overall combination of these areas with other less studied areas through bioinformatics analysis. Furthermore, I will discuss the initial list of candidate major space genes that are most frequently altered by microgravity environments through

microarray based assays and cross-platform, cross-species bioinformatics analysis.

**1.1 An overview of two relevant biotechnologies in space bioscience research:** 

As we depend on the unit gravity (1g) on earth for our daily lives, most of the cells in our body depend on the gravity for proper growth and function. When the cells are placed in reduced gravity environment, many of their functions are affected at various degrees. A clear understanding of microgravity effects on our genes of cells in our organ systems is essential for extraterrestrial health of space travelers. Because of the high-cost and low efficiency of space flown experiments, ground-based methods for simulating the microgravity environment have been developed. These simulated microgravity studies include head down bed rest for humans (LeBlanc et al, 2007), tail suspension for rodents (Sonnenfeld & Shearer 2002; Sonnenfeld 2005), cell and microorganism cultures with high aspect ratio wall vessel bioreactors (RWV) (Schwarz et al, 1992; Tsao et al 1992; Hammond & Hammond 2001; Nickerson et al, 2003) and random positioning machines (RPM) (Hoson et al 1993; Walther et

**Simulated microgravity and gene expression microarrays** 

al, 1998; Pardo et al, 2005), denervation (Nikawa et al, 2004), and diamagnetic levitation (Dai et al, 2009; Hammer et al, 2009). Bed rest with the head tilted down at ~ 6 has been found to induce physiological alterations similar to those experienced in the space environment. In a similar way, tail suspension for mice and rats presents physiological effects analogous to those observed in a microgravity environment. To date, most of the microarray based studies of microgravity effects on gene expressions have used the ground-based RWV and RPM bioreactors to simulate microgravity environment (Figure 1 & Table 1). The following section in the introduction gives a brief overview of the major kinds of simulated microgravity models.

Fig. 1. The pie chart shows the percentage of each kind of microgravity used for the microarray studies discussed in this chapter.

#### **1.1.1 Some ground-based simulated microgravity in bioscience research**

Ground-based gravity-simulation experiments at the cellular and molecular levels have gained much insight into the underlying molecular and cellular alterations induced by microgravity stress as well as the mechanisms of the microgravity effects. In ground-based microgravity bioscience research, most simulation models simulate reduced gravity in the range of 10-4 ~ 10-6 g which is very small, close to "micro" (10-6) g level (Klaus 2001). The most commonly used devices for simulating a microgravity environment are the RWV (Figure 2A) and the RPM (Figure 2B), which are also known as the 3D clinostat (van Loon 2007). The RWV and RPM bioreactors were developed to simulate microgravity by mimicking a functional weightless state. The RWV bioreactor rotates cells in a zero head space suspension culture that keeps the cells in a near free fall state (Figure 2A), which we have used for our studies (Clement et al 2007; 2008). The RPM is constructed of two independently rotating frames; one inside the other (Figure 2B). The frames are computer controlled and rotated at random rates (Hoson et al, 1997). This allows samples to continuously randomly position resulting in a vector-averaged simulation of near weightlessness (van Loon, 2007). Both of these systems have been designed to attempt to mimic the weightlessness experienced by objects in orbit around the Earth. Neither system eliminates gravity, but they do make a time-averaged g-vector close to zero (Klaus 2001). Both devices do not allow the cells to receive gravitational loads in any fixed direction.

Fig. 2. A Simplified Schematic View of How the Three Ground-Based Simulated Microgravity Bioreactor Models Work. a). Rotating Wall Vessel bioreactor, rotating along horizontal axis. b) Random Positioning Machine: The blue arrow shows the direction of rotation of the outer frame and red arrows shows the rotation of the inner frame. c) Diamagnetic levitation model: A simple schematic of a variable magnetic force apparatus, a superconducting solenoid encased in a liquid nitrogen shielded liquid helium dewar with a room temperature bore passing through the center.

In addition to the RWV and RPM models, another ground-based simulated microgravity model that has been used less frequently is the diamagnetic levitation model (Figure 2C). Diamagnetic levitation is a method that uses magnetic force to create a near weightless state for ground based gravity studies. Such a variable magnetic force apparatus (VMF) is used to simulate a gravity environment from 0g to 2g (Valles & Guevorkian 2002; Coleman et al, 2007; Hammer et al, 2009). The device is essentially a superconducting solenoid encased in a liquid nitrogen shielded liquid helium dewar with a room temperature bore passing through the center (Figure 2C). Samples placed in the bore experience different gravitational force depending on the vertical position in the bore (Valles & Guevorkian 2002; Coleman et al, 2007).

#### **1.1.2 Genomic technology of gene expression microarrays**

Since the first microarray studies were published over 15 years ago, DNA microarrays have been used in many areas of biomedical sciences. Gene expression DNA microarrays have the potential to become key tools in space bioscience research because gene activity regulation is mostly controlled at the RNA level which is mainly determined by transcription initiation step. There are essentially four main manufacturing techniques for DNA microarrays: photolithography, contact printed, non-contact printed (inkjet), and bead arrays. Photolithography is a form of photochemical synthesis. A main advantage is the ability to put millions of features on one chip (Dalma-Weizhausz et al, 2006). Contact printing involves using robotically controlled print heads that spot or "print" the cDNA or oligonucleotides on a glass slide. Contact printed microarrays are what are commonly used for creating in-house microarrays. Non-contact printed works in a similar fashion to an inkjet printer. This technology does not have the capacity of arrays manufactured through photolithography, but it is improving every year. In 2006, around 180,000 features could be printed on one glass slide (Wolber et al, 2006). Today that has risen to close to 500,000 (Agilent website). With bead arrays the oligonucleotides are attached to 3μm silicon beads

(a) (b) (c)

Microgravity Bioreactor Models Work. a). Rotating Wall Vessel bioreactor, rotating along horizontal axis. b) Random Positioning Machine: The blue arrow shows the direction of rotation of the outer frame and red arrows shows the rotation of the inner frame. c)

Diamagnetic levitation model: A simple schematic of a variable magnetic force apparatus, a superconducting solenoid encased in a liquid nitrogen shielded liquid helium dewar with a

In addition to the RWV and RPM models, another ground-based simulated microgravity model that has been used less frequently is the diamagnetic levitation model (Figure 2C). Diamagnetic levitation is a method that uses magnetic force to create a near weightless state for ground based gravity studies. Such a variable magnetic force apparatus (VMF) is used to simulate a gravity environment from 0g to 2g (Valles & Guevorkian 2002; Coleman et al, 2007; Hammer et al, 2009). The device is essentially a superconducting solenoid encased in a liquid nitrogen shielded liquid helium dewar with a room temperature bore passing through the center (Figure 2C). Samples placed in the bore experience different gravitational force depending on the vertical position in the bore (Valles & Guevorkian 2002; Coleman et al, 2007).

Since the first microarray studies were published over 15 years ago, DNA microarrays have been used in many areas of biomedical sciences. Gene expression DNA microarrays have the potential to become key tools in space bioscience research because gene activity regulation is mostly controlled at the RNA level which is mainly determined by transcription initiation step. There are essentially four main manufacturing techniques for DNA microarrays: photolithography, contact printed, non-contact printed (inkjet), and bead arrays. Photolithography is a form of photochemical synthesis. A main advantage is the ability to put millions of features on one chip (Dalma-Weizhausz et al, 2006). Contact printing involves using robotically controlled print heads that spot or "print" the cDNA or oligonucleotides on a glass slide. Contact printed microarrays are what are commonly used for creating in-house microarrays. Non-contact printed works in a similar fashion to an inkjet printer. This technology does not have the capacity of arrays manufactured through photolithography, but it is improving every year. In 2006, around 180,000 features could be printed on one glass slide (Wolber et al, 2006). Today that has risen to close to 500,000 (Agilent website). With bead arrays the oligonucleotides are attached to 3μm silicon beads

Fig. 2. A Simplified Schematic View of How the Three Ground-Based Simulated

room temperature bore passing through the center.

**1.1.2 Genomic technology of gene expression microarrays** 

which are randomly deposited on a substrate such as a glass slide. The technology allows for hundreds of thousands of features to be attached to one slide (Fan et al, 2006). Both the photolithography and bead array are proprietary process of Affymetrix and Illumina, respectively. Adoption of these kinds of arrays, means adopting a complete system including the microarray scanner. In contrast, the contact and non-contact printed arrays are printed on glass microscope slides and can be used in a wide variety of microarray scanners and with a wide variety of software packages.

Although the high throughput gene expression microarray analysis is tremendously time efficient in that genes from the entire genome can be analyzed simultaneously in one experiment, there have been a number of concerns that have called into question the validity of microarray technology. With spotted (contact printed) microarrays there is evidence that cross-hybridization can happen (Handley et al, 2004), which reduces the specificity of the detection power. There have also been major concerns about the reproducibility of microarray data (Tan et al, 2003) as well as the bias and lack of interpretation that is present in many microarray studies (Richard, 2010). However, high fidelity tends to occur from experiments or studies performed using the same platform within a research laboratory. Tan et al reported a study that used the same samples and conditions, but three different microarray platforms. They found there was a high correlation (>0.9) between the data using the same platform, but when the data was compared between platforms it was as low as 0.47 and only reached 0.59 at best (Tan et al, 2003). Despite the justified concerns over the repeatability of results and cross-platform correlations, microarrays still hold tremendous potential for application to research into areas involving changes in environmental conditions such as microgravity research. In recent years, the MicroArray Quality Control (MAQC) consortium has shown that if standards are met and maintained, microarrays can yield a wealth of reliable data. They were able to show that if proper standards are set and followed inter-platform and intra-platform results are reproducible (MAQC Consortium, 2006). In addition to standardization of protocols, it is important to perform biochemical assays (such as Northern blotting, RT-PCR, etc.) to validate some of the microarray data obtained in individual research labs (Clement, 2010). Most microarrays that are currently being used are high-density, whole genome and multiplexed microarrays. Regardless of microarray platforms, the general procedure for a gene expression microarray assay is more or less the same towards evaluating mRNA abundance.

Once an experimental design has been decided upon, the procedure can be divided into bench work (wet lab) and desk work (computer analysis). A more detailed discussion of gene expression microarray and experimental design can be found from recent publications (Stekel et al 2003; Clement 2010).

#### **1.1.3 Combined use of microarrays and simulated microgravity biotechnologies in the search for gravity sensitive genes**

With the advent of high-throughput genomic technology such as gene expression microarrays, large scale genome-wide search for gravity sensitive genes have been carried out using mRNAs from a variety of organisms such as human, rat, mouse, xenopus, yeast, C. elegans, Drosophila, and several types of plants and microorganisms. The majority (about two thirds) of these studies have been on gene expression in cells grown in some form of simulated microgravity (Table 1).


Table 1. Publications on Microarray Based Analysis of Microgravity Effects on Cells and Organisms

Table 1. Publications on Microarray Based Analysis of Microgravity Effects on Cells and

Organisms

To create the table of published microarray based microgravity studies, I started by doing two searches in PubMed using the search terms "microgravity and microarray" and "spaceflown and microarray". This yielded 47 and 36 citations respectively. I then analyzed the lists to remove redundancies, review articles, and articles that did not directly relate to analyzing the microgravity environment. This yielded a list of 49 published articles. After reviewing the literature, I was able to identify 7 more published microarray studies that did not appear in the PubMed searches. I added these to create the final list of 56 microarray based microgravity studies. Microarray data from these microgravity studies will be subjected to bioinformatics analysis towards the identification of major space genes.

The main aim of this chapter is therefore to review the current status of gene expression microarray technology in space bioscience research.

#### **2. Microarray analysis of microgravity exposed cells of the immune system**

The complex immune system evolved on earth has many windows of opportunity for a sudden switch to the space environment to dysregulate it. Hopefully, with dedicated research efforts of the space life sciences, especially with the application of advanced biotechnologies, a better understanding of immunology in the space environment will lead to effective countermeasures.

Study of the immune system is very important since it is known that astronauts have a much higher rate of infection during and after spaceflight (Sonnenfeld 1988; Sonnefeld 2005; Klaus and Howard 2006). It has been shown that inhibition of T cell activation in microgravity was a result of microgravity itself; factors other than microgravity can be excluded from the depressed activation of lymphocytes during spaceflight (Cogoli et al 1983; Ullrich et al, 2008). To understand the molecular mechanisms for the reduced activation of T cells during microgravity, many experiments have been documented from various laboratories using a variety of cell lines or animal models exposed to spaceflight and different simulated microgravity models (reviewed by Cogoli 1997; Sonnefeld 2005; Aponte et al, 2006). Although variable and contradictory results are common, overall evidence indicates that many functions such as signal transduction, cell-cell contact, cytoskeleton, and cell migration tend to be altered in microgravity. Experiments with human and mouse lymphocytes demonstrated a significant decrease in cell proliferation and in IL-2 and IL-Ra synthesis (Cogoli, 1997; Walther et al, 1998). An increase in urinary IL-6 excretion was observed on space flight and after landing (Stein and Schluter, 1994). IL-2 production decreased after space flight for three different T cell subsets (CD3+, CD4+ and CD8+) and IFN-γ production decreased in the CD4+ subset (Crucian et al, 2000). Spaceflight studies performed with rodents have found that microgravity caused rodents to be more susceptible to infection (Sonnenfeld et al, 1988), inhibited NK cell activity (Rykova et al, 1992), reduced capability for wound healing (Davidson et al., 1999), inhibited INF-γ production (Gould et al., 1987) and a reduction in lymphoid organ size (Congdon et al, 1996). A study using rhesus monkeys, microgravity inhibited IL-1 production and decreased response to colony stimulating factor (CSF) on bone marrow cells (Sonnenfeld et al, 1996). Similar to the in flight experiments, antiorthostatic suspension experiments on rodents have shown a increased levels of corticosterone, a reduced ability to clear bacteria from organs and a increased rate of mortality (Aviles et al, 2003). The important function of cytoskeleton in sensing of microgravity during spaceflight is reviewed (HughesFulford, 2003). Over the past three decades, many more (thousands) publications have documented various results toward elucidation of microgravity mediated immune dysfunction. The most consistently observed effects of microgravity on the immune system have been a reduction in T cell and NK cell populations and functions, especially a reduction in cell-mediated immunity, altered cytokine production, as well as an increased susceptibility to infection under space flight conditions. Decreases in the reactivity of T cells, T cell cytotoxicity, and T cell helper activities have been documented for both spaceflight and ground based simulated microgravity studies.

Towards deciphering why T-cell activation is inhibited in microgravity, genome-wide microarray based analysis has become increasingly used within the last decade. Cell response to microgravity has been studied in relatively well-controlled clinostats and bioreactor cell cultures which are particularly convenient for time-course or multiplexed microarray analysis. The first report on cDNA microarray analysis of space flown T cells was documented in 2001(Lewis et al, 2001). In this study, human T cells (Jurkat, human acute leukemic T cell line, E6-1) were space-flown for 24h and 48h. cDNA microarray (GeneFilterTM) analysis was performed to evaluate gene expression of 4,324 human genes at the 24h time point and 20,000 genes at the 48h time point. They identified differentially expressed genes that encode proteins for cytoskeletal organization, growth and metabolism, adhesion and signal transduction, transcription, apoptosis and tumor suppression (Lewis et al, 2001). The cDNA microarray (GeneFilterTM) analysis of Jurkat cells flown on STS-95 in 1998 found that around 98% the genes examined had similar expression patterns when the space-flown cells were compared to ground control cells. They were the first to report that cytoskeletal genes were differentially regulated: calponin, dynamin, tropomodulin, keratin8, myosins, ankyrin, an actinlike protein, plectin, and C-NAP-1 were upregulated; gelsolin was downregulated. Their data indicated that the expression of genes functioning in interconnect cytoskeletal elements to each other and to cell membrane, regulate filament polymerization and microtubule organization centers were altered by spaceflight (Lewis et al, 2001).

Another earlier microarray based analysis of microgravity effects on T cell activation was reported by Meloni et al (2002). They intended to discriminate between effects of microgravity and cosmic radiations on the influence of microgravity on mitogenic activation of T cells and studied the effects of high cosmic radiations on the gene expression in human T cells boarded in a stratospheric balloon (22 hours flight). They used cDNA microarray hybridization technology for the gene expression analysis and found that activated cells react to the ionizing stress by activating genes involved in cell cycle check-point, oxidative stress response, heat shock protein production or by repressing genes involved in antigen recognition.

Aiming to examine the roles of early genes in initiating and maintaining T cell activity, Hughes-Fulford's lab performed a microarray analysis of simulated microgravity (4h RPM) effect on human peripheral lymphocytes and found that PKA was a key player in the loss of T cell activation in microgravity (Boonyaratanakornkit, et al. 2005). For this study they used Human Genome Focus Arrays (Affymetrix) and identified 91 down-regulated genes as a result of exposure to simulated microgravity. The expression of early genes regulated primarily by transcription factors NF-κB, CREB, ELK, AP-1, and STAT were impaired in microgravity, suggesting that microgravity either slows, impedes, or fully blocks key

Fulford, 2003). Over the past three decades, many more (thousands) publications have documented various results toward elucidation of microgravity mediated immune dysfunction. The most consistently observed effects of microgravity on the immune system have been a reduction in T cell and NK cell populations and functions, especially a reduction in cell-mediated immunity, altered cytokine production, as well as an increased susceptibility to infection under space flight conditions. Decreases in the reactivity of T cells, T cell cytotoxicity, and T cell helper activities have been documented for both

Towards deciphering why T-cell activation is inhibited in microgravity, genome-wide microarray based analysis has become increasingly used within the last decade. Cell response to microgravity has been studied in relatively well-controlled clinostats and bioreactor cell cultures which are particularly convenient for time-course or multiplexed microarray analysis. The first report on cDNA microarray analysis of space flown T cells was documented in 2001(Lewis et al, 2001). In this study, human T cells (Jurkat, human acute leukemic T cell line, E6-1) were space-flown for 24h and 48h. cDNA microarray (GeneFilterTM) analysis was performed to evaluate gene expression of 4,324 human genes at the 24h time point and 20,000 genes at the 48h time point. They identified differentially expressed genes that encode proteins for cytoskeletal organization, growth and metabolism, adhesion and signal transduction, transcription, apoptosis and tumor suppression (Lewis et al, 2001). The cDNA microarray (GeneFilterTM) analysis of Jurkat cells flown on STS-95 in 1998 found that around 98% the genes examined had similar expression patterns when the space-flown cells were compared to ground control cells. They were the first to report that cytoskeletal genes were differentially regulated: calponin, dynamin, tropomodulin, keratin8, myosins, ankyrin, an actinlike protein, plectin, and C-NAP-1 were upregulated; gelsolin was downregulated. Their data indicated that the expression of genes functioning in interconnect cytoskeletal elements to each other and to cell membrane, regulate filament polymerization and microtubule organization centers were altered by spaceflight

Another earlier microarray based analysis of microgravity effects on T cell activation was reported by Meloni et al (2002). They intended to discriminate between effects of microgravity and cosmic radiations on the influence of microgravity on mitogenic activation of T cells and studied the effects of high cosmic radiations on the gene expression in human T cells boarded in a stratospheric balloon (22 hours flight). They used cDNA microarray hybridization technology for the gene expression analysis and found that activated cells react to the ionizing stress by activating genes involved in cell cycle check-point, oxidative stress response, heat shock protein production or by

Aiming to examine the roles of early genes in initiating and maintaining T cell activity, Hughes-Fulford's lab performed a microarray analysis of simulated microgravity (4h RPM) effect on human peripheral lymphocytes and found that PKA was a key player in the loss of T cell activation in microgravity (Boonyaratanakornkit, et al. 2005). For this study they used Human Genome Focus Arrays (Affymetrix) and identified 91 down-regulated genes as a result of exposure to simulated microgravity. The expression of early genes regulated primarily by transcription factors NF-κB, CREB, ELK, AP-1, and STAT were impaired in microgravity, suggesting that microgravity either slows, impedes, or fully blocks key

spaceflight and ground based simulated microgravity studies.

(Lewis et al, 2001).

repressing genes involved in antigen recognition.

signaling pathways in early T cell activation (Boonyaratanakornkit et al, 2005). They showed that IL-2 was among the down-regulated genes, which correlates well with previous nonarray based reports (Cogoli et al, 1993; Pippia et al, 1996; Walther et al, 1998; Hughes-Fulford et al, 2005). Interestingly, IL-2 was also significantly decreased (and IL-10 expression was increased) in a recent real time PCR based analysis of gene expression in T-cells of mice after being flown in space aboard STS-118 for 13 days (Gridley et al, 2009).

Ward et al used Affymetrix Human U133A arrays to study activated human peripheral lymphocytes exposed to simulated microgravity in RWV (22 rpm) for 24h (Ward et al, 2006). From their triplicate experiments they identified 89 (10 up, 79 down) genes that were statistically significant (P≤ 0.01) and at least 1.5 fold up or down-regulated in an all of the arrays. A larger proportion of these affected genes are found to be players in fundamental cellular processes such as immune responses, signal transduction, DNA repair and apoptosis, and metabolic pathways (Ward et al, 2006).

A microarray based study of mRNA expression in murine thymus tissue extracted from C57BL/6NTac female mice that had been flown aboard the space shuttle Endeavour (STS118) for thirteen days was reported recently by Lebsack et al (2010). They used Affymetrix gene chips for this study and found 970 genes significantly differentially regulated (644 up and 326 down). Among the identified genes in stress response, RNA binding motif 3(RBM3) and cold inducible binding protein (CIRBP) were up regulated, while HSP90, HSP110, STIp1, FKBP4 were down regulated. More importantly, genes that regulate immune response were affected by space flight: CD44 and CXCL10 that promote T cell development were down regulated; whereas CTLA-4 (negative regulator of T cell activation) mRNA was upregulated. Overall, the genes identified in this study were involved in stress regulation, glucocorticoid receptor metabolism, and T cell signaling and activity (Lebsack et al, 2010).

The above five studies made good use of the biotechnologies in their respective microgravity studies. Although impressive findings are documented in each individual report, widely varied results in terms of the type of genes or trends in expressions are noted among these microgravity studies using microarrays in the immune system. For a systematic view of these studies, I attempted to compile the data into a tabulated form and was hampered by obstacles, mainly due to a lack of overlap in the identified gravity sensitive genes. The only "overlap" was two genes (STAT1 and XCL1) found to be differentially regulated in two studies: in one study they were both down-regulated (Boonyarantankornkit et al, 2005), while in the other they were both up-regulated (Ward et al, 2006). The large variation in the resulting gravity sensitive genes may in part be due to numerous variables and the overall complexity of the immune system itself (Gridley et al, 2003). In addition, variations of the types of microgravity, different cell lines or cell types, various microarray platforms, etc., could also contribute to the differences in the results. Furthermore, how the data was documented and reported can also contribute to the lack of consistency in resulting sensitive genes. Another contributing factor is the fact that these few studies span a large time frame (2001 ~ 2010) for the relatively young microarray biotechnology, the time period the technology itself undergoes development and is still in the process of standardization. With more standardization in the genome wide assays, meaningful compilation of data can be applied efficiently in a statistical analysis toward the identification of major space genes.

Nonetheless, it is still of interest to see which pathways or molecular functions the gravity sensitive genes from afore mentioned studies. To generate the KEGG Pathways for immune system related cells, I needed to prepare the data for analysis in DAVID (Database for Annotation, Visualization, and Integrated Discovery)(Huang et al, 2009a; Huang et al, 2009b). Out of the five microarray studies, four had gene tables, or lists of genes that were relatively easy to use. Three of these lists used human cells (Lewis et al, 2001; Boonyarantankornkit et al, 2005; Ward et al, 2006), one used mouse tissue (Lebsack et al, 2010). Since the majority of the studies were done with human cells, I chose those three gene lists to compile a master gene list by manually combining the genes from the three microarray studies and using the DAVID conversion tool to convert them all into the same format. This conversion is necessary because of inconsistency in the way gene tables are presented: some of the gene lists included gene bank accession numbers and gene symbols, some included gene symbols but no accession numbers, and others included accession numbers but no symbols, still others with no gene list at all. To limit confusion and for ease of references, both accession numbers and gene symbols should be included in gene lists whenever possible. The converted gene list resulted in 142 genes after redundant, unknown, or unable to define genes were eliminated. This combined gene list was uploaded to the DAVID Functional Annotation tool to identify the statistically significant KEGG Pathways (Table 2). Interestingly but not surprisingly, most of the functional pathways identified using the combined gravity sensitive genes are key pathways for innate and adaptive immunity (Table 2). This is indeed significant, despite the fact that such a large variation in the type and trends of gene expressions were found among these studies.


Table 2. Pathway Identification of Microarray Identified Gravity Sensitive Genes in Immune System. Legend: red means up regulated; green means downregulated; purple means opposite trends between studies; underlined means it appears in more than one study.

Does the cell use "many roads lead to Rome" approach to adapt to microgravity environment? It may be possible that major space pathways rather than specific major space genes are key determinants for adaptation to microgravity. More studies would be needed for statistically based cross-laboratory and cross-platform analysis of microarray based data, which will provide key insight into the molecular mechanism of microgravity mediated immune dysfunction. An altered immune response to microgravity is attributed to be a key factor for bone loss because the altered production and action of cytokines in the immune system could affect bone remolding (Zayzafoon et al, 2005).

Nonetheless, it is still of interest to see which pathways or molecular functions the gravity sensitive genes from afore mentioned studies. To generate the KEGG Pathways for immune system related cells, I needed to prepare the data for analysis in DAVID (Database for Annotation, Visualization, and Integrated Discovery)(Huang et al, 2009a; Huang et al, 2009b). Out of the five microarray studies, four had gene tables, or lists of genes that were relatively easy to use. Three of these lists used human cells (Lewis et al, 2001; Boonyarantankornkit et al, 2005; Ward et al, 2006), one used mouse tissue (Lebsack et al, 2010). Since the majority of the studies were done with human cells, I chose those three gene lists to compile a master gene list by manually combining the genes from the three microarray studies and using the DAVID conversion tool to convert them all into the same format. This conversion is necessary because of inconsistency in the way gene tables are presented: some of the gene lists included gene bank accession numbers and gene symbols, some included gene symbols but no accession numbers, and others included accession numbers but no symbols, still others with no gene list at all. To limit confusion and for ease of references, both accession numbers and gene symbols should be included in gene lists whenever possible. The converted gene list resulted in 142 genes after redundant, unknown, or unable to define genes were eliminated. This combined gene list was uploaded to the DAVID Functional Annotation tool to identify the statistically significant KEGG Pathways (Table 2). Interestingly but not surprisingly, most of the functional pathways identified using the combined gravity sensitive genes are key pathways for innate and adaptive immunity (Table 2). This is indeed significant, despite the fact that such a large variation in the type and trends of gene expressions were found

Table 2. Pathway Identification of Microarray Identified Gravity Sensitive Genes in Immune System. Legend: red means up regulated; green means downregulated; purple means opposite trends between studies; underlined means it appears in more than one study.

Does the cell use "many roads lead to Rome" approach to adapt to microgravity environment? It may be possible that major space pathways rather than specific major space genes are key determinants for adaptation to microgravity. More studies would be needed for statistically based cross-laboratory and cross-platform analysis of microarray based data, which will provide key insight into the molecular mechanism of microgravity mediated immune dysfunction. An altered immune response to microgravity is attributed to be a key factor for bone loss because the altered production and action of cytokines in the immune

system could affect bone remolding (Zayzafoon et al, 2005).

among these studies.

#### **3. Microarray analysis of microgravity exposed bone cells**

Physical inactivity or mechanical unloading to the skeletal system is an underlying cause for bone density loss in clinical disorders such as spinal cord injury, stroke, prolonged bed-rest, aging and osteoporosis as well as in spaceflight microgravity environments (LeBlanc et al, 2005; Beller et al, 2011). Therefore, continued effort in deciphering the mechanism and finding a remedy for bone density loss is beneficial to human health both in space and on earth. Spaceflight caused bone density reduction specifically involves weight-bearing bones (Mack et al, 1967; Vose et al, 1974; Schneider et al, 1995; Collet et al, 1997; Lang et al, 2006; Keyak et al., 2009). In the space environment, bone density in the lower extremities and spine of crew member is lost at a rate of 1% to 2% per month (LeBlanc et al, 2007; Amin, 2010). Microgravity disturbs the balance between bone formation and resorption in bone remolding process: it tends to increase bone resorption functions of osteoclast and decrease bone formation functions of osteoblast. A net loss of calcium in-flight of similar magnitude to that observed in earlier studies from Skylab as well as an increase in bone resorption markers (Smith et al, 2005). Studies of spaceflights showed decreased serum levels of bone formation markers such as alkaline phosphatase (ALP), osteocalcin (OCN), and the C-terminal peptide of pro-collagen I (Collet et al, 1997; Caillot-Augusseau et al, 2000). Increased resorption with little change in formation is the main finding in space flight caused uncoupled bone remodeling (reviewed by LeBlanc et al, 2005). The unbalanced bone resorption and formation resulted in increased Ca2+secretion. Ground-based simulated microgravity studies showed decreased bone formation (Nakamura et al, 2003; Zayzafoon et al, 2004; Pardo et al, 2005; Patel et al, 2007; 2009). Although a gene-specific approach has identified several key genes involved in bone cell growth and development that are affected in microgravity, a comprehensive genome-wide search allows for the identification of more genes as well as possible pathways through which the bone loss occurs.

A number of microarray based analysis of microgravity effect on bone cells have been published relatively recently. Pardo et al showed that gene expression of 140 genes (88 down and 52 up) were significantly altered after exposure of 2T3 murine preosteoblast to 3 days simulated microgravity in RPM (Pardo et al, 2005). They used CodeLink Uniset Mouse 1 Bioarrays (Amersham Biosciences) and the median intensity of all the probes to normalize the intensity of the individual probes. In agreement with spaceflight data and simulated microgravity studies, their microarray data showed genes important for bone density such as alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteomodulin, parathyroid hormone-related protein(PTHrP), parathyroid receptor 1 (PthR1), and platelet derived growth factor (PDGF) were significantly down-regulated. In contrast, cathepsin K (responsible for bone resorption in osteoclasts) is upregulated in the 2T3 cells in response to simulated microgravity. ALP is a known marker for bone formation, and Runx2 is involved in osteoblastic differentiation and skeletal morphogenesis. Both interact with secreted bone morphogenic proteins and with insulin like growth factor 1 (IGF1), which has been shown to regulate Runx2 in endothelial cells (Qiao et al 2004). In their data, IGF1 was shown to be slightly down-regulated although it did not pass the significance test (Pardo et al 2005). PthR1 promotes the release of Ca2+ and it has already been shown to be gravity sensitive in bone cells (Torday 2003). Overall, this study shed much light on the mechanism of microgravity mediated bone loss through the use of microarray analysis. Patel et al (2007), the same research group as the aforementioned study by Pardo et al, published a further study on 2T3 murine preosteoblast cells grown in RWV for 3 days. The cells were seeded on microcarrier beads and rotated on the RWV at 22 rpm. Microarray analysis of the simulated microgravity treated preosteoblasts showed that the microgravity downregulated 61 and upregulated 45 genes by more than twofold compared to static 1 g controls. Comparison of the RWV/microarray data with the data from previous RPM/microarray analysis they found 14 mechanosensitive genes that were changed in the same direction. Once again ALP, runx2, PthR1, and PDGF were shown to be significantly down-regulated. Thus, the two different simulators of microgravity on the same cell line assayed by microarray kits from two different vendors produce similar results with regard to bone cell differentiation and osteoblast function. In a further non-microarray based study (Patel et al, 2009), they cultured 2T3 cells in SMG using RPM and PCR-based analysis. They again found that ALP, Runx2, PthR1 were down-regulated in simulated microgravity. They also found that low magnitude and high frequency (LMHF) mechanical loading (0.1 ~ 0.4 g at 30 Hz for 10 ~ 60 min/day) prevented a decrease in ALP, Runx2, PthR1, but static conditions had no effect (Patel et al, 2009).

Yamada et al studied osteoblasts (NOS-1 cells derived from a human osteosarcoma) that were exposed to 3 days of simulated microgravity in RPM. They used AtlasTM Human 3.8K microarray for the mRNA analysis. As with the previous studies, ALP activity was significantly reduced in the cells exposed to simulated microgravity. However, there is not a gene list for the microarray analysis. It is significant to find that the addition of chitosan (a natural polyaminosaccharide) significantly increased ALP activity in the cells exposed to simulated microgravity (Yamada et al 2007).

A recent study by Capulli et al (2009) used primary mouse calvarial osteoblasts grown in simulated microgravity (RWV) for 5 days and used Agilent microarrays for the analysis of simulated microgravity exposed bone cells. The cells were seeded to microcarrier beads and grown in RWV at 16 rpm to simulate microgravity. They found that 133 genes were differentially regulated, 45 genes were significantly up-regulated and 88 were downregulated. The significantly differentially regulated genes were presented in tables of clusters and molecular function classifications. Among the differentially regulated genes are genes involved in osteoblast differentiation, function, and osteoblast–osteoclast cross-talk, genes of extracellular matrix, glycosaminoglycan/heparin-binding activity, and growth factor activity. The findings concerning FN1 are consistent with other studies in different cell lines (Dapp et al, 2004, Nikawa et al, 2004, Sheyn et al, 2010). The finding with CTGF is also consistent with other findings (Sheyn et al, 2010).

Qian et al (2009) examined gene expression profile changes of human osteoblast-like cell line MG-63 in response to 24 h simulated microgravity, highmagneto-gravitational environment (HMGE). They used 35 mm cell culture plates seeded with cells and placed them into the HMGE at special positions to achieve the gravity effects of 0, 1, and 2 g by the object stage. They used a self-made circulating water-bath as a control for temperature of 37±0.5oC. High-density human genome (HG) U133 Plus 2.0 Arrays (Affymetrix) were used for the gene expression analysis. Among the total of 54,613 gene probes examined with the microarray, they found 53 genes were statistically down-regulated and 55 genes were statistically up-regulated compared with the 0 g with the temperature control. They presented the identified genes in tables according to cellular functions. They specifically

aforementioned study by Pardo et al, published a further study on 2T3 murine preosteoblast cells grown in RWV for 3 days. The cells were seeded on microcarrier beads and rotated on the RWV at 22 rpm. Microarray analysis of the simulated microgravity treated preosteoblasts showed that the microgravity downregulated 61 and upregulated 45 genes by more than twofold compared to static 1 g controls. Comparison of the RWV/microarray data with the data from previous RPM/microarray analysis they found 14 mechanosensitive genes that were changed in the same direction. Once again ALP, runx2, PthR1, and PDGF were shown to be significantly down-regulated. Thus, the two different simulators of microgravity on the same cell line assayed by microarray kits from two different vendors produce similar results with regard to bone cell differentiation and osteoblast function. In a further non-microarray based study (Patel et al, 2009), they cultured 2T3 cells in SMG using RPM and PCR-based analysis. They again found that ALP, Runx2, PthR1 were down-regulated in simulated microgravity. They also found that low magnitude and high frequency (LMHF) mechanical loading (0.1 ~ 0.4 g at 30 Hz for 10 ~ 60 min/day) prevented a decrease in ALP, Runx2, PthR1, but static conditions had no

Yamada et al studied osteoblasts (NOS-1 cells derived from a human osteosarcoma) that were exposed to 3 days of simulated microgravity in RPM. They used AtlasTM Human 3.8K microarray for the mRNA analysis. As with the previous studies, ALP activity was significantly reduced in the cells exposed to simulated microgravity. However, there is not a gene list for the microarray analysis. It is significant to find that the addition of chitosan (a natural polyaminosaccharide) significantly increased ALP activity in the cells exposed to

A recent study by Capulli et al (2009) used primary mouse calvarial osteoblasts grown in simulated microgravity (RWV) for 5 days and used Agilent microarrays for the analysis of simulated microgravity exposed bone cells. The cells were seeded to microcarrier beads and grown in RWV at 16 rpm to simulate microgravity. They found that 133 genes were differentially regulated, 45 genes were significantly up-regulated and 88 were downregulated. The significantly differentially regulated genes were presented in tables of clusters and molecular function classifications. Among the differentially regulated genes are genes involved in osteoblast differentiation, function, and osteoblast–osteoclast cross-talk, genes of extracellular matrix, glycosaminoglycan/heparin-binding activity, and growth factor activity. The findings concerning FN1 are consistent with other studies in different cell lines (Dapp et al, 2004, Nikawa et al, 2004, Sheyn et al, 2010). The finding with CTGF is

Qian et al (2009) examined gene expression profile changes of human osteoblast-like cell line MG-63 in response to 24 h simulated microgravity, highmagneto-gravitational environment (HMGE). They used 35 mm cell culture plates seeded with cells and placed them into the HMGE at special positions to achieve the gravity effects of 0, 1, and 2 g by the object stage. They used a self-made circulating water-bath as a control for temperature of 37±0.5oC. High-density human genome (HG) U133 Plus 2.0 Arrays (Affymetrix) were used for the gene expression analysis. Among the total of 54,613 gene probes examined with the microarray, they found 53 genes were statistically down-regulated and 55 genes were statistically up-regulated compared with the 0 g with the temperature control. They presented the identified genes in tables according to cellular functions. They specifically

effect (Patel et al, 2009).

simulated microgravity (Yamada et al 2007).

also consistent with other findings (Sheyn et al, 2010).

noted that cytoskeleton-related genes such as WASF2 and WIPF1 genes were the most mechanosensitive.

Hammer et al used diamagnetic levitation to conduct experiments of how gravity affects MC3T3-E1 osteoblastic cell line. In the experiment, they exposed cells to 0g, 1g, and 2g gravity. The cells were exposed to the magnetic field for two days after which RNA was extracted and microarrays were run. They used Affymetrix Mouse genome arrays and the intensity of housekeeping genes for normalization, Robust Multichip Average (RMA) for normalization. The data was then scaled to a median of 100; genes below 50 were filtered out. They used a 3 fold cut-off to determine the gene list for further bioinformatics analysis. Based on this criterion they identified 2270 genes that were upregulated and 135 genes that were down-regulated when 1g samples were compared to 0g samples. The focus of the paper is more on testing the diamagnetic levitation biotechnology rather than on the genes that are differentially regulated (Hammer et al, 2009). .

Sambandam et al (2010) published a recent study of osteoclast grown in RWV SMG (16 rpm to simulate 0.0008g environment) for 24 hours. For the study they used Agilent whole genome arrays. They followed standard statistical procedures to identify their list of differentially regulated genes. They identified 3,404 differentially expressed genes. They have followed MIAME standards by depositing the microarray profile data in the GEO database. Some genes of interest that were up-regulated include CTSK, CTSL, and CTSB, as well as several MMPs, bone matrix degrading proteases. Their microarray data agrees with previous finding that stimulation of osteoclastogenesis in microgravity environment.

All seven studies also made good use of the biotechnologies in their respective studies. To further examine all the microarray-identified genes from bone cells exposed to microgravity, I performed pathway analysis using DAVID. As in the immune system section, the use of gene bank accession numbers and gene symbols are not standardized here also. In order to find out which pathways the altered genes are involved, I first used the DAVID conversion tool to convert them all into the same format. Out of the seven microarray studies involving bone cells, five studies have the gene lists published in the paper or supplemental tables. Since the majority (four out of five) were studies done in mice (Pardo et al, 2005; Patel et al, 2007; Capulli et al, 2009; Sambandam et al 2010) and one was in human (Qian et al, 2007), I chose to use those four gene lists to compile into one master list in the same format, and uploaded them to the DAVID Functional Annotation tool for pathway analysis. The list was run as species Mouse and Identified the statistically significant KEGG Pathways (data not shown).

Within the seven microarray analysis of microgravity exposed bone cells discussed above, there are certain consistency or overlap in the identified gravity sensitive genes among these different studies. Are there potential major space genes or bone-specific space genes among these studies? To this end, I performed further cross-laboratory, cross-species, crossmicrogravity-platform, and cross-microarray-platform comparative analysis here. In order to identify the major gravity sensitive genes from these microarray/microgravity studies, I manually examined and picked out the genes that appeared to be significant in more than one of the five studies (two out of the seven studies do not have a gene list with the publications nor available in supplement). Essentially, any gene that was identified in two or more of the five studies were compiled into a table along with the data values (fold changes) from each specific source of origin for direct comparison (Table 3).


Table 3. Microgravity Sensitive Genes Identified by Microarray analysis of Bone Cells It is more apparent now with this table that the data from the same research group tends to be more consistent (compare Pardo et al 2005 and Patel et al 2007). The highest overlapped (in three of the five studies) genes were osteoglycin(OGN), osteomodulin (OMD), and secreted Frizzle-Related Protein 2 (SFRP2) and all three genes were down-regulated in all the microarray/microgravity studies that identified them (Pardo et al, 2005; Patel et al, 2007; Capulli et al, 2009). Osteoglycin(OGN) encodes a protein (a small proteoglycan) which induces bone formation in conjunction with transforming growth factor beta. Osteomodulin (OMD) is implicated in biomineralization processes. In addition to the reduction of expression of the two genes important in bone formation, the majority of the gravity sensitive genes shown in Table 3 are genes encode proteins in pathways involving ECMreceptor interaction and focal adhesion: Integrin β5 (ITGB5), thrombospondins (THBS1, THBS2), collagen (COL11A1), and fibronectin (FN1). In addition, four components in the TGF-β signaling pathway were also found in this table: bone morphogenic protein (BMP4), decorin (DCN), and the thrombospondins (THBS1, THBS2). Furthermore, the two cytokines, interleukin-1(IL-1) and interleukin-6(IL-6), that coordinate and regulate many cellular activities of the innate immunity were among the more frequently identified microgravity sensitive genes in bone (Table 3).

Table 3. Microgravity Sensitive Genes Identified by Microarray analysis of Bone Cells It is more apparent now with this table that the data from the same research group tends to be more consistent (compare Pardo et al 2005 and Patel et al 2007). The highest overlapped (in three of the five studies) genes were osteoglycin(OGN), osteomodulin (OMD), and secreted Frizzle-Related Protein 2 (SFRP2) and all three genes were down-regulated in all the microarray/microgravity studies that identified them (Pardo et al, 2005; Patel et al, 2007; Capulli et al, 2009). Osteoglycin(OGN) encodes a protein (a small proteoglycan) which induces bone formation in conjunction with transforming growth factor beta. Osteomodulin

(OMD) is implicated in biomineralization processes. In addition to the reduction of expression of the two genes important in bone formation, the majority of the gravity sensitive genes shown in Table 3 are genes encode proteins in pathways involving ECMreceptor interaction and focal adhesion: Integrin β5 (ITGB5), thrombospondins (THBS1, THBS2), collagen (COL11A1), and fibronectin (FN1). In addition, four components in the TGF-β signaling pathway were also found in this table: bone morphogenic protein (BMP4), decorin (DCN), and the thrombospondins (THBS1, THBS2). Furthermore, the two cytokines, interleukin-1(IL-1) and interleukin-6(IL-6), that coordinate and regulate many cellular activities of the innate immunity were among the more frequently identified microgravity

sensitive genes in bone (Table 3).

#### **4. Microarray analysis of microgravity exposed muscle cells**

Muscle atrophy as a result of spaceflight has been a condition that was identified early on in the space program. As with bone cells and cells in the immune system, spaceflight also has a major effect on muscle cells. A study of an exercise program showed that crewmembers while aboard the International Space Station (ISS) for 6 months calf muscle volume and peak power decreased significantly, and there was a redistribution among the faster phenotypes, despite the fact that the crewmembers exercised regularly (Trappe et al, 2009). Microgravity effect on muscle has been one of the most studied areas in space life science research. Ground based analogs such as bed rest or unilateral leg suspension (ULLS) have been used in humans and HLS has been used in rats. Bed rest and ULLS have shown results that seem to be similar to spaceflight (Narici & deBoer, 2011). However, it has been cautioned that the HLS using rodent models may not be the best choice when extrapolating results to humans; the processes involved in muscle atrophy and disuse in rodent and humans are very different (Rennie et al, 2010). Currently, little is even known about mechanisms of muscle atrophy and disuse in general (Rennie et al, 2010). In studying microgravity effect on muscle cells, most commonly used model systems are rodents and space flight experiments have been performed extensively using both mice and rats flown in space. The most commonly used ground based simulated microgravity models are hind limb-suspension of rodents. These have been combined with microarray biotechnology for genome wide gene expression analysis of the microgravity effect. Here I will focus my discussion on the microarray based studies and apply DAVID bioinformatics tools to examine the genes that have been identified in these studies.

Taylor et al (2002) used Clontech Atlas DNA expression array to study the alteration of gene expression profiles in skeletal muscles (tibialis anterior and gastrocnemius) of male rats flown on the STS 90 Neurolab for 17 days. They found that 50 genes showed differential regulation: 38 genes were downregulated and 12 were upregulated. Genes related to cell proliferation and growth factor cascades (such as p21cip1, Rb, cyclins G1/S, -E, -D3, MAP kinase 3, MDA3, and ras related protein RAB2) were down-regulated during spaceflight. The microarray data indicates that genes involved in regulation of muscle satellite cell replication are down regulated by microgravity (Taylor et al 2002). Thus, this experiment gives further insight into the mechanisms underlying muscle atrophy and diminished muscle repair capability associated with the space environment.

Another 2002 study (Wittwer et al 2002) using Clontech Atlas 1.2K Rat arrays identified 105 genes out of 1200 tested that were significantly differentially regulated after 35 days hindlimb suspension. Much of their microarray data agreed with similar studies reported previously. Data suggested a coordinated increase in the expression of genes (PFKM, ALDOA, and GAPDH) in the glycolic pathway involved in the cytoplasmic conversion of glucose to pyruvate in 35-day HS *m. solei*. In addition, the mRNA of an enzyme that controls glycolytic flux, adenylate kinase 1 (AK1), was also increased. mRNA of proteins (LDL receptor, SR-BI, FATP, and H-FABP) involved in the uptake and transport of fatty acids from the blood into the muscle fibers decreased. mRNAs of vesicle transport proteins (IRAP, M6P/IGFR2, and VAMP3) involving mitrochondrial energy conversion were increased. These genes have been implicated in glucose uptake, mediated by the major insulinmediated glucose transporter of skeletal muscle, glut-4. Data suggests the decreased fatty acid import proteins as a regulation towards reduced fatty acid uptake and transport concomitant with adaptation toward greater glycogen utilization and a generally reduced energy demand in the atrophied muscle. The gene activities for intracellular protein degradation such as lysosomal protease mRNAs (cathepsin C, L, and D) and some enzymes involved in cytosolic protein degradation by proteasomes (i.e., TPPII, UBE2B, rP28 alpha, and carboxypeptidase D) were increased. Extracellular proteases (MMP-2, u-PA) as well as protease inhibitors (TIMP-2 and -3) were also increased at mRNA level. The findings gave much insight towards the understanding of skeletal muscle atrophy.

Stein et al (2002) studied soleus muscle isolated from 5 control and 5 hindlimb suspended rats (21 days) with the Affymetrix microarray system for assessing gene expression on fuel pathways within the muscle. Similar to the Wittwer et al report, they observed a consistent decrease in expression of genes involved in fatty acid oxidation and an increase in expression of genes in the glycolytic activity in the suspended group. Their microarray data further confirms that disuse atrophy is associated with a change in mRNA levels of enzymes involved in energy metabolism, a shift away from use of fat to use of glucose.

Dapp et al (2004) applied the mouse hindlimb suspension (HS) model for gene expressional alterations underlying loaddependent muscular adaptations. Gene expression was assessed from total RNA by a muscle-specific low-density cDNA microarray (custom designed Atlas cDNA arrays with 222 double spots on each nylon array). Immediate early genes such as FRA1, JUND, and JUN were induced as were IGFs, while myosin heavy chain was reduced in mouse soleus muscle after 7 days hind-limb suspension.

Nikawa et al (2004) published a study comparing gene expression patterns in gastrocnemius muscle (the largest muscle of the calf of the leg that acts by extending the foot and bending the knee) cells from rats flown in space, rats exposed to hind-limb suspension, and denervated rats. Using Affymetrix Rat Genome U34 GeneChips, they found that most gene expression changes were unique to spaceflight (Nikawa et al 2004). The DNA microarray data indicated that spaceflight specifically caused altered expression of some mitochondrial genes and cytosleletal genes (such as A-kinase anchoring protein and cytoplasmic dynein) as well as up-regulated ubiquitin ligase genes (MuRF-1, Cbl-b, and Siah-1A ). Several oxidative stress-inducible genes were also upregulated and hightly expressed in the muscle of the spaceflown rats (Nikawa et al 2004). They proposed that mitochondrial dislocation during spaceflight may have caused atrophy in the form of insufficient energy supply and leakage of reactive oxygen species from the mitochondria. Although only a fraction of the genes are discussed in limited space of the original publication, comprehensive tables of microarray data are presented in the paper, which provides a very valuable source of reference information. The same research group published another research paper using human 20 day bed rest to study muscle disuse atrophy mechanism and used DNA microarray for their genome wide gene expression analysis (Ogawa et al, 2006). Their data suggested that Cbl-b or atrogin-1 mediated ubiquitination pathway could be important in unloading induced muscle atrophy in humans.

Mazzatti et al 2008 reported a study using gene arrays (Agilent Mouse Oligo Arrays) to determine the acute effects of short-term HLS on metabolic consequences of unloading. They used Agilent whole-genome arrays to examine mRNA expression in mouse soleus and gastronemius muscle cells after 24 hours hind-limb suspension and identified 600 genes with a FDR of 0.05 and at least 1.5 fold differential regulation in both cell types. Several proteins (PPARδ, UCP-3, AMPK, and CPT1/2) that have putative roles in the maintenance

concomitant with adaptation toward greater glycogen utilization and a generally reduced energy demand in the atrophied muscle. The gene activities for intracellular protein degradation such as lysosomal protease mRNAs (cathepsin C, L, and D) and some enzymes involved in cytosolic protein degradation by proteasomes (i.e., TPPII, UBE2B, rP28 alpha, and carboxypeptidase D) were increased. Extracellular proteases (MMP-2, u-PA) as well as protease inhibitors (TIMP-2 and -3) were also increased at mRNA level. The findings gave

Stein et al (2002) studied soleus muscle isolated from 5 control and 5 hindlimb suspended rats (21 days) with the Affymetrix microarray system for assessing gene expression on fuel pathways within the muscle. Similar to the Wittwer et al report, they observed a consistent decrease in expression of genes involved in fatty acid oxidation and an increase in expression of genes in the glycolytic activity in the suspended group. Their microarray data further confirms that disuse atrophy is associated with a change in mRNA levels of enzymes

Dapp et al (2004) applied the mouse hindlimb suspension (HS) model for gene expressional alterations underlying loaddependent muscular adaptations. Gene expression was assessed from total RNA by a muscle-specific low-density cDNA microarray (custom designed Atlas cDNA arrays with 222 double spots on each nylon array). Immediate early genes such as FRA1, JUND, and JUN were induced as were IGFs, while myosin heavy chain was reduced

Nikawa et al (2004) published a study comparing gene expression patterns in gastrocnemius muscle (the largest muscle of the calf of the leg that acts by extending the foot and bending the knee) cells from rats flown in space, rats exposed to hind-limb suspension, and denervated rats. Using Affymetrix Rat Genome U34 GeneChips, they found that most gene expression changes were unique to spaceflight (Nikawa et al 2004). The DNA microarray data indicated that spaceflight specifically caused altered expression of some mitochondrial genes and cytosleletal genes (such as A-kinase anchoring protein and cytoplasmic dynein) as well as up-regulated ubiquitin ligase genes (MuRF-1, Cbl-b, and Siah-1A ). Several oxidative stress-inducible genes were also upregulated and hightly expressed in the muscle of the spaceflown rats (Nikawa et al 2004). They proposed that mitochondrial dislocation during spaceflight may have caused atrophy in the form of insufficient energy supply and leakage of reactive oxygen species from the mitochondria. Although only a fraction of the genes are discussed in limited space of the original publication, comprehensive tables of microarray data are presented in the paper, which provides a very valuable source of reference information. The same research group published another research paper using human 20 day bed rest to study muscle disuse atrophy mechanism and used DNA microarray for their genome wide gene expression analysis (Ogawa et al, 2006). Their data suggested that Cbl-b or atrogin-1 mediated ubiquitination pathway could be important in

Mazzatti et al 2008 reported a study using gene arrays (Agilent Mouse Oligo Arrays) to determine the acute effects of short-term HLS on metabolic consequences of unloading. They used Agilent whole-genome arrays to examine mRNA expression in mouse soleus and gastronemius muscle cells after 24 hours hind-limb suspension and identified 600 genes with a FDR of 0.05 and at least 1.5 fold differential regulation in both cell types. Several proteins (PPARδ, UCP-3, AMPK, and CPT1/2) that have putative roles in the maintenance

much insight towards the understanding of skeletal muscle atrophy.

in mouse soleus muscle after 7 days hind-limb suspension.

unloading induced muscle atrophy in humans.

involved in energy metabolism, a shift away from use of fat to use of glucose.

of metabolic flexibility were upregulated. They also concluded that there was increased reliance on glucose as an energy source or a loss of metabolic flexibility. Muscle unloading appears to result in reduced fatty acid oxidation, decreased transcription of genes involved in lipid metabolism and increased expression of genes involved in glycogen synthesis (Mazzatti et al 2008). The findings that increased reliance on glucose fit with previous studies that have shown that atrophied muscle rely more on glucose for energy (Fitts et al, 2000; Henriksen and Tishler 1988; Stein et al, 2002; Martin et al, 1988).

A more recent study (Allen et al 2009) of murine skeletal muscles(gastrocnemius from mice) compared space flown mice (11-day, 19-h on STS-108 shuttle flight) with the ground-based unloading model of hindlimb suspension (one group of pure suspension and one of suspension followed by 3.5 h of reloading). They found that spaceflight causes differential regulation in genes involved in muscle growth and fiber type. Their microarray data showed that 272 mRNAs significantly differentially regulated by spaceflight, of which many genes were found to belong to pathways involved in muscle growth and adaptation. Spaceflight significantly altered the levels of mRNAs involved with the PI3 kinase/Akt/mTOR pathway: the PI3-kinase regulatory subunit polypeptide 1, pi3kr1/p85α, the forkhead box O1 (FoxO1), transcription factor, the muscle-specific ubiquitin ligase F-box only protein 32 (MAFbx/atrogin1), and the ubiquitin-conjugating enzyme E2 variant 2 mRNAs were increased; Insulin receptor substrate-1 (IRS-1) mRNA levels were decreased in space flown mouse gastrocnemius muscles. Genes in the TNF-α/NF-κB signaling pathway and the calcineurin/nuclear factor of activated T cells (NFAT) pathway were also affected: the TNF-α downstream target TNF-α-induced protein 2 mRNA and the NF-kB inhibitor nuclear factor B light chain gene enhancer in B cells inhibitor α(Nfkbia/IκBα) mRNA were significantly increased, whereas mRNA levels of the NFAT cytoplasmic, calcineurindependent 3 (Nfatc3) transcription factor were significantly decreased in SF gastrocnemius. In addition, mRNAs for three members of the CAAT/enhancer binding protein (C/EBP) family of transcription factors, C/EBP-α, C/EBP-β, and C/EBP-δ were also increased in SF gastrocnemius. They found that space flight increased myostatin (which limits muscle growth) mRNA and decrease the mRNA levels of myostatin inhibitor FSTL3. They also found that mRNA levels of the slow oxidative fiber-associated transcriptional coactivators, peroxisome proliferator activated receptor alpha (PPAR-α) and the PPAR-γ coactivator 1α (PGC1-α) decreased in space-flight. They concluded that spaceflight induced significant changes in mRNA expression of genes associated with muscle growth and fiber type toward a less oxidative phenotype (Allen et al 2009).

Chopard et al (2009) performed a genome-wide gene expression analysis of female skeletal muscles during 60 days of bed rest with and without exercise or dietary protein supplementation as countermeasures. They investigated the effects of long-term bed rest on the gene expression of soleus (SOL) and vastus lateralis (VL) muscles in healthy women using a customized microarray containing 6,681 muscles-relevant genes. They found clusters of genes involved in nucleic acid and protein metabolism were upregulated and that encoding components involved in energy metabolism were downregulated. Counter measures (exercise and nutrition) had some compensatory effects on gene expression profiles.

Reich et al (2010) examined the global gene expression patterns of the left vastus lateralis muscle in seven sedentary men following 48 h unloading via unilateral lower limb suspension and 24 h reloading. Microarray analysis of gene expression changes were used for the identification of enriched functions and canonical pathways. They found that the highest ranked canonical pathways were related to protein ubiquitination and oxidative stress response pathways. Gene functions related to mitochondrial metabolism were the most significantly downregulated. The increases in mRNA for ubiquitin proteasome pathway-related E3 ligase and stress response gene heme oxygenase-1 as well as extracellular matrix (ECM) component COL4A3 were confirmed by qRT-PCR. The unloading associated gene expression patterns were not reversed on reloading.

The ten studies discussed above made good use of microarray biotechnology in their respective studies. Out of the ten studies, we found seven that had gene lists of some form that could be further analyzed. Among the seven gene lists, a few genes were reported in more than one study. Fibronectin (FN1) was identified by several microarray studies in muscle. In space-flown rat muscles, FN1 was down-regulated (Nikawa et al, 2004) and it was down-regulated in HLS rats as well (Dapp et al, 2004). Interestingly, FN1 was upregulated in the recovery time points in Dapp et al. FN1 was also up-regulated in women exposed to bed rest (Chopard et al, 2009). ACADVL was found to be down-regulated both in rat (Stein et al, 2002) and human muscles (Chopard et al, 2009). Another four genes were identified in the same directions between a study in mouse and a study in rat: MT1, MT2, and PIM3 were upregulated, whereas MARCKS was down regulated in both studies (Nikawa et al, 2004; Allen et al, 2009). In addition, CYR61 gene was identified to be upregulated by a study in mouse (Allen et al, 2009) and a study in rat muscles (Nikawa et al, 2004). I also did DAVID analysis for all the genes pooled from the ten studies and found that 32 pathways were involved (data not shown). This large variation could at least in part be due to the heterogeneity of the study conditions. The factors discussed in the immune and bone sections also apply.

#### **5. Microarray analysis of microgravity exposed other cells**

Exposure of rat bone marrow mesenchymal stems cells (rBMSC) to HMGE simulated microgravity study found that BMSC proliferation and osteogenesis decreased and the cells were growth arrested in the G0/G1 phase of cell cycle (Dai et al 2007). Data from their microarray (CapitalBio Corporation) analysis confirmed that rBMSC proliferation and osteogenesis gene activities decreased under simulated microgravity. Insulin-like growth factor-I, epidermal growth factor, and basic fibroblastic growth factor that normally stimulated rBMSC proliferation had only a marginal effect in the simulated microgravity..

Sheyn et al (2010) using Affymetrix microarrays examined simulated microgravity (RWV) effect on human mesenchymal stem cells. The cells were seeded on microcarrier beads and placed in a RWV bioreactor, which was rotated at 16 rpm to simulate a microgravity environment. They identified 882 genes that were down regulated and 505 genes were upregulated by 2 fold or above and with a P value of 0.05 or less. They identified a large number of gene clusters responded to microgravity. In agreement with previous studies, their microarray data showed a general trend of less osteogenesis and more adipogenesis when hMSCs cells were cultured in simulated microgravity. Most of the extracellular matrix related genes were downregulated. They identified many genes that were involved in the actin cytoskeleton such as COL1A1, COL1A2, FN1, SPARC, CTGF, and IGFBP3, were downregulated. The mRNA of several growth and differention factors such as fibroblast growth factor, vascular endothelial growth factor, insulin-like growth factor-related proteins, and

for the identification of enriched functions and canonical pathways. They found that the highest ranked canonical pathways were related to protein ubiquitination and oxidative stress response pathways. Gene functions related to mitochondrial metabolism were the most significantly downregulated. The increases in mRNA for ubiquitin proteasome pathway-related E3 ligase and stress response gene heme oxygenase-1 as well as extracellular matrix (ECM) component COL4A3 were confirmed by qRT-PCR. The

The ten studies discussed above made good use of microarray biotechnology in their respective studies. Out of the ten studies, we found seven that had gene lists of some form that could be further analyzed. Among the seven gene lists, a few genes were reported in more than one study. Fibronectin (FN1) was identified by several microarray studies in muscle. In space-flown rat muscles, FN1 was down-regulated (Nikawa et al, 2004) and it was down-regulated in HLS rats as well (Dapp et al, 2004). Interestingly, FN1 was upregulated in the recovery time points in Dapp et al. FN1 was also up-regulated in women exposed to bed rest (Chopard et al, 2009). ACADVL was found to be down-regulated both in rat (Stein et al, 2002) and human muscles (Chopard et al, 2009). Another four genes were identified in the same directions between a study in mouse and a study in rat: MT1, MT2, and PIM3 were upregulated, whereas MARCKS was down regulated in both studies (Nikawa et al, 2004; Allen et al, 2009). In addition, CYR61 gene was identified to be upregulated by a study in mouse (Allen et al, 2009) and a study in rat muscles (Nikawa et al, 2004). I also did DAVID analysis for all the genes pooled from the ten studies and found that 32 pathways were involved (data not shown). This large variation could at least in part be due to the heterogeneity of the study conditions. The factors discussed in the immune and

Exposure of rat bone marrow mesenchymal stems cells (rBMSC) to HMGE simulated microgravity study found that BMSC proliferation and osteogenesis decreased and the cells were growth arrested in the G0/G1 phase of cell cycle (Dai et al 2007). Data from their microarray (CapitalBio Corporation) analysis confirmed that rBMSC proliferation and osteogenesis gene activities decreased under simulated microgravity. Insulin-like growth factor-I, epidermal growth factor, and basic fibroblastic growth factor that normally stimulated rBMSC proliferation had only a marginal effect in the simulated microgravity.. Sheyn et al (2010) using Affymetrix microarrays examined simulated microgravity (RWV) effect on human mesenchymal stem cells. The cells were seeded on microcarrier beads and placed in a RWV bioreactor, which was rotated at 16 rpm to simulate a microgravity environment. They identified 882 genes that were down regulated and 505 genes were upregulated by 2 fold or above and with a P value of 0.05 or less. They identified a large number of gene clusters responded to microgravity. In agreement with previous studies, their microarray data showed a general trend of less osteogenesis and more adipogenesis when hMSCs cells were cultured in simulated microgravity. Most of the extracellular matrix related genes were downregulated. They identified many genes that were involved in the actin cytoskeleton such as COL1A1, COL1A2, FN1, SPARC, CTGF, and IGFBP3, were downregulated. The mRNA of several growth and differention factors such as fibroblast growth factor, vascular endothelial growth factor, insulin-like growth factor-related proteins, and

unloading associated gene expression patterns were not reversed on reloading.

**5. Microarray analysis of microgravity exposed other cells** 

bone sections also apply.

A recent study using hematopoietic stem cells by Fridley et al (2010) was not a microgravity study per se. Instead it was a comparative study of stirred tank bioreactors and RWV bioreactors. They studied how these devices would work in the formation of embryoid bodies and the generation of hematopoietic progenitor cells. For the RWV study they ran the bioreactor samples at speeds ranging from 10 rpm to 40 rpm. They used Roche Nimblegene 4 plex microarrays (72k) for the study. They did discuss some of the genes that were differentially regulated, but they did not publish a full gene list. One gene that was significantly down-regulated was E-Cadherin (Fridley et al, 2010).

Clement et al, using Agilent 22K Human 1A microarrays, examined skin cells (HEK001) for their response to simulated microgravity over the course of 3, 4, 9, and 10 days followed by recovery from SMG in normal cell culture conditions for 15, 50, and 60 days. A total of 162 genes (P < 0.05) were differentially regulated by at least 2 fold at some point during the time course. They also used Northern blotting to both qualitatively and quantitatively verify their data. The genes that were analyzed showed a statistical correlation with the microarray data ranging from 0.69 to 0.97. Interestingly, 80% of the differentially expressed genes recovered from 4 days SMG exposure after 15 days in normal cell culture conditions. Genes that were exposed to 9 days SMG required more than 50 days to recover to pre-exposure levels. Also of interest, HLA-G, a key gene in cellular immune response suppression, was found to be significantly up-regulated during the recovery phase (Clement et al 2008). This study indicates that longer term exposure to microgravity may have long term affects on the body even after returning to normal gravity. Further studies on microgravity exposure and subsequent recovery should be carried out.

The effect of microgravity on the nervous system and brain at the molecular level is still not well understood. Frigeri et al examined gene expression in mouse brain after 2 weeks hindlimb supesension (Frigeri et al 2008). Using 27K cDNA microarrays, they found 592 statistically significant genes with 1.5 fold or higher differential regulation. Hind-limb suspension also seems to affect the pathways involved in learning and memory as well as blood coagulation. They found that hind-limb suspension causes a more hyper-coagulative state and an increased risk of venous thrombosis (Frigeri et al 2008). In a recent study PC12 cells were exposed to 4 days SMG to analyze oxidation sensitive genes in microgravity (Kwon et al 2006). They found that 65 genes were up-regulated and 39 were down-regulated as a result of exposure to SMG. They found that genes involved in DNA repair and replication, cell proliferation, apoptosis, molecular transport, and oxidative phosphorylation were affected by exposure to SMG.

A 2002 study of human fibroblast grown onboard the space shuttle (STS-93) in 1999 identified changes in expression of 10 genes in the TNF or IL gene families. For this study, they chose 202 genes which were then spotted on to a nylon membrane. They did verify their results on some genes with semi-quantitative PCR with a correlation of 0.89. The genes identified are involved in the regulation of bone density or proinflammatory status (Semova et al 2002).

Some of the first microarray studies of space flown cells was done using renal cortical cells flown on STS-90 (Hammond et al, 1999). They applied a high throughput microarray system to study gravity-induced gene-expression changes in human renal cells and found 1632 out of 10,000 genes changed as a result of exposure to microgravity (Hammond et al, 2000).

Kitamoto et al studied Xenopus A6 cells grown in RPM simulated microgravity environment and examined gene expression using customized Agilent 8K Xenopus laevis DNA microarrays (Kitamoto et al, 2004, Kitamoto et al, 2005). They found that 52 out of 8091 genes examined showed differential regulation. Their time course data showed interesting gene expression patterns of microgravity effect and cellular adaptation. They also found that SPARC was down-regulated Xenopus A8 cell lines (Ikuzawa and Asashima 2008). Their data combined with previous observations (Kitamoto et al, 2005) lead them to conclude that SPARC might play a key role in response to microgravity (Ikuzawa and Asashima 2008). Down-regulation of SPARC and SPARC related genes has also been observed in human mesenchymel stem cells grown in SMG (Sheyn et al, 2010).

Liver cells have also been studied in SMG. Both of the SMG studies on liver cells were carried out using HepG2 cells (Clement et al, 2007; Khaoustov 2001). Both studies showed that liver cells grown in a RWV caused altered gene expression patterns. The Clement study used Agilent 22K human 1A microarrays to study HepG2 cells. They identified 139 genes which were differentially regulated with a fold change of >1.5 and P<0.01. Khaoustov et al used 6K Human Array containing 6144 genes showed that 95 genes were differentially regulated. More systematic studies would be needed in further studying microgravity effects on liver cells.

#### **6. Towards the identification of major space genes**

A main focus of this chapter was to try to identify potential major space genes by reviewing gene expression profiles, compare and contrast the expression profiles of cells from different lineages of different organisms exposed to various microgravity conditions. My original hypothesis was that it could be possible that a common set of key microgravity sensitive genes in different cells would be preferentially altered in microgravity conditions. The identification of this common set of genes might lead to the identification of "major space genes" that together play a major check-and-balance role ultimately determining the outcome of a cell, or an organism such as an individual person, in the response to microgravity conditions. This is analogous to areas of cancer research resulting in the identification of growing numbers of major cancer genes (oncogenes and tumor suppressor genes) that are directly involved in the determination the fate of a cell---whether a cell becomes cancerous or not. I therefore believed that a systematic examination of molecular alterations of cells under microgravity conditions would enhance our knowledge of cellular responses to microgravity and the underlying mechanisms. Since cells of different organ or tissue systems may be affected by this environmental factor differently, studying and comparing the cellular response to such gravity changes using various cell lines of different lineage or tissue origin would generate a comprehensive database of cellular activity alteration associated with microgravity, and the identification of major space genes. This would provide scientific basis for therapeutic intervention aimed to prevent and correct abnormalities resulting from space flight**.**

Examining high throughput microarray data as well as other gene expression data from cells and organisms exposed to microgravity, there were a great many differentially

to study gravity-induced gene-expression changes in human renal cells and found 1632 out of 10,000 genes changed as a result of exposure to microgravity (Hammond et al, 2000).

Kitamoto et al studied Xenopus A6 cells grown in RPM simulated microgravity environment and examined gene expression using customized Agilent 8K Xenopus laevis DNA microarrays (Kitamoto et al, 2004, Kitamoto et al, 2005). They found that 52 out of 8091 genes examined showed differential regulation. Their time course data showed interesting gene expression patterns of microgravity effect and cellular adaptation. They also found that SPARC was down-regulated Xenopus A8 cell lines (Ikuzawa and Asashima 2008). Their data combined with previous observations (Kitamoto et al, 2005) lead them to conclude that SPARC might play a key role in response to microgravity (Ikuzawa and Asashima 2008). Down-regulation of SPARC and SPARC related genes has also been observed in human

Liver cells have also been studied in SMG. Both of the SMG studies on liver cells were carried out using HepG2 cells (Clement et al, 2007; Khaoustov 2001). Both studies showed that liver cells grown in a RWV caused altered gene expression patterns. The Clement study used Agilent 22K human 1A microarrays to study HepG2 cells. They identified 139 genes which were differentially regulated with a fold change of >1.5 and P<0.01. Khaoustov et al used 6K Human Array containing 6144 genes showed that 95 genes were differentially regulated. More systematic studies would be needed in further studying microgravity

A main focus of this chapter was to try to identify potential major space genes by reviewing gene expression profiles, compare and contrast the expression profiles of cells from different lineages of different organisms exposed to various microgravity conditions. My original hypothesis was that it could be possible that a common set of key microgravity sensitive genes in different cells would be preferentially altered in microgravity conditions. The identification of this common set of genes might lead to the identification of "major space genes" that together play a major check-and-balance role ultimately determining the outcome of a cell, or an organism such as an individual person, in the response to microgravity conditions. This is analogous to areas of cancer research resulting in the identification of growing numbers of major cancer genes (oncogenes and tumor suppressor genes) that are directly involved in the determination the fate of a cell---whether a cell becomes cancerous or not. I therefore believed that a systematic examination of molecular alterations of cells under microgravity conditions would enhance our knowledge of cellular responses to microgravity and the underlying mechanisms. Since cells of different organ or tissue systems may be affected by this environmental factor differently, studying and comparing the cellular response to such gravity changes using various cell lines of different lineage or tissue origin would generate a comprehensive database of cellular activity alteration associated with microgravity, and the identification of major space genes. This would provide scientific basis for therapeutic intervention aimed to prevent and correct

Examining high throughput microarray data as well as other gene expression data from cells and organisms exposed to microgravity, there were a great many differentially

mesenchymel stem cells grown in SMG (Sheyn et al, 2010).

**6. Towards the identification of major space genes** 

abnormalities resulting from space flight**.**

effects on liver cells.

regulated genes that showed little or no commonality across closely related studies. In most study areas such as immune, bone, and muscle response to microgravity, there was overall very little commonality or overlap among the reports at the individual gene expression level. However, when looked at gene pathways through DAVID analysis, relatively focused and very insightful pathways were identified for the microarray based analysis of microgravity effects on the immune and bone cells. Would it be that major space pathways rather than major space genes are playing key roles in microgravity response? Typically, there are many gene components in a pathway. It is possible that genes in a pathway can have various levels of activity in response to an environmental challenge and the level of activity of the pathway is optimized to be able to best adapt to environmental change by adjusting component genes activities. Whatever the case, it is too early to conclude from such an initial attempt of cross platform and cross species examinations.

Nonetheless, as part of the identification of space genes, I decided to make a further attempt to compare the gene lists from the microarray studies in mammals that were discussed in this chapter. I compared gene lists from these studies to determine what genes might be common among these studies. There were a large number of differences between the gene lists. This has been shown to be the case in other studies examining microarray experiments between labs and across platforms. These variations are not surprising considering the significant variations between experiments. First, the type of cells used for the studies were very different. These cells included rat muscle, mouse muscle, Xenopus renal, and human liver, renal, keratinocyte, fibroblasts, etc. The methods for studying microgravity were different. These included Random Positioning Machine (RPM), Rotating Wall Vessel (RWV) bioreactors, bed rest, hind limb suspension, as well as space flown. Finally, and perhaps more importantly, the microarray platforms had major variations. All of these variables make it particularly difficult to compare data. For the purpose of this discussion, we decided to include any gene on our final list of potential "space genes" if it appeared on two or more gene lists of the studies we examined. Using this loosely defined criterion, the initial potential candidates for "space genes" were subjected to bioinformatics analysis.

Using the DAVID Bioinformatics Resources, I processed these genes together to identify relevant KEGG pathways (Table 4). The KEGG Pathways and Gene Ontologies were included in the tables if they were considered to be statistically significant (P≤0.05).

The pathway table shows that Extracellular Matrix-Receptor Interaction and Focal Adhesion Pathways are two important pathways. Both have been identified as pathways that may be affected by Microgravity (Ingber 1998).

As part of the attempt to identify and create a list of potential space genes from the microarray based microgravity studies, I decided to analyze a variety of gene lists from these studies. First, I limited the scope of the bioinformatics analysis to mammalian cells. I then examined all of these studies to identify the ones that had useable gene lists. Out of a total of 36 mammalian studies, I identified 26 studies that could be used for this purpose. If a gene appeared as differentially regulated in at least four of these studies, it would be included in the candidate gene list. This yielded a list of 8 potential space genes, name Putative Space Genes (Table 5).

The initial candidate major space genes thus identified in Table 5 (for which I briefly discussed while reviewing each of the involved studies) will provide a clue for further scrutiny in future more systematic studies. I believe that systematic examination of molecular alterations of cells under microgravity conditions will enhance our knowledge of cellular responses to gravity changes and the underlying mechanisms.


Table 4. KEGG Pathways identified using the DAVID Bioinformatics Resources.


Table 5. List of putative space genes.

#### **7. References**


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### **Biotechnology Patents: Safeguarding Human Health**

Rajendra K. Bera *International Institute of Information Technology, Bangalore India* 

#### **1. Introduction**

348 Innovations in Biotechnology

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Prolonged unloading of rat soleus muscle causes distinct adaptations of the gene

alkaline phosphatase activity on human osteoblastic cells under hypofunctional

troponin I that is up-regulated in simulated microgravity. *Journal of Biological* 

osteogenic differentiation of human mesenchymal stem cells and increases

Health related problems affect every human being in an interconnected way, between generations and between societies, through spatial and temporal transitions. This timeless and all-pervasive aspect of health makes biotechnology unique among all technologies. While the science that drives biotechnology has far to go before it reaches a comparable level of maturity of eighteenth century physics, nevertheless biology is now deeply rooted in science; it has taken huge strides from its humble beginnings as a classification science to cell biology, to molecular biology, and now modestly to quantum biology. Erwin Schrödinger, a pioneer of quantum mechanics, was among the first scientists to suggest, in his book *What is Life*? (Schrödinger, 1944), that quantum mechanics can provide deep insights into life's mechanisms. However, our current understanding of how such quantum phenomena as superposition, entanglement, collapse of the wavefunction, etc. affect the chemistry of life is nascent (Ball, 2011).

The links in the supply chain that support biotechnology products and services include knowledge creation in R&D laboratories, product creation in biotechnology and pharmaceutical industries, and the ultimate receiver of therapeutic remedies—a human patient, *inter alia*, communally bound by morality and ethics. The fact that this supply chain, in principle, must cater to every human being on our planet, demands that it be protected with utmost care. Perhaps the most vulnerable link in this chain is the patentable knowledge created through privately funded R&D, which, unless diligently protected, is easy prey to infringement and theft. The maintenance of this chain is astronomically expensive and complex as it must balance some extreme needs: huge funding for exploration-intensive, curiosity-driven, 'blue-sky' R&D; highly risky upfront investments by industry before going to market; enormous funds to protect its intellectual property, if necessary, through litigation; and the need to provide safe and affordable remedies to very large populations of indigent people in the world to keep them healthy.

This chapter is written for biotechnologists who wish to get an understanding of the role patents could play in protecting and advancing their research output in the service of mankind when commercial applications of that research is the optimal means of doing so. Here we discuss general principles of biotechnology patents and related issues, rather than country specific ones.

#### **2. The promise of biotechnology**

Biotechnology took roots more than 10,000 years ago. The 'old or traditional' methods of biotechnology were mainly fermentation (through the unwitting use of microbes) to produce such products as beer, wine, and cheese, and cross-breeding (through the unwitting use of genetic material) to modify plants and animals through progressive selection for desired traits. These methods were developed empirically, patiently, and over countless years (Darwin, 1872; Smith 2009). It was only during 1857 and 1876 that the fermentative ability of microorganisms was demonstrated by Louis Pasteur (Smith, 2009). The discovery by Alexander Fleming of the antibiotic penicillin in 1929 and its large-scale production in the 1940s created major advances in fermentation technology. Since then the technology has advanced rapidly not just in the production of antibiotics but in many other biochemical products including organic acids, polysaccharides, enzymes, vaccines, and hormones. Modern breeding methods now selectively move genes within the same species or between species.

The 'new or modern' biotechnology that emerged in the 1970s is (Lilly, 1997) "the application of scientific and engineering principles to the processing of materials by biological agents to provide goods and services." Its methods use microbial, animal or plant cells or enzymes for the purposes of breaking down, synthesizing, or transforming materials. The scientific foundation of biotechnology was laid in a remarkable paper by James D. Watson and Francis H. C. Crick in *Nature* (Watson & Crick, 1953), which elucidated the double-helix structure of cellular DNA1 (deoxyribonucleic acid). It gave birth to molecular biology and paved the way for developing recombinant DNA and cell fusion techniques along with scientific versions of 'old' biotechnological processes of modern biotechnology. Advances such as the transformation of *Escherichia coli*2; cutting and joining DNA strands (recombinant DNA technology) (Cohen, et al, 1973)3; the rapid cloning of DNA strands (PCR technique) (Mullis, et al, 1986); the ability to make monoclonal antibodies (hybridoma technique) (Köhler & Milstein, 1975)4, etc. have made possible the creation of genetically engineered life forms capable of manufacturing new and improved drugs, such as, human insulin, interferons, vaccines, and treatments for a host of human afflictions such as septic shock, anemia, diabetes, AIDS, cancer, hepatitis, and heart attack. Genetically engineered transgenic animals such as the Harvard mouse5 and the SCID mouse6 play an immensely important role in cancer and immunology research, respectively. Many other transgenic life forms such as bacteria, cows, pigs, goats, etc. play a crucial role in

<sup>1</sup> Formally known as B-DNA. Other forms of DNA, e.g., A-DNA, C-DNA, D-DNA, Z-DNA, DNAtriplex, DNA-quadruplex, etc. also exist. B-DNA is the most stable helical form adopted by random sequence DNA under physiological conditions

<sup>2</sup> This bacterium was discovered by Theodor Escherich (and named after him) in 1885.

<sup>3</sup> The method was protected by three patents, which have now expired.

<sup>4</sup> Amazingly, the method was never patented.

<sup>5</sup> In 1988, Philip Leder and Timothy Stewart of Harvard University inserted a cancer gene into mouse egg cells and produced the patented transgenic mouse (U.S. Patent No. 4,736,866, now expired).

Transgenic mice have become an incredibly powerful cancer research tool. 6 The SCID (*s*evere *c*ombined *i*mmuno*d*eficiency) mouse lacks T and B lymphocytes and immuno-

globulins, either from inbreeding with an autosomal-recessive trait or from genetic engineering. It is used as a model for studies of the immune system.

Biotechnology took roots more than 10,000 years ago. The 'old or traditional' methods of biotechnology were mainly fermentation (through the unwitting use of microbes) to produce such products as beer, wine, and cheese, and cross-breeding (through the unwitting use of genetic material) to modify plants and animals through progressive selection for desired traits. These methods were developed empirically, patiently, and over countless years (Darwin, 1872; Smith 2009). It was only during 1857 and 1876 that the fermentative ability of microorganisms was demonstrated by Louis Pasteur (Smith, 2009). The discovery by Alexander Fleming of the antibiotic penicillin in 1929 and its large-scale production in the 1940s created major advances in fermentation technology. Since then the technology has advanced rapidly not just in the production of antibiotics but in many other biochemical products including organic acids, polysaccharides, enzymes, vaccines, and hormones. Modern breeding methods now selectively move genes within the same

The 'new or modern' biotechnology that emerged in the 1970s is (Lilly, 1997) "the application of scientific and engineering principles to the processing of materials by biological agents to provide goods and services." Its methods use microbial, animal or plant cells or enzymes for the purposes of breaking down, synthesizing, or transforming materials. The scientific foundation of biotechnology was laid in a remarkable paper by James D. Watson and Francis H. C. Crick in *Nature* (Watson & Crick, 1953), which elucidated the double-helix structure of cellular DNA1 (deoxyribonucleic acid). It gave birth to molecular biology and paved the way for developing recombinant DNA and cell fusion techniques along with scientific versions of 'old' biotechnological processes of modern biotechnology. Advances such as the transformation of *Escherichia coli*2; cutting and joining DNA strands (recombinant DNA technology) (Cohen, et al, 1973)3; the rapid cloning of DNA strands (PCR technique) (Mullis, et al, 1986); the ability to make monoclonal antibodies (hybridoma technique) (Köhler & Milstein, 1975)4, etc. have made possible the creation of genetically engineered life forms capable of manufacturing new and improved drugs, such as, human insulin, interferons, vaccines, and treatments for a host of human afflictions such as septic shock, anemia, diabetes, AIDS, cancer, hepatitis, and heart attack. Genetically engineered transgenic animals such as the Harvard mouse5 and the SCID mouse6 play an immensely important role in cancer and immunology research, respectively. Many other transgenic life forms such as bacteria, cows, pigs, goats, etc. play a crucial role in

1 Formally known as B-DNA. Other forms of DNA, e.g., A-DNA, C-DNA, D-DNA, Z-DNA, DNAtriplex, DNA-quadruplex, etc. also exist. B-DNA is the most stable helical form adopted by random

5 In 1988, Philip Leder and Timothy Stewart of Harvard University inserted a cancer gene into mouse egg cells and produced the patented transgenic mouse (U.S. Patent No. 4,736,866, now expired). Transgenic mice have become an incredibly powerful cancer research tool. 6 The SCID (*s*evere *c*ombined *i*mmuno*d*eficiency) mouse lacks T and B lymphocytes and immunoglobulins, either from inbreeding with an autosomal-recessive trait or from genetic engineering. It is

2 This bacterium was discovered by Theodor Escherich (and named after him) in 1885.

3 The method was protected by three patents, which have now expired.

**2. The promise of biotechnology** 

species or between species.

sequence DNA under physiological conditions

4 Amazingly, the method was never patented.

used as a model for studies of the immune system.

the development of therapies and the manufacture of pharmaceuticals. Thus one of the main objectives of biotechnology is to find means of scaling-up biological processes.

The mapping of the human genome independently by the Human Genome Project and by Celera Genomics in 2001 was a remarkable breakthrough in data collection to aid studies of the human body.7 A crucial step in providing personalized medical care was thus taken. The breakthrough creation of a bacterial cell controlled by a chemically synthesized genome was reported by Craig Ventor's team in May 2010 in *Science* (Gibson, et al, 2010). The team reported synthesizing the genome of the bacterium *Mycoplasma mycoides*, comprising some 1.1 million base pairs as a proof of principle that cells can be produced based upon genome sequences designed in the computer. There is, of course, much to be learnt before one can construct and transplant whole computer-designed genomes of higher life forms. Important limiting factors are insufficient scientific knowledge of gene structure and function, and of microRNAs.

The potential curative abilities of stem cells come from their remarkable ability to renew themselves through cell division, sometimes even after long periods of inactivity, and to develop into many different cell types in the body. When a stem cell divides, each new cell, under appropriate circumstances, may either remain a stem cell or become a specialized cell such as a muscle cell, a red blood cell, or a brain cell. This unique regenerative ability of stem cells offers new opportunities for treating diseases such as diabetes and heart disease. However, much remains to be understood about them before reliable cell-based therapies can be designed to treat diseases.

The strength of modern biotechnology comes from its interdisciplinary nature and the interactions it orchestrates between various parts of biology and engineering. It draws insights and knowledge from a wide range of fields: biochemistry, microbiology, molecular biology, cell biology, immunology, protein engineering, enzymology, breeding techniques, chemical engineering, mechanical engineering, computational methods, mathematical simulation, bioinformatics, etc. The products and processes it spawns are the results of intense R&D, astronomical funding, and the unique entrepreneurial spirit of the biotechnology community in converting R&D results into therapies and cures, diagnostic tools and tests for disease detection, etc.

At present, biotechnology produces a range of embryonic enabling technologies for which some applications are known and many more expected. It is sustained by an enormous faith that suites of these enabling technologies, when further refined and augmented, will eventually find vast new applications of tremendous value to society. So the main benefits essentially lie in the future. The expected benefits include novel personalized pharmaceutical drugs and therapies for many diseases based on individual genomic information, genetically engineered healthier food with longer shelf-life, and new energyefficient techniques for protecting the environment. The biotechnology industry's

<sup>7</sup> The first analyses of the working draft human genome sequence were reported in the February 16, 2001 issue of *Science* and February 15, 2001 issue of *Nature*. The papers from *Nature* included initial sequence analyses generated by the publicly sponsored Human Genome Project, while *Science* publications focused on the draft sequence reported by the private company, Celera Genomics. The papers can be found at

http://www.ornl.gov/sci/techresources/Human\_Genome/project/journals/journals.shtml.

dependence on multi-nation patent protection for survival in the marketplace is therefore not at all surprising. In fact, it is imperative that every biotechnology researcher understands the circumstances when acquiring and protecting his research results by patents is crucial. Breakthrough R&D results by themselves are not enough; to serve society they must lead to commercially viable products and processes or find philanthropic hosts or find federal support. Of particular research interest are genes and their corresponding proteins as they are believed to represent the future of diagnostic and therapeutic medicine.

#### **3. Basics of patent law**

A patent is a limited period monopoly intellectual property right granted to an inventor of any country by a Government of any other or same country for an invention that fulfills prescribed statutory requirements of the granting country. Patent monopoly differs from market monopoly; a patent is a right to exclude, a right to prevent trespassing. In this sense it is no different from, say, the right to keep our house or car or any other personal possession free from trespassers. A patent grants inventors the right to exclude others from making, using, selling or offering to sell, and importing the claimed invention in the country of grant; it does not confer any right to practice the invention. This is because in practicing the invention, one may well need complementary patents held by others unwilling to cooperate or there may be other laws, rules or regulations that prevent its practice.

Patents are issued only to the first inventor (or group of joint inventors) of an invention who files a legally valid patent application; all others are barred, even if they independently created the invention. Consequently, those other inventors must get a license from the first inventor if they wish to practice the invention. Patents granted by a country, like its laws, have no extraterritorial effect; hence patents are unenforceable, if infringed, in another country where the invention in question is not patented. If multi-country patent protection is required, the invention must be patented in each desired country. There is no such thing as a "world-wide patent".

The modern concept of patents dates back to the year 1421, when the Italian city-state Florence granted the first recorded patent to Fillippo Brunelleschi, for the design and use of a ship, the Badalone (seagoing monster; it was used to carry marble along the Arno river), for three years.8 The Venetian Senate passed the first patent law on March 14, 1474, granting limited duration monopoly for original devices. That same Venice in 1594 granted Galileo9 a "privilege" (what we know as a patent) on a machine which he had invented10 "for raising water and irrigating land with small expense and great convenience," on the condition that it had never before been thought of or made by others. In his petition for the privilege he

http://www.lse.ac.uk/collections/economicHistory/GEHN/GEHNPDF/PotentiallyGlobal-IInkster.pdf.

<sup>8</sup> Christine MacLeod, Inventing the Industrial Revolution: The English Patent System. 1660-1800, Cambridge University Press, 2002, p. 11.

<sup>9</sup> Galileo Galilei (February 18, 1564 – January 8, 1642) is known as the father of modern science. He is perhaps the only scientist who is known by his first name rather than his last. In life sciences, Leonardo da Vinci, who preceded Galileo, is actually the unacknowledged "father of modern science" because of his remarkable studies of the human anatomy, and his empirical approach to science. See, e.g., Fritjof Capra, The Science of Leonardo, Doubleday, New York, 2007.

<sup>9</sup> Inkster, I., Potentially Global: A Story of Useful and Reliable Knowledge and Material Progress in Europe circa 1474-1912. Available at

dependence on multi-nation patent protection for survival in the marketplace is therefore not at all surprising. In fact, it is imperative that every biotechnology researcher understands the circumstances when acquiring and protecting his research results by patents is crucial. Breakthrough R&D results by themselves are not enough; to serve society they must lead to commercially viable products and processes or find philanthropic hosts or find federal support. Of particular research interest are genes and their corresponding proteins as they are believed to represent the future of diagnostic and therapeutic medicine.

A patent is a limited period monopoly intellectual property right granted to an inventor of any country by a Government of any other or same country for an invention that fulfills prescribed statutory requirements of the granting country. Patent monopoly differs from market monopoly; a patent is a right to exclude, a right to prevent trespassing. In this sense it is no different from, say, the right to keep our house or car or any other personal possession free from trespassers. A patent grants inventors the right to exclude others from making, using, selling or offering to sell, and importing the claimed invention in the country of grant; it does not confer any right to practice the invention. This is because in practicing the invention, one may well need complementary patents held by others unwilling to

cooperate or there may be other laws, rules or regulations that prevent its practice.

Patents are issued only to the first inventor (or group of joint inventors) of an invention who files a legally valid patent application; all others are barred, even if they independently created the invention. Consequently, those other inventors must get a license from the first inventor if they wish to practice the invention. Patents granted by a country, like its laws, have no extraterritorial effect; hence patents are unenforceable, if infringed, in another country where the invention in question is not patented. If multi-country patent protection is required, the invention must be patented in each desired country. There is no such thing

The modern concept of patents dates back to the year 1421, when the Italian city-state Florence granted the first recorded patent to Fillippo Brunelleschi, for the design and use of a ship, the Badalone (seagoing monster; it was used to carry marble along the Arno river), for three years.8 The Venetian Senate passed the first patent law on March 14, 1474, granting limited duration monopoly for original devices. That same Venice in 1594 granted Galileo9 a "privilege" (what we know as a patent) on a machine which he had invented10 "for raising water and irrigating land with small expense and great convenience," on the condition that it had never before been thought of or made by others. In his petition for the privilege he

8 Christine MacLeod, Inventing the Industrial Revolution: The English Patent System. 1660-1800,

9 Galileo Galilei (February 18, 1564 – January 8, 1642) is known as the father of modern science. He is perhaps the only scientist who is known by his first name rather than his last. In life sciences, Leonardo da Vinci, who preceded Galileo, is actually the unacknowledged "father of modern science" because of his remarkable studies of the human anatomy, and his empirical approach to science. See, e.g., Fritjof

9 Inkster, I., Potentially Global: A Story of Useful and Reliable Knowledge and Material Progress in

http://www.lse.ac.uk/collections/economicHistory/GEHN/GEHNPDF/PotentiallyGlobal-IInkster.pdf.

**3. Basics of patent law** 

as a "world-wide patent".

Cambridge University Press, 2002, p. 11.

Europe circa 1474-1912. Available at

Capra, The Science of Leonardo, Doubleday, New York, 2007.

said, "it not being fit that this invention, which is my own, discovered by me with great labor and expense, be made the common property of everyone," adding further, that if he were granted the privilege, "I shall the more attentively apply myself to new inventions for universal benefit." Clearly, even the great scientist Galileo was not willing to divulge his invention for free exploitation by others without just compensation for his efforts. The Venetian Council granted Galileo a "privilege" for 21 years.

An invention is the creation of a new technical idea *and* of the physical means to accomplish or embody it. An idea *per se* is not an invention; a useful and successful implementation of an idea is. Four types of inventions are eligible for patents: process, machine, manufacture, or composition of matter, collectively known as statutory subject matter. They are subject to certain limitations that vary from country to country. However, there is universal agreement among nations that abstract ideas (e.g., mathematical formulas), laws of nature, natural phenomena, and products of nature are ineligible, but their application to a known structure or process may be eligible. What to exclude from patent monopoly is a national prerogative, largely derived from government policy decisions that accrue from the prevailing socioeconomic conditions it must manage, and international treaty obligations.

Inventions that qualify as statutory subject matter must then face additional stringent statutory tests of substantial and credible utility (industrial application) in the eyes of an expert (such as a patent examiner in a patent office) in the field of the invention, novelty with respect to prior art (state-of-the-art) as it exists on the date of filing the patent application (in the United States there is some relaxation available), and nonobviousness, i.e., the invention has an inventive step that is unlikely to have been made by a person having ordinary skill in the art (PHOSITA), if required, assuming he would make the effort to study relevant prior art.

There is a *quid pro quo* attached to the grant of patents. To get a patent the inventor must put the invention in the public's possession. He must therefore fully describe his invention (written description requirement) in the patent application *before* the invention is formally examined by a patent examiner (examination typically takes two or more years). This description must be so clear and detailed as to enable a person skilled in the technologies related to the invention in question (an expert) to independently reproduce the invention (enablement requirement) without undue extra-solution activity, such as further research, data gathering, etc. on his part. In fact, this description should leave no doubt that the patent applicant was in possession of the claimed invention at the time of filing his application. Patents may be granted on improvements over existing inventions.

Patent laws of a country do not over-ride its other laws that might regulate the invention's use. For example, a new pharmaceutical cannot be marketed without the approval of appropriate authorities. Patent laws of a country may take into account moral, cultural, ethical, social, environmental, or scientific concerns of society.

In most countries with a patent regime, a pending patent application is placed in the public's possession 18 months after the first "priority" filing date11 of the application or the

<sup>11</sup> The priority date of a patent application is the filing date of the first patent application (the priority document), which discloses the invention, and to which priority is properly claimed in the country of interest. The written description of the invention in the priority document should be detailed enough so as to enable one skilled in the relevant art to make and use the invention.

date of patent grant, whichever is earlier, by means of publication in print and world-wideweb enabled media. This gives an opportunity to others to improve upon the invention and possibly patent improvements (or focus on something else), without unduly stifling innovation. Limited period monopoly (usually 20 years from the priority filing date) is meant to prevent undue concentration of economic power, yet allow inventors (a rare breed) an opportunity to recover costs and earn profit from their long and expensive effort, not otherwise possible if others could reverse engineer the invention (often a far less expensive process) and duplicate it without penalty. The goal has been to get as many useful inventions into the public domain and in free use as soon as possible and thus enrich society as a whole without being unfair to the inventor. Patent protection is therefore a bargain struck by society on the premise that, in its absence there would be insufficient invention and innovation. Patent and other laws do not forbid an altruistic inventor (unless bound by a legal contract, say, to his employer) from freely placing his patentable inventions in the public domain without patent protection.

Patents are granted to inventors. The rights attached to a patent may be exercised by the patentee, his or her heirs or assigns during the term of the patent. A patent may be assigned (e.g., to one's employer) or licensed, with or without conditions attached, to one or more legal entities. A patent license to a licensee is an agreement that the patent owner will not enforce certain or all rights of exclusion against the licensee. Anyone else infringing the patent can be sued in a court of law by the patent's owner.

Limited period patent monopoly may provide an enormous first mover advantage to an entrepreneur, especially if it involves new technology. Alexander Graham Bell's two telephone patents—"Improvement in Telegraphy" (U.S. 174,465), granted on March 7, 1876, provided a monopoly on the basic principle of telephony, and "Improvement in Electric Telegraphy" (U.S. 186,787) granted on January 30, 1877, provided a monopoly on the telephone hardware—are outstanding examples. By the time the patents expired, American Bell (later to become AT&T) had acquired a "natural monopoly" in the telephone business.

An alternative to patent protection is to keep the invention a trade secret, which lasts as long as the secret is kept. This works if the invention's independent discovery is so unlikely that it can be monopolized indefinitely. Otherwise, independent discoverers of the secret can practice their invention with impunity, and worse, one of them may patent it and deny all others the use of the invention if not licensed from him. If one is keen to commercialize the invention, patent protection is much safer than trade secret, especially during negotiations with investors when detailed exposure of the invention may be necessary. At times, keeping marginal improvements of a patented invention as trade secrets may be preferred, especially if constrained by patenting costs.

#### **3.1 Filing and prosecuting a patent application**

To get a patent one must file a patent application at an appropriate office designated for the purpose, usually the patent office of the selected country. Each country has its own rules and regulations for filing and these must be strictly followed. To claim priority over others for an invention, it is necessary to be the "first to file" the application in accordance with the country's statutory requirements, which may include statutory grace periods. To claim priority, one may file a provisional application which, at the minimum, fulfills the written description and enablement requirements, but it must be followed, within a year, by a proper application for the same invention as described in the provisional application, else the priority date is lost. Patent offices act only on proper applications. Provisional applications remain dormant during their life.

Prosecution is the process by which a (proper) patent application is defended before a patent office. The process often lasts several years. The application includes a complete description of the invention, a list of claims on statutory subject matter sought to be protected, and the requisite filled-in patent office forms, along with a filing fee. As prosecution proceeds, there may be other fees to be paid at various stages. For filing and prosecution details, visit the website of the desired patent office.

#### **3.2 Patent claims**

354 Innovations in Biotechnology

date of patent grant, whichever is earlier, by means of publication in print and world-wideweb enabled media. This gives an opportunity to others to improve upon the invention and possibly patent improvements (or focus on something else), without unduly stifling innovation. Limited period monopoly (usually 20 years from the priority filing date) is meant to prevent undue concentration of economic power, yet allow inventors (a rare breed) an opportunity to recover costs and earn profit from their long and expensive effort, not otherwise possible if others could reverse engineer the invention (often a far less expensive process) and duplicate it without penalty. The goal has been to get as many useful inventions into the public domain and in free use as soon as possible and thus enrich society as a whole without being unfair to the inventor. Patent protection is therefore a bargain struck by society on the premise that, in its absence there would be insufficient invention and innovation. Patent and other laws do not forbid an altruistic inventor (unless bound by a legal contract, say, to his employer) from freely placing his patentable inventions in the

Patents are granted to inventors. The rights attached to a patent may be exercised by the patentee, his or her heirs or assigns during the term of the patent. A patent may be assigned (e.g., to one's employer) or licensed, with or without conditions attached, to one or more legal entities. A patent license to a licensee is an agreement that the patent owner will not enforce certain or all rights of exclusion against the licensee. Anyone else infringing the

Limited period patent monopoly may provide an enormous first mover advantage to an entrepreneur, especially if it involves new technology. Alexander Graham Bell's two telephone patents—"Improvement in Telegraphy" (U.S. 174,465), granted on March 7, 1876, provided a monopoly on the basic principle of telephony, and "Improvement in Electric Telegraphy" (U.S. 186,787) granted on January 30, 1877, provided a monopoly on the telephone hardware—are outstanding examples. By the time the patents expired, American Bell (later to become AT&T) had acquired a "natural monopoly" in the

An alternative to patent protection is to keep the invention a trade secret, which lasts as long as the secret is kept. This works if the invention's independent discovery is so unlikely that it can be monopolized indefinitely. Otherwise, independent discoverers of the secret can practice their invention with impunity, and worse, one of them may patent it and deny all others the use of the invention if not licensed from him. If one is keen to commercialize the invention, patent protection is much safer than trade secret, especially during negotiations with investors when detailed exposure of the invention may be necessary. At times, keeping marginal improvements of a patented invention as trade secrets may be preferred, especially

To get a patent one must file a patent application at an appropriate office designated for the purpose, usually the patent office of the selected country. Each country has its own rules and regulations for filing and these must be strictly followed. To claim priority over others for an invention, it is necessary to be the "first to file" the application in accordance with the country's statutory requirements, which may include statutory grace periods. To claim

public domain without patent protection.

telephone business.

if constrained by patenting costs.

**3.1 Filing and prosecuting a patent application** 

patent can be sued in a court of law by the patent's owner.

The legal core of a patent application is the list of claims. Each claim in this list covers and secures a process, a machine, a manufacture, a composition of matter, or a design, but never the function or result of either, nor the scientific explanation of their operation. The claims define the scope of a patent grant and function to forbid not only exact copies of an invention but also products that go to the heart of the invention but avoid the literal language of the claim by making a non-critical change. (See Section 4 below.) Whether a claim is allowed by a patent office is judged on the basis of novelty, nonobviousness, and utility (industrial applicability) of the invention being considered. Of course, claims are interpreted in light of the description of the invention provided in the patent application and information elicited during prosecution from the inventor, prior art and other sources. Almost all litigation related to patent infringement centers on the validity and scope of the claims.

In biotechnology, claims may be product claims, process claims, or product-by-process claims. Product claims may include such things as novel protein products, known but purified protein products, DNA sequence of a gene that encodes a particular protein, etc. Process claims may include preparation or use of recombinant DNA, the use of bacteria or cultured cells transformed with vectors containing DNA encoding a desired protein product, methods of use for proteins, methods for production or use of monoclonal antibodies, etc. Product-by-process claims deal with products that are too complex to be described conventionally (e.g., with reference to its composition, structure or some other testable parameter) and hence are described by the process with which it is made. By such claims it is not possible to use a new process to claim an old product. The focus here is the patentability of the product itself, not on the process used to describe it since the reference to a process serves only the purpose of defining the product.

There have been attempts by biotechnology inventors to get "reach-through claims" granted. Such claims seek to protect things which may not have been identified by the applicant in his patent application but which *may be* identified in the future by others by carrying out the invented process. This is different from the product-by-process claims as the products claimed in reach-through claims are speculative and hence do not fulfill the statutory requirements of disclosure and enablement. The purported justification for such broad extra-legal claims is that a pioneering invention paves the way for subsequent inventions and hence its inventor is "entitled" to capture some of the follow-on value based on the relative contribution of his pioneering invention. (Christie & Lim, 2005; IPO, 2009)

### **4. Infringement**

Protecting an active patent when infringed can be a nightmare. It is time consuming, and hugely expensive (usually measured in millions of U.S. dollars) if it involves litigation. Alleged infringers, if challenged, are quite likely to counter-challenge by questioning the validity of the disputed patent. It is therefore imperative, especially in biotechnology where patents underpin business, that patent applications are prepared by experienced patent attorneys and that inventors work closely with them to minimize litigation possibilities. Considerations that go into the preparation of a fortified patent application include the doctrine of equivalent, prosecution history estoppel, reverse doctrine of equivalents, prior art or state-of-the-art, and the anticipated profile of the imaginary PHOSITA.

#### **4.1 Doctrine of equivalents**

Literal infringement of a valid active patent where the alleged infringer exactly or nearly exactly copies an invention without a licence from the patent owner is understandably rare. Generally, one tries to work around a patented invention by introducing differences and variations to avoid infringement. The question then is whether the modified product or process is remote enough from the patent that it will not infringe. Inadvertent infringement may arise if a product or process is invented in ignorance of an active patent whose existence is discovered only later, say, after a business commitment has been made to produce the product or use the process.

Such situations are partially dealt with by the judicially created doctrine of equivalents. This is a rule of claim interpretation under which a product or process, although not a literal infringement, is an infringement if it performs substantially the same function in substantially the same way to obtain the same result as a patented product or process. This doctrine, which has universal appeal, expands patent protection beyond the literal language of the claim. To determine what counts as an equivalent one must find a balance between two opposing public policies: (1) the importance of providing public notice as to what infringes by requiring clear and distinct claims, and (2) the need to prevent an infringer from avoiding liability by merely playing semantic games or by making only minor changes in the accused product or process to avoid the literal language of the claims (Belvis, 2003). In litigation, courts may seek expert opinion as to scientific or engineering facts and the decision may well depend on the most believable expert. Note that things that are equivalent for one purpose may not be so for other purposes. The Supreme Court of the United States sums it succinctly in *Graver Tank*12:

What constitutes equivalency must be determined against the context of the patent, the prior art, and the particular circumstances of the case. Equivalence, in the patent law, is not the prisoner of a formula and is not an absolute to be considered in a vacuum. It does not require complete identity for every purpose and in every respect. In determining equivalents, things equal to the same thing may not be equal to each other and, by the same token, things for most purposes different may sometimes be equivalents. Consideration must be given to the purpose for which an ingredient is used in a patent, the qualities it has when combined with the other ingredients, and the

 <sup>12</sup> *Graver Tank & Mfg. Co.* v. *Linde Air Products*, 339 U.S. 605 (1950). Available at http://supreme.justia.com/us/339/605/case.html.

Protecting an active patent when infringed can be a nightmare. It is time consuming, and hugely expensive (usually measured in millions of U.S. dollars) if it involves litigation. Alleged infringers, if challenged, are quite likely to counter-challenge by questioning the validity of the disputed patent. It is therefore imperative, especially in biotechnology where patents underpin business, that patent applications are prepared by experienced patent attorneys and that inventors work closely with them to minimize litigation possibilities. Considerations that go into the preparation of a fortified patent application include the doctrine of equivalent, prosecution history estoppel, reverse doctrine of equivalents, prior

Literal infringement of a valid active patent where the alleged infringer exactly or nearly exactly copies an invention without a licence from the patent owner is understandably rare. Generally, one tries to work around a patented invention by introducing differences and variations to avoid infringement. The question then is whether the modified product or process is remote enough from the patent that it will not infringe. Inadvertent infringement may arise if a product or process is invented in ignorance of an active patent whose existence is discovered only later, say, after a business commitment has been made to

Such situations are partially dealt with by the judicially created doctrine of equivalents. This is a rule of claim interpretation under which a product or process, although not a literal infringement, is an infringement if it performs substantially the same function in substantially the same way to obtain the same result as a patented product or process. This doctrine, which has universal appeal, expands patent protection beyond the literal language of the claim. To determine what counts as an equivalent one must find a balance between two opposing public policies: (1) the importance of providing public notice as to what infringes by requiring clear and distinct claims, and (2) the need to prevent an infringer from avoiding liability by merely playing semantic games or by making only minor changes in the accused product or process to avoid the literal language of the claims (Belvis, 2003). In litigation, courts may seek expert opinion as to scientific or engineering facts and the decision may well depend on the most believable expert. Note that things that are equivalent for one purpose may not be so for other purposes. The Supreme Court of the

What constitutes equivalency must be determined against the context of the patent, the prior art, and the particular circumstances of the case. Equivalence, in the patent law, is not the prisoner of a formula and is not an absolute to be considered in a vacuum. It does not require complete identity for every purpose and in every respect. In determining equivalents, things equal to the same thing may not be equal to each other and, by the same token, things for most purposes different may sometimes be equivalents. Consideration must be given to the purpose for which an ingredient is used in a patent, the qualities it has when combined with the other ingredients, and the

art or state-of-the-art, and the anticipated profile of the imaginary PHOSITA.

**4. Infringement** 

**4.1 Doctrine of equivalents** 

produce the product or use the process.

United States sums it succinctly in *Graver Tank*12:

http://supreme.justia.com/us/339/605/case.html.

<sup>12</sup> *Graver Tank & Mfg. Co.* v. *Linde Air Products*, 339 U.S. 605 (1950). Available at

function which it is intended to perform. An important factor is whether persons reasonably skilled in the art would have known of the interchangeability of an ingredient not contained in the patent with one that was.

In *Warner-Jenkinson*13 the same Court then clarified and restricted the application of the doctrine of equivalents, holding that:

Each element contained in a patent claim is deemed material to defining the scope of the patented invention, and *thus the doctrine of equivalents must be applied to individual elements of the claim, not to the invention as a whole*. It is important to ensure that the application of the doctrine, even as to an individual element, is not allowed such broad play as to effectively eliminate that element in its entirety. [Emphasis added]

This restriction on the doctrine of equivalents serves to eliminate one of the great mischiefs that could be played in patent law. Absent this rule, one could attempt to use the doctrine of equivalents to subvert patent claims. Rather than focusing on specific claim language and elements of the claim, the case could be tried based on how the accused device was equivalent to that claim as a whole. The Court further held that the equivalence determination was to be made at the time of the alleged infringement and not at the time the patent issued. It is likely that the less certain and more complex the courts perceive a scientific field underlying a technology to be (as is the case with biotechnology), the less scope will be given to patents under the doctrine of equivalents. If the patent is a pioneer in a whole new field, it will generally receive a broader range of equivalents than one for a narrow improvement to existing technology (Blenko, 1990). There are a few other restrictions that circumscribe the doctrine of equivalents: prosecution history estoppel, the reverse doctrine of equivalents, and prior art.

#### **4.2 Prosecution history estoppel**

During prosecution, quite likely, one or more claims will be rejected or require amendment to become narrower and detailed, in view of prior art. If a claim is allowed after being narrowed to avoid prior art, the patentee is barred from asserting the narrowed claim in its earlier broader sense under the doctrine of equivalents. This means that broad claims that have to be amended during prosecution can be difficult to enforce, if infringed. In short, rejected or narrowed claims cannot be expanded to their earlier scope under the doctrine of equivalents. In fact, such claims practically forego any benefit that could have accrued under the doctrine of equivalents in infringement cases.

#### **4.3 Reverse doctrine of equivalents**

A further restriction on the doctrine of equivalents is the *reverse doctrine of equivalents*. As noted by the Supreme Court of the United States in the *Graver Tank* case:

The wholesome realism of this doctrine [of equivalence] is not always applied in favor of a patentee but is sometimes used against him. Thus, where a device is so far changed

 <sup>13</sup> *Warner-Jenkinson Co.* v. *Hilton Davis Chemical Co.*, 520 U.S. 17 (1997). Available at http://supreme.justia.com/us/520/17/case.html.

in principle from a patented article that it performs the same or similar function in a substantially different way, but nevertheless falls within the literal words of the claim, the doctrine of equivalents may be used to restrict the claim and defeat the patentee's action for infringement. [Citations omitted]

Thus, where an invention relies on the fundamental concept embodied in a patent but is more sophisticated than the patented device due to "a significant advance," the accused device does not infringe by virtue of the reverse doctrine of equivalents. Once a patentee establishes literal infringement, the burden is on the alleged infringer to establish noninfringement under the reverse doctrine of equivalents. This is an untested area of patent law but may become important in biotechnology with respect to certain pioneering technologies, such as, synthetic cell technology.

#### **4.4 Prior art**

Prior art or state-of-the-art is all information, available in any form, in the public domain. It does not include secret information, such as trade secrets. The existing reservoir of ideas and their expression form the foundation on which new intellectual property is built. Normally, prior art does not include unpublished work or mere conversations (although in the European Patent Convention, oral disclosures do form prior art14). There is a continuing effort by various countries to document their respective traditional knowledge, such as medicinal properties of plants, and make that knowledge available as searchable prior art. The doctrine of equivalents excludes whatever is already prior art.

#### **4.5 The PHOSITA in biotechnology**

In examining a patent application, the patent examiner faces an immediate problem. How to define the relevant PHOSITA? In patent law the PHOSITA is a legal fictional character or a team of characters analogous to the "reasonable person" in the common law of torts. The PHOSITA is a statistical concept in the sense that there is a very high probability that no one from the community of ordinarily skilled persons in the relevant technical field(s) will be able to come up with the invention in question or its close equivalent or a superior one if the community was required to do so. So the PHOSITA, by definition, is neither a genius nor a layperson, but one possessing normal skills and knowledge in the required technical field. In this sense he serves as a litmus test for deciding if an invention is nonobvious or involves an inventive step. If the PHOSITA is deemed capable of coming up with the invention by applying his mind, knowledge, skill, and common sense, that particular invention is deemed unpatentable. In short, a "person of ordinary skill is also a person of ordinary creativity, not an automaton."15

Unfortunately, "ordinary skill" must be determined on a case-by-case basis, depending on the sophistication and technological features of the invention. Clearly, the ordinary skills of

<sup>14</sup> Art. 54(2) EPC: "The state of the art shall be held to comprise everything made available to the public by means of a written or oral description, by use, or in any other way, before the date of filing of the European patent application."

<sup>15</sup> *KSR International Co.* v. *Teleflex Inc. et al*, 550 U.S.\_\_ (2007). Availlable at http://supreme.justia.com/us/550/04-1350/

Thus, where an invention relies on the fundamental concept embodied in a patent but is more sophisticated than the patented device due to "a significant advance," the accused device does not infringe by virtue of the reverse doctrine of equivalents. Once a patentee establishes literal infringement, the burden is on the alleged infringer to establish noninfringement under the reverse doctrine of equivalents. This is an untested area of patent law but may become important in biotechnology with respect to certain pioneering

Prior art or state-of-the-art is all information, available in any form, in the public domain. It does not include secret information, such as trade secrets. The existing reservoir of ideas and their expression form the foundation on which new intellectual property is built. Normally, prior art does not include unpublished work or mere conversations (although in the European Patent Convention, oral disclosures do form prior art14). There is a continuing effort by various countries to document their respective traditional knowledge, such as medicinal properties of plants, and make that knowledge available as searchable prior art.

In examining a patent application, the patent examiner faces an immediate problem. How to define the relevant PHOSITA? In patent law the PHOSITA is a legal fictional character or a team of characters analogous to the "reasonable person" in the common law of torts. The PHOSITA is a statistical concept in the sense that there is a very high probability that no one from the community of ordinarily skilled persons in the relevant technical field(s) will be able to come up with the invention in question or its close equivalent or a superior one if the community was required to do so. So the PHOSITA, by definition, is neither a genius nor a layperson, but one possessing normal skills and knowledge in the required technical field. In this sense he serves as a litmus test for deciding if an invention is nonobvious or involves an inventive step. If the PHOSITA is deemed capable of coming up with the invention by applying his mind, knowledge, skill, and common sense, that particular invention is deemed unpatentable. In short, a "person of ordinary skill is also a person of ordinary

Unfortunately, "ordinary skill" must be determined on a case-by-case basis, depending on the sophistication and technological features of the invention. Clearly, the ordinary skills of

14 Art. 54(2) EPC: "The state of the art shall be held to comprise everything made available to the public by means of a written or oral description, by use, or in any other way, before the date of filing of the

action for infringement. [Citations omitted]

technologies, such as, synthetic cell technology.

**4.5 The PHOSITA in biotechnology** 

creativity, not an automaton."15

European patent application."

http://supreme.justia.com/us/550/04-1350/

The doctrine of equivalents excludes whatever is already prior art.

<sup>15</sup> *KSR International Co.* v. *Teleflex Inc. et al*, 550 U.S.\_\_ (2007). Availlable at

**4.4 Prior art** 

in principle from a patented article that it performs the same or similar function in a substantially different way, but nevertheless falls within the literal words of the claim, the doctrine of equivalents may be used to restrict the claim and defeat the patentee's a nuclear physicist are different from those of a chef or a cobbler or a molecular biologist. Factors used in determining ordinary skill include the time frame of the invention; education level of the inventor, education level of active workers in the field of the invention, and the type of problems generally encountered in the field; prior art solutions relevant to the invention; rapidity with which innovations are made in the field; sophistication of the technology; etc. Further, with time, the profile of a PHOSITA, in advancing technologies, will only improve due to the infusion of new knowledge and skills. Therefore, in rapidly advancing fields, such as biotechnology, determining the profile of a PHOSITA requires great skill and frequent revision.

The PHOSITA's role is crucial in several places—in the enablement requirement, the nonobviousness requirement, the utility requirement (the invention must operate as described if he is to be enabled), and the written description requirement, as compliance with these requirements is measured from his perspective. Therefore, claims must be written so that a PHOSITA would understand the bounds of the patent, including the territory covered by the doctrine of equivalents. A fundamental test for the doctrine of equivalents is whether a PHOSITA would reasonably interchange the elements in a claim at issue in an infringement case. What is not clear is that as higher education spreads and the PHOSITAs learn to solve problems at conceptual levels, how that will affect the doctrine of equivalents.

The enablement and non-obviousness questions arise before the issuance of a patent while the question of interchangeability arises at the time of infringement. Note that while biotechnology patent examiners are experts in biotechnology, infringement and validity cases are decided by judges who are not. So there is often a misalignment of the PHOSITA's profile separately conjured by the examiner and the judge in any given biotechnology patent case. In fact, it is rather difficult for courts to insert, in their decisions, the role of "common sense" a PHOSITA might routinely employ in his day-to-day work.

In infringement cases, the cut-off date chosen to ascertain prior art and the PHOSITA's profile can become a critical factor even when the dates differ by only a few weeks. Scientific breakthroughs and pioneer inventions suddenly appearing on the scene around the cut-off date can complicate matters tremendously. In the fast changing world of biotechnology, what is nonobvious today may well be obvious next year or next week!

#### **5. Patentability conditions in biotechnology**

Large scale patenting of living matter is recent. Indeed, prior to 1980, few patents had been granted on non-living biological matter and biologically pure cultures of micro-organisms as they did not exist in nature in their pure form; they could only be produced in carefully controlled laboratory environments. Patent laws around the world till then had assumed that higher life forms were not patentable as they were deemed products of nature. An abrupt change in legal thinking occurred when the Supreme Court of the United States in its June 16, 1980 decision in *Diamond* v. *Chakrabarty* held that "a live, human-made microorganism is patentable subject matter" under the U.S. Patent Act of 1952. Recall that recombinant DNA technology was already well known in 1980. The Court reasoned that Chakrabarty's microorganism was a "nonnaturally occurring manufacture or composition of matter—a product of human ingenuity" worthy of liberal encouragement under the patent system. It declared that "the relevant distinction was not between living and inanimate things, but between products of nature, whether living or not, and human-made inventions." The floodgates for biotechnology patents were thus opened in the United States,16 and eventually, using similar reasoning, patents on living matter were allowed in other countries. In 1988, the United States Patent and Trademark Office (USPTO) issued the first transgenic animal patent on the now famous Harvard mouse17, a mouse genetically engineered to be particularly susceptible to tumor growth. Patents have since been issued on many other genetically engineered plants and animals.

The nature of biotechnology and its close working association with bioinformatics and molecular biology has added a new and complex dimension in patenting. The DNA is both a material molecule as well as a literal embodiment of coded information (the book of life). The courts are still trying to understand this deep two-facedness of DNA. For example, can artificially created DNA or gene sequences be copyrighted?

Finally, when filing a biotechnology patent application, particular attention should be paid to meeting the legal requirements of (1) statutory subject matter, (2) utility, (3) novelty, (4) nonobviousness, and (5) specification (description, enablement, and claims). The vast majority of litigation cases revolve around these statutory requirements.

#### **5.1 Statutory subject matter**

Biotechnology deals with bio-matter itself (including products of biotechnology living or non-living) and processes of making bio-matter. Examples of non-living bio-matter are amino acids, peptides, proteins, fats and fatty acids, and nucleic acids. They are all chemical compounds and are usually better known in the form of antibodies, hormones, enzymes, antibiotics, steroids, cholesterol, DNA molecules, etc.

The primary entity in living bio-matter is the cell, the smallest reproducible unit of life. A wide range of biotechnology product inventions, e.g., proteins, antibodies, intracellular components of plant and animal cells (DNA fragments, DNA constructs, DNA promoters, plasmids, vectors, RNAs, ribosomes, chloroplasts, mitochondria, Golgi bodies, etc.) and living matter *per se*, such as cell lines, fused cells, plant seeds, tissue cultures, microorganisms, plants and nonhuman animals, are patentable subject matter.

Biotechnology process inventions include processes for sequencing DNA, RNA or proteins; processes for genetically manipulating cells, plants or animals; processes for recovering proteins produced by cell lines or animals; processes for detecting and characterizing mutagenic agents; processes for culturing tissue or cells; processes for diagnosing or detecting biological states; fermentation, chemical and diagnostics methods; methods of treating human or animal bodies; methods of controlling pests; etc. Biotechnology processes also provide the potential for creating genetically altered bio-matter itself. In the early to mid-1980s researchers were already creating genetically altered transgenic mice, hamsters, rats, hogs, poultry, cattle, sheep, and fish. In 2010, the first synthetic cell capable of

 16 Chakrabarty was granted U.S. Patent No. 4,259,444, Microorganisms having multiple compatible degradative energy-generating plasmids and preparation thereof, filed on June 7, 1972, issued on March 31, 1981. The patent has now expired.

<sup>17</sup> This was U.S. Patent No. 4,736,866, Transgenic non-human mammals, issued April 12, 1988 to Philip Leder and Timothy A. Stewart. The patent has now expired.

patent system. It declared that "the relevant distinction was not between living and inanimate things, but between products of nature, whether living or not, and human-made inventions." The floodgates for biotechnology patents were thus opened in the United States,16 and eventually, using similar reasoning, patents on living matter were allowed in other countries. In 1988, the United States Patent and Trademark Office (USPTO) issued the first transgenic animal patent on the now famous Harvard mouse17, a mouse genetically engineered to be particularly susceptible to tumor growth. Patents have since been issued on

The nature of biotechnology and its close working association with bioinformatics and molecular biology has added a new and complex dimension in patenting. The DNA is both a material molecule as well as a literal embodiment of coded information (the book of life). The courts are still trying to understand this deep two-facedness of DNA. For example, can

Finally, when filing a biotechnology patent application, particular attention should be paid to meeting the legal requirements of (1) statutory subject matter, (2) utility, (3) novelty, (4) nonobviousness, and (5) specification (description, enablement, and claims). The vast

Biotechnology deals with bio-matter itself (including products of biotechnology living or non-living) and processes of making bio-matter. Examples of non-living bio-matter are amino acids, peptides, proteins, fats and fatty acids, and nucleic acids. They are all chemical compounds and are usually better known in the form of antibodies, hormones, enzymes,

The primary entity in living bio-matter is the cell, the smallest reproducible unit of life. A wide range of biotechnology product inventions, e.g., proteins, antibodies, intracellular components of plant and animal cells (DNA fragments, DNA constructs, DNA promoters, plasmids, vectors, RNAs, ribosomes, chloroplasts, mitochondria, Golgi bodies, etc.) and living matter *per se*, such as cell lines, fused cells, plant seeds, tissue cultures,

Biotechnology process inventions include processes for sequencing DNA, RNA or proteins; processes for genetically manipulating cells, plants or animals; processes for recovering proteins produced by cell lines or animals; processes for detecting and characterizing mutagenic agents; processes for culturing tissue or cells; processes for diagnosing or detecting biological states; fermentation, chemical and diagnostics methods; methods of treating human or animal bodies; methods of controlling pests; etc. Biotechnology processes also provide the potential for creating genetically altered bio-matter itself. In the early to mid-1980s researchers were already creating genetically altered transgenic mice, hamsters, rats, hogs, poultry, cattle, sheep, and fish. In 2010, the first synthetic cell capable of

16 Chakrabarty was granted U.S. Patent No. 4,259,444, Microorganisms having multiple compatible degradative energy-generating plasmids and preparation thereof, filed on June 7, 1972, issued on March

17 This was U.S. Patent No. 4,736,866, Transgenic non-human mammals, issued April 12, 1988 to Philip

microorganisms, plants and nonhuman animals, are patentable subject matter.

many other genetically engineered plants and animals.

artificially created DNA or gene sequences be copyrighted?

antibiotics, steroids, cholesterol, DNA molecules, etc.

**5.1 Statutory subject matter** 

31, 1981. The patent has now expired.

Leder and Timothy A. Stewart. The patent has now expired.

majority of litigation cases revolve around these statutory requirements.

continuous self-replication was created, a cell that was completely controlled by a computer designed synthetic chromosome.

#### **5.2 The utility requirement (genetic materials)**

A patent examiner will accept a utility asserted by an applicant unless there is evidence or sound scientific reasoning against it. Clinical trials of a new pharmaceutical are not required to establish its utility18. Transgenic animals are generally created with a specific use in mind, so their utility is usually obvious. For gene sequences a nontrivial utility of the protein it will produce must be shown. Citing generic useful functions such as a marker, probe, or primer for various genetic researchers may likely be considered trivial given the present state-ofthe-art in gene research.

New processes related to genetic materials must show utility for the product of a process as well as the process itself, otherwise one could end in patenting a process which yielded an unpatentable product. "Until the process claim has been reduced to production of a product shown to be useful, the metes and bounds of that monopoly are not capable of precise delineation. It may engross a vast, unknown, and perhaps unknowable area."19 Less this constraint, the patentable field would be too broad. Therefore, to assert utility, say, for a process for making a protein, one must establish that the protein itself has substantial and specific utility in currently available form.

General utility is disallowed because it would embrace a broad class of an invention. For example, regarding ESTs (expressed sequence tags), "a claim to a polynucleotide whose use is disclosed simply as a 'gene probe' or 'chromosome marker' would not be considered to be *specific* in the absence of a disclosure of a specific DNA target."20 An EST does not explain the purpose and use of the gene. Therefore an EST patent "would amount to a hunting license"21 for performing research that may lead nowhere. Likewise, cDNA fragments used as probes for finding full-length genes lacks specific utility because, "[a]ny partial nucleic acid prepared from any cDNA may be used as a probe in the preparation and or identification of a full-length cDNA." 22 Biotechnology patents must present a higher degree of utility than for most other types of patents, say, in mechanical or electrical engineering.

#### **5.3 The novelty requirement**

Non-naturally occurring life forms such as transgenic animals that are "man-made" or "man-altered" for the first time satisfy the novelty requirement. Gene sequences, either artificially created or purified and altered from their natural state, say, by deleting the introns and retaining the protein coding exons, may fulfill the novelty requirement.

<sup>18</sup> See, e.g., *In Re Brana*, 51 F.3d 1560 (Fed. Cir. 1995). Available at

http://law.justia.com/cases/federal/appellate-courts/F3/51/1560/618133/.

<sup>19</sup> *Brenner* v. *Mansion*, 383 U.S. 519 (1966). Available at

http://supreme.justia.com/us/383/519/case.html.

<sup>20</sup> USPTO, Revised Interim Utility Guidelines Training Materials (1999) at 5. Available at

http://www.uspto.gov/web/menu/utility.pdf.

<sup>21</sup> *In re Fisher*, 421 F.3d 1365, 1376-77 (Fed. Cir. 2005). Available at

http://www.cafc.uscourts.gov/images/stories/opinions-orders/04-1465.pdf.

<sup>22</sup> USPTO, Revised Interim Utility Guidelines Training Materials (1999) at 51.

However, the current state-of-the-art in human gene sequencing and its rate of advance are such that in future litigation courts may well set a more stringent criterion for novelty. This is perhaps inevitable given that many of the products of interest to the biotechnology industry are synthetic versions of substances that already exist in nature, and creating those synthetic versions is within the capabilities of a PHOSITA. Under these circumstances, can a synthetic version be called "new"? While methods of use of "new" sequences may be novel, claiming those sequences as new compositions may not be easily allowed.23

#### **5.4 The nonobviousness requirement**

The nonobviousness requirement is an important policy lever by which governments can efficiently control the transfer of intellectual wealth to promote industrial products and processes. This is particularly important when dealing with innovative medical products and diagnostic tools being produced and mass marketed by multinational pharmaceutical and biotechnology firms.

The core of a researcher's activity is hypotheses testing. This is what many biotechnology PHOSITA do routinely. Scientific inventions in biotechnology rarely come about *de novo*. Thus how much of the experimental research or testing conducted in the lead-up to an invention is attributable to a PHOSITA is central to the obviousness test. Routine, ordinary, logical, or workshop activity is not deserving of patent monopoly. "The results of ordinary innovations are not the subject of exclusive rights under patent law;" otherwise "patents might stifle rather than promote the progress of useful arts."24 Thus routine testing in the lead-up period to invention in anticipation of reasonable expectation of success should be expected of an ordinarily creative PHOSITA.

Setting obviousness standards for gene patents is difficult because scientists use similar techniques to isolate different gene sequences, even though the gene may be new. A related question immediately arises. If homology-based utility satisfies the requirement of utility, would the invention be considered obvious? The USPTO's view25, obviously in the context of U.S. patent law, is that nonobviousness and utility requirements are separate. This is because even though a claim to a nucleic acid is supported by a homology-based utility over a set of nucleic acids, that utility is not *prima facie* obvious. Homology-based deductions may provide a reason or motivation to make the claimed composition, but it would still be necessary to establish a fact-intensive comparison of the claim with the prior art rather than the mechanical application of one or another *per se* rule. In short, "obvious-to-try" and obviousness is not always the same thing (rules of thumb are not rules of law). The mere fact that something is "obvious to try" in view of known prior art does not automatically imply that the invention resulting therefrom is obvious. This is especially true where the prior art does not contain any suggestion or teaching that might suggest how the invention might be accomplished or any basis for reasonable expectation that beneficial results will accrue by proceeding along the lines taken by an inventor.

 <sup>23</sup> *In re Gleave*, 560 F.3d 1331 (Fed. Cir. 2009). Available at

http://www.cafc.uscourts.gov/images/stories/opinions-orders/08-1453.pdf.

<sup>24</sup> *KSR International Co.* v. *Teleflex Inc. et al*, 550 U.S. \_\_ (2007), at 24.

<sup>25</sup> Utility Examination Guidelines, USPTO, Federal Register, Vol. 66, No. 4, January 5, 2001, Notices, pp. 1092-1099. Available at http://www.uspto.gov/web/offices/com/sol/notices/utilexmguide.pdf.

However, the current state-of-the-art in human gene sequencing and its rate of advance are such that in future litigation courts may well set a more stringent criterion for novelty. This is perhaps inevitable given that many of the products of interest to the biotechnology industry are synthetic versions of substances that already exist in nature, and creating those synthetic versions is within the capabilities of a PHOSITA. Under these circumstances, can a synthetic version be called "new"? While methods of use of "new" sequences may be novel,

The nonobviousness requirement is an important policy lever by which governments can efficiently control the transfer of intellectual wealth to promote industrial products and processes. This is particularly important when dealing with innovative medical products and diagnostic tools being produced and mass marketed by multinational pharmaceutical

The core of a researcher's activity is hypotheses testing. This is what many biotechnology PHOSITA do routinely. Scientific inventions in biotechnology rarely come about *de novo*. Thus how much of the experimental research or testing conducted in the lead-up to an invention is attributable to a PHOSITA is central to the obviousness test. Routine, ordinary, logical, or workshop activity is not deserving of patent monopoly. "The results of ordinary innovations are not the subject of exclusive rights under patent law;" otherwise "patents might stifle rather than promote the progress of useful arts."24 Thus routine testing in the lead-up period to invention in anticipation of reasonable expectation of success should be

Setting obviousness standards for gene patents is difficult because scientists use similar techniques to isolate different gene sequences, even though the gene may be new. A related question immediately arises. If homology-based utility satisfies the requirement of utility, would the invention be considered obvious? The USPTO's view25, obviously in the context of U.S. patent law, is that nonobviousness and utility requirements are separate. This is because even though a claim to a nucleic acid is supported by a homology-based utility over a set of nucleic acids, that utility is not *prima facie* obvious. Homology-based deductions may provide a reason or motivation to make the claimed composition, but it would still be necessary to establish a fact-intensive comparison of the claim with the prior art rather than the mechanical application of one or another *per se* rule. In short, "obvious-to-try" and obviousness is not always the same thing (rules of thumb are not rules of law). The mere fact that something is "obvious to try" in view of known prior art does not automatically imply that the invention resulting therefrom is obvious. This is especially true where the prior art does not contain any suggestion or teaching that might suggest how the invention might be accomplished or any basis for reasonable expectation that beneficial results will accrue by

25 Utility Examination Guidelines, USPTO, Federal Register, Vol. 66, No. 4, January 5, 2001, Notices, pp. 1092-1099. Available at http://www.uspto.gov/web/offices/com/sol/notices/utilexmguide.pdf.

claiming those sequences as new compositions may not be easily allowed.23

**5.4 The nonobviousness requirement** 

expected of an ordinarily creative PHOSITA.

proceeding along the lines taken by an inventor.

<sup>23</sup> *In re Gleave*, 560 F.3d 1331 (Fed. Cir. 2009). Available at

<sup>24</sup> *KSR International Co.* v. *Teleflex Inc. et al*, 550 U.S. \_\_ (2007), at 24.

http://www.cafc.uscourts.gov/images/stories/opinions-orders/08-1453.pdf.

and biotechnology firms.

To assess nonobviousness requires profiling a PHOSITA, who in biotechnology usually holds a PhD and is therefore an expert in common perception. Moreover, this PHOSITA is more likely to be a team of experts rather than a "mythical individual". So how does one reasonably determine this mythical PHOSITA at a point in time in an area of technology which is advancing so rapidly that the profile would need to be updated, at times, on a weekly basis? How are questions related to the doctrine of equivalents to be handled, especially if the invention is a synthetic version of a 'product of nature'? After all, Nature too is experimenting constantly with its own creations, including the destruction and creation of new species in the predator-prey game of "survival of the fittest" or "natural selection". These are extremely difficult questions to deal with in litigation.

#### **5.5 The specification requirement**

A specification is targeted at an expert in the field of the invention. Therefore, it is unnecessary for an applicant to spell out every detail but only enough to convince an expert that the inventor possessed the invention as of the filing date, and to enable a PHOSITA to make and use the invention without undue experimentation26. For example, in a gene related patent, a written description doesn't need a recitation or incorporation by reference of genes and sequences that are well documented in the prior art. An adequate description of the invention therefore depends on the nature and scope of the invention, not the description's length. Furthermore, an actual reduction to practice is *not* required. An invention can be "complete" even without an actual reduction. The Court of Appeals for the Federal Circuit in the United States, in *Falkner v. Inglis*, 448 F.3d 1357, 1366, 79 USPQ2d 1001, 1007 (Fed. Cir. 2006)27 has succinctly stated: "(1) examples are not necessary to support the adequacy of a written description; (2) the written description standard may be met … even where actual reduction to practice of an invention is absent; and (3) there is no per se rule that an adequate written description of an invention that involves a biological macromolecule must contain a recitation of known structure."

Our current knowledge depicts living matter as incredibly complex (almost like a black-box) and therefore not describable either completely or accurately as required under the written description requirement. Inventions involving biological materials such as cell lines, cloning vectors, hybridomas, plasmids, microorganisms, etc., are sometimes impossible to describe adequately in words and reproducing them is not always a completely repeatable process. This problem is addressed by depositing appropriate biological materials with a recognized repository which provides permanence and availability to other researchers on demand (Berns, et al, 1996). This removes any uncertainty regarding the precise characterization of the material, such as a microorganism or cell line claimed in the invention, while ensuring that others will be able to practice the invention completely. In cases where the deposit requirement applies to higher-life forms, such as transgenic animals, the requirements may be satisfactorily fulfilled if a deposit of the lowest common denominator of a higher life form, e.g., the sperm, egg, fertilized egg, embryo, etc. is deposited. As on June 03, 2011, 75 Contracting Parties had signed the Budapest Treaty on the International Recognition of the

<sup>26</sup> If the profiled PHOSITA is generally expected to perform complex experimental tasks, then such tasks will not be considered as "undue".

<sup>27</sup> Available at http://law.justia.com/cases/federal/appellate-courts/F3/448/1357/637048/.

Deposit of Micro-organisms for the Purposes of Patent Procedure28 (done at Budapest on April 28, 1977, and amended on September 26, 1980). The Treaty specifically requires the following:

Contracting States which allow or require the deposit of microorganisms for the purposes of patent procedure shall recognize, for such purposes, the deposit of a microorganism with any international depositary authority. Such recognition shall include the recognition of the fact and date of the deposit as indicated by the international depositary authority as well as the recognition of the fact that what is furnished as a sample is a sample of the deposited microorganism. (Article 3(1)(a)) As far as matters regulated in this Treaty and the Regulations are concerned, no Contracting State may require compliance with requirements different from or additional to those which are provided in this Treaty and the Regulations. (Article 3(2))

The satisfaction of the specification requirement is largely a procedural matter and depends on the skill of the patent counsel and the inventor's cooperation in preparing the patent application. On the other hand, fulfilling the requirements of utility, novelty, and nonobviousness depend more on the substantive merits of the invention itself.

#### **6. Patent related treaties & agreements**

Grant of patents and their enforcement, if infringed, rests with national governments. Differing national economic and geopolitical needs have resulted in wide differences among national patent systems that, at times, have led to odious disharmonies in patent enforcement and flow of trade and commerce. There has been a long-felt need for harmonized patent laws, especially by those who need their inventions protected concurrently in major world markets. Since the 1880s, limited harmonization among groups of nations, mainly related to procedural matters, has been achieved through various international treaties. The important ones are: (1) Paris Convention for the Protection of Industrial Property (1883),29 (2) Patent Cooperation Treaty (1970),30 (3) Agreement on Trade-Related Aspects of Intellectual Property Rights (TRIPS) (The Agreement is Annex 1C of the Marrakesh Agreement Establishing the World Trade Organization, signed in Marrakesh, Morocco on 15 April 1994),31 and (4) The Trilateral Cooperation (1983)32 agreement among the patent offices of the United States, Europe, and Japan.

#### **6.1 The Paris Convention**

The Paris Convention (1883), now administered by the World Intellectual Property Organization (WIPO)33, has shaped the patent laws of various countries, especially those of its member States called Contracting Parties. It was the first important international treaty designed to help people of one country obtain protection in another for their intellectual

<sup>28</sup> Available at http://www.wipo.int/treaties/en/registration/budapest. 29 Available at http://www.wipo.int/treaties/en/ip/paris/trtdocs\_wo020.html.

<sup>30</sup> Available at http://www.wipo.int/pct/en/texts/pdf/pct.pdf. 31 Available at http://www.wto.org/english/docs\_e/legal\_e/27-trips.pdf. 32 Website: http://www.trilateral.net/index.html.

<sup>33</sup> WIPO "is responsible for the promotion of the protection of intellectual property throughout the world through cooperation among States". Website: http://www.wipo.int//portal/index.html.en.

Deposit of Micro-organisms for the Purposes of Patent Procedure28 (done at Budapest on April 28, 1977, and amended on September 26, 1980). The Treaty specifically requires the

The satisfaction of the specification requirement is largely a procedural matter and depends on the skill of the patent counsel and the inventor's cooperation in preparing the patent application. On the other hand, fulfilling the requirements of utility, novelty, and non-

Grant of patents and their enforcement, if infringed, rests with national governments. Differing national economic and geopolitical needs have resulted in wide differences among national patent systems that, at times, have led to odious disharmonies in patent enforcement and flow of trade and commerce. There has been a long-felt need for harmonized patent laws, especially by those who need their inventions protected concurrently in major world markets. Since the 1880s, limited harmonization among groups of nations, mainly related to procedural matters, has been achieved through various international treaties. The important ones are: (1) Paris Convention for the Protection of Industrial Property (1883),29 (2) Patent Cooperation Treaty (1970),30 (3) Agreement on Trade-Related Aspects of Intellectual Property Rights (TRIPS) (The Agreement is Annex 1C of the Marrakesh Agreement Establishing the World Trade Organization, signed in Marrakesh, Morocco on 15 April 1994),31 and (4) The Trilateral Cooperation (1983)32 agreement among

The Paris Convention (1883), now administered by the World Intellectual Property Organization (WIPO)33, has shaped the patent laws of various countries, especially those of its member States called Contracting Parties. It was the first important international treaty designed to help people of one country obtain protection in another for their intellectual

33 WIPO "is responsible for the promotion of the protection of intellectual property throughout the world through cooperation among States". Website: http://www.wipo.int//portal/index.html.en.

obviousness depend more on the substantive merits of the invention itself.

**6. Patent related treaties & agreements** 

the patent offices of the United States, Europe, and Japan.

28 Available at http://www.wipo.int/treaties/en/registration/budapest. 29 Available at http://www.wipo.int/treaties/en/ip/paris/trtdocs\_wo020.html.

30 Available at http://www.wipo.int/pct/en/texts/pdf/pct.pdf. 31 Available at http://www.wto.org/english/docs\_e/legal\_e/27-trips.pdf. 32 Website: http://www.trilateral.net/index.html.

**6.1 The Paris Convention** 

Contracting States which allow or require the deposit of microorganisms for the purposes of patent procedure shall recognize, for such purposes, the deposit of a microorganism with any international depositary authority. Such recognition shall include the recognition of the fact and date of the deposit as indicated by the international depositary authority as well as the recognition of the fact that what is furnished as a sample is a sample of the deposited microorganism. (Article 3(1)(a)) As far as matters regulated in this Treaty and the Regulations are concerned, no Contracting State may require compliance with requirements different from or additional to those which are provided in this Treaty and the Regulations. (Article 3(2))

following:

creations in the form of inventions (patents), trademarks, and industrial designs. As on July 15, 2011, there were 173 member States34. The Convention does not allow Contracting Parties to discriminate between their own nationals and nationals of other Contracting Parties as regards the protection of industrial property. *Inter alia*, the Convention lays down the common basic structure for patent protection to which the Contracting Parties are bound. This basic structure does not unduly trespass on the sovereign rights of Contracting Parties or compromise their national interests. In fact, Article 19, of the Convention states that Contracting Parties "reserve the right to make separately between themselves special agreements for the protection of industrial property, in so far as these agreements do not contravene the provisions of this Convention." Because of this, patent laws of respective member States share a substantial common core.

The Paris Convention forms the foundation for two other important treaties related to patents—the Patent Cooperation Treaty (1970) and the Agreement on Trade-Related Aspects of Intellectual Property Rights (TRIPS) (1995).

#### **6.2 Patent Cooperation Treaty (PCT)**

The *Patent Cooperation Treaty* (PCT), administered by WIPO, became operational on June 1, 1978. As of September 23, 2011, the PCT had 144 signatories35. While the Paris Convention provides a means of access into different countries' patent systems, a patent application once filed, must be prosecuted through each national patent system. Under the PCT, patent applicants from Contracting States enjoy a relatively simple way of commencing patent applications in a number of countries simultaneously. This provision has since been encoded in the patent laws of most Contracting States.

The PCT provides a centralized mechanism for filing patent applications, prior art search and preliminary examination of the patent application; it does *not* grant patents. PCT Contracting States are bound by Chapter II of the PCT relating to the international preliminary examination of patent applications. An applicant can designate specific countries or regional conventions for grant of patent by filing an international patent application in the appropriate receiving office. After an international search report and a non-binding preliminary opinion on patentability has been provided, the applicant must still apply separately and individually to each jurisdiction where patent protection is required. While the search and the preliminary opinion might reduce subsequent searchrelated workload of national patent offices examining the patent application, the main and substantial workload still belongs to the national patent office. Every biotechnologist should become familiar with the process of filing a patent application under the PCT.

#### **6.3 TRIPS**

Of all the treaties in force, TRIPS is the most ambitious. Ratification of TRIPS is a prerequisite for a country to become a member of the World Trade Organization (WTO)36. As on October 4, 2011, WTO had 153 members. TRIPS entered into force on January 1, 1996, and covers various forms of intellectual property rights, including patents. It introduced

 34 Visit http://www.wipo.int/export/sites/www/treaties/en/documents/pdf/paris.pdf for updates.

<sup>35</sup> Visit http://www.wipo.int/pct/guide/en/gdvol1/annexes/annexa/ax\_a.pdf for updates.

<sup>36</sup> Web site: http://www.wto.org/.

intellectual property law into the international trading system for the first time. It was negotiated at the end of the Uruguay Round of the General Agreement on Tariffs and Trade (GATT) in 1994. TRIPS nudged signatory countries towards a level of uniformity, which most are still struggling to cope with even though it was sweetened with some concessions for developing and underdeveloped countries. For example, art. 1.1 leaves member states "free to determine the appropriate method of implementing the provisions of this Agreement within their own legal system and practice" and a November 2005 decision of the Council for TRIPS allowed least-developed country members to postpone implementation of many TRIPS obligations until 2013.37 The difficulties faced by developing countries are not just due to their inferior stages of technological advancement but also due to social, administrative, infrastructural, and other costs incurred in implementing TRIPS. This is particularly visible in the case of pharmaceutical products.38

Under the TRIPS Agreement, member countries are required to make patents available "for any invention, whether products or processes, in all fields of technology" without discrimination, subject to certain legal requirements being met. These requirements are that they must fulfill the member country's legislated criteria for novelty, inventiveness, and industrial applicability. Further, once patent rights are granted, the owner of the patent should be able to enjoy those rights in the member country without discrimination as to the place of invention and whether products are imported or locally produced. The above is subject to three exceptions: (1) the invention should not be contrary to *ordre public* or morality; (2) inventions related to diagnostic, therapeutic and surgical methods for the treatment of humans or animals may be excluded from being patented; and (3) inventions related to plants and animals other than micro-organisms and essentially biological processes for the production of plants or animals other than non-biological and microbiological processes may be excluded. However, when such exclusions are made in the patent system for plant varieties, an effective *sui generis* system of protection must be provided. Subsequent to the Doha Round which took several years to negotiate, TRIPS permits countries to issue compulsory licenses to meet the health needs of nations unable to produce locally needed medicines.39 This, however, means little to countries which lack the ability to manufacture pharmaceuticals locally.

Several TRIPS articles remain open to wide interpretation to allow each member country freedom to tailor its patent system according to its domestic needs, present state of development, and growth potential. For example, while TRIPS lists an "inventive step" as one of the requirements for patentable subject matter (art. 27(1)), it does not define the term. Likewise it defines the scope of a patent in terms of the nature of the rights conferred (art. 28), but does not set out the breadth of the technological terrain a patent must cover. This allows member states to supply their own definitions of "inventive step" and determine the scope of patent protection.

<sup>37</sup> For developing countries, the patent standards (articles 27-34) of the TRIPS Agreement became generally operational on January 1, 2000. Those developing countries that did not allow product patents on pharmaceutical and agricultural chemical products were given a grace period of five years to cover them, subject to a "mail box" provision for patents arising in the meantime.

<sup>38</sup> See, *e.g.*, Janice M. Mueller, *Taking TRIPS to India—Novartis, Patent Law, and Access to Medicines*, 356 New England Journal of Medicine, 541, 541 (2007).

<sup>39</sup> Declaration on the TRIPS Agreement and Public Health (adopted on November 14, 2001). Available at http://docsonline.wto.org/DDFDocuments/t/WT/Min01/DEC2.doc.

#### **6.4 The Trilateral Cooperation**

366 Innovations in Biotechnology

intellectual property law into the international trading system for the first time. It was negotiated at the end of the Uruguay Round of the General Agreement on Tariffs and Trade (GATT) in 1994. TRIPS nudged signatory countries towards a level of uniformity, which most are still struggling to cope with even though it was sweetened with some concessions for developing and underdeveloped countries. For example, art. 1.1 leaves member states "free to determine the appropriate method of implementing the provisions of this Agreement within their own legal system and practice" and a November 2005 decision of the Council for TRIPS allowed least-developed country members to postpone implementation of many TRIPS obligations until 2013.37 The difficulties faced by developing countries are not just due to their inferior stages of technological advancement but also due to social, administrative, infrastructural, and other costs incurred in implementing TRIPS. This is particularly visible in

Under the TRIPS Agreement, member countries are required to make patents available "for any invention, whether products or processes, in all fields of technology" without discrimination, subject to certain legal requirements being met. These requirements are that they must fulfill the member country's legislated criteria for novelty, inventiveness, and industrial applicability. Further, once patent rights are granted, the owner of the patent should be able to enjoy those rights in the member country without discrimination as to the place of invention and whether products are imported or locally produced. The above is subject to three exceptions: (1) the invention should not be contrary to *ordre public* or morality; (2) inventions related to diagnostic, therapeutic and surgical methods for the treatment of humans or animals may be excluded from being patented; and (3) inventions related to plants and animals other than micro-organisms and essentially biological processes for the production of plants or animals other than non-biological and microbiological processes may be excluded. However, when such exclusions are made in the patent system for plant varieties, an effective *sui generis* system of protection must be provided. Subsequent to the Doha Round which took several years to negotiate, TRIPS permits countries to issue compulsory licenses to meet the health needs of nations unable to produce locally needed medicines.39 This, however, means little to countries which lack the

Several TRIPS articles remain open to wide interpretation to allow each member country freedom to tailor its patent system according to its domestic needs, present state of development, and growth potential. For example, while TRIPS lists an "inventive step" as one of the requirements for patentable subject matter (art. 27(1)), it does not define the term. Likewise it defines the scope of a patent in terms of the nature of the rights conferred (art. 28), but does not set out the breadth of the technological terrain a patent must cover. This allows member states to supply their own definitions of "inventive step" and determine the

37 For developing countries, the patent standards (articles 27-34) of the TRIPS Agreement became generally operational on January 1, 2000. Those developing countries that did not allow product patents on pharmaceutical and agricultural chemical products were given a grace period of five years to cover

38 See, *e.g.*, Janice M. Mueller, *Taking TRIPS to India—Novartis, Patent Law, and Access to Medicines*, 356

39 Declaration on the TRIPS Agreement and Public Health (adopted on November 14, 2001). Available at

them, subject to a "mail box" provision for patents arising in the meantime.

http://docsonline.wto.org/DDFDocuments/t/WT/Min01/DEC2.doc.

the case of pharmaceutical products.38

ability to manufacture pharmaceuticals locally.

New England Journal of Medicine, 541, 541 (2007).

scope of patent protection.

The Trilateral Cooperation was set up in 1983 between the USPTO, the European Patent Office and the Japan Patent Office (collectively known as the Trilateral Offices) to overcome certain problems arising due to a dramatic rise in the number of patent filings in the early 1980s. These Offices process the greater part of all patent applications filed worldwide including PCT applications. Under the Cooperation, the Offices focus on addressing global patent workload challenges, e.g., decreasing pendency and examination backlogs, improving patent quality, and leveraging IT solutions to accelerate processing of patent applications. Through work sharing arrangements the Offices leverage work done earlier by another Office to improve their own search and examination practices. One of their goals is to eventually develop a paperless administration of the patent procedure, the exchange of documents, and electronic filing of applications.

#### **6.5 Hurdles in the path of harmonization**

Attempts to harmonize different national patent systems face major hurdles: the standards to be followed for utility, novelty, and nonobviousness; defining circumstances when research exemption and compulsory licensing are appropriate; setting objective standards for analyzing infringement and award of relief; etc. These issues are dealt with in widely differing ways by different countries. Any debate on global harmonization must also consider alternative mechanisms for encouraging technological innovation, not just the patent system, and account for the fact that different countries are at different stages of transition—from the industrial age to the information age. A recent paper (Reichman and Dreyfuss, 2007) succinctly notes:

[T]he worldwide intellectual property system has entered a brave new scientific epoch, in which experts have only tentative, divergent ideas about how best to treat a daunting array of emerging new technologies. The existing system has become increasingly dysfunctional because it operates with a set of rudimentary working hypothesis that have not kept pace with technical change.

Any attempt to push harmonization beyond TRIPS would require great care. At the least, individual nations must be clear about the patent system that would best serve their interests in the new knowledge economy. The daunting nature of the task becomes evident from the experience of the United States, which after six years of feet-dragging and several aborted attempts at reforming its Patent Act, finally enacted the Leahy-Smith America Invents Act, 201140 on September 16, 2011. It is seen as "a jobs creation bill." The Act, most importantly, changes the earlier "first-to-invent" system to a "first-to-file" system to make it compatible with the rest of the world, raises patentability standards, makes injunctions and damages harder to obtain, provides new options for challenging bad patents, provides enhanced funding of PTO operations, provides for expedited examination of patent applications (for a fee), etc. Full implementation of the new law will take several months. A lack of political will to sink differences to bring about change was evident throughout.

<sup>40</sup> Available at

http://frwebgate.access.gpo.gov/cgi-bin/getdoc.cgi?dbname=112\_cong\_bills&docid=f:s23es.txt.pdf.

#### **7. Societal impact of biotechnology patents**

Since the 1970s the spotlight has shifted from technological advances of the industrial revolution (driven by Newton's laws of motion and Maxwell's laws of electromagnetism) to advances in biotechnology (driven by molecular biology and biochemistry). Towards the end of the twentieth century, conventional wisdom asserted that while that century belonged to physics and chemistry, which led to huge industrialization and consequent megacities, the twenty-first century will belong to biology and associated technologies. Its impact on society is expected to be phenomenal, affecting every inhabitant on our planet in an intimate way. The nature of the impact will crucially depend on how society accepts or rejects new technical innovations in biotechnology (Smith, 2009). Even though, since the 1980s, biotechnology has been recognized and welcomed as a highly promising strategic technology by many industrialized nations (Bera, 2009c), it has not resulted in automatic acceptance by society. The rate at which R&D results can be assimilated will depend less on scientific or technical considerations but more on such factors as availability of venture capital, the ability to acquire and protect patents, marketing skills, the efficacy and costeffectiveness of new technologies, and possibly of far greater importance, public perception and acceptance. Perhaps no other industry is as heavily dependent on patents as the biotechnology industry and on public trust.

The emergence of the modern biotechnology industry in the 1970s as an intermediate sector between academic research institutions and Big Pharma was novel. Academic researchers played an important role in the founding of many biotechnology companies; some participated in both worlds, some turned into entrepreneurs. University-industry collaboration became a critical factor in commercial success as did the foresight of some venture capitalists who were willing and able to support inexperienced companies entering a market with a seven- to ten-year product development cycle. Indeed, without patent rights in areas such as isolation and purification of proteins, DNA sequences, monoclonal antibodies, transgenic organisms and gene expression systems, etc., many biotechnology companies could not exist (Bera, 2009b; Williams, 2005).

#### **7.1 New technologies spark patenting debates**

Historically, the birth of each new technology tends to spark a patenting debate. In the early 20th century the debate was whether agricultural inventions could be patented on the grounds that agriculture was not an industry. In the 1970s, it was argued that pharmaceutical patents were unethical. In the 1980s, the biotechnology industry faced hostility over "patenting of life". Even now, bioethical, social, and legal questions related to biotechnology patents are far from being over, as are issues related to intellectual property, scientific integrity, and conflicts of interest in research. For a satisfactory resolution, the debate must involve experts from diverse fields: science, engineering, theology, and philosophy. Finally, since the 1990s, the computer revolution and the Internet have produced many controversies related to software and business method patents (Poynder, 2000). An apparently persuasive argument against biotechnology patents is the field's rapid pace of development. For example, a few decades ago, finding a gene may have taken ten years, but now one can be found within seconds using a computer search and gene maps (Demaine & Fellmeth, 2002). If invention is inevitable, does it merit reward of exclusivity? If yes, for how long? A long period may spur innovation but also limit the spread of new and useful products and processes and make them more expensive. On the other hand, rapid

Since the 1970s the spotlight has shifted from technological advances of the industrial revolution (driven by Newton's laws of motion and Maxwell's laws of electromagnetism) to advances in biotechnology (driven by molecular biology and biochemistry). Towards the end of the twentieth century, conventional wisdom asserted that while that century belonged to physics and chemistry, which led to huge industrialization and consequent megacities, the twenty-first century will belong to biology and associated technologies. Its impact on society is expected to be phenomenal, affecting every inhabitant on our planet in an intimate way. The nature of the impact will crucially depend on how society accepts or rejects new technical innovations in biotechnology (Smith, 2009). Even though, since the 1980s, biotechnology has been recognized and welcomed as a highly promising strategic technology by many industrialized nations (Bera, 2009c), it has not resulted in automatic acceptance by society. The rate at which R&D results can be assimilated will depend less on scientific or technical considerations but more on such factors as availability of venture capital, the ability to acquire and protect patents, marketing skills, the efficacy and costeffectiveness of new technologies, and possibly of far greater importance, public perception and acceptance. Perhaps no other industry is as heavily dependent on patents as the

The emergence of the modern biotechnology industry in the 1970s as an intermediate sector between academic research institutions and Big Pharma was novel. Academic researchers played an important role in the founding of many biotechnology companies; some participated in both worlds, some turned into entrepreneurs. University-industry collaboration became a critical factor in commercial success as did the foresight of some venture capitalists who were willing and able to support inexperienced companies entering a market with a seven- to ten-year product development cycle. Indeed, without patent rights in areas such as isolation and purification of proteins, DNA sequences, monoclonal antibodies, transgenic organisms and gene expression systems, etc., many biotechnology

Historically, the birth of each new technology tends to spark a patenting debate. In the early 20th century the debate was whether agricultural inventions could be patented on the grounds that agriculture was not an industry. In the 1970s, it was argued that pharmaceutical patents were unethical. In the 1980s, the biotechnology industry faced hostility over "patenting of life". Even now, bioethical, social, and legal questions related to biotechnology patents are far from being over, as are issues related to intellectual property, scientific integrity, and conflicts of interest in research. For a satisfactory resolution, the debate must involve experts from diverse fields: science, engineering, theology, and philosophy. Finally, since the 1990s, the computer revolution and the Internet have produced many controversies related to software and business method patents (Poynder, 2000). An apparently persuasive argument against biotechnology patents is the field's rapid pace of development. For example, a few decades ago, finding a gene may have taken ten years, but now one can be found within seconds using a computer search and gene maps (Demaine & Fellmeth, 2002). If invention is inevitable, does it merit reward of exclusivity? If yes, for how long? A long period may spur innovation but also limit the spread of new and useful products and processes and make them more expensive. On the other hand, rapid

**7. Societal impact of biotechnology patents** 

biotechnology industry and on public trust.

companies could not exist (Bera, 2009b; Williams, 2005).

**7.1 New technologies spark patenting debates** 

discovery of genes does not imply rapid availability of useful and safe applications based on those genes. Those applications come from creative geniuses. Should they not be rewarded with patents? For inventions, such as vaccines for public health, the balancing act is never easy, given the enormous R&D costs of developing them and the crucial role of Nobel class researchers who make them possible. As Todd Dickinson (former director of the USPTO) once remarked, "there are so many chemicals in the human body that, if we ruled them all off limits to patenting, we would rule out an extraordinary number of valuable and important inventions. … Without the funding and incentives that are provided by the patent system, research into the basis of genetic diseases and the development of tools for the diagnosis and treatment of such diseases would be significantly curtailed." (Dickinson, 2000)

Another objection is that the "current model rewards particular kinds of creative effort, namely those which result in commercial gain. It is therefore likely to hinder innovation of products that have limited market value, but which have huge social benefit."41 This flawed argument overlooks the obvious fact that intellectual property laws *were* meant to encourage commercial gain. There are other laws which encourage social benefit and one does not criticize those laws for hindering innovation that lead to commercial gains. The correct approach, if providing social benefit which have limited market value is the objective, is to elect governments that will act more enthusiastically in providing social benefits (of course, the government will have to increase taxes to do so), encourage the general population to contribute to philanthropic activity by donating time and service to community activities, including creating intellectual property. There are no laws against such philanthropic activities, but there is a huge lack of enthusiasm on the part of the general population to help itself. That same population works more energetically when it gets a share of "commercial gains" in terms of employment opportunities and wages. Commenting on the allegation that the global intellectual property regime denies poor people access to drugs, Alasdair Poore said, "Without an effective patent system, who would have made the necessary investment to discover and manufacture those drugs? It's politics and economics that block access to drugs for the world's poor, not the IP system." (Prowse, 2009)

On closer inspection, one finds that the broadest debates concern ethical and societal aspects of patenting genetic materials, the perceived rights of indigenous communities that have shaped their environment and its organisms and thus the genetic resource embodied therein, and the manner in which the bioindustry prospects (or allegedly pirates) biological resources of poor countries and commercializes the products it derives through patents (Koopman, 2003). At another level, while patenting of biotechnology inventions is being criticized, it is really the science behind it that the opponents seem to be against.

#### **7.2 Knowledge is commercial power**

An important, although not the only, measure of a technology's success is its embodiment in products and processes that generate a profitable commercial market through public acceptance. Public acceptance is a factor *only* if there is a supplier willing to assume business risks and enter the market. One might then assume that if a suite of patentable products and

<sup>41</sup> See, e.g., Who Owns Science? The Manchester Manifesto, Institute for Science, Ethics and Innovation, The University of Manchester, 2009. Available at

http://www.isei.manchester.ac.uk/TheManchesterManifesto.pdf.

processes, paid for out of the public purse, were owned by the government and made available free or at a nominal cost for commercial exploitation, business risks would be lowered. That such is not the case was the genesis of the Bayh-Dole Act42 of 1980 in the United States. What the government found was that discoveries made in the universities with federal funds were grossly underutilized43 because government policy required that it take title to all such inventions and license them non-exclusively. The vast majority of university discoveries, as expected, were early stage discoveries that required substantial additional investment to turn them into a marketable product. It was estimated in 2002 that a "dollar's worth of academic invention or discovery require[d] upwards of \$10,000 of private capital to bring to market."44 (The government funds the 'inspiration' while the private sector funds the 'perspiration'!) "New drug development costs have risen from \$0.8 billion (1997) to an expected \$1.9 billion (2013)."45 Without the protection of an exclusive license, companies were reluctant to invest huge sums when the resulting products could easily be appropriated by competitors.46

The Bayh-Dole Act was a bold, against-the-grain, initiative meant to rejuvenate the U.S. economy. Under Bayh-Dole, the government relinquished its intellectual property rights on the outputs of federally funded research and permitted universities and small businesses to acquire title to inventions created with federal funds. It also allowed exclusive licensing of patents thus acquired, to industry since, without it, companies were wary of investing in the further development of university developed technologies. In addition, descriptions of inventions were given legal protection from public dissemination and from requests under the *Freedom of Information Act*47 for a reasonable period to enable patent applications to be filed. In return, the government retained a royalty-free, non-exclusive license to practice the patented inventions coming out of federally funded research throughout the world (including use by government contractors) and held 'march-in rights', which allowed the government to take back the title if the patent owner failed to commercialize the invention. However, the exercise of march-in rights was made substantially difficult and appealable in courts. The march-in rights were basically introduced to prevent companies from licensing university patents with the sole intention of blocking rival companies from doing so. The

<sup>42</sup> University and Small Business Patent Procedure Act of 1980, (Pub. L. 96-517), §6(a), Dec. 12, 1980, 94 Stat. 3018 (35 U.S.C. 200 et seq.). Also known as the Bayh-Dole Act of 1980, it was given effect from July 1, 1981, "to use the patent system to promote the utilization of inventions arising from federally funded research or development."

<sup>43</sup> In 1980, the Federal Government held title to approximately 28,000 patents. Fewer than 5% of these were licensed to industry for development of commercial products. (See The Bayh-Dole Act: A Guide to the Law and Implementing Regulations, Council on Governmental Relations, October 1999, p. 2. Available at

http://www.cogr.edu/docs/Bayh\_Dole.pdf.)

<sup>44</sup> Innovation's golden goose, The Economist, December 14, 2002, p. 3.

<sup>45</sup> Wai Lang Chu, CRO's drug R&D contribution never been more significant, 25 September 2006. Available at http://www.outsourcing-pharma.com/Preclinical-Research/CRO-s-drug-R-Dcontribution-never-been-more-significant.

<sup>46</sup> The Bayh-Dole Act: Important to our Past, Vital to our Future, 2006. Sense of Congress resolution passed by the U.S. House of Representatives on December 6, 2006. Available at

http://www.autm.net/Content/NavigationMenu/About/PublicPolicy/BDTalkPts031407.pdf.

<sup>47</sup> Available at http://usgovinfo.about.com/library/foia/blfoiacode.htm.

processes, paid for out of the public purse, were owned by the government and made available free or at a nominal cost for commercial exploitation, business risks would be lowered. That such is not the case was the genesis of the Bayh-Dole Act42 of 1980 in the United States. What the government found was that discoveries made in the universities with federal funds were grossly underutilized43 because government policy required that it take title to all such inventions and license them non-exclusively. The vast majority of university discoveries, as expected, were early stage discoveries that required substantial additional investment to turn them into a marketable product. It was estimated in 2002 that a "dollar's worth of academic invention or discovery require[d] upwards of \$10,000 of private capital to bring to market."44 (The government funds the 'inspiration' while the private sector funds the 'perspiration'!) "New drug development costs have risen from \$0.8 billion (1997) to an expected \$1.9 billion (2013)."45 Without the protection of an exclusive license, companies were reluctant to invest huge sums when the resulting products could

The Bayh-Dole Act was a bold, against-the-grain, initiative meant to rejuvenate the U.S. economy. Under Bayh-Dole, the government relinquished its intellectual property rights on the outputs of federally funded research and permitted universities and small businesses to acquire title to inventions created with federal funds. It also allowed exclusive licensing of patents thus acquired, to industry since, without it, companies were wary of investing in the further development of university developed technologies. In addition, descriptions of inventions were given legal protection from public dissemination and from requests under the *Freedom of Information Act*47 for a reasonable period to enable patent applications to be filed. In return, the government retained a royalty-free, non-exclusive license to practice the patented inventions coming out of federally funded research throughout the world (including use by government contractors) and held 'march-in rights', which allowed the government to take back the title if the patent owner failed to commercialize the invention. However, the exercise of march-in rights was made substantially difficult and appealable in courts. The march-in rights were basically introduced to prevent companies from licensing university patents with the sole intention of blocking rival companies from doing so. The

42 University and Small Business Patent Procedure Act of 1980, (Pub. L. 96-517), §6(a), Dec. 12, 1980, 94 Stat. 3018 (35 U.S.C. 200 et seq.). Also known as the Bayh-Dole Act of 1980, it was given effect from July 1, 1981, "to use the patent system to promote the utilization of inventions arising from federally funded

43 In 1980, the Federal Government held title to approximately 28,000 patents. Fewer than 5% of these were licensed to industry for development of commercial products. (See The Bayh-Dole Act: A Guide to the Law and Implementing Regulations, Council on Governmental Relations, October 1999, p. 2.

45 Wai Lang Chu, CRO's drug R&D contribution never been more significant, 25 September 2006. Available at http://www.outsourcing-pharma.com/Preclinical-Research/CRO-s-drug-R-D-

46 The Bayh-Dole Act: Important to our Past, Vital to our Future, 2006. Sense of Congress resolution

http://www.autm.net/Content/NavigationMenu/About/PublicPolicy/BDTalkPts031407.pdf.

easily be appropriated by competitors.46

research or development."

http://www.cogr.edu/docs/Bayh\_Dole.pdf.)

contribution-never-been-more-significant.

44 Innovation's golden goose, The Economist, December 14, 2002, p. 3.

passed by the U.S. House of Representatives on December 6, 2006. Available at

47 Available at http://usgovinfo.about.com/library/foia/blfoiacode.htm.

Available at

rights were meant to ensure fair competition and to meet the needs of U.S. citizens; they were not meant for the government to set prices, as some have tried to claim.48

So, post-Bayh-Dole, we now witness the hitherto unimagined situation where innovations, already paid for by the public, can be brought to the market only if those innovations are privatized and resold to the public via patents acquired by commercial entities. Otherwise certain markets will likely vanish on their own because the risks are too high! The biotechnology industry, through university-industry collaboration, has shown that knowledge is a phenomenal commercial power. The emulation of the Act by other countries, although common now, will not necessarily have the same impact that has been visible in the U.S. because of dissimilar national circumstances, or the absence of world-class research universities.

Patent laws never anticipated that together "blue sky" research *and* living matter would play such a fundamental role in late 20th century commerce with such speed, and economists and policy makers never imagined the deep post-1980 university-industry (or generally, public-private) collaborations that rapidly gained momentum in the biotechnology sector following three signal events in 1980 in the United States: the Bayh-Dole Act, grant of the Cohen-Boyer patent, and the Chakrabarty court decision (Bera, 2009d). These rapid changes have bewildered lawmakers, patent offices, the judiciary, and relevant enforcement agencies. To older generations it is sacrilege that "even the pure quest for knowledge is subverted by the need for profit."49 To the new generation it is the welcome emergence of a new and refreshing paradigm where pure knowledge is rapidly converted to applications to serve consumers through conventional market mechanisms of demand and supply. The future may see the emergence of other market mechanisms, whose advent no economist will likely anticipate, because they will occur to accommodate needs triggered by innovation alongside need inspired innovations.

The writing on the wall is clear; the times are changing, and so must the way we teach, create, use, and protect knowledge and the innovations they spawn. Initially, important chunks of that knowledge in biotechnology coming from universities will be tacit and scarce, hence university-industry collaboration will be crucial for technology transfer and commercial success. The relative youth of the biotechnology industry and its dependence on scientific breakthroughs means that star scientists—their accessibility, location, motivation to collaborate at the bench-science level with scientists in industry in converting basic scientific knowledge into commercially viable products and processes—will be crucial in determining the pace of diffusion of tacit scientific knowledge (Zucker & Darby, 1996). To remain relevant in an economically global world, the social role of universities and government research laboratories must change as must our understanding of morality, ethics, and citizenship.

It is mainly due to the university-industry collaboration example set by the United States that universities elsewhere are now expected to transform themselves into engines of economic growth, rather than remain as not-for-profit ivory towers. This is an enormous social transformation, and an enormous opportunity for universities to help the world settle down in the new era of a knowledge-intensive global economy. In this world, university-

<sup>48</sup> Statement of Senator Birch Bayh to the National Institute's of Health, May 25, 2004. Available at http://www.ott.nih.gov/policy/meeting/Senator-Birch-Bayh.pdf.

<sup>49</sup> The quote is from John Sulston, How science is shackled by intellectual property, The Guardian, 26 November 2009.

industry collaborative research is a natural means of providing continuing education to knowledge workers throughout their professionally productive life. It is also a natural means of mutual technology transfers between academic researchers and applied industry researchers, especially of tacit knowledge. Donald Kennedy of Stanford University was spot on when he said, "Technology transfer is the movement of ideas in people."50 This movement in biotechnology must frequently happen under the protective cover of patents. One of the outstanding examples of technology transfer between university and industry is the licensing policy adopted by Stanford University with respect to the Cohen-Boyer patents51 (Bera, 2009a). No doubt, other technology transfer models to fulfill emerging needs will evolve as the biotechnology sector matures.

Modern science-based industries (SBIs) critically depend on monetary funds, star scientists (human capital), and protected intellectual property (intellectual capital). Where and when star scientists publish also has a determining effect on the commercial adoption of new technologies. Geographically, SBIs tend to nucleate in close proximity of universities hosting a star group of scientists active in the relevant science, as it greatly improves mutual accessibility of both people and research facilities. This has generally been the case for biotechnology, especially in the United States (Zucker & Darby, 1996). Governments must bear this in mind when framing policies for economic and industrial growth and providing infrastructure. Once nucleation is complete and substantial diffusion of tacit knowledge of the stars has taken place, further expansion of the industry can spread to far-off places, especially of manufacturing units and support R&D groups.

#### **7.3 Gene patents (unresolved issues)**

A recent gene patent case, *Association for Molecular Pathology* v. *the USPTO and Myriad Genetics*, No. 10-1406 (Fed. Cir. 2011)52, decided by the United States Court of Appeals for the Federal Circuit, has gained extraordinary attention. In this case, the validity of a series of patents that claim isolated DNA compositions and methods for testing the presence of genetic mutations that are correlated with an increased risk of certain breast and ovarian cancers (the BRCA1 and BRCA2 genes) was challenged. Myriad owns or is the exclusive licensee of these patents. Opponents of the patents argued that the patent claims encompass patent-ineligible subject matter, e.g., products of nature. At issue were such fundamental questions as to whether isolated DNA should be eligible for a patent, and whether the patenting of genes promotes or stifles innovation and development of new diagnostics and therapies. The Court stated expressly that an isolated partial DNA fragment, not just cDNA has a "markedly different structure to native DNA" and so reaffirmed that isolated gene sequences are patentable. Patentability of DNA sequences as diagnostics remains uncertain. Whether the Supreme Court of the United States will entertain an appeal in the Myriad case is not yet known.

Quite independent of how this case eventually ends, patent law will need to revisit the grant of gene patents. We now know that the one-gene-one-protein assumption of yore is no

<sup>50</sup> As quoted in Zucker & Darby, 1996.

<sup>51</sup> Stanford University, which owned the patents, granted non-exclusive licenses to 467 companies and amassed licensing revenues of \$255 million. The patents expired in 1997.

<sup>52</sup> Available at http://www.cafc.uscourts.gov/images/stories/opinions-orders/10-1406.pdf.

industry collaborative research is a natural means of providing continuing education to knowledge workers throughout their professionally productive life. It is also a natural means of mutual technology transfers between academic researchers and applied industry researchers, especially of tacit knowledge. Donald Kennedy of Stanford University was spot on when he said, "Technology transfer is the movement of ideas in people."50 This movement in biotechnology must frequently happen under the protective cover of patents. One of the outstanding examples of technology transfer between university and industry is the licensing policy adopted by Stanford University with respect to the Cohen-Boyer patents51 (Bera, 2009a). No doubt, other technology transfer models to fulfill emerging needs

Modern science-based industries (SBIs) critically depend on monetary funds, star scientists (human capital), and protected intellectual property (intellectual capital). Where and when star scientists publish also has a determining effect on the commercial adoption of new technologies. Geographically, SBIs tend to nucleate in close proximity of universities hosting a star group of scientists active in the relevant science, as it greatly improves mutual accessibility of both people and research facilities. This has generally been the case for biotechnology, especially in the United States (Zucker & Darby, 1996). Governments must bear this in mind when framing policies for economic and industrial growth and providing infrastructure. Once nucleation is complete and substantial diffusion of tacit knowledge of the stars has taken place, further expansion of the industry can spread to far-off places,

A recent gene patent case, *Association for Molecular Pathology* v. *the USPTO and Myriad Genetics*, No. 10-1406 (Fed. Cir. 2011)52, decided by the United States Court of Appeals for the Federal Circuit, has gained extraordinary attention. In this case, the validity of a series of patents that claim isolated DNA compositions and methods for testing the presence of genetic mutations that are correlated with an increased risk of certain breast and ovarian cancers (the BRCA1 and BRCA2 genes) was challenged. Myriad owns or is the exclusive licensee of these patents. Opponents of the patents argued that the patent claims encompass patent-ineligible subject matter, e.g., products of nature. At issue were such fundamental questions as to whether isolated DNA should be eligible for a patent, and whether the patenting of genes promotes or stifles innovation and development of new diagnostics and therapies. The Court stated expressly that an isolated partial DNA fragment, not just cDNA has a "markedly different structure to native DNA" and so reaffirmed that isolated gene sequences are patentable. Patentability of DNA sequences as diagnostics remains uncertain. Whether the Supreme Court of the United States will entertain an appeal in the Myriad case

Quite independent of how this case eventually ends, patent law will need to revisit the grant of gene patents. We now know that the one-gene-one-protein assumption of yore is no

51 Stanford University, which owned the patents, granted non-exclusive licenses to 467 companies and

52 Available at http://www.cafc.uscourts.gov/images/stories/opinions-orders/10-1406.pdf.

amassed licensing revenues of \$255 million. The patents expired in 1997.

will evolve as the biotechnology sector matures.

especially of manufacturing units and support R&D groups.

**7.3 Gene patents (unresolved issues)** 

is not yet known.

50 As quoted in Zucker & Darby, 1996.

longer true. The proteome is larger than the genome—there are more proteins than genes due to alternative splicing of genes and post-translation modifications like glycosylation and phosphorylation. The cause of most disorders and diseases is a combination of genetic and environmental factors and this raises important questions about the adequacy, scope and purpose of patent law in view of rapidly advancing knowledge in biotechnology and related fields. It is amply possible that under present laws, a single gene or a short DNA sequence, if patented, could result in a near monopoly on diagnostic tests and treatments for widespread and serious ailments, such as, diabetes, cancer, multiple sclerosis, and Alzheimer's disease. Can such patents be considered as serving human society if the patent owner cannot pursue all known downstream opportunities and blocks others from pursuing those or new ones? If such patents are inevitable, what steps should be taken to ensure that they do not obstruct others ready to pursue opportunities not pursued by the patent owner. Even otherwise, courts and administrative agencies continue to struggle with issues raised by gene patents and their predecessors—chemical patents—as to when and how patents should be granted on biochemicals in their natural and modified states under existing patent laws.

The fact that creation of transgenic humans is, in principle, possible, inevitably raises questions of human dignity, and moral and ethical issues. There appears to be a general consensus that transgenic humans are not patentable. Yet, no unambiguous definition of a transgenic human exists. Given that the genomic DNA differences between human and chimpanzee is only about 1.2%, the possibility of creating a patentable transgenic talking chimpanzee that can communicate with humans is not a fantasy. Such a chimpanzee might actually be able to speak and be capable of making connections between human words, objects, and even emotions. For the first time we may then be able to establish verbal communication with another species and derive remarkable insights about the animal kingdom. Should this possibility be denied to the human race because the transgenic chimpanzee is also a transgenic human? (Bera, 2009d)

The owner of a gene patent does not "own" any organism containing that gene. Thus, a person whose body contained a patented gene would not infringe the patent. However, if a gene, patented or not, is inserted into a living organism that organism may become patentable and then commercially exploited. Ownership and commercial exploitation of plants and animals, such as buying and selling them, is widely accepted in our society, but not of humans in *today's* world. Finally, animal breeding is not new and has been practiced since virtually the beginning of agriculture. Human breeding through marriage customs is also not new. Clearly, when one discusses moral issues related to patents, it is not the invention that is morally repugnant but its use in certain unintended ways. No one in the patent system—inventors, patent examiners, judges, or even legislatures (representing the people) can anticipate all uses of a particular patent that may eventually turn out to be, on a statistical balance, detrimental or beneficial to society. Any premeditated restrictions on the grant of patents must carefully consider the possibility that such restrictions may undermine the patent law's primary objective of promoting technical innovation.

#### **8. Concerns over biopiracy**

A paradigm shift inevitably entails shifts of power. The rise of biotechnology has sharpened the divide between the science-based industrial nations, and the genetically endowed but less-developed nations whose genetic resources are prospected by the former. The conflicting issues being debated include the proprietary character of natural genetic material and the nature of commercial exploitation of the value added to those materials through R&D. Natural genetic resources abound in developing countries with a tropical climate, e.g., Brazil, Peru, Costa Rica, and India, in the form of gene pools, organisms, and ecosystems. Biotechnologists are obviously interested in these resources and related 'traditional knowledge' held by indigenous communities as inputs to research while the biotechnology industry is interested in prospecting those resources for potential commercial exploitation.

The methodology and approach of traditional knowledge is holistic and applied according to notions of biocentricism (a political or ethical stance which asserts the value of nonhuman life in nature), co-evolution and equality; it does not rely on empirical verification, rather it seeks connections between the physiological characteristics of organisms (visible phenotypic properties) with their spiritual ones. In contrast, modern biotechnology concentrates on biochemical genotypic properties. Nevertheless, traditional knowledge can be a valuable starting point for biotechnologists by indicating to them specific organisms and their known medical usage (Koopman, 2003). The enormous gap in terms of effectiveness and use between products and processes derived from traditional knowledge and from modern biotechnology must be filled by very expensive R&D, which is clearly outside the capabilities of indigenous communities. These are uncontested facts.

The biopiracy debate then essentially revolves around ethical and societal values as viewed from two widely different cultures over the patenting of genetic material whose natural inputs were prospected in and transported from indigenous communities on the basis of their traditional knowledge, with next to nothing in return in terms of acknowledgement or affordable products and processes or sharing of R&D knowledge. In short, the indigenous communities see this as blatant biopiracy. This is a clash between two cultures—of shared community rights against privately held individual rights. Indigenous communities seldom recognize the concepts of individual ownership, exclusion and competition that underlie the Western concepts of property law regimes.

Such irreconcilable differences have found palatable compromises in the form of the Convention on Biological Diversity53 and TRIPS, where each culture makes concessions to the other. Countries providing access to genetic resources or traditional knowledge are permitted, and some have implemented, *sui generis* systems where they provide access on certain conditions, such as, getting prior informed consent of a national office dealing with such matters, benefit sharing arrangements (e.g., sharing of proceeds derived from commercial exploitation, training in R&D, transfer of technology under 'fair and most favorable terms' consistent with the 'adequate and effective protection of intellectual property rights'), and treating certain violations of the statutes as criminal offenses, in exchange for biological samples and traditional knowledge. In a subtle way, these *sui generis* systems are enforcing "reach through claims" on others for advantages nature has endowed indigenous communities with and the traditional knowledge they have developed long ago. The debate never mentions the tremendous unpatentable scientific knowledge of Newton, Maxwell, Einstein, and others which has been freely bestowed on the world without seeking rents, and which has allowed such dreams as putting a man on the moon possible.

<sup>53</sup> Available at http://www.cbd.int/doc/legal/cbd-en.pdf.

#### **9. Conclusions**

374 Innovations in Biotechnology

conflicting issues being debated include the proprietary character of natural genetic material and the nature of commercial exploitation of the value added to those materials through R&D. Natural genetic resources abound in developing countries with a tropical climate, e.g., Brazil, Peru, Costa Rica, and India, in the form of gene pools, organisms, and ecosystems. Biotechnologists are obviously interested in these resources and related 'traditional knowledge' held by indigenous communities as inputs to research while the biotechnology industry is interested in prospecting those resources for potential commercial exploitation. The methodology and approach of traditional knowledge is holistic and applied according to notions of biocentricism (a political or ethical stance which asserts the value of nonhuman life in nature), co-evolution and equality; it does not rely on empirical verification, rather it seeks connections between the physiological characteristics of organisms (visible phenotypic properties) with their spiritual ones. In contrast, modern biotechnology concentrates on biochemical genotypic properties. Nevertheless, traditional knowledge can be a valuable starting point for biotechnologists by indicating to them specific organisms and their known medical usage (Koopman, 2003). The enormous gap in terms of effectiveness and use between products and processes derived from traditional knowledge and from modern biotechnology must be filled by very expensive R&D, which is clearly

outside the capabilities of indigenous communities. These are uncontested facts.

Western concepts of property law regimes.

53 Available at http://www.cbd.int/doc/legal/cbd-en.pdf.

The biopiracy debate then essentially revolves around ethical and societal values as viewed from two widely different cultures over the patenting of genetic material whose natural inputs were prospected in and transported from indigenous communities on the basis of their traditional knowledge, with next to nothing in return in terms of acknowledgement or affordable products and processes or sharing of R&D knowledge. In short, the indigenous communities see this as blatant biopiracy. This is a clash between two cultures—of shared community rights against privately held individual rights. Indigenous communities seldom recognize the concepts of individual ownership, exclusion and competition that underlie the

Such irreconcilable differences have found palatable compromises in the form of the Convention on Biological Diversity53 and TRIPS, where each culture makes concessions to the other. Countries providing access to genetic resources or traditional knowledge are permitted, and some have implemented, *sui generis* systems where they provide access on certain conditions, such as, getting prior informed consent of a national office dealing with such matters, benefit sharing arrangements (e.g., sharing of proceeds derived from commercial exploitation, training in R&D, transfer of technology under 'fair and most favorable terms' consistent with the 'adequate and effective protection of intellectual property rights'), and treating certain violations of the statutes as criminal offenses, in exchange for biological samples and traditional knowledge. In a subtle way, these *sui generis* systems are enforcing "reach through claims" on others for advantages nature has endowed indigenous communities with and the traditional knowledge they have developed long ago. The debate never mentions the tremendous unpatentable scientific knowledge of Newton, Maxwell, Einstein, and others which has been freely bestowed on the world without seeking

rents, and which has allowed such dreams as putting a man on the moon possible.

Our new understanding of living matter is leading us to uncharted territories. Recombinant DNA technology, transgenic animals, synthetic cells are just the tip of the iceberg. Creation of computer designed, engineered life is no longer science fiction but a potential reality. Will surreptitiously created transgenic humans one day enslave natural humans and rule the world. Can such an event be stalled? What will a world dominated by transgenic humans be like? Will it be more humane than ours? Will they rule more by the head and less by the heart or the other way round? We have no way of knowing.

While patent laws forbid patenting of abstract ideas, thought processes, laws of nature, natural phenomena, and products of nature so as not to stifle advancement of knowledge, the laws do recognize, as they did for Galileo, that certain down-to-earth inventions, conjured through human ingenuity by applying these forbidden things, go beyond philosophical musings and have potential commercial value, because of their utility to humans. In such cases, a *quid pro quo* system that provides limited period monopoly with commercial advantage in the form of patents, in exchange for a full public disclosure of the invention not later than the date of patent grant, encourages further creation of new inventions or improvements over old ones. Patent laws were meant to encourage commercial gain in an equitable manner. There are other laws and practices that encourage material and spiritual contributions to society through raised taxes, philanthropy, free social service, open-source, etc., which are not motivated by commercial gain. Patent laws do not interfere with these other laws and practices. Outside of contractual obligations to, say, his employer, a biotechnologist can choose to patent or not patent. Patent laws do not operate in isolation. Their purpose is to benefit society on the whole so that the positives outweigh the negatives in a statistical sense. There is no denying that countries with a thriving patent system, with all its faults, have advanced technologically more rapidly than all other countries and provided a better quality of life to their citizens. It is hoped that this chapter will help the reader decide when patenting is appropriate in light of other alternatives.

#### **10. References**

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**Part 5** 

**General Biotechnology** 

376 Innovations in Biotechnology

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Dickinson, T. (2000). Gene Patents and Other Genomic Inventions: Hearing before the

IPO (2009). Examination Guidelines for Patent Applications relating to Biotechnological Inventions in the Intellectual Property Office, *IPO*, U.K., (April 2009). Koopman, J. (2003). Biotechnology, Patent Law and Piracy: Mirroring the Interests in

Köhler, G, & Milstein C. (1975). Continuous cultures of fused cells secreting antibody of predefined specificity, *Nature*, Vol. 256, Issue 5517, (07 August 1975), pp. 495-7. Lilly, M.D. (1997). The development of biochemical engineering science in Europe, *Journal of* 

Mullis, K.B. et al. (1986). Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction, *Cold Spring Harbor Symp. Quant. Biol.*, Vol. 51, pp. 263-73. Poynder, R. (2000). Internet sparks patenting controversy, *IP Matters*, (30 April 2001), Available from http://www.richardpoynder.co.uk/internet\_sparks.htm Prowse, P. (2009). Patent profession welcomes Manchester Manifesto on science but slams 'misleading' comments on IP, (November 27, 2009), Retrieved from http://www.cipa.org.uk/pages/press/article?D5C2CBED-894B-488B-ACD2-

Reichman, J.H. & Dreyfuss, R.C. (2007). Harmonization without Consensus: Critical

Schrödinger, E. (1944). What is Life? Cambridge University Press, Cambridge, Retrieved

Williams, A. (2005). The New Innovation: Rethinking Intellectual Property for the ONE Big

Zucker, L.G., & Darby, M.R. (1996) Star scientists and institutional transformation: Patterns

of invention and innovation in the formation of the biotechnology industry, *Proc.* 

from http://whatislife.stanford.edu/LoCo\_files/What-is-Life.pdf Smith, J.E. (2009). *Biotechnology*, (5th edition), Cambridge University Press, Cambridge. Watson, J.D., and Crick, F.H.C. (1953). Molecular structure of nucleic acids, *Nature*, Vol. 171,

Idea, New Paradigm Learning Corporation, (June 2005).

*Natl. Acad. Sci. USA*, Vol. 93, November 1996, pp. 12709-12716.

Reflections on Drafting a Substantive Patent Law Treaty, *Duke Law Journal*, Vol. 57,

*Biotechnology*, Vol. 59, Issues 1-2, (17 December 1997), pp. 11-18.

House of Representative, *106th Congress*, (July 13, 2000), Retrieved from http://commdocs.house.gov/committees/judiciary/hju66043.000/hju66043\_0f.htm Gibson, D.G., *et al*. (2010). Creation of a Bacterial Cell Controlled by a Chemically

Offices. *Intellectual Property Quarterly*, Vol. 3, (2005), pp. 236-266. Darwin, C. (1872). *The Origin of Species*, 6th edition, Modern Library, ISBN 0451625587. Demaine, L.J., & Fellmeth, A.X. (2002). Reinventing the double helix: a novel and

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Synthesized Genome, *Science*, (20 May 2010), pp. 1-12.

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of the Examination Practices of the United States, European and Japanese Patent

nonobvious reconceptualization of the biotechnology patent, *Stanford Law Review*,

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Resources of Life and Culture, *Electronic Journal of Comparative Law*, Vol. 7.5, (December 2003), pp. 1-19, Available from http://www.ejcl.org/ejcl/75/art75-7.html

### **Biotechnology Virtual Labs: Facilitating Laboratory Access Anytime-Anywhere for Classroom Education**

Shyam Diwakar, Krishnashree Achuthan, Prema Nedungadi and Bipin Nair *Amrita Vishwa Vidyapeetham (Amrita University) India* 

#### **1. Introduction**

Biotechnology is becoming more popular and well identified as a mainline industry. Students have shown greater interest in learning the techniques. As a discipline, biotechnology has led to new advancements in many areas. Criminal investigation has changed dramatically thanks to DNA fingerprinting. Significant advances in forensic medicine, anthropology and wildlife management have been noticed in the last few years. Biotechnology has brought out hundreds of medical diagnostic tests that keep the blood safe from infectious diseases such as HIV and also aid detection of other conditions early enough to be successfully treated. Medical kits for diabetes, blood cholesterol and home pregnancy tests are also biotechnology diagnostic products. Industrial biotech applications have led to cleaner processes that produce less waste and use less energy and water in such industrial sectors as chemicals, pulp and paper, textiles, food, energy, and metals and minerals. Laundry detergents produced in many countries contain biotechnology-based enzymes making them nature friendly and safer. Agricultural biotechnology benefits farmers, consumers and the environment by increasing yields and farm income, decreasing pesticide applications and improving soil and water quality, and providing healthful foods for consumers. Biotechnology has created more than 200 new therapies and vaccines, including products to treat cancer, diabetes, HIV/ AIDS and autoimmune disorders.

This rise in application has led to an increased rise in the number of students undertaking University-level biotechnology courses. However, biotechnology education requires an eclectic approach of combining various sub-disciplines. Biology courses and chemistry courses in biotechnology have diversified the approach of the topic. Most common courses that biotechnology degree programs focus at the University level in India consist of cell biology, molecular biology, microbiology, immunology, ecology, statistics and biophysics.

A brief description of the courses will be sketched so a better picture can be understood on the university-level curriculum at most places in India and abroad. Cell biology is a course that focuses on theoretical fundamentals behind the structure, function, and biosynthesis of cellular membranes and organelles; cell growth and oncogenic transformation; transport, receptors, and cell signaling; the cytoskeleton, the extracellular matrix, and cell movements; chromatin structure and RNA synthesis. Molecular biology course covers a detailed analysis of the biochemical mechanisms that control the maintenance, expression, and evolution of prokaryotic and eukaryotic genomes. The topics also include gene regulation, DNA replication, genetic recombination, and mRNA translation. In particular, the logic of experimental design and data analysis is emphasized. Microbiology course introduces students to the principles of infectious agents. Fundamental techniques in microbiological researches, such as sterilization, isolation, morphological observation, and cultivation are usually covered. Immunology courses focus on the mechanisms which govern the immune response. This will usually include the cells, organs and molecules that mediate the innate and adaptive aspects of the immune system as they apply to infection, tumor recognition, autoimmune diseases, immunodeficiency, cancer and hypersensitivity. Population ecology courses introduce students to major concepts in population ecology including topics such as mathematical models of population growth, population viability analysis, habitat fragmentation and meta-populations, dispersal, population harvesting, predation and population cycles, competition, and estimation of population parameters in the field. Biochemistry course explores the roles of essential biological molecules focusing on protein chemistry, while covering lipids and carbohydrates. It provides a systematic and methodical application of general and organic chemistry principles. Students examine the structure of proteins, their function, their binding to other molecules and the methodologies for the purification and characterization of proteins. Enzymes and their kinetics and mechanisms are covered in detail. Metabolic pathways are examined from thermodynamic and regulatory perspectives. A typical course in biochemistry provides the linkage between the inanimate world of chemistry and the living world of biology. Biophysics is a course that usually links to the study of underlying physical phenomenon in biology and their function. Biophysics course usually cover techniques, methods and applications besides molecular structure and function. Topics in biophysics covered will include an introduction to cell and molecular biology, biorheology, Brownian motion, molecular interactions in macromolecules, protein and nucleic acid structure, physics of biopolymers, chemical kinetics, mechanical and adhesive properties of biomolecules, molecular manipulation techniques, cell membrane structure, membrane channels and pumps, molecular motors, neuronal biophysics and related biophysical mechanisms. A very significant yet seemingly unrelated course is biostatistics. A single introductory course in biostatistics involves an emphasis on principles of statistical reasoning, underlying assumptions, and careful interpretation of results. Topics covered include descriptive statistics, graphical displays of data, introduction to probability, expectations and variance of random variables, confidence intervals and tests for means, differences of means, proportions, differences of proportions, chi-square tests for categorical variables, regression and multiple regressions, an introduction to analysis of variance.

Using software technologies for education has become a new trend. Computer-based technologies developed by academic institutions as well as industries worldwide are revolutionizing the educational system. A new field involving the use of virtual reality techniques is becoming the training environment. Through virtual labs, a new interdisciplinary field of science brings together biologists and physicists to tackle this grand challenge through quantitative experiments and models. Using several pro-learning even

transformation; transport, receptors, and cell signaling; the cytoskeleton, the extracellular matrix, and cell movements; chromatin structure and RNA synthesis. Molecular biology course covers a detailed analysis of the biochemical mechanisms that control the maintenance, expression, and evolution of prokaryotic and eukaryotic genomes. The topics also include gene regulation, DNA replication, genetic recombination, and mRNA translation. In particular, the logic of experimental design and data analysis is emphasized. Microbiology course introduces students to the principles of infectious agents. Fundamental techniques in microbiological researches, such as sterilization, isolation, morphological observation, and cultivation are usually covered. Immunology courses focus on the mechanisms which govern the immune response. This will usually include the cells, organs and molecules that mediate the innate and adaptive aspects of the immune system as they apply to infection, tumor recognition, autoimmune diseases, immunodeficiency, cancer and hypersensitivity. Population ecology courses introduce students to major concepts in population ecology including topics such as mathematical models of population growth, population viability analysis, habitat fragmentation and meta-populations, dispersal, population harvesting, predation and population cycles, competition, and estimation of population parameters in the field. Biochemistry course explores the roles of essential biological molecules focusing on protein chemistry, while covering lipids and carbohydrates. It provides a systematic and methodical application of general and organic chemistry principles. Students examine the structure of proteins, their function, their binding to other molecules and the methodologies for the purification and characterization of proteins. Enzymes and their kinetics and mechanisms are covered in detail. Metabolic pathways are examined from thermodynamic and regulatory perspectives. A typical course in biochemistry provides the linkage between the inanimate world of chemistry and the living world of biology. Biophysics is a course that usually links to the study of underlying physical phenomenon in biology and their function. Biophysics course usually cover techniques, methods and applications besides molecular structure and function. Topics in biophysics covered will include an introduction to cell and molecular biology, biorheology, Brownian motion, molecular interactions in macromolecules, protein and nucleic acid structure, physics of biopolymers, chemical kinetics, mechanical and adhesive properties of biomolecules, molecular manipulation techniques, cell membrane structure, membrane channels and pumps, molecular motors, neuronal biophysics and related biophysical mechanisms. A very significant yet seemingly unrelated course is biostatistics. A single introductory course in biostatistics involves an emphasis on principles of statistical reasoning, underlying assumptions, and careful interpretation of results. Topics covered include descriptive statistics, graphical displays of data, introduction to probability, expectations and variance of random variables, confidence intervals and tests for means, differences of means, proportions, differences of proportions, chi-square tests for categorical variables, regression and

multiple regressions, an introduction to analysis of variance.

Using software technologies for education has become a new trend. Computer-based technologies developed by academic institutions as well as industries worldwide are revolutionizing the educational system. A new field involving the use of virtual reality techniques is becoming the training environment. Through virtual labs, a new interdisciplinary field of science brings together biologists and physicists to tackle this grand challenge through quantitative experiments and models. Using several pro-learning even distance education courses have started using virtual laboratories to enable students to access equipment since they are independent from opening hours and the work schedule of the staff. In many engineering courses within India, simulation is the most effective tool in training students in the use of sophisticated as well as complicated instruments that are routinely employed in modern biological and chemical laboratories. For the life sciences, this also circumvents the use of expensive and hazardous biological and chemical agents which toxic to the experimentalists as well as to the environment. Above all, the virtual lab technology is cheap as well as cost effective.

Education in many universities and research institutes include their own virtual laboratories on the web, which are accessible to people around the world. Although some laboratory practice requires getting one's hands 'dirty', it has already been established that the Virtual Lab enables the students to understand the underlying principles and the theory behind laboratory experiments. E-learning plays and will play an important role in diverse regions such as India where the traditional lab facilities at Universities are not very well localized to suit requirements of all sub-regions. With multi-campus scenarios as in some Universities such as ours, offering cross-disciplinary courses needs to exploit the use of extensive elearning facilities (Bijlani et al., 2008).

Biotechnology lab courses richly rely upon new up-to-date content and various techniques that require a new synergy of knowledge and experimental implementation. Hence a new kind of experimental science that can be brought as a virtual simulation based laboratory is necessary. The developments of the virtual labs include mathematical techniques in biology to study, to hypothesize and to demonstrate complex biological functions. However virtual labs in heavy engineering topics such as analyzing nanomaterials with high-power microscopes and lab courses in biotechnology or biology will also have to exploit multiple techniques besides simulators alone as many scenarios cannot be reproduced mathematically while retaining the "real" lab-like feel.

In this chapter, we focus on the development and use on the virtual biotechnology laboratory courses through a combination of techniques to try completing the learning experience as that of a regular University laboratory.

#### **2. Why virtual labs?**

There are many main reasons to focus on creating virtual labs for University education (Auer et al., 2003). Among the primary reasons include the cost and lack of sufficient skillset for facing the current growth in biotechnology sector. The setup cost of laboratories puts a large overhead on the educators. The Universities also need to setup laboratories to educate sufficient target group with the details of common biotechnological techniques and protocols (O'donoghue et al., 2001).

Another new motivation is the need to introduce and focus well-explored potential virtual lab areas which use computational methods, mathematical modeling and biophysics, computational biology and computational neuroscience. Computational biology and biophysics are upcoming areas and most techniques derive basis from real laboratory experiments. Another intention of using virtual labs via a computational approach is to train young scientists in the field of the mathematical thinking for life sciences and related environments. Main goals of cross-disciplinary sciences include the need to ensure that the students will be able to integrate different exhaustive models into a larger framework, i.e. in the perspective of comprehensive biological systems such as cells and biological networks. Such a role will also give an overview of the modeling approaches that are most appropriate to describe life-science processes. For the everyday biologist, the major use of virtual labs will also be in the learning perspective of advanced but common-to-use simulation tools.

Virtual labs and use of virtual tools should lead to an increase the awareness of a crucial need for standard model descriptions. Most simulators and common-use tools require various formats and schema and with the explosion of data, the use of virtual labs across the country or across multiple countries is also intended to unite educators to work towards common model descriptions and standardization of their data.

For the biotechnology sector, a highly favoring motivation for the shift to the virtual lab paradigm is the explosion of data-rich information sets, due to the genomics revolution, which are difficult to understand without the use of analytical tools. Also, recent development of mathematical tools such as chaos theory to help understand complex, nonlinear mechanisms in biology seems to push the need for information-rich virtual labs in simulation domain.

To aid further, an increase in computing power which enables calculations and simulations to be performed that were not previously possible, have set a new trend in the concept and use of computing. Simulations in the past that needed more intensive computers now can plainly be run through long battery-life laptops (Aycock et al., 2008), given that in many cases laptops today even host servers.

A slightly different reason that also pushes the concept of virtual labs for undergraduate and master level education at the Universities also seems to be an increasing interest in in silico experimentation due to ethical considerations, risk, unreliability and other complications involved in human and animal research.

Given all above reasons and motivation, virtual labs are today's experimental approach towards a newer trend in future education. However the virtual lab environments are still under severe testing and newer models seems to switch to more intelligent and adaptive platforms that can yield efficient knowledge dissipation. One such common model is the adaptive learning system (ALS) currently employed by many e-learning applications strewed on the internet.

#### **3. Other virtual labs and online courses in biosciences**

Very little work has been actually done in the biology sector. There are online "dissections" of frog tutorials by Mable Kinzie developed in 1994 and an improved version of the same was hosted in 2002 (http://curry.edschool.Virginia.EDU/go/frog/menu.html). Quick "movies": http://www.bio.unc.edu/faculty/goldstein/lab/movies.html Virtual "experiments": Biology Labs On-Line (BLOL) is a collaboration of the California State University system Center for Distributed Learning and Addison Wesley Longman, with partial funding provided by the National Science Foundation (http://biologylab.awlonline.com). A project titled "BIOTECH Project" developed by University of Arizona, with aim of supporting Arizona teachers to conduct molecular genetics (DNA science) experiments with their students and assists teachers in developing new activities for their classroom (http://biotech.bio5.org/home). "Protein Lab" by A.J. Booth, is a computer simulation of protein purification. These labs are extremely helpful for beginners in the art of protein purification. It gives them a chance to get beyond the details of individual techniques and get a sense of the overall process of a protein purification strategy. (http://www.booth1.demon.co.uk/archive).To enhance education, there is a great need for individualized courseware to provide educational content that fits to the learner's learning style and knowledge base. University of Utah's genetic science learning center has its very animated genetics labs at http://learn.genetics.utah.edu/gslc. The labs were developed with the mission in making science easy for everyone to understand. Similar projects at Howard Hughes http://www.hhmi.org/biointeractive/vlabs and at Pearson's http://www.phschool.com/science/biology\_place/labbench have been useful as virtual education websites.

Online biotechnology courses are available through several leading universities around the world, including the Massachusetts Institute of Technology (MIT), Osaka University and the Open University. OpenCourseWare (OCW) from TUFTS and MIT offer courses on the Web that containing all or some of the materials from the university's original oncampus classrooms. Many biotechnology courses on OCW make use of several different learning materials available online or by download, lab notes, assignments, lectures on scientific communications and study materials. Online biotechnology courses are known to be very helpful for students to study/prepare for the positions as lab technicians, research assistants and quality assurance analysts in such fields as agriculture, pharmaceuticals and manufacturing.

#### **4. Amrita VL**

382 Innovations in Biotechnology

the students will be able to integrate different exhaustive models into a larger framework, i.e. in the perspective of comprehensive biological systems such as cells and biological networks. Such a role will also give an overview of the modeling approaches that are most appropriate to describe life-science processes. For the everyday biologist, the major use of virtual labs will also be in the learning perspective of advanced but common-to-use

Virtual labs and use of virtual tools should lead to an increase the awareness of a crucial need for standard model descriptions. Most simulators and common-use tools require various formats and schema and with the explosion of data, the use of virtual labs across the country or across multiple countries is also intended to unite educators to work towards

For the biotechnology sector, a highly favoring motivation for the shift to the virtual lab paradigm is the explosion of data-rich information sets, due to the genomics revolution, which are difficult to understand without the use of analytical tools. Also, recent development of mathematical tools such as chaos theory to help understand complex, nonlinear mechanisms in biology seems to push the need for information-rich virtual labs in

To aid further, an increase in computing power which enables calculations and simulations to be performed that were not previously possible, have set a new trend in the concept and use of computing. Simulations in the past that needed more intensive computers now can plainly be run through long battery-life laptops (Aycock et al., 2008), given that in many

A slightly different reason that also pushes the concept of virtual labs for undergraduate and master level education at the Universities also seems to be an increasing interest in in silico experimentation due to ethical considerations, risk, unreliability and other

Given all above reasons and motivation, virtual labs are today's experimental approach towards a newer trend in future education. However the virtual lab environments are still under severe testing and newer models seems to switch to more intelligent and adaptive platforms that can yield efficient knowledge dissipation. One such common model is the adaptive learning system (ALS) currently employed by many e-learning applications

Very little work has been actually done in the biology sector. There are online "dissections" of frog tutorials by Mable Kinzie developed in 1994 and an improved version of the same was hosted in 2002 (http://curry.edschool.Virginia.EDU/go/frog/menu.html). Quick "movies": http://www.bio.unc.edu/faculty/goldstein/lab/movies.html Virtual "experiments": Biology Labs On-Line (BLOL) is a collaboration of the California State University system Center for Distributed Learning and Addison Wesley Longman, with partial funding provided by the National Science Foundation (http://biologylab.awlonline.com). A project titled "BIOTECH Project" developed by University of Arizona, with aim of supporting Arizona teachers to conduct molecular genetics (DNA science) experiments with their students and assists teachers

common model descriptions and standardization of their data.

simulation tools.

simulation domain.

strewed on the internet.

cases laptops today even host servers.

complications involved in human and animal research.

**3. Other virtual labs and online courses in biosciences** 

Amrita University's Virtual and Accessible Laboratories Universalizing Education (VALUE) initiative was initially targeted towards making biotechnology, physics and chemistry courses virtually accessible for undergraduate and postgraduate education. The project led to the development of 14 labs in biotechnology and 13 labs in physical and chemical sciences. The schema of virtual labs was based on one of our studies.

An average survey of the VL framework software was performed and the tests were shown (see Table 1 in Diwakar et al., 2011). The developed virtual labs are available for public use (See http://amrita.edu/virtuallabs). Any user may login with an open-id or Google's gmail account and access the authentication-compulsory regions such as the remote-panel, simulator and animations. The website uses the name and email address that provider gives only to set up an user account.

#### **5. Techniques – Animation, simulations and remote-triggered experiments**

The key learning component in many biological laboratories is the complexity of the procedure and details of the step-by-step protocol carried out in the laboratory. Although some of these biological processes can be replaced by mathematical equations modeling the system, most of the "feel" is in performing the detailed procedure which is not derived from sets of equations. Graphical animations deliver a high degree of the reality to the virtual labs through their seeming closeness to the appearance and feel of the lab. Graphical animations also cut out the complexity of the modeling process by increasing the "feel" of experiment. Like the proverb goes, "a picture is worth a thousand words", animations reveal better information that cannot be easily conveyed via text alone or static illustration.

In our biotechnology virtual labs, the animation type of experiments include the use of 2D flash based animations for illustrating detailed procedures such as wet lab protocols and heavy engineering techniques that are out of scope for simulation due to various reasons like complicated equations, numerical issues in simulation, lack of modeling data etc. Besides animation, another common technique in our virtual labs included engineeringbased approaches such as remote-triggered experiments or remote-controlled experiments.

The very common and research-inspiring approach is the use of mathematical simulators to model biological and biotechnological processes or sub-processes. Although mathematics has long been intertwined with the biological sciences, an explosive synergy between biology and mathematics seems poised to enrich and extend both fields greatly in the coming decades. Among the various scenarios to study biology and disseminate information effectively and efficiently, includes the use of e-learning as a medium to offer courses.

Applying mathematics to biotechnology for virtual lab creation has recently turned into an explosion of interest in the field. The NASA virtual laboratory or the HHMI virtual labs at Howard Hughes Medical Institute or the Utah genetics virtual laboratory are some examples.

For our labs, a combination of user-interactive animation, mathematical simulations, remote-trigger of actual equipment and the use of augmented perception haptic devices are used to deploy effectively the real laboratory feel of a biotech lab online.

#### **6. Models in biology – As virtual labs**

Design of simulation labs requires basic mathematical models. Some models that were used to develop the virtual labs are listed below.

#### **6.1 Neurophysiology and neuronal biophysics**

In order to understand neuronal biophysics and simulations on voltage clamp and current clamp in detail, we modeled a section of excitable neuronal membrane using the Hodgkin-Huxley equations (Hodgkin and Huxley, 1952) that can be accessed a graphical web-based simulator. Various experiments using this simulator deal with the several parameters of Hodgkin-Huxley equations and will model resting and action potentials, voltage and current clamp, pharmacological effects of drugs that block specific channels etc. This lab complements some of the exercises in the Virtual Neurophysiology lab.

#### **6.2 Population ecology**

As part of population ecology virtual labs, we developed a set of mathematical ecology models to understand the basic dynamics and behavior of population in various aspects. Some models include:

the "feel" of experiment. Like the proverb goes, "a picture is worth a thousand words", animations reveal better information that cannot be easily conveyed via text alone or static

In our biotechnology virtual labs, the animation type of experiments include the use of 2D flash based animations for illustrating detailed procedures such as wet lab protocols and heavy engineering techniques that are out of scope for simulation due to various reasons like complicated equations, numerical issues in simulation, lack of modeling data etc. Besides animation, another common technique in our virtual labs included engineeringbased approaches such as remote-triggered experiments or remote-controlled

The very common and research-inspiring approach is the use of mathematical simulators to model biological and biotechnological processes or sub-processes. Although mathematics has long been intertwined with the biological sciences, an explosive synergy between biology and mathematics seems poised to enrich and extend both fields greatly in the coming decades. Among the various scenarios to study biology and disseminate information effectively and efficiently, includes the use of e-learning as a medium to offer

Applying mathematics to biotechnology for virtual lab creation has recently turned into an explosion of interest in the field. The NASA virtual laboratory or the HHMI virtual labs at Howard Hughes Medical Institute or the Utah genetics virtual laboratory are some

For our labs, a combination of user-interactive animation, mathematical simulations, remote-trigger of actual equipment and the use of augmented perception haptic devices are

Design of simulation labs requires basic mathematical models. Some models that were used

In order to understand neuronal biophysics and simulations on voltage clamp and current clamp in detail, we modeled a section of excitable neuronal membrane using the Hodgkin-Huxley equations (Hodgkin and Huxley, 1952) that can be accessed a graphical web-based simulator. Various experiments using this simulator deal with the several parameters of Hodgkin-Huxley equations and will model resting and action potentials, voltage and current clamp, pharmacological effects of drugs that block specific channels etc. This lab

As part of population ecology virtual labs, we developed a set of mathematical ecology models to understand the basic dynamics and behavior of population in various aspects.

used to deploy effectively the real laboratory feel of a biotech lab online.

complements some of the exercises in the Virtual Neurophysiology lab.

**6. Models in biology – As virtual labs** 

to develop the virtual labs are listed below.

**6.2 Population ecology** 

Some models include:

**6.1 Neurophysiology and neuronal biophysics** 

illustration.

experiments.

courses.

examples.


#### **6.3 Biochemistry, cell biology, microbiology, immunology and molecular biology**

Simple linear equations were used to understand molecular mass flow in AGE, PAGE exercises. No differential equations were used in biology oriented virtual labs where the focus was on the look and feel. In many cases, animation played a major role in these areas rather than mathematical simulations. As in the case of realistically animating experiments there are a lot of advantages; although it cannot be considered as a complete replacement of real labs due to its limitations. One solution was to provide the necessary details of the instruments we were using for the lab. Per say, if we use cooling centrifuges for an experiment in the virtual lab, one may not fully show all details corresponding to the operating methods of the centrifuge. But in the case of a real laboratory the student gets an opportunity to have a hands-on experience on the equipment while doing the experiment. Also, many of the experiments require instrumentation facilities. Also instruments from different companies have slight differences in design and operating mechanisms, which may not be shown in the virtual labs. Thus even though virtual lab meets the major target, it shadows the minor details of the experiments. Not all parameters such as changes in temperature during an experiment especially (where small changes do not matter) may not be included in the virtual lab for the sake of simplicity. In a real lab, curious students can perform these kinds of interesting experiments but to do the same in virtualized experiments is difficult.

#### **7. Major challenges**

Setting and developing AMRITA virtual labs (see Fig. 2) as a complete learning experience has not been an easy task. Amongst the major challenges we faced included usage/design scalability, deliverability efficiency, network connectivity issues, security and speed of adaptability to incorporate and update changes into existing experiments.

Owing to the scientific domain, biotechnology lends the following challenges to establishing virtual labs:


In order to address some of these issues and to overcome restrictions, we deployed virtual lab experiments as web-client based animations or simulators besides remote triggered experiments. The virtual lab was based on a website that was designed for favorable use within intranets and internets. However, efficiency depended on the internet bandwidth and connectivity. Our target was any campus with a download link of 256kbps should suffice. To retain this compatibility the animations had to be size-delimited. To overcome the problem, longer experiments had to be sliced to smaller portions, each loading in sequence. This was possible as we maintained the virtual lab experiments as flash animations (Adobe, USA). Having labs in flash environments allowed the scalability and access although flash based action script programming needed additional programmers and training.

Other e-learning issues such as student-teacher collaboration via chat, video interfacing etc. were overcome via AVIEW-like environment (Bijlani et al, 2008). The intention of the virtual labs was also to extend the facility to develop an applied computational laboratory.

#### **8. Methodology**

Amongst others, the focus of having and designing virtual labs was also based on John Keller's ARCS model of motivation. Design of courses, simulations and models for computational approaches in biology will be the highlight. A lot of attention was on courses whose content will be applicable to the existing P.G. programs.

perform these kinds of interesting experiments but to do the same in virtualized

Setting and developing AMRITA virtual labs (see Fig. 2) as a complete learning experience has not been an easy task. Amongst the major challenges we faced included usage/design scalability, deliverability efficiency, network connectivity issues, security and speed of

Owing to the scientific domain, biotechnology lends the following challenges to establishing

• The development of analytical solutions in the arena is limited as biological processes are typically non-linear and are coupled systems of differential equations in various

• The mathematics behind models is hidden by their complexity and appears refined

• Most simulation platforms need direct hands-on experience between teachers and

• Besides, such courses also need simultaneous theoretical explanations which may need classroom-like scenarios with video presentations, white-board and other tools. We could overcome the issue here using a collaborative suit, AVIEW (Bijlani et al., 2008).

In order to address some of these issues and to overcome restrictions, we deployed virtual lab experiments as web-client based animations or simulators besides remote triggered experiments. The virtual lab was based on a website that was designed for favorable use within intranets and internets. However, efficiency depended on the internet bandwidth and connectivity. Our target was any campus with a download link of 256kbps should suffice. To retain this compatibility the animations had to be size-delimited. To overcome the problem, longer experiments had to be sliced to smaller portions, each loading in sequence. This was possible as we maintained the virtual lab experiments as flash animations (Adobe, USA). Having labs in flash environments allowed the scalability and access although flash based action script programming needed additional programmers and

Other e-learning issues such as student-teacher collaboration via chat, video interfacing etc. were overcome via AVIEW-like environment (Bijlani et al, 2008). The intention of the virtual

Amongst others, the focus of having and designing virtual labs was also based on John Keller's ARCS model of motivation. Design of courses, simulations and models for computational approaches in biology will be the highlight. A lot of attention was on courses

labs was also to extend the facility to develop an applied computational laboratory.

whose content will be applicable to the existing P.G. programs.

• The number of students that can be catered at any given time is restricted.

• There are not many courses in India developed for this scenario.

adaptability to incorporate and update changes into existing experiments.

experiments is difficult.

**7. Major challenges** 

virtual labs:

forms.

students.

training.

**8. Methodology** 

through simulation platforms.

Fig. 1. Sakshat Amrita virtual labs. Accessible at http://amrita.edu/virtuallabs

For all biotech virtual labs, we had set the following lab-level objectives as general guidelines.


The implementation of animation and simulation based virtual labs was mainly done in Action Script 3 in Adobe flash in order to bring better definition to 2-D graphics. Action script allowed flash swf files as output thereby allowing both a better look-and-feel and an enhanced interactivity with the software. The physics simulator tools worked reasonably well. We did not use java as a programming medium in our learning tool to make sure we have complete cross-OS, cross-browser compatibility, to reduce initial loading time and also to consider support for the commercial operating systems such as Microsoft's Windows platform that support flash better than Java plug-in.

We used a new VLCOP platform (Nedungadi et al., 2011) in its full functionality for the virtual labs. The minor intention was to deploy preliminary platform with a learning environment and later render the environment adaptive and intelligent as per the user-audience. The main reason to precursor with such a test was cost-efficiency. Cost-efficiency of e-learning programs has been increasingly important because some institutions have failed due to the lack of wellthought out financial plans (Wentling et al, 2002; Morgan, 2000).

Virtual Labs use self-assessment based on questionnaire to evaluate user's experience. Although not implemented, an advanced form of the lab is being planned to include teacher's assessment, peer-assessment and collaborative assessment. Teacher assessment will actually have a "real" instructor on the deployment site to evaluate the lab user/student. Peer-assessment will include any student or teacher to assess another. Collaborative assessment will include both the instructor and the student to perform assessment on the completion of an experiment.

For our installation and deployment, we focused to reduce internet downtime. A 2004 study indicated that overall downtime costs companies an average of 3.6% of annual revenue (internet sources, see www.sentinelbussiness.it) indicating leading causes for downtime being software failure and human error. Through our studies, we managed to reduce unnecessary events and maintain downtime to less than 27 minutes for 6 months (not as in Amrita Learning software, see Table I in Diwakar et al., 2011). However, this could be because of our lack of full incorporation of the complex adaptive learning system as it was done for the schools where it was tested. However a test on real-time upgrade to such a model based on our previous experiences (data not shown) with Amrita learning (Nedungadi and Raman, 2010) indicated that overall loss of virtual lab in terms of downtime will be significantly less.

#### **9. Feedback and assessment**

Feedback is usually not used as an evaluator but an assessment tool for student quality. With that in mind, the virtual lab evealuation criterion was focussed on measuring and estimating the student's involvement in the particular experiment of a particular lab. A way to increment the quantity and timing of feedback is to provide enough detail. Through animation, we have also increased evaluatory criterion and details in the virtual environments. It was noted that in more than 95 experiments performed by more than 30 people within a particular time-window there were more than 91% of appreciation (further statistics pending, data not shown) when two experiments, one with detail oriented interactive animation and other without interaction were delivered to assess the involvement of the students in terms of their self-assessment.

The implementation of animation and simulation based virtual labs was mainly done in Action Script 3 in Adobe flash in order to bring better definition to 2-D graphics. Action script allowed flash swf files as output thereby allowing both a better look-and-feel and an enhanced interactivity with the software. The physics simulator tools worked reasonably well. We did not use java as a programming medium in our learning tool to make sure we have complete cross-OS, cross-browser compatibility, to reduce initial loading time and also to consider support for the commercial operating systems such as Microsoft's Windows

We used a new VLCOP platform (Nedungadi et al., 2011) in its full functionality for the virtual labs. The minor intention was to deploy preliminary platform with a learning environment and later render the environment adaptive and intelligent as per the user-audience. The main reason to precursor with such a test was cost-efficiency. Cost-efficiency of e-learning programs has been increasingly important because some institutions have failed due to the lack of well-

Virtual Labs use self-assessment based on questionnaire to evaluate user's experience. Although not implemented, an advanced form of the lab is being planned to include teacher's assessment, peer-assessment and collaborative assessment. Teacher assessment will actually have a "real" instructor on the deployment site to evaluate the lab user/student. Peer-assessment will include any student or teacher to assess another. Collaborative assessment will include both the instructor and the student to perform

For our installation and deployment, we focused to reduce internet downtime. A 2004 study indicated that overall downtime costs companies an average of 3.6% of annual revenue (internet sources, see www.sentinelbussiness.it) indicating leading causes for downtime being software failure and human error. Through our studies, we managed to reduce unnecessary events and maintain downtime to less than 27 minutes for 6 months (not as in Amrita Learning software, see Table I in Diwakar et al., 2011). However, this could be because of our lack of full incorporation of the complex adaptive learning system as it was done for the schools where it was tested. However a test on real-time upgrade to such a model based on our previous experiences (data not shown) with Amrita learning (Nedungadi and Raman, 2010) indicated that overall loss of virtual lab in terms of downtime

Feedback is usually not used as an evaluator but an assessment tool for student quality. With that in mind, the virtual lab evealuation criterion was focussed on measuring and estimating the student's involvement in the particular experiment of a particular lab. A way to increment the quantity and timing of feedback is to provide enough detail. Through animation, we have also increased evaluatory criterion and details in the virtual environments. It was noted that in more than 95 experiments performed by more than 30 people within a particular time-window there were more than 91% of appreciation (further statistics pending, data not shown) when two experiments, one with detail oriented interactive animation and other without interaction were delivered to assess the

platform that support flash better than Java plug-in.

assessment on the completion of an experiment.

will be significantly less.

**9. Feedback and assessment** 

thought out financial plans (Wentling et al, 2002; Morgan, 2000).

involvement of the students in terms of their self-assessment.

Fig. 2. Neuron simulator. The Neuron simulator lab uses Hodgkin-Huxley equations to study and analyze the action potential properties. The simulator allows some pharmacological studies and complements the neurophysiology virtual lab.

#### **10. Case study: Virtual neurophysiology laboratory**

Our preliminary studies in the biotech sector were on neurophysiology techniques. The virtual neurophysiology laboratory provides an opportunity for students to substitute classroom physiology course with detailed techniques and protocols of a real laboratory. Besides the material like chemicals, physiology demands extensive knowledge and experience from the instructor. For example, rat brain slicing protocol which is the first experiment (in the virtual lab) takes approximately 6-10 hours to complete training and about 2-3 weeks to train one student in a real laboratory.

With the focus on time (Rohrig et al., 1999) and learning know-how, we adapted the usual lab experimental protocols as user-interactive animations of the neurophysiology lab experience. The work involved both animators and programmers. For some experiments such as brain slice preparation, animations were sufficient whereas for some others such as Hodgkin-Huxley neuronal model (Hodgkin et al., 1952, see Fig. 3.) for demonstrating behavior of single neurons, we used Java based simulator. The same simulator was embedded into other experiments such as voltage clamp protocol and current clamp protocol to allow the student to see the corresponding behavior as seen in real neurons (Koch, 1999).

A new set of experiments developed included the use of electronic resistance-capacitance (RC) circuits that could be remotely triggered as mimicking the electrical dynamics of a passive neuronal membrane. Passive neuronal membranes are modeled as RC-circuits with high resistance and low capacitance (for more details see Koch, 1999). In the simulation lab that was developed to complement the exercises of the VL, a model detailed study was added. Some of the main objectives and experiments using a neuron simulator included:


Also as part of the labs, we follow a particular formatting for each experiment within the lab. The goal was to allow the student to study the theory, the approach and do a self-test before actually going into the simulator or the virtual experiment. Covering some explanations and incorporating the same theory into the actual "lab" part of the experiment has been one of the primary goals. Each experiment in the labs (especially in Biotechnology) opens by default with the textual theory, which can also be randomly accessed by clicking on the icon "theory".

All the control and experimental parameters are explained in the "manual". The instructor and the student are informed on how various parameters change in the experiment in the very context of the virtual experimental lab procedure. For those experiments that have both an animation learning component and simulator component, each of the user controls and the variable parameters are explained. Also included in the manual is a help that actually explains the usage of radio controls and icons covered by the experiment. The intention was to evaluate the basic info that once the student completes the familiarization process by going through the theory and manual sections, he/she can take a "self-evaluatory" quiz module that chooses to test the student on some questions based on the theory background of the experiment.

The "simulator" tab actually leads to the experiment workbench. "Protocol for brain slicing" that is actually a detailed lab process that would take 6-10 weeks for post-master's student to learn and about 3-10 hours per procedure. That experiment we have virtualized by means of an interactive action script based animation. The second neurophysiology experiment concerns the modeling of a neuronal cell. In this case we have used a Flash based learning component along with a HH-simulator of a biophysical neuron.

The "assignment" icon is the lab experiment question with which intention the student performs the experiment. An instructor version of the assignment will include a model

A new set of experiments developed included the use of electronic resistance-capacitance (RC) circuits that could be remotely triggered as mimicking the electrical dynamics of a passive neuronal membrane. Passive neuronal membranes are modeled as RC-circuits with high resistance and low capacitance (for more details see Koch, 1999). In the simulation lab that was developed to complement the exercises of the VL, a model detailed study was added. Some of the main objectives and experiments using a neuron simulator included:

Also as part of the labs, we follow a particular formatting for each experiment within the lab. The goal was to allow the student to study the theory, the approach and do a self-test before actually going into the simulator or the virtual experiment. Covering some explanations and incorporating the same theory into the actual "lab" part of the experiment has been one of the primary goals. Each experiment in the labs (especially in Biotechnology) opens by default with the textual theory, which can also be randomly

All the control and experimental parameters are explained in the "manual". The instructor and the student are informed on how various parameters change in the experiment in the very context of the virtual experimental lab procedure. For those experiments that have both an animation learning component and simulator component, each of the user controls and the variable parameters are explained. Also included in the manual is a help that actually explains the usage of radio controls and icons covered by the experiment. The intention was to evaluate the basic info that once the student completes the familiarization process by going through the theory and manual sections, he/she can take a "self-evaluatory" quiz module that chooses to test the student on some

The "simulator" tab actually leads to the experiment workbench. "Protocol for brain slicing" that is actually a detailed lab process that would take 6-10 weeks for post-master's student to learn and about 3-10 hours per procedure. That experiment we have virtualized by means of an interactive action script based animation. The second neurophysiology experiment concerns the modeling of a neuronal cell. In this case we have used a Flash based learning

The "assignment" icon is the lab experiment question with which intention the student performs the experiment. An instructor version of the assignment will include a model

• Modeling sodium ion channel and its effect on neural signaling

• Understanding pharmacological implications of ionic currents

questions based on the theory background of the experiment.

component along with a HH-simulator of a biophysical neuron.

• Modeling delayed rectifier potassium channels • Modeling passive membrane properties

• Capacitive transients using Voltage Clamp • Effect of temperature on neuronal dynamics

accessed by clicking on the icon "theory".

• Modeling action potential • Modeling resting potential

• Current clamp protocol • Voltage clamp protocol

• Plotting F-vs-I curve • Plotting V-vs-I curve solved question or key tips in case of a protocol-like experiment. Additional reading material and reference information and other details will be found in our "misc info" icon.

Among the various methodologies the lab covers simulation-based, animation-based and remote-triggered experiments. The simulator was that of a bio-realistic model cell and was combined with an interactive animation-based learning-tool made using Flash. Maldarelli et al. (2009) report the advantage of virtual lab demonstration as an effective lab tool. The remote-triggered experiments were based on real electronic circuits that mimic the phenomenon observed in neuronal cells. The basic behavior of Resistancecapacitance circuits that can be modified remotely by a user to study and imitate real neuronal circuits as he/she does in a neuronal biophysics laboratory on a patch of a neuronal membrane.

Fig. 3. Remote-triggered Experiment. Remote panel is also made with re-configurable panels and control options. This experiment emulates action potential generation using analog neurons.

We also tested the virtual lab via a questionnaire-based feedback for overall quality. Among the major questions, several virtualizations related questions were presented in the questionnaire. The general developer/designer related questions included in the lab were to rate the experiment that was most recently completed, extent of control on the interface, closeness to lab environment and feel, measurement and analysis of data, user-manual quality, adequacy of bibliography and references, results interpretation, whether any clear information was gained by using the virtual lab, any problems faced, how helpful the lab was and overall motivation.

Fig. 4. Tiger population study. Using exponential growth patterns to predict tiger population in India.

#### **11. Taking project tiger to the classroom: A virtual lab case study**

Using a virtual lab was not our only objective. We wanted to test a real scenario and see if the virtual lab could be used a as research tool. Tiger population study uses virtual labs to take India's Project tiger to the classroom. Half of the tiger population in the world is in India. Due to reduction in their population in large numbers, from 1969 onwards the 'tiger' was declared as an endangered species (by CITES). Educating about tiger populations is vital. Typically courses in population ecology deal about population variations. In this section, we suggest on the applicative use of population ecology simulators as classroom models to complete the learning experience for a population ecology laboratory course. This section also reports the analysis, interpretation and some preliminary predictions in variations of tiger population in India.

Here we used the exponential growth model experiment in population ecology lab 1 (at http://amrita.edu/virtuallabs). First step is to select an experiment followed by selecting a mathematical model which is described in the experiment from the set of experiments (see Fig. 4). Some existing data tested indicated the validation of the technique. (Fig 5B).

#### **11.1 Data collection**

392 Innovations in Biotechnology

quality, adequacy of bibliography and references, results interpretation, whether any clear information was gained by using the virtual lab, any problems faced, how helpful the lab

Fig. 4. Tiger population study. Using exponential growth patterns to predict tiger

**11. Taking project tiger to the classroom: A virtual lab case study** 

Using a virtual lab was not our only objective. We wanted to test a real scenario and see if the virtual lab could be used a as research tool. Tiger population study uses virtual labs to take India's Project tiger to the classroom. Half of the tiger population in the world is in India. Due to reduction in their population in large numbers, from 1969 onwards the 'tiger' was declared as an endangered species (by CITES). Educating about tiger populations is vital. Typically courses in population ecology deal about population variations. In this section, we suggest on the applicative use of population ecology simulators as classroom models to complete the learning experience for a population ecology laboratory course. This section also reports the analysis, interpretation and some preliminary predictions in

Here we used the exponential growth model experiment in population ecology lab 1 (at http://amrita.edu/virtuallabs). First step is to select an experiment followed by selecting a

was and overall motivation.

population in India.

variations of tiger population in India.

Statistical data for this study was collected from Project Tiger which includes the tiger population from 1972 – 2002 of various tiger reserves (see Table 1). And the second data set was the crime reported for the numbers of tiger that have been killed in past few years were from WPSI's Wildlife Crime Database (14. WPSI's Tiger Poaching Statistics, http://www.wpsi-india.org/statistics/index.php).

Fig. 5. Virtual Lab model for tiger population study. Note that exponential model was chosen based on decline in populations. Predictions with other models such as Lotka-Volterra and Logistic growth were inappropriate or had errors.

Growth rate has been calculated by using the formula, Growth rate (t 1) (t 1) t ( ) <sup>N</sup> *g t* + + − <sup>=</sup> . where

N( 1) *<sup>t</sup>*<sup>+</sup> is the total number of individuals at t+1, t' represent the time in years.

#### **11.2 On-screen methods**

We have used an adaptive growth rate for different periods as shown in Table 1. Simulator's viewable window contain three main tabs, 1) Statistics button will show the growth of population while the simulation is running 2) Data plots button, will Population size Vs Time, 3) Worksheet button is an implementation of the model in excel.

#### **11.3 Assumptions with tiger populations and growth model**

Population ecology models include several assumptions, in order to realistically apply the model on data. Growth of prey population is exponential in absence of predators;


Although the assumptions make it difficult to actually call the simulation 'realistic', the validations showed a realistic trend and hence for this model, a simple exponential growth simulator was used.


Calculated average growth rate was = 0.1545

Table 1. Shows the statistical data for tiger population from 1972 – 2002 and extended (prediction column) the curve to 2012 using continuous growth model simulator (data collected from Tiger project India at http://projecttiger.nic.in/populationinstate.asp).

#### **11.4 Results**

Taking real data to class rooms have been very difficult with population ecology due to its high unpredictability and model-related unreliability. However, using a simple growth rate simulator and using patterns from a short period, the model shows promise.

The simulation showed predictability in the growth of tiger population in India for the years 2003- 2012 by extending the behavioral pattern of tiger population in India for last 30 years (see Fig.6). Predicted tiger population for the year 2007 was 1664. According to National Tiger Conservation Authority (on 2008), the total tiger population reaches 1,411 (i.e. ranging between 1,165 and 1,657). The difference in number of tigers from predicted to this statistical report may be because of some environmental factors, number of tigers that have been killed in past few years and the census by National Tiger Conservation Authority was only partially included West Bengal.

#### **11.5 Conclusion**

394 Innovations in Biotechnology

We have used an adaptive growth rate for different periods as shown in Table 1. Simulator's viewable window contain three main tabs, 1) Statistics button will show the growth of population while the simulation is running 2) Data plots button, will Population size Vs

Population ecology models include several assumptions, in order to realistically apply the

• During the process, the environment does not change in favour of one species and the

Although the assumptions make it difficult to actually call the simulation 'realistic', the validations showed a realistic trend and hence for this model, a simple exponential growth

> **Total number of tigers predicted by the model**

Table 1. Shows the statistical data for tiger population from 1972 – 2002 and extended (prediction column) the curve to 2012 using continuous growth model simulator (data collected from Tiger project India at http://projecttiger.nic.in/populationinstate.asp).

simulator and using patterns from a short period, the model shows promise.

Taking real data to class rooms have been very difficult with population ecology due to its high unpredictability and model-related unreliability. However, using a simple growth rate

The simulation showed predictability in the growth of tiger population in India for the years 2003- 2012 by extending the behavioral pattern of tiger population in India for last 30 years (see Fig.6). Predicted tiger population for the year 2007 was 1664. According to National

1972 268 - - 1979 711 - 0.6230 1984 1121 - 0.3657 1989 1327 - 0.1552 1993 1366 - 0.0285 1995 1333 - -0.0247 1997 1498 1521 0.1236 2002 1576 1586 0.0409 2007 - 1664 0.0468 2012 - 1718 0.0314

**Growth rate** 

model on data. Growth of prey population is exponential in absence of predators;

• Tiger population grows/declines exponentially within a short duration (10 years)

Time, 3) Worksheet button is an implementation of the model in excel.

**11.3 Assumptions with tiger populations and growth model** 

• The rate of change of tiger population is proportional to its size.

**Total number of tigers**

genetic adaptation is sufficiently slow.

**Year** 

Calculated average growth rate was = 0.1545

**11.2 On-screen methods** 

simulator was used.

**11.4 Results** 

The predicted data (Table.1, third column) for tiger population in India showed standard deviation of 10% from real data. With some assumptions, it was possible to use simple models like exponential growth models for studying tiger populations. For a very short duration (such as in the data shown in Table 1), basic growth show a slowly saturating exponential and hence data matched the predictions (see Fig. 6). Online population ecology experiments developed on the basis of mathematical equations could help students to get a deeper understanding on model dynamics by exploring the parameter space provided by the model. Also it is always feasible for the user to supply the real data as input and observe the corresponding dynamics. The possibility to study such experiments has value. Biotechnology studies often include data collection and such models allow building simple hypothesis based on the dynamics. This new e-learning environment engaged and motivated the students to practice and explore the parametric space provided for the population ecology experiments.

Newer studies for analyzing fish populations and deer populations are being developed as part of the ongoing process. Such data will be made available as a virtual lab for continued use and study. We also noticed that the undergraduate and postgraduate students show an increased attention to details when we trained them on virtual labs instead of plainly explaining the theory. There was a 23% (metric not shown) improvement in interest to critically analyze population models among students who were introduced to population ecology studies directly virtual labs.

#### **12. Cost of virtual labs**

In order to estimate the true financial cost of our virtual lab project, we had to include both project development and delivery and maintenance costs. As indicated by Kruse (see http://www.e-learningguru.com/articles/art5\_2.htm), design of courseware needed more initial costs than instructor-led learning but delivery and maintenance is affordably cheaper. We estimate, based on Amrita learning software experience that there will be negligible costs for maintain web-based experiments. The main post-deployment costs included administration and maintenance. The administration and maintenance estimates included tracking of user-behavior, technical support, content updates and technology updates. Student material development, instructor costs and subject expert costs were included in the development expenses.

#### **13. Some evaluatory setbacks and associated feedback**

What we know from the Virtual Lab studies performed is that user-involvement in assessment is vital for improving the knowledge-experience for the user. Self-assessment hints preliminary results but are not comprehensive. Users tend to show implicit behavior patterns indicating favor of the tool rather than the experiment for their choice of vote. Interactional voting behavior is also dependent on age and other characteristic learner attitudes. In our studies, younger students mostly at the undergraduate level evaluated the tool using mid-range scores compared to the varying yet favorably high votes of the Master's and Graduate students in the feedback assessment. Although this may need further testing, we believe that scores from the higher age-experience level indicated statistically relevant reliability much more than undergraduates (data not shown due to pending experiments).

Overall, 27 Master's level students who helped in intensive evaluation of the Virtual lab platform as part of their regular class-room course, appeared predominantly positive about the value of virtual labs in e-learning, but anxieties were also expressed about the potential for e-labs to replace face-to-face teaching and labs in the economically challenged regions of the country apart an indication either of the value on the personal and face-to-face tutoring through an expressed preference for it. Students who were positive about their experiences of virtual labs indicated that they had received appropriate introductions and felt supported by staff, indicating the importance of sound inductions into the use of institutional systems and technologies.

Fig. 6. Growth of tigers with predictions. A. The plot shows the nature / pattern of statistical data for tiger population in India from 1972 – 2002 (blue line) and an extended prediction (red line) of the curve to 2012 using continuous growth model simulator. The model assumes a 10% standard deviation shown by the error bar. B. The plot shows the enlarged curve of predicted piece-wise continuous growth of tiger populations in India

#### **14. Conclusion and further remarks**

Education using VL has been the new venture to better education and provide extensible laboratory experience to University students. The virtual lab protocols for neurophysiology and related sciences have been a successful complement to the usual theoretical education that happens at our school of biotechnology at the level of masters and undergraduate education. Although the elements can be improved, our approach to virtualization has answered many key results in establishing the virtual lab features such as teacherindependent/teacher-friendly approach to e-learning.

patterns indicating favor of the tool rather than the experiment for their choice of vote. Interactional voting behavior is also dependent on age and other characteristic learner attitudes. In our studies, younger students mostly at the undergraduate level evaluated the tool using mid-range scores compared to the varying yet favorably high votes of the Master's and Graduate students in the feedback assessment. Although this may need further testing, we believe that scores from the higher age-experience level indicated statistically relevant reliability much more than undergraduates (data not shown due to

Overall, 27 Master's level students who helped in intensive evaluation of the Virtual lab platform as part of their regular class-room course, appeared predominantly positive about the value of virtual labs in e-learning, but anxieties were also expressed about the potential for e-labs to replace face-to-face teaching and labs in the economically challenged regions of the country apart an indication either of the value on the personal and face-to-face tutoring through an expressed preference for it. Students who were positive about their experiences of virtual labs indicated that they had received appropriate introductions and felt supported by staff, indicating the importance of sound

Fig. 6. Growth of tigers with predictions. A. The plot shows the nature / pattern of statistical data for tiger population in India from 1972 – 2002 (blue line) and an extended prediction (red line) of the curve to 2012 using continuous growth model simulator. The model assumes a 10% standard deviation shown by the error bar. B. The plot shows the enlarged

Education using VL has been the new venture to better education and provide extensible laboratory experience to University students. The virtual lab protocols for neurophysiology and related sciences have been a successful complement to the usual theoretical education that happens at our school of biotechnology at the level of masters and undergraduate education. Although the elements can be improved, our approach to virtualization has answered many key results in establishing the virtual lab features such as teacher-

curve of predicted piece-wise continuous growth of tiger populations in India

**14. Conclusion and further remarks** 

independent/teacher-friendly approach to e-learning.

inductions into the use of institutional systems and technologies.

pending experiments).

We tried to avoid the most usual failures in e-learning labs (Romiszowski, 2004) by focusing to avoid the common failures. Our design issues were based on a successfully tested e-learning software environment (Nedungadi and Raman, 2010) and included a clear identification and analysis of the real problem associated with University laboratory courses. Each virtual lab included overall strategic design decision such as structure of the courses, technologies employed and mode of experiment. Each experiment and the lab was considered with instructional design and elements that were evaluated so to motivate the learner experience. Such elements included the choice of graphical front-ends and authoring tools. We had also previously estimated issues related to dissemination for rapid, efficient and cost-effective usability taking into consideration both pedagogical and infrastructural complicacy.

Large scale tests will be needed to analyse and provide the assessment. These tests will also require both learners and educators (lab faculty) to use the software platform. Some tests in biotechnology are already underway via the VALUE initiative (Diwakar et al. 2011).

Several users raised the issue of how to support learners using VL. In real-world labs, learners work in the same place at the same time so there is teacher or peer support available. This kind of support is not immediately available to remote learners.

From our experience, the most vital requirement for each virtual lab is that of technical coordinators and subject matter experts whose inputs improve the lab's knowledge bank and usability. The Virtual lab project is already online for public preview via http://amrita.edu/virtuallabs or the National mission site http://vlab.co.in

#### **15. Acknowledgment**

This project derives direction and ideas from the Chancellor of Amrita University, Sri Mata Amritanandamayi Devi. The authors would also like to acknowledge the contributions of Mr. Raghu Raman, Director, CREATE, Amrita University and CREATE team. This project is funded by NMEICT, MHRD, Government of India.

#### **16. References**


### **Gender, Knowledge, Scientific Expertise, and Attitudes Toward Biotechnology: Technological Salience and the Use of Knowledge to Generate Attitudes**

Richard M. Simon *Department of Sociology, Rice University USA* 

#### **1. Introduction**

398 Innovations in Biotechnology

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Education in Developing Nations via Virtual Labs: Analysis, Implementation and Case-studies, International Journal of Computer Theory and Engineering vol. 3, no.

Distributed Virtual Labs, Proceedings of 2011 IAJC-ASEE International Conference,

Since the advent of recombinant DNA techniques in the early 1970s, biotechnologies have received much attention both for their potential to help and to harm individuals and society. Through the development of gene therapies, stem cell technologies, reproductive technologies and genetically modified crops, biotechnologies have promised to help the sick, the barren, and the poor live more fulfilling lives. At the same time governments, the general public, and scientists themselves have recognized the risks involved with biotechnologies. Since the very beginning of modern biotechnological techniques, scientists have warned that there is "serious concern that some of these artificial recombinant DNA molecules could prove biologically hazardous" (Berg et al., 1974: 303). Risks include the unknown consequences of consuming organisms that have been genetically manipulated, and the release of novel genetic material into wild populations (Torgersen et al., 2002). But aside from the technical risks involved with biotechnologies, concerns have also arisen over the ethical implications of manipulating basic life processes. Critics have rebuked scientists for "taking a technological and reductionist perception of life itself" (Torgersen et al., 2002: 39), and the seemingly imminent development of human cloning and "designer babies" (McGee, 2000; Hughes, 2004) were added to the already controversial issues of embryonic stem cell research and genetically modified organisms. The potential to simultaneously do great help, and great harm, has made biotechnology one of the most important science policy issues of our time.

Often the views of scientists, industry, government, and the public have been at odds. Because of their enormous commercial potential, industry has largely downplayed the risks and emphasized the benefits of biotechnologies (see, for example, Rampton's and Stauber's (1998) exposé of Monsanto's attempts to suppress evidence that one of its products, recombinant bovine growth hormone, causes cancer). National governments, recognizing the importance of biotechnology as a key technology in the post-industrial marketplace, have simultaneously sought to protect industry from an overzealous public, yet appear responsive to public concerns. The general public has been especially critical of biotechnologies, though the level of skepticism varies by region and the particular issue at stake (Allum et al., 2002).

In the case of biotechnology, public concern has turned out to have significant influence. In the arenas of politics and economy, public concerns about biotechnology have translated into real transformations. Bauer and Gaskell observe that,

In contemporary times, public opinion is not merely a perspective "after the fact"; it is a crucial constraint, in the dual sense of the limitations and opportunities for governments and industries to exploit the new technology. Whereas the biotechnology industry assumed that regulatory processes were the sole hurdle prior to commercialisation, it is now apparent that a second hurdle, national and international public opinion, must be taken into account (2002: 1).

Both by voting with their dollars, and by making it difficult for biotech firms to bring products to market (Weber et al., 2009), "public opinion" has affected the development and distribution of biotechnologies (for a more extended discussion of the efficacy of public opinion see Page and Shapiro, 1992). Furthermore, governmental bodies at the national level, as well as the European Union, have heard concerns and adjusted policy accordingly (Torgersen et al., 2002). Clearly, if the future of biotechnology is to be grasped, public opinion, and the processes by which it is formed, must be understood.

One issue that is frequently referred to when considering the public's attitudes is knowledge of biotechnology. Despite initial hesitance, "an increasing number of scientists concluded that the risks had been exaggerated" (Torgersen et al., 2002: 34). Yet public attitudes have become more negative, and more ambivalent, over time (Shanahan et al., 2001). Despite some studies that suggest a link between genetically modified organisms and health problems (e.g., a recent study that found a connection between the consumption of genetically modified corn and organ failure in rats; Vendômois et al., 2009), scientists have largely dismissed public outcry over biotechnologies as reactionary and a consequence of a public that remains uninformed (see, for example, McHughen, 2007). This very basic hypothesis – that people will become more accepting of biotechnology once they better understand it – has received some support in studies of public opinion (Allum et al., 2008; Bak, 2001; Wright & Nerlich, 2006). However, not all people use knowledge in the same way. The *contexts* in which knowledge is called forth have been shown to play a crucial role in how it is used to formulate opinions toward science and technology. Studies that test for interaction effects show that the effect of knowledge on attitudes varies by region (Allum et al., 2002), levels of political knowledge (Sturgis & Allum, 2004), and by gender (von Roten, 2004; Simon, 2010, 2011).

Gender differences in the use of knowledge to formulate attitudes are particularly intriguing because of the persistent gender gap in support for science and technology. Whether asked about environmental issues (Stern et al., 1993; Hayes, 2001; McCright, 2010), nuclear power (Freudenburg & Davidson, 2007; Davidson & Freudenburg, 1996; Krannich & Albrecht, 1995), biotechnologies (Qin & Brown, 2007; Bryant & Pini, 2006; Simon 2010, 2011) including reproductive technologies (Napolitano & Ogunseitan, 1999), or science in general (Trankina, 1993; Barke et al., 1997; von Roten, 2004; Mallow et al., 2010; Breakwell & Robertson 2001; Hayes & Tariq 2000), women express more skepticism toward science and technology than men do. Gender is an important determinant of people's attitudes toward science and

biotechnologies, though the level of skepticism varies by region and the particular issue at

In the case of biotechnology, public concern has turned out to have significant influence. In the arenas of politics and economy, public concerns about biotechnology have translated

Both by voting with their dollars, and by making it difficult for biotech firms to bring products to market (Weber et al., 2009), "public opinion" has affected the development and distribution of biotechnologies (for a more extended discussion of the efficacy of public opinion see Page and Shapiro, 1992). Furthermore, governmental bodies at the national level, as well as the European Union, have heard concerns and adjusted policy accordingly (Torgersen et al., 2002). Clearly, if the future of biotechnology is to be grasped, public

One issue that is frequently referred to when considering the public's attitudes is knowledge of biotechnology. Despite initial hesitance, "an increasing number of scientists concluded that the risks had been exaggerated" (Torgersen et al., 2002: 34). Yet public attitudes have become more negative, and more ambivalent, over time (Shanahan et al., 2001). Despite some studies that suggest a link between genetically modified organisms and health problems (e.g., a recent study that found a connection between the consumption of genetically modified corn and organ failure in rats; Vendômois et al., 2009), scientists have largely dismissed public outcry over biotechnologies as reactionary and a consequence of a public that remains uninformed (see, for example, McHughen, 2007). This very basic hypothesis – that people will become more accepting of biotechnology once they better understand it – has received some support in studies of public opinion (Allum et al., 2008; Bak, 2001; Wright & Nerlich, 2006). However, not all people use knowledge in the same way. The *contexts* in which knowledge is called forth have been shown to play a crucial role in how it is used to formulate opinions toward science and technology. Studies that test for interaction effects show that the effect of knowledge on attitudes varies by region (Allum et al., 2002), levels of political knowledge (Sturgis & Allum, 2004), and by gender (von Roten,

Gender differences in the use of knowledge to formulate attitudes are particularly intriguing because of the persistent gender gap in support for science and technology. Whether asked about environmental issues (Stern et al., 1993; Hayes, 2001; McCright, 2010), nuclear power (Freudenburg & Davidson, 2007; Davidson & Freudenburg, 1996; Krannich & Albrecht, 1995), biotechnologies (Qin & Brown, 2007; Bryant & Pini, 2006; Simon 2010, 2011) including reproductive technologies (Napolitano & Ogunseitan, 1999), or science in general (Trankina, 1993; Barke et al., 1997; von Roten, 2004; Mallow et al., 2010; Breakwell & Robertson 2001; Hayes & Tariq 2000), women express more skepticism toward science and technology than men do. Gender is an important determinant of people's attitudes toward science and

In contemporary times, public opinion is not merely a perspective "after the fact"; it is a crucial constraint, in the dual sense of the limitations and opportunities for governments and industries to exploit the new technology. Whereas the biotechnology industry assumed that regulatory processes were the sole hurdle prior to commercialisation, it is now apparent that a second hurdle, national and international

stake (Allum et al., 2002).

2004; Simon, 2010, 2011).

into real transformations. Bauer and Gaskell observe that,

public opinion, must be taken into account (2002: 1).

opinion, and the processes by which it is formed, must be understood.

technology, and the fact that there is some evidence that indicates that men and women use knowledge differently to form attitudes suggests this is of critical importance for understanding public attitudes toward science and technology.

In a previous study of gender differences in attitudes toward biotechnology (Simon, 2010), I found that the more knowledge that men had on the subject, the less inclined they were to be pessimistic about its effects on society. The effect I found for men clearly supports the knowledge deficit hypothesis: the more men know, the more likely they are to support biotechnology. However, the effect of knowledge on attitudes proved to be the *opposite* for women: women became more *critical* of biotechnology with more knowledge. The results of that study suggest scientific knowledge persuades men to be supportive of biotechnology, but that same knowledge causes women to be critical of it.

What is causing men and women to use knowledge in such radically different ways? One hypothesis is that science and technology, and biotechnologies in particular, have radically different implications for men and women. Because of gender roles associated with childbearing and childrearing, women are more likely to be directly affected by some of the negative consequences of science and technology. As Dorothy Nelkin suggests regarding attitudes toward nuclear power, women are its "most active and outspoken critics" because of "the special effects radiation has on the health of women and on future generations" (1981: 15). This perspective is echoed by ecofeminists such as Mallory (2006) who argue for a parallel between the exploitation of nature characteristic of science and technology and the exploitation of women, and Bryant and Pini who theorize "a relationship between chemicals and women's reproductive bodies" (2006: 268). Biotechnologies may be especially relevant to women because the implications for bodily contact and harm are an integral element of the controversies surrounding biotechnology (DuPuis, 2000). With respect to gender differences in attitudes toward biotechnology, Napolotano and Ogunseitan remark, "many of the applications [of biotechnologies] towards human health issues will likely affect fetuses, mothers, and young children more than adult males and non-childbearing female members of society" (1999: 202).

The implication of these arguments is that biotechnology is more *salient* for women than it is for men. I submit that the gender difference in *technological salience* produces the circumstances that facilitate a unique set of evaluative criteria for women that are not utilized by those with more distance from biotechnology. The hypothesis is that, for women, biotechnology is *personal* in a way that it is not for men, and the more women understand about biotechnology the better they are able to grasp this.

The technological salience explanation of gender differences in attitudes toward science and technology (developed with my colleague Katherine Johnson; Johnson and Simon, n.d.) suggests certain hypotheses. The theory states that the criteria that people will use to form attitudes toward science and technology will be determined by how salient the particular issue is to their own lived experiences. The hypotheses I intend to explore have to do with how men and women use knowledge differently with respect to their views on the public's role in science policy. It is intuitive that when people think the public should decide science policy, their own knowledge of a topic should be an important determinant of their attitude toward it. However, when people think experts should decide science policy, it is unclear how knowledge might relate to attitudes toward biotechnology. If biotechnology is really a personal matter for women, a matter relevant to their bodies, values, and roles in a way it is not for men, then knowledge of it should be absolutely crucial for determining their attitudes under nearly all circumstances. Even when women are willing to abdicate responsibility to scientific experts or government to regulate other forms of science and technology, because biotechnology has an especial salience for women, they should be unlikely to disregard their own knowledge when evaluating it. On the other hand, I predict that men's use of knowledge to generate attitudes will be conditional upon their willingness to let scientific experts and government regulate science and technology. Because biotechnology is not especially salient for men compared to women, they should categorize it alongside other issues in science and technology that are not especially salient for them. When men abdicate responsibility for science policy to experts, their own knowledge of biotechnology should be less important in determining their attitudes compared to men who think science policy should be decided by the public.

In technical terms, I am hypothesizing a three-way interaction of gender, knowledge, and willingness to leave science policy to experts in determining attitudes toward biotechnology. For women, the effect of their knowledge on their attitudes should be the same no matter what their opinions on the role of experts are (Hypothesis 1), because even if they are generally willing to let experts decide science policy, the especial salience of biotechnology for women will make their understanding of it relevant under any circumstances. But because biotechnology is not especially salient for men, when the responsibility for determining a positive or negative attitude is externalized to experts, the importance of one's own knowledge about the topic should be less important because responsibility is transferred to an external source (Hypothesis 2). Because of the particular salience of biotechnology for women, the effect of their knowledge on their attitudes should be unconditional. Because biotechnology is less salient for men, the effect of their knowledge on their attitudes should be more likely to be affected by other circumstances; in particular, it should become less relevant when they believe the public should stay out of scientific affairs. Stated formally:

**H1**: Controlling for other relevant variables, the effect of knowledge on attitudes toward biotechnology will be *unconditional* upon the opinion that experts should decide science policy issues for females (i.e., the knowledge-experts interaction will *not* significantly predict attitudes for females).

**H2**: Controlling for other relevant variables, the effect of knowledge on attitudes toward biotechnology will be weaker when males hold the opinion that experts should decide science policy issues, and stronger when males hold the opinion that the public should decide science policy issues. (i.e., the knowledge-experts interaction *will* significantly predict attitudes for males, and it will be negative).

#### **2. Data and methods**

#### **2.1 Sample**

The Eurobarometer 63.1 is used to test these hypotheses because it features items on attitudes toward biotechnology, the role of experts in scientific decision-making, and a twelve-question quiz designed to measure scientific knowledge. The Eurobarometer 63.1 is a representative sample of thirty-two European nations and includes household respondents aged 15 and over. These nations include 25 European Union member countries: Austria, Belgium, Republic of Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, and the United Kingdom, plus the EU candidate countries of Bulgaria, Croatia, Romania, Turkey, as well as three European Free Trade Association countries: Iceland, Norway, and Switzerland (Papacostas, 2006).

Data for the Eurobarometer 63.1 were collected between the 3rd of January and the 15th of February 2005 by TNS Opinion and Social on behalf of the European Commission. It utilized a probabilistic, stratified sample design in which each nation was stratified first by region, then municipality. In each country, a number of sampling points were drawn with probability proportional to population size and to population density. Finally, households were chosen within each municipality. The survey was administered at the homes of the respondents in face-to-face interviews (Papacostas, 2006).

#### **2.2 Dependent variable**

402 Innovations in Biotechnology

not for men, then knowledge of it should be absolutely crucial for determining their attitudes under nearly all circumstances. Even when women are willing to abdicate responsibility to scientific experts or government to regulate other forms of science and technology, because biotechnology has an especial salience for women, they should be unlikely to disregard their own knowledge when evaluating it. On the other hand, I predict that men's use of knowledge to generate attitudes will be conditional upon their willingness to let scientific experts and government regulate science and technology. Because biotechnology is not especially salient for men compared to women, they should categorize it alongside other issues in science and technology that are not especially salient for them. When men abdicate responsibility for science policy to experts, their own knowledge of biotechnology should be less important in determining their attitudes compared to men

In technical terms, I am hypothesizing a three-way interaction of gender, knowledge, and willingness to leave science policy to experts in determining attitudes toward biotechnology. For women, the effect of their knowledge on their attitudes should be the same no matter what their opinions on the role of experts are (Hypothesis 1), because even if they are generally willing to let experts decide science policy, the especial salience of biotechnology for women will make their understanding of it relevant under any circumstances. But because biotechnology is not especially salient for men, when the responsibility for determining a positive or negative attitude is externalized to experts, the importance of one's own knowledge about the topic should be less important because responsibility is transferred to an external source (Hypothesis 2). Because of the particular salience of biotechnology for women, the effect of their knowledge on their attitudes should be unconditional. Because biotechnology is less salient for men, the effect of their knowledge on their attitudes should be more likely to be affected by other circumstances; in particular, it should become less relevant when they believe the public should stay out of scientific

**H1**: Controlling for other relevant variables, the effect of knowledge on attitudes toward biotechnology will be *unconditional* upon the opinion that experts should decide science policy issues for females (i.e., the knowledge-experts interaction will *not*

**H2**: Controlling for other relevant variables, the effect of knowledge on attitudes toward biotechnology will be weaker when males hold the opinion that experts should decide science policy issues, and stronger when males hold the opinion that the public should decide science policy issues. (i.e., the knowledge-experts interaction *will*

The Eurobarometer 63.1 is used to test these hypotheses because it features items on attitudes toward biotechnology, the role of experts in scientific decision-making, and a twelve-question quiz designed to measure scientific knowledge. The Eurobarometer 63.1 is a representative sample of thirty-two European nations and includes household respondents aged 15 and over. These nations include 25 European Union member countries: Austria, Belgium, Republic of Cyprus, Czech Republic, Denmark, Estonia,

who think science policy should be decided by the public.

significantly predict attitudes for females).

significantly predict attitudes for males, and it will be negative).

affairs. Stated formally:

**2. Data and methods** 

**2.1 Sample** 

The purpose of this study is to investigate gender differences in attitudes toward biotechnology. Hence, the dependent variable is derived from a question asking respondents whether they think biotechnology "will have a positive, negative, or no effect on our way of life over the next twenty years." This variable was recoded so that all respondents indicating that they think biotechnology will have a negative effect "on our way of life" were coded as 1 and all other respondents were coded as 0. The dependent variable is therefore a measure of *pessimism* about biotechnology.

#### **2.3 Independent variables**

Independent variables of interest are gender, scientific knowledge, and a variable derived from the question, "Which of the two following views is closest to your own? Decisions about science and technology should be based primarily on the advice of experts about risks and benefits involved, or on the general public's views of risks and benefits?" All respondents who indicated that decisions should be left to experts were coded as 1 and all respondents who indicated that the general public should make decisions were coded as 0.

To create the gender variable used in the analysis, all males were coded 1, and all females were coded 0.

The knowledge variable is a scale constructed from a twelve question factual test on scientific knowledge administered with the Eurobarometer. This test asked questions about science in general, not about biotechnology specifically. The items were recoded so that correct responses were equal to 1, and incorrect responses were equal to 0. Each respondent's score on the knowledge variable is equal to the proportion of correct answers they made on the knowledge test (e.g., someone who earned eight correct answers would be coded as .67). The knowledge scale obtained an alpha = .72.

Control variables included variables capturing political attitudes, age, education, and religiosity. The political attitudes variable was derived from responses to the question: "In political matters people talk of 'the left' and 'the right'. How would you place your views on this scale?" Respondents were asked to rate their political attitudes on a scale of 1 to 10, with lower values indicating that they are more "left", and higher values indicating that they are more to the "right". Respondents' age was coded in years. The Eurobarometer's education item does not directly measure respondents' educational attainment; instead respondents are asked to give the age at which they left school. This variable was recoded into dummies that roughly correspond to stages in the educational career: respondents who left school before high school age ("Edu. 0-14"), those of high school age ("Edu. 15-18"), those of college age but who have not reached the typical age of college graduation ("Edu. 19-21"), and those who left school after the age of 22 ("Edu. 22+"), assumedly college graduates. Another dummy was created for respondents who had not yet left school ("still studying"). This education measure is not ideal, but previous work (Simon, 2010, 2011) with the Eurobarometer's education measure has showed that it possesses criterion validity when predicting attitudes toward biotechnology. Respondents' religiosity was measured with an ordinal item asking respondents, "Apart from weddings or funerals, about how often do you attend religious services?", with responses ranging from "never" to "more than once a week". Higher values indicate more frequent attendance of religious services, and lower values indicate less frequent attendance.

#### **2.4 Analytic sample**

The total number of respondents included in the Eurobarometer 63.1 is 31,390. Missing values were addressed using pairwise deletion. After deleting missing values the sample was reduced to 26,621. Because of the complex sampling design, the data were weighted with a weight designed to accommodate analyses that make use of all the nations in the study. Weighting the data further reduced the analytic sample to 24,630. Splitting the sample by gender resulted in an *n* = 12,379 for males, and an *n* = 12,251 for females.

#### **2.5 Analytic approach**

Hypothesis 1 predicts that the experts-knowledge interaction will not be significant in the female model. Hypothesis 2 predicts that the experts-knowledge interaction will be significant and negative in the male model. To test the hypotheses, the sample was split by gender, and separate logistic regression models predicting attitudes toward biotechnology (with "it will have a negative effect on our way of life" coded as "1") were run for males and females, including a test of the "experts-knowledge" interaction in both models.

#### **3. Results**

Table 1 shows means (or proportions) and standard deviations for all variables by gender. A few items are of note. First, while slightly less than 20 percent of males thought biotechnology will have a negative effect "on our way of life over the next twenty years", slightly more than 22 percent of females had the same opinion. While the gender difference in attitudes is slight, it is consistent with previous research that has shown females to be more skeptical of biotechnology than males. Also consistent with previous research, females possess less scientific knowledge than males. (averaging .59 on the knowledge test vs. .66, respectively, a difference of nearly one entire correct answer). Approximately three-quarters of both males and females believe that experts, not the general public, should decide science policy issues.



Males: *n* = 12,379

404 Innovations in Biotechnology

lower values indicating that they are more "left", and higher values indicating that they are more to the "right". Respondents' age was coded in years. The Eurobarometer's education item does not directly measure respondents' educational attainment; instead respondents are asked to give the age at which they left school. This variable was recoded into dummies that roughly correspond to stages in the educational career: respondents who left school before high school age ("Edu. 0-14"), those of high school age ("Edu. 15-18"), those of college age but who have not reached the typical age of college graduation ("Edu. 19-21"), and those who left school after the age of 22 ("Edu. 22+"), assumedly college graduates. Another dummy was created for respondents who had not yet left school ("still studying"). This education measure is not ideal, but previous work (Simon, 2010, 2011) with the Eurobarometer's education measure has showed that it possesses criterion validity when predicting attitudes toward biotechnology. Respondents' religiosity was measured with an ordinal item asking respondents, "Apart from weddings or funerals, about how often do you attend religious services?", with responses ranging from "never" to "more than once a week". Higher values indicate more frequent attendance of religious services, and lower

The total number of respondents included in the Eurobarometer 63.1 is 31,390. Missing values were addressed using pairwise deletion. After deleting missing values the sample was reduced to 26,621. Because of the complex sampling design, the data were weighted with a weight designed to accommodate analyses that make use of all the nations in the study. Weighting the data further reduced the analytic sample to 24,630. Splitting the

Hypothesis 1 predicts that the experts-knowledge interaction will not be significant in the female model. Hypothesis 2 predicts that the experts-knowledge interaction will be significant and negative in the male model. To test the hypotheses, the sample was split by gender, and separate logistic regression models predicting attitudes toward biotechnology (with "it will have a negative effect on our way of life" coded as "1") were run for males and

Table 1 shows means (or proportions) and standard deviations for all variables by gender. A few items are of note. First, while slightly less than 20 percent of males thought biotechnology will have a negative effect "on our way of life over the next twenty years", slightly more than 22 percent of females had the same opinion. While the gender difference in attitudes is slight, it is consistent with previous research that has shown females to be more skeptical of biotechnology than males. Also consistent with previous research, females possess less scientific knowledge than males. (averaging .59 on the knowledge test vs. .66, respectively, a difference of nearly one entire correct answer). Approximately three-quarters of both males and females believe that experts, not the

sample by gender resulted in an *n* = 12,379 for males, and an *n* = 12,251 for females.

females, including a test of the "experts-knowledge" interaction in both models.

general public, should decide science policy issues.

values indicate less frequent attendance.

**2.4 Analytic sample** 

**2.5 Analytic approach** 

**3. Results** 

Females: *n* = 12,251

Table 1. Mean or proportion and standard deviation by gender.

Table 2 features results of a series of logistic regressions predicting attitude toward biotechnology for females only. Odds ratios are shown. Model 1 features only the knowledge scale as an independent variable; it is positive and significant. Without controlling for any other variables, each additional correct answer on the knowledge test increases the likelihood of being pessimistic about biotechnology by about 33 percent. Model 2 adds the experts variable as a predictor; it is negative and significant. Controlling for level of scientific knowledge, feeling that experts and not the public should be responsible for science policy decreases the odds of being pessimistic toward biotechnology by about 31 percent. Model 3 retains the knowledge and experts variables as predictors, but adds the knowledge-experts interaction term. Recall that Hypothesis 1 predicted that, for females, the effect of knowledge on attitudes toward biotechnology would be about the same regardless of the respondent's opinion about the role experts should play in science policy issues. Model 3 reveals that the effect of knowledge on attitude is not affected by opinion about the role of experts in science policy. Model 4 shows that this is true even when controlling for political attitude, age, religiosity, and education, confirming Hypothesis 1.

Table 3 shows logistic regression results for the male sample only. Model 1 uses only the knowledge scale to predict pessimism toward biotechnology. Without accounting for any other variables, each additional correct answer on the knowledge test results a reduction in the odds of being pessimistic about biotechnology of about 20 percent for males. However, adding the experts variable reduces the effect of knowledge to insignificance. Like their female counterparts, when males leave policy decisions up to experts, they are much less likely to oppose biotechnology. Model 3 adds the interaction term. The effect is strong, negative, and highly significant. The correlation between knowledge and attitude is much weaker for males who think science policy should be decided by experts compared to males who think it should be decided by the public. Model 4 includes the control variables. The experts-knowledge interaction effect on attitude is slightly reduced when controlling for political ideology, age, religiosity, and education, though it is still strong, negative, and significant, confirming Hypothesis 2. This is a much different scenario than for females, for which the experts-knowledge interaction did not predict biotechnology attitude.

To further illustrate how opinion on the role of experts in science policy tempers the effect of knowledge on biotechnology attitude for males but not for females, each male and female sample was further split by whether the respondent thought science policy decisions should be made by experts or by the public. Table 4 shows results of logistic regressions predicting biotechnology attitude by gender and science policy opinion. Model 1 displays the coefficients only for females who believe that science policy decisions should be left to experts. With all of the control variables in the equation, each additional correct answer on the knowledge test results in a 53 percent increase in the odds of being pessimistic about biotechnology. Model 2 shows results only for females who think the public should decide science policy issues. For these women, greater knowledge also leads greater odds of rejecting biotechnology. Model 3 features the same regression model, but this time only for males who put their faith in experts. Controlling for political views, age, religiosity, and education, the effect of knowledge on attitude is strong and negative: with each additional correct answer on the knowledge test, the odds of being pessimistic about biotechnology are reduced by about 47 percent.


\*sig. at p.<.05

\*\*sig. at p.<.01

\*\*\*sig. at p.<.001

However, the effect of knowledge on attitude is drastically different for males who think science policy should be the domain of the public. For these males, the effect of knowledge is strong and *positive*, nearly doubling the odds of being pessimistic about biotechnology with each additional correct answer on the knowledge test. For females, more knowledge leads to a greater likelihood of rejecting biotechnology no matter who they think should be making science policy decisions, and in both cases the effect of knowledge on attitude is about the same. For males, the effect of knowledge on attitude cannot be predicted unless the respondent's opinion on science policy decisions is known.


\*sig. at p.<.05 \*\*sig. at p.<.01

\*\*\*sig. at p.<.001

Table 3. Logistic regressions predicting whether or not biotechnology will make things worse, male sample only

#### **4. Discussion**

406 Innovations in Biotechnology

is much weaker for males who think science policy should be decided by experts compared to males who think it should be decided by the public. Model 4 includes the control variables. The experts-knowledge interaction effect on attitude is slightly reduced when controlling for political ideology, age, religiosity, and education, though it is still strong, negative, and significant, confirming Hypothesis 2. This is a much different scenario than for females, for which the experts-knowledge interaction did not predict

To further illustrate how opinion on the role of experts in science policy tempers the effect of knowledge on biotechnology attitude for males but not for females, each male and female sample was further split by whether the respondent thought science policy decisions should be made by experts or by the public. Table 4 shows results of logistic regressions predicting biotechnology attitude by gender and science policy opinion. Model 1 displays the coefficients only for females who believe that science policy decisions should be left to experts. With all of the control variables in the equation, each additional correct answer on the knowledge test results in a 53 percent increase in the odds of being pessimistic about biotechnology. Model 2 shows results only for females who think the public should decide science policy issues. For these women, greater knowledge also leads greater odds of rejecting biotechnology. Model 3 features the same regression model, but this time only for males who put their faith in experts. Controlling for political views, age, religiosity, and education, the effect of knowledge on attitude is strong and negative: with each additional correct answer on the knowledge test, the odds of being pessimistic about biotechnology are

> Knowledge 1.325\*\* 1.539\*\*\* 1.354 1.421\* Experts .686\*\*\* .609\*\* .698 KnowXExperts 1.208 1.029 Politics .964\*\* Age 1.003 Religiosity 1.017 Edu 0-14 .956 Edu 15-18 1.134 Edu 19-21 1.068 Edu Still Studying .753\*

Table 2. Logistic regressions predicting whether or not biotechnology will make things

Model 1 Model 2 Model 3 Model 4

biotechnology attitude.

reduced by about 47 percent.

(Edu 22+ ref cat)

\*sig. at p.<.05 \*\*sig. at p.<.01 \*\*\*sig. at p.<.001

worse, female sample only.

This paper made two predictions about how males and females use knowledge differently to generate attitudes toward biotechnology based on the idea of *technological salience*. It was predicted that because biotechnologies are more salient to females compared to males, the effect of their knowledge on their attitudes should hold no matter what their opinions on the role of experts are, because even if they are generally willing to let experts decide science policy, the especial salience of biotechnology for women will make their understanding of it relevant under a more robust set of circumstances.

But because biotechnology is not especially salient for men, when the responsibility for determining a positive or negative attitude is externalized to experts, the importance of one's own knowledge about the topic should be less important because responsibility is transferred to an external source. Because of the particular salience of biotechnology for women, the effect of their knowledge on their attitudes should be unconditional. Because biotechnology is less salient for men, the effect of their knowledge on their attitudes should be more likely to be affected by other circumstances; in particular, it should become less relevant when they believe the public should stay out of scientific affairs. Hypothesis 1 predicted that the experts-knowledge interaction would not significantly predict biotechnology attitude for females; that is, the effect of knowledge on attitude should be the same regardless of their opinion about who should make science policy decisions. Table 2 showed that, indeed, the experts-knowledge interaction was not significant for females. Hypothesis 2 predicted that the effect of knowledge on attitudes toward biotechnology should be weaker when males hold the opinion that experts should decide science policy issues, and stronger when males hold the opinion that the public should decide science policy issues; that is, the knowledge-experts interaction should be *negative* and significant for males. The evidence in Table 3 supports this hypothesis. When males do not trust the general public to make good policy decisions, their own knowledge is less important in determining their attitude toward biotechnology. Females, however, seem equally insistent upon using the knowledge they have under any circumstances.


\*sig. at p.<.05

(Edu 22+ ref cat)

\*\*sig. at p.<.01

\*\*\*sig. at p.<.001

Table 4. Logistic regressions predicting whether or not biotechnology will make things worse, by gender and opinion on expertise.

But because biotechnology is not especially salient for men, when the responsibility for determining a positive or negative attitude is externalized to experts, the importance of one's own knowledge about the topic should be less important because responsibility is transferred to an external source. Because of the particular salience of biotechnology for women, the effect of their knowledge on their attitudes should be unconditional. Because biotechnology is less salient for men, the effect of their knowledge on their attitudes should be more likely to be affected by other circumstances; in particular, it should become less relevant when they believe the public should stay out of scientific affairs. Hypothesis 1 predicted that the experts-knowledge interaction would not significantly predict biotechnology attitude for females; that is, the effect of knowledge on attitude should be the same regardless of their opinion about who should make science policy decisions. Table 2 showed that, indeed, the experts-knowledge interaction was not significant for females. Hypothesis 2 predicted that the effect of knowledge on attitudes toward biotechnology should be weaker when males hold the opinion that experts should decide science policy issues, and stronger when males hold the opinion that the public should decide science policy issues; that is, the knowledge-experts interaction should be *negative* and significant for males. The evidence in Table 3 supports this hypothesis. When males do not trust the general public to make good policy decisions, their own knowledge is less important in determining their attitude toward biotechnology. Females, however, seem equally insistent

> Females Males Experts Public Experts Public

Knowledge 1.563\* 1.171\* .526\*\*\* 1.925\* Politics .967\* .961 .952\*\*\* .950\* Age 1.000 1.012\*\*\* .998 .996 Religiosity 1.027 .988 .981 1.021 Edu 0-14 1.091 .702 1.290\* 1.590\* Edu 15-18 1.155 1.071 1.178 1.166 Edu 19-21 1.105 1.000 1.343\*\* 1.257 Edu Still Studying .606\*\*\* 1.314 1.477\*\* .784

Table 4. Logistic regressions predicting whether or not biotechnology will make things

upon using the knowledge they have under any circumstances.

(Edu 22+ ref cat)

\*sig. at p.<.05 \*\*sig. at p.<.01 \*\*\*sig. at p.<.001

worse, by gender and opinion on expertise.

Table 4 lends further support to the theory of technological salience, and complicates the way we understand how men's and women's knowledge differently affects their attitudes toward biotechnology. Consistent with previous research on how males and females use knowledge to generate attitudes (Simon, 2010), this study found that women become more likely to reject biotechnology with greater knowledge of it, and this relationship holds regardless of their opinion about who should make decisions about science in society. It is intuitive that when people think science should be controlled by the public, their own knowledge of a topic should be an important determinant of their attitude toward it. However, for women, even when they believe that science is best run by the experts, their knowledge is just as important for determining their attitude toward biotechnology, and in both cases they become more skeptical of it the more they understand. Table 4 also shows the dramatic difference in the way males who trust experts use knowledge compared to males who believe science policy decisions should be a public affair. Though Hypothesis 2 – that knowledge would be less important in determining attitude for males who abdicate to experts compared to males who think the public should be responsible for science policy decisions – was supported in by the results displayed in Table 3, Table 4 revealed that knowledge still significantly predicted attitude for males who defer to experts. While the effect of knowledge on attitude is stronger for males who think science policy should be decided by the public, Table 4 shows another telling gender difference. Males who trust experts become *less* likely to reject biotechnology with increasing levels of knowledge; males who insist that science should be controlled by the public become *more* likely to reject biotechnology with increasing levels of knowledge.

I propose that the explanation that best accommodates these findings is the differential salience of biotechnologies for women compared to men. Previous research has suggested that because of gender roles associated with childbearing and childrearing, women are more likely to be directly affected by some of the negative consequences of biotechnologies (Napolitano & Ogunseitan 1999; Bryant & Pini 2006). The more women understand biotechnology, the more they see the personal implications, and the more skeptical they become. When a technology is particularly salient to one's lived experiences, it is likely to be viewed from a much more personal perspective, one in which opinions about the role of experts in policy decisions become of secondary importance compared to how the technology is likely to affect one's own life.

Because biotechnologies are not as salient to males, they use knowledge to generate their attitudes in a different way. This study has suggested that when males award experts responsibility for making important decisions about science in society, the way they use knowledge largely conforms to the deficit model (Allum et al., 2008; Bak, 2001; Wright & Nerlich, 2006): the more they know, the more supportive they become. This seems to typify the kind of male frequently described in the literature on gender and science, one that has much invested in hegemony of scientific institutions, and the one described so eloquently by Hayes:

Because men have historically commanded the technoscientific components of society, they have not only acquired the necessary scientific and technological knowledge to dominate nature but have also been socialised to unecological attitudes toward the environment. Denied access to the technoscientific realm, not only have women been traditionally prevented from acquiring this knowledge but they have also been socialised to the more ecologically benign roles of mother and nurturer as reflected in their reproductive and greater child-rearing responsibilities within society (2001: 658).

However, not all males fit this stereotype. Although only about 27 percent of males in this study thought that science policy should be decided by the public, those who did became more critical of biotechnology with increasing knowledge of it, an effect of knowledge typical of females.

This study has several implications for the future of biotechnology and the study of its relations with society. If public opinion has been as instrumental in the development of biotechnologies as Bauer and Glaskel (2002) insist that it has been, then it seems that gendered experiences of biotechnologies have been instrumental in equal measure. In Bauer and Glaskel's edited volume on public perceptions of biotechnology – the most comprehensive and influential single text on this subject to date – gender is hardly mentioned at all, and when it is it is utilized as a control variable, not a focus of sustained analysis. If the future of biotechnology depends on its public reception, then it is also dependent upon gender roles and experiences, and how they relate to biotechnologies.

What do these results tell us about the relationship between understanding biotechnology and its acceptance? As Priest remarks, "It remains tempting for the scientific community and those who speak in public on its behalf to assume that dissent generally represents ignorance, and that it therefore can be reduced or eliminated by education" (2001: 98). But charges by scientific experts that resistance to biotechnologies is a consequence of an illinformed public are only half true. It seems that, rather than providing the general public with accurate information, advocates of biotechnologies would have an interest in keeping women ignorant. Proponents of the "public understanding of science" have largely missed the point when it comes to women because they tend to see science and technology in terms of an objective cost-benefit analysis (e.g., Wertz et al., 1986) rather than as objects of lived experience. Embryonic stem cell techniques may sound innocuous to men, who may view the experimental material simply as a collection of cells. But when you have had a fetus in your own body, or even simply have the potential to carry a child, then embryonic stem cell techniques become more about powerful social institutions doing things to your body, and less about the greater good for humanity.

While this study is suggestive, it is also limited in some respects. The argument rests on claims about women's knowledge of biotechnology, though the knowledge variable used in the analysis measures general scientific knowledge, not knowledge about biotechnology specifically. It is assumed that general scientific knowledge can be taken as a faithful proxy for knowledge of biotechnology. There are surely arguments that could be summoned to challenge this assumption. However, it should be noted that the gender-knowledge interaction found here is similar to that found in a previous study (Simon, 2010) that made use of a knowledge test focused exclusively on genetics and biotechnologies. Another limitation is that the salience of biotechnology to respondents was not directly tested, but merely inferred from previous research on gender and technology and the gender differences in attitude formation observed from analyses of the Eurobarometer 63.1. I have elsewhere begun to perform more direct tests of technological salience with Katherine Johnson (Johnson and Simon, n.d.), but more research needs to be done to rule out alternative hypotheses. Perhaps there is some other gender division that is causing men and women to use knowledge in such disparate ways. The technological salience hypothesis is consistent with the findings presented here, but more direct tests need to be performed before it can be accepted with confidence.

#### **5. Conclusions**

410 Innovations in Biotechnology

However, not all males fit this stereotype. Although only about 27 percent of males in this study thought that science policy should be decided by the public, those who did became more critical of biotechnology with increasing knowledge of it, an effect of knowledge

This study has several implications for the future of biotechnology and the study of its relations with society. If public opinion has been as instrumental in the development of biotechnologies as Bauer and Glaskel (2002) insist that it has been, then it seems that gendered experiences of biotechnologies have been instrumental in equal measure. In Bauer and Glaskel's edited volume on public perceptions of biotechnology – the most comprehensive and influential single text on this subject to date – gender is hardly mentioned at all, and when it is it is utilized as a control variable, not a focus of sustained analysis. If the future of biotechnology depends on its public reception, then it is also dependent upon gender roles and experiences, and how they relate to biotechnologies.

What do these results tell us about the relationship between understanding biotechnology and its acceptance? As Priest remarks, "It remains tempting for the scientific community and those who speak in public on its behalf to assume that dissent generally represents ignorance, and that it therefore can be reduced or eliminated by education" (2001: 98). But charges by scientific experts that resistance to biotechnologies is a consequence of an illinformed public are only half true. It seems that, rather than providing the general public with accurate information, advocates of biotechnologies would have an interest in keeping women ignorant. Proponents of the "public understanding of science" have largely missed the point when it comes to women because they tend to see science and technology in terms of an objective cost-benefit analysis (e.g., Wertz et al., 1986) rather than as objects of lived experience. Embryonic stem cell techniques may sound innocuous to men, who may view the experimental material simply as a collection of cells. But when you have had a fetus in your own body, or even simply have the potential to carry a child, then embryonic stem cell techniques become more about powerful social institutions doing things to your body, and

While this study is suggestive, it is also limited in some respects. The argument rests on claims about women's knowledge of biotechnology, though the knowledge variable used in the analysis measures general scientific knowledge, not knowledge about biotechnology specifically. It is assumed that general scientific knowledge can be taken as a faithful proxy for knowledge of biotechnology. There are surely arguments that could be summoned to challenge this assumption. However, it should be noted that the gender-knowledge interaction found here is similar to that found in a previous study (Simon, 2010) that made use of a knowledge test focused exclusively on genetics and biotechnologies. Another limitation is that the salience of biotechnology to respondents was not directly tested, but merely inferred from previous research on gender and technology and the gender differences in attitude formation observed from analyses of the Eurobarometer 63.1. I have

society (2001: 658).

less about the greater good for humanity.

typical of females.

women been traditionally prevented from acquiring this knowledge but they have also been socialised to the more ecologically benign roles of mother and nurturer as reflected in their reproductive and greater child-rearing responsibilities within

> This chapter has sought to utilize the technological salience hypothesis to better understand gender differences in attitudes toward biotechnology. The analyses presented here support the idea that the salience of technologies to one's lived experiences engender unique criteria with which to evaluate those technologies. Based on previous research that has suggested that biotechnologies are especially salient for women compared to men, it was predicted that women would be less likely to discount their own knowledge in forming attitudes than men. This prediction was affirmed. Further analyses of the Eurobarometer 63.1 indicated that for men who concede science policy to experts, higher levels of scientific knowledge lead to a lesser likelihood of being pessimistic of biotechnology, while for men who insist that science policy should be the public's responsibility, higher levels of scientific knowledge lead to a greater likelihood of being pessimistic about biotechnology. While the analyses support the technological salience hypothesis, further research should use more direct measures of salience, rather than assuming gender automatically determines salience, as I have done.

#### **6. References**


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### **Structural Bioinformatics for Protein Engineering**

Davi S. Vieira, Marcos R. Lourenzoni, Carlos A. Fuzo, Richard J. Ward and Léo Degrève *University of São Paulo - FFCLRP, Departament of Chemistry Ribeirão Preto, São Paulo Brazil* 

#### **1. Introduction**

414 Innovations in Biotechnology

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Proteins are amongst the most abundant and functionally diverse macromolecules present in all living cells, and are linear heteropolymers synthesized using the same set of 20 amino acids found in all organisms from the archebacteria to more complex forms of life. By virtue of their vast diversity of amino acid sequences, a single cell may contain a huge variety of proteins, ranging from small peptides to large protein complexes with molecular weights in the range of 103-106 Daltons. Consequently, proteins show impressive diversity in biological function which includes enzyme catalysis, signal transmission via hormones, antibodies, transport, muscle contraction, antibiotics, toxic venom components and many others. Among these proteins, it may be argued that enzymes present the greatest variety and specialization, since virtually all the chemical reactions in the cell are catalyzed by enzymes (Aehle, 2004).

Due to their biotechnological and biomedical potential, enzymes and antimicrobial peptides have been the focus of extensive research efforts. This interest has been driven by a growing market for enzymes for various applications such as food supplements for humans and animals, enzymes to replace organochlorine compounds in some industrial processes, production of biofuels, vaccines, drugs, etc. (Aehle, 2004). Furthermore, the applications of enzymes in health, food, energy, materials and environment is likely to increase.

The elucidation of the three-dimensional structure of a protein is a fundamental step to understand its biological function. The 3D structure can be obtained by experimental techniques such as X-ray crystallography, nuclear magnetic resonance spectroscopy (NMR) and more recently by cryo-electron microscopy. As of mid-May 2011 there were more than 73,000 protein structures deposited in the Protein Data Bank (PDB - www.rcsb.org/pdb), of which approximately 90% have been determined by X-ray diffraction, 9% by NMR and 1% by other techniques.

Despite the availability of established experimental methodologies for the determination and analysis of three-dimensional structures of proteins to elucidate their structure-activity relationships, there are inherent limitations which can restrict their application. X-ray diffraction determines the spatial electron density distribution in the biomolecule and thereby provides the Cartesian coordinates of all atoms in the protein, except hydrogens. NMR spectroscopy is limited to low molecular weight proteins, generally smaller than 30kDa, although with the introduction of TROSY-based experiments (transverse relaxationoptimized spectroscopy) the molecular weight limitation has been extended to approximately 800kD (Fernández & Wider, 2003). Cryo-electron microscopy is a lowresolution technique that is a powerful tool for the determination of the structure of large proteins or protein complexes, such as virus structures (Mancini *et. al.,* 2000).

Conformational changes and dynamics are key processes to understand biological mechanisms and functions. Many biological processes involve functionally important changes in the protein 3D structure, however techniques for structure determination do not describe (or describe poorly) protein dynamics. Examples of the importance of protein dynamics in structural biology include the conformational changes associated with protein folding, catalytic functions of enzymes and signal transduction. In this context, bioinformatics techniques, such as molecular dynamics (MD) simulation, together with homology and statistical analysis of protein structures have become useful tools in structural biology not only for understanding protein function (van Gunsteren et. al., 2008), but also for protein engineering (Vieira & Degrève, 2009). Growing interest in bioinformatics techniques and advances in experimental methodologies have led to many successes in engineering enzyme function (Aehle, 2004).

The purpose of this chapter is to introduce the use of MD simulations in a biological context to target proteins of biotechnological interest, such as xylanases, antibody/antigen interactions and antimicrobial peptides. Using relevant models we discuss the use of MD simulations to study properties such as enzyme thermostability, protein conformational changes and functional movements, intra and intermolecular interactions through hydrogen bonding analysis, protein-protein interaction and modelling of peptidemembrane interactions. The main goal is to demonstrate how the three-dimensional structure, function, interaction potential and dynamics are highly correlated and together comprise a valuable data set to explore the broad research field of structure/function relationships of proteins.

#### **2. An introduction to molecular dynamics simulations**

Molecular dynamics (MD) simulation is one of the main computational methodologies which can provide detailed microscopic information at an atomic and molecular level for a variety of systems. The behaviour of matter can be understood by the structure and dynamics of its constituent atoms or atomic groups, and is considered as an *N*-body problem. Due to the lack of general analytical algorithms, a solution to the classical *N*-body problem is exclusively a numerical task. Therefore, the study of matter at this level requires the computational resources to allow the investigation of the movements of individual particles or molecules, and the MD approach aims to reproduce the properties of a real system using a microscopic model system. The continual increase in computational power enables the investigation of increasingly large model systems, and consequently to approach broader and more complex biological questions. MD simulation is not limited to biological and biochemical systems, and MD has a broad range of applications in material science, physics, chemistry and engineering (Leach, 2001).

thereby provides the Cartesian coordinates of all atoms in the protein, except hydrogens. NMR spectroscopy is limited to low molecular weight proteins, generally smaller than 30kDa, although with the introduction of TROSY-based experiments (transverse relaxationoptimized spectroscopy) the molecular weight limitation has been extended to approximately 800kD (Fernández & Wider, 2003). Cryo-electron microscopy is a lowresolution technique that is a powerful tool for the determination of the structure of large

Conformational changes and dynamics are key processes to understand biological mechanisms and functions. Many biological processes involve functionally important changes in the protein 3D structure, however techniques for structure determination do not describe (or describe poorly) protein dynamics. Examples of the importance of protein dynamics in structural biology include the conformational changes associated with protein folding, catalytic functions of enzymes and signal transduction. In this context, bioinformatics techniques, such as molecular dynamics (MD) simulation, together with homology and statistical analysis of protein structures have become useful tools in structural biology not only for understanding protein function (van Gunsteren et. al., 2008), but also for protein engineering (Vieira & Degrève, 2009). Growing interest in bioinformatics techniques and advances in experimental methodologies have led to many successes in

The purpose of this chapter is to introduce the use of MD simulations in a biological context to target proteins of biotechnological interest, such as xylanases, antibody/antigen interactions and antimicrobial peptides. Using relevant models we discuss the use of MD simulations to study properties such as enzyme thermostability, protein conformational changes and functional movements, intra and intermolecular interactions through hydrogen bonding analysis, protein-protein interaction and modelling of peptidemembrane interactions. The main goal is to demonstrate how the three-dimensional structure, function, interaction potential and dynamics are highly correlated and together comprise a valuable data set to explore the broad research field of structure/function

Molecular dynamics (MD) simulation is one of the main computational methodologies which can provide detailed microscopic information at an atomic and molecular level for a variety of systems. The behaviour of matter can be understood by the structure and dynamics of its constituent atoms or atomic groups, and is considered as an *N*-body problem. Due to the lack of general analytical algorithms, a solution to the classical *N*-body problem is exclusively a numerical task. Therefore, the study of matter at this level requires the computational resources to allow the investigation of the movements of individual particles or molecules, and the MD approach aims to reproduce the properties of a real system using a microscopic model system. The continual increase in computational power enables the investigation of increasingly large model systems, and consequently to approach broader and more complex biological questions. MD simulation is not limited to biological and biochemical systems, and MD has a broad range of applications in material science,

proteins or protein complexes, such as virus structures (Mancini *et. al.,* 2000).

engineering enzyme function (Aehle, 2004).

**2. An introduction to molecular dynamics simulations** 

physics, chemistry and engineering (Leach, 2001).

relationships of proteins.

MD simulations are applied to systems that are in states in which quantum effects are disregarded, which imposes a limit on the types of problem that can be addressed. For example, chemical reactions and transitions between different electronic states present a quantum nature due to their dimensional scale and their high energies, and cannot be treated by MD. MD simulation employs the classical equations to describe the motion of atoms and molecules as a function of the time under determined conditions (pH, temperature and pressure) taking into account an intermolecular interaction potential. Hybrid methodologies (Quantum Mechanics/Molecular Mechanics, QM/MM) are capable of treating quantum phenomena in MD simulations to describe, for example, chemical reactions using Valence Bond MD. Other well established hybrid methods are Carr-Parrinelo and *ab initio* MD (Leach, 2001), however these are limited by the intensive computing power required.

The use of MD simulation allows the study of the temporal evolution of molecular motions, generating a series of complete atomic coordenates (ie. Cartesian *x,y,z* coordinates for each atom) that permit the prediction of microscopic and macroscopic properties of the system from Statistical Mechanics analysis (McQuarrie, 1976). Starting from a pre-defined atomic arrangement that defines the initial configuration of the system, the individual atoms move under the influence of their intermolecular potentials. Given the atomic positions and velocities at a given time (*t*), the resulting forces can be calculated for each particle in the system permitting calculation of the atomic positions and velocities at a later time (*t+dt*). This procedure generates molecular trajectories for the whole system over the total simulation time.

The choice of the number of atoms in a system depends on the properties that are to be studied with the available computational capacity, and must be representative of the macroscopic real system. The current computational power with present methods allows simulations of systems constituted by up to 108 atoms. Recently, a 1-milliseconds all-atom MD simulation of a folded protein has been reported (Shaw et. al., 2010). The equilibrium properties of the system are computed as time averages over given time intervals that must be somewhat longer than the corresponding event observed at the atomic scale. Temperature, radial pair distribution functions, potential and kinetic energies are examples of fast convergence properties, while surface tension, diffusion coefficient and pressure are properties of slow convergence. An important characteristic of MD simulation is that information on the system is gained on a time scale that is often not easily obtained by experimental approaches (Rapaport, 2005).

#### **2.1 Molecular dynamics and statistical mechanics**

Statistical mechanics deals with ensemble averages, and is well suited to analysis of the data produced by MD methods. During MD simulations, the mechanical, thermal and chemical thermodynamic variables must be fixed and simulations are usually conducted under constant *NPT*, *NVT*, *NVE* or *μNT* conditions. (where *N* is the number of atoms, *P* the pressure, *T* the temperature, *V* the volume, *E* the internal energy and *μ* the chemical potential). An ensemble is a large set of replicas generated under the defined external conditions (McQuarrie, 1976), and in the *NVT* (or canonical) ensemble, *N*, *V* and *T* are fixed external parameters and therefore the instantaneous temperature (obtained from the kinetic energy) must be controlled to a fixed value by an thermostat algorithm, which multiplies the instantaneous atomic velocities by a coefficient defined by the difference between instantaneous and desired temperatures. Several algorithms can be used to control the temperature including the Berendsen, Nosé-Hoover and V-rescale thermostats (Allen & Tildesley, 1992; Rapaport 2005). *NVT* and *NPT* ensembles are widely used in thermal treatments such as annealing in order to promote thermal denaturation of proteins in unphysical conditions (Rocco et. al.; 2008). *NPT* or isobaric-isothermal ensembles present better characteristics than the canonical ensemble, since the lack of the thermodynamic information about the equilibrium conditions are circumvented by the adjustment of the pressure to the imposed external conditions. This allows the volume to fluctuate by scaling the dimensions of the system by a coefficient defined by the instantaneous and desired pressures.

The equilibrium average of a given property *A* is expressed by:

$$\left\langle A \right\rangle\_{ensemble} = \frac{\iint A(\vec{p}^N, \vec{r}^N) e^{-\beta \mathbb{E}(\vec{p}^N, \vec{r}^N)} d\vec{p}^N d\vec{r}^N}{Q} \tag{1}$$

where *Np* and *Nr* are the momenta and positions of the *N* particles at time t, *N N <sup>E</sup>*(,) *<sup>p</sup> <sup>r</sup>* is the sum of the potential, *<sup>N</sup> U r*( ) , and kinetic energy, *<sup>N</sup> <sup>K</sup>*( ) *<sup>p</sup>* , and β = 1 /*kT* . The denominator term, *Q*, is the partition function. For a system of *N* identical particles the partition function for the canonical ensemble is:

$$Q\_{NVT} = \frac{1}{N!} \frac{1}{h^{\otimes N}} \iint e^{-\beta E(\vec{p}^N, \vec{r}^N)} d\vec{p}^N d\vec{r}^N \tag{2}$$

where *h* is the Planck's constant. The partition function is central to the statistical mechanics analysis since it contains all the information to determine the macroscopic thermodynamics properties, which are crucial for correlating the microscopic and macroscopic levels (McQuarrie, 1976). Generally, the partition function cannot be computed since it depends on the interatomic forces and positions.

The most fundamental axiom of statistical mechanics, the ergodic hypothesis, states that the ensemble average equals the time average.

$$\left\langle A \right\rangle\_{ensemble} = \left\langle A \right\rangle\_{time} \tag{3}$$

This theory is based on the fact that if one allows the system to evolve indefinitely in time it will pass through all possible energy states, therefore the MD simulation should generate enough configurations in time to ensure that the equality is satisfied. The average in eq.(1) is an average over all the possible positions of the particles. During a MD simulation, the averages are calculated over the configurations obtained as a function of time so that in accord with the ergodicity hypothesis:

$$\left\langle A \right\rangle\_{\text{time}} = \lim\_{\tau \to \infty} \frac{1}{\tau} \int\_{t=0}^{t} A[\vec{p}^{N}(t), \vec{r}^{N}(t)] dt = \frac{1}{M} \sum\_{m=1}^{M} A\_{m}(\vec{p}^{N}, \vec{r}^{N}) \tag{4}$$

where *M* is the number of *N N A p <sup>m</sup>*(,) *<sup>r</sup>* values produced by the MD simulation.

A variety of thermodynamic properties can be calculated from the configurations generated by the MD simulations. Comparison with experimental results is an important way to check

temperature including the Berendsen, Nosé-Hoover and V-rescale thermostats (Allen & Tildesley, 1992; Rapaport 2005). *NVT* and *NPT* ensembles are widely used in thermal treatments such as annealing in order to promote thermal denaturation of proteins in unphysical conditions (Rocco et. al.; 2008). *NPT* or isobaric-isothermal ensembles present better characteristics than the canonical ensemble, since the lack of the thermodynamic information about the equilibrium conditions are circumvented by the adjustment of the pressure to the imposed external conditions. This allows the volume to fluctuate by scaling the dimensions of the system by a coefficient defined by the instantaneous and

*A p r e dp dr <sup>A</sup>*

denominator term, *Q*, is the partition function. For a system of *N* identical particles the

*NVT <sup>N</sup> <sup>Q</sup> e dp dr <sup>N</sup> <sup>h</sup>*

−β

where *h* is the Planck's constant. The partition function is central to the statistical mechanics analysis since it contains all the information to determine the macroscopic thermodynamics properties, which are crucial for correlating the microscopic and macroscopic levels (McQuarrie, 1976). Generally, the partition function cannot be computed since it depends on

The most fundamental axiom of statistical mechanics, the ergodic hypothesis, states that the

This theory is based on the fact that if one allows the system to evolve indefinitely in time it will pass through all possible energy states, therefore the MD simulation should generate enough configurations in time to ensure that the equality is satisfied. The average in eq.(1) is an average over all the possible positions of the particles. During a MD simulation, the averages are calculated over the configurations obtained as a function of time so that in

*time m*

τ

where *M* is the number of *N N A p <sup>m</sup>*(,) *<sup>r</sup>* values produced by the MD simulation.

τ τ→∞

*t m A A p t r t dt A p r*

A variety of thermodynamic properties can be calculated from the configurations generated by the MD simulations. Comparison with experimental results is an important way to check

= =

<sup>0</sup> *M* <sup>1</sup> 1 1 lim [ ( ), ( )] (,)

*N N N N Ep r N N*

are the momenta and positions of the *N* particles at time t, *N N <sup>E</sup>*(,) *<sup>p</sup> <sup>r</sup>* is

*N N Ep r N N*

<sup>=</sup> (2)

*ensemble time A A* <sup>=</sup> (3)

*M N N N N*

= ≈ (4)

(,)

, and

β

= 1 /*kT* . The

(1)

*Q* (,) (,) <sup>−</sup>β

<sup>=</sup>

, and kinetic energy, *<sup>N</sup> <sup>K</sup>*( ) *<sup>p</sup>*

The equilibrium average of a given property *A* is expressed by:

*ensemble*

desired pressures.

and *Nr*

the sum of the potential, *<sup>N</sup> U r*( )

the interatomic forces and positions.

ensemble average equals the time average.

accord with the ergodicity hypothesis:

partition function for the canonical ensemble is:

where *Np*

the accuracy of the simulation and the validity of the model and method. Simulation techniques can also be used for prediction of thermodynamic properties for which there is no experimental data. For example, the internal energy can be calculated as follows:

$$
\left< E \right> = \frac{1}{M} \sum\_{i=1}^{M} E\_i \tag{5}
$$

and the heat capacity at constant volume, *CV* , as given by the internal energy fluctuation is obtained from:

$$C\_V = \frac{\left\langle E^2 \right\rangle - \left\langle E \right\rangle^2}{kT^2} \tag{6}$$

where *E*2 and *E* <sup>2</sup> can be calculated at the end of simulation.

Heat capacity is an important quantity to investigate phase transitions, because it shows a characteristic dependence upon temperature. A first-order phase transition shows an infinite heat capacity at the transition temperature and a discontinuity for a second-order phase transition. The heat capacity can also be calculated from a series of simulations at different temperatures followed by the differentiation of the energy with respect to the temperature.

#### **2.2 Molecular interactions and equations of motion**

The main influence of a molecular simulation is the potential energy, which determines how the particles of the system interact. The simplest model of a particle is a sphere, and the simplest model of interaction is that between pairs of atoms. Such simple models are capable of reproducing two main features of real systems, *(i)* the resistance to compression (short range) and *(ii)* the capability of keeping atoms together in a condensed phase since atoms are attracted to each other over longer ranges (Rapaport, 2005). MD techniques use analytical expressions for the energy potential functions that are obtained from experimental data or quantum calculations. These analytical expressions are generally effective pair interaction potentials that allow the molecular systems to exhibit the correct characteristics of real systems (Maitland et. al., 1981).

The most widely used of these interaction potentials between monoatomic molecules was initially proposed to reproduce structural properties of liquid argon (Maitland & Smith, 1971), written in the form of the Lennard-Jones (LJ) potential:

$$\mathcal{U}I(r\_{ij}) = 4\varepsilon \left[ \left( \frac{\sigma}{r\_{ij}} \right)^{12} - \left( \frac{\sigma}{r\_{ij}} \right)^{6} \right] \tag{7}$$

where *ij r* is the distance between atom *i* and *j*, -ε is the depth of the potential well and σ the radius of the particles. An intermolecular potential energy must reproduce the attractive (long-range) and repulsive (short-range) behaviours, and in the case of LJ potential the repulsive and attractive behaviours are represented by the first and second terms respectively. More complete expressions describing the total intra and intermolecular potential energies for complex molecules will be described in section 2.5.

The intermolecular interactions are generally cut off at the separation distance *cr* , i.e.

$$
\mathcal{L}I(r\_{\vec{\eta}}) = 0 \qquad r\_{\vec{\eta}} \ge r\_c \tag{8}
$$

The force between the atoms *i* and *j* is given by:

$$\vec{F}\_{i\dot{j}} = -\frac{\partial II(r\_{\dot{j}})}{\partial r\_{i\dot{j}}} \tag{9}$$

Special care must be taken with the treatment of the long range interactions, since the distance cut-off may introduce a discontinuity in the potential energy and the force near the given value. This artefact creates problems due to the requirement for energy conservation in MD simulations, and efficient mathematical approaches have been suggested to circumvent this problem, such as the use of switching functions (Allen & Tildesley, 1992).

The coordinates of the atoms of the system are obtained by solving the equation for Newton's second law;

$$m\_i \frac{d^2 \vec{r\_i}}{dt^2} = m\_i \overrightarrow{a\_i} = \vec{F\_i} = \frac{1}{2} \sum\_{j=1 \atop j \neq i}^{N} \vec{F\_{ij}} \tag{10}$$

where *Fi* is the force acting on the atom *i* of mass *mi* and acceleration *<sup>i</sup> a* . Each pair of atoms need only be identified once because *F F ij j* = − *<sup>i</sup>* (Newton's third law).

The differential equation (10) is solved by integration using standard numerical techniques, such as finite difference or predictor-corrector. Finite difference methods are widely employed in MD simulation to integrate the equation of motion, for example the Verlet and leap-frog algorithms (Allen & Tildesley, 1992).

The Verlet algorithm is based on positions *r t*( ) and accelerations *a t*( ) . The positions at times *t dt* <sup>±</sup> are obtained by Taylor expansion of*r t*( ) :

$$
\vec{r}\_i(t+dt) = \vec{r}\_i(t) + \vec{v}\_i(t)dt + \vec{a}\_i(t)\frac{dt^2}{2!} + \dots \tag{11}
$$

$$
\vec{r}\_i(t - dt) = \vec{r}\_i(t) - \vec{v}\_i(t)dt + \vec{a}\_i(t)\frac{dt^2}{2!} - \dots \tag{12}
$$

where *<sup>i</sup> v t*( ) is the velocity of the atom *i*, *dt* is the time step and, *ir t dt* ( ) <sup>+</sup> and *ir t dt* ( ) <sup>−</sup> are the later and earlier positions relative to *ir t*( ) . By addition of equations (11) and (12) and taking into account equation (10), we obtain the equation for advancing the positions:

$$
\nabla \vec{r}\_i(t+dt) = \mathcal{Z}\vec{r}\_i(t) - \vec{r}\_i(t-dt)dt + \frac{dt^2}{m\_i}\vec{F}\_i(t) \tag{13}
$$

Although the velocities are not necessary to compute the trajectories, they are useful to estimate the kinetic energy and hence the total energy of the sytem. The atomic velocities can be calculated from:

*ij*

*N*

*j j i*

1 2 <sup>=</sup> ≠

(Newton's third law).

1

and accelerations *a t*( )

2

2

*i*

*m* 2 ( ) 2 () ( ) () += −− + (13)

−= − + − (12)

. By addition of equations (11) and (12) and

+= + + + (11)

*ij U r*

*r* <sup>∂</sup> ( ) = − <sup>∂</sup>

Special care must be taken with the treatment of the long range interactions, since the distance cut-off may introduce a discontinuity in the potential energy and the force near the given value. This artefact creates problems due to the requirement for energy conservation in MD simulations, and efficient mathematical approaches have been suggested to circumvent this problem, such as the use of switching functions (Allen & Tildesley, 1992). The coordinates of the atoms of the system are obtained by solving the equation for

*i ii i ij*

The differential equation (10) is solved by integration using standard numerical techniques, such as finite difference or predictor-corrector. Finite difference methods are widely employed in MD simulation to integrate the equation of motion, for example the Verlet and

:

( ) ( ) ( ) ( ) .... 2!

( ) ( ) ( ) ( ) .... 2!

where *<sup>i</sup> v t*( ) is the velocity of the atom *i*, *dt* is the time step and, *ir t dt* ( ) <sup>+</sup> and *ir t dt* ( ) <sup>−</sup> are

*i ii i dt r t dt r t v t dt a t*

*i ii i dt r t dt r t v t dt a t*

taking into account equation (10), we obtain the equation for advancing the positions:

*i ii i*

*dt r t dt r t r t dt dt F t*

Although the velocities are not necessary to compute the trajectories, they are useful to estimate the kinetic energy and hence the total energy of the sytem. The atomic velocities

= ==

*d r m ma F F*

*U rij* ()0 = *ij <sup>c</sup> r r* ≥ (8)

(9)

(10)

. Each pair of atoms

. The positions at times

The intermolecular interactions are generally cut off at the separation distance *cr* , i.e.

*ij*

*F*

*i*

is the force acting on the atom *i* of mass *mi* and acceleration *<sup>i</sup> a*

*dt* 2 2

The force between the atoms *i* and *j* is given by:

need only be identified once because *F F ij j* = − *<sup>i</sup>*

leap-frog algorithms (Allen & Tildesley, 1992). The Verlet algorithm is based on positions *r t*( )

*t dt* ± are obtained by Taylor expansion of*r t*( )

the later and earlier positions relative to *ir t*( )

can be calculated from:

Newton's second law;

where *Fi* 

$$
\vec{v}\_i(t) = \frac{\vec{r}\_i(t+dt) - \vec{r}\_i(t-dt)}{2dt} \tag{14}
$$

Other integration algorithms are available (Allen & Tildesley, 1992). The numerical integration of the motion equations encompasses numerical approximations so that the time step, *dt*, must be chosen to be at least one order of magnitude smaller than the highfrequency motions and generally of the order of a few femtoseconds. The high-frequency motions, such as bond stretching involving hydrogen atoms that can impair the use of *dt ≈ 1fs*, are avoided by maintaining constant bond lengths, since generally these bonds are not interesting and have a negligible effect on the overall stability of the system.

#### **2.3 Initial configuration of the system**

The MD simulations must be capable to sample the regions of the phase space. A MD simulation must be insensitive to the initial state, therefore any appropriate initial state is permissible. A simple method is to build the system from a random distribution of particles or from a regular lattice obeying the numerical density (*N/V*) of the real system. The initial velocities can be also assigned in different ways, either randomly or by fixing the value based on temperature (Maxwell-Boltzmann distribution). Velocities are adjusted to ensure that the center of mass of the system is at rest. In MD simulation of biomolecules, the initial structure must be known and the solvent molecules and ions are added to fulfil the system and neutralize it. Initial protein structures can be found in the PDB data bank or using specific methods of bioinformatics that predict and validate three-dimensional structures.

The set-up of the simulation box is the important first step in the definition of the system. The limits of the simulation box should not produce interface effects in homogeneous systems, and for this reason periodic boundary conditions (PBC) are applied to extend the system to the thermodynamic limit. PBC consists of building replicas of the around of the main box in order to obtain a system that mimics a macroscopic thermodynamic system. All the particles in the replica move identically so that the motion of the atoms is not limited by the walls of the box. During the course of a simulation, when a molecule moves in the central box, all its images move in the same manner (Allen & Tildesley, 1992). In other words, a system which is bounded but free of physical walls can be simulated by the application of PBC.

Five shapes of cells produce periodic images: the cube, the parallelepiped, the truncated octahedron, the rhombic dodecahedron and the hexagonal prism. The choice of the simulation box should be made based on the geometry of the system of interest, for example a globular protein is suitable to be simulated in a cubic or dodecahedron periodic cell, but inadequate for a parallelepiped.

#### **2.4 Controlling the simulations**

Many changes occur in the structure of the system during a simulation process. As the initial structure undergoes modifications as a function of time, the MD data allow examination of the changes at each step and consequently, the evaluation of the equilibrium conditions. The equilibrium condition, *i.e.* small fluctuations near mean values of a given property, is fundamental to control the MD simulations. Stabilization conditions can be identified by accompanying determined properties that are calculated during the simulation. Many properties can be used to control the MD simulations such as the energy profiles, the stability of the temperature, etc. After establishing that the system is stabilized, the calculation phase can be initiated in which the properties of interest can be computed.

In a system constituted by a protein in water, the stability is established by a delicate energetic balance involving intramolecular and intermolecular interactions and kinetic energy. Despite the importance of the intramolecular interactions, the stability of biomolecules in general is strongly influenced by protein-water interactions. The solvent molecules make significant contributions to the conformational changes of the protein, and at equilibrium the protein/solvent interactions are completely relaxed. An important tool to detect equilibrium conformations in MD simulation of proteins is the Root Mean Square Deviation (RMSD), which is defined by;

$$RMSSD(t) = \sqrt{\frac{\sum\_{i=1}^{n} |\vec{R}\_i - \vec{r}\_i(t)|^2}{n}} \tag{15}$$

where *n* is the number of atoms (frequently only the Cα atoms) used in the calculation. | ( )| *R rt i i* <sup>−</sup> is the distance between the atom *i* at time *t* and the same atom in the reference structure. The reference structure can be the experimentally determined initial structure or any other structure, for example the last structure generated by the simulation. The RMSD calculation is made after the superposition of the entire structures or may be applied to more limited groups of atoms or residues. The RMSD provides information about the structural stability as a function of time and the changes of the structures during the simulation process. Figure 1 shows two different cases of the RMSD of a protein which is not stabilized even after 100.0 ns as compared with the RMSD of a protein that reaches equilibrium in only 5.0 ns. These results illustrate the important point that the equilibration time is inherent to each protein, to its environment and to the quality of the initial threedimensional structure.

Fig. 1. Root mean square deviations (RMSD) for two different MD simulations. The black line corresponds to a protein not stabilized in 100.0 ns and the gray line to the protein that shows a fast equilibration.

stability of the temperature, etc. After establishing that the system is stabilized, the calculation phase can be initiated in which the properties of interest can be computed.

In a system constituted by a protein in water, the stability is established by a delicate energetic balance involving intramolecular and intermolecular interactions and kinetic energy. Despite the importance of the intramolecular interactions, the stability of biomolecules in general is strongly influenced by protein-water interactions. The solvent molecules make significant contributions to the conformational changes of the protein, and at equilibrium the protein/solvent interactions are completely relaxed. An important tool to detect equilibrium conformations in MD simulation of proteins is the Root Mean Square

*n*

*i*

( ) <sup>=</sup>

=

1

where *n* is the number of atoms (frequently only the Cα atoms) used in the calculation. | ( )| *R rt i i* <sup>−</sup> is the distance between the atom *i* at time *t* and the same atom in the reference structure. The reference structure can be the experimentally determined initial structure or any other structure, for example the last structure generated by the simulation. The RMSD calculation is made after the superposition of the entire structures or may be applied to more limited groups of atoms or residues. The RMSD provides information about the structural stability as a function of time and the changes of the structures during the simulation process. Figure 1 shows two different cases of the RMSD of a protein which is not stabilized even after 100.0 ns as compared with the RMSD of a protein that reaches equilibrium in only 5.0 ns. These results illustrate the important point that the equilibration time is inherent to each protein, to its environment and to the quality of the initial three-

Fig. 1. Root mean square deviations (RMSD) for two different MD simulations. The black line corresponds to a protein not stabilized in 100.0 ns and the gray line to the protein that

*RMSD t*

*i i*

*n*

*R rt*

−


2

(15)

Deviation (RMSD), which is defined by;

dimensional structure.

shows a fast equilibration.

#### **2.5 Force fields and molecular simulations software packages**

The Force Field in MD simulations is the name given to the set of parameters that define the potential interaction energies. These parameters are adjusted to fully describe the molecular behaviour and its interactions. Intra- and intermolecular potential interactions can be expressed in terms of relatively reduced number of types of components. The interaction between bonded atoms can be subdivided into bond stretching, angle bending and dihedralangle bending (torsion) components, while the interactions between non-bonded atoms are by electrostatic and van der Waals interactions. The full expression can be described by:

$$V\_{\text{bonds}} = \sum\_{\text{bonds 2}} \frac{k\_i}{2} (l\_i - l\_{i,0})^2 \tag{16}$$

$$V\_{angles} = \sum\_{angles} \frac{k\_i}{2} (\theta\_i - \theta\_{i,0})^2 \tag{17}$$

$$V\_{\text{bosous}} = \frac{V\_n}{2} [1 + \cos(n\phi - \gamma)] \tag{18}$$

$$V\_{non-bonded} = \sum\_{i=1}^{N} \sum\_{j=i+1}^{N} \left[ \left( \frac{A\_{12}}{r^{12}} - \frac{B\_6}{r^6} \right) + \frac{q\_i q\_j}{4 \pi \varepsilon\_0 r\_{ij}} \right] \tag{19}$$

$$V(\vec{r}^N) = V\_{bonds} + V\_{angles} + V\_{torsions} + V\_{non-bonded} \tag{20}$$

*<sup>N</sup> V r*( ) denotes the total potential energy as a function of the positions *<sup>r</sup>* of *N* particles or atoms. Bonds and angles are modelled by harmonic potentials (eq. 16 and 17) that give the change in energy as *<sup>i</sup> xx l* ( ,) ≡ θ deviates from the reference value, *i i l* ,0 ,0 (, ) θ . The third term, eq. 18, is the torsional contribution that evaluates how the energy changes in the vibrations of structures of four atoms (planar, like aromatic rings or angular such as in the sp3 hybridization) and finally, the fourth term is the interaction between non-bonded atoms, including van der Waals and electrostatic contributions, as calculated between the nonbonded pairs of atoms in the same or different molecules. The terms *A*<sup>12</sup> and *B*6 in eq. 19 are the Lennard-Jones parameters which consider the size of the molecules and the distance between atoms at zero energy potential.

Electrostatic interactions are important long-range forces that influence protein stability and present difficulties in their treatment in MD simulations, and are integral parts of MD simulation software packages. Some methods can be cited as the most important for protein systems, such as the Reaction Field method and the Ewald Sum. Excellent books are available for consultation on this issue (Allen & Tildesley, 1992; Leach, 2001). Several Force Fields are available and all of them are, or at least should be equivalent, independently of the methodology used to compute their parameters.

An increasing number of force fields and MD software packages are available, many of which are free and can be obtained from the web sites of the research group responsible for their development. MD software packages have been employed in research in theoretical chemistry and biology, protein engineering and industry (pharmaceutical and materials). Some of the most important and referenced MD packages are listed below, and all are well documented and many offer parallelization options.

*GROMACS (Groningem Machine for Chemical Simulation):* Currently available in version 4.5.3 (2011) at the website www.gromacs.org. It refers to both the MD software package and the GROMACS Force Field that is updated like other force fields. It is parameterized to several solvent, proteins, nucleic acids and carbohydrates. *AMBER* (*Assisted Model Building with Energy Refinement*): refers to both the Force Field and the MD program specially developed for biomolecules, and can be obtained at symbolic price. *CHARMM* (*Chemistry at Harvard Macromolecular Mechanics*): is also a Force Field and MD program which although is not fully available for academic purposes, can be obtained from Accelrys. Inc through a commercial license. *NAMD (Not Another Molecular Dynamics):* has as its main feature the high degree of scalability in multi-processed computational systems, up to around one hundred processors with no loss performance. *Q Program:* is a MD software package designed for free energy calculations in biological systems. It is fully available for academic purposes from the Åqvist group website (Åqvist, 2007). Other MD simulation packages include TINKER, LAMMPS, AMMP, BOSS, CERIUS2, CPMD, INSIGHT2, ORAC and OPLS.

#### **3. Biotechnological applications**

#### **3.1 Xylanases**

Xylanases (EC 3.2.1.8) are enzymes produced by a wide variety of bacteria and fungi that hydrolyze the 1,4-β-D-xylosidic linkages of xylans, a mixed family of plant cell wall polysaccharides that are abundant in nature. The enzymatic hydrolysis of xylan has attracted considerable biotechnological interest with applications in the food engineering, bio-ethanol fuel production and cellulose pulp industries (Beg et al., 2001). Ongoing efforts have been directed toward both the identification of new xylanases and the improvement of the catalytic properties of existing enzymes with the goal of enhancing their compatibility for industrial applications.

Based on aminoacid sequence similarity and three-dimensional structural homology, xylanases are classified as family GH10 or GH11 hydrolases. GH11 xylanases present compact globular three-dimensional structures with a conserved scaffold comprised of a single α-helix and two extended pleated β-sheets forming a jellyroll fold. The major surface feature is a long cleft that spans the entire molecule and which contains the active site. The overall shape of GH11 xylanases resembles a right-hand, and as shown in Figure 2 the various structural features have accordingly been denominated as fingers, palm, thumb, cord and helix regions.

The globular structure and relatively small size (~200 amino acids) of the GH11 xylanases are good models for molecular simulation which can be used to investigate important physicochemical and biochemical properties, such as thermostability.

Thermostable proteins are of biotechnological interest because they can be employed in industrial processes which use high temperatures, such as cellulose pulp bleaching. Several strategies have been attempted with the aim of enhancing thermostability of proteins, including the introduction of disulfide bridges (Wakarchuk et al., 1994), increasing the hydrophobic contacts by addition of aromatic interactions (Georis et al., 2000), optimization of

chemistry and biology, protein engineering and industry (pharmaceutical and materials). Some of the most important and referenced MD packages are listed below, and all are well

*GROMACS (Groningem Machine for Chemical Simulation):* Currently available in version 4.5.3 (2011) at the website www.gromacs.org. It refers to both the MD software package and the GROMACS Force Field that is updated like other force fields. It is parameterized to several solvent, proteins, nucleic acids and carbohydrates. *AMBER* (*Assisted Model Building with Energy Refinement*): refers to both the Force Field and the MD program specially developed for biomolecules, and can be obtained at symbolic price. *CHARMM* (*Chemistry at Harvard Macromolecular Mechanics*): is also a Force Field and MD program which although is not fully available for academic purposes, can be obtained from Accelrys. Inc through a commercial license. *NAMD (Not Another Molecular Dynamics):* has as its main feature the high degree of scalability in multi-processed computational systems, up to around one hundred processors with no loss performance. *Q Program:* is a MD software package designed for free energy calculations in biological systems. It is fully available for academic purposes from the Åqvist group website (Åqvist, 2007). Other MD simulation packages include TINKER, LAMMPS,

Xylanases (EC 3.2.1.8) are enzymes produced by a wide variety of bacteria and fungi that hydrolyze the 1,4-β-D-xylosidic linkages of xylans, a mixed family of plant cell wall polysaccharides that are abundant in nature. The enzymatic hydrolysis of xylan has attracted considerable biotechnological interest with applications in the food engineering, bio-ethanol fuel production and cellulose pulp industries (Beg et al., 2001). Ongoing efforts have been directed toward both the identification of new xylanases and the improvement of the catalytic properties of existing enzymes with the goal of enhancing their compatibility

Based on aminoacid sequence similarity and three-dimensional structural homology, xylanases are classified as family GH10 or GH11 hydrolases. GH11 xylanases present compact globular three-dimensional structures with a conserved scaffold comprised of a single α-helix and two extended pleated β-sheets forming a jellyroll fold. The major surface feature is a long cleft that spans the entire molecule and which contains the active site. The overall shape of GH11 xylanases resembles a right-hand, and as shown in Figure 2 the various structural features have accordingly been denominated as fingers, palm, thumb, cord and helix regions. The globular structure and relatively small size (~200 amino acids) of the GH11 xylanases are good models for molecular simulation which can be used to investigate important

Thermostable proteins are of biotechnological interest because they can be employed in industrial processes which use high temperatures, such as cellulose pulp bleaching. Several strategies have been attempted with the aim of enhancing thermostability of proteins, including the introduction of disulfide bridges (Wakarchuk et al., 1994), increasing the hydrophobic contacts by addition of aromatic interactions (Georis et al., 2000), optimization of

documented and many offer parallelization options.

AMMP, BOSS, CERIUS2, CPMD, INSIGHT2, ORAC and OPLS.

physicochemical and biochemical properties, such as thermostability.

**3. Biotechnological applications** 

**3.1 Xylanases** 

for industrial applications.

the electrostatic surface potential (Torrez et al., 2003), and optimization of intramolecular and protein/solvent hydrogen bonding (Viera & Degrève, 2009). Due to the important enthalpic contribution of hydrogen bonding in biological systems, hydrogen bond optimization is a particularly interesting strategy to improve protein thermostability. Formally, hydrogen bonds are rigorously evaluated by quantum mechanics methodologies using the Morokuma Analysis that deconstructs the energy of hydrogen bonds into five components: electrostatic, polarisation, exchange repulsion, charge transfer and mixing (Morokuma, 1977). In contrast, molecular simulation obtains a reasonable estimative of hydrogen bond occurrence and stabilization by evaluating geometric criteria. For example, intramolecular hydrogen bonds are identified by interactions with a donor-receptor distance shorter than 0.24 nm, the (A-H···O) angle larger than 110º (A≡ N or O) and the fractional occurrence larger than 20% of the total simulation time. The intermolecular hydrogen bonds can be detected by two criteria; *(i)* based on the radial distribution functions, *g(r)*, and *(ii)* distribution of the interaction energies between the protein atoms and the solvent, F(E), (Degrève et al., 2004).

Fig. 2. Two different views of three-dimensional structure of the GH11 xylanase from *Bacillus subtilis*, PDB code 1XXN. The regions based in the right-hand analogy are indicated.

The dynamics and energetic properties of MD simulations are well suited to monitor interatomic distances and angles over time, and can characterize all atom pairs involved in hydrogen bonds, hydrophobic clusters or salt bridges. Figure 3 show the hydrogen bonding network in a three-dimensional structure of the *Bacillus subtilis* xylanase from a MD simulation identified by the geometric criteria describe above.

Fig. 3. (a) A snapshot at 10.0ns of GH11 xylanase from the MD simulation in canonical ensemble at physiological pH. The intramolecular hydrogen bonding network is showed as the green dotted lines. (b) The intermolecular hydrogen bonding interactions of the first solvation shell of two Glu residues (red) and one Arg residue (blue).

Intermolecular hydrogen bonds may be identified by considering the first solvation shell of a given residue. As shown in Figure 3(b), the *g(r) ≡ gAW(r)* function must display a peak in an appropriate region, approximately 2Å, defined by the nature of the protein atom. Furthermore, the *F(E)* should display a peak, or a shoulder, in the attractive energy region (negative energies) as observed in the example shown in Figure 4.

Fig. 4. (a) Radial distribution functions *gAW(r)*, where *A* is the protein atoms and *W* the water molecule. (b) Distribution of the interaction energies between atoms of protein and the water molecules. Protein atoms are defined in the legend.

As shown in Figs 4(a) and 4(b), each atom or a group of atoms has a defined influence on the protein energetics. These interactions are unique to each protein, and provide an individual map of the protein enabling us to make predictions with respect to the thermostabilization mechanism adopted by proteins. Recently, Vieira & Degrève have published an MD simulation study of a thermophilic-mesophilic pair of GH11 xylanases, in which a difference of 20°C in thermostability could be interpreted in terms of different patterns of intra and intermolecular hydrogen bonds exhibited by these proteins (Vieira & Degrève, 2009).

MD studies by Vieira & Degrève (2009) estimate the total interaction potential energy for the thermophilic xylanase to be around -600kcal.mol-1 more stable than its mesophilic counterpart, and approximately 20% of this difference is due to intermolecular hydrogen bonds energies. Although the backbone of the thermophilic xylanase is more rigid than the mesophilic enzyme (as determined by the intramolecular hydrogen bonds and salt bridges), the increased thermostability seems to be a consequence of the greater degree of solvation. Although both xylanases are highly solvated, the intermolecular hydrogen bonding network in the thermophilic enzyme is energetically more attractive than those at mesophilic counterpart. In other words, the highly solvated surface characterized by optimization of strong protein-water intermolecular hydrogen bonds is clearly a thermostabilization factor. The main residues which contribute to the thermostability of thermophilic xylanase were located in the finger region and have been targeted for site-directed mutagenesis. This insight as to a major contribution to protein thermostability would not be available from the analysis of a structure determined by X-ray crystallography, in which water molecules are poorly resolved. Therefore, MD simulations can complement the experimental studies in the development and of design process of new proteins.

#### **3.2 Antibody engineering**

426 Innovations in Biotechnology

Intermolecular hydrogen bonds may be identified by considering the first solvation shell of a given residue. As shown in Figure 3(b), the *g(r) ≡ gAW(r)* function must display a peak in an appropriate region, approximately 2Å, defined by the nature of the protein atom. Furthermore, the *F(E)* should display a peak, or a shoulder, in the attractive energy region

Fig. 4. (a) Radial distribution functions *gAW(r)*, where *A* is the protein atoms and *W* the water molecule. (b) Distribution of the interaction energies between atoms of protein and the

As shown in Figs 4(a) and 4(b), each atom or a group of atoms has a defined influence on the protein energetics. These interactions are unique to each protein, and provide an individual map of the protein enabling us to make predictions with respect to the thermostabilization mechanism adopted by proteins. Recently, Vieira & Degrève have published an MD simulation study of a thermophilic-mesophilic pair of GH11 xylanases, in which a difference of 20°C in thermostability could be interpreted in terms of different patterns of intra and intermolecular hydrogen bonds exhibited by these proteins (Vieira &

MD studies by Vieira & Degrève (2009) estimate the total interaction potential energy for the thermophilic xylanase to be around -600kcal.mol-1 more stable than its mesophilic counterpart, and approximately 20% of this difference is due to intermolecular hydrogen bonds energies. Although the backbone of the thermophilic xylanase is more rigid than the mesophilic enzyme (as determined by the intramolecular hydrogen bonds and salt bridges), the increased thermostability seems to be a consequence of the greater degree of solvation. Although both xylanases are highly solvated, the intermolecular hydrogen bonding network in the thermophilic enzyme is energetically more attractive than those at mesophilic counterpart. In other words, the highly solvated surface characterized by optimization of

(negative energies) as observed in the example shown in Figure 4.

water molecules. Protein atoms are defined in the legend.

Degrève, 2009).

Antibodies or immunoglobulins (Ig) are globular proteins that play an important role in protecting the host organism against infectious diseases. The primary function of an antibody is to bind molecules that are foreign to the host (antigens), and understanding the interactions between an antibody and its antigen is important for the design and development of new or improved antibodies. The host typically produces several types of antibody that have similar structures, consisting of four polypeptides (two heavy chains and two light chains) that are linked by disulphide bonds to form a molecular complex. Five heavy chain isotypes are known, giving rise to five different Ig classes (IgG1-4, IgA1-2, IgD, IgM, IgE), each with a distinct function in humans. Each class of heavy chain can combine with one of two light chain isotypes (kappa and lambda). As a result of these molecular combinations, antibodies differ in size, electric charge, amino acid composition and carbohydrate content (Roitt, 1997).

The most common class of IgG antibodies present a basic structure of two light chains (each of which has two domains) and two heavy chains (each having four domains). The two N-terminal domains of the heavy chain are linked by disulphide bonds to the two domains of the light chains to form a "Y"-shaped structure. The N-terminal domain of both the heavy and the light chain show pronounced amino acid sequence (and are appropriately called the variable domains, VL and VH), while the other domains are called constant domains (CL, CH1, CH2 and CH3) (Alberts et al, 1989). The variable domains have three regions of hypervariability in the amino acid sequence and are termed Complementarity Determining Regions (CDRs). These regions are responsible for the recognition and binding of the antibodies to a specific antigen, and determine the affinity and specificity to the antibody-antigen interaction. The "Y"-shaped antibody structure can be divided into three fragments: Fv (variable fragment), Fab (antigen binding fragment) and Fc (crystallization fragment). An engineered antibody may be obtained from separate segments of Fv heavy and light chains joined by a flexible peptide linker to form a single-chain Fv (scFv).

Molecular modeling by homology uses experimentally determined protein structures to predict the three-dimensional conformation of a protein with a similar amino acid sequence whose structure is unknown. Molecular modeling can be used with reasonable success if the amino acid sequences of the known and the unknown proteins share at least 40% identity, and within these limits more than 90% of the atoms in the main chain can be modeled with an accuracy of about 0.1 nm (Sanchez & Šali, 1997). The program *Modeller* (Šali & Blundell, 1993) can be used for homology modeling in conjunction with the programs *PROCHECK* (Laskowski et al., 1993) and *VERIFY\_3D* (Luthy et al., 1992) that can be used to assess the quality of the generated structures. These tools can be used to model both the variable regions of antibodies and antigen.

Starting from the three-dimensional structures of antibody and antigen, the next step is to identify the epitopes on the antigen molecule that are recognized by the antibody. Docking techniques can be used to explore the regions of best fit between antibody and antigen, and the more information about the epitopes better the final solution generated by the docking program. For identification and optimization of regions of the antibody that enter in contact with the antigen, the use of available structural, biological and biochemical information is essential to obtain the most reliable results. Furthermore, the docking approach should not simply focus on solving a problem of rigid body fitting of geometric shapes between the two components, but should recognize that interatomic forces in the context of complex dynamics can give rise to structural changes that can maximize the contacts at the interface of the antibody-antigen complex.

Starting from predictions of antibody-antigen interfaces derived from docking studies, MD simulation can be used for the evaluation of the structures of the proposed antibody-antigen complexes. MD is a powerful tool that can be used to study the structural movements of antibody and antigen and to monitor interatomic interactions in a physiological aqueous environment. With this strategy, a new scFv (Ab) for a particular antigen (Ag) has been developed at the Brazilian company, Cientistas Associados (Cientistas Associados, 2008). It has been demonstrated that the binding between Ag and Ab is dynamic, with structural variations observed in both molecules, which can be improved by changing the contact specificity through the mutation of key residues at the Ag|Ab interface.

Crotoxin is a phospholipase A2 neurotoxin, and is a major component of the venom of the rattlesnake *Crotalus durissus terrificus*, and the lethal effects are due to blockage of neuromuscular transmission. Crotoxin is comprised of two non-identical subunits (CA and CB), which separately present present low toxicity. The CA subunit (acidic protein) and CB (basic phospholipase) spontaneously associate in a 1:1 complex, giving rise to the particularly lethal neurotoxin (Faure et al., 1991). Studies reported that three scFv could bind crotoxin (Cardoso et al., 2000). These scFvs recognized distinct epitopes close to CB, however only one scFv was able to effectively neutralize the activity induced by crotoxin.

In order to study this antibody|antigen complex, the structures of crotoxin and scFv were subjected to homology modelling. The template for the crotoxin was the phospholipase A2 from *Crotalus atrox*, which has 50% identity when compared to CB. In the case of a template for the scFv, a search was made of the "framework" (BLAST) and the structure of the CDRs with highest resolution and structural identity was selected. The Ag|Ab complex was obtained with the program HEX (Ritchie, 2003), and since the binding site of the antibody (CDR) is known through epitope mapping (Choumet et al. 2003), the crotoxin was in the correct orientation in relation to the CDRs.

Four MD simulations were performed with the GROMACS software package using a time step of 2 fs at 298 K in the NVT ensemble. A cut-off radius of 1.3 nm was applied. The simulated systems were electrically neutralized by the addition of counter ions and the simulation boxes were designed to avoid overlap of the cut-off radius through the sides of the boxes. The systems include a free Ab in solution (System 1), a free Ag in solution (System 2), Ag|Ab complex (System 3) and mutate Ab in Ag|Ab complex (System 4). In

quality of the generated structures. These tools can be used to model both the variable

Starting from the three-dimensional structures of antibody and antigen, the next step is to identify the epitopes on the antigen molecule that are recognized by the antibody. Docking techniques can be used to explore the regions of best fit between antibody and antigen, and the more information about the epitopes better the final solution generated by the docking program. For identification and optimization of regions of the antibody that enter in contact with the antigen, the use of available structural, biological and biochemical information is essential to obtain the most reliable results. Furthermore, the docking approach should not simply focus on solving a problem of rigid body fitting of geometric shapes between the two components, but should recognize that interatomic forces in the context of complex dynamics can give rise to structural changes that can maximize the contacts at the interface

Starting from predictions of antibody-antigen interfaces derived from docking studies, MD simulation can be used for the evaluation of the structures of the proposed antibody-antigen complexes. MD is a powerful tool that can be used to study the structural movements of antibody and antigen and to monitor interatomic interactions in a physiological aqueous environment. With this strategy, a new scFv (Ab) for a particular antigen (Ag) has been developed at the Brazilian company, Cientistas Associados (Cientistas Associados, 2008). It has been demonstrated that the binding between Ag and Ab is dynamic, with structural variations observed in both molecules, which can be improved by changing the contact

Crotoxin is a phospholipase A2 neurotoxin, and is a major component of the venom of the rattlesnake *Crotalus durissus terrificus*, and the lethal effects are due to blockage of neuromuscular transmission. Crotoxin is comprised of two non-identical subunits (CA and CB), which separately present present low toxicity. The CA subunit (acidic protein) and CB (basic phospholipase) spontaneously associate in a 1:1 complex, giving rise to the particularly lethal neurotoxin (Faure et al., 1991). Studies reported that three scFv could bind crotoxin (Cardoso et al., 2000). These scFvs recognized distinct epitopes close to CB, however only one scFv was able to effectively neutralize the activity induced by crotoxin.

In order to study this antibody|antigen complex, the structures of crotoxin and scFv were subjected to homology modelling. The template for the crotoxin was the phospholipase A2 from *Crotalus atrox*, which has 50% identity when compared to CB. In the case of a template for the scFv, a search was made of the "framework" (BLAST) and the structure of the CDRs with highest resolution and structural identity was selected. The Ag|Ab complex was obtained with the program HEX (Ritchie, 2003), and since the binding site of the antibody (CDR) is known through epitope mapping (Choumet et al. 2003), the crotoxin was in the

Four MD simulations were performed with the GROMACS software package using a time step of 2 fs at 298 K in the NVT ensemble. A cut-off radius of 1.3 nm was applied. The simulated systems were electrically neutralized by the addition of counter ions and the simulation boxes were designed to avoid overlap of the cut-off radius through the sides of the boxes. The systems include a free Ab in solution (System 1), a free Ag in solution (System 2), Ag|Ab complex (System 3) and mutate Ab in Ag|Ab complex (System 4). In

specificity through the mutation of key residues at the Ag|Ab interface.

regions of antibodies and antigen.

of the antibody-antigen complex.

correct orientation in relation to the CDRs.

System 3, the antigen and antibody are initially separated by 0.7 nm. The initial structure of the Ab in the System 4 was mutated in four positions as compared to the Ab in System 3 to enhance the Ag|Ab interaction.

The Ag|Ab complex was formed by superposition of the final simulated antibody and antigen structures by docking. In the System 3, formation of the complex was monitored through the intermolecular interaction potential (IIP) between the Ag and Ab calculated during the simulation (Figure 5).

Fig. 5. Intermolecular Interaction Potential between the antigen and the antibody. A) System 3 and B) System 4 (see text for details).

The IIP shows that when Ag and Ab are separated by 0.7 nm (Figure 6a, time = 0 ns), the potential is almost zero and there is no effective interaction. Time around 1.0 ns and IIP = - 60 kcal. mol-1 marks the start of the Ag|Ab complex formation (Figure 6b), and from that moment the attractive interaction at the Ag|Ab interface increases, and the potential decreases to approximately -170 kcal.mol-1 in the equilibrium structure at the end of the simulation (Figure 6c). The Ag|Ab complexes obtained from MD simulation are presented in Figure 7. It is evident that the Ag|Ab interface can be extended since the Tyr and Thr residues are distant from the antigen.

Fig. 6. Configurations of System 3 complex formation between the antibody (left) and antigen (right), at times 0, 1 and 10 ns.

Fig. 7. Final configurations of Systems 3 and 4 from MD simulations, showing the Ag|Ab complex (left and right molecule respectively). Potential isocontours shown at +1 (kT/e) (in blue) and -1 (kT/e) (in red) for permittivities equal to 2 (solute) and to 78.5 (solvent). a) System 3 and b) System 4.

To study the contribution of electrostatic and van der Waals interactions of individual residues to the stability of the interface, four amino acids in the antibody were mutated with the aim of enhancing the Ag|Ab interaction. The substitutions were chosen to optimize the interface by choosing amino acids with opposite charges to those observed in the potential isocontours of the antigen. Figure 8 shows the residues mutated; Thr/Glu, Tyr/Lys, Tyr/Asp and Glu/Ser (System 4). Except the Tyr/Glu mutation, all other residues show a strong residue complementarity, and the polar Ser and Thr were replaced by negatively charged residues.

The most significant results of reduction of the IIP were observed for Thr/Glu and Ser/Asp mutations, which resulted in the addition of two charged residues. IIP for Thr/Glu and Ser/Asp mutations presented a reduction of approximately -10 and -20 kcal.mol-1, respectively. Panels C and G, in Figure 8, show that there was a local change in the potential isocontours of the antigen in System 4. The final structures collected for the System 4 show that the complex is more compact, with a more complementary interface in relation to System 3 (Figures 7a and 7b).

The phenolic side chain of the Tyr residues have both aromatic and polar characteristics, which result in different interaction potential. The electrostatic potential is predominant in the hydroxyl group interaction, while in the aromatic ring the intermolecular interaction is predominantly van der Waals. In the case of Tyr/Glu mutation, the IIP increased as a result of repulsive interaction on the site. In System 3, the IIP of two Tyr residues are attractive, approximately -20 kcal. mol-1, a sufficient energy for Tyr hydroxyl group to form hydrogen bonds. In this context, the mutations and Tyr to Lys and Tyr to Glu proved to be inefficient, since the IIP for the Lys is similar to that of Glu and Tyr shows positive IIP as a result of a local repulsion.

We conclude that uncharged residues at the Ag|Ab interface, such as Tyr residues (System 3), may result in more attractive interactions in relation to charged residues when in a favorable conformation with a good fit, showing that van der Waals forces play an important role in interface stabilization. In addition, charged residues on the antibody interact with antigen residues over longer distances, strengthening the binding between antigen and antibody. Therefore, the formation of Ag|Ab complex is mediated by residues

Fig. 7. Final configurations of Systems 3 and 4 from MD simulations, showing the Ag|Ab complex (left and right molecule respectively). Potential isocontours shown at +1 (kT/e) (in blue) and -1 (kT/e) (in red) for permittivities equal to 2 (solute) and to 78.5 (solvent). a)

To study the contribution of electrostatic and van der Waals interactions of individual residues to the stability of the interface, four amino acids in the antibody were mutated with the aim of enhancing the Ag|Ab interaction. The substitutions were chosen to optimize the interface by choosing amino acids with opposite charges to those observed in the potential isocontours of the antigen. Figure 8 shows the residues mutated; Thr/Glu, Tyr/Lys, Tyr/Asp and Glu/Ser (System 4). Except the Tyr/Glu mutation, all other residues show a strong residue complementarity, and the polar Ser and Thr were replaced

The most significant results of reduction of the IIP were observed for Thr/Glu and Ser/Asp mutations, which resulted in the addition of two charged residues. IIP for Thr/Glu and Ser/Asp mutations presented a reduction of approximately -10 and -20 kcal.mol-1, respectively. Panels C and G, in Figure 8, show that there was a local change in the potential isocontours of the antigen in System 4. The final structures collected for the System 4 show that the complex is more compact, with a more complementary interface in relation to

The phenolic side chain of the Tyr residues have both aromatic and polar characteristics, which result in different interaction potential. The electrostatic potential is predominant in the hydroxyl group interaction, while in the aromatic ring the intermolecular interaction is predominantly van der Waals. In the case of Tyr/Glu mutation, the IIP increased as a result of repulsive interaction on the site. In System 3, the IIP of two Tyr residues are attractive, approximately -20 kcal. mol-1, a sufficient energy for Tyr hydroxyl group to form hydrogen bonds. In this context, the mutations and Tyr to Lys and Tyr to Glu proved to be inefficient, since the IIP for the Lys is similar to that of Glu and Tyr

We conclude that uncharged residues at the Ag|Ab interface, such as Tyr residues (System 3), may result in more attractive interactions in relation to charged residues when in a favorable conformation with a good fit, showing that van der Waals forces play an important role in interface stabilization. In addition, charged residues on the antibody interact with antigen residues over longer distances, strengthening the binding between antigen and antibody. Therefore, the formation of Ag|Ab complex is mediated by residues

System 3 and b) System 4.

by negatively charged residues.

System 3 (Figures 7a and 7b).

shows positive IIP as a result of a local repulsion.

whose interactions are dominated by van der Waals interactions and which are important for specificity (antigen recognition), while the charged residues are important for the enhancing affinity (binding). A MD strategy can therefore be used in antibody engineering for the evaluation of the proposed changes both locally to evaluate each residue for specificity, and globally to evaluate the effect of residue changes on affinity.

Fig. 8. Final configurations from MD simulations of Systems 3 and 4, showing the local environments of each residue of the antibody (left) compared to the antigen (right), represented as potential isocontours. Panels A, B, C, and D show the antibody residue in System 3 and panels E, F, G and H show the mutated residues in System 4.

#### **3.3 Antimicrobial peptides**

Antimicrobial peptides (AMP) play an important role in the innate immune defense system in all organisms and display activity against a wide variety of microorganisms. AMP are generally comprised of fewer than 50 amino acid residues, and are characterized both by an overall positive charge due the presence of multiple lysine and arginine residues and by a large proportion (at least 50 %) of hydrophobic residues. The Antimicrobial Peptide Database (APD) (Wang & Wang, 2004) contains more than 1750 AMPs, despite this experimental effort, knowledge with respect to the structures of these peptides remains limited since 3D structures are available for only 229 (13% of the total) of these AMPs. Therefore, it is evident that the investigation of AMPs structure and dynamics will yield knowledge that is important for the understanding of the mode of action of these biomolecules.

During the last decade, the accumulation of a large body of experimental data has demonstraed that AMPs act by predominantly affecting the integrity of cell membranes through their interaction with phospholipids. The cytoplasmic membranes of multicellular organisms and bacteria have distinct lipid compositions, and the specidicity of AMPs activity is therefore determined not only by the physico-chemical properties of the peptide but also by the composition of the target cell membranes. An understanding of the interactions between AMP and cell membrane at a molecular level is therefore of importance for the development of AMPs as therapeutic agents, which ideally would have a potent antimicrobial activity with low toxicity against host cells. The cytoplasmic membrane of the Gram negative bacteria *Escherichia coli* contains 70 to 80% neutral lipids with phosphatidylethanolamine (PE) head groups, 20 to 25% with phosphatidylglycerol (PG) head groups, which imparts a negative charge to the membrane, and other lipids that are present in smaller quantities (Dowhan, 1997). In eukaryotic cells, the extracellular leaflet of the cytoplasmic membrane is composed predominantly of lipid with phosphatidylcholine (PC) head groups (Mateo et al., 2006). It is crucial that these differences in lipid composition between eukaryotic and bacterial cell membranes be taken into account for investigations with respect to the toxic and antimicrobial activities of AMPs.

AMPs with altered amino acid sequences have been synthesized with the aim of decreasing toxicity and increasing cell selectivity, for example, the indolicidin (Selsted et al., 1992). Indolicidin (IND) is a peptide containing 13 amino acid residues (ILPWKWPWWPWRR-NH2) and contains a large portion of tryptophan residues (39%). The IND is amidated at its C-terminus, displays activity against a broad range of microorganisms however is toxic to lymphocytes and erythrocytes. Two mutants of IND, a Pro/Ala mutant (CP10A ILAWKWAWWAWRR-NH2) and the mutant CP11 (ILKKWPWWPWRRK-NH2) that contains two extra positive charges, have been the target of several functional studies (Zhang et al., 2001; Halevy et al., 2003). This set of peptides is of interest as a model system since the toxic activity of the CP10A mutant is higher against human erythrocytes when compared to the native IND, while the CP11 has the lowest hemolytic activity among the three peptides (Halevy et al., 2003). Conversely, the antimicrobial activity is greatest for the CP11 and lowest for the CP10A (CP11>IND>CP10A) (Zhang et al., 2001). The functional properties of these mutants is therefore influenced by the physico-chemical characteristics of the peptide, and provides an excellent model system to study the peptide|membrane interaction using MD simulations.

MD studies were carried out with the IND and the two mutants CP10A and CP11 were used as model peptides to study the interaction of the peptides with two membrane models: one containing dipalmitoylphosphatidylcholine (DPPC) with 64 lipids in each layer, and the other containing a mixed bilayer with 64 lipids in each layer formed by 75 % of dipalmitoylphosphatidylethanolamine (DPPE) and 25 % of dipalmitoylphosphatidylglycerol (DPPG). Both bilayer systems were solvated and electrical neutrality of the mixed bilayer was maintained by the addition of one sodium ion for each DPPG molecule. Six systems were studied by introducing one of the peptides (IND, CP10A or CP11) into the bulk solution within a distance of about 4 nm from the center of the mass (CM) of one of the bilayers (DPPC or DPPE/DPPG). The simulations were initiated by energy minimization using the steepest

investigation of AMPs structure and dynamics will yield knowledge that is important for the

During the last decade, the accumulation of a large body of experimental data has demonstraed that AMPs act by predominantly affecting the integrity of cell membranes through their interaction with phospholipids. The cytoplasmic membranes of multicellular organisms and bacteria have distinct lipid compositions, and the specidicity of AMPs activity is therefore determined not only by the physico-chemical properties of the peptide but also by the composition of the target cell membranes. An understanding of the interactions between AMP and cell membrane at a molecular level is therefore of importance for the development of AMPs as therapeutic agents, which ideally would have a potent antimicrobial activity with low toxicity against host cells. The cytoplasmic membrane of the Gram negative bacteria *Escherichia coli* contains 70 to 80% neutral lipids with phosphatidylethanolamine (PE) head groups, 20 to 25% with phosphatidylglycerol (PG) head groups, which imparts a negative charge to the membrane, and other lipids that are present in smaller quantities (Dowhan, 1997). In eukaryotic cells, the extracellular leaflet of the cytoplasmic membrane is composed predominantly of lipid with phosphatidylcholine (PC) head groups (Mateo et al., 2006). It is crucial that these differences in lipid composition between eukaryotic and bacterial cell membranes be taken into account for investigations

AMPs with altered amino acid sequences have been synthesized with the aim of decreasing toxicity and increasing cell selectivity, for example, the indolicidin (Selsted et al., 1992). Indolicidin (IND) is a peptide containing 13 amino acid residues (ILPWKWPWWPWRR-NH2) and contains a large portion of tryptophan residues (39%). The IND is amidated at its C-terminus, displays activity against a broad range of microorganisms however is toxic to lymphocytes and erythrocytes. Two mutants of IND, a Pro/Ala mutant (CP10A ILAWKWAWWAWRR-NH2) and the mutant CP11 (ILKKWPWWPWRRK-NH2) that contains two extra positive charges, have been the target of several functional studies (Zhang et al., 2001; Halevy et al., 2003). This set of peptides is of interest as a model system since the toxic activity of the CP10A mutant is higher against human erythrocytes when compared to the native IND, while the CP11 has the lowest hemolytic activity among the three peptides (Halevy et al., 2003). Conversely, the antimicrobial activity is greatest for the CP11 and lowest for the CP10A (CP11>IND>CP10A) (Zhang et al., 2001). The functional properties of these mutants is therefore influenced by the physico-chemical characteristics of the peptide, and provides an excellent model system to

MD studies were carried out with the IND and the two mutants CP10A and CP11 were used as model peptides to study the interaction of the peptides with two membrane models: one containing dipalmitoylphosphatidylcholine (DPPC) with 64 lipids in each layer, and the other containing a mixed bilayer with 64 lipids in each layer formed by 75 % of dipalmitoylphosphatidylethanolamine (DPPE) and 25 % of dipalmitoylphosphatidylglycerol (DPPG). Both bilayer systems were solvated and electrical neutrality of the mixed bilayer was maintained by the addition of one sodium ion for each DPPG molecule. Six systems were studied by introducing one of the peptides (IND, CP10A or CP11) into the bulk solution within a distance of about 4 nm from the center of the mass (CM) of one of the bilayers (DPPC or DPPE/DPPG). The simulations were initiated by energy minimization using the steepest

understanding of the mode of action of these biomolecules.

with respect to the toxic and antimicrobial activities of AMPs.

study the peptide|membrane interaction using MD simulations.

descent algorithm, in order to eliminate bad contacts and undesirable forces. For each system, an initial simulation was performed for 0.5 ns applying restrictions to the peptides and bilayer atomic coordinates to equilibrate the systems using a *dt = 0.5 fs*. The restrictions were removed and the systems were initially simulated by 30 fast heating and cooling cycles of simulated annealing (Fuzo et al., 2008) with *dt =2 fs*. In the next stage, the systems were submitted to 20 ns NpT simulations with *dt* of *2 fs*, with 50 ns total simulation time for each system. Full details of the simulation parameters are given in Fuzo & Degrève (2009, 2011).

At the start of the simulations, the peptides were positioned in the solvent phase in order to allow a free interaction with the bilayer. During the simulation the peptides diffused through the solvent toward the membrane, before associating with the bilayers as observed in Figure 9. Differences were observed when the peptides were inserted into the two types of bilayers, and the mean position of the peptide CMs in relation to the bilayer CMs, given by a value *d* as measured along the axis perpendicular to bilayer plane, and which is lower when the peptide is inserted into the bilayer.

Fig. 9. Final configurations of systems containing peptides inserted into DPPC (top) and DPPE/DPPG (bottom) bilayers.

Table 1 shows that in the case of DPPC bilayers, all *d* values were less than the average position of the phosphorus atoms of DPPC (1.8 nm) in the order CP11>IND>CP10A, showing that the CP10A peptide inserted almost to the centre of the bilayer, the CP11 peptide remained close to the interface of the bilayer with the aqueous phase. The behavior of the IND peptide was intermediate between the two extremes. In the DPPE/DPPG systems the peptides also inserted into the bilayer giving *d* values that were lower than the phosphorous atoms, however it was observed that the order of insertion of the peptides was altered (ie. CP11>CP10A>IND) as compared to the DPPC bilayer. In all cases, the peptide that inserts deeper into a given bilayer is that which has the highest activity against a cell membrane of the same composition, thus in the systems studied with the DPPC bilayer the peptides that show a deeper insertion are those that have the highest hemolytic activity (Halevy et al., 2003), whereas for the DPPE/DPPG systems those peptides with higher antimicrobial activity were more deeply inserted (Zhang et al., 2001). This correlation between membrane activity and peptide insertion may have an important impact for the design of new AMPs, since more effective mutants can be designed rationallybased on the information obtained from MD simulation.


Table 1. Distances between the CM of peptides and bilayers along the axis perpendicular to bilayer plane (*d*), and the number of hydrogen bonds between the peptides and bilayers (*NHB*).

Two important differences were obseved between the interactions of the peptides and the two different bilayers. The first was a greater number of hydrogen bonds (NHB) between the peptides and oxygen atoms of the DPPC bilayers (Table 1), which was due to a stretched conformation of the phosphotidylcholine groups of the DPPC molecule (Figure 10A**)** in which the distance between the CM of the phosphate and choline groups was 0.47 ± 0.06 nm, which compares with the phosphotidylethanolamine groups in the **DPPE/DPPG bilayer** where the distances between the phosphate and amine groups was 0.37 ± 0.01 nm. The proximity between the amine and phosphate groups in the DPPE molecules makes causes greater difficulty for the positively charged groups in peptides to approach the lipid headgroup phosphate group due to electrostatic repulsion with the positively charged amino group, thereby hindering the formation of HBs between the peptides and oxygen atoms. The second difference was the presence of cation-π interactions between choline groups of the DPPC and the side-chains of some Trp residues of the peptides. The cation-π interactions were evaluated by determining the radial distribution functions between the CM of the tryptophan side chains and nitrogen atoms of the choline groups, where the presence of the first peak (Dmax) at distances smaller than 0.5 nm and a number of choline groups greater than one (NChol) is indicative of cation-π interactions. These cation-π interactions were found with 1 to 2 choline groups for some tryptophan residues for all three peptides (as shown in Table 2). Examples of the proximity of the choline headgroups for each peptide are shown in Figures 10C, 10D, and 10E for IND, CP10A, and CP11, respectivelly. The cation-π interactions observed between the choline groups and the tryptophan residues do not occur in lipid bilayers containing PE due the geometry of this group, in which the amine and phosphate groups are closer, thereby impeding the approximation of the tryptophan side chains to the amine groups to form cation-π interactions.

The simulations of the three peptides have shown that cation-π interactions between the choline headgroup and the tryptophan make an important contribution to the recognition of eukaryotic membranes by these peptides, thereby indicating that this type of interaction must be considered when designing new AMPs.

peptides that show a deeper insertion are those that have the highest hemolytic activity (Halevy et al., 2003), whereas for the DPPE/DPPG systems those peptides with higher antimicrobial activity were more deeply inserted (Zhang et al., 2001). This correlation between membrane activity and peptide insertion may have an important impact for the design of new AMPs, since more effective mutants can be designed rationallybased on the

**d (nm) NHB**

IND 1.43 ± 0.06 2.02 ± 0.05 24.4 ± 1.6 (13.8 ± 1.8)/(4.1 ± 0.8) CP10A 1.24 ± 0.08 1.84 ± 0.08 20.8 ± 1.6 (8.1 ± 1.5)/(10.0 ± 1.6) CP11 1.61 ± 0.07 1.70 ± 0.06 25.7 ± 2.1 (12.5 ± 1.5)/(4.0 ± 1.0)

Table 1. Distances between the CM of peptides and bilayers along the axis perpendicular to bilayer plane (*d*), and the number of hydrogen bonds between the peptides and bilayers (*NHB*).

Two important differences were obseved between the interactions of the peptides and the two different bilayers. The first was a greater number of hydrogen bonds (NHB) between the peptides and oxygen atoms of the DPPC bilayers (Table 1), which was due to a stretched conformation of the phosphotidylcholine groups of the DPPC molecule (Figure 10A**)** in which the distance between the CM of the phosphate and choline groups was 0.47 ± 0.06 nm, which compares with the phosphotidylethanolamine groups in the **DPPE/DPPG bilayer** where the distances between the phosphate and amine groups was 0.37 ± 0.01 nm. The proximity between the amine and phosphate groups in the DPPE molecules makes causes greater difficulty for the positively charged groups in peptides to approach the lipid headgroup phosphate group due to electrostatic repulsion with the positively charged amino group, thereby hindering the formation of HBs between the peptides and oxygen atoms. The second difference was the presence of cation-π interactions between choline groups of the DPPC and the side-chains of some Trp residues of the peptides. The cation-π interactions were evaluated by determining the radial distribution functions between the CM of the tryptophan side chains and nitrogen atoms of the choline groups, where the presence of the first peak (Dmax) at distances smaller than 0.5 nm and a number of choline groups greater than one (NChol) is indicative of cation-π interactions. These cation-π interactions were found with 1 to 2 choline groups for some tryptophan residues for all three peptides (as shown in Table 2). Examples of the proximity of the choline headgroups for each peptide are shown in Figures 10C, 10D, and 10E for IND, CP10A, and CP11, respectivelly. The cation-π interactions observed between the choline groups and the tryptophan residues do not occur in lipid bilayers containing PE due the geometry of this group, in which the amine and phosphate groups are closer, thereby impeding the approximation of the tryptophan side

The simulations of the three peptides have shown that cation-π interactions between the choline headgroup and the tryptophan make an important contribution to the recognition of eukaryotic membranes by these peptides, thereby indicating that this type of interaction

**DPPC DPPE/DPPG DPPC DPPE/DPPG** 

information obtained from MD simulation.

chains to the amine groups to form cation-π interactions.

must be considered when designing new AMPs.

**Peptide**

Fig. 10. Typical geometries of DPPC (A) and DPPE (B) molecules obtained from simulations. In (C), (D), and (E) are shown some examples of choline group neighbors of Trp residues for peptides IND, CP10A, and CP11, respectively. Choline groups are circled by a dotted line.


Table 2. Parameters extracted from radial distribution functions between the CM of Trp side chains and nitrogen atoms of choline groups.

#### **4. Trends in structural bioinformatics**

One of the most important tools in the theoretical studies of bio(macro)molecule is the MD simulation. MD calculates the time dependent properties of a (bio)molecular system. Current MD simulations of biomolecules are directed to investigate structural and dynamics properties of systems composed of very large number of atoms over timescales of many nanoseconds to microseconds (case of peptides folding). The continued growth of computer power and the advent of new algorithms like GPU-accelerated calculations are closely related to the more realistic approach of the molecular systems that allows chemical processes in condensed phases to be studied in an accurate and unbiased way. For example, MD trajectories can be generated with forces obtained from accurate electronic structure details (*ab initio* MD), as well as the improvement of the calculation efficiency when dealing with the complexity of biological problems. In this context we can cite a pharmacological interesting system, the case of quaternary structures of virus proteins and their interactions with active biological molecules, and the proteincarbohydrate interactions in glycoproteins. Future bioinformatics oriented studies will likely provide us with valuable tools for validating, improvement, prediction and development of a variety of biotechnological applications.

#### **5. References**


of many nanoseconds to microseconds (case of peptides folding). The continued growth of computer power and the advent of new algorithms like GPU-accelerated calculations are closely related to the more realistic approach of the molecular systems that allows chemical processes in condensed phases to be studied in an accurate and unbiased way. For example, MD trajectories can be generated with forces obtained from accurate electronic structure details (*ab initio* MD), as well as the improvement of the calculation efficiency when dealing with the complexity of biological problems. In this context we can cite a pharmacological interesting system, the case of quaternary structures of virus proteins and their interactions with active biological molecules, and the proteincarbohydrate interactions in glycoproteins. Future bioinformatics oriented studies will likely provide us with valuable tools for validating, improvement, prediction and

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### **Monoclonal Antibody Development and Physicochemical Characterization by High Performance Ion Exchange Chromatography**

Jennifer C. Rea, Yajun Jennifer Wang, Tony G. Moreno, Rahul Parikh, Yun Lou and Dell Farnan *Genentech, Inc., Protein Analytical Chemistry, South San Francisco, CA USA* 

#### **1. Introduction**

438 Innovations in Biotechnology

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Monoclonal antibodies (mAbs) represent a significant portion of products in the biopharmaceuticals market (Reichert, 2011; Scolnik, 2009). mAbs have been developed to treat a variety of indications to address significant unmet medical needs (Waldmann, 2003; Reichert & Valge-Archer, 2007; Ziegelbauer et al., 2008), and are generally target-specific and well tolerated with a relatively long half-life, contributing to the success of the molecule class for drug development (Scolnik, 2009). Of the classes of antibodies, or immunoglobulins, IgG1 is the most common immunoglobulin used for pharmaceutical and biomedical purposes (Reichert et al., 2005); however, other immunoglobulin types (e.g., IgG2, IgG4) and mAb-related products (e.g., Fc-fusion proteins, Fabs, etc.) are also being used for therapeutic purposes (Hudson & Souriau, 2003).

While a successful mAb product can generate upwards of a billion dollars or more in sales annually (Reichert, 2009), it takes a significant amount of time and resources to develop a new therapeutic mAb; current estimates indicate that it can take about 10-15 years (Dickson & Gagnon, 2004; DiMasi et al., 2003) and over \$1 billion to bring a new biologic drug to market (DiMasi & Grabowski, 2007). Addressing bottlenecks and making improvements in the development process is essential to save time and money, expediting the delivery of new drugs to the clinic.

This chapter will briefly cover monoclonal antibody development, production and purification, and then focus on antibody characterization, particularly charge-species analysis using high performance ion exchange chromatography (IEC). Method development strategies, method robustness, validation and automation will be discussed. This chapter aims to be a reference text demonstrating the utility of IEC as well as providing strategies for developing rugged IEC methods for the characterization of therapeutic mAbs.

#### **2. Monoclonal antibody development**

Antibodies are physiological blood components that are produced by B lymphocytes, intended to bind to and neutralize foreign antigens and pathogens. Antibodies bind to a corresponding antigen in a highly specific manner. Although not covered in this chapter, potential mechanisms of action for mAbs have been described (Green et al., 2000). Polyclonal antibodies are a combination of immunoglobulin molecules secreted against a specific antigen, each identifying a different epitope. In contrast, monoclonal antibodies are derived from a single cell line that are all clones of a unique parent cell.

The first monoclonal antibody product was approved in 1986 and was a murine antibody for the prevention of kidney transplant rejection; however, patients frequently developed antibodies against the mouse-derived mAbs, which limited their effectiveness (Jones et al., 2007; Kuus-Reichel et al., 1994; Shawler et al., 1985). Advances in antibody engineering yielded techniques for generating chimeric mAbs that contain sequences from both human and murine sources (Morrison et al., 1984; Reichmann et al., 1988). Many of the mAbs approved for commercialization in the 1990s and early 2000s were chimeric antibodies. Chimeric antibody products are superior to murine antibodies, but they still pose a risk of immunogenicity to patients from their residual murine components (Carter, 2001). New technologies were developed to produce humanized mAbs, which contain approximately 95% human components and 5% murine components (Carter & Presta, 2000, 2002). In these humanized mAbs, the CDRs of a human antibody gene were replaced by those from the CDR of a murine mAb gene (Figure 1). The resulting humanized antibody has the same antigen binding properties as the original murine antibody but contains minimal murine sequences (Co & Queen, 1991).

Fig. 1. Schematic representation of the sequence composition of murine, chimeric, humanized, and human antibodies.

More recently, less immunogenic therapeutic antibody products were developed by creating fully human mAbs. Several technologies exist to develop fully human antibodies, each falling into one of the two general classes—*in vivo* approaches using a murine system in which the immunoglobulin genes have been replaced by their human counterparts, or *in vitro* approaches such as phage display libraries containing millions of variations of antibody sequences coupled with a mechanism to express and screen these antibodies *in vitro* (Lonberg, 2005; McCafferty et al., 1990). Combining *in vivo* and *in vitro* discovery and molecular engineering technologies allows exquisite control of the antibody sequences and properties that was not possible when mAbs were first developed. Because of these advanced antibody engineering technologies, almost all antibody products currently in development are humanized or fully human mAbs and their derivatives.

#### **3. Monoclonal antibody production**

440 Innovations in Biotechnology

corresponding antigen in a highly specific manner. Although not covered in this chapter, potential mechanisms of action for mAbs have been described (Green et al., 2000). Polyclonal antibodies are a combination of immunoglobulin molecules secreted against a specific antigen, each identifying a different epitope. In contrast, monoclonal antibodies are

The first monoclonal antibody product was approved in 1986 and was a murine antibody for the prevention of kidney transplant rejection; however, patients frequently developed antibodies against the mouse-derived mAbs, which limited their effectiveness (Jones et al., 2007; Kuus-Reichel et al., 1994; Shawler et al., 1985). Advances in antibody engineering yielded techniques for generating chimeric mAbs that contain sequences from both human and murine sources (Morrison et al., 1984; Reichmann et al., 1988). Many of the mAbs approved for commercialization in the 1990s and early 2000s were chimeric antibodies. Chimeric antibody products are superior to murine antibodies, but they still pose a risk of immunogenicity to patients from their residual murine components (Carter, 2001). New technologies were developed to produce humanized mAbs, which contain approximately 95% human components and 5% murine components (Carter & Presta, 2000, 2002). In these humanized mAbs, the CDRs of a human antibody gene were replaced by those from the CDR of a murine mAb gene (Figure 1). The resulting humanized antibody has the same antigen binding properties as the original murine antibody but contains minimal murine

Fig. 1. Schematic representation of the sequence composition of murine, chimeric,

More recently, less immunogenic therapeutic antibody products were developed by creating fully human mAbs. Several technologies exist to develop fully human antibodies, each falling into one of the two general classes—*in vivo* approaches using a murine system in which the immunoglobulin genes have been replaced by their human counterparts, or *in vitro* approaches such as phage display libraries containing millions of variations of antibody sequences coupled with a mechanism to express and screen these antibodies *in vitro* (Lonberg, 2005; McCafferty et al., 1990). Combining *in vivo* and *in vitro* discovery and molecular engineering technologies allows exquisite control of the antibody sequences and properties that was not possible when mAbs were first developed. Because of these

derived from a single cell line that are all clones of a unique parent cell.

sequences (Co & Queen, 1991).

humanized, and human antibodies.

Recombinant monoclonal antibodies are typically produced in mammalian cell lines under defined cell culture conditions. Commercial scale production processes vary depending on the mAb, but generally, cells are taken from a master cell bank and inoculated into smallscale bioreactors. The cell culture is transferred to increasingly larger bioreactors until it reaches the final commercial scale bioreactor. Currently, final scale reactors have volumes ranging from 12,000 L to 24,000 L (Gottschalk, 2009). The cells are cultured in a controlled environment for days to weeks, and then the cell culture fluid is harvested by centrifugation (Shukla & Kandula, 2008). In mammalian cells, the product monoclonal antibodies are secreted from the cells into the supporting fluid medium. Centrifugation separates the cells from the fluids and facilitates simpler recovery procedures downstream.

Commercial mAb production requires considerable preproduction effort to ensure that the cell line is stable and can produce commercially appropriate quantities of antibody. In addition, the commercial production process must produce a product that meets the quality expectations of regulatory authorities. In the past few years, improvements have been made in critical areas, such as cell line generation and large-scale cell culture production, to maximize specific antibody productivity from a given cell line and improve overall productivity in bioreactors. These advances include the use of new expression vectors and transfection technology, high-throughput, robust screening technologies to select the highest producing clones rapidly and more effectively, improvements in cell culture and optimized bioreactor processes (Li et al., 2010; Schlatter et al., 2005). As a result, the production of cell lines expressing multigram quantities of antibody per liter of culture medium is now routine.

The product quality and product heterogeneity of every mAb is highly dependent on its manufacturing process (Abu-Absi et al., 2010; Horvath et al., 2010). The ideal manufacturing conditions would have optimal production levels of product in conjunction with the desired product quality profile. Attributes that are typically deemed critical in selecting stable clones and cell culture conditions are the product titer and product heterogeneity, including charged species and aggregates. Production titers directly correlate to the costs of the process and are desired to be as high as possible with minimal impact to other quality attributes of the product (Kelley, 2009). Critical quality attributes of the product, such as the level of aggregation, are carefully monitored, as failure to control critical quality attributes may pose a safety risk to the patient (Rosenberg, 2006).

#### **4. Monoclonal antibody purification and formulation**

Once monoclonal antibodies are produced in cells, the mAbs must be recovered and purified. Recovery and purification processes vary widely depending on the manufacturing process and specific mAb characteristics, but generally, the isolation and purification of mAbs involve a centrifugation step to separate the cells from the cell culture fluid containing the mAb product, one or more chromatography steps, which can include affinity chromatography, cation or anion exchange chromatography, hydrophobic interaction chromatography (HIC) and displacement chromatography (Shukla et al., 2007), and filtration or precipitation steps (Gottschalk, 2009). Many of the purification steps are designed to remove contaminants and adventitious agents (e.g., bacteria, fungi, viruses, and mycoplasma).

After elution from the final chromatographic purification step, a unit operation is required to exchange the components of the chromatography elution buffer with the chosen formulation components. The predominant technology that has been used in the industry for buffer exchange and concentration is ultrafiltration/diafiltration using tangential-flow filtration (Genovesi, 1983; Shiloach, 1988; van Reis, 2001). After this step, the drug substance is filtered and typically frozen as bulk for storage until filling occurs to produce the final drug product.

The formulation of the mAb therapeutic is chosen in part to ensure product quality during shelf life. Formulations are designed to minimize protein aggregation, decrease viscosity, and increase shelf life through preventing degradation (Shire, 2009). High protein concentration formulations are being developed to allow for subcutaneous or intramuscular delivery of mAb products (Shire et al., 2004). Historically, the most conventional route of delivery for protein drugs has been intravenous administration because of poor bioavailability by most other routes, greater control during clinical administration, and faster pharmaceutical development. Subcutaneous delivery allows for home administration and improved patient compliance. However, development of high protein concentration formulations involves unique manufacturing challenges compared to low concentration formulations, such as higher viscosities and necessary changes to unit operation steps.

#### **5. Monoclonal antibody characterization and release testing**

Biopharmaceutical manufacturing of monoclonal antibodies produces a heterogeneous product of structurally related species. Antibody speciation can occur throughout the manufacturing process at various steps, including cell culture, harvest, purification, formulation, filling and during shelf life. Full-length monoclonal antibodies are high molecular weight proteins (around 150,000 Da), and have highly complex secondary and tertiary structures, subject to post-translational modifications. Therefore, product characterization and quality control testing are required at critical points throughout clinical development and manufacturing to control for these species (Harris et al., 2004). Figure 2 depicts the structure of a monoclonal antibody compared to a small molecule drug, illustrating the increased complexity of a biologic compared to a small molecule therapeutic.

Antibodies can be characterized by many physicochemical properties including hydrated size (Stokes radius), molecular weight, charge, hydrophobicity, electrophoretic mobility, isoelectric point (pI), sedimentation velocity, glycosylation, and spectral properties. The nature of each species can be related to differences in their primary, secondary, tertiary, or quaternary protein structures. In addition, monoclonal antibodies are susceptible to chemical or enzymatic modification, particularly at sites that are exposed to the proteinliquid interface. Product heterogeneity can be caused by a number of modifications, such as C-terminal processing of lysine residues (Harris, 1995; Santora et al., 1999; Weitzhandler et al., 1998), deamidation (Di Donato et al., 1993; Hsu et al., 1998), glycation (nonenzymatic glucose addition) (Quan et al., 2008), amino acid sequence variations (Yang et al., 2010), and noncovalent complexes (Santora et al., 2001).

filtration or precipitation steps (Gottschalk, 2009). Many of the purification steps are designed to remove contaminants and adventitious agents (e.g., bacteria, fungi, viruses, and

After elution from the final chromatographic purification step, a unit operation is required to exchange the components of the chromatography elution buffer with the chosen formulation components. The predominant technology that has been used in the industry for buffer exchange and concentration is ultrafiltration/diafiltration using tangential-flow filtration (Genovesi, 1983; Shiloach, 1988; van Reis, 2001). After this step, the drug substance is filtered and typically frozen as bulk for storage until filling occurs to produce the final

The formulation of the mAb therapeutic is chosen in part to ensure product quality during shelf life. Formulations are designed to minimize protein aggregation, decrease viscosity, and increase shelf life through preventing degradation (Shire, 2009). High protein concentration formulations are being developed to allow for subcutaneous or intramuscular delivery of mAb products (Shire et al., 2004). Historically, the most conventional route of delivery for protein drugs has been intravenous administration because of poor bioavailability by most other routes, greater control during clinical administration, and faster pharmaceutical development. Subcutaneous delivery allows for home administration and improved patient compliance. However, development of high protein concentration formulations involves unique manufacturing challenges compared to low concentration formulations, such as higher viscosities and necessary changes to unit operation steps.

Biopharmaceutical manufacturing of monoclonal antibodies produces a heterogeneous product of structurally related species. Antibody speciation can occur throughout the manufacturing process at various steps, including cell culture, harvest, purification, formulation, filling and during shelf life. Full-length monoclonal antibodies are high molecular weight proteins (around 150,000 Da), and have highly complex secondary and tertiary structures, subject to post-translational modifications. Therefore, product characterization and quality control testing are required at critical points throughout clinical development and manufacturing to control for these species (Harris et al., 2004). Figure 2 depicts the structure of a monoclonal antibody compared to a small molecule drug, illustrating the increased complexity of a biologic compared to a small molecule therapeutic. Antibodies can be characterized by many physicochemical properties including hydrated size (Stokes radius), molecular weight, charge, hydrophobicity, electrophoretic mobility, isoelectric point (pI), sedimentation velocity, glycosylation, and spectral properties. The nature of each species can be related to differences in their primary, secondary, tertiary, or quaternary protein structures. In addition, monoclonal antibodies are susceptible to chemical or enzymatic modification, particularly at sites that are exposed to the proteinliquid interface. Product heterogeneity can be caused by a number of modifications, such as C-terminal processing of lysine residues (Harris, 1995; Santora et al., 1999; Weitzhandler et al., 1998), deamidation (Di Donato et al., 1993; Hsu et al., 1998), glycation (nonenzymatic glucose addition) (Quan et al., 2008), amino acid sequence variations (Yang et al., 2010), and

**5. Monoclonal antibody characterization and release testing** 

noncovalent complexes (Santora et al., 2001).

mycoplasma).

drug product.

Antibody products are characterized by physicochemical, immunochemical, and biological methods. Guidance documents have been issued by regulatory agencies and industry representatives recommending approaches for protein characterization (International Conference on Harmonisation of Technical Requirements for the Registration of Pharmaceuticals for Human Use (ICH), 1999; Schnerman et al., 2004). These orthogonal assays include potency, identity and purity assays, which evaluate "critical quality attributes" such as size and charge heterogeneity. These critical quality attributes are part of the overall target product profile, which is based on the desired clinical performance. The extent of characterization is linked to the level of risk associated with each phase of drug development. For example, while there may not be sufficient time or resources for extensive characterization of an antibody during early stage development, it is expected that the molecule will be well-characterized before the Biologic License Application (BLA) is submitted to the regulatory agencies.

Fig. 2. Comparison of the structures of a mAb (Herceptin) and a small molecule therapeutic (Tarceva).

Many of the recommended protein characterization assays are based on liquid chromatography methods, such as ion exchange chromatography (IEC) for charge heterogeneity analysis, size exclusion chromatography (SEC) for size heterogeneity, and reversed-phase high performance liquid chromatography (RP-HPLC) for peptide mapping (Chirino & Mire-Sluis, 2004). The remainder of this chapter will primarily focus on ion exchange chromatography methods for analyzing charge heterogeneity for characterization and support of formulation and process development, as well as for lot release testing of drug substance and drug product (Schnerman et al., 2004).

#### **5.1 Analyzing mAb charge heterogeneity using IEC**

As mentioned previously, monoclonal antibodies are large proteins that are quite complex. While the light chain and heavy chain sequences of a particular mAb may be known, a number of modifications can introduce heterogeneity in the product. Thus, it is important to develop appropriate analytical methods to resolve the minor forms of the product. Analytical biochemists routinely use IEC for resolving charge variants of the protein. The scientist must then utilize orthogonal analytical methods to characterize the separated peaks of the ion exchange chromatogram. The characterization of a mAb is particularly important if the modifications occur in the complementarity-determining regions (CDR), as modifications in the CDR can affect the binding activity and potency of the mAb.

A strategy for the assignment of peaks from a weak cation exchange (WCX) mAb separation using a salt gradient has been published (Harris et al., 2001). Seven forms of a therapeutic recombinant antibody were resolved by cation-exchange chromatography. The peak fractions were collected, and structural differences were assigned by peptide mapping, which involves digesting the mAb with an enzyme and injecting the digest onto a reverse-phase column coupled to a mass spectrometer, and by hydrophobic interaction chromatography (HIC) after papain digestion. The peaks in this particular case were attributed to deamidation, isomerization, and succinimide intermediates. Other orthogonal analytical methods were used to characterize the IEC peaks; one of these methods—potency testing—determined that one minor peak demonstrated much lower potency than the main peak.

In another study, a recombinant humanized monoclonal IgG1 antibody with different states of glycosylation on the conserved asparagine residue in the CH2 domain was analyzed by cation exchange chromatography (Gaza-Bulseco et al., 2008). Two major peaks were observed and were further characterized by enzymatic digestion and mass spectrometry. It was found that this recombinant monoclonal antibody contained three glycosylation states—zero, one or two glycosylated heavy chains. The peak that eluted earlier on the cation exchange column contained antibodies with two glycosylated heavy chains containing fucosylated biantennary complex oligosaccharides with zero, one or two terminal galactose residues. The peak that eluted later from the column contained antibodies with zero, one or two glycosylated heavy chains. The oligosaccharide on the antibodies that eluted in the later peak was composed of only two GlcNAc residues. These results indicate that conformational changes, caused by different types of neutral oligosaccharides as well as the absence of certain oligosaccharides, can be differentiated by cation exchange column chromatography.

#### **5.2 Lot release testing of mAbs**

Once the mAb is purified and formulated, the resulting drug substance must be tested prior to lot release. A set of tests and acceptance criteria are established based on mAb characterization and regulatory requirements in order to ensure product quality (Food & Drug Administration (FDA), 1999). These tests typically include appearance, identity, purity, protein concentration, potency of the molecule, microbial limits or bioburden, and bacterial endotoxins (Table 1). IEC is one of the most frequently used lot release methods for purity for mAbs (Schnerman et al., 2004). Once these tests are performed and the results

As mentioned previously, monoclonal antibodies are large proteins that are quite complex. While the light chain and heavy chain sequences of a particular mAb may be known, a number of modifications can introduce heterogeneity in the product. Thus, it is important to develop appropriate analytical methods to resolve the minor forms of the product. Analytical biochemists routinely use IEC for resolving charge variants of the protein. The scientist must then utilize orthogonal analytical methods to characterize the separated peaks of the ion exchange chromatogram. The characterization of a mAb is particularly important if the modifications occur in the complementarity-determining regions (CDR), as

A strategy for the assignment of peaks from a weak cation exchange (WCX) mAb separation using a salt gradient has been published (Harris et al., 2001). Seven forms of a therapeutic recombinant antibody were resolved by cation-exchange chromatography. The peak fractions were collected, and structural differences were assigned by peptide mapping, which involves digesting the mAb with an enzyme and injecting the digest onto a reverse-phase column coupled to a mass spectrometer, and by hydrophobic interaction chromatography (HIC) after papain digestion. The peaks in this particular case were attributed to deamidation, isomerization, and succinimide intermediates. Other orthogonal analytical methods were used to characterize the IEC peaks; one of these methods—potency testing—determined that one minor peak demonstrated much lower

In another study, a recombinant humanized monoclonal IgG1 antibody with different states of glycosylation on the conserved asparagine residue in the CH2 domain was analyzed by cation exchange chromatography (Gaza-Bulseco et al., 2008). Two major peaks were observed and were further characterized by enzymatic digestion and mass spectrometry. It was found that this recombinant monoclonal antibody contained three glycosylation states—zero, one or two glycosylated heavy chains. The peak that eluted earlier on the cation exchange column contained antibodies with two glycosylated heavy chains containing fucosylated biantennary complex oligosaccharides with zero, one or two terminal galactose residues. The peak that eluted later from the column contained antibodies with zero, one or two glycosylated heavy chains. The oligosaccharide on the antibodies that eluted in the later peak was composed of only two GlcNAc residues. These results indicate that conformational changes, caused by different types of neutral oligosaccharides as well as the absence of certain oligosaccharides, can be differentiated

Once the mAb is purified and formulated, the resulting drug substance must be tested prior to lot release. A set of tests and acceptance criteria are established based on mAb characterization and regulatory requirements in order to ensure product quality (Food & Drug Administration (FDA), 1999). These tests typically include appearance, identity, purity, protein concentration, potency of the molecule, microbial limits or bioburden, and bacterial endotoxins (Table 1). IEC is one of the most frequently used lot release methods for purity for mAbs (Schnerman et al., 2004). Once these tests are performed and the results

modifications in the CDR can affect the binding activity and potency of the mAb.

**5.1 Analyzing mAb charge heterogeneity using IEC** 

potency than the main peak.

by cation exchange column chromatography.

**5.2 Lot release testing of mAbs** 

meet the established acceptance criteria, a Certificate of Analysis (COA) is generated and the lot is released for use. Finally, adequate stability studies should be performed on the mAb drug substance (e.g. frozen bulk for storage) and drug product (e.g. final vial) according to regulatory guidelines (Food & Drug Administration (FDA), 2003).


Table 1. Commonly used tests found on a Certificate of Analysis for lot release; a selected subset is used for stability testing of mAbs.

#### **6. Mechanism of ion exchange chromatography of mAbs**

Ion exchange chromatography (IEC) has been a platform for monoclonal antibody purification and characterization for many years. For the analysis of charged species of proteins, IEC is a popular method due to the fact that it preserves the native conformation and maintains bioactivity of the protein, is relatively easy of use, is supported by the maturity of the equipment and consumables market, and has widespread use in the biopharmaceutical industry (Rea et al., 2010).

Charge-based methods are an integral component of characterization studies and quality control strategies because they are sensitive to many types of modifications. Charge profiling of intact antibodies can resolve species related to protein conformation, size, sequence species, glycosylation and post-translational modifications (Gaza-Bulseco et al., 2008; Harris et al., 2001; He et al., 2010). Although IEC can be used to track specific species, it is common to group all species not associated with the main peak and report them as either acidic or basic species (Figure 3). In addition, fractions collected from an IEC run can often be directly injected onto orthogonal columns for further analysis, such as reverse-phase and size exclusion chromatography columns, or submitted for potency testing.

IEC separates proteins based on differences in the surface charge of the molecules, with separation being dictated by the protein interaction with the stationary phase. The two main categories of ion exchange chromatography are cation exchange (CEX) and anion exchange (AEX). Cation exchange chromatography retains biomolecules by the interaction of the negatively-charged resin with histidine (pK ~ 6.5), lysine (pK ~ 10) and arginine (pK ~ 12) in the protein. Anion exchange chromatography primarily retains biomolecules by the interaction of the positively-charged resin with aspartic or glutamic acid side chains, which have pKa of ~4.4. In addition to the amino acid residues, cation exchange columns can also separate deamidated, glycated and other charged variants. Anion exchange columns have also been useful for separating phosphorylated and hydroxyl modified amino acids. When the pH equals the pI value of the protein, the net charge on the molecule is zero. However, significant retention can occur for proteins even when the pH of the mobile phase is equal to the pI of the molecule; despite an overall net charge of zero, only a portion of the mAb molecule will interact with the stationary phase, and there will be a net charge on that portion of the molecule because of an uneven distribution of charged groups throughout the molecule (Vlasak & Ionescu, 2008). Thus, it is possible to separate proteins having very similar charge (Figure 4), or even structural isomers with identical pI values, by ion exchange chromatography.

Fig. 3. Typical cation exchange chromatogram for analytical characterization of a mAb. Integration is shown, and main peak, acidic and basic regions are denoted.

There are two ways to elute the protein from the IEC column: 1) increasing salt concentration with time or 2) by varying the mobile phase pH value as a function of time. Increasing the salt concentration elutes the protein by increasing the ionic strength of the mobile phase, thus affecting the charge interaction of the mAb and the stationary phase. A pH gradient elutes the protein by changing the charge on the molecule, thus affecting the binding of the molecule to the stationary phase. While conventional salt gradient cation exchange chromatography is regarded as the gold standard for charge sensitive antibody analysis (Vlasak & Ionescu, 2008), method parameters such as column type, mobile phase pH, and salt concentration gradient often need to be optimized for each individual antibody. A recent publication described a multi-product pH gradient IEC method for the separation of mAb charge species for a variety of mAbs using a single method (Farnan & Moreno, 2009). The following sections will discuss both salt-gradient and pH-gradient based elution methods, and the combination of the two modalities (hybrid methods).

IEC separates proteins based on differences in the surface charge of the molecules, with separation being dictated by the protein interaction with the stationary phase. The two main categories of ion exchange chromatography are cation exchange (CEX) and anion exchange (AEX). Cation exchange chromatography retains biomolecules by the interaction of the negatively-charged resin with histidine (pK ~ 6.5), lysine (pK ~ 10) and arginine (pK ~ 12) in the protein. Anion exchange chromatography primarily retains biomolecules by the interaction of the positively-charged resin with aspartic or glutamic acid side chains, which have pKa of ~4.4. In addition to the amino acid residues, cation exchange columns can also separate deamidated, glycated and other charged variants. Anion exchange columns have also been useful for separating phosphorylated and hydroxyl modified amino acids. When the pH equals the pI value of the protein, the net charge on the molecule is zero. However, significant retention can occur for proteins even when the pH of the mobile phase is equal to the pI of the molecule; despite an overall net charge of zero, only a portion of the mAb molecule will interact with the stationary phase, and there will be a net charge on that portion of the molecule because of an uneven distribution of charged groups throughout the molecule (Vlasak & Ionescu, 2008). Thus, it is possible to separate proteins having very similar charge (Figure 4), or even structural

isomers with identical pI values, by ion exchange chromatography.

Fig. 3. Typical cation exchange chromatogram for analytical characterization of a mAb.

There are two ways to elute the protein from the IEC column: 1) increasing salt concentration with time or 2) by varying the mobile phase pH value as a function of time. Increasing the salt concentration elutes the protein by increasing the ionic strength of the mobile phase, thus affecting the charge interaction of the mAb and the stationary phase. A pH gradient elutes the protein by changing the charge on the molecule, thus affecting the binding of the molecule to the stationary phase. While conventional salt gradient cation exchange chromatography is regarded as the gold standard for charge sensitive antibody analysis (Vlasak & Ionescu, 2008), method parameters such as column type, mobile phase pH, and salt concentration gradient often need to be optimized for each individual antibody. A recent publication described a multi-product pH gradient IEC method for the separation of mAb charge species for a variety of mAbs using a single method (Farnan & Moreno, 2009). The following sections will discuss both salt-gradient and pH-gradient based elution

Integration is shown, and main peak, acidic and basic regions are denoted.

methods, and the combination of the two modalities (hybrid methods).

Fig. 4. Separation of mAbs differing by only one charge, a single amino acid change to primary structure. The elution buffer (0.5 M NaCl in 20 mM Tris, pH 7.3) was increased linearly on a ProPac WCX-10 column (4 x 250 mm), which was held at 50 ºC and had a flow rate of 1 mL min-1.

#### **7. Developing a salt-based IEC method**

Salt-based IEC separations are developed by choosing a cation or anion exchange column and varying the buffer system, mobile phase pH value, and ionic strength gradient of the elution buffer. Figure 5 shows a typical development workflow for salt-based IEC and pHbased IEC development, and can serve as a guide for initial IEC method development. The following sections will cover in more detail the outputs to consider when screening various parameters during development. More general considerations regarding HPLC method development can be found in various texts (Kastner, 2000; Snyder et al., 1997).

Fig. 5. Sequential salt-gradient IEC and pH-gradient IEC method development and optimization work flow.

#### **7.1 Column selection, buffers and operating parameters for salt gradient IEC**

Column selection is perhaps the most subjective part of the optimization process; picking between the different vendor offerings and functionalities can be difficult. Prior experience, data in the literature or unpublished results within the organization are often the best starting points.

Analytical ion exchange chromatography of proteins is typically carried out using mobile phases that are relatively neutral in pH values, 5.5 to 8.5 This general practice is recommended because at pH extremes, the protein is more likely to degrade. The selection of whether to use anion or cation exchange chromatography is also driven by the isoelectric point of the protein (pI) and the species to be resolved, e.g., phosphorylated species, Cterminal lysine variants, etc.

If the pI value of the mAb is greater than 8, a CEX column is evaluated at pH 6-7 initially. CEX primarily retains mAbs by the interaction of acid groups on the CEX resin with lysine, arginine and histidine side chains on the mAb. Since mAbs are positively charged at a mobile phase pH below their pI, the mAb species would likely be retained and resolved on a CEX column under the recommended mobile phase pH range.

If the pI value of the mAb is less than 6, an AEX column is evaluated at a pH above 6 initially. AEX primarily retains biomolecules by the interaction of amine groups on the ion exchange resin with aspartic or glutamic acid side chains. Since mAbs are negatively charged at a mobile phase pH above their pI, the mAb species would likely be retained and resolved on an AEX column.

For intermediate pI values of 6-8, both CEX and AEX are evaluated because of the possibility that the portion of the mAb that interacts with the stationary phase, typically the side chains that are exposed to the mobile phase, has a different charge than the pI would suggest, e.g., the surface charge of the mAb is positive despite the entire mAb having an overall negative charge. Ultimately, the species of interest that are to be resolved determine whether CEX or AEX is chosen for molecules with intermediate pI's; the separation mode that better separates the species of interest is usually the one that is chosen for mAb analysis.

Figure 6 shows CEX and AEX chromatograms of a Fab (mAb fragment) reference sample and thermally stressed sample. In this case, the Fab molecule has a nominal pI value for the main species of 7.6. It should be noted that the separations on the AEX and CEX columns were each optimized independently for column type, pH value and salt gradient. It should also be noted that the terms "strong" and "weak" (in SAX, strong anion exchange, and WCX, weak cation exchange) refer to the extent of variation of ionization with pH due to the functional groups on the resin and not the strength of binding. Strong ion exchangers are completely ionized over a wide pH range whereas with weak ion exchangers, the degree of dissociation and thus exchange capacity varies much more markedly with pH. For this example, SAX results in significantly more peaks and much better resolution of the charge species in comparison to the WCX chromatogram. Particularly interesting is that the difference between the WCX and SAX elution profiles are much more vivid for the stressed samples than for the reference materials. We have seen examples where the converse is true and the CEX separation is better than that observed on the AEX. This contrast between the AEX and CEX profiles highlights an important feature of IEC that electrophoretic methods don't exhibit, which is the ability to magnify particular aspects of the protein structure and accentuate the separation of species relating to particular motifs (Vlasak & Ionescu, 2008).

Column selection is perhaps the most subjective part of the optimization process; picking between the different vendor offerings and functionalities can be difficult. Prior experience, data in the literature or unpublished results within the organization are often the best

Analytical ion exchange chromatography of proteins is typically carried out using mobile phases that are relatively neutral in pH values, 5.5 to 8.5 This general practice is recommended because at pH extremes, the protein is more likely to degrade. The selection of whether to use anion or cation exchange chromatography is also driven by the isoelectric point of the protein (pI) and the species to be resolved, e.g., phosphorylated species, C-

If the pI value of the mAb is greater than 8, a CEX column is evaluated at pH 6-7 initially. CEX primarily retains mAbs by the interaction of acid groups on the CEX resin with lysine, arginine and histidine side chains on the mAb. Since mAbs are positively charged at a mobile phase pH below their pI, the mAb species would likely be retained and resolved on a

If the pI value of the mAb is less than 6, an AEX column is evaluated at a pH above 6 initially. AEX primarily retains biomolecules by the interaction of amine groups on the ion exchange resin with aspartic or glutamic acid side chains. Since mAbs are negatively charged at a mobile phase pH above their pI, the mAb species would likely be retained and

For intermediate pI values of 6-8, both CEX and AEX are evaluated because of the possibility that the portion of the mAb that interacts with the stationary phase, typically the side chains that are exposed to the mobile phase, has a different charge than the pI would suggest, e.g., the surface charge of the mAb is positive despite the entire mAb having an overall negative charge. Ultimately, the species of interest that are to be resolved determine whether CEX or AEX is chosen for molecules with intermediate pI's; the separation mode that better separates the species of interest is usually the one that is chosen for mAb analysis. Figure 6 shows CEX and AEX chromatograms of a Fab (mAb fragment) reference sample and thermally stressed sample. In this case, the Fab molecule has a nominal pI value for the main species of 7.6. It should be noted that the separations on the AEX and CEX columns were each optimized independently for column type, pH value and salt gradient. It should also be noted that the terms "strong" and "weak" (in SAX, strong anion exchange, and WCX, weak cation exchange) refer to the extent of variation of ionization with pH due to the functional groups on the resin and not the strength of binding. Strong ion exchangers are completely ionized over a wide pH range whereas with weak ion exchangers, the degree of dissociation and thus exchange capacity varies much more markedly with pH. For this example, SAX results in significantly more peaks and much better resolution of the charge species in comparison to the WCX chromatogram. Particularly interesting is that the difference between the WCX and SAX elution profiles are much more vivid for the stressed samples than for the reference materials. We have seen examples where the converse is true and the CEX separation is better than that observed on the AEX. This contrast between the AEX and CEX profiles highlights an important feature of IEC that electrophoretic methods don't exhibit, which is the ability to magnify particular aspects of the protein structure and accentuate the separation of species relating to particular motifs (Vlasak & Ionescu, 2008).

CEX column under the recommended mobile phase pH range.

**7.1 Column selection, buffers and operating parameters for salt gradient IEC** 

starting points.

terminal lysine variants, etc.

resolved on an AEX column.

Fig. 6. Separation of Fab charge species using a weak cation exchange column (WCX) and a strong anion exchange column (SAX). Thermally stressed samples are labeled by incubation time and temperature of incubation.

In general, we have observed that for the separation of mAb variants using ion-exchange chromatography, the optimized chromatogram has a relatively shallow gradient over a narrow range of salt concentration. A typical method results in 100 mM NaCl as the center point of the gradient, with salt concentration increasing over 70 mM NaCl in a linear gradient. It is recommended to perform iterative gradient optimizations to narrow the NaCl gradient down to around 2 mM/mL min-1. Iterative cycles are quicker and more predictive than performing a very long shallow gradient.

Chromatograms obtained during the mobile phase pH value optimization for a mAb with a pI value around 9.5 are shown in Figure 7. Buffer species and buffer concentration for saltgradient IEC are generally not significant factors, but should be chosen considering target pH and buffer pKa.

Although temperature does not significantly affect electrostatic interactions, it often affects the pH value of the mobile phase. This is particularly of concern for a Good's buffer system (group of buffers described in the research of Dr. Norman Good et al. in 1966, often used for IEC and other biochemistry applications) (Good et al., 1966), which can exhibit a change in pH value of around 0.02 per °C temperature change. This sensitivity creates a need to control the column temperature carefully. A column compartment is always used, typically set at a value greater than 30°C to ensure good temperature stability in compartments that can only apply heat. Above 30°C, temperature control within +/- 1°C is readily achievable with commercially available equipment.

Fig. 7. Effect of mobile phase pH on mAb separation by WCX. The elution buffer (0.5 M NaCl) was increased linearly at 1 mM min-1 at a flow rate of 1 mL min-1 on a ProPac WCX-10 column (4 x 250 mm), which was held at 30 ºC. Different initial salt concentrations were optimized for each pH value. Integration is shown, and main peak, acidic and basic regions are denoted.

Subtle variations in selectivity with temperature may result from temperature-induced changes in mobile phase pH value (Figure 8). In Figure 8, the elution profile changes in two distinct regions as a function of temperature. Below 40°C, subtle changes in elution profile and retention times are observed consistent with minor changes to the mobile phase pH value as a function of temperature. However, above 40°C, the profiles exhibit much more radical changes with increasing temperature. This is interpreted to be related to the mAb having lost higher order structure at those elevated temperatures due to protein denaturing. For the mAb in Figure 8, it is clear that moderately elevated temperatures are not possible while maintaining the higher order structure; in general for IgG1 mAbs, chromatography at temperatures up to 55°C is readily possible. In summary, while mobile phase temperature does not affect protein charge directly, temperature can affect mobile phase pH and the structure of the protein, which can affect chromatographic separations. Thus, column temperature should be optimized considering these temperature effects.

Fig. 7. Effect of mobile phase pH on mAb separation by WCX. The elution buffer (0.5 M NaCl) was increased linearly at 1 mM min-1 at a flow rate of 1 mL min-1 on a ProPac WCX-10 column (4 x 250 mm), which was held at 30 ºC. Different initial salt concentrations were optimized for each pH value. Integration is shown, and main peak, acidic and basic regions

Subtle variations in selectivity with temperature may result from temperature-induced changes in mobile phase pH value (Figure 8). In Figure 8, the elution profile changes in two distinct regions as a function of temperature. Below 40°C, subtle changes in elution profile and retention times are observed consistent with minor changes to the mobile phase pH value as a function of temperature. However, above 40°C, the profiles exhibit much more radical changes with increasing temperature. This is interpreted to be related to the mAb having lost higher order structure at those elevated temperatures due to protein denaturing. For the mAb in Figure 8, it is clear that moderately elevated temperatures are not possible while maintaining the higher order structure; in general for IgG1 mAbs, chromatography at temperatures up to 55°C is readily possible. In summary, while mobile phase temperature does not affect protein charge directly, temperature can affect mobile phase pH and the structure of the protein, which can affect chromatographic separations. Thus, column

temperature should be optimized considering these temperature effects.

are denoted.

Fig. 8. Effect of temperature on mAb separation by CEX. The elution buffer (0.2 M sodium sulfate) was increased linearly on a ProPacWCX-10 column (4 x 250 mm).

#### **8. Developing a pH gradient-based IEC method**

Despite good resolving power and robustness, salt-based ion exchange separations are usually protein-specific and time-consuming to develop. A novel pH-based separation of proteins by cation exchange chromatography that was multi-product, high-resolution, and robust against variations in sample matrix salt concentration and pH was recently reported (Farnan & Moreno, 2009). A pH gradient-based separation method using cation exchange chromatography was also evaluated in a mock validation and deemed highly robust (Rea et al., 2011). Figure 9 depicts the separation of 16 mAbs by pH gradient IEC (pH-IEC). Each mAb was injected sequentially, demonstrating that in contrast to salt-based IEC, pH-IEC can be used to analyze multiple mAbs with a single method.

Fig. 9. Separation of 16 mAbs using a ProPac WCX-10 column by pH gradient IEC. Each mAb was analyzed using the same pH-IEC method, and each mAb was injected sequentially. mAb pI values ranged from pI 7.3 to pI 9.4.

Similar to salt-gradient IEC methods, pH-IEC separations are developed by choosing a cation or anion exchanging column and varying the buffer system, pH of the mobile phases, and other operating parameters, such as temperature and flow rate. Figure 5 shows a typical development workflow for pH-IEC, and can serve as a guide for initial pH-IEC method development.

#### **8.1 Column selection, buffers and operating parameters for pH gradient IEC**

Like conventional IEC, the conditions chosen for pH-IEC separations, such as buffer, pH, column temperature, and sample load, are dependent on the type of column selected. To choose a column, the pI of the mAb and the expected charge species should be considered. Considerations for column selection may differ slightly for pH-IEC compared to conventional IEC. For example, because the column will be exposed to a pH gradient, the column must be able to perform adequately over a large pH range, i.e., the charged groups on the chromatography resin must maintain their charge over the operating pH range. Also, buffer strength can affect resolution, and pH-IEC mobile phases typically have lower buffer strengths than conventional salt-gradient IEC. Several pH-IEC buffer systems have been published for mAb separations; these buffer systems can be used as starting points for formulating buffers for pH-IEC methods (Farnan and Moreno, 2009; Rea et al., 2011; Rozhkova, 2009).

#### **8.2 High-throughput multi-product separations using pH-IEC**

To increase the throughput of the analytical methods, smaller particle sizes and shorter column lengths are being utilized to reduce run time. In Figure 10, the utilization of a 3 µm particle size column reduced analysis time 16-fold compared to a 10 µm particle size column. Analysis times are greatly reduced using smaller particle sizes because as the particle size decreases, there a significant gain in column efficiency, and the efficiency does not decrease at increased flow rates or linear velocities (Swartz, 2005). In addition, because different mAbs can be analyzed using the same pH-IEC method in the same sequence, these high-throughput methods are capable of analyzing hundreds of mAbs per day, which is not possible with conventional, product-specific salt-based IEC.

Fig. 10. Separation of a mAb using (A) a WCX column, 10 µm and (B) a SCX column, 3 µm, by pH gradient IEC. Each mAb was analyzed using the same buffers and gradient volume.

#### **9. Hybrid/combination modes of IEC**

452 Innovations in Biotechnology

Like conventional IEC, the conditions chosen for pH-IEC separations, such as buffer, pH, column temperature, and sample load, are dependent on the type of column selected. To choose a column, the pI of the mAb and the expected charge species should be considered. Considerations for column selection may differ slightly for pH-IEC compared to conventional IEC. For example, because the column will be exposed to a pH gradient, the column must be able to perform adequately over a large pH range, i.e., the charged groups on the chromatography resin must maintain their charge over the operating pH range. Also, buffer strength can affect resolution, and pH-IEC mobile phases typically have lower buffer strengths than conventional salt-gradient IEC. Several pH-IEC buffer systems have been published for mAb separations; these buffer systems can be used as starting points for formulating buffers

To increase the throughput of the analytical methods, smaller particle sizes and shorter column lengths are being utilized to reduce run time. In Figure 10, the utilization of a 3 µm particle size column reduced analysis time 16-fold compared to a 10 µm particle size column. Analysis times are greatly reduced using smaller particle sizes because as the particle size decreases, there a significant gain in column efficiency, and the efficiency does not decrease at increased flow rates or linear velocities (Swartz, 2005). In addition, because different mAbs can be analyzed using the same pH-IEC method in the same sequence, these high-throughput methods are capable of analyzing hundreds of mAbs per day, which is not

Fig. 10. Separation of a mAb using (A) a WCX column, 10 µm and (B) a SCX column, 3 µm, by pH gradient IEC. Each mAb was analyzed using the same buffers and gradient volume.

**8.1 Column selection, buffers and operating parameters for pH gradient IEC** 

for pH-IEC methods (Farnan and Moreno, 2009; Rea et al., 2011; Rozhkova, 2009).

**8.2 High-throughput multi-product separations using pH-IEC** 

possible with conventional, product-specific salt-based IEC.

Salt and pH may be combined to elute proteins from IEC columns. Combination or hybrid methods can be employed if either salt-based or pH-based methods prove inadequate for resolving species of interest, especially at extreme pHs. When pH increases above their pKa, amines, as used exclusively in the pH-IEC piperazine/imidazole/tris buffer system, become deprotonated and uncharged, resulting in decreased ionic strength. The bound proteins will also deprotonate and carry less charge. However, adequate amounts of positively charged ions are required to displace the bound proteins and to elute them off the cation exchange resin. Since the buffer salts alone can not provide enough positively charged ions at higher pH, additional salt is added to the pH-IEC elution buffers to maintain ionic strength. Figure 11 depicts measured conductivity as a function of elution time in pH-IEC with and without salt. In this case, adding salt to the elution buffer will compensate for the loss of ionic strength (represented by conductivity) due to deprotonation of buffer ions.

In Figure 12, separation of the charge species of three mAbs using pH-IEC with and without salt is compared. Without salt, mAb-1 with a pI of 9.4 did not elute, and mAb-3, with a low pI of 6.2, showed a very broad peak with significant tailing and no resolution of charge species. With the addition of salt, adequate separation of charge species is obtained for both high pI and low pI mAbs.

Fig. 11. Measured conductivity as a function of elution time in pH-IEC A) without, and B) with salt. Buffers are A) 11.6 mM piperazine, 1.5 mM imidazole, 2.4 mM Tris, B) 4 mM piperazine, 4 mM imidazole, 4 mM Tris, 16 mM NaCl.

#### **10. Equipment configurations to accelerate development**

Ionic strength gradient ion-exchange methods are typically product-specific, with each method requiring a unique pair of mobile phases and experimental conditions. As discussed above, a significant number of mobile phase pH values and gradient profiles need to be evaluated. Changing mobile phase pH values normally requires user intervention to supply new mobile phase pairs; the time needed to manually change the system can slow down development. In order to more efficiently develop analytical IEC methods, alternative equipment configurations have been utilized to accelerate the selection of operational parameters, including quaternary buffer systems and customized solvent selection valves on the pump inlets to allow selection from an array of available solvents. Such modifications to the equipment and workflows can allow consecutive performance of significantly more experiments without requiring user intervention.

Fig. 12. Separation of the charge species of three mAbs using pH gradient with and without salt in a ProPac WCX-10 column. A) pH 5 to 9.5 in 45 minutes, gradient 0.1 pH unit/min; B) pH 5 - 10.8 in 58 minutes, gradient 0.1 pH unit/min.

A quaternary buffer system can be utilized to develop a method and reduce the amount of user intervention by using a pair of buffer solvents (solvent lines A and B) to allow the pump to admix to achieve the desired mobile phase pH, and using two other solvents (solvent lines C and D) to generate the ionic strength gradient for elution. The quaternary system can apply different combinations of salt and pH by automatically programming the percentages of the four solvents to be mixed and applied to the column. Thus, programs can be generated to screen a variety of salt and pH conditions in a single sequence using only four buffers.

Another approach, which is particularly important for binary pump systems, is to add a multi-port solvent selection valve to the system prior to the pump. Although the customized

development. In order to more efficiently develop analytical IEC methods, alternative equipment configurations have been utilized to accelerate the selection of operational parameters, including quaternary buffer systems and customized solvent selection valves on the pump inlets to allow selection from an array of available solvents. Such modifications to the equipment and workflows can allow consecutive performance of significantly more

0 10 20 30 40 50

0 10 20 30 40 50 60

Fig. 12. Separation of the charge species of three mAbs using pH gradient with and without salt in a ProPac WCX-10 column. A) pH 5 to 9.5 in 45 minutes, gradient 0.1 pH unit/min; B)

A quaternary buffer system can be utilized to develop a method and reduce the amount of user intervention by using a pair of buffer solvents (solvent lines A and B) to allow the pump to admix to achieve the desired mobile phase pH, and using two other solvents (solvent lines C and D) to generate the ionic strength gradient for elution. The quaternary system can apply different combinations of salt and pH by automatically programming the percentages of the four solvents to be mixed and applied to the column. Thus, programs can be generated to

Another approach, which is particularly important for binary pump systems, is to add a multi-port solvent selection valve to the system prior to the pump. Although the customized

screen a variety of salt and pH conditions in a single sequence using only four buffers.

Time (min)

Time (min)

A: without salt

B: with salt

experiments without requiring user intervention.

Piperazine 11.6 mM Imidazole: 1.5 mM Tris: 2.4 mM

mAb-3 pI 6.2 mAb-2 pI 8.2 mAb-1 pI 9.4

Piperazine 4 mM Imidazole: 4 mM Tris: 4 mM

16mM NaCl in Buffer B

blank

blank

mAb-3 pI 6.2 mAb-2 pI 8.2 mAb-1 pI 9.4

pH 5 - 10.8 in 58 minutes, gradient 0.1 pH unit/min.

Absorbance (mAu)

Absorbance (mAu)

valve system requires the production of many buffers, the multiple valve configurations can allow users to further customize buffer components and concentrations (as opposed to only salt gradient and pH) compared to the quaternary system. Such a system allows up to a dozen more buffer combinations to be evaluated without intervention.

Because pH-IEC is performed by using a pH gradient and not a salt gradient, simply reversing the pH gradient allows for the chromatography mode to be switched between CEX and AEX. This reversal of gradient can be automatically performed through the chromatography software. Thus, multiple CEX and AEX columns can be screened by using only two buffers at different pH's and using a column switching valve to screen different column types (Figure 13). During development, it is helpful to have online pH and conductivity meters to ensure that the pH gradient is roughly linear and that the conductivity does not interfere with the separation efficiency.

Fig. 13. HPLC column compartment equipped with a 6-port column switching valve, for screening of up to six different columns for pH-IEC without the need to change buffers or columns.

#### **11. Method robustness and validation**

The robustness of an analytical procedure is a measure of its capability to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. For IEC, robustness can be evaluated by varying parameters such as injection volume, buffer pH, flow rate, and column temperature. In addition to robustness, intermediate precision can be demonstrated by evaluating inter-laboratory variations, such as different days of analysis and different analysts. Furthermore, the ability to use different instrument and column manufacturers for a particular method greatly reduces the business risk of the method; if a column supplier cannot meet demand or if an instrument manufacturer ceases production of a particular instrument model, method transfer to other instruments and columns can occur without loss of performance.

#### **11.1 Obtaining robust performance**

Obtaining robust performance of an IEC method often goes beyond the design of the method itself, and involves good equipment hygiene, elimination of metal corrosion (e.g. formation of iron oxide) and contamination (e.g. presence of metal ions such as Fe3+ ions), and mitigates the differences between instrument types. Problematic metal contamination typically results from corrosion of the fluid-contacting metal parts and can be avoided by using PEEK or titanium materials in the fluid paths. Good practices on obtaining robust method performance are discussed in the following sections.

#### **11.1.1 Equipment hygiene**

Maintaining good equipment hygiene is important in order to achieve robust performance. The following are good practices to ensure instrument hygiene:


#### **11.1.2 Metal contamination**

Metals can negatively affect the ion-exchange chromatography of proteins. Protein chelation with metals are a secondary retention mechanism to the primary electrostatic interaction of ion-exchange chromatography. This secondary interaction results in peak tailing. These interactions can either occur with metal contaminating the column or with corroded surfaces within the HPLC. In addition to affecting separation, corrosion can result in physical damage to system, such pump seal failure and compromised performance of the detector cells. Halide containing eluents readily corrode HPLC systems manufactured from stainless steel, as stainless steel has the propensity to form rust (Collins et al., 2000a). Sodium acetate or sodium sulfate can be used as an eluting salt instead of halides; however, sulfate is divalent, thus concentrations in the eluting mobile phase would be different compared to using a halide, as halides are monovalent.

Metal contamination may be reversed by flushing with chelating agents such as oxalic acid dihydrate (Rao & Pohl, 2011). Also, stainless steel systems may require periodic passivation for reliable usage (Collins et al., 2000b). In light of the drawbacks of using a stainless steel HPLC system, more manufacturers are including biocompatible equipment (e.g. Titanium or PEEK) for analyzing mAbs and other protein products.

#### **11.1.3 Transferring methods between instrument types**

Transferring methods between instruments from different manufacturers can pose challenges due to the differences between instruments. As mentioned previously, equipment composition (e.g. stainless steel vs. titanium) is one of the factors to be considered when transferring a method between instrument types, in addition to gradient delay, mixing volumes, pump capabilities, and column compartment temperature ranges. Gradient delay and mixing volumes can differ between instruments, but they are generally

and mitigates the differences between instrument types. Problematic metal contamination typically results from corrosion of the fluid-contacting metal parts and can be avoided by using PEEK or titanium materials in the fluid paths. Good practices on obtaining robust

Maintaining good equipment hygiene is important in order to achieve robust performance.

1. Filter mobile phases that are amenable to microbial growth with 0.2 mm filters prior to use; replace solvent reservoir filters (sinkers) each time mobile phase bottles are

2. Flush and store HPLC system in 10% isopropanol in water when not in use, to prevent

6. Follow manufacturer's instructions regarding proper maintenance of HPLC

Metals can negatively affect the ion-exchange chromatography of proteins. Protein chelation with metals are a secondary retention mechanism to the primary electrostatic interaction of ion-exchange chromatography. This secondary interaction results in peak tailing. These interactions can either occur with metal contaminating the column or with corroded surfaces within the HPLC. In addition to affecting separation, corrosion can result in physical damage to system, such pump seal failure and compromised performance of the detector cells. Halide containing eluents readily corrode HPLC systems manufactured from stainless steel, as stainless steel has the propensity to form rust (Collins et al., 2000a). Sodium acetate or sodium sulfate can be used as an eluting salt instead of halides; however, sulfate is divalent, thus concentrations in the eluting mobile phase would be different

Metal contamination may be reversed by flushing with chelating agents such as oxalic acid dihydrate (Rao & Pohl, 2011). Also, stainless steel systems may require periodic passivation for reliable usage (Collins et al., 2000b). In light of the drawbacks of using a stainless steel HPLC system, more manufacturers are including biocompatible equipment (e.g. Titanium

Transferring methods between instruments from different manufacturers can pose challenges due to the differences between instruments. As mentioned previously, equipment composition (e.g. stainless steel vs. titanium) is one of the factors to be considered when transferring a method between instrument types, in addition to gradient delay, mixing volumes, pump capabilities, and column compartment temperature ranges. Gradient delay and mixing volumes can differ between instruments, but they are generally

3. Leave the system running at low flow rates to prevent salt build up and clogging;

5. Flush auto-sampler components as needed with 10% isopropanol in water;

method performance are discussed in the following sections.

The following are good practices to ensure instrument hygiene:

4. Keep all lines flushing as opposed to just a single channel;

compared to using a halide, as halides are monovalent.

or PEEK) for analyzing mAbs and other protein products.

**11.1.3 Transferring methods between instrument types** 

**11.1.1 Equipment hygiene** 

replenished;

growth of microbes;

instrumentation.

**11.1.2 Metal contamination** 

only a significant concern for very fast gradient separations. In addition, shallow IEC gradients can challenge the performance of an HPLC; however, most gradients are >70 mM salt and can be proportioned over 30-40% of the pump range, well within the capabilities of modern pumps. Often a gradient hold for 5 minutes at the initial salt concentration is included, just in case a method is particularly sensitive when being transferred from one equipment type to another. In such cases the hold time can be adjusted to compensate for differences in the gradient delay volume between the instruments.

Temperatures inside the column are dependent on oven design and plumbing configuration. Having a pre-column heat exchanger in line or out of line could make a several degree difference in the temperature at which the column chemistry occurs. This is particularly concerning for buffers with which the pH can change rapidly with temperature. Figure 14 shows a comparison of column compartment temperature settings for two different instruments from different manufacturers. To make the correlation, thermocouples were fitted into T-pieces in the fluid path inside the column oven, but just prior to the column, and temperatures were measured for a range of column compartment set points and mobile phase flow rates. These measurements were used to estimate the temperature of mobile phase going through the column for each set point. By equating the measured fluid temperatures for each flow rate, the correlation of column compartment temperatures were plotted. It is noted in this correlation that there was also a significant effect of the mobile phase flow rate on the correlation.

Different detectors can sometimes yield differences in baseline slope. This can occur when moving from a single/double wavelength detector with a reference beam to a photodiode array (PDA) detector. The selection of an appropriate reference wavelength and bandwidth on the PDA can overcome detector variance.

Fig. 14. Comparison of column compartment temperature settings required to achieve the same columns compartment temperature for two different HPLC models at different flow rates. Results are shown for a 4 x 250 mm Dionex ProPac column. The Agilent 1100 HPLC was configured using only the left hand side heat exchanger. The Waters 2695 HPLC was configured with the solvent pre-heater in-line.

#### **11.2 Method validation**

Before an analytical method can be incorporated into a characterization platform or a quality control system, it must first be demonstrated that the method is suitable for its intended purpose. Guidelines for validation of analytical methods have been published in the United States Pharmacopeia, by the International Conference on Harmonization (ICH), US Food and Drug Administration (FDA), and in published reviews (Bakshi & Singh, 2002). Methods must be evaluated considering regulatory requirements and validation procedures. In other words, the "validatability" of these methods must be assessed before implementation. Validation tests include precision, accuracy, and linearity. Intermediate precision is tested by using multiple instruments, multiple analysts, and multiple column lots. Methods must be validated and documented according to regulatory requirements prior to implementation into a control system for lot release of drug substance and drug product. Robustness studies can also be performed in conjunction with method validation. It has been our experience that the most significant effects on method robustness are: mobile phase pH value, column temperature, metal contamination and column age.

A system suitability range can be obtained from robustness studies. This range is often based on the standard deviation of the mean for a particular measured component, such as main peak relative area. The system suitability range indicates the precision of the method. pH-IEC may demonstrate an improvement in precision over conventional salt-based IEC (Rea et al., 2011). The 6σ ranges in Figure 15, which predicts a 99% method success rate, demonstrate the improved precision of the pH-gradient IEC method over conventional IEC, which can have a 6σ range of up to 8% main peak relative area (Figure 15).

Fig. 15. Six sigma range (±3SD) for main peak relative area for salt gradient IEC (diamonds) and pH gradient IEC (circles) for a variety of mAbs.

### **12. Automation in sample preparation and data handling**

In addition to high-throughput and multi-product analytical methods, the use of robotics for sample preparation automation may further reduce sample analysis time and cost. There are several companies that provide liquid handling automation instruments, including LEAP Technologies and TECAN. The LEAP Technologies CTC PAL liquid handling system is capable of on-the-fly sample preparation, such as protein dilution and digestion. On-the-fly sample preparations are viable if the sample preparation takes less time than the analytical method. For sample preparations that take longer than the analytical run time, batch sample preparation can be performed using robotic liquid handling systems such as the TECAN Freedom EVO, which can handle multi-well plates for increased sample throughput. Robotic liquid samplers can increase reproducibility, efficiency and safety compared to manual handling of samples.

The final steps to most characterization workflows include data analysis and report generation. Several software packages are available that are designed to reduce the time necessary to complete post-data acquisition tasks. For liquid chromatography applications, commercially available chromatography data software, such as Dionex's Chromeleon Chromatography Management Software and Waters Corporation's Empower Chromatography Data Software, include features such as automated peak integration and one-click report generation. In addition, laboratories are increasingly implementing electronic laboratory notebooks, which has advantages over traditional laboratory notebooks, including ease of data sharing and collaboration, streamlined review and witnessing processes, standardized documentation, and long-term data preservation.

#### **13. Conclusion**

458 Innovations in Biotechnology

Before an analytical method can be incorporated into a characterization platform or a quality control system, it must first be demonstrated that the method is suitable for its intended purpose. Guidelines for validation of analytical methods have been published in the United States Pharmacopeia, by the International Conference on Harmonization (ICH), US Food and Drug Administration (FDA), and in published reviews (Bakshi & Singh, 2002). Methods must be evaluated considering regulatory requirements and validation procedures. In other words, the "validatability" of these methods must be assessed before implementation. Validation tests include precision, accuracy, and linearity. Intermediate precision is tested by using multiple instruments, multiple analysts, and multiple column lots. Methods must be validated and documented according to regulatory requirements prior to implementation into a control system for lot release of drug substance and drug product. Robustness studies can also be performed in conjunction with method validation. It has been our experience that the most significant effects on method robustness are: mobile phase pH value, column

A system suitability range can be obtained from robustness studies. This range is often based on the standard deviation of the mean for a particular measured component, such as main peak relative area. The system suitability range indicates the precision of the method. pH-IEC may demonstrate an improvement in precision over conventional salt-based IEC (Rea et al., 2011). The 6σ ranges in Figure 15, which predicts a 99% method success rate, demonstrate the improved precision of the pH-gradient IEC method over conventional IEC,

Fig. 15. Six sigma range (±3SD) for main peak relative area for salt gradient IEC (diamonds)

which can have a 6σ range of up to 8% main peak relative area (Figure 15).

**11.2 Method validation** 

temperature, metal contamination and column age.

and pH gradient IEC (circles) for a variety of mAbs.

Monoclonal antibodies are valuable therapeutic products that are approved for a variety of indications. In this chapter, mAb development, production, purification, formulation, characterization and regulatory requirements were discussed, followed by a more detailed discussion on charge species analysis using IEC. Method development strategies, method robustness, validation and automation, as well as applications of salt-gradient and pHgradient IEC methodologies for the analysis of mAbs were also covered. This chapter is intended to be a reference text for scientists such that a concise strategy can be implemented for developing robust IEC methods for the characterization of therapeutic mAbs, resulting in shorter method development times and enabling faster analysis of mAb products to support biopharmaceutical pipelines.

#### **14. Acknowledgment**

The authors would like to acknowledge Liangyi Zhang at Genentech and Mark van Gils at Dionex (A Thermo-Fisher Company) for contributions to this work.

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### *Edited by Eddy C. Agbo*

Innovations in Biotechnology provides an authoritative crystallization of some of the evolving leading-edge biomedical research topics and developments in the field of biotechnology. It is aptly written to integrate emerging basic research topics with their biotechnology applications. It also challenges the reader to appreciate the role of biotechnology in society, addressing clear questions relating to biotech policy and ethics in the context of the research advances. In an era of interdisciplinary collaboration, the book serves an excellent indepth text for a broad range of readers ranging from social scientists to students, researchers and policy makers. Every topic weaves back to the same bottom line: how does this discovery impact society in a positive way?

Innovations in Biotechnology

Innovations in Biotechnology

*Edited by Eddy C. Agbo*

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