Scale-Up and Bioprocessing of Phages

*John Maxim Ward, Steven Branston, Emma Stanley and Eli Keshavarz-Moore*

#### **Abstract**

A profusion of new applications for phage technologies has been developed within the last few years, stimulating investigations into the large-scale production of different phages. Applications such as antibiotic replacement, phages as gene therapy vectors, phages as vaccines, diagnostics using filamentous phages and novel optical applications such as the phage laser may need grams to kilogrammes of phage in the future. However, many of the techniques that are used for the growth and purification of bacteriophage at small scale are not transferable to large-scale production facilities of phage in industrial processes. In this chapter, the stages of production that need to be carried out at scale are examined for the efficient large-scale fermentation of the filamentous phage M13 and the *Siphoviridae* phage lambda (λ). A number of parameters are discussed: the multiplicity of infection (MOI) of phage to host cells, the impact of agitation on the initial infection stages, the co-growth with phage rather than static attachment, the use of engineered host cells expressing nuclease, the optimisation of both the quantity and the physiology of the *E. coli* inoculum and phage precipitation methods.

**Keywords:** phage, PEG precipitation, nuclease, filamentous phage, lambda, phage diagnostics, phage laser, fermenter

#### **1. Introduction**

Bacteriophages, often shortened to just phages, are viruses that infect bacteria. Their discovery and characterisation in the early days of bacterial molecular biology has led to certain phages being very well understood in terms of their life cycle, and several phages that infect *Escherichia coli* have become tools in molecular biology techniques such as cloning [1–3]. There has been a resurgence recently in the use of bacteriophages as therapeutics, as vectors for the delivery of vaccines [4], for the killing of pathogenic bacteria as an alternative to antibiotics [5] and for gene therapy to transfer DNA to target human or animal cells [6]. Some of these uses would need the production of many millions of doses of a vaccine, for example, or very large quantities for use as an antibacterial. This has increased demand for investigation into the large-scale production of bacteriophage which would necessitate volumes from hundreds to thousands of litres. The use of phage as biotherapeutics such as vaccines or for gene therapy may be advantageous as phage is considered cheap to manufacture, with large quantities of the product being rapidly produced. But the large-scale production of wild type or genetically modified bacteriophages

for use in the biotherapeutics industry provides significant process and regulatory challenges. Bacteriophages, like any virus, are dependent on a host organism to propagate, in the examples here it is *E. coli*; consequently, the generation of progeny bacteriophage is unequivocally linked to the physiology, molecular biology and growth needs of the host which are important to understand in order to maximise production.

Methods for the production of phages, e.g. λ and M13 bacteriophage, at laboratory production scale have remained unchanged for many years [7]. However, aspects of these protocols are either not practical or unsuitable for large-scale production of phages. Therefore, it is highly desirable to consider early on in the development of phage technologies how any successful bacteriophage therapeutic would be produced at large scale at an industrial level.

One of the problems associated with producing and using λ as a biotherapeutic is the issue of host-derived nucleic acid. The λ lifecycle [8] involves the phage progeny escaping from the host cell by lysis of the bacteriophage host, whereupon the cell contents including high-molecular-weight host chromosomal DNA and RNA are released into the culture supernatant. This significant quantity of host cell-derived nucleic acid can cause important problems for both downstream processing [9] and from a regulatory point of view [10], so reducing the presence of bacterial host nucleic acid in the first stages of the process stream would remove these issues.

M13 is an unusual phage because it does not lyse its host and the entire phage is secreted from the host bacterium through special pores spanning the cell wall [11] although this does make the culture supernatant relatively free of contaminating host cell material, unlike the supernatant in a λ fermentation. In both lytic and secreted phage production the first downstream stage is the concentration of the phage from whatever volume of growth medium was used to grow the infected cells. Filamentous phage such as M13 has a very asymmetric shape with wild-type M13 having a length to width ratio of 138:1 and this extreme asymmetry allows a mild precipitation using polyethylene glycol (PEG) [12].

In this paper we present initial studies into the parameters that need to be manipulated for scaling up fermentation of M13 phage for industrial production.

#### **2. Lambda phage**

Lambda (λ) bacteriophage is a temperate phage with a double-stranded (ds) genome of approximately 48 kb [13]. This is encapsulated in an icosahedral capsid (~50 nm in diameter) with a long flexible non-contractile tail (~150 nm in length). The host for λ production is *E. coli* with infection by lambda phage taking place via the maltose binding protein, LamB. Lambda bacteriophage is one of the most intensely studied bacteriophages and has been used for many studies on uncovering basic molecular biology [14] and in biotechnology for phage display of peptides and proteins [15], vaccine [16] and gene transfer and therapy [6].

The large-scale production of genetically modified lambda bacteriophage for use in the biotherapeutics industry provides significant process and regulatory challenges.

One of the problems associated with producing and using lambda bacteriophage as a biotherapeutic is the issue of host-derived nucleic acid. The lambda lifecycle involves the cell lysis of the bacteriophage host, whereupon high-molecular-weight host chromosomal DNA is liberated into the culture supernatant. The presence of large quantities of host cell-derived nucleic acid can cause significant problems during processing as high-molecular-weight chromosomal DNA causes an increase in the cell lysate viscosity [9]. Furthermore, the presence of nucleic acid in the final

**79**

*Scale-Up and Bioprocessing of Phages*

processing protocol.

binant antibodies [22].

fewer contaminants than phage λ cultures.

**4. Multiplicity of infection (MOI)**

*DOI: http://dx.doi.org/10.5772/intechopen.88275*

**3. M13: a filamentous bacteriophage**

product is non-desirable from a regulatory perspective [10], and thus reducing its presence in the first stages of the process stream would alleviate these issues. The lysis of the host *E. coli* cell and the release of intracellular contents (DNA, RNA and proteins) as well as fragments of cell wall will have detrimental effects when processing lambda. For example, intracellular contents can co-precipitate with the phage, can compete for binding sites on chromatography material and can block membranes and chromatography columns. These contaminants need to be taken into consideration when planning a large-scale purification and downstream

M13 is an unusual phage as it has a filamentous structure of 900 nm in length and 6.5 nm in width. It is a member of a small group of closely related phages including F1 and Fd [17] that infect *E. coli.* The genome is a single-stranded circular DNA molecule, and the length of the phage (but not its width) is simply determined by the size of the page genome. Short phage particles can be made using plasmids that contain just the replication origin and packaging signals, and phage particles longer than the wild type can be made by inserting DNA sequences into the phage genome. It was thought that the very long but thin shape of M13 and other filamentous phages would increase their shear sensitivity in the various kinds of industrialscale processing equipment of pumps, continuous centrifuges and membrane filters. This was seen not to be the case [18] which is highly advantageous for largescale downstream processing of this and other filamentous phages. Filamentous phages have a rather special property in that they do not lyse their host, but set up a permanently infected state, and new, progeny phage is extruded through special structures in the cell wall. Derivatives of M13 phage were extensively used in the early years of DNA sequencing by the Sanger method [19] and in the techniques of site-directed mutagenesis [20] and phage display [21] for the maturation of recom-

The unusual mode of growth of filamentous phage by secretion from the host without lysis has considerable advantages for these molecular biology methods because the phage in the supernatant of growing cells is relatively free of any cellular contaminants [23] such as intracellular proteins, genomic DNA and RNA. This makes the purification of filamentous phage a relatively simple matter with many

The multiplicity of infection or MOI is the number of phage particles added per host cell to initiate infection and thus production. The methods developed for the uses of phage λ and M13 at small scale or a few mL generally tend to use a high MOI of 5–10 or more. The MOI is important for scale-up as it defines how large the culture that provides the phage for the scale-up needs to be. It is neither desirable nor sensible to have to grow a fermenter of phage to inoculate a slightly larger fermenter in the final preparation. Also with a large MOI of just under 10, we reach the point where there will be enough phage for every cell to be infected, and at that MOI we can only expect a single burst of phage particles for lytic phage and therefore only a small increase in the number of phage added. With an MOI of 1, only 63% of cells will be infected by one or more phages because there is a Poisson distribution of MOI to infected cells [24]. But some phages have many binding sites per cell, e.g.

#### *Scale-Up and Bioprocessing of Phages DOI: http://dx.doi.org/10.5772/intechopen.88275*

*Bacteriophages - Perspectives and Future*

would be produced at large scale at an industrial level.

mild precipitation using polyethylene glycol (PEG) [12].

proteins [15], vaccine [16] and gene transfer and therapy [6].

In this paper we present initial studies into the parameters that need to be manipulated for scaling up fermentation of M13 phage for industrial production.

Lambda (λ) bacteriophage is a temperate phage with a double-stranded (ds) genome of approximately 48 kb [13]. This is encapsulated in an icosahedral capsid (~50 nm in diameter) with a long flexible non-contractile tail (~150 nm in length). The host for λ production is *E. coli* with infection by lambda phage taking place via the maltose binding protein, LamB. Lambda bacteriophage is one of the most intensely studied bacteriophages and has been used for many studies on uncovering basic molecular biology [14] and in biotechnology for phage display of peptides and

The large-scale production of genetically modified lambda bacteriophage for use in the biotherapeutics industry provides significant process and regulatory

One of the problems associated with producing and using lambda bacteriophage as a biotherapeutic is the issue of host-derived nucleic acid. The lambda lifecycle involves the cell lysis of the bacteriophage host, whereupon high-molecular-weight host chromosomal DNA is liberated into the culture supernatant. The presence of large quantities of host cell-derived nucleic acid can cause significant problems during processing as high-molecular-weight chromosomal DNA causes an increase in the cell lysate viscosity [9]. Furthermore, the presence of nucleic acid in the final

production.

**2. Lambda phage**

for use in the biotherapeutics industry provides significant process and regulatory challenges. Bacteriophages, like any virus, are dependent on a host organism to propagate, in the examples here it is *E. coli*; consequently, the generation of progeny bacteriophage is unequivocally linked to the physiology, molecular biology and growth needs of the host which are important to understand in order to maximise

Methods for the production of phages, e.g. λ and M13 bacteriophage, at laboratory production scale have remained unchanged for many years [7]. However, aspects of these protocols are either not practical or unsuitable for large-scale production of phages. Therefore, it is highly desirable to consider early on in the development of phage technologies how any successful bacteriophage therapeutic

One of the problems associated with producing and using λ as a biotherapeutic is the issue of host-derived nucleic acid. The λ lifecycle [8] involves the phage progeny escaping from the host cell by lysis of the bacteriophage host, whereupon the cell contents including high-molecular-weight host chromosomal DNA and RNA are released into the culture supernatant. This significant quantity of host cell-derived nucleic acid can cause important problems for both downstream processing [9] and from a regulatory point of view [10], so reducing the presence of bacterial host nucleic acid in the first stages of the process stream would remove these issues. M13 is an unusual phage because it does not lyse its host and the entire phage is secreted from the host bacterium through special pores spanning the cell wall [11] although this does make the culture supernatant relatively free of contaminating host cell material, unlike the supernatant in a λ fermentation. In both lytic and secreted phage production the first downstream stage is the concentration of the phage from whatever volume of growth medium was used to grow the infected cells. Filamentous phage such as M13 has a very asymmetric shape with wild-type M13 having a length to width ratio of 138:1 and this extreme asymmetry allows a

**78**

challenges.

product is non-desirable from a regulatory perspective [10], and thus reducing its presence in the first stages of the process stream would alleviate these issues. The lysis of the host *E. coli* cell and the release of intracellular contents (DNA, RNA and proteins) as well as fragments of cell wall will have detrimental effects when processing lambda. For example, intracellular contents can co-precipitate with the phage, can compete for binding sites on chromatography material and can block membranes and chromatography columns. These contaminants need to be taken into consideration when planning a large-scale purification and downstream processing protocol.

#### **3. M13: a filamentous bacteriophage**

M13 is an unusual phage as it has a filamentous structure of 900 nm in length and 6.5 nm in width. It is a member of a small group of closely related phages including F1 and Fd [17] that infect *E. coli.* The genome is a single-stranded circular DNA molecule, and the length of the phage (but not its width) is simply determined by the size of the page genome. Short phage particles can be made using plasmids that contain just the replication origin and packaging signals, and phage particles longer than the wild type can be made by inserting DNA sequences into the phage genome. It was thought that the very long but thin shape of M13 and other filamentous phages would increase their shear sensitivity in the various kinds of industrialscale processing equipment of pumps, continuous centrifuges and membrane filters. This was seen not to be the case [18] which is highly advantageous for largescale downstream processing of this and other filamentous phages. Filamentous phages have a rather special property in that they do not lyse their host, but set up a permanently infected state, and new, progeny phage is extruded through special structures in the cell wall. Derivatives of M13 phage were extensively used in the early years of DNA sequencing by the Sanger method [19] and in the techniques of site-directed mutagenesis [20] and phage display [21] for the maturation of recombinant antibodies [22].

The unusual mode of growth of filamentous phage by secretion from the host without lysis has considerable advantages for these molecular biology methods because the phage in the supernatant of growing cells is relatively free of any cellular contaminants [23] such as intracellular proteins, genomic DNA and RNA. This makes the purification of filamentous phage a relatively simple matter with many fewer contaminants than phage λ cultures.

### **4. Multiplicity of infection (MOI)**

The multiplicity of infection or MOI is the number of phage particles added per host cell to initiate infection and thus production. The methods developed for the uses of phage λ and M13 at small scale or a few mL generally tend to use a high MOI of 5–10 or more. The MOI is important for scale-up as it defines how large the culture that provides the phage for the scale-up needs to be. It is neither desirable nor sensible to have to grow a fermenter of phage to inoculate a slightly larger fermenter in the final preparation. Also with a large MOI of just under 10, we reach the point where there will be enough phage for every cell to be infected, and at that MOI we can only expect a single burst of phage particles for lytic phage and therefore only a small increase in the number of phage added. With an MOI of 1, only 63% of cells will be infected by one or more phages because there is a Poisson distribution of MOI to infected cells [24]. But some phages have many binding sites per cell, e.g.

T4 has 105 molecules of OmpC to bind to [25], while M13 only has approximately 3 [26], so the kinetics of phage finding and attaching to bacteria and forming a productive infection are quite complex. There are 30,000 LamB proteins in the outer membrane which is the initial receptor for λ but many fewer copies of the mannose receptor, ManY, in the inner membrane which is where the DNA of λ crosses the inner membrane [27, 28]. For M13 the receptor is the tip of the F plasmid's transfer pilus, and there are usually one or two F pili per cell. But once M13 has established its replication inside an *E. coli* host, the cell is then permanently infected and will continue to secrete phage from these intact cells.

Under ideal conditions the burst size of λ phage particles is 170 +/− 10 which takes 51 min [29], and during this time uninfected cells will still be growing and dividing, providing new hosts for the phage that are released. To get repeated rounds of replication, the ideal cell numbers and MOI for each phage are different and take into account cell division rates, numbers of receptors, the choice between a lytic and lysogenic cycle in phages where those can take place, the burst size and rate of replication and maturation of the phage. This complex interaction of several parameters means that it is difficult to say a priori what the combination of cells, phage and time of addition is the most appropriate for a given size of growth chamber.

**Figure 1** shows the relationship between the host *E. coli* and MOI of M13.

The graph of MOI and final phage production in **Figure 1** shows that from a 106 range of MOI added at the start of growth in **Figure 1**, all three cultures reach the same final M13 phage titre. It just takes slightly longer for the lower MOI cultures to reach the final of approx. 5 × 1011 phage per mL. This has important consequences for scaleup of M13 production. For example, if a large fermenter of, say, 100 L were needed and the MOI of 50:1 was needed, we would need to have 500 mL of the equivalent inoculum used here. The information from **Figure 1** shows that we can use just 0.5 mL of the same titre inoculum or much less, e.g. down to a few microlitres. For convenience it is best to inoculate a fermenter with enough in terms of volume that will reach the medium in the fermenter, so a few millilitres of a phage dilution are all that is needed. This means that one phage stock can be used for multiple fermentation runs.

#### **Figure 1.**

*Titre of M13 produced from different MOI on E. coli TOP10 F′. M13 phage was prepared from a 400 mL culture of E. coli TOP10 F′ and precipitated with PEG 6000 at a final concentration of 3.3% and 330 mM NaCl. The precipitated phage was centrifuged at 14,000 ×g for 10 min at 4°C and resuspended in 8 mL of 10 mM Tris.HCl pH 7.5 and filtered through a 0.22 μm filter. The stock of M13 was approximately 1 × 1012 pfu/mL. E. coli TOP10 F′ was grown in 400 mL of Nutrient Broth No. 2 (Oxoid) and a 40-mL inoculum in 2-L shake flask at 37°C. Around 2 mL of M13 was added at the appropriate dilutions to achieve the three different MOI of 0.00005:1, 0.05:1 and 50:1. Each point is the average of three flasks.*

**81**

**Figure 2.**

**6. Nuclease-producing** *E. coli*

*Scale-Up and Bioprocessing of Phages*

MOI ranging over a 106

*DOI: http://dx.doi.org/10.5772/intechopen.88275*

**5. Initial phage binding and infection in fermenters**

comparing this with a static attachment/incubation of 30 min.

from what is done at small scale in the molecular biology lab.

range.

The standard way of initiating infection of λ or M13 is to mix the phage and cells and allow a static incubation for usually 5–15 min for phage attachment to the phage binding target on the cell surface. The phage/cell mixture is then added to broth if liquid growth is desired or to 3 mL of soft agar and poured onto an agar plate if plaques are wanted. This static incubation is in the recipes for all phages being handled at the lab scale and probably came about because researchers thought it would maximise the attachment of phage to their host. However, in a fermenter it is not sensible to turn off the stirrer and let the cells sit for 15 min while phage attachment takes place. We tested what would happen if M13 phage were simply added directly to shaking cultures of *E. coli* JM107 without stopping the shaking and

**Figure 2** shows this experiment with three different inocula of M13 phage giving

It is clear from **Figure 2** that the static incubation is not needed for the initial attachment of M13 to sensitive *E. coli*, and so the required dose of M13 phage can be directly added to a fermenter with the *E. coli* host already growing with the impeller stirring. The culture which will be stirred at a high rate *does not* need to be stopped and left to go static for 15–30 min. This would compromise the growth of the cells in a large fermenter, and so our findings give a positive help for the scale-up of phage and the way one can run a large fermenter where procedures have to be different

The above sections on MOI and removing the necessity for a static attachment of phage were shown with M13 as examples. The M13 phage does not lyse its host, so the culture medium after infection is uncontaminated by the bulk of the host cellular contents and largely contains just the filamentous M13 bacteriophage particles. We have examined the supernatants from M13-infected cultures and determined the levels of host cell contaminants of DNA and protein [23] which are quite low

*Static versus shaking incubation of M13 with E. coli TOP10 F′. The conditions and culture quantities were identical to those shown in Figure 1 with the addition of three conditions at each MOI where the M13 phage was added and left for 30 min static incubation. These cultures for the static incubation were then grown with* 

*shaking at 200 rpm in a 37°C orbital incubator. Each point is the average of three flasks.*

*Bacteriophages - Perspectives and Future*

continue to secrete phage from these intact cells.

molecules of OmpC to bind to [25], while M13 only has approximately 3

[26], so the kinetics of phage finding and attaching to bacteria and forming a productive infection are quite complex. There are 30,000 LamB proteins in the outer membrane which is the initial receptor for λ but many fewer copies of the mannose receptor, ManY, in the inner membrane which is where the DNA of λ crosses the inner membrane [27, 28]. For M13 the receptor is the tip of the F plasmid's transfer pilus, and there are usually one or two F pili per cell. But once M13 has established its replication inside an *E. coli* host, the cell is then permanently infected and will

Under ideal conditions the burst size of λ phage particles is 170 +/− 10 which takes 51 min [29], and during this time uninfected cells will still be growing and dividing, providing new hosts for the phage that are released. To get repeated rounds of replication, the ideal cell numbers and MOI for each phage are different and take into account cell division rates, numbers of receptors, the choice between a lytic and lysogenic cycle in phages where those can take place, the burst size and rate of replication and maturation of the phage. This complex interaction of several parameters means that it is difficult to say a priori what the combination of cells, phage and time

of addition is the most appropriate for a given size of growth chamber.

This means that one phage stock can be used for multiple fermentation runs.

*Titre of M13 produced from different MOI on E. coli TOP10 F′. M13 phage was prepared from a 400 mL culture of E. coli TOP10 F′ and precipitated with PEG 6000 at a final concentration of 3.3% and 330 mM NaCl. The precipitated phage was centrifuged at 14,000 ×g for 10 min at 4°C and resuspended in 8 mL of 10 mM Tris.HCl pH 7.5 and filtered through a 0.22 μm filter. The stock of M13 was approximately 1 × 1012 pfu/mL. E. coli TOP10 F′ was grown in 400 mL of Nutrient Broth No. 2 (Oxoid) and a 40-mL inoculum in 2-L shake flask at 37°C. Around 2 mL of M13 was added at the appropriate dilutions to achieve the three* 

*different MOI of 0.00005:1, 0.05:1 and 50:1. Each point is the average of three flasks.*

**Figure 1** shows the relationship between the host *E. coli* and MOI of M13. The graph of MOI and final phage production in **Figure 1** shows that from a 106 range of MOI added at the start of growth in **Figure 1**, all three cultures reach the same final M13 phage titre. It just takes slightly longer for the lower MOI cultures to reach the final of approx. 5 × 1011 phage per mL. This has important consequences for scaleup of M13 production. For example, if a large fermenter of, say, 100 L were needed and the MOI of 50:1 was needed, we would need to have 500 mL of the equivalent inoculum used here. The information from **Figure 1** shows that we can use just 0.5 mL of the same titre inoculum or much less, e.g. down to a few microlitres. For convenience it is best to inoculate a fermenter with enough in terms of volume that will reach the medium in the fermenter, so a few millilitres of a phage dilution are all that is needed.

T4 has 105

**80**

**Figure 1.**

### **5. Initial phage binding and infection in fermenters**

The standard way of initiating infection of λ or M13 is to mix the phage and cells and allow a static incubation for usually 5–15 min for phage attachment to the phage binding target on the cell surface. The phage/cell mixture is then added to broth if liquid growth is desired or to 3 mL of soft agar and poured onto an agar plate if plaques are wanted. This static incubation is in the recipes for all phages being handled at the lab scale and probably came about because researchers thought it would maximise the attachment of phage to their host. However, in a fermenter it is not sensible to turn off the stirrer and let the cells sit for 15 min while phage attachment takes place. We tested what would happen if M13 phage were simply added directly to shaking cultures of *E. coli* JM107 without stopping the shaking and comparing this with a static attachment/incubation of 30 min.

**Figure 2** shows this experiment with three different inocula of M13 phage giving MOI ranging over a 106 range.

It is clear from **Figure 2** that the static incubation is not needed for the initial attachment of M13 to sensitive *E. coli*, and so the required dose of M13 phage can be directly added to a fermenter with the *E. coli* host already growing with the impeller stirring. The culture which will be stirred at a high rate *does not* need to be stopped and left to go static for 15–30 min. This would compromise the growth of the cells in a large fermenter, and so our findings give a positive help for the scale-up of phage and the way one can run a large fermenter where procedures have to be different from what is done at small scale in the molecular biology lab.

#### **Figure 2.**

*Static versus shaking incubation of M13 with E. coli TOP10 F′. The conditions and culture quantities were identical to those shown in Figure 1 with the addition of three conditions at each MOI where the M13 phage was added and left for 30 min static incubation. These cultures for the static incubation were then grown with shaking at 200 rpm in a 37°C orbital incubator. Each point is the average of three flasks.*

#### **6. Nuclease-producing** *E. coli*

The above sections on MOI and removing the necessity for a static attachment of phage were shown with M13 as examples. The M13 phage does not lyse its host, so the culture medium after infection is uncontaminated by the bulk of the host cellular contents and largely contains just the filamentous M13 bacteriophage particles. We have examined the supernatants from M13-infected cultures and determined the levels of host cell contaminants of DNA and protein [23] which are quite low

compared to the large amount of cellular DNA, RNA and protein released by lytic phage. A phage such as λ is a representative of lytic phage which is the majority of the types of phages used in therapy and biological control for the replacement of antibiotics. At each cycle of replication and release of the new phage particles, the host is lysed, and the cell contents are released into the medium along with insoluble debris from the cell wall and membrane. This leads to problems in subsequent downstream purification due to the large amount of different cellular molecules competing in the subsequent downstream processing steps.

The host RNA and DNA represent major contaminants that need to be removed especially for gene therapy applications. The release of host cell DNA also increases the viscosity of the medium, and this has an adverse effect on clarification by centrifugation and membrane concentration due to blocking of the membranes. For lab-scale molecular biology work, it is normal to add the enzymes pancreatic RNAseI and pancreatic DNAseI from bovine pancreas preparations. With the advent of bovine spongiform encephalopathy (BSE) which peaked in the 1990s, the addition of any bovine or animal proteins into the growth or purification train of material destined for human therapy was banned. These regulatory restrictions removed the ability to use these cheap nucleic acid-degrading enzymes, and the substitutes from bacterial sources were much more expensive. A strategy to overcome this problem was developed by us, and this was to express a broad-spectrum nuclease in the periplasm of *E. coli* which would be released as cells were lysed [30, 31]. The enzyme Staphylococcal nuclease has been extensively characterised and used from the 1960s onwards [32] and can degrade both DNA and RNA. The expression of this in *E. coli* where it is secreted into the periplasm does not affect the growth of *E. coli* because the enzyme cannot get access to its substrate while the cell is growing normally. When the cells are lysed by a bacteriophage such as λ or by homogenisation, the nuclease can gain access to the released DNA and RNA and degrade them. This 'cell engineering' approach to assisting bioprocessing was developed at UCL and has been shown to give considerable gains in the centrifugation steps and other downstream purification steps in bioprocessing of proteins such as Fab fragments [33–35].

We sought to apply this cell engineering strategy for the production of λ phage and to help solve the problem of the large amount of cellular contaminants released into the media when λ phage cultures need to be harvested and processed.

*E. coli* JM107 [3] was grown in 2 L fermenters with either JM107 or JM107 containing the plasmid pMMBompnucB which is a broad host-range plasmid vector based on an Inc. Q plasmid RSF1010 and contains the Staphylococcal nuclease which has been altered by the addition of the *E. coli* ompA signal sequence for secretion [31]. **Figure 3** shows the growth of the two hosts with no added λ phage and the same hosts with 8 × 1010 λ phage particles added after 2 hours when the OD600 had reached 10.

The addition of λ to the fermenter used the strategy that we had developed where a low MOI is used and without a static initial incubation of the host cells and the phage. In this way we can add the phage directly to a growing fermenter of host with the impeller (single shaft with three top-driven, equally spaced, sixbladed turbines) and four diametrically opposed baffles in the fermenter, running throughout. The growth profiles of the two uninfected cultures show no difference in their growth profiles which means that the presence of the expressed nuclease enzyme in the periplasm has no effect on growth rate or final OD. In the two cultures with added λ phage, the OD drop is the same for both hosts showing that λ replication and cell lysis are the same in both.

**83**

**Figure 4.**

*plaque counting.*

expressing the periplasmic nuclease.

*Scale-Up and Bioprocessing of Phages*

**Figure 3.**

*DOI: http://dx.doi.org/10.5772/intechopen.88275*

Both the nuclease and the non-nuclease-expressing *E. coli* JM107 produced the same 5-log increase in phage particles with the same time profile. The efficacy of the nuclease in the removal of the host nucleic acid was assessed by the electrophoresis of samples from each time point on agarose gels and the visualisation of the released nucleic acid (both DNA and RNA). **Figure 5** shows the complete degradation and removal of the released host genomic DNA and host RNA in the strain of *E. coli*

*Production of λ phage in 2 L fermenters with and without periplasmic nuclease expression. λ phage was added at 2 hours in Figure 3, and the graph here shows λ phage amounts from the 2 hours onwards. Samples from the fermenters shown in Figure 3 were diluted in phage buffer and titred on E. coli JM107 using soft top agar and* 

*Growth of E. coli JM107 and λ production with and without a nuclease-expressing plasmid. E. coli JM107 containing pMMBompnucB and with no plasmid was grown in 2 L (working volume 1.5 L) LH 210 series fermenter (Bioprocess Engineering Services, Charing, Kent, UK) with 150 mL of E. coli inoculum and a final working volume of 1.5 L in phage media containing 100 μg/mL ampicillin and 20 μg/mL IPTG. λ phage particles were added at 2 hours to give an MOI of 0.05 (4 mL of 2 × 1010 pfu/mL). Impeller stirring continued* 

*throughout the growth and addition of phage, and the OD600 was monitored.*

The presence of the expressed periplasmic nuclease is apparent from the difference in the samples in **Figure 5B** compared to the samples from the same time points in the fermenter with no expression plasmid for the Staphylococcal nuclease.

The production of λ was monitored throughout growth and is shown in **Figure 4**.

#### **Figure 3.**

*Bacteriophages - Perspectives and Future*

such as Fab fragments [33–35].

OD600 had reached 10.

replication and cell lysis are the same in both.

compared to the large amount of cellular DNA, RNA and protein released by lytic phage. A phage such as λ is a representative of lytic phage which is the majority of the types of phages used in therapy and biological control for the replacement of antibiotics. At each cycle of replication and release of the new phage particles, the host is lysed, and the cell contents are released into the medium along with insoluble debris from the cell wall and membrane. This leads to problems in subsequent downstream purification due to the large amount of different cellular molecules

The host RNA and DNA represent major contaminants that need to be removed especially for gene therapy applications. The release of host cell DNA also increases the viscosity of the medium, and this has an adverse effect on clarification by centrifugation and membrane concentration due to blocking of the membranes. For lab-scale molecular biology work, it is normal to add the enzymes pancreatic RNAseI and pancreatic DNAseI from bovine pancreas preparations. With the advent of bovine spongiform encephalopathy (BSE) which peaked in the 1990s, the addition of any bovine or animal proteins into the growth or purification train of material destined for human therapy was banned. These regulatory restrictions removed the ability to use these cheap nucleic acid-degrading enzymes, and the substitutes from bacterial sources were much more expensive. A strategy to overcome this problem was developed by us, and this was to express a broad-spectrum nuclease in the periplasm of *E. coli* which would be released as cells were lysed [30, 31]. The enzyme Staphylococcal nuclease has been extensively characterised and used from the 1960s onwards [32] and can degrade both DNA and RNA. The expression of this in *E. coli* where it is secreted into the periplasm does not affect the growth of *E. coli* because the enzyme cannot get access to its substrate while the cell is growing normally. When the cells are lysed by a bacteriophage such as λ or by homogenisation, the nuclease can gain access to the released DNA and RNA and degrade them. This 'cell engineering' approach to assisting bioprocessing was developed at UCL and has been shown to give considerable gains in the centrifugation steps and other downstream purification steps in bioprocessing of proteins

We sought to apply this cell engineering strategy for the production of λ phage and to help solve the problem of the large amount of cellular contaminants released

The addition of λ to the fermenter used the strategy that we had developed where a low MOI is used and without a static initial incubation of the host cells and the phage. In this way we can add the phage directly to a growing fermenter of host with the impeller (single shaft with three top-driven, equally spaced, sixbladed turbines) and four diametrically opposed baffles in the fermenter, running throughout. The growth profiles of the two uninfected cultures show no difference in their growth profiles which means that the presence of the expressed nuclease enzyme in the periplasm has no effect on growth rate or final OD. In the two cultures with added λ phage, the OD drop is the same for both hosts showing that λ

The production of λ was monitored throughout growth and is shown in

into the media when λ phage cultures need to be harvested and processed. *E. coli* JM107 [3] was grown in 2 L fermenters with either JM107 or JM107 containing the plasmid pMMBompnucB which is a broad host-range plasmid vector based on an Inc. Q plasmid RSF1010 and contains the Staphylococcal nuclease which has been altered by the addition of the *E. coli* ompA signal sequence for secretion [31]. **Figure 3** shows the growth of the two hosts with no added λ phage and the same hosts with 8 × 1010 λ phage particles added after 2 hours when the

competing in the subsequent downstream processing steps.

**82**

**Figure 4**.

*Growth of E. coli JM107 and λ production with and without a nuclease-expressing plasmid. E. coli JM107 containing pMMBompnucB and with no plasmid was grown in 2 L (working volume 1.5 L) LH 210 series fermenter (Bioprocess Engineering Services, Charing, Kent, UK) with 150 mL of E. coli inoculum and a final working volume of 1.5 L in phage media containing 100 μg/mL ampicillin and 20 μg/mL IPTG. λ phage particles were added at 2 hours to give an MOI of 0.05 (4 mL of 2 × 1010 pfu/mL). Impeller stirring continued throughout the growth and addition of phage, and the OD600 was monitored.*

#### **Figure 4.**

*Production of λ phage in 2 L fermenters with and without periplasmic nuclease expression. λ phage was added at 2 hours in Figure 3, and the graph here shows λ phage amounts from the 2 hours onwards. Samples from the fermenters shown in Figure 3 were diluted in phage buffer and titred on E. coli JM107 using soft top agar and plaque counting.*

Both the nuclease and the non-nuclease-expressing *E. coli* JM107 produced the same 5-log increase in phage particles with the same time profile. The efficacy of the nuclease in the removal of the host nucleic acid was assessed by the electrophoresis of samples from each time point on agarose gels and the visualisation of the released nucleic acid (both DNA and RNA). **Figure 5** shows the complete degradation and removal of the released host genomic DNA and host RNA in the strain of *E. coli* expressing the periplasmic nuclease.

The presence of the expressed periplasmic nuclease is apparent from the difference in the samples in **Figure 5B** compared to the samples from the same time points in the fermenter with no expression plasmid for the Staphylococcal nuclease.

#### **Figure 5.**

*Degradation of host nucleic acid from l infected E. coli JM107 and JM107 containing pMMBompnucB. (A) Agarose gel with samples from fermenter in Figure 3 growing E. coli JM107 with λ. 1, 1 kb ladder; 2, 0 h; 3, 1 h; 4, 2 h; 5, 3 h; 6, 4 h; 7, 5 h; 8, 6 h; 9, 7 h; 10, 8 h; 11 λ HindIII ladder. (B) Agarose gel with samples from fermenter in Figure 3 growing E. coli JM107 containing pMMBompnucB infected with λ. 1, 1 kb ladder; 2, 0 h; 3, 1 h; 4, 2 h; 5, 3 h; 6, 4 h; 7, 5 h; 8, 6 h; 9, 7 h; 10, 8 h; 11 λ HindIII ladder. The same volume of sample from each fermenter time point was loaded onto each lane.*

Almost all of the released host genomic DNA and the large majority of the RNA has been degraded in the culture that expresses the nuclease. **Figure 4** shows that the production of λ phage particles is identical in both fermenters, and the presence of the nuclease does not impinge on λ production and leads to a removal of the majority of the nucleic acid that would normally need the addition of bovine pancreatic DNAseI and RNAseA or more expensive bacterial equivalents such as Benzonase™ [36] to reduce the amount of nucleic acids. This cell engineering approach means that no animal-derived enzymes need be added, no costly commercial bacterial enzymes need be added and the engineered cells provide their own nuclease which degrades the nucleic acid in situ, so no additional time for incubation of any added enzyme is needed. Therefore a saving of both time and money is achieved via cell engineering for bioprocessing.

#### **7. Precipitation of M13**

Bacteriophages produced at any scale need to be concentrated by some method after the growth and production have taken place. The properties of phages allow some precipitation methods that are milder than conditions needed to precipitate host soluble protein or nucleic acid. Phages are large multicomponent entities usually many hundreds of times larger than a medium-sized soluble protein. Their asymmetric shape also enables mild precipitation methods. Polyethylene glycol (PEG) precipitation is a mild method of precipitating biological material and is very efficacious in the precipitation of large asymmetric material such as DNA or macromolecular assemblies, e.g. virus-like particles such as phages [37, 38]. The larger and more asymmetric the particle, the lower the amount of PEG is needed to precipitate the particle and leave behind other smaller soluble materials. An exploration of different average molecular weights of PEG from 600 to 20,000 for M13 precipitation showed that PEG 6000 and PEG 8000 combined the best properties of precipitation at low % concentration with lower viscosities than PEG 12,000 and PEG 20,000 [23] and 2% PEG 6000 with 330 mM NaCl gave >95% precipitation of M13. The relationship between PEG and NaCl is shown in **Figure 6** where the increasing PEG molecular weight and PEG concentration with % of M13 recovered were investigated.

**85**

*Scale-Up and Bioprocessing of Phages*

*DOI: http://dx.doi.org/10.5772/intechopen.88275*

in large-scale M13 phage precipitation.

**8. Conclusion**

**Figure 6.**

*for 10 min at 8000 ×g at 4°C.*

The diagram in **Figure 6** shows the relationship between increasing chain length/molecular weight of PEG, the % of PEG and the amount of M13 phage recovered. Higher-molecular-weight PEG preparations can be used at low final percentages, but there is significant increase in the viscosity of high-molecular-weight PEG solutions in the stock solutions needed to add to the phage-containing solution, making PEG 20,000 very difficult to use. PEG 6000 and PEG 8000 achieve virtually the same precipitation profile as PEG 12,000 and the lower viscosity of stock solutions of PEG 6000 make this chain length the best for precipitation with low viscosity. A concentration of 2% w/v PEG 6000 is ideal with 330 mM NaCl [23]. It was discovered that the nutrient media commonly used for growth such as NB2 contains sufficient salt (Na, K and NH3 ions) that the added NaCl can be reduced to only 135 mM which would make a saving in materials and disposal costs

*The relationship between M13 recovery and PEG molecular weight and percentage PEG. All precipitations were carried out at 10 mL scale with additions of PEG to the final % concentration shown in the diagram with 4 × 1012 CsCl purified M13 and 330 mM NaCl. The mixtures were incubated on ice for 1 hour and centrifuged* 

The strategies for the scale-up of phage growth and primary downstream purification are still in their infancy, but we have shown that there are significant gains to be made from the work described here. The amount of phage that needs to be added to large-scale growth volumes can be reduced by several orders of magnitude from what is common at the lab scale. Phage can be added directly to fermenters where rapidly stirring impellers are needed to maintain aeration and correct physiology of the host with no cessation of the stirring, and the phage will find their host and attach with no difficulty. The use of cells engineered to produce their own broadspectrum periplasmic nuclease gives significant gains in the destruction of host DNA and RNA release on lysis, and this prevents the contamination of the phage with nucleic acid when the phages are concentrated by precipitation. The gentle precipitation method of using low concentrations of PEG can then be used to give relatively pure preparations of phage in one step. These methods can be used together and will

#### **Figure 6.**

*Bacteriophages - Perspectives and Future*

engineering for bioprocessing.

*each fermenter time point was loaded onto each lane.*

**7. Precipitation of M13**

**Figure 5.**

Almost all of the released host genomic DNA and the large majority of the RNA has been degraded in the culture that expresses the nuclease. **Figure 4** shows that the production of λ phage particles is identical in both fermenters, and the presence of the nuclease does not impinge on λ production and leads to a removal of the majority of the nucleic acid that would normally need the addition of bovine pancreatic DNAseI and RNAseA or more expensive bacterial equivalents such as Benzonase™ [36] to reduce the amount of nucleic acids. This cell engineering approach means that no animal-derived enzymes need be added, no costly commercial bacterial enzymes need be added and the engineered cells provide their own nuclease which degrades the nucleic acid in situ, so no additional time for incubation of any added enzyme is needed. Therefore a saving of both time and money is achieved via cell

*Degradation of host nucleic acid from l infected E. coli JM107 and JM107 containing pMMBompnucB. (A) Agarose gel with samples from fermenter in Figure 3 growing E. coli JM107 with λ. 1, 1 kb ladder; 2, 0 h; 3, 1 h; 4, 2 h; 5, 3 h; 6, 4 h; 7, 5 h; 8, 6 h; 9, 7 h; 10, 8 h; 11 λ HindIII ladder. (B) Agarose gel with samples from fermenter in Figure 3 growing E. coli JM107 containing pMMBompnucB infected with λ. 1, 1 kb ladder; 2, 0 h; 3, 1 h; 4, 2 h; 5, 3 h; 6, 4 h; 7, 5 h; 8, 6 h; 9, 7 h; 10, 8 h; 11 λ HindIII ladder. The same volume of sample from* 

Bacteriophages produced at any scale need to be concentrated by some method after the growth and production have taken place. The properties of phages allow some precipitation methods that are milder than conditions needed to precipitate host soluble protein or nucleic acid. Phages are large multicomponent entities usually many hundreds of times larger than a medium-sized soluble protein. Their asymmetric shape also enables mild precipitation methods. Polyethylene glycol (PEG) precipitation is a mild method of precipitating biological material and is very efficacious in the precipitation of large asymmetric material such as DNA or macromolecular assemblies, e.g. virus-like particles such as phages [37, 38]. The larger and more asymmetric the particle, the lower the amount of PEG is needed to precipitate the particle and leave behind other smaller soluble materials. An exploration of different average molecular weights of PEG from 600 to 20,000 for M13 precipitation showed that PEG 6000 and PEG 8000 combined the best properties of precipitation at low % concentration with lower viscosities than PEG 12,000 and PEG 20,000 [23] and 2% PEG 6000 with 330 mM NaCl gave >95% precipitation of M13. The relationship between PEG and NaCl is shown in **Figure 6** where the increasing PEG molecular weight and PEG concentration with % of M13 recovered were investigated.

**84**

*The relationship between M13 recovery and PEG molecular weight and percentage PEG. All precipitations were carried out at 10 mL scale with additions of PEG to the final % concentration shown in the diagram with 4 × 1012 CsCl purified M13 and 330 mM NaCl. The mixtures were incubated on ice for 1 hour and centrifuged for 10 min at 8000 ×g at 4°C.*

The diagram in **Figure 6** shows the relationship between increasing chain length/molecular weight of PEG, the % of PEG and the amount of M13 phage recovered. Higher-molecular-weight PEG preparations can be used at low final percentages, but there is significant increase in the viscosity of high-molecular-weight PEG solutions in the stock solutions needed to add to the phage-containing solution, making PEG 20,000 very difficult to use. PEG 6000 and PEG 8000 achieve virtually the same precipitation profile as PEG 12,000 and the lower viscosity of stock solutions of PEG 6000 make this chain length the best for precipitation with low viscosity. A concentration of 2% w/v PEG 6000 is ideal with 330 mM NaCl [23]. It was discovered that the nutrient media commonly used for growth such as NB2 contains sufficient salt (Na, K and NH3 ions) that the added NaCl can be reduced to only 135 mM which would make a saving in materials and disposal costs in large-scale M13 phage precipitation.

#### **8. Conclusion**

The strategies for the scale-up of phage growth and primary downstream purification are still in their infancy, but we have shown that there are significant gains to be made from the work described here. The amount of phage that needs to be added to large-scale growth volumes can be reduced by several orders of magnitude from what is common at the lab scale. Phage can be added directly to fermenters where rapidly stirring impellers are needed to maintain aeration and correct physiology of the host with no cessation of the stirring, and the phage will find their host and attach with no difficulty. The use of cells engineered to produce their own broadspectrum periplasmic nuclease gives significant gains in the destruction of host DNA and RNA release on lysis, and this prevents the contamination of the phage with nucleic acid when the phages are concentrated by precipitation. The gentle precipitation method of using low concentrations of PEG can then be used to give relatively pure preparations of phage in one step. These methods can be used together and will

allow the large-scale uses of phage in the future in medical and clinical applications and then beyond into biotechnological applications such as the uses of filamentous phage in electronics like phage batteries [39] and the phage laser [40].

### **Acknowledgements**

We acknowledge and thank the Engineering and Physical Sciences Research Council (EPSRC) for support via the Life Science IMRC for Bioprocessing and the EPSRC for the PhD studentship to SB. We thank the Biotechnology and Biological Sciences Research Council (BBSRC) for grant funding to JMW and EKM to support ES under research grant BBD521465/1.

### **Conflict of interest**

There are no conflicts of interest.

### **Author details**

John Maxim Ward\*, Steven Branston, Emma Stanley and Eli Keshavarz-Moore Department of Biochemical Engineering, University College London, London, UK

\*Address all correspondence to: j.ward@ucl.ac.uk

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**87**

*Scale-Up and Bioprocessing of Phages*

[1] Karn J, Brenner S, Barnett L, Cesareni G. Novel bacteriophage lambda cloning vector. Proceedings of the National Academy of Sciences of the United States of America.

[2] Chauthaiwale VM, Therwath A, Deshpande VV. Bacteriophage lambda as a cloning vector. Microbiological

[3] Yanisch-Perron C, Vieira J, Messing J. Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mp18 and pUC19 vectors.

[4] Lankes HA, Zanghi CN, Santos K, Capella C, Duke CM, Dewhurst S. *In vivo* gene delivery and expression by bacteriophage lambda vectors. Journal of Applied Microbiology.

[5] Azeredo J, Sutherland IW. The use of phages for the removal of infectious biofilms. Current Pharmaceutical Biotechnology. 2008;**9**:261-266

[6] Jepson CD, March JB. Bacteriophage

[7] Green MR, Sambrook J. Molecular Cloning. New York: Cold Spring Harbor

[9] Boynton ZL, Koon JJ, Brennan EM, Clouart JD, Horowitz DM, Gerngross TU, et al. Reduction of cell lysate viscosity during processing of poly (3-hydroxyalkanoates) by chromosomal integration of the Staphylococcal nuclease gene in *Pseudomonas* 

lambda is a highly stable DNA vaccine delivery vehicle. Vaccine.

**References**

1980;**77**:5172-5176

Reviews. 1992;**56**:577-591

Gene. 1985;**33**:103-119

2007;**102**:1337-1349

2004;**22**:2413-2419

Laboratory Press; 2012

2015;**479-480**:310-330

[8] Casjens SR, Hendrix RW. Bacteriophage lambda: Early pioneer and still relevant. Virology.

*DOI: http://dx.doi.org/10.5772/intechopen.88275*

*putida*. Applied and Environmental Microbiology. 1999;**65**:1524-1529

[11] Smeal SW, Schmitt MA, Pereira RR,

Automated fluorescent DNA sequencing

[13] Sanger F, Coulson AR, Hong GF, Hill DF, Petersen GB. Nucleotide sequence of bacteriophage λ

DNA. Journal of Molecular Biology.

and Molecular Biology Reviews.

[15] Maruyama IN1, Maruyama HI, Brenner S. Lambda foo: A lambda phage vector for the expression of foreign proteins. Proceedings of the National Academy of Sciences of the United States of America 1994;

[16] Razazan A, Nicastro J, Slavcev R, Barati N, Arab A, Mosaffa F, et al. Lambda bacteriophage nanoparticles displaying GP2, a HER2/neu derived peptide, induce prophylactic and therapeutic activities against TUBO tumor model in mice. Scientific Reports.

[14] Gottesman ME, Weisberg RA. Little Lambda, who made thee? Microbiology

Prasad A, Fisk JD. Simulation of the M13 life cycle I: Assembly of a genetically-structured deterministic chemical kinetic simulation. Virology.

[12] Du Z, Hood L, Wilson RK.

of polymerase chain reaction products. Methods in Enzymology.

2017;**500**:259-274

1993;**218**:104-121

1982;**162**:729-773

2004;**68**:796-813

**91**:8273-8277

2019;**9**:2221

[10] World Health Organisation. Guidelines on the quality, safety, and efficacy of biotherapeutic protein products prepared by recombinant DNA technology. World Health Organisation. Replacement of Annex 3 of WHO Technical Report Series, No. 814; 2014

*Scale-Up and Bioprocessing of Phages DOI: http://dx.doi.org/10.5772/intechopen.88275*

#### **References**

*Bacteriophages - Perspectives and Future*

ES under research grant BBD521465/1.

There are no conflicts of interest.

**Acknowledgements**

**Conflict of interest**

allow the large-scale uses of phage in the future in medical and clinical applications and then beyond into biotechnological applications such as the uses of filamentous

We acknowledge and thank the Engineering and Physical Sciences Research Council (EPSRC) for support via the Life Science IMRC for Bioprocessing and the EPSRC for the PhD studentship to SB. We thank the Biotechnology and Biological Sciences Research Council (BBSRC) for grant funding to JMW and EKM to support

John Maxim Ward\*, Steven Branston, Emma Stanley and Eli Keshavarz-Moore Department of Biochemical Engineering, University College London, London, UK

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: j.ward@ucl.ac.uk

provided the original work is properly cited.

phage in electronics like phage batteries [39] and the phage laser [40].

**86**

**Author details**

[1] Karn J, Brenner S, Barnett L, Cesareni G. Novel bacteriophage lambda cloning vector. Proceedings of the National Academy of Sciences of the United States of America. 1980;**77**:5172-5176

[2] Chauthaiwale VM, Therwath A, Deshpande VV. Bacteriophage lambda as a cloning vector. Microbiological Reviews. 1992;**56**:577-591

[3] Yanisch-Perron C, Vieira J, Messing J. Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;**33**:103-119

[4] Lankes HA, Zanghi CN, Santos K, Capella C, Duke CM, Dewhurst S. *In vivo* gene delivery and expression by bacteriophage lambda vectors. Journal of Applied Microbiology. 2007;**102**:1337-1349

[5] Azeredo J, Sutherland IW. The use of phages for the removal of infectious biofilms. Current Pharmaceutical Biotechnology. 2008;**9**:261-266

[6] Jepson CD, March JB. Bacteriophage lambda is a highly stable DNA vaccine delivery vehicle. Vaccine. 2004;**22**:2413-2419

[7] Green MR, Sambrook J. Molecular Cloning. New York: Cold Spring Harbor Laboratory Press; 2012

[8] Casjens SR, Hendrix RW. Bacteriophage lambda: Early pioneer and still relevant. Virology. 2015;**479-480**:310-330

[9] Boynton ZL, Koon JJ, Brennan EM, Clouart JD, Horowitz DM, Gerngross TU, et al. Reduction of cell lysate viscosity during processing of poly (3-hydroxyalkanoates) by chromosomal integration of the Staphylococcal nuclease gene in *Pseudomonas* 

*putida*. Applied and Environmental Microbiology. 1999;**65**:1524-1529

[10] World Health Organisation. Guidelines on the quality, safety, and efficacy of biotherapeutic protein products prepared by recombinant DNA technology. World Health Organisation. Replacement of Annex 3 of WHO Technical Report Series, No. 814; 2014

[11] Smeal SW, Schmitt MA, Pereira RR, Prasad A, Fisk JD. Simulation of the M13 life cycle I: Assembly of a genetically-structured deterministic chemical kinetic simulation. Virology. 2017;**500**:259-274

[12] Du Z, Hood L, Wilson RK. Automated fluorescent DNA sequencing of polymerase chain reaction products. Methods in Enzymology. 1993;**218**:104-121

[13] Sanger F, Coulson AR, Hong GF, Hill DF, Petersen GB. Nucleotide sequence of bacteriophage λ DNA. Journal of Molecular Biology. 1982;**162**:729-773

[14] Gottesman ME, Weisberg RA. Little Lambda, who made thee? Microbiology and Molecular Biology Reviews. 2004;**68**:796-813

[15] Maruyama IN1, Maruyama HI, Brenner S. Lambda foo: A lambda phage vector for the expression of foreign proteins. Proceedings of the National Academy of Sciences of the United States of America 1994; **91**:8273-8277

[16] Razazan A, Nicastro J, Slavcev R, Barati N, Arab A, Mosaffa F, et al. Lambda bacteriophage nanoparticles displaying GP2, a HER2/neu derived peptide, induce prophylactic and therapeutic activities against TUBO tumor model in mice. Scientific Reports. 2019;**9**:2221

[17] Mai-Prochnow A, Gee J, Hui K, Kjelleberg S, Rakonjac J, McDougald D, et al. Big things in small packages: The genetics of filamentous phage and effects on fitness of their host. FEMS Microbiology Reviews. 2015;**39**:465-487

[18] Branston S, Stanley E, Ward J, Keshavarz-Moore E. Study of robustness of filamentous bacteriophages for industrial applications. Biotechnology and Bioengineering. 2011;**108**:1468-1472

[19] Gardner RC, Howarth AJ, Messing J, Shepherd RJ. Cloning and sequencing of restriction fragments generated by Eco RI\*. DNA. 1982;**1**:109-115

[20] Norrander J, Kempe T, Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 1983;**26**:101-106

[21] Smith GP. Filamentous fusion phage: Novel expression vectors that display cloned antigens on the virion surface. Science. 1985;**228**:1315-1317

[22] Clackson T, Hoogenboom HR, Griffiths AD, Winter G. Making antibody fragments using phage display libraries. Nature. 1991;**352**:624-628

[23] Branston S, Stanley E, Keshavarz-Moore E, Ward J. Precipitation of filamentous bacteriophages for their selective recovery in primary purification. Biotechnology Progress. 2011;**28**:129-136

[24] Ellis EL, Delbruck MJ. The growth of bacteriophage. The Journal of General Physiology. 1939;**22**:365-384

[25] Xu Z, Lee SY. Display of polyhistidine peptides on the *Escherichia coli* cell surface by using outer membrane protein C as an anchoring motif. Applied and Environmental Microbiology. 1999;**65**:5142-5147

[26] Clarke M, Maddera L, Harris RL, Silverman PM. F-pili dynamics

by live-cell imaging. PNAS. 2008;**105**:17978-17981

[27] Gibbs K, Isaac D, Xu J, Hendrix R, Silhavy T, Theriot J. Complex spatial distribution and dynamics of an abundant *Escherichia coli* outer membrane protein, LamB. Molecular Microbiology. 2004;**53**:1771-1783

[28] Edgar R, Rokney A, Feeney M, Semsey S, Kessel M, Goldberg MB, et al. Bacteriophage infection is targeted to cellular poles. Molecular Microbiology. 2008;**68**:1107-1116

[29] Wang I-N. Lysis timing and bacteriophage fitness. Genetics. 2006;**172**:17-26

[30] Cooke GD, Cranenburgh RM, Hanak JAJ, Dunnill P, Thatcher DR, Ward JM. Purification of essentially RNA free plasmid DNA using a modified *Escherichia coli* host strain expressing Ribonuclease A. Journal of Biotechnology. 2001;**85**:297304

[31] Cooke GD, Cranenburgh RM, Hanak JAJ, Ward JM. A modified *Escherichia coli* protein production strain expressing staphylococcal nuclease, capable of auto-hydrolysing host nucleic acid. Journal of Biotechnology. 2003;**101**:229-239

[32] Heins JN, Suriano JR, Taniuchi H, Anfinsen CB. Characterization of a nuclease produced by *Staphylococcus aureus*. The Journal of Biological Chemistry. 1967;**242**:1016-1020

[33] Balasundaram B, Nesbeth D, Ward JM, Keshavarz-Moore E, Bracewell DG. Step change in the efficiency of centrifugation through cell engineering: Coexpression of Staphylococcal nuclease to reduce the viscosity of the bioprocess feedstock. Biotechnology and Bioengineering. 2009;**104**:134-142

[34] Nesbeth D, Pardo MA, Ali S, Ward J, Keshavarz-Moore E. Growth and

**89**

2013

*Scale-Up and Bioprocessing of Phages*

2012;**109**:517-527

2017;**39**:1865-1873

0229866; 1992

1960;**11**:553-571

2009;**324**:1051-1055

1960

*DOI: http://dx.doi.org/10.5772/intechopen.88275*

productivity impacts of periplasmic nuclease expression in an *Escherichia coli* fab' fragment production strain. Biotechnology and Bioengineering.

[35] Schofield DM, Sirka E, Keshavarz-Moore E, Ward JM, Nesbeth DN. Improving Fab' fragment retention in an autonucleolytic *Escherichia coli* strain by swapping periplasmic nuclease translocation signal from OmpA to DsbA. Biotechnology Letters.

[36] Molin S, Givskov M, Riise E. Production in *Escherichia coli* of Extracellular Serratia spp. Hydrolase. Benzon Pharma, A/S, Hvidovre, Denmark. European Patent No.

[37] Albertsson PA. Partitions of Cell Particles and Macro-Molecules. New York: John Wiley & Sons, Inc.;

[38] Philipson L, Albertsson PA, Frick G. The purification and concentration of viruses by aqueous polymer phase systems. Virology.

[39] Lee YJ, Yi H, Kim WJ, Kang K, Yun DS, Strano MS, et al. Fabricating

[40] Hales JE, Ward J, Aeppli G and Dafforn T. Fluorescent Composition. WO2013093499 PCT/GB2012/053236.

genetically engineered highpower lithium-ion batteries using multiple virus genes. Science.

*Scale-Up and Bioprocessing of Phages DOI: http://dx.doi.org/10.5772/intechopen.88275*

*Bacteriophages - Perspectives and Future*

[17] Mai-Prochnow A, Gee J, Hui K, Kjelleberg S, Rakonjac J, McDougald D, et al. Big things in small packages: The genetics of filamentous phage and effects on fitness of their host. FEMS Microbiology Reviews. 2015;**39**:465-487 by live-cell imaging. PNAS. 2008;**105**:17978-17981

Silhavy T, Theriot J. Complex spatial distribution and dynamics of an abundant *Escherichia coli* outer membrane protein, LamB. Molecular Microbiology. 2004;**53**:1771-1783

[27] Gibbs K, Isaac D, Xu J, Hendrix R,

[28] Edgar R, Rokney A, Feeney M, Semsey S, Kessel M, Goldberg MB, et al. Bacteriophage infection is targeted to cellular poles. Molecular Microbiology.

[29] Wang I-N. Lysis timing and bacteriophage fitness. Genetics.

[30] Cooke GD, Cranenburgh RM, Hanak JAJ, Dunnill P, Thatcher DR, Ward JM. Purification of essentially RNA free plasmid DNA using a modified *Escherichia coli* host strain expressing Ribonuclease A. Journal of Biotechnology. 2001;**85**:297304

[31] Cooke GD, Cranenburgh RM, Hanak JAJ, Ward JM. A modified

*Escherichia coli* protein production strain expressing staphylococcal nuclease, capable of auto-hydrolysing host nucleic acid. Journal of Biotechnology.

[32] Heins JN, Suriano JR, Taniuchi H, Anfinsen CB. Characterization of a nuclease produced by *Staphylococcus aureus*. The Journal of Biological Chemistry. 1967;**242**:1016-1020

[33] Balasundaram B, Nesbeth D,

Coexpression of Staphylococcal nuclease to reduce the viscosity of the bioprocess feedstock. Biotechnology and Bioengineering. 2009;**104**:134-142

Keshavarz-Moore E. Growth and

Ward JM, Keshavarz-Moore E, Bracewell DG. Step change in the efficiency of centrifugation through cell engineering:

[34] Nesbeth D, Pardo MA, Ali S, Ward J,

2008;**68**:1107-1116

2006;**172**:17-26

2003;**101**:229-239

[18] Branston S, Stanley E, Ward J, Keshavarz-Moore E. Study of robustness of filamentous bacteriophages for industrial applications. Biotechnology and Bioengineering. 2011;**108**:1468-1472

[19] Gardner RC, Howarth AJ, Messing J, Shepherd RJ. Cloning and sequencing of restriction fragments generated by Eco

[20] Norrander J, Kempe T, Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 1983;**26**:101-106

[21] Smith GP. Filamentous fusion phage: Novel expression vectors that display cloned antigens on the virion surface. Science. 1985;**228**:1315-1317

[22] Clackson T, Hoogenboom HR, Griffiths AD, Winter G. Making

antibody fragments using phage display libraries. Nature. 1991;**352**:624-628

[23] Branston S, Stanley E, Keshavarz-Moore E, Ward J. Precipitation of filamentous bacteriophages for their selective recovery in primary purification. Biotechnology Progress.

[24] Ellis EL, Delbruck MJ. The growth of bacteriophage. The Journal of General Physiology. 1939;**22**:365-384

polyhistidine peptides on the *Escherichia* 

[26] Clarke M, Maddera L, Harris RL, Silverman PM. F-pili dynamics

[25] Xu Z, Lee SY. Display of

*coli* cell surface by using outer membrane protein C as an anchoring motif. Applied and Environmental Microbiology. 1999;**65**:5142-5147

2011;**28**:129-136

RI\*. DNA. 1982;**1**:109-115

**88**

productivity impacts of periplasmic nuclease expression in an *Escherichia coli* fab' fragment production strain. Biotechnology and Bioengineering. 2012;**109**:517-527

[35] Schofield DM, Sirka E, Keshavarz-Moore E, Ward JM, Nesbeth DN. Improving Fab' fragment retention in an autonucleolytic *Escherichia coli* strain by swapping periplasmic nuclease translocation signal from OmpA to DsbA. Biotechnology Letters. 2017;**39**:1865-1873

[36] Molin S, Givskov M, Riise E. Production in *Escherichia coli* of Extracellular Serratia spp. Hydrolase. Benzon Pharma, A/S, Hvidovre, Denmark. European Patent No. 0229866; 1992

[37] Albertsson PA. Partitions of Cell Particles and Macro-Molecules. New York: John Wiley & Sons, Inc.; 1960

[38] Philipson L, Albertsson PA, Frick G. The purification and concentration of viruses by aqueous polymer phase systems. Virology. 1960;**11**:553-571

[39] Lee YJ, Yi H, Kim WJ, Kang K, Yun DS, Strano MS, et al. Fabricating genetically engineered highpower lithium-ion batteries using multiple virus genes. Science. 2009;**324**:1051-1055

[40] Hales JE, Ward J, Aeppli G and Dafforn T. Fluorescent Composition. WO2013093499 PCT/GB2012/053236. 2013

**91**

**Chapter 6**

**Abstract**

Cas9, phage

**1. Introduction**

Surveillance and Elimination of

Bacteriophage Contamination in

*James A. Zahn and Mathew C. Halter*

in a commercial fermentation process.

an Industrial Fermentation Process

Commercial fermentation processes are often vulnerable to bacteriophage due to the lack of genetic diversity and use of high cell density cultures. Bacteriophage infections in these fermentations can have adverse impacts on operability of the production facility and product quality and prevent recovery of valuable bioproducts in the downstream process. Prevention strategies have been developed and optimized through feedback from bacteriophage diagnostic tests, which inform improvements to process design for elimination of entry points, as well as modification of the biocatalyst to reduce or eliminate bacteriophage virulence. In this chapter, we provide case studies for successful elimination of bacteriophage virulence via host modifications, including bacteriophage binding-site modifications on the outer membrane of an *Escherichia coli* production host, used for commercial manufacture of 1,3-propanediol, as well as application of CRISPR-associated protein 9 (Cas9) for bacteriophage immunity. Finally, we report application of bacteriophage diagnostic methods to fully characterize and eliminate bacteriophage entry points

**Keywords:** bacteriophage, white biotechnology, industrial fermentation, CRISPR,

Increasing awareness of our dependence on petroleum, coupled with the negative effects of this dependency on oil supply, price volatility, and gas emissions, is the driver behind the growing global market for biorenewable chemicals made through white biotechnology processes. In 2011, revenue from biorenewable chemicals exceeded \$2.4 billion, and revenue continues to grow at a compound annual growth rate of 14.8%, with glycerin and lactic acid accounting for 79% of the market share [1]. Growth for biorenewable chemicals that are used as monomers the manufacture of bioplastics represents a fast-growing segment of this industry [1]. One example of a biorenewable chemical that has displaced petrochemical manufacturing routes is 1,3-propanediol or BioPDO™. This chemical had historically been manufactured using petroleum-derived ethylene oxide or acrolein [2, 3]. In 2006, DuPont Tate & Lyle Bio Products, a joint venture formed between DuPont and Tate & Lyle, commercialized an aerobic fermentation process for production of 1,3-propanediol (BioPDO™) from glucose derived from yellow dent field corn.

#### **Chapter 6**

## Surveillance and Elimination of Bacteriophage Contamination in an Industrial Fermentation Process

*James A. Zahn and Mathew C. Halter*

#### **Abstract**

Commercial fermentation processes are often vulnerable to bacteriophage due to the lack of genetic diversity and use of high cell density cultures. Bacteriophage infections in these fermentations can have adverse impacts on operability of the production facility and product quality and prevent recovery of valuable bioproducts in the downstream process. Prevention strategies have been developed and optimized through feedback from bacteriophage diagnostic tests, which inform improvements to process design for elimination of entry points, as well as modification of the biocatalyst to reduce or eliminate bacteriophage virulence. In this chapter, we provide case studies for successful elimination of bacteriophage virulence via host modifications, including bacteriophage binding-site modifications on the outer membrane of an *Escherichia coli* production host, used for commercial manufacture of 1,3-propanediol, as well as application of CRISPR-associated protein 9 (Cas9) for bacteriophage immunity. Finally, we report application of bacteriophage diagnostic methods to fully characterize and eliminate bacteriophage entry points in a commercial fermentation process.

**Keywords:** bacteriophage, white biotechnology, industrial fermentation, CRISPR, Cas9, phage

#### **1. Introduction**

Increasing awareness of our dependence on petroleum, coupled with the negative effects of this dependency on oil supply, price volatility, and gas emissions, is the driver behind the growing global market for biorenewable chemicals made through white biotechnology processes. In 2011, revenue from biorenewable chemicals exceeded \$2.4 billion, and revenue continues to grow at a compound annual growth rate of 14.8%, with glycerin and lactic acid accounting for 79% of the market share [1]. Growth for biorenewable chemicals that are used as monomers the manufacture of bioplastics represents a fast-growing segment of this industry [1].

One example of a biorenewable chemical that has displaced petrochemical manufacturing routes is 1,3-propanediol or BioPDO™. This chemical had historically been manufactured using petroleum-derived ethylene oxide or acrolein [2, 3]. In 2006, DuPont Tate & Lyle Bio Products, a joint venture formed between DuPont and Tate & Lyle, commercialized an aerobic fermentation process for production of 1,3-propanediol (BioPDO™) from glucose derived from yellow dent field corn.

A life cycle analysis (LCA) showed that the BioPDO™ manufacturing process consumed 42% less energy and emitted 56% less greenhouse gas emissions than petrochemical manufacturing routes. BioPDO™ is included as a raw material in polyester manufacture for textiles, carpet (Sorona®), and thermoplastic resins (Sorona®EP). Furthermore, BioPDO™ has direct uses in foods, cosmetics, and personal care applications through the Zemea® brand, and heat transfer, homopolymer, polyurethane, and other industrial applications through the Susterra® brand [3, 4].

In addition to a more favorable life cycle analyses, White Biotechnology processes like the BioPDO™ process demonstrate many advantages over petrochemical processes, such as the ability of the process to maintain high specificity [5], high yield, and ability to maintain lower concentrations of chemical intermediates that are generally recognized to form undesirable impurities in the final product [6].

Although many advantages exist for bioprocesses, disadvantages include (a) low to moderate productivity rates requiring intensification capital investments related to equipment capacity, (b) process susceptibility to bacterial and bacteriophage contamination, which has a negative impact on operability factor, (c) generally higher variable cost of manufacturing due to high electrical and related energy costs, (d) high water use, (e) odors, (f) costs associated with the production and filtration of large volumes of gases required for the fermentation process, and treatment of waste gases to remove odors, viable cells, and regulated or unregulated chemicals, (g) higher separation and refining costs due to the presence of large amounts of water, and (h) disposal of large volumes of nonhazardous waste.

In this chapter, we provide a detailed analysis of one of the most challenging issues for white biotechnology processes, which is the prevention of lytic bacteriophage events in commercial fermentors. Bacteriophages can cause rapid lytic infections of the highly clonal bacterial populations that are used in white biotechnology processes. Lytic infections of bacteria by bacteriophage reduce or abolish product productivity, and reduce the efficiency of cell recovery methods, which causes reduced product quality or a complete loss of the product. Batches affected by lytic bacteriophage infections cause a loss in production capacity or asset utilization, and financial losses to a business [7]. Lytic events in industrial fermentation may necessitate temporary shutdown of the facility for cleaning and elimination of bacteriophage, or even prolonged shutdown periods for cleaning and modification of aseptic barriers in the facility. In addition to surveillance and elimination best practices, this chapter outlines facility design considerations that are important in the prevention of bacteriophage in a biomanufacturing facility.

#### **2. Prevalence of bacteriophage contamination in industrial fermentation processes**

With the steady increase in the use of prokaryotic biocatalysts over the course of the last several decades for protein, small molecule, and chemical production, a focus has been placed on maintaining a bacteriophage-free environment in the manufacturing facility. The prevalence of bacteriophage in commercial fermentation processes varies considerably within the industry. For example, there have been no reported bacteriophage-related losses in certain industrial process, including: (a) syngas fermentations utilizing *Clostridium ljungdahlii*, *Clostridium autoethanogenum*, and *Clostridium coskatii*; (b) the commercial process for production of Spinosad and Spinetoram using *Saccharopolyspora spinosa*; and the commercial process for (c) production of xanthan gum by *Xanthomonas campestris*. In contrast, moderate to severe fermentative losses have been observed for certain

**93**

growth.

*Surveillance and Elimination of Bacteriophage Contamination in an Industrial Fermentation…*

bacterial-mediated processes, including the acetone-butanol-ethanol process that uses *Clostridium acetobutylicum*, *Clostridium beijerinckii*, or *Clostridium* sp. [8]; the *Escherichia coli*-mediated NutraSweet® process [9]; the *E. coli*-mediated BioPDO™ process [10]; the vinegar fermentation processes that uses *Acetobacter europaeus*, or

a.Tanji et al. [14] proposed that "the most likely source of phage contamination in the *E. coli* culturing process is human, since *E. coli* is one of the main inhabitants of the gastrointestinal tracts of warm-blooded animals." Infection of microflora present in fermentation plant personnel may serve as a reservoir to maintain bacteriophage in the plant environment for extended periods of time [15]. Production hosts that are incapable of human colonization or that exhibit distinct phylogenetic differences to the natural human microflora may

b.Certain processes using *E. coli* have a distinct disadvantage that aseptic design and operation principles are unknowingly sacrificed by continuous improvement programs, which focus on minimizing process cycle time, or related cost reductions. Processes that produce end products that are inhibitory to foreign bacterial growth are especially vulnerable since a false sense of security is provided by the fact that the *E. coli* production hosts exhibit improved competitiveness over most other microorganisms. Under these conditions, foreign growth, which may harbor bacteriophage, can go undetected or unreported. Process cycle time improvements typically target a reduction in maintenance tasks supporting sterile barriers, or a reduction in time allotted for sterilization and clean-in-place (CIP) procedures to clean piping, valves, and vessels. These

*Acetobacter* sp. [11]; and food processes that utilize various bacteria [12, 13]. Although no single factor appears to link processes that are susceptible to

bacteriophage-induced fermentative losses, some common themes exist:

be advantaged over *E. coli* production hosts for these processes.

efforts can negatively impact bacteriophage elimination programs.

c.*Xanthomonas campestris* has a number of established bacteriophages that have been registered as biocontrol agents to minimize its impact as a plant pathogen [16]. Therefore, the absence of bacteriophage-induced losses for the commercial xanthan gum process is aligned with the absence of an appropriate reservoir in the manufacturing facility that ecologically supports *X. campestris*

d.Microorganisms with doubling times greater than 2–3 hours, which includes *Saccharopolyspora spinosa*, *Clostridium ljungdahlii*, and *Clostridium autoethanogenum*, often exhibit effective DNA restriction-modification systems that serve to destroy foreign DNA that enters the cell. These microorganisms are typically referred to as recalcitrant to recombinant DNA technology, as measured by low or unmeasurable transformation efficiency [17]. More recently, a number of widespread bacteriophage immunity systems have been described, some of which contain elements that act similarly to restriction-modification systems, to modify or destroy both foreign (i.e., non-native plasmid or cosmid-associated DNA) and bacteriophage-associated DNA [18, 19]. Interestingly, the *E. coli* production host used for the BioPDO™ processes was found to lack a functional bacteriophage exclusion operon (BREX [18]), as well as a functional CRISPR-Cas restriction-modification system [7, 20]. Highly optimized strains utilized in commercial manufacturing process often contain intended and unintended modifications to the host chromosome that contribute to improved rate,

*DOI: http://dx.doi.org/10.5772/intechopen.81151*

*Surveillance and Elimination of Bacteriophage Contamination in an Industrial Fermentation… DOI: http://dx.doi.org/10.5772/intechopen.81151*

bacterial-mediated processes, including the acetone-butanol-ethanol process that uses *Clostridium acetobutylicum*, *Clostridium beijerinckii*, or *Clostridium* sp. [8]; the *Escherichia coli*-mediated NutraSweet® process [9]; the *E. coli*-mediated BioPDO™ process [10]; the vinegar fermentation processes that uses *Acetobacter europaeus*, or *Acetobacter* sp. [11]; and food processes that utilize various bacteria [12, 13].

Although no single factor appears to link processes that are susceptible to bacteriophage-induced fermentative losses, some common themes exist:


*Bacteriophages - Perspectives and Future*

A life cycle analysis (LCA) showed that the BioPDO™ manufacturing process consumed 42% less energy and emitted 56% less greenhouse gas emissions than petrochemical manufacturing routes. BioPDO™ is included as a raw material in polyester manufacture for textiles, carpet (Sorona®), and thermoplastic resins (Sorona®EP). Furthermore, BioPDO™ has direct uses in foods, cosmetics, and personal care applications through the Zemea® brand, and heat transfer, homopolymer, polyure-

In addition to a more favorable life cycle analyses, White Biotechnology processes like the BioPDO™ process demonstrate many advantages over petrochemical processes, such as the ability of the process to maintain high specificity [5], high yield, and ability to maintain lower concentrations of chemical intermediates that are generally recognized to form undesirable impurities in the final product [6].

Although many advantages exist for bioprocesses, disadvantages include (a) low to moderate productivity rates requiring intensification capital investments related to equipment capacity, (b) process susceptibility to bacterial and bacteriophage contamination, which has a negative impact on operability factor, (c) generally higher variable cost of manufacturing due to high electrical and related energy costs, (d) high water use, (e) odors, (f) costs associated with the production and filtration of large volumes of gases required for the fermentation process, and treatment of waste gases to remove odors, viable cells, and regulated or unregulated chemicals, (g) higher separation and refining costs due to the presence of large amounts of water, and (h) disposal of large volumes of nonhazardous waste. In this chapter, we provide a detailed analysis of one of the most challenging issues for white biotechnology processes, which is the prevention of lytic bacteriophage events in commercial fermentors. Bacteriophages can cause rapid lytic infections of the highly clonal bacterial populations that are used in white biotechnology processes. Lytic infections of bacteria by bacteriophage reduce or abolish product productivity, and reduce the efficiency of cell recovery methods, which causes reduced product quality or a complete loss of the product. Batches affected by lytic bacteriophage infections cause a loss in production capacity or asset utilization, and financial losses to a business [7]. Lytic events in industrial fermentation may necessitate temporary shutdown of the facility for cleaning and elimination of bacteriophage, or even prolonged shutdown periods for cleaning and modification of aseptic barriers in the facility. In addition to surveillance and elimination best practices, this chapter outlines facility design considerations that are important in

thane, and other industrial applications through the Susterra® brand [3, 4].

the prevention of bacteriophage in a biomanufacturing facility.

**2. Prevalence of bacteriophage contamination in industrial fermentation** 

With the steady increase in the use of prokaryotic biocatalysts over the course

of the last several decades for protein, small molecule, and chemical production, a focus has been placed on maintaining a bacteriophage-free environment in the manufacturing facility. The prevalence of bacteriophage in commercial fermentation processes varies considerably within the industry. For example, there have been no reported bacteriophage-related losses in certain industrial process, including: (a) syngas fermentations utilizing *Clostridium ljungdahlii*, *Clostridium autoethanogenum*, and *Clostridium coskatii*; (b) the commercial process for production of Spinosad and Spinetoram using *Saccharopolyspora spinosa*; and the commercial process for (c) production of xanthan gum by *Xanthomonas campestris*. In contrast, moderate to severe fermentative losses have been observed for certain

**92**

**processes**

**Figure 1.**

*Alignment of the E. coli K12 CRISPR operon (upper panel) with the native nonfunctional CRISPR-Cas operon in the BioPDO™ E. coli production host (lower panel).*

titer, and yield. Although the BioPDO™ production host contains nearly a complete *E. coli* K12 CRISPR-Cas operon (**Figure 1**), upstream regulatory modifications, which were correlated with improved product production rate resulted in the loss of function for this operon. Vale et al. [19] found that "Cas protein expression is particularly costly, as Cas-deficient mutants achieved higher competitive abilities than the wild-type strain with functional Cas proteins." Smaller spacer libraries of approximately four spacers or less were not associated with fitness costs, suggesting that the genetically engineered spacer library approach of Halter and Zahn may serve to minimize energy and ATP burden on the cell, as indicated by the minimal impact of recombinant seven-spacer system on 1,3-propanediol biosynthesis [7].

#### **2.1 Isolation and identification of bacteriophage DNA from lytic production samples in the BioPDO™ process**

Lytic bacteriophage infection with the BioPDO™ process is characterized by sudden cellular lysis, which coincides with a sudden and rapid increase in dissolved oxygen (to 100% dissolved oxygen), a complete loss of oxygen uptake rate, and a complete loss of carbon dioxide evolution rate during the fed-batch fermentation. Optical density (OD) of the fermentation decreases rapidly from an OD550 nm at approximately 11 hours of 42 ± 2 absorbance units to less than 1.4 ± 0.5 in a period of 30–40 minutes. Viable cell counts for the process show that a nearly complete 10-log reduction occurs within this time period. As in-house molecular techniques for bacteriophage detection were not initially developed at the time of facility start-up, these early events were poorly characterized. However, procedures existed to sample and preserve fermentor samples in segregated freezers to support future investigative efforts. Fermentor samples that were collected consisted of a crude mixture of cleared (lysed) *E. coli* cells, nucleic acids, proteins, and cell membrane components. These samples were subsequently determined to contain bacteriophage particles, which were directly observed by transmission electron microscopy (**Figure 2**).

#### *2.1.1 DNA isolation methodology*

Samples were syringe-filtered through a 0.2-μm filter to separate the larger cell debris, and bacteriophage particles were precipitated and concentrated for transmission electron microscopy (**Figure 2**). To improve DNA sequencing efforts,

**95**

*Surveillance and Elimination of Bacteriophage Contamination in an Industrial Fermentation…*

bacteriophage nucleic acids were separated from lysed *E. coli* DNA. The bacteriophage DNA is protected by the proteinaceous capsid head, allowing for DNAse digest of exogenous *E. coli* DNA. Filtered samples were incubated with DNAse I according to the manufacturers recommended protocol overnight, ensuring complete digestion of all DNA present that could interfere with bacteriophage DNA sequencing. After complete digestion, the DNAse was deactivated by heat denaturation. The next step was the removal of the bacteriophage capsid protecting the DNA. This was performed by treatment with proteinase K according to the manufacturer's protocol for 3 hours. The proteinase K treatment removes the capsid protein shell, allowing the bacteriophage DNA to enter solution. Now that the bacterial DNA was fully degraded and the bacteriophage DNA removed from the particle capsid, the final remaining steps were simply precipitating the remaining protein from solution, separation from the aqueous phase by centrifugation, and precipitation of nucleic acids. Protein precipitation was performed by addition of 2 M potassium hydroxide. Immediate flocculent formation was evident, but the samples were stored on ice for a short period to encourage further protein precipitation. Chilled samples were centrifuged in a tabletop centrifuge at 13,000 × *g* for 10 minutes to separate the protein flocculent from the nucleic acid-containing supernatant. Upon separation, nucleic acids were precipitated through a 50% isopropyl alcohol wash. Samples were again stored on ice to allow precipitation to progress more efficiently, followed by centrifugation at 13,000 × *g* for 5 minutes. The visible, cloudy DNA pellet was resuspended

The isolated DNA was sent for 454 pyrosequencing, which revealed an approximately 46,000 base pair circular genome. The genomic characterization of the plasmid was described previously [7]. Sequence analysis of genomes isolated from lytic batches ranging back to the manufacturing facility start-up in 2006 revealed an interesting fact that every lytic bacteriophage event was caused by the same bacteriophage. This bacteriophage shared sequence homology with a T1-like bacteriophage, referred to as RTP bacteriophage [21], but sequence homology of tail fibers and other open reading frames in the genome indicated it was a new representative of this group [7]. To reflect the source of the novel bacteriophage, it was named "*DTL-*phage,*"* for the location of its discovery at the DuPont Tate & Lyle Bio Products fermentation manufacturing site. It is noteworthy that with the significant diversity of coliphages in nature, only one specific type of bacteriophage was

*DOI: http://dx.doi.org/10.5772/intechopen.81151*

*Transmission electron micrograph of bacteriophage DTL.*

in ddH2O and quantified for sequencing.

*2.1.2 Bacteriophage typing*

**Figure 2.**

*Surveillance and Elimination of Bacteriophage Contamination in an Industrial Fermentation… DOI: http://dx.doi.org/10.5772/intechopen.81151*

#### **Figure 2.**

*Bacteriophages - Perspectives and Future*

**Figure 1.**

titer, and yield. Although the BioPDO™ production host contains nearly a complete *E. coli* K12 CRISPR-Cas operon (**Figure 1**), upstream regulatory modifications, which were correlated with improved product production rate resulted in the loss of function for this operon. Vale et al. [19] found that "Cas protein expression is particularly costly, as Cas-deficient mutants achieved higher competitive abilities than the wild-type strain with functional Cas proteins." Smaller spacer libraries of approximately four spacers or less were not associated with fitness costs, suggesting that the genetically engineered spacer library approach of Halter and Zahn may serve to minimize energy and ATP burden on the cell, as indicated by the minimal impact of recombinant

*Alignment of the E. coli K12 CRISPR operon (upper panel) with the native nonfunctional CRISPR-Cas operon* 

**2.1 Isolation and identification of bacteriophage DNA from lytic production** 

Lytic bacteriophage infection with the BioPDO™ process is characterized by sudden cellular lysis, which coincides with a sudden and rapid increase in dissolved oxygen (to 100% dissolved oxygen), a complete loss of oxygen uptake rate, and a complete loss of carbon dioxide evolution rate during the fed-batch fermentation. Optical density (OD) of the fermentation decreases rapidly from an OD550 nm at approximately 11 hours of 42 ± 2 absorbance units to less than 1.4 ± 0.5 in a period of 30–40 minutes. Viable cell counts for the process show that a nearly complete 10-log reduction occurs within this time period. As in-house molecular techniques for bacteriophage detection were not initially developed at the time of facility start-up, these early events were poorly characterized. However, procedures existed to sample and preserve fermentor samples in segregated freezers to support future investigative efforts. Fermentor samples that were collected consisted of a crude mixture of cleared (lysed) *E. coli* cells, nucleic acids, proteins, and cell membrane components. These samples were subsequently determined to contain bacteriophage particles, which were directly observed by transmission electron microscopy

Samples were syringe-filtered through a 0.2-μm filter to separate the larger cell debris, and bacteriophage particles were precipitated and concentrated for transmission electron microscopy (**Figure 2**). To improve DNA sequencing efforts,

seven-spacer system on 1,3-propanediol biosynthesis [7].

**samples in the BioPDO™ process**

*in the BioPDO™ E. coli production host (lower panel).*

**94**

(**Figure 2**).

*2.1.1 DNA isolation methodology*

*Transmission electron micrograph of bacteriophage DTL.*

bacteriophage nucleic acids were separated from lysed *E. coli* DNA. The bacteriophage DNA is protected by the proteinaceous capsid head, allowing for DNAse digest of exogenous *E. coli* DNA. Filtered samples were incubated with DNAse I according to the manufacturers recommended protocol overnight, ensuring complete digestion of all DNA present that could interfere with bacteriophage DNA sequencing. After complete digestion, the DNAse was deactivated by heat denaturation. The next step was the removal of the bacteriophage capsid protecting the DNA. This was performed by treatment with proteinase K according to the manufacturer's protocol for 3 hours. The proteinase K treatment removes the capsid protein shell, allowing the bacteriophage DNA to enter solution. Now that the bacterial DNA was fully degraded and the bacteriophage DNA removed from the particle capsid, the final remaining steps were simply precipitating the remaining protein from solution, separation from the aqueous phase by centrifugation, and precipitation of nucleic acids. Protein precipitation was performed by addition of 2 M potassium hydroxide. Immediate flocculent formation was evident, but the samples were stored on ice for a short period to encourage further protein precipitation. Chilled samples were centrifuged in a tabletop centrifuge at 13,000 × *g* for 10 minutes to separate the protein flocculent from the nucleic acid-containing supernatant. Upon separation, nucleic acids were precipitated through a 50% isopropyl alcohol wash. Samples were again stored on ice to allow precipitation to progress more efficiently, followed by centrifugation at 13,000 × *g* for 5 minutes. The visible, cloudy DNA pellet was resuspended in ddH2O and quantified for sequencing.

#### *2.1.2 Bacteriophage typing*

The isolated DNA was sent for 454 pyrosequencing, which revealed an approximately 46,000 base pair circular genome. The genomic characterization of the plasmid was described previously [7]. Sequence analysis of genomes isolated from lytic batches ranging back to the manufacturing facility start-up in 2006 revealed an interesting fact that every lytic bacteriophage event was caused by the same bacteriophage. This bacteriophage shared sequence homology with a T1-like bacteriophage, referred to as RTP bacteriophage [21], but sequence homology of tail fibers and other open reading frames in the genome indicated it was a new representative of this group [7]. To reflect the source of the novel bacteriophage, it was named "*DTL-*phage,*"* for the location of its discovery at the DuPont Tate & Lyle Bio Products fermentation manufacturing site. It is noteworthy that with the significant diversity of coliphages in nature, only one specific type of bacteriophage was

detected over a period of 12 years. The prevalence of this particular bacteriophage at this facility was likely enhanced by the lack of significant changes in bacteriophage resistance in the *E. coli* host strain, and the massive amount of bacteriophage particles that was released from the site's five—600,000-L production fermentors. With a burst size of approximately 300 new phage particles with each cell lysis, infected fermentors were estimated to release approximately 2 × 1015 viral particles per affected fermentor, which were then distributed to vent and broth deactivation systems where these phage particles were presumed to be deactivated. Subsequent viral load studies showed that large amount (40–65%) of the bacteriophage present in liquid and respiratory off-gas analysis streams remained active. Although a majority of flow in these systems was contained, broth from in-process fermentor sampling efforts, and a majority of excess flow from off-gas analysis streams for respiratory gas analysis was not contained, and contributed to bacteriophagecontaining aerosols, and dried particulate matter, which was disseminated in the production facility.

While we have shown that bacteriophage *DTL* is highly selective for *E. coli* (**Figure 3**), it is capable of lysing several different *E. coli* Group A representatives and related subspecies (**Figure 4**), suggesting that bacteriophage particles could infect related *E. coli* subspecies present in the local manufacturing environment, since these representatives are common human and animal inhabitants [14].

Since the bacteriophage plaque assays provide slow (34–48 hours) feedback for the presence of bacteriophage contamination in process samples, development of a rapid diagnostic test was considered a critical path for effectively mitigating fermentative losses in the facility. To this end, polymerase chain reaction (PCR) assays were developed to detect *DTL*-phage nucleic acids in fermentation broth (**Table 1**). Primer sets were designed based on the genome sequence ([22]; Genbank accession number MG050172), and endpoint reactions performed on both lysed and nonlysed fermentation samples were run on 1% agarose gel, providing a binary visual confirmation of the presence or absence of *DTL*-phage genomic DNA (**Figure 5**).

#### **Figure 3.**

*Plaque assays using DTL-phage on phylogenetically diverse bacteria. From the top panel—left to right: Escherichia coli FM5, E. coli ATCC 8739, Enterobacter aerogenes ATCC 13048: from the lower panel—left to right, Klebsiella pneumoniae ATCC 15574, Pseudomonas aeruginosa ATCC 9027, and Pseudomonas fluorescens ATCC 13525. Background spotting, which is most notable with P. aeruginosa is due to nutrient carry over in the phage preparation.*

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*Surveillance and Elimination of Bacteriophage Contamination in an Industrial Fermentation…*

*Susceptibility of Escherichia coli group A representatives and related E. coli subspecies to DTL-phage. Susceptible = BL21, DH5α, B178, W2244, FM5, and W1485. Nonsusceptible = BW25113 and K802.*

*DTL*-phage 43 qRT F GAAGAGGTGTTTAATTTCGCCG *DTL*-phage 43 qRT R GCCAAACCCGTTAATGTGAAC *DTL*-phage 40 qRT F AGAGGTAGTGGTAGGTTCCG *DTL*-phage 40 qRT R TCAAGAATCGCAGAGTAACCG *DTL*-phage 19 qRT F GCACGCTGGTTAATGGAATG *DTL*-phage 19 qRT R TTCTTGATGGAGATTGTCGGG *DTL*-phage 6 qRT F GGCGTAAAACAGTAATTCAGGTC *DTL*-phage 6 qRT R TTCACACCATCACTACCATCAG *DTL*-phage 66 qRT F GCAGTAAGCCAGAGATTAGCG *DTL*-phage 66 qRT R CTATCCAGTGACCCAACCTTG

After optimization of the assay, early PCR screening of every fermenter was implemented in the manufacturing facility for transfer of seed fermentors. It was soon clear that endpoint PCR, followed by gel-electrophoresis was still too slow to effectively meet the 2-hour transfer window in the manufacturing process, and secondly, the technical tasks and decisions were too complex to effectively implement for the nontechnical manufacturing facility workforce. Real-time quantitative PCR is an assay that provides a more rapid result (<1 hour), and also produces a threshold cycle (Ct) count that can be used as a straightforward quality control parameter to set a simple quantitative threshold for amplicon copy numbers. The use of real-time quantitative PCR to track fermenter contamination provides a more rapid screening procedure and reduces the time from fermentor sampling to a formation of a transfer decision by slightly over 1 hour. Primer sets were optimized to amplify targets within the bacteriophage *DTL* genome with above 95% efficiency, and premade reaction cocktails were also developed to minimize low-volume pipetting steps needed to be performed by nontechnical staff. This procedure had the following steps: (a) A seed fermenter sample would be collected by the fermentation technician and transferred into

*DOI: http://dx.doi.org/10.5772/intechopen.81151*

**Primer Sequence**

*DTL-phage primer sets for PCR diagnostic testing.*

**Figure 4.**

**Table 1.**

*Surveillance and Elimination of Bacteriophage Contamination in an Industrial Fermentation… DOI: http://dx.doi.org/10.5772/intechopen.81151*

#### **Figure 4.**

*Bacteriophages - Perspectives and Future*

production facility.

detected over a period of 12 years. The prevalence of this particular bacteriophage at this facility was likely enhanced by the lack of significant changes in bacteriophage resistance in the *E. coli* host strain, and the massive amount of bacteriophage particles that was released from the site's five—600,000-L production fermentors. With a burst size of approximately 300 new phage particles with each cell lysis, infected fermentors were estimated to release approximately 2 × 1015 viral particles per affected fermentor, which were then distributed to vent and broth deactivation systems where these phage particles were presumed to be deactivated. Subsequent viral load studies showed that large amount (40–65%) of the bacteriophage present in liquid and respiratory off-gas analysis streams remained active. Although a majority of flow in these systems was contained, broth from in-process fermentor sampling efforts, and a majority of excess flow from off-gas analysis streams for respiratory gas analysis was not contained, and contributed to bacteriophagecontaining aerosols, and dried particulate matter, which was disseminated in the

While we have shown that bacteriophage *DTL* is highly selective for *E. coli* (**Figure 3**), it is capable of lysing several different *E. coli* Group A representatives and related subspecies (**Figure 4**), suggesting that bacteriophage particles could infect related *E. coli* subspecies present in the local manufacturing environment, since these representatives are common human and animal inhabitants [14].

Since the bacteriophage plaque assays provide slow (34–48 hours) feedback for the presence of bacteriophage contamination in process samples, development of a rapid diagnostic test was considered a critical path for effectively mitigating fermentative losses in the facility. To this end, polymerase chain reaction (PCR) assays were developed to detect *DTL*-phage nucleic acids in fermentation broth (**Table 1**). Primer sets were designed based on the genome sequence ([22]; Genbank accession number MG050172), and endpoint reactions performed on both lysed and nonlysed fermentation samples were run on 1% agarose gel, providing a binary visual confir-

mation of the presence or absence of *DTL*-phage genomic DNA (**Figure 5**).

*Plaque assays using DTL-phage on phylogenetically diverse bacteria. From the top panel—left to right: Escherichia coli FM5, E. coli ATCC 8739, Enterobacter aerogenes ATCC 13048: from the lower panel—left to right, Klebsiella pneumoniae ATCC 15574, Pseudomonas aeruginosa ATCC 9027, and Pseudomonas fluorescens ATCC 13525. Background spotting, which is most notable with P. aeruginosa is due to nutrient carry over in the* 

**96**

**Figure 3.**

*phage preparation.*

*Susceptibility of Escherichia coli group A representatives and related E. coli subspecies to DTL-phage. Susceptible = BL21, DH5α, B178, W2244, FM5, and W1485. Nonsusceptible = BW25113 and K802.*


#### **Table 1.**

*DTL-phage primer sets for PCR diagnostic testing.*

After optimization of the assay, early PCR screening of every fermenter was implemented in the manufacturing facility for transfer of seed fermentors. It was soon clear that endpoint PCR, followed by gel-electrophoresis was still too slow to effectively meet the 2-hour transfer window in the manufacturing process, and secondly, the technical tasks and decisions were too complex to effectively implement for the nontechnical manufacturing facility workforce. Real-time quantitative PCR is an assay that provides a more rapid result (<1 hour), and also produces a threshold cycle (Ct) count that can be used as a straightforward quality control parameter to set a simple quantitative threshold for amplicon copy numbers. The use of real-time quantitative PCR to track fermenter contamination provides a more rapid screening procedure and reduces the time from fermentor sampling to a formation of a transfer decision by slightly over 1 hour. Primer sets were optimized to amplify targets within the bacteriophage *DTL* genome with above 95% efficiency, and premade reaction cocktails were also developed to minimize low-volume pipetting steps needed to be performed by nontechnical staff. This procedure had the following steps: (a) A seed fermenter sample would be collected by the fermentation technician and transferred into

#### **Figure 5.**

*DTL-phage amplicons generated from PCR (DTL-phage 40 set,* **Table 1***) and separated by 1% agarose gel electrophoresis. Lane 2 is a positive control of purified DTL-phage DNA, lane 3 is a seed fermenter sample containing 2 × 104 particles of DTL-phage per mL, and lane 4 is a sterile water negative control. Lane 1: Invitrogen E-Gel 1 Kb Plus Express DNA Ladder (part #10488091) 100–5000 bp.*

a class 2 biological safety cabinet. (b) The sample would be filtered through a 0.2-μm sterile syringe filter to remove intact cells, and then diluted 1:10 in sterile water. This dilution was performed to dilute inhibitory substances present in the fermentation media, allowing for elimination of the DNA extraction step. (c) Load the processed sample with positive and negative controls into the quantitative PCR instrument, and initiate the analysis. This qPCR assay was the cornerstone in a strategy to screen seed fermentors prior to transfer to the production fermentor as a safeguard to limit bacteriophage-infected seeds from contaminating production fermentors. This new procedure included control charting intensities for Ct trends, which aided in the tracking of bacteriophage contamination levels (**Figure 6**), which increased the operability factor for the

#### **Figure 6.**

*Quantitative PCR (qPCR) amplification curves of seed media screened for DTL-phage. Blue colored curves represent a positive control for presence of DTL-phage (equivalent to 180 plaque forming units per milliliter), green curves represent a negative control, and red curves represent the seed media being screened. The left panel shows a negative result for the seed fermentation sample with no detectable DTL-phage, and the right panel shows a positive response for a seed fermentation sample that contained an internal addition of 80 plaque forming units per milliliter.*

**99**

*Surveillance and Elimination of Bacteriophage Contamination in an Industrial Fermentation…*

fermentation process by ensuring that only bacteriophage-free seed material was

There is a clear incentive for businesses that utilize fermentation-based manufacturing technologies to invest in the development of production strains that are resistant to potential bacteriophage threats. There are at least four target areas that have been described in literature for enhancing bacteriophage resistance in prokaryotic organisms that are sensitive to lytic bacteriophage infections [23]: (a) prevention of phage adsorption, (b) preventing phage entry, (c) cutting bacteriophage nucleic acids, and (d) abortive infection systems. The following analysis will summarize efforts in two of the four areas and prevention of phage adsorption

Classical strain improvement programs for the generation of bacteriophage resistance often start with acquiring genotypic diversity in a host population through spontaneous or induced mutation, followed by challenge and selection of survivors to bacteriophage infection. Several studies utilizing this strategy have reported resistance through single nucleotide polymorphisms (SNPs) or insertional mutagenesis, which often alters the structure of bacteriophage binding sites on the cell surface. Additionally, significant deletions of chromosomal DNA have been described that involve structural changes of one or more cell surface

The initial step in the bacteriophage virulence cycle is the adsorption of the bacteriophage particle to the outer surface of the bacterial cell [23]. Once bound, the bacteriophage infects the cell by transferring genetic material into the cell, where it then utilizes host cellular translation systems to generate additional bacteriophage particles. This binding of the bacteriophage to the outer membrane is typically mediated by a highly specific receptor on the outside of the cell, is typically a membrane protein, a specific class of lipid, or a carbohydrate moiety, and is extremely specific to a bacteriophage and its host. Common genetic techniques can, therefore, be used to target these binding sites to reduce binding affinity or

Over several years, classical strain improvement programs for the BioPDO™ process have generated and screened strains that were selected through challenge for resistance to *DTL*-phage [7]. These screens included strains that demonstrated a 3–5-log improvement in the reduced sensitivity as measured by the standard bacteriophage plaque assay (**Figure 7**). In all cases, bacteriophage resistance was associated with a 63–96% reduction in 1,3-propanediol production titer. Further analysis showed that these strains had a lower solvent tolerance, which indicated that the bacteriophage resistance mechanism(s) were presumably linked to changes in cellular ultrastructure. Genomic sequencing efforts confirmed that SNPs occurred in a series of genes involved in synthesis and glycosylation of lipopolysaccharide, including glycosylation with heptose residues [27]. Heptose residues appear to be essential for high-affinity binding

The second strategy, cutting bacteriophage nucleic acids is a rapidly evolving field that has been heavily influenced by recent discoveries in bacterial-acquired immunity [12]. Clustered regularly interspaced short palindromic repeats (CRISPR) are a molecular system by which prokaryotes obtain acquired resistance to bacteriophages. The CRISPR operon is widely distributed in prokaryotes and represents the most abundant form of innate immunity in these organisms [29]. Upon injection of the genetic material, the cell recognizes foreign DNA, and the first gene involved

**2.2 Efforts to produce a bacteriophage-resistant production strain**

and cutting phage nucleic acids with a heterologous CRISPR-Cas system.

*DOI: http://dx.doi.org/10.5772/intechopen.81151*

transferred into production fermentors.

biomolecules [14, 24–26].

eliminate the site [24, 26].

of *DTL*-phage to the *E. coli* cell [28].

fermentation process by ensuring that only bacteriophage-free seed material was transferred into production fermentors.

#### **2.2 Efforts to produce a bacteriophage-resistant production strain**

There is a clear incentive for businesses that utilize fermentation-based manufacturing technologies to invest in the development of production strains that are resistant to potential bacteriophage threats. There are at least four target areas that have been described in literature for enhancing bacteriophage resistance in prokaryotic organisms that are sensitive to lytic bacteriophage infections [23]: (a) prevention of phage adsorption, (b) preventing phage entry, (c) cutting bacteriophage nucleic acids, and (d) abortive infection systems. The following analysis will summarize efforts in two of the four areas and prevention of phage adsorption and cutting phage nucleic acids with a heterologous CRISPR-Cas system.

Classical strain improvement programs for the generation of bacteriophage resistance often start with acquiring genotypic diversity in a host population through spontaneous or induced mutation, followed by challenge and selection of survivors to bacteriophage infection. Several studies utilizing this strategy have reported resistance through single nucleotide polymorphisms (SNPs) or insertional mutagenesis, which often alters the structure of bacteriophage binding sites on the cell surface. Additionally, significant deletions of chromosomal DNA have been described that involve structural changes of one or more cell surface biomolecules [14, 24–26].

The initial step in the bacteriophage virulence cycle is the adsorption of the bacteriophage particle to the outer surface of the bacterial cell [23]. Once bound, the bacteriophage infects the cell by transferring genetic material into the cell, where it then utilizes host cellular translation systems to generate additional bacteriophage particles. This binding of the bacteriophage to the outer membrane is typically mediated by a highly specific receptor on the outside of the cell, is typically a membrane protein, a specific class of lipid, or a carbohydrate moiety, and is extremely specific to a bacteriophage and its host. Common genetic techniques can, therefore, be used to target these binding sites to reduce binding affinity or eliminate the site [24, 26].

Over several years, classical strain improvement programs for the BioPDO™ process have generated and screened strains that were selected through challenge for resistance to *DTL*-phage [7]. These screens included strains that demonstrated a 3–5-log improvement in the reduced sensitivity as measured by the standard bacteriophage plaque assay (**Figure 7**). In all cases, bacteriophage resistance was associated with a 63–96% reduction in 1,3-propanediol production titer. Further analysis showed that these strains had a lower solvent tolerance, which indicated that the bacteriophage resistance mechanism(s) were presumably linked to changes in cellular ultrastructure. Genomic sequencing efforts confirmed that SNPs occurred in a series of genes involved in synthesis and glycosylation of lipopolysaccharide, including glycosylation with heptose residues [27]. Heptose residues appear to be essential for high-affinity binding of *DTL*-phage to the *E. coli* cell [28].

The second strategy, cutting bacteriophage nucleic acids is a rapidly evolving field that has been heavily influenced by recent discoveries in bacterial-acquired immunity [12]. Clustered regularly interspaced short palindromic repeats (CRISPR) are a molecular system by which prokaryotes obtain acquired resistance to bacteriophages. The CRISPR operon is widely distributed in prokaryotes and represents the most abundant form of innate immunity in these organisms [29]. Upon injection of the genetic material, the cell recognizes foreign DNA, and the first gene involved

*Bacteriophages - Perspectives and Future*

a class 2 biological safety cabinet. (b) The sample would be filtered through a 0.2-μm sterile syringe filter to remove intact cells, and then diluted 1:10 in sterile water. This dilution was performed to dilute inhibitory substances present in the fermentation media, allowing for elimination of the DNA extraction step. (c) Load the processed sample with positive and negative controls into the quantitative PCR instrument, and initiate the analysis. This qPCR assay was the cornerstone in a strategy to screen seed fermentors prior to transfer to the production fermentor as a safeguard to limit bacteriophage-infected seeds from contaminating production fermentors. This new procedure included control charting intensities for Ct trends, which aided in the tracking of bacteriophage contamination levels (**Figure 6**), which increased the operability factor for the

*Quantitative PCR (qPCR) amplification curves of seed media screened for DTL-phage. Blue colored curves represent a positive control for presence of DTL-phage (equivalent to 180 plaque forming units per milliliter), green curves represent a negative control, and red curves represent the seed media being screened. The left panel shows a negative result for the seed fermentation sample with no detectable DTL-phage, and the right panel shows a positive response for a seed fermentation sample that contained an internal addition of 80 plaque* 

*Invitrogen E-Gel 1 Kb Plus Express DNA Ladder (part #10488091) 100–5000 bp.*

*DTL-phage amplicons generated from PCR (DTL-phage 40 set,* **Table 1***) and separated by 1% agarose gel electrophoresis. Lane 2 is a positive control of purified DTL-phage DNA, lane 3 is a seed fermenter sample* 

 *particles of DTL-phage per mL, and lane 4 is a sterile water negative control. Lane 1:* 

**98**

**Figure 6.**

*forming units per milliliter.*

**Figure 5.**

*containing 2 × 104*

#### **Figure 7.**

*Bacteriophage plaque assay plate showing the presence of E. coli colonies growing in the zone of clearing that are resistant to DTL-phage challenge.*

in the CRISPR pathway creates an approximately 20–25 base pair cut (this varies between species and CRISPR subtypes). This snippet of DNA is then integrated into a library of recognized bacteriophage genetic elements, flanked on each side by a palindromic repeat. This genetic element can then be transcribed, producing a single-stranded transcript that is homologous to the DNA of the intruding bacteriophage. The palindromic repeats flanking the element serve to bind the short fragment of transcript to a nuclease, which is then guided by the homologous element to potential binding sites on the intruding segment of DNA, where a single cut is made rendering it nonfunctional.

Since it was established earlier that the BioPDO™ *E. coli* production host did not carry a functional CRISPR operon in its genome, and the specific binding site of the *DTL*-phage was originally unknown, work was initiated with CRISPR to generate a spacer library specific to *DTL*-phage infection. The well-characterized CRISPR cassette of *Streptococcus thermophilus* was amplified, and its 12 bacteriophage spacers (remnants of previously acquired resistance in this species) were replaced with seven spacers that were homologous to different regions of the *DTL-*phage genome. These seven spacers were specifically chosen to deliver CRISPR-nuclease-delivered cuts in the middle of important open reading frames within the bacteriophage genome, thereby ensuring overwhelming and targeted knockout of its genetic functionality.

Upon incorporation of this tailored cassette to the BioPDO™ *E. coli* production strain, plaque assays and phage challenges indicated the organism had obtained full resistance to this bacteriophage, as indicated by the lack of plaques formed in experiments using phage titers as high as 106 phage particles per milliliter. The utilization of multiple homologous spacers was an important aspect of this project to reduce the potential for resistance. Because CRISPR resistance depends on homologous binding of spacers bound to the CRISPR nuclease to the intruding phage DNA, a single SNP acquired within the homologous stretch of DNA in the phage genome could disrupt the DNA-nuclease interaction, rendering that spacer no longer active. The use of seven targeted spacers minimizes the potential for resistance, and no significant energy or ATP burden related to the lack of fitness hypothesis, proposed by Vale et al., was observed [19]. Although Vale et al., proposed a threshold number of four spacers as the limit regarding negative impacts on cell fitness, the slight increase to a seven-spacer library appeared to have no negative consequences on 1,3-propanediol synthesis rate [7].

**101**

*Surveillance and Elimination of Bacteriophage Contamination in an Industrial Fermentation…*

**2.3 Reservoirs and process entry points for bacteriophage in a manufacturing** 

Host resistance to bacteriophage infection is only a partial solution to the prevention of bacteriophage in fermentation processes. This approach must be balanced with effective facility sanitation procedures, active bacteriophage surveillance programs that are coupled with reservoir elimination efforts, and finally, the validation, maintenance, and monitoring of aseptic barriers utilized in the process

As mentioned previously, bacteriophage-infected production fermentors for the BioPDO™ process were estimated to release approximately 2 × 1015 viral particles per affected fermentor based on a 300-burst size model. Bacteriophagecontaminated air and liquid in fermentors was transferred to vent and broth deactivation systems where the viral particles were presumed to be deactivated. Earlier pilot plant studies of microbial and biological materials deactivated by a high-temperature short-time sterilizer (HTST) did not consider bacteriophage inactivation because (a) bacteriophage was considered an addressable nuisance in the operation of the facility, (b) bacteriophage libraries specific to the BioPDO™ production microorganism had not been assembled, and (c) risks to a commercial process remained largely speculative. Furthermore, the closest-related bacteriophage, *RTP*-phage had not been isolated and described until after the plant start-up in 2006. Subsequent heat-inactivation studies on phage-contaminated batches after facility start-up were successful in showing that bacteriophage particles were not completely deactivated in the HTST broth deactivation system, mainly due to

Other weaknesses in containment systems were gas and liquid sampling systems,

The ecological aspects of DTL bacteriophage are poorly characterized due to the lack of historical environmental samples from the fermentation facility during the period of the initial infection. We are in agreement with the proposal of Tanji et al. [14] that the most likely source of phage contamination for an *E. coli* culturing process is human, and the initial source of DTL-phage in the BioPDO™ facility, was most likely also human. The fact that DTL phage is now prevalent in the plant environment is thought to be mainly a result of a series of lytic events in production fermentors that established localized concentrations of the bacteriophage in reservoirs that are inhabited by natural populations of *E. coli*, or closely related

One of the most significant failure points in the commercial fermentation facility process for bacteriophage entry was through sterile filtration barriers. The small size of DTL-phage (capsid diameter = 0.075 μm) supports passage of the virus through liquid-service filtration systems (0.2–0.45 μm cutoff size), and conditional passage through gas-phase filtration systems (0.2 μm cutoff size) when entrained liquid is present. Aerial transfer of bacteriophage particles is well-established as a route for contamination of industrial fermentation processes [29, 30]; therefore,

which were commonly used in the fermentation industry for respiratory gas analysis and control through feedback loops, and in-process analytical for measuring broth parameters associated with growth and product production, respectively. Once free of containment systems, the bacteriophage particles were unintentionally disseminated by human activity and other environmental mechanisms. This disseminated material served to challenge sterile barriers that were in place for air filtration, filters used for sterile filtration of liquids used in the process, and cross contamination of microbiology process laboratories, which impacted the seed train,

*DOI: http://dx.doi.org/10.5772/intechopen.81151*

**setting**

for bacteriophage exclusion.

resistance to heat inactivation [30, 31].

and laboratory diagnostic tests.

microorganisms that are susceptible to this bacteriophage.

*Surveillance and Elimination of Bacteriophage Contamination in an Industrial Fermentation… DOI: http://dx.doi.org/10.5772/intechopen.81151*

#### **2.3 Reservoirs and process entry points for bacteriophage in a manufacturing setting**

Host resistance to bacteriophage infection is only a partial solution to the prevention of bacteriophage in fermentation processes. This approach must be balanced with effective facility sanitation procedures, active bacteriophage surveillance programs that are coupled with reservoir elimination efforts, and finally, the validation, maintenance, and monitoring of aseptic barriers utilized in the process for bacteriophage exclusion.

As mentioned previously, bacteriophage-infected production fermentors for the BioPDO™ process were estimated to release approximately 2 × 1015 viral particles per affected fermentor based on a 300-burst size model. Bacteriophagecontaminated air and liquid in fermentors was transferred to vent and broth deactivation systems where the viral particles were presumed to be deactivated. Earlier pilot plant studies of microbial and biological materials deactivated by a high-temperature short-time sterilizer (HTST) did not consider bacteriophage inactivation because (a) bacteriophage was considered an addressable nuisance in the operation of the facility, (b) bacteriophage libraries specific to the BioPDO™ production microorganism had not been assembled, and (c) risks to a commercial process remained largely speculative. Furthermore, the closest-related bacteriophage, *RTP*-phage had not been isolated and described until after the plant start-up in 2006. Subsequent heat-inactivation studies on phage-contaminated batches after facility start-up were successful in showing that bacteriophage particles were not completely deactivated in the HTST broth deactivation system, mainly due to resistance to heat inactivation [30, 31].

Other weaknesses in containment systems were gas and liquid sampling systems, which were commonly used in the fermentation industry for respiratory gas analysis and control through feedback loops, and in-process analytical for measuring broth parameters associated with growth and product production, respectively. Once free of containment systems, the bacteriophage particles were unintentionally disseminated by human activity and other environmental mechanisms. This disseminated material served to challenge sterile barriers that were in place for air filtration, filters used for sterile filtration of liquids used in the process, and cross contamination of microbiology process laboratories, which impacted the seed train, and laboratory diagnostic tests.

The ecological aspects of DTL bacteriophage are poorly characterized due to the lack of historical environmental samples from the fermentation facility during the period of the initial infection. We are in agreement with the proposal of Tanji et al. [14] that the most likely source of phage contamination for an *E. coli* culturing process is human, and the initial source of DTL-phage in the BioPDO™ facility, was most likely also human. The fact that DTL phage is now prevalent in the plant environment is thought to be mainly a result of a series of lytic events in production fermentors that established localized concentrations of the bacteriophage in reservoirs that are inhabited by natural populations of *E. coli*, or closely related microorganisms that are susceptible to this bacteriophage.

One of the most significant failure points in the commercial fermentation facility process for bacteriophage entry was through sterile filtration barriers. The small size of DTL-phage (capsid diameter = 0.075 μm) supports passage of the virus through liquid-service filtration systems (0.2–0.45 μm cutoff size), and conditional passage through gas-phase filtration systems (0.2 μm cutoff size) when entrained liquid is present. Aerial transfer of bacteriophage particles is well-established as a route for contamination of industrial fermentation processes [29, 30]; therefore,

*Bacteriophages - Perspectives and Future*

rendering it nonfunctional.

*are resistant to DTL-phage challenge.*

**Figure 7.**

ments using phage titers as high as 106

in the CRISPR pathway creates an approximately 20–25 base pair cut (this varies between species and CRISPR subtypes). This snippet of DNA is then integrated into a library of recognized bacteriophage genetic elements, flanked on each side by a palindromic repeat. This genetic element can then be transcribed, producing a single-stranded transcript that is homologous to the DNA of the intruding bacteriophage. The palindromic repeats flanking the element serve to bind the short fragment of transcript to a nuclease, which is then guided by the homologous element to potential binding sites on the intruding segment of DNA, where a single cut is made

*Bacteriophage plaque assay plate showing the presence of E. coli colonies growing in the zone of clearing that* 

Since it was established earlier that the BioPDO™ *E. coli* production host did not carry a functional CRISPR operon in its genome, and the specific binding site of the *DTL*-phage was originally unknown, work was initiated with CRISPR to generate a spacer library specific to *DTL*-phage infection. The well-characterized CRISPR cassette of *Streptococcus thermophilus* was amplified, and its 12 bacteriophage spacers (remnants of previously acquired resistance in this species) were replaced with seven spacers that were homologous to different regions of the *DTL-*phage genome. These seven spacers were specifically chosen to deliver CRISPR-nuclease-delivered cuts in the middle of important open reading frames within the bacteriophage genome, thereby ensuring overwhelming and targeted knockout of its genetic functionality. Upon incorporation of this tailored cassette to the BioPDO™ *E. coli* production strain, plaque assays and phage challenges indicated the organism had obtained full resistance to this bacteriophage, as indicated by the lack of plaques formed in experi-

of multiple homologous spacers was an important aspect of this project to reduce the potential for resistance. Because CRISPR resistance depends on homologous binding of spacers bound to the CRISPR nuclease to the intruding phage DNA, a single SNP acquired within the homologous stretch of DNA in the phage genome could disrupt the DNA-nuclease interaction, rendering that spacer no longer active. The use of seven targeted spacers minimizes the potential for resistance, and no significant energy or ATP burden related to the lack of fitness hypothesis, proposed by Vale et al., was observed [19]. Although Vale et al., proposed a threshold number of four spacers as the limit regarding negative impacts on cell fitness, the slight increase to a seven-spacer library appeared to have no negative consequences on 1,3-propanediol synthesis rate [7].

phage particles per milliliter. The utilization

**100**

air filtration systems play a large role in protecting the fermentation process from airborne bacteriophage particles.

Our empirical studies on the Donaldson sterile air filter, model P-SRF N 30/30 (0.2 μm absolute, expanded PTFE filter membrane) indicated that the filter was 100% effective in rejection of DTL-phage particles if the filter remained above the system pressure dew point temperature of 102.5°F (**Figure 8**). Since the design temperature of air present in the header was 122, the 102.5°F pressure dew point was considered to have sufficient safety factor to prevent formation of condensate. Wetting of the filter was correlated strongly with lytic infections by DTL-phage [7]. Wetting of the sterile filter due to entrained water in the process air header was infrequently observed when: (a) low ambient temperatures cooled areas of the process air header that were not adequately protected with heat tape and/or insulation (**Figure 9**), (b) cooling tower water or chilled water from the intercooler and/or aftercooler heat exchangers on the air compressor leaked into the air stream and traveled through the air header to prefilters and sterile filters, (c) low-point zones in the process header not adequately drained with autopurging condensate traps, and (d) high-temperature excursions of the chilled water system, which was used to condense water in the compressed air product stream. A critical factor for surveillance and prevention of bacteriophage contamination was the integration of dew point sensors at upstream locations from the sterile filter, and more frequent sterilizations of the filters in the sterile filter housing. Since sterile filters have a limitation of approximately 170 steam cycles, more frequent sterilization served to increase the number of filter replacements and overall process costs.

Bubble column bioreactors used in aerobic fermentation processes require large volumes of air to meet the oxygen uptake requirements for *E. coli* cells in the fermentation, as well as a means for mixing to drive heat transfer, gas solubility, CO2 ventilation or removal, and mixing of substrates, and other feeds. Due to the expense of water vapor removal in very large volumes of air, many fermentation systems utilize ANSI/ISA-7.0.01-1996 air quality class 6 or lower specifications, which establish a dew point of 50°F at atmosphere pressure. This type of air compressor system, which contains chilled fluid after-coolers, passes the compressed air through a cooled heat exchanger, causing water vapor to condense out of the air stream, and typically produces air with a dew point not lower than 41°F (5°C). The pressure dew point refers to the dew point temperature of a gas under pressure, which in this case is increased to a value of 102.5°F at a pressure of approximately 84.7 psia (**Figure 9**). The main issues with this system are (a) the potential for

#### **Figure 8.**

*Sterile process of destructive disassembly a Donaldson sterile air filter, model P-SRF N 30/30 (0.2 μm absolute, expanded PTFE filter membrane) for bacteriophage diagnostic testing. Note the dark water staining on protective prefilter barrier layer on the filter.*

**103**

0.2-μm air filter.

**Figure 9.**

*Surveillance and Elimination of Bacteriophage Contamination in an Industrial Fermentation…*

heat exchanger leakage into the air stream and (b) temperature excursions of the cooling fluid (chilled water), due to its use in other areas of the manufacturing facility. Temperature excursions for chilled water were found to reduce the ability of the after-cooler to remove water vapor in air supplied to the fermentors, which increased the potential for condensate to form in the process air header. This condensate was found to cause passage of bacteriophage particles through the sterile,

*Diagram of the process air system that provided a sterile-filtered air to a production fermentor.*

Deviations from aseptic design principles are nearly always a root cause for introduction of foreign bacterial growth and bacteriophage into axenic fermentation systems. A deviation in these principles occurred as part of the risk mitigation effort to prevent condensate formation in the process air header. As part of this project, sterile air bypass lines were installed on the air header line to eliminate zero air flow conditions that occurred during the sterilization cycle for fermentors. During fermentor sterilization, sterile air flow was stopped, and a 45 psig steam feed line between the sterile filter housing and fermentor was opened. During the 90-minute sterilization cycle, the temperature of the air header was found to drop well below the pressure dew point, especially during cold weather months, which introduced condensate upstream of the sterile air filters. Introduction of a process steam line upstream of the sterile filter housing, as is done more commonly in the industry, was not considered a best practice because the sterile air filter would remain wetted for a short period after the steam sterilization process was completed, and nonsterile air flow was reinitiated as a feed stream to the filter housing. The wetted filter was believed to promote passage of bacteriophage particles into the fermentor. One solution, practiced at a separate facility, was to have a secondary low-flow sterile air header upstream of the main sterile filter housing that provided sterile dry air to the housing after the steam sterilization to dry the wet sterile filters. As these former solutions required significant down time to perform repiping, two other solutions were prioritized: (1) ensure that heat tracing and insulation of the air header was sufficient to maintain temperature above the pressure dew point and (2) prevent the low or no-air-flow condition by adding a small diameter purge line directly after the sterile air filter housing to provide constant flow in the air header. Due to the potential for ammonia gas in the bypass air stream, piping effort focused on connection of this line to the fermentor exhaust header, which contained an ammonia scrubber system. The new piping system did not contain proper aseptic barriers and steam-purged interlocks, which then permitted backflow of

*DOI: http://dx.doi.org/10.5772/intechopen.81151*

*Surveillance and Elimination of Bacteriophage Contamination in an Industrial Fermentation… DOI: http://dx.doi.org/10.5772/intechopen.81151*

**Figure 9.** *Diagram of the process air system that provided a sterile-filtered air to a production fermentor.*

heat exchanger leakage into the air stream and (b) temperature excursions of the cooling fluid (chilled water), due to its use in other areas of the manufacturing facility. Temperature excursions for chilled water were found to reduce the ability of the after-cooler to remove water vapor in air supplied to the fermentors, which increased the potential for condensate to form in the process air header. This condensate was found to cause passage of bacteriophage particles through the sterile, 0.2-μm air filter.

Deviations from aseptic design principles are nearly always a root cause for introduction of foreign bacterial growth and bacteriophage into axenic fermentation systems. A deviation in these principles occurred as part of the risk mitigation effort to prevent condensate formation in the process air header. As part of this project, sterile air bypass lines were installed on the air header line to eliminate zero air flow conditions that occurred during the sterilization cycle for fermentors. During fermentor sterilization, sterile air flow was stopped, and a 45 psig steam feed line between the sterile filter housing and fermentor was opened. During the 90-minute sterilization cycle, the temperature of the air header was found to drop well below the pressure dew point, especially during cold weather months, which introduced condensate upstream of the sterile air filters. Introduction of a process steam line upstream of the sterile filter housing, as is done more commonly in the industry, was not considered a best practice because the sterile air filter would remain wetted for a short period after the steam sterilization process was completed, and nonsterile air flow was reinitiated as a feed stream to the filter housing. The wetted filter was believed to promote passage of bacteriophage particles into the fermentor. One solution, practiced at a separate facility, was to have a secondary low-flow sterile air header upstream of the main sterile filter housing that provided sterile dry air to the housing after the steam sterilization to dry the wet sterile filters. As these former solutions required significant down time to perform repiping, two other solutions were prioritized: (1) ensure that heat tracing and insulation of the air header was sufficient to maintain temperature above the pressure dew point and (2) prevent the low or no-air-flow condition by adding a small diameter purge line directly after the sterile air filter housing to provide constant flow in the air header. Due to the potential for ammonia gas in the bypass air stream, piping effort focused on connection of this line to the fermentor exhaust header, which contained an ammonia scrubber system. The new piping system did not contain proper aseptic barriers and steam-purged interlocks, which then permitted backflow of

*Bacteriophages - Perspectives and Future*

airborne bacteriophage particles.

air filtration systems play a large role in protecting the fermentation process from

increase the number of filter replacements and overall process costs.

Bubble column bioreactors used in aerobic fermentation processes require large volumes of air to meet the oxygen uptake requirements for *E. coli* cells in the fermentation, as well as a means for mixing to drive heat transfer, gas solubility, CO2 ventilation or removal, and mixing of substrates, and other feeds. Due to the expense of water vapor removal in very large volumes of air, many fermentation systems utilize ANSI/ISA-7.0.01-1996 air quality class 6 or lower specifications, which establish a dew point of 50°F at atmosphere pressure. This type of air compressor system, which contains chilled fluid after-coolers, passes the compressed air through a cooled heat exchanger, causing water vapor to condense out of the air stream, and typically produces air with a dew point not lower than 41°F (5°C). The pressure dew point refers to the dew point temperature of a gas under pressure, which in this case is increased to a value of 102.5°F at a pressure of approximately 84.7 psia (**Figure 9**). The main issues with this system are (a) the potential for

*Sterile process of destructive disassembly a Donaldson sterile air filter, model P-SRF N 30/30 (0.2 μm absolute, expanded PTFE filter membrane) for bacteriophage diagnostic testing. Note the dark water staining on* 

Our empirical studies on the Donaldson sterile air filter, model P-SRF N 30/30 (0.2 μm absolute, expanded PTFE filter membrane) indicated that the filter was 100% effective in rejection of DTL-phage particles if the filter remained above the system pressure dew point temperature of 102.5°F (**Figure 8**). Since the design temperature of air present in the header was 122, the 102.5°F pressure dew point was considered to have sufficient safety factor to prevent formation of condensate. Wetting of the filter was correlated strongly with lytic infections by DTL-phage [7]. Wetting of the sterile filter due to entrained water in the process air header was infrequently observed when: (a) low ambient temperatures cooled areas of the process air header that were not adequately protected with heat tape and/or insulation (**Figure 9**), (b) cooling tower water or chilled water from the intercooler and/or aftercooler heat exchangers on the air compressor leaked into the air stream and traveled through the air header to prefilters and sterile filters, (c) low-point zones in the process header not adequately drained with autopurging condensate traps, and (d) high-temperature excursions of the chilled water system, which was used to condense water in the compressed air product stream. A critical factor for surveillance and prevention of bacteriophage contamination was the integration of dew point sensors at upstream locations from the sterile filter, and more frequent sterilizations of the filters in the sterile filter housing. Since sterile filters have a limitation of approximately 170 steam cycles, more frequent sterilization served to

**102**

**Figure 8.**

*protective prefilter barrier layer on the filter.*

contaminated waste gas into the fermentor air header (poststerile filter) under infrequent vent header over-pressure events.

One of the more surprising discoveries in this work was the level of bacteriophage contamination (up to 7 × 103 plaque forming units per square centimeter) in the process air header, as well as the surface of sterile air filters between sterilization cycles. This result indicated that the extremely short, but elevated air temperature of 260°F between the air compressor and after-cooler was insufficient in deactivating *DTL*-phage (**Figure 9**). Based on this finding, air header sanitation procedures were developed to infrequently steam sterilize the air header for the location between the preair filter housing and the air compressors. While this practice reduced phage burden in the air header, it could not be operated in a continuous mode, and therefore, the sterile air filters served as the only barrier to bacteriophage entry. Additional design improvements related to a secondary barrier for airborne phage entry include: (a) a heat-sterilization system on the product side of the air compressor to elevate the temperature to 300°F, and increase temperature hold time by 10-fold to 1 second, and (b) filtration/precipitation systems for pretreatment of supply air to the air compressors.

Generation of bacteriophage aerosols in the BioPDO™ manufacturing facility is largely driven by failure points in containment systems, including the industrystandard water-web hypochlorous acid scrubber system for fermentor vent stream treatment, as well as the use of open liquid transfer systems, such as trench drains that are used for streams originating from steam interlocks on sample systems, drains from sample sinks, drains from vapor–liquid separators, and unintended leaks from heat exchangers and piping. Efforts to ensure that these streams are transferred by closed piping systems to chemical or steam deactivation systems is critical for achieving low airborne concentrations of bacteriophage in the manufacturing environment. Frequent cleaning of these areas with disinfectants has been shown to be critical for limiting dispersion in the manufacturing facility environment [32].

#### **3. Conclusions**

As the most abundant biological entity on the planet, bacteriophages play an important ecological role in ecosystems and have also been exploited for the development of many modern technologies, including gene transfer and treatment of bacterial infections [7, 14, 21, 23]. The presence of bacteriophage in an industrial fermentation facility can be a serious problem, resulting in reduced product quality, loss in production facility operability, and financial losses to a business. In this chapter, we have described basic and applied research efforts around our goal of reducing the impact of bacteriophage-related losses in a commercial process for the manufacture of 1,3-propanediol. The key to success in these efforts was the development of rapid diagnostic methods that were subsequently leveraged by a diverse team to quickly diagnose and eliminate sources of bacteriophage in a fast-paced manufacturing environment.

Many of the solutions related to operating a manufacturing facility bacteriophage-free require participation of a cross-disciplinary team that encompass many areas of expertise, including virology, microbiology, microbial ecology, chemical, and mechanical engineering. As with any new biomanufacturing process, designers utilize a basic set of assumptions that serve in the design and construction of the facility. Some of these assumptions are not fully tested in a pilot plant, and the issues that arise are often seen for the first time in the commercial fermentation facility. In this chapter, we have characterized issues related to the use of 0.2 μm (absolute)

**105**

**Author details**

provided the original work is properly cited.

James A. Zahn\* and Mathew C. Halter

DuPont Tate & Lyle Bio Products, Loudon, TN, USA

\*Address all correspondence to: james.a.zahn@dupont.com

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Surveillance and Elimination of Bacteriophage Contamination in an Industrial Fermentation…*

sterile filters as the only mechanism for exclusion of bacteriophage from the fermentation process. This issue is especially problematic since these filters were not specifically designed for removal of irregular bacteriophage particles with diameters

The authors declare that there are no conflict of interests with this work.

Our studies indicate that CRISPR/Cas9-mediated resistance to bacteriophage DTL in the *E. coli* PDO production strain is a highly effective strategy for eliminating bacteriophage virulence. The CRISPR spacer customization strategy further ensures that spacers are not generated against foreign DNA that that is inserted into the production host as part of future host improvement (transformation) projects. Furthermore, the metabolic burden hypothesis proposed by Vale et al. [19] is avoided since we have limited the spacer library to a total of seven spacers. The main disadvantage of this approach is that this recombinant CRISPR system has a narrow range of bacteriophage specificity, and that there is a significant selection pressure for sequence modifications that are not recognized by this existing spacer library. In this regard, there is a difficult balance between spacer library size as an assurance of bacteriophage resistance, and acceptance of lower 1,3-propanediol productivity due to the metabolic burden

(0.075 μm), which is significantly smaller than the stated filter cutoff size.

*DOI: http://dx.doi.org/10.5772/intechopen.81151*

due to the increase in spacer library size.

**Conflict of interest**

*Surveillance and Elimination of Bacteriophage Contamination in an Industrial Fermentation… DOI: http://dx.doi.org/10.5772/intechopen.81151*

sterile filters as the only mechanism for exclusion of bacteriophage from the fermentation process. This issue is especially problematic since these filters were not specifically designed for removal of irregular bacteriophage particles with diameters (0.075 μm), which is significantly smaller than the stated filter cutoff size.

Our studies indicate that CRISPR/Cas9-mediated resistance to bacteriophage DTL in the *E. coli* PDO production strain is a highly effective strategy for eliminating bacteriophage virulence. The CRISPR spacer customization strategy further ensures that spacers are not generated against foreign DNA that that is inserted into the production host as part of future host improvement (transformation) projects. Furthermore, the metabolic burden hypothesis proposed by Vale et al. [19] is avoided since we have limited the spacer library to a total of seven spacers. The main disadvantage of this approach is that this recombinant CRISPR system has a narrow range of bacteriophage specificity, and that there is a significant selection pressure for sequence modifications that are not recognized by this existing spacer library. In this regard, there is a difficult balance between spacer library size as an assurance of bacteriophage resistance, and acceptance of lower 1,3-propanediol productivity due to the metabolic burden due to the increase in spacer library size.

#### **Conflict of interest**

*Bacteriophages - Perspectives and Future*

phage contamination (up to 7 × 103

supply air to the air compressors.

environment [32].

**3. Conclusions**

manufacturing environment.

infrequent vent header over-pressure events.

contaminated waste gas into the fermentor air header (poststerile filter) under

One of the more surprising discoveries in this work was the level of bacterio-

the process air header, as well as the surface of sterile air filters between sterilization cycles. This result indicated that the extremely short, but elevated air temperature of 260°F between the air compressor and after-cooler was insufficient in deactivating *DTL*-phage (**Figure 9**). Based on this finding, air header sanitation procedures were developed to infrequently steam sterilize the air header for the location between the preair filter housing and the air compressors. While this practice reduced phage burden in the air header, it could not be operated in a continuous mode, and therefore, the sterile air filters served as the only barrier to bacteriophage entry. Additional design improvements related to a secondary barrier for airborne phage entry include: (a) a heat-sterilization system on the product side of the air compressor to elevate the temperature to 300°F, and increase temperature hold time by 10-fold to 1 second, and (b) filtration/precipitation systems for pretreatment of

Generation of bacteriophage aerosols in the BioPDO™ manufacturing facility is largely driven by failure points in containment systems, including the industrystandard water-web hypochlorous acid scrubber system for fermentor vent stream treatment, as well as the use of open liquid transfer systems, such as trench drains that are used for streams originating from steam interlocks on sample systems, drains from sample sinks, drains from vapor–liquid separators, and unintended leaks from heat exchangers and piping. Efforts to ensure that these streams are transferred by closed piping systems to chemical or steam deactivation systems is critical for achieving low airborne concentrations of bacteriophage in the manufacturing environment. Frequent cleaning of these areas with disinfectants has been shown to be critical for limiting dispersion in the manufacturing facility

As the most abundant biological entity on the planet, bacteriophages play an important ecological role in ecosystems and have also been exploited for the development of many modern technologies, including gene transfer and treatment of bacterial infections [7, 14, 21, 23]. The presence of bacteriophage in an industrial fermentation facility can be a serious problem, resulting in reduced product quality, loss in production facility operability, and financial losses to a business. In this chapter, we have described basic and applied research efforts around our goal of reducing the impact of bacteriophage-related losses in a commercial process for the manufacture of 1,3-propanediol. The key to success in these efforts was the development of rapid diagnostic methods that were subsequently leveraged by a diverse team to quickly diagnose and eliminate sources of bacteriophage in a fast-paced

Many of the solutions related to operating a manufacturing facility bacteriophage-free require participation of a cross-disciplinary team that encompass many areas of expertise, including virology, microbiology, microbial ecology, chemical, and mechanical engineering. As with any new biomanufacturing process, designers utilize a basic set of assumptions that serve in the design and construction of the facility. Some of these assumptions are not fully tested in a pilot plant, and the issues that arise are often seen for the first time in the commercial fermentation facility. In this chapter, we have characterized issues related to the use of 0.2 μm (absolute)

plaque forming units per square centimeter) in

**104**

The authors declare that there are no conflict of interests with this work.

#### **Author details**

James A. Zahn\* and Mathew C. Halter DuPont Tate & Lyle Bio Products, Loudon, TN, USA

\*Address all correspondence to: james.a.zahn@dupont.com

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Sanford K, Chotani G, Danielson N, Zahn JA. Scaling up of renewable chemicals. Current Opinion in Biotechnology. 2016;**38**:112-122

[2] Kraus GA. Synthetic methods for the preparation of 1,3-propanediol. CLEAN—Soil, Air, Water. 2008;**36**(8):648-651

[3] Urban RA, Bakshi BR. 1,3-Propanediol from fossils versus biomass: A life cycle evaluation of emissions and ecological resources. Industrial and Engineering Chemistry Research. 2009;**48**:8068-8082

[4] DuPont Tate & Lyle Bio Products. Life Cycle Analysis Overview– Susterra® Propanediol. 2016. Available from: http://www.duponttateandlyle. com/sites/default/files/Susterra%20 LCA.pdf

[5] Pauling L. Molecular basis of biological specificity. Nature. 1974;**248**:769-771

[6] Weider PR, Powell JB, Slaugh LH, Forschner TC, Semple TC. Process for Preparing 1,3-Propanediol. United States Patent Number 5,545,767. United States Patent and Trademark Office; 1996

[7] Halter MC, Zahn JA. Characterization of a novel lytic bacteriophage from an industrial *Escherichia coli* fermentation process and elimination of virulence using a heterologous CRISPR–Cas9 system. Journal of Industrial Microbiology & Biotechnology. 2018;**45**:153-163. DOI: 10.1007/ s10295-018-2015-7

[8] Jones DT, Shirley M, Wu X, Keis S. Bacteriophage infections in the industrial acetone butanol (AB) fermentation process.

Journal of Molecular Microbiology and Biotechnology. 2000;**2**(1): 21-26

[9] Guiness R. 2006. Personal communication

[10] Nakamura CE, Whited GM. Metabolic engineering for the microbial production of 1,3-propanediol. Current Opinion in Biotechnology. 2003;**14**: 454-459

[11] Sellmer S, Sievers M, Teuber M. Morphology, virulence and epidemiology of bacteriophage particles isolated from industrial vinegar fermentations. Systematic and Applied Microbiology. 1992;**15**: 610-616

[12] Sturino JM, Klaenhammer TR. Engineered bacteriophage-defense systems in bioprocessing. Nature Reviews Microbiology. 2006;**4**: 395-404

[13] Whitehead HR, Hunter GJE. Bacteriophage infection in cheese manufacture. The Journal of Dairy Research. 1945;**14**:64-80

[14] Tanji Y, Hattori K, Suzuki K, Miyanaga K. Spontaneous deletion of a 209-kilobase-pair fragment from the *Escherichia coli* genome occurs with acquisition of resistance to an assortment of infectious phages. Applied and Environmental Microbiology. 2008;**74**(14):4256-4263. DOI: 10.1128/AEM.00243-08 Epub 2008 May 23

[15] Bennett AM. Health hazards in biotechnology. In: Hambleton P, Melling J, Salusbury TT, editors. Biosafety in Industrial Biotechnology. Dordrecht: Springer; 1994. DOI: 10.1007/978-94-011-1352-6\_7

**107**

1436.2006

MG050172

*Surveillance and Elimination of Bacteriophage Contamination in an Industrial Fermentation…*

[23] Labrie SJ, Samson JE, Moineau S. Bacteriophage resistance mechanisms.

[24] Le S, Yao X, Lu S, Tan Y, Rao X, Li M, et al. Chromosomal DNA deletion confers phage resistance to *Pseudomonas* 

2014;**4**:4738. Published online 2014 Apr

[25] Duerkopa BA, Huoc W, Bhardwajc P, Palmerc KL, Hooper LV. Molecular

2016;**7**(4):e01304-e01316. DOI: 10.1128/

[26] Lucchini S, Sidoti J, Brüssow H. Broad-range bacteriophage resistance in *Streptococcus thermophilus* by insertional mutagenesis. Virology.

[27] Benz I, Schmidt MA. Glycosylation with heptose residues mediated by the *aah* gene product is essential for adherence of the AIDA-I adhesion. Molecular Microbiology.

[28] Gronow S, Brabetz W, Brade H.

[29] Koonin EV, Makarova KS, Zhang F. Diversity, classification and evolution of CRISPR-Cas systems. Current Opinion

[30] Sing EL, Elliker PR, Sandine WE. A method for evaluating the destruction of air-borne bacteriophages. Journal of Milk and Food Technology.

Nature Reviews Microbiology.

*aeruginosa*. Scientific Reports.

28. DOI: 10.1038/srep04738

basis for lytic bacteriophage resistance in enterococci. MBio.

2010;**8**:317-327

mBio.01304-16

2000;**275**(2):267-277

2001;**40**(6):1403-1413

Comparative functional characterization in vitro of heptosyltransferase I (WaaC) and II (WaaF) from *Escherichia coli*. European Journal of Biochemistry.

2000;**267**:6602-6611

in Microbiology. 2017;**37**:

1964;**27**(5):125-128

67-78

*DOI: http://dx.doi.org/10.5772/intechopen.81151*

[16] Jones JB, Jackson LE, Balogh B, Obradovic A, Iriarte FB, Momol MT. Bacteriophages for plant disease control. Annual Review of Phytopathology. 2007;**45**(1):

[17] Matsushima P, Broughton MC, Turner JR, Baltz RH. Conjugal transfer of cosmid DNA from *Escherichia coli* to *Saccharopolyspora spinosa*: Effects of chromosomal insertions on macrolide A83543 production. Gene.

[18] Goldfarb T, Sberro H, Weinstock E, Cohen O, Doron S, Charpak-Amikam Y, et al. BREX is a novel phage resistance system widespread in microbial genomes. The EMBO Journal. 2015;**34**(2):169-183. Published online 2014 Dec 1. DOI:

[19] Vale PF, Lafforgue G, Gatchitch F, Gardan R, Moineau S, Gandon S. Costs of CRISPR-Cas-mediated resistance in *Streptococcus thermophiles*. Proceedings of the Biological Sciences. 2015;**282**(1812):20151270. DOI: 10.1098/

[20] Tamulaitis G, Venclovas C, Siksnys V. Type III CRISPR-Cas immunity: Major differences brushed aside. Trends in Microbiology. 2017;**25**(1):49-61. DOI:

245-262

1994;**146**(1):39-45

10.15252/embj.201489455

10.1016/j.tim.2016.09.012

[21] Wietzorrek A, Schwarz H, Herrmann C, Braun V. The Genome of the novel phage Rtp, with a Rosette-Like tail tip, is homologous to the genome of phage T1. Journal of Bacteriology. 2006;**188**(4):1419-1436.

DOI: 10.1128/JB.188.4.1419-

[22] Genbank accession number MG050172. *Escherichia* phage DTL, complete genome. Available from: https://www.ncbi.nlm.nih.gov/nuccore/

rspb.2015.1270

*Surveillance and Elimination of Bacteriophage Contamination in an Industrial Fermentation… DOI: http://dx.doi.org/10.5772/intechopen.81151*

[16] Jones JB, Jackson LE, Balogh B, Obradovic A, Iriarte FB, Momol MT. Bacteriophages for plant disease control. Annual Review of Phytopathology. 2007;**45**(1): 245-262

[17] Matsushima P, Broughton MC, Turner JR, Baltz RH. Conjugal transfer of cosmid DNA from *Escherichia coli* to *Saccharopolyspora spinosa*: Effects of chromosomal insertions on macrolide A83543 production. Gene. 1994;**146**(1):39-45

[18] Goldfarb T, Sberro H, Weinstock E, Cohen O, Doron S, Charpak-Amikam Y, et al. BREX is a novel phage resistance system widespread in microbial genomes. The EMBO Journal. 2015;**34**(2):169-183. Published online 2014 Dec 1. DOI: 10.15252/embj.201489455

[19] Vale PF, Lafforgue G, Gatchitch F, Gardan R, Moineau S, Gandon S. Costs of CRISPR-Cas-mediated resistance in *Streptococcus thermophiles*. Proceedings of the Biological Sciences. 2015;**282**(1812):20151270. DOI: 10.1098/ rspb.2015.1270

[20] Tamulaitis G, Venclovas C, Siksnys V. Type III CRISPR-Cas immunity: Major differences brushed aside. Trends in Microbiology. 2017;**25**(1):49-61. DOI: 10.1016/j.tim.2016.09.012

[21] Wietzorrek A, Schwarz H, Herrmann C, Braun V. The Genome of the novel phage Rtp, with a Rosette-Like tail tip, is homologous to the genome of phage T1. Journal of Bacteriology. 2006;**188**(4):1419-1436. DOI: 10.1128/JB.188.4.1419- 1436.2006

[22] Genbank accession number MG050172. *Escherichia* phage DTL, complete genome. Available from: https://www.ncbi.nlm.nih.gov/nuccore/ MG050172

[23] Labrie SJ, Samson JE, Moineau S. Bacteriophage resistance mechanisms. Nature Reviews Microbiology. 2010;**8**:317-327

[24] Le S, Yao X, Lu S, Tan Y, Rao X, Li M, et al. Chromosomal DNA deletion confers phage resistance to *Pseudomonas aeruginosa*. Scientific Reports. 2014;**4**:4738. Published online 2014 Apr 28. DOI: 10.1038/srep04738

[25] Duerkopa BA, Huoc W, Bhardwajc P, Palmerc KL, Hooper LV. Molecular basis for lytic bacteriophage resistance in enterococci. MBio. 2016;**7**(4):e01304-e01316. DOI: 10.1128/ mBio.01304-16

[26] Lucchini S, Sidoti J, Brüssow H. Broad-range bacteriophage resistance in *Streptococcus thermophilus* by insertional mutagenesis. Virology. 2000;**275**(2):267-277

[27] Benz I, Schmidt MA. Glycosylation with heptose residues mediated by the *aah* gene product is essential for adherence of the AIDA-I adhesion. Molecular Microbiology. 2001;**40**(6):1403-1413

[28] Gronow S, Brabetz W, Brade H. Comparative functional characterization in vitro of heptosyltransferase I (WaaC) and II (WaaF) from *Escherichia coli*. European Journal of Biochemistry. 2000;**267**:6602-6611

[29] Koonin EV, Makarova KS, Zhang F. Diversity, classification and evolution of CRISPR-Cas systems. Current Opinion in Microbiology. 2017;**37**: 67-78

[30] Sing EL, Elliker PR, Sandine WE. A method for evaluating the destruction of air-borne bacteriophages. Journal of Milk and Food Technology. 1964;**27**(5):125-128

**106**

*Bacteriophages - Perspectives and Future*

[1] Sanford K, Chotani G, Danielson N, Zahn JA. Scaling up of renewable chemicals. Current Opinion in Biotechnology. 2016;**38**:112-122

Journal of Molecular Microbiology and Biotechnology. 2000;**2**(1):

[9] Guiness R. 2006. Personal

[10] Nakamura CE, Whited GM. Metabolic engineering for the microbial production of 1,3-propanediol. Current Opinion

[11] Sellmer S, Sievers M, Teuber M.

in Biotechnology. 2003;**14**:

Morphology, virulence and epidemiology of bacteriophage particles isolated from industrial vinegar fermentations. Systematic and

Applied Microbiology. 1992;**15**:

[12] Sturino JM, Klaenhammer TR. Engineered bacteriophage-defense systems in bioprocessing. Nature Reviews Microbiology. 2006;**4**:

[13] Whitehead HR, Hunter GJE. Bacteriophage infection in cheese manufacture. The Journal of Dairy

[14] Tanji Y, Hattori K, Suzuki K, Miyanaga K. Spontaneous deletion of a 209-kilobase-pair fragment from the *Escherichia coli* genome occurs with acquisition of resistance to an assortment of infectious phages. Applied and Environmental Microbiology. 2008;**74**(14):4256-4263. DOI: 10.1128/AEM.00243-08 Epub 2008

[15] Bennett AM. Health hazards in biotechnology. In: Hambleton P, Melling J, Salusbury TT, editors. Biosafety in Industrial Biotechnology. Dordrecht: Springer; 1994. DOI: 10.1007/978-94-011-1352-6\_7

Research. 1945;**14**:64-80

21-26

454-459

610-616

395-404

May 23

communication

[2] Kraus GA. Synthetic methods for the preparation of 1,3-propanediol.

1,3-Propanediol from fossils versus biomass: A life cycle evaluation of emissions and ecological resources. Industrial and Engineering Chemistry

[4] DuPont Tate & Lyle Bio Products. Life Cycle Analysis Overview–

Susterra® Propanediol. 2016. Available from: http://www.duponttateandlyle. com/sites/default/files/Susterra%20

CLEAN—Soil, Air, Water. 2008;**36**(8):648-651

**References**

[3] Urban RA, Bakshi BR.

Research. 2009;**48**:8068-8082

[5] Pauling L. Molecular basis of biological specificity. Nature.

[6] Weider PR, Powell JB, Slaugh LH, Forschner TC, Semple TC. Process for Preparing 1,3-Propanediol. United States Patent Number 5,545,767. United States Patent and Trademark Office;

LCA.pdf

1996

1974;**248**:769-771

[7] Halter MC, Zahn JA.

s10295-018-2015-7

Characterization of a novel lytic bacteriophage from an industrial *Escherichia coli* fermentation

[8] Jones DT, Shirley M, Wu X, Keis S. Bacteriophage infections in the industrial acetone butanol (AB) fermentation process.

process and elimination of virulence using a heterologous CRISPR–Cas9 system. Journal of Industrial Microbiology & Biotechnology. 2018;**45**:153-163. DOI: 10.1007/

[31] Jonczyk E, Klak M, Miedzybrodzki R, Gorski A. The influence of external factors on bacteriophages—review. Folia Microbiologica. 2011;**56**:191-200

[32] Martinez JE. The rotation of disinfectants principle: True or false. Pharmaceutical Technology. 2009;**33**(2):58-71. Available at: http:// www.pharmtech.com/rotationdisinfectants-principle-true-or-false

**109**

**Chapter 7**

**Abstract**

Targeting Peptides Derived from

*Supang Khondee and Wibool Piyawattanametha*

ized medicine, and image-guided therapy.

**1. Introduction**

Phage Display for Clinical Imaging

Phage display is a high-throughput technology used to identify peptides or proteins with high and specific binding affinities to a target, which is usually a protein biomarker or therapeutic receptor. In general, this technique allows peptides with a particular sequence to be presented on a phage particle. Peptides derived from phage display play an important role in drug discovery, drug delivery, cancer imaging, and treatment. Phage peptides themselves can act as sole therapeutics, for example, drugs, gene therapeutic, and immunotherapeutic agents that are comprehensively described elsewhere. In this chapter, we discuss phage selection and screening procedures in detail including some modifications to reduce nonspecific binding. In addition, the rationale for discovery and utilization of phage peptides as molecular imaging probes is focused upon. Molecular imaging is a new paradigm that uses advanced imaging instruments integrated with specific molecular imaging probes. Applications include monitoring of metabolic and molecular functions, therapeutic response, and drug efficacy, as well as early cancer detection, personal-

**Keywords:** peptides, membrane receptors, imaging, phage display, endoscopy

One of the most important practices in modern era clinical imaging is imaging at the molecular level which can help characterize and measure in vivo biological processes at the cellular level [1]. Thus, the technique provides unambiguous and high-resolution real-time information for disease diagnoses and therapies. In addition, molecular imaging is usually noninvasive as a biologically active, receptor-specific, targeting vector conjugated to a radioligand, nanoparticle, and/or fluorescent/magnetic resonance imaging (MRI) probe is administered first, and then the probe signals can be quantified by positron emission tomography (PET), singlephoton emission computed tomography (SPECT), magnetic resonance imaging (MRI), fluorescence imaging, or ultrasound. The specificity of probes may be contributed from targeting peptides, small proteins, and antibodies linked to the probes [2]. One of the most important aspects in successful molecular imaging is the development of imaging probes. Initial efforts focused on probes that are radiolabeled small molecules or macromolecules, e.g., monoclonal antibodies and their fragments [3]. Most such probes are unsuccessful because small molecules provide low specificity, whereas the antibodies have low target permeability. Taken into account altogether, these probes have low contrast between target tissues and background, leading to poor imaging qualities. Compared to small molecules and antibodies,

#### **Chapter 7**

*Bacteriophages - Perspectives and Future*

Microbiologica. 2011;**56**:191-200

[32] Martinez JE. The rotation of disinfectants principle: True or false. Pharmaceutical Technology. 2009;**33**(2):58-71. Available at: http:// www.pharmtech.com/rotationdisinfectants-principle-true-or-false

[31] Jonczyk E, Klak M, Miedzybrodzki R, Gorski A. The influence of external factors on bacteriophages—review. Folia

**108**

## Targeting Peptides Derived from Phage Display for Clinical Imaging

*Supang Khondee and Wibool Piyawattanametha*

#### **Abstract**

Phage display is a high-throughput technology used to identify peptides or proteins with high and specific binding affinities to a target, which is usually a protein biomarker or therapeutic receptor. In general, this technique allows peptides with a particular sequence to be presented on a phage particle. Peptides derived from phage display play an important role in drug discovery, drug delivery, cancer imaging, and treatment. Phage peptides themselves can act as sole therapeutics, for example, drugs, gene therapeutic, and immunotherapeutic agents that are comprehensively described elsewhere. In this chapter, we discuss phage selection and screening procedures in detail including some modifications to reduce nonspecific binding. In addition, the rationale for discovery and utilization of phage peptides as molecular imaging probes is focused upon. Molecular imaging is a new paradigm that uses advanced imaging instruments integrated with specific molecular imaging probes. Applications include monitoring of metabolic and molecular functions, therapeutic response, and drug efficacy, as well as early cancer detection, personalized medicine, and image-guided therapy.

**Keywords:** peptides, membrane receptors, imaging, phage display, endoscopy

#### **1. Introduction**

One of the most important practices in modern era clinical imaging is imaging at the molecular level which can help characterize and measure in vivo biological processes at the cellular level [1]. Thus, the technique provides unambiguous and high-resolution real-time information for disease diagnoses and therapies. In addition, molecular imaging is usually noninvasive as a biologically active, receptor-specific, targeting vector conjugated to a radioligand, nanoparticle, and/or fluorescent/magnetic resonance imaging (MRI) probe is administered first, and then the probe signals can be quantified by positron emission tomography (PET), singlephoton emission computed tomography (SPECT), magnetic resonance imaging (MRI), fluorescence imaging, or ultrasound. The specificity of probes may be contributed from targeting peptides, small proteins, and antibodies linked to the probes [2].

One of the most important aspects in successful molecular imaging is the development of imaging probes. Initial efforts focused on probes that are radiolabeled small molecules or macromolecules, e.g., monoclonal antibodies and their fragments [3]. Most such probes are unsuccessful because small molecules provide low specificity, whereas the antibodies have low target permeability. Taken into account altogether, these probes have low contrast between target tissues and background, leading to poor imaging qualities. Compared to small molecules and antibodies,

peptide imaging probes are more promising. The peptide length varies from several to approximately 50 amino acids [4]; thus they are usually more specific than small molecules and also more permeable than antibodies. The peptides have high capillary permeability, which allows efficient penetration into tissues. In addition, they also have high uptake rates in the target and rapid clearance from blood [1]. These distinctive advantages facilitate peptides as popular imaging probes. Such a probe is usually composed of a targeting peptide, a linker, and an imaging moiety. The linkers commonly are organic spacers, macrocyclic or branched chelators, and polymers, which link peptides with appropriate moieties. Different moieties render the probes observable by various devices, e.g., near-infrared (NIR) fluorescent dyes or quantum dots for optical imaging, radionuclides for PET or SPECT, and paramagnetic agents for MRI.

Phage display technology is a powerful approach to screen for peptides with high affinities and specificities to biomarkers. This technology was established by Smith et al. in 1985 to display polypeptides on the surface of filamentous M13-derived bacteriophage (phage) [5]. This technique modifies the phage genome to fuse the deoxyribonucleic acid (DNA) encoding a peptide to a gene encoding a protein comprising the phage coat; thus the peptide appears on the surface of the phage. In this way, each phage contains a single-peptide variant and its encoding DNA sequence, thus retaining a genotype-phenotype linkage. A library or pool of phages normally contains 109 –1011 peptide variants for screening. The selection procedure consists of three main steps: (1) panning the pool of phages on the immobilized biomarker, (2) removing unbound phages by washing, and (3) elution of bound phages. After several rounds of such selections, the peptide sequences with high affinities to the biomarker are determined by sequencing the encoding DNAs in the phages. Specificity of the peptides can be also improved by adding extra negative selection steps [6].

Since its inception nearly 45 years ago, phage display has been widely used in thousands of research papers to isolate peptides that bind various targets [7]. The phage peptides are labeled with imaging agents such as radioactives, fluorescences, and nanoparticles. These probes have been successfully used to image tumors and cancers [8–10]. Moreover, phage display also influenced many other scientific fields such as drug discovery, vaccine development, and targeted drug delivery and gene therapy. With advances in molecular biology, the number of disease-associated biomarkers at the molecular level is ever-increasing. These new discoveries are motivating the applications of phage display to diagnostic imaging and targeted drug delivery.

#### **2. Phage biology and phage selection screening methods**

#### **2.1 Phage biology**

Bacteriophages (or phages) are viruses that infect bacteria. Phage virions vary widely in size, shape, and complexity, and phage genomes range in size from 3.4 kb to almost 500 kb [11]. Most phage genomes (>95%) discovered to date are linear, double-stranded DNAs (dsDNAs), but there are also genomes that are singlestranded DNAs (ssDNA), circular DNAs, and RNAs [12]. The phage genome is packed into a protein capsid, which together form a phage particle. Some phages, such as pleomorphic phages, are further covered by bacterial lipoprotein membrane during budding [13]. As a parasite, the phage life cycle is intertwined with that of the host cell, i.e., bacteria. The phage particle first attaches itself to a host cell by specific recognition of a receptor or other surface moiety of the host; then the phage

**111**

**Figure 1.**

*A scheme presents lytic and lysogenic stages of phage.*

*Targeting Peptides Derived from Phage Display for Clinical Imaging*

nucleic acids are inserted into the host cell. Inside the host cell, the phage genome shuts down defense mechanisms of the host and hijacks host cellular components to replicate phage genome and express capsid genes from the phage genome. Eventually, phage genomes and capsids are assembled into progeny phage particles. These phage particles emerge from the host cell, which usually results in cell lysis by

Phages were discovered by Twort in 1915 and d'Hérelle in 1917 [12], respectively.

In phage display, the most widely used phage is the M13 strain of filamentous bacteriophage. This type of phage infects F plasmid-containing gram-negative bacteria, such as *Escherichia coli*. Besides M13, other members of filamentous phages such as f1 and fd strains have also been used for phage display [14]. These phages have circular single-stranded DNA (ssDNA). The genome is composed of 11 genes [15], which are classified into three groups by functions. The first group comprises

Now, it is clear that phages are the most abundant organisms on the earth; they have been found in every environment with bacteria. An estimated 1031 phage particles exist on earth. Since the 1940s, phages have been model organisms and have contributed to molecular biology substantially. The most remarkable contributions include revealing the random nature of mutation, the discovery of DNA as the genetic material, and the understanding of gene expression control. With advances

in biotechnology, phage display was established by Smith in 1985.

*DOI: http://dx.doi.org/10.5772/intechopen.84281*

phage proteins (**Figure 1**).

*Targeting Peptides Derived from Phage Display for Clinical Imaging DOI: http://dx.doi.org/10.5772/intechopen.84281*

*Bacteriophages - Perspectives and Future*

magnetic agents for MRI.

normally contains 109

selection steps [6].

drug delivery.

**2.1 Phage biology**

peptide imaging probes are more promising. The peptide length varies from several to approximately 50 amino acids [4]; thus they are usually more specific than small molecules and also more permeable than antibodies. The peptides have high capillary permeability, which allows efficient penetration into tissues. In addition, they also have high uptake rates in the target and rapid clearance from blood [1]. These distinctive advantages facilitate peptides as popular imaging probes. Such a probe is usually composed of a targeting peptide, a linker, and an imaging moiety. The linkers commonly are organic spacers, macrocyclic or branched chelators, and polymers, which link peptides with appropriate moieties. Different moieties render the probes observable by various devices, e.g., near-infrared (NIR) fluorescent dyes or quantum dots for optical imaging, radionuclides for PET or SPECT, and para-

Phage display technology is a powerful approach to screen for peptides with high affinities and specificities to biomarkers. This technology was established by Smith et al. in 1985 to display polypeptides on the surface of filamentous M13-derived bacteriophage (phage) [5]. This technique modifies the phage genome to fuse the deoxyribonucleic acid (DNA) encoding a peptide to a gene encoding a protein comprising the phage coat; thus the peptide appears on the surface of the phage. In this way, each phage contains a single-peptide variant and its encoding DNA sequence, thus retaining a genotype-phenotype linkage. A library or pool of phages

consists of three main steps: (1) panning the pool of phages on the immobilized biomarker, (2) removing unbound phages by washing, and (3) elution of bound phages. After several rounds of such selections, the peptide sequences with high affinities to the biomarker are determined by sequencing the encoding DNAs in the phages. Specificity of the peptides can be also improved by adding extra negative

Since its inception nearly 45 years ago, phage display has been widely used in thousands of research papers to isolate peptides that bind various targets [7]. The phage peptides are labeled with imaging agents such as radioactives, fluorescences, and nanoparticles. These probes have been successfully used to image tumors and cancers [8–10]. Moreover, phage display also influenced many other scientific fields such as drug discovery, vaccine development, and targeted drug delivery and gene therapy. With advances in molecular biology, the number of disease-associated biomarkers at the molecular level is ever-increasing. These new discoveries are motivating the applications of phage display to diagnostic imaging and targeted

Bacteriophages (or phages) are viruses that infect bacteria. Phage virions vary widely in size, shape, and complexity, and phage genomes range in size from 3.4 kb to almost 500 kb [11]. Most phage genomes (>95%) discovered to date are linear, double-stranded DNAs (dsDNAs), but there are also genomes that are singlestranded DNAs (ssDNA), circular DNAs, and RNAs [12]. The phage genome is packed into a protein capsid, which together form a phage particle. Some phages, such as pleomorphic phages, are further covered by bacterial lipoprotein membrane during budding [13]. As a parasite, the phage life cycle is intertwined with that of the host cell, i.e., bacteria. The phage particle first attaches itself to a host cell by specific recognition of a receptor or other surface moiety of the host; then the phage

**2. Phage biology and phage selection screening methods**

–1011 peptide variants for screening. The selection procedure

**110**

nucleic acids are inserted into the host cell. Inside the host cell, the phage genome shuts down defense mechanisms of the host and hijacks host cellular components to replicate phage genome and express capsid genes from the phage genome. Eventually, phage genomes and capsids are assembled into progeny phage particles. These phage particles emerge from the host cell, which usually results in cell lysis by phage proteins (**Figure 1**).

Phages were discovered by Twort in 1915 and d'Hérelle in 1917 [12], respectively. Now, it is clear that phages are the most abundant organisms on the earth; they have been found in every environment with bacteria. An estimated 1031 phage particles exist on earth. Since the 1940s, phages have been model organisms and have contributed to molecular biology substantially. The most remarkable contributions include revealing the random nature of mutation, the discovery of DNA as the genetic material, and the understanding of gene expression control. With advances in biotechnology, phage display was established by Smith in 1985.

In phage display, the most widely used phage is the M13 strain of filamentous bacteriophage. This type of phage infects F plasmid-containing gram-negative bacteria, such as *Escherichia coli*. Besides M13, other members of filamentous phages such as f1 and fd strains have also been used for phage display [14]. These phages have circular single-stranded DNA (ssDNA). The genome is composed of 11 genes [15], which are classified into three groups by functions. The first group comprises

**Figure 1.**

*A scheme presents lytic and lysogenic stages of phage.*

capsid proteins: protein III (pIII), pVI, pVII, pVIII, and pIX; the second group is for DNA replication, pII, pV, and pX; the last group consists of proteins for assembly, pI, pIV, and pXI. This genome is contained in a protein coat to form a phage particle of 6.5 nm in width and ~900 nm in length. Normally, filamentous phage is not lytic; thus phages are released from bacteria without bacterial lysis. Instead of phage M13, phage display can also use phagemid, which is simply a plasmid with a phage origin of replication so that the plasmid can be replicated and packaged into phage particles. The phagemid as a cloning vector needs helper phage to complete its infection process and virion packaging [16].

#### **2.2 Phage selection screening methods**

#### *2.2.1 Peptide library*

Peptide library construction is the first step in selecting peptides with high affinities to the target of interest. Each of the 20 amino acids is encoded by codons, and each codon consists of three consecutive nucleotides. There are four types of nucleotides, denoted as A (adenine), G (guanine), C (cytosine), and T (thymine). A random peptide is constructed by synthesizing an oligonucleotide containing (NNK)n, where N stands for any of the four nucleotides, K stands for either G or T, and n indicates the desired length of the peptide. Note that only G or T is introduced to the third position of a codon because this reduces the frequency of stop codons (NNN generates three stop codons: TAA, TGA and TAG, whereas NNK generates only one codon: TAG). To add a site with N, simply provide an equimolar mixture of A, T, G, and C, and randomly one of them is added at the end of the nucleotide chain. As for a site with K, just provide a mixture of G and T to the reaction [17]. In this way, numerous oligonucleotides are synthesized in parallel, and each oligonucleotide encodes a random peptide. Note that the NNK codon pattern is generated by controlling nucleotide types to be added in reactions. This simple rule can be modified to create particular codons, e.g., allowing no stop codon or creating codons of charged residues.

Peptide libraries often have short peptide lengths, approximately less than 50 residues. The optimum length required for the randomized displayed peptide is often unknown before selection and varies with many factors including the folding properties of the displayed peptide and the target of interest. For a library of random peptides with seven residues, the maximum number of different peptides is 207 . However, this number is usually unapproachable due to codon degeneracy and early stop codon. In other words, a library usually contains redundant peptides and peptides shorter than desired. On the other hand, the capacity of selection is limited by transfection, i.e., only 108 –1010 phages, each encloses one peptide, can be transformed into *E. coli* by electroporation or other techniques. Taken together, the diversity of a library is important for the success of selection and screening for high affinity peptides. A typical commercially available library archives peptide diversity at the level of 109 [4].

In M13 phage, the oligonucleotides encoding random peptides are mostly fused to the N-terminus of pIII, with a spacer as a linker to generate a phage library. Another widely used gene for peptide display is the one encoding pVIII. Both pIII and pVIII are the major and minor capsid proteins, accessible from the outer surface of the phage. pIII has 406 residues, and for each phage, there are in total 3–5 pIII proteins which form a knob-like structure at one end of the phage. This structure is responsible for infection of bacteria via the F-pilus, virion stability, and assembly termination. The peptides linked to pIII for display almost have no restriction on length, facilitating pIII as the mostly targeted for peptide display applications. As for pVIII, it is a short helical protein (50 residues) [18], and about 2700 pVIII molecules are present on the capsid. However, only the three residues

**113**

*2.2.3 In vivo screening*

*Targeting Peptides Derived from Phage Display for Clinical Imaging*

at the N-terminus are accessible from the outer surface of the phage. Unlike pIII, pVIII can tolerate only short peptides with less than ten residues to be linked and successfully displayed, which is likely due to interrupted assembly by long peptides. However, this problem can be alleviated by reducing the density of pVIII mutants [19]. Interestingly, some other capsid proteins such as pV1, pVII, and pIX have poorer accessibilities on the phage surface but nevertheless successfully display

In a phage library, each of the trillion phages displays a single variant of random peptides on its surface. Many methods have been developed to identify the peptides that have high affinities to the target of interest, e.g., a biomarker protein. To this end, biopanning is the most commonly used method, and it has many variations to improve the performance of selection and screening or to accommodate for special targets [20]. Biopanning consists of four basic steps: (i) target immobilization, (ii) phage binding, (iii) washing, and (iv) phage elution. In the first step, the target of interest is purified and immobilized on plates. Some targets are not able to maintain structural integrity after being separated from cells, e.g., some transmembrane proteins. Therefore, whole cells or engineered cells may instead be immobilized on plates or suspended in solutions in this step [21]. In phage binding, the phages in a library are incubated with the plate, and appropriate buffers are also added to facilitate the binding reactions between the displayed peptides on phages and the targets on plates. After that, the plates are washed to remove unbound phages. Phage elution is to acquire the bound phages by disrupting the interaction between the peptides and the targets. This disruption is conducted by changing pH or adding competing ligands, denaturant, or protease. The eluted phages are amplified by infecting *E. coli*. The phages may go through another round of biopanning or are subject to DNA sequencing to determine the peptide sequences (**Figure 2**). These peptides are

Following several rounds of biopanning, the selected peptides have high affinities to the target but may not have high specificities, i.e., the peptides may also bind to nontargets with high affinities. To this end, subtractive screening can be added to the basic biopanning steps. The subtractive strategy allows phages to interact with nontargets, and thus the unbound phages are candidates that are specific to the target of interest. For example, to identify the peptides that specifically bind to esophageal cancer cells, Zhang et al. use normal human esophageal epithelial cells as the nontarget to perform subtractive screening, followed by screening against esophageal cancer cells. They repeated this procedure for three rounds and discovered two peptides that exhibited higher binding affinities and specificities for the cancer cells, which were validated by enzyme-linked immunosorbent assays (ELISAs), immunofluorescence assays, and immunohistochemistry assays [6]. Improvement of peptide library screening could be performed through small modifications. A stepwise reduction of elution buffer pH in the final round of biopanning reduces low affinity phages, thus effectively further enriching for high affinity phages. Optimized commercial kits are also available, for example, solidphase screening, solution-sorting screening, kinetic antibody binding screening,

In vivo screening of phage display is designed to isolate tissue-specific binders in living animals. Unlike in vitro screening, this in vivo approach considers the

*DOI: http://dx.doi.org/10.5772/intechopen.84281*

considered to possess high affinities to the target.

and capture-sandwich ELISA screening [22, 23].

peptides for screening [19].

*2.2.2 In vitro screening*

at the N-terminus are accessible from the outer surface of the phage. Unlike pIII, pVIII can tolerate only short peptides with less than ten residues to be linked and successfully displayed, which is likely due to interrupted assembly by long peptides. However, this problem can be alleviated by reducing the density of pVIII mutants [19]. Interestingly, some other capsid proteins such as pV1, pVII, and pIX have poorer accessibilities on the phage surface but nevertheless successfully display peptides for screening [19].

#### *2.2.2 In vitro screening*

*Bacteriophages - Perspectives and Future*

process and virion packaging [16].

*2.2.1 Peptide library*

**2.2 Phage selection screening methods**

capsid proteins: protein III (pIII), pVI, pVII, pVIII, and pIX; the second group is for DNA replication, pII, pV, and pX; the last group consists of proteins for assembly, pI, pIV, and pXI. This genome is contained in a protein coat to form a phage particle of 6.5 nm in width and ~900 nm in length. Normally, filamentous phage is not lytic; thus phages are released from bacteria without bacterial lysis. Instead of phage M13, phage display can also use phagemid, which is simply a plasmid with a phage origin of replication so that the plasmid can be replicated and packaged into phage particles. The phagemid as a cloning vector needs helper phage to complete its infection

Peptide library construction is the first step in selecting peptides with high affinities to the target of interest. Each of the 20 amino acids is encoded by codons, and each codon consists of three consecutive nucleotides. There are four types of nucleotides, denoted as A (adenine), G (guanine), C (cytosine), and T (thymine). A random peptide is constructed by synthesizing an oligonucleotide containing (NNK)n, where N stands for any of the four nucleotides, K stands for either G or T, and n indicates the desired length of the peptide. Note that only G or T is introduced to the third position of a codon because this reduces the frequency of stop codons (NNN generates three stop codons: TAA, TGA and TAG, whereas NNK generates only one codon: TAG). To add a site with N, simply provide an equimolar mixture of A, T, G, and C, and

randomly one of them is added at the end of the nucleotide chain. As for a site with K, just provide a mixture of G and T to the reaction [17]. In this way, numerous oligonucleotides are synthesized in parallel, and each oligonucleotide encodes a random peptide. Note that the NNK codon pattern is generated by controlling nucleotide types to be added in reactions. This simple rule can be modified to create particular codons,

Peptide libraries often have short peptide lengths, approximately less than 50 residues. The optimum length required for the randomized displayed peptide is often unknown before selection and varies with many factors including the folding properties of the displayed peptide and the target of interest. For a library of random peptides with seven residues, the maximum number of different peptides is 207

this number is usually unapproachable due to codon degeneracy and early stop codon. In other words, a library usually contains redundant peptides and peptides shorter than desired. On the other hand, the capacity of selection is limited by transfection,

by electroporation or other techniques. Taken together, the diversity of a library is important for the success of selection and screening for high affinity peptides. A typi-

In M13 phage, the oligonucleotides encoding random peptides are mostly fused

cal commercially available library archives peptide diversity at the level of 109

to the N-terminus of pIII, with a spacer as a linker to generate a phage library. Another widely used gene for peptide display is the one encoding pVIII. Both pIII and pVIII are the major and minor capsid proteins, accessible from the outer surface of the phage. pIII has 406 residues, and for each phage, there are in total 3–5 pIII proteins which form a knob-like structure at one end of the phage. This structure is responsible for infection of bacteria via the F-pilus, virion stability, and assembly termination. The peptides linked to pIII for display almost have no restriction on length, facilitating pIII as the mostly targeted for peptide display applications. As for pVIII, it is a short helical protein (50 residues) [18], and about 2700 pVIII molecules are present on the capsid. However, only the three residues

–1010 phages, each encloses one peptide, can be transformed into *E. coli*

. However,

[4].

e.g., allowing no stop codon or creating codons of charged residues.

**112**

i.e., only 108

In a phage library, each of the trillion phages displays a single variant of random peptides on its surface. Many methods have been developed to identify the peptides that have high affinities to the target of interest, e.g., a biomarker protein. To this end, biopanning is the most commonly used method, and it has many variations to improve the performance of selection and screening or to accommodate for special targets [20].

Biopanning consists of four basic steps: (i) target immobilization, (ii) phage binding, (iii) washing, and (iv) phage elution. In the first step, the target of interest is purified and immobilized on plates. Some targets are not able to maintain structural integrity after being separated from cells, e.g., some transmembrane proteins. Therefore, whole cells or engineered cells may instead be immobilized on plates or suspended in solutions in this step [21]. In phage binding, the phages in a library are incubated with the plate, and appropriate buffers are also added to facilitate the binding reactions between the displayed peptides on phages and the targets on plates. After that, the plates are washed to remove unbound phages. Phage elution is to acquire the bound phages by disrupting the interaction between the peptides and the targets. This disruption is conducted by changing pH or adding competing ligands, denaturant, or protease. The eluted phages are amplified by infecting *E. coli*. The phages may go through another round of biopanning or are subject to DNA sequencing to determine the peptide sequences (**Figure 2**). These peptides are considered to possess high affinities to the target.

Following several rounds of biopanning, the selected peptides have high affinities to the target but may not have high specificities, i.e., the peptides may also bind to nontargets with high affinities. To this end, subtractive screening can be added to the basic biopanning steps. The subtractive strategy allows phages to interact with nontargets, and thus the unbound phages are candidates that are specific to the target of interest. For example, to identify the peptides that specifically bind to esophageal cancer cells, Zhang et al. use normal human esophageal epithelial cells as the nontarget to perform subtractive screening, followed by screening against esophageal cancer cells. They repeated this procedure for three rounds and discovered two peptides that exhibited higher binding affinities and specificities for the cancer cells, which were validated by enzyme-linked immunosorbent assays (ELISAs), immunofluorescence assays, and immunohistochemistry assays [6].

Improvement of peptide library screening could be performed through small modifications. A stepwise reduction of elution buffer pH in the final round of biopanning reduces low affinity phages, thus effectively further enriching for high affinity phages. Optimized commercial kits are also available, for example, solidphase screening, solution-sorting screening, kinetic antibody binding screening, and capture-sandwich ELISA screening [22, 23].

#### *2.2.3 In vivo screening*

In vivo screening of phage display is designed to isolate tissue-specific binders in living animals. Unlike in vitro screening, this in vivo approach considers the

**Figure 2.**

*General scheme of affinity selection of target-specific peptides.*

complexity and heterogeneity of the living organism and thus is one step closer to clinical applications. The phage library is administered directly into a living animal and allowed to circulate for a period of time, and then the animal is sacrificed with the desired organ extracted and homogenized in saline. The lysates or pelleted cells of the organ are used to infect *E. coli* so that the phages with high-affinity binding peptides are amplified. This procedure is repeated several rounds, and the resultant phages are sequenced to determine the binding peptide sequences. Similar to in vitro screening, in vivo screening can have a step to wash away unbound phages. It is achieved usually by perfusion of the left ventricle of the animal with saline [24].

In vivo screening faces a more complicated environment than in vitro screening and thus requires extra considerations. Filamentous bacteriophages are often used for in vitro screening; however these viral particles are quite long (>500 nm) and thus may have problems during extravasation to some tissues. For example, M13 phage cannot be used to target liver parenchymal cells due to the impermeability problem [25]. Instead, T7 bacteriophage is used to identify peptides targeting livers because it is smaller in size. An even more challenging issue for in vivo phages is how to avoid immune surveillance for accurate phage displays. Host immune system, particularly reticuloendothelial system, degrades phages quickly and increases nonspecific uptake in the liver and spleen. As expected, the severity of these phenomena reduces within immune-compromised nude and SCID mouse strains. Glycosylation and succinylation to wild-type M13 phages substantially reduce its half-life in murine bloodstream from 4.5 hours to 18 and 1.5 minutes, respectively [26]. This indicates that phages modified to display peptides should have a similarly short half-life, which is consistent with existing data. Therefore, the optimal time of phage library circulation needs to be determined before the actual in vivo phage display experiments. The time varies with phage modifications and targeted tissues.

Phage library administration is another concern for in vivo screening. Administration approaches determine circulation routes of phages in living animals.

**115**

*Targeting Peptides Derived from Phage Display for Clinical Imaging*

The most widely used approach is intravenous delivery. It enables rapid exposure of the phages to vascular receptors of any organ or tissue. However, this approach is inappropriate to the discovery of peptides targeting brain tissues due to the blood-brain barrier. To this end, Wan et al. administered phage library intranasally and identified a peptide targeting the brain, which performs 50-fold better than random control peptides. Other "bloodless" approaches including site-directed phage administrations and transdermal delivery have been used successfully to identify targeting peptides [27].

**3. General considerations for phage display to target membrane** 

factor receptor 2 (HER2) membrane protein in 20-25% of breast cancers.

Despite being popular targets, membrane proteins present a limitation for phage display. The key to a successful phage display is the presentation of correctly folded targets, i.e., native structures of extracellular domains (ectodomains) in membrane proteins. This is fairly easy for soluble proteins because they fold correctly in solution while being immobilized on a surface, whereas membrane proteins usually fail to fold into their native structure in solution without membranes [29]. For example, G protein-coupled receptors (GPCRs) constitute the largest class of drug targets but have had limited success in phage display due to their hydrophobic regions and complicated ectodomains, which are usually comprised of the N-terminal chain, parts of the seven transmembrane helices, and their connecting loops. To stabilize the membrane proteins, several methods have been developed. The principle of this method is to engineer the membrane proteins by mutations or adding hydrophilic domains so that they maintain stability in solution for phage display. Note that such engineering must not interfere with the native fold of the ectodomain of membrane proteins. Another way to circumvent the limitation of unstable membrane proteins in solution is directly biopanning on whole cells instead of immobilizing membrane proteins on a surface as targets for phage display screening. Although this whole cell panning provides membrane proteins in native state as screening targets, it causes extra difficulties. First, the membrane protein may be present at low density on the cell surface; thus other nontargeted membrane proteins generate a high background noise. Second, other components on the surface of cells, such as sugar or lipid polymers, may sequester phage particles for nonspecific uptake into a cell. Taken together, both of these possibilities facilitate the selection of nonspecific ligands, rendering phage display inefficient. To address these newly introduced problems, Jones et al. have reported a modified phage display screening for antibodies. The key innovation is that the cell is

Membrane proteins are the most popular targets for diagnostic and therapeutic applications. The structures of membrane proteins are generally composed of three parts, i.e., the extracellular, transmembrane, and intracellular domains. The extracellular domains are the primary targets of drug discovery and diagnosis. About 60% of drug targets are membrane proteins [28]. More specifically, membrane proteins are targeted by the 61% monoclonal antibodies approved or under review as therapeutic drugs throughout Europe and the United States. This prevalence is all attributed to the unique properties of membrane proteins. First, they have various important biological functions such as in signaling and cell channels; thus drugs targeting them can manipulate cellular functions effectively. Second, membrane proteins are presented on cell surface and thus are more accessible than cellular proteins. Some membrane proteins can define cell types. For example, various cluster of differentiation (CD) markers are membrane proteins and define immune cell types. More importantly, diseases usually alter expression levels of membrane proteins. A well-known example is the overexpression of human epidermal growth

*DOI: http://dx.doi.org/10.5772/intechopen.84281*

**receptors**

*Targeting Peptides Derived from Phage Display for Clinical Imaging DOI: http://dx.doi.org/10.5772/intechopen.84281*

*Bacteriophages - Perspectives and Future*

complexity and heterogeneity of the living organism and thus is one step closer to clinical applications. The phage library is administered directly into a living animal and allowed to circulate for a period of time, and then the animal is sacrificed with the desired organ extracted and homogenized in saline. The lysates or pelleted cells of the organ are used to infect *E. coli* so that the phages with high-affinity binding peptides are amplified. This procedure is repeated several rounds, and the resultant phages are sequenced to determine the binding peptide sequences. Similar to in vitro screening, in vivo screening can have a step to wash away unbound phages. It is achieved usually by perfusion of the left ventricle of the animal with saline [24]. In vivo screening faces a more complicated environment than in vitro screening and thus requires extra considerations. Filamentous bacteriophages are often used for in vitro screening; however these viral particles are quite long (>500 nm) and thus may have problems during extravasation to some tissues. For example, M13 phage cannot be used to target liver parenchymal cells due to the impermeability problem [25]. Instead, T7 bacteriophage is used to identify peptides targeting livers because it is smaller in size. An even more challenging issue for in vivo phages is how to avoid immune surveillance for accurate phage displays. Host immune system, particularly reticuloendothelial system, degrades phages quickly and increases nonspecific uptake in the liver and spleen. As expected, the severity of these phenomena reduces within immune-compromised nude and SCID mouse strains. Glycosylation and succinylation to wild-type M13 phages substantially reduce its half-life in murine bloodstream from 4.5 hours to 18 and 1.5 minutes, respectively [26]. This indicates that phages modified to display peptides should have a similarly short half-life, which is consistent with existing data. Therefore, the optimal time of phage library circulation needs to be determined before the actual in vivo phage display experiments. The time varies with phage modifications and targeted tissues.

*General scheme of affinity selection of target-specific peptides.*

Phage library administration is another concern for in vivo screening.

Administration approaches determine circulation routes of phages in living animals.

**114**

**Figure 2.**

The most widely used approach is intravenous delivery. It enables rapid exposure of the phages to vascular receptors of any organ or tissue. However, this approach is inappropriate to the discovery of peptides targeting brain tissues due to the blood-brain barrier. To this end, Wan et al. administered phage library intranasally and identified a peptide targeting the brain, which performs 50-fold better than random control peptides. Other "bloodless" approaches including site-directed phage administrations and transdermal delivery have been used successfully to identify targeting peptides [27].

#### **3. General considerations for phage display to target membrane receptors**

Membrane proteins are the most popular targets for diagnostic and therapeutic applications. The structures of membrane proteins are generally composed of three parts, i.e., the extracellular, transmembrane, and intracellular domains. The extracellular domains are the primary targets of drug discovery and diagnosis. About 60% of drug targets are membrane proteins [28]. More specifically, membrane proteins are targeted by the 61% monoclonal antibodies approved or under review as therapeutic drugs throughout Europe and the United States. This prevalence is all attributed to the unique properties of membrane proteins. First, they have various important biological functions such as in signaling and cell channels; thus drugs targeting them can manipulate cellular functions effectively. Second, membrane proteins are presented on cell surface and thus are more accessible than cellular proteins. Some membrane proteins can define cell types. For example, various cluster of differentiation (CD) markers are membrane proteins and define immune cell types. More importantly, diseases usually alter expression levels of membrane proteins. A well-known example is the overexpression of human epidermal growth factor receptor 2 (HER2) membrane protein in 20-25% of breast cancers.

Despite being popular targets, membrane proteins present a limitation for phage display. The key to a successful phage display is the presentation of correctly folded targets, i.e., native structures of extracellular domains (ectodomains) in membrane proteins. This is fairly easy for soluble proteins because they fold correctly in solution while being immobilized on a surface, whereas membrane proteins usually fail to fold into their native structure in solution without membranes [29]. For example, G protein-coupled receptors (GPCRs) constitute the largest class of drug targets but have had limited success in phage display due to their hydrophobic regions and complicated ectodomains, which are usually comprised of the N-terminal chain, parts of the seven transmembrane helices, and their connecting loops. To stabilize the membrane proteins, several methods have been developed. The principle of this method is to engineer the membrane proteins by mutations or adding hydrophilic domains so that they maintain stability in solution for phage display. Note that such engineering must not interfere with the native fold of the ectodomain of membrane proteins.

Another way to circumvent the limitation of unstable membrane proteins in solution is directly biopanning on whole cells instead of immobilizing membrane proteins on a surface as targets for phage display screening. Although this whole cell panning provides membrane proteins in native state as screening targets, it causes extra difficulties. First, the membrane protein may be present at low density on the cell surface; thus other nontargeted membrane proteins generate a high background noise. Second, other components on the surface of cells, such as sugar or lipid polymers, may sequester phage particles for nonspecific uptake into a cell. Taken together, both of these possibilities facilitate the selection of nonspecific ligands, rendering phage display inefficient. To address these newly introduced problems, Jones et al. have reported a modified phage display screening for antibodies. The key innovation is that the cell is

transfected so that the target membrane protein and green fluorescent protein (GFP) are highly expressed simultaneously. For phage display, the green fluorescence provides a means to select only the cells with the target membrane protein highly expressed on cell surface using fluorescence-activated cell sorting (FACS) [29]. In other steps of phage display, small but nontrivial modifications are also adopted to improve or overcome the problems of using whole cells. For example, a low pH wash is found effective in removing phage that is present through nonantibody binding. Details of these many small modifications in phage display protocols are reviewed by Alfaleh et al. [30].

A third category of methods has also been developed to create cell membranelike microenvironments that preserve the stability and integrity of membrane proteins for phage display. In the first method, the membrane protein is purified in the presence of detergents, which forms micelles after exceeding their critical micellar concentration. These micelles mimic cell membrane and have been used in phage display to identify antibodies to the sodium-citrate cotransporter and the fluoride ion channel. However, detergent micelles themselves are unstable and heterogeneous in size, which may cause membrane proteins to unfold or aggregate. In another method, the membrane protein is inserted into a liposome, which is a bilayer structure that more closely mimics the cell membrane than detergent micelles. Recently, nanodiscs and virus-like particles (VLP) have been developed to mimic host membrane protein. Nanodiscs are macromolecular structures that spontaneously assemble when lipids and apolipoprotein A1 or B are mixed. In general, one nanodisc structure contains a lipid bilayer of ~1000 phospholipids bundled by two apolipoproteins. The diameter of a nanodisc is ~10 nm and thus houses only one membrane protein approximately. Nanodiscs can take in a membrane protein through coupled in vitro transcription-translation. A VLP contains viral capsid proteins, lipids, and membrane proteins on its surface, but not a viral genome. It is much more stable than native cells, micelles, and liposomes and thus can withstand wash buffers with detergents, which decreases nonspecific binding in phage display.

In summary, some simple ectodomains of membrane proteins can be directly immobilized on plastic surfaces and treated as soluble proteins in phage display, whereas some other membrane proteins need to be engineered to increase ectodomain stability and integrity before phage display. Various nanoparticles such as detergent micelle, nanodisc, and VLP have been developed to create membrane-like environments to house membrane proteins. Panning directly on wild-type cells or on engineered cells, e.g., with membrane proteins highly expressed, is another way to represent membrane proteins for phage display. Such methods have to manage high background noise from cells. However, these methods have a unique advantage, i.e., consistent with native binding mechanisms. Many membrane receptors, e.g., cytokine receptors, exert their biological functions through ligand binding and dimerization. The ligand may interact with the monomer first and then mediate dimerization for function or bind to the dimer directly and then carry out its function. These two scenarios may both occur depending on the ligand concentrations [31]. Therefore, in those methods without whole cells, the monomer immobilized in phage display may not mimic the targets in vivo, and thus the ligand selected by phage display may not bind to in vivo receptors with high affinities.

#### **4. Phage display provides potential therapeutic and diagnostic agents**

Peptide phage display has played an important role in the development of clinically useful therapeutics and diagnostic agents. Peptide-based therapeutics have attracted a significant level of interest in the drug discovery and development industry. First, phage particles themselves can be used as the therapeutic agent. For

**117**

*Targeting Peptides Derived from Phage Display for Clinical Imaging*

the treatment of acute hereditary angioedema [35].

receptor-positive tumors in humans [36].

**5.1 Magnetic resonance imaging**

**imaging**

by delivering DNA encoding for bactericidal toxin proteins [32].

example, the M13 bacteriophage was used successfully to treat a bacterial infection

Second, peptides derived from phage display can be used as therapeutic drugs. In 2017, peptide drug annual market was approximately \$300–500 million and is estimated to increase 25% each year [33]. Compared to proteins and antibodies, peptides have numerous advantages, for example, low manufacturing costs, better activity and stability, negligible immunogenicity, and superior organ penetration. A number of peptide drugs developed from phage display technique have been approved or are currently in clinical trials. For example, DX-890, an inhibitor of human neutrophil elastase, with potential application in the treatment of pulmonary diseases such as cystic fibrosis and chronic obstructive pulmonary disease, was originated from phage display [34]. Ecallantide, a highly potent inhibitor of human plasma kallikrein, has been approved by the US Food and Drug Administration for

Filamentous phage has also been used as an immunogenic carrier useful in vaccine development, with high immunogenicity, low production cost, and high stability. In addition, phage can also act as a gene-delivery vehicle. For example, phage can deliver functional genes to mammalian cells through receptor-mediated endocytosis. Phage-derived peptides that bind protein targets with high affinity and specificity can be used as molecular imaging probes. The classic example is octreotide, an eight amino acid cyclized peptide that binds the somatostatin receptor. 111In-DTPAoctreotide (OctreoScan®) has been used successfully to image somatostatin

**5. Applications of targeting peptides derived from phages in clinical** 

Targeted molecular imaging of disease processes, particularly tumor growth and metastasis, has been a focus of many investigations recently. Molecular imaging probes have assisted in the understanding of fundamental biological processes, disease pathologies, as well as pharmaceutical development. Enormous progress has been made in both discovery of imaging probes and development of imaging instruments. Additionally, optical imaging methods provide many advantages over other imaging modalities that include high sensitivity, the use of nonradioactive materials, and safe detection using readily available instruments at moderate cost. Today, in vivo imaging can be applied at preclinical and clinical settings due to significant improvements in engineering technologies, optical systems, and advanced imaging instruments. These technologies in a combination with cutting-edge optical imaging probes provide noninvasive, real-time imaging at macroscopic and cellular levels. Indeed, the combination of numerous NIR probes and targeted ligands, such as antibodies, aptamers, and engineered peptides, has significantly enhanced the performance of optical imaging systems. Recent progress in clinical imaging and the utilization of phage-derived targeting peptides are reviewed below.

MRI is a medical imaging technique used in radiology to form pictures of the anatomy and the physiological processes of the body, including in healthy and diseased tissues and organs. MRI scanners use strong magnetic fields, electric field gradients, and radio waves to generate images of the organs in the body. After a radiofrequency pulse, MRI detects the relaxation times of magnetic dipoles, such as hydrogen atoms in water and organic compounds, and generates MR signals. MRI offers spatial resolution

*DOI: http://dx.doi.org/10.5772/intechopen.84281*

#### *Targeting Peptides Derived from Phage Display for Clinical Imaging DOI: http://dx.doi.org/10.5772/intechopen.84281*

*Bacteriophages - Perspectives and Future*

transfected so that the target membrane protein and green fluorescent protein (GFP) are highly expressed simultaneously. For phage display, the green fluorescence provides a means to select only the cells with the target membrane protein highly expressed on cell surface using fluorescence-activated cell sorting (FACS) [29]. In other steps of phage display, small but nontrivial modifications are also adopted to improve or overcome the problems of using whole cells. For example, a low pH wash is found effective in removing phage that is present through nonantibody binding. Details of these many small modifications in phage display protocols are reviewed by Alfaleh et al. [30]. A third category of methods has also been developed to create cell membranelike microenvironments that preserve the stability and integrity of membrane proteins for phage display. In the first method, the membrane protein is purified in the presence of detergents, which forms micelles after exceeding their critical micellar concentration. These micelles mimic cell membrane and have been used in phage display to identify antibodies to the sodium-citrate cotransporter and the fluoride ion channel. However, detergent micelles themselves are unstable and heterogeneous in size, which may cause membrane proteins to unfold or aggregate. In another method, the membrane protein is inserted into a liposome, which is a bilayer structure that more closely mimics the cell membrane than detergent micelles. Recently, nanodiscs and virus-like particles (VLP) have been developed to mimic host membrane protein. Nanodiscs are macromolecular structures that spontaneously assemble when lipids and apolipoprotein A1 or B are mixed. In general, one nanodisc structure contains a lipid bilayer of ~1000 phospholipids bundled by two apolipoproteins. The diameter of a nanodisc is ~10 nm and thus houses only one membrane protein approximately. Nanodiscs can take in a membrane protein through coupled in vitro transcription-translation. A VLP contains viral capsid proteins, lipids, and membrane proteins on its surface, but not a viral genome. It is much more stable than native cells, micelles, and liposomes and thus can withstand wash buffers with detergents, which decreases nonspecific binding in phage display. In summary, some simple ectodomains of membrane proteins can be directly immobilized on plastic surfaces and treated as soluble proteins in phage display, whereas some other membrane proteins need to be engineered to increase ectodomain stability and integrity before phage display. Various nanoparticles such as detergent micelle, nanodisc, and VLP have been developed to create membrane-like environments to house membrane proteins. Panning directly on wild-type cells or on engineered cells, e.g., with membrane proteins highly expressed, is another way to represent membrane proteins for phage display. Such methods have to manage high background noise from cells. However, these methods have a unique advantage, i.e., consistent with native binding mechanisms. Many membrane receptors, e.g., cytokine receptors, exert their biological functions through ligand binding and dimerization. The ligand may interact with the monomer first and then mediate dimerization for function or bind to the dimer directly and then carry out its function. These two scenarios may both occur depending on the ligand concentrations [31]. Therefore, in those methods without whole cells, the monomer immobilized in phage display may not mimic the targets in vivo, and thus the ligand selected by

phage display may not bind to in vivo receptors with high affinities.

**4. Phage display provides potential therapeutic and diagnostic agents**

Peptide phage display has played an important role in the development of clinically useful therapeutics and diagnostic agents. Peptide-based therapeutics have attracted a significant level of interest in the drug discovery and development industry. First, phage particles themselves can be used as the therapeutic agent. For

**116**

example, the M13 bacteriophage was used successfully to treat a bacterial infection by delivering DNA encoding for bactericidal toxin proteins [32].

Second, peptides derived from phage display can be used as therapeutic drugs. In 2017, peptide drug annual market was approximately \$300–500 million and is estimated to increase 25% each year [33]. Compared to proteins and antibodies, peptides have numerous advantages, for example, low manufacturing costs, better activity and stability, negligible immunogenicity, and superior organ penetration. A number of peptide drugs developed from phage display technique have been approved or are currently in clinical trials. For example, DX-890, an inhibitor of human neutrophil elastase, with potential application in the treatment of pulmonary diseases such as cystic fibrosis and chronic obstructive pulmonary disease, was originated from phage display [34]. Ecallantide, a highly potent inhibitor of human plasma kallikrein, has been approved by the US Food and Drug Administration for the treatment of acute hereditary angioedema [35].

Filamentous phage has also been used as an immunogenic carrier useful in vaccine development, with high immunogenicity, low production cost, and high stability. In addition, phage can also act as a gene-delivery vehicle. For example, phage can deliver functional genes to mammalian cells through receptor-mediated endocytosis.

Phage-derived peptides that bind protein targets with high affinity and specificity can be used as molecular imaging probes. The classic example is octreotide, an eight amino acid cyclized peptide that binds the somatostatin receptor. 111In-DTPAoctreotide (OctreoScan®) has been used successfully to image somatostatin receptor-positive tumors in humans [36].

#### **5. Applications of targeting peptides derived from phages in clinical imaging**

Targeted molecular imaging of disease processes, particularly tumor growth and metastasis, has been a focus of many investigations recently. Molecular imaging probes have assisted in the understanding of fundamental biological processes, disease pathologies, as well as pharmaceutical development. Enormous progress has been made in both discovery of imaging probes and development of imaging instruments. Additionally, optical imaging methods provide many advantages over other imaging modalities that include high sensitivity, the use of nonradioactive materials, and safe detection using readily available instruments at moderate cost. Today, in vivo imaging can be applied at preclinical and clinical settings due to significant improvements in engineering technologies, optical systems, and advanced imaging instruments. These technologies in a combination with cutting-edge optical imaging probes provide noninvasive, real-time imaging at macroscopic and cellular levels. Indeed, the combination of numerous NIR probes and targeted ligands, such as antibodies, aptamers, and engineered peptides, has significantly enhanced the performance of optical imaging systems. Recent progress in clinical imaging and the utilization of phage-derived targeting peptides are reviewed below.

#### **5.1 Magnetic resonance imaging**

MRI is a medical imaging technique used in radiology to form pictures of the anatomy and the physiological processes of the body, including in healthy and diseased tissues and organs. MRI scanners use strong magnetic fields, electric field gradients, and radio waves to generate images of the organs in the body. After a radiofrequency pulse, MRI detects the relaxation times of magnetic dipoles, such as hydrogen atoms in water and organic compounds, and generates MR signals. MRI offers spatial resolution on the millimeter scale with simultaneous physiological and anatomical correlation. However, MRI requires long scan and postprocessing times and has relatively low sensitivity, thus requiring high doses of magnetic contrast agents.

In a reparative or reactive process, excess fibrous connective tissue (type I collagen) or fibrosis could be formed in a tissue. This is a common consequence in many chronic heart, kidney, liver, lung, or vasculature diseases. The high levels of collagen in fibrosis make it an attractive MRI target. Therefore, a collagenspecific MRI contrast agent was developed. A type I collagen-specific peptide was identified using phage display and subsequently modified to improve affinity for collagen. Conjugate EP-3533 consists of a peptide of 16 amino acids, with 3 amino acids flanking a cyclic peptide of 10 amino acids that is formed through a disulfide bond. The peptide was modified with biphenylalanine and gadolinium to improve collagen binding and sensitivity. EP-3533 was evaluated in a mouse model of aged myocardial infarction. From MR images, EP-3533 was able to enhance the collagenrich scar, providing specific, high-contrast images compared to adjacent viable myocardium tissues and blood vessels [37].

Another example of the utilization of peptides from phage with MRI in early detection of colorectal cancer (CRC) was reported. Human gastric mucin (MUC5AC) is secreted in the colonic mucus of cancer patients. MUC5AC is a specific marker of precancerous lesions called aberrant crypt foci. MRI can detect the accumulation of MUC5AC in xenograft and mouse stomach. To enhance MRI visualization, peptides that specifically bound MUC5AC were developed using an M13 phage library. Once, the peptide binding MUC5AC (C-PSIYPLL-C, 60C) was identified; it was synthesized and conjugated to biotin and finally to ultra-small particles of iron oxide (USPIOs). The ability of USPIO-60C to detect MUC5AC in vivo was investigated on two xenograft mouse models. A heterogeneous but significant negative enhancement was observed in MUC5AC-secreting tumor postcontrast images 1 hour after intravenous USPIO-60C administration [38]. The results provided in this study supply a proof of concept that targeted contrast agents can be used to detect pathologies earlier than allowed by conventional MRI approaches or clinical assessment.

#### **5.2 Positron emission tomography/single-photon emission computed tomography**

PET and SPECT are nuclear medicine tomographic imaging techniques using gamma (γ) rays. Both techniques require the injection of a radioactive tracer. There are three main tracers used in SPECT imaging: technetium-99m, iodine-123, and iodine-131. The radioactive tracer emits gamma rays from the patient. PET and SPECT record high- and low-energy γ-rays emitted from within the body. These imaging modalities have very high sensitivity but relatively low spatial resolution.

VRPMPLQ (VQ ) is a heptapeptide sequence first identified by Hsiung and colleagues by screening phage display peptide libraries against fresh human colonic adenomas [39]. In a later study, Shi and team synthesized and evaluated 99mTc-HYNIC-VQ (HYNIC 5,6-hydrazinonicotinamide) as a SPECT radiotracer for tumor imaging in five different xenograft mouse models (HT-29 human colon cancer, CL187 human colon cancer, BGC823 human gastric cancer, U87MG human glioma, and UM-SCC-22B human head and neck cancer). The images were acquired 1 and 2 hours postinjection. The tumors were clearly visualized at 1 hour postinjection with excellent target-to-background (T/B) contrast. The studies demonstrated that 99mTc-HYNIC-VQ could provide high-contrast images in different tumor models and an inflammation model [40].

In another study, a radioactive probe targeted to a dysplastic lesion in inflammatory bowel disease (IBD) or early CRC was developed. The cyclic peptide

**119**

*Targeting Peptides Derived from Phage Display for Clinical Imaging*

feasibility of TCP-1-targeted detection of colorectal tumor [42].

rapidly, leading to exciting findings and applications.

detect HCC xenograft tumors in vivo with photoacoustic imaging.

biopsy or resection to be performed concurrently with diagnosis.

c[Cys-Thr-Pro-Ser-Pro-Phe-Ser-His-Cys]OH (TCP-1) was originally identified in an orthotopic mouse CRC model using phage display selection [41]. TCP-1 peptide was labeled with radioisotope technetium-99 m (99mTc) and the NIR fluorophore cyanine-7 (Cy7) for molecular imaging. The in vivo images of 99mTc-TCP-1 in xenografted HCT116 and PC3 prostate cancer models were collected using dynamic or static SPECT. The 99mTc-TCP-1 or control peptides were administered via intravenous or tail vein injection. Dynamic images of 99mTc-TCP-1 in HCT116 colon cancer xenograft mice exhibited that the tumor could be detected in 15–30 minutes after injection and remained visible until 180 minutes. The data demonstrated the

Photoacoustic tomography (PAT) is an emerging imaging technique that demonstrates great potential for preclinical research and clinical applications. PAT is a hybrid system that detects the acoustic energy of endogenous chromophore or exogenous contrast agent optical absorption. PAT generates high-resolution images in both the optical ballistic and diffusive regimes due to less ultrasound scatter in tissue. Over the past decade, the photoacoustic technique has been developing

Epidermal growth factor receptor (EGFR) is highly overexpressed in hepatocellular carcinoma (HCC). Therefore, it is a potential cell surface molecule for in vivo targeted imaging of HCC. A peptide specific for EGFR previously reported by Zhou and team was conjugated to Cy5.5 dye. Nude mice were injected with EGFR overexpressed human HCC cells. A 2D ultrasound scanner and MRI system were used to monitor tumor growth in the mice. Cy5.5-labeled EGFR and control peptides were injected to the mice separately. Photoacoustic images were recorded periodically for 24 hours. At 3 hours postinjection, the maximum photoacoustic signal in tumors was seen and results in high-contrast images of tumors beneath the skin. The T/B ratio was significantly different between the EGFR and control peptide. The signal was diminished by 24 hours [43]. From the data, a peptide specific for EGFR can

Optical imaging offers several unique advantages. Optics is nonionizing and provides resolution on the micron scale. Another advantage of optical imaging is the ability to collect images in real time in comparison to other imaging modalities,

Endoscopes are thin, flexible instruments that provide a macroscopic view of the large mucosal surfaces in hollow organs internal to the human body. Endomicroscopy employs high numerical aperture (NA) optics to provide a small field-of-view (FOV) with micron-level resolution for observing subcellular features. It commonly requires scaling down the size of a conventional microscope design into a miniature package. Novel optical designs and scanning mechanisms have been developed to improve imaging performance for both endoscopy and endomicroscopy. These instruments provide a unique opportunity for early cancer detection and prevention by allowing

Previously, all CRCs were believed to arise from adenomas that progress through the traditional adenocarcinoma sequence. Recently, this pathway has been found

*DOI: http://dx.doi.org/10.5772/intechopen.84281*

**5.3 Photoacoustic tomography**

**5.4 Optical endomicroscopy**

*5.4.1 Wide-field fluorescence endoscopy*

such as MRI and PET.

*Targeting Peptides Derived from Phage Display for Clinical Imaging DOI: http://dx.doi.org/10.5772/intechopen.84281*

c[Cys-Thr-Pro-Ser-Pro-Phe-Ser-His-Cys]OH (TCP-1) was originally identified in an orthotopic mouse CRC model using phage display selection [41]. TCP-1 peptide was labeled with radioisotope technetium-99 m (99mTc) and the NIR fluorophore cyanine-7 (Cy7) for molecular imaging. The in vivo images of 99mTc-TCP-1 in xenografted HCT116 and PC3 prostate cancer models were collected using dynamic or static SPECT. The 99mTc-TCP-1 or control peptides were administered via intravenous or tail vein injection. Dynamic images of 99mTc-TCP-1 in HCT116 colon cancer xenograft mice exhibited that the tumor could be detected in 15–30 minutes after injection and remained visible until 180 minutes. The data demonstrated the feasibility of TCP-1-targeted detection of colorectal tumor [42].

#### **5.3 Photoacoustic tomography**

*Bacteriophages - Perspectives and Future*

myocardium tissues and blood vessels [37].

on the millimeter scale with simultaneous physiological and anatomical correlation. However, MRI requires long scan and postprocessing times and has relatively low

In a reparative or reactive process, excess fibrous connective tissue (type I collagen) or fibrosis could be formed in a tissue. This is a common consequence in many chronic heart, kidney, liver, lung, or vasculature diseases. The high levels of collagen in fibrosis make it an attractive MRI target. Therefore, a collagenspecific MRI contrast agent was developed. A type I collagen-specific peptide was identified using phage display and subsequently modified to improve affinity for collagen. Conjugate EP-3533 consists of a peptide of 16 amino acids, with 3 amino acids flanking a cyclic peptide of 10 amino acids that is formed through a disulfide bond. The peptide was modified with biphenylalanine and gadolinium to improve collagen binding and sensitivity. EP-3533 was evaluated in a mouse model of aged myocardial infarction. From MR images, EP-3533 was able to enhance the collagenrich scar, providing specific, high-contrast images compared to adjacent viable

Another example of the utilization of peptides from phage with MRI in early detection of colorectal cancer (CRC) was reported. Human gastric mucin (MUC5AC) is secreted in the colonic mucus of cancer patients. MUC5AC is a specific marker of precancerous lesions called aberrant crypt foci. MRI can detect the accumulation of MUC5AC in xenograft and mouse stomach. To enhance MRI visualization, peptides that specifically bound MUC5AC were developed using an M13 phage library. Once, the peptide binding MUC5AC (C-PSIYPLL-C, 60C) was identified; it was synthesized and conjugated to biotin and finally to ultra-small particles of iron oxide (USPIOs). The ability of USPIO-60C to detect MUC5AC in vivo was investigated on two xenograft mouse models. A heterogeneous but significant negative enhancement was observed in MUC5AC-secreting tumor postcontrast images 1 hour after intravenous USPIO-60C administration [38]. The results provided in this study supply a proof of concept that targeted contrast agents can be used to detect pathologies earlier than allowed by conventional MRI approaches or clinical assessment.

**5.2 Positron emission tomography/single-photon emission computed** 

PET and SPECT are nuclear medicine tomographic imaging techniques using gamma (γ) rays. Both techniques require the injection of a radioactive tracer. There are three main tracers used in SPECT imaging: technetium-99m, iodine-123, and iodine-131. The radioactive tracer emits gamma rays from the patient. PET and SPECT record high- and low-energy γ-rays emitted from within the body. These imaging modalities have very high sensitivity but relatively low spatial resolution. VRPMPLQ (VQ ) is a heptapeptide sequence first identified by Hsiung and colleagues by screening phage display peptide libraries against fresh human colonic adenomas [39]. In a later study, Shi and team synthesized and evaluated 99mTc-HYNIC-VQ (HYNIC 5,6-hydrazinonicotinamide) as a SPECT radiotracer for tumor imaging in five different xenograft mouse models (HT-29 human colon cancer, CL187 human colon cancer, BGC823 human gastric cancer, U87MG human glioma, and UM-SCC-22B human head and neck cancer). The images were acquired 1 and 2 hours postinjection. The tumors were clearly visualized at 1 hour postinjection with excellent target-to-background (T/B) contrast. The studies demonstrated that 99mTc-HYNIC-VQ could provide high-contrast images in different tumor models

In another study, a radioactive probe targeted to a dysplastic lesion in inflammatory bowel disease (IBD) or early CRC was developed. The cyclic peptide

sensitivity, thus requiring high doses of magnetic contrast agents.

**118**

**tomography**

and an inflammation model [40].

Photoacoustic tomography (PAT) is an emerging imaging technique that demonstrates great potential for preclinical research and clinical applications. PAT is a hybrid system that detects the acoustic energy of endogenous chromophore or exogenous contrast agent optical absorption. PAT generates high-resolution images in both the optical ballistic and diffusive regimes due to less ultrasound scatter in tissue. Over the past decade, the photoacoustic technique has been developing rapidly, leading to exciting findings and applications.

Epidermal growth factor receptor (EGFR) is highly overexpressed in hepatocellular carcinoma (HCC). Therefore, it is a potential cell surface molecule for in vivo targeted imaging of HCC. A peptide specific for EGFR previously reported by Zhou and team was conjugated to Cy5.5 dye. Nude mice were injected with EGFR overexpressed human HCC cells. A 2D ultrasound scanner and MRI system were used to monitor tumor growth in the mice. Cy5.5-labeled EGFR and control peptides were injected to the mice separately. Photoacoustic images were recorded periodically for 24 hours. At 3 hours postinjection, the maximum photoacoustic signal in tumors was seen and results in high-contrast images of tumors beneath the skin. The T/B ratio was significantly different between the EGFR and control peptide. The signal was diminished by 24 hours [43]. From the data, a peptide specific for EGFR can detect HCC xenograft tumors in vivo with photoacoustic imaging.

#### **5.4 Optical endomicroscopy**

Optical imaging offers several unique advantages. Optics is nonionizing and provides resolution on the micron scale. Another advantage of optical imaging is the ability to collect images in real time in comparison to other imaging modalities, such as MRI and PET.

Endoscopes are thin, flexible instruments that provide a macroscopic view of the large mucosal surfaces in hollow organs internal to the human body. Endomicroscopy employs high numerical aperture (NA) optics to provide a small field-of-view (FOV) with micron-level resolution for observing subcellular features. It commonly requires scaling down the size of a conventional microscope design into a miniature package. Novel optical designs and scanning mechanisms have been developed to improve imaging performance for both endoscopy and endomicroscopy. These instruments provide a unique opportunity for early cancer detection and prevention by allowing biopsy or resection to be performed concurrently with diagnosis.

#### *5.4.1 Wide-field fluorescence endoscopy*

Previously, all CRCs were believed to arise from adenomas that progress through the traditional adenocarcinoma sequence. Recently, this pathway has been found

to account for approximately 60% of CRCs, and up to 35% are now attributed to the serrated pathway [44]. White-light endoscopy (WLE) that is normally used in colonoscopy is sensitive to gross morphologic abnormalities, such as polyps. Dysplasia that is flat in morphology, focal in size, and patchy in distribution appears "invisible" on conventional wide-field endoscopy. Therefore, imaging methods with improved contrast and sensitivity to molecular rather than morphological properties that could improve early detection and prevention of CRC are in needed. There are several reports on discovery and validation of targeted peptides derived from phage display for early detection of CRC [45].

One group used phage display to identify a peptide that binds to dysplastic colonic mucosa in vivo in a genetically engineered mouse model of colorectal tumorigenesis, *CPC;Apc* [46, 47]. A peptide, QPIHPNNM, was isolated after several rounds of in vivo T7 library biopanning. The peptide was synthesized, fluorescently labeled, and purified. The peptide was sprayed topically in mouse distal colon. The wide-field fluorescent videos were recorded. After quantitative image analysis, the fluorescent-labeled peptide was significantly bound twofold greater to the colonic adenomas when compared to the control peptide. The target peptide also showed minimal binding to an activated KrasG12D mutant mouse model that demonstrates hyperplastic polyp-like features used as a control hyperplastic model that does not progress to carcinoma (**Figure 3A**) and the lumen of a *CPC;Apc* bred mouse negative (**Figure 3B**).

c-Met overexpression has been shown to occur as an early event in colorectal adenocarcinoma. A peptide that has high affinity for the extracellular domain of human c-Met was discovered using an M-13 phage display library. GE-137 is a water-soluble cyclic peptide (AGSCYCSGPPRFECWCYETEGT) labeled with a cyanine dye with a high affinity for human c-Met. The quantitative biodistribution, pharmacokinetics, binding specificity, and qualitative fluorescence of GE-137 were assessed in a CRC xenograft mouse model using subcutaneous injection of the c-Met-expressing human CRC cell line HT29. Intravenously administered GE-137 accumulated in the c-Met-expressing tumor xenografts and left a fluorescent signal in the tumors and kidneys 120 and 240 minutes

#### **Figure 3.**

*Images from wide-field endoscopy videos after topical application of fluorescence-labeled peptides. The top and bottom panels represent frames from white light and fluorescence, respectively. (A) The hyperplastic epithelium after QPI peptide application, (B) the lumen of a CPC;Apc bred mouse negative for Cre recombinase (control litter mate), (C) single adenoma after control peptide application, (D) single adenoma, and (E) multiple adenomas in a CPC;Apc mouse after QPI peptide application. Used with permission [47].*

**121**

**Figure 4.**

*visible in FL. Used with permission [48].*

*Targeting Peptides Derived from Phage Display for Clinical Imaging*

to dysplasia than normal was found with a 19.4-fold difference.

after injection (**Figure 4**). In a pilot study in 15 patients at high risk of colorectal neoplasia, a total of 101 lesions were detected during first inspection with white light (WL), and an additional 22 were detected during second inspection with dual WL/fluorescence (FL). After immunohistochemical analysis, 36 hyperplastic lesions and 8 serrated polyps were identified. Most of these were visible in fluorescent mode (94 and 100%, respectively), and the majority (78 and 87%, respectively) showed increased fluorescence. From the data, GE-137 peptide showed some specificity to hyperplasia and serrated lesions in CRC mouse model

Overexpression of EGFR has been reported in as high as 97% of colonic adenocarcinomas, and it is a validated biomarker for CRC. In one study, the extracellular domain of EGFR (EGFR-ECD) was expressed and purified [49]. A library of M13 bacteriophage was used to select peptide candidates that bind specifically to EGFR-ECD. A peptide, QRHKPRE, that is specific for EGFR was developed and validated. Peptide binding to cells occurred within 2.46 minutes and had an affinity of 50 nM. A NIR fluorescence endoscope was used to perform *in vivo* imaging to validate peptide binding to spontaneous colonic adenomas in a *CPC;Apc* mouse model via topical administration (**Figure 5**). T/B ratios of polyps and flat lesions were 4.0 ± 1.7 and 2.7 ± 0.7, respectively. Subsequently, specific peptide binding to human colonic adenomas was assessed on immunohistochemistry and immunofluorescence. On human colonic specimens, greater intensity from peptide binding

In another study, a phage-derived peptide was tested for specific binding to sessile serrated adenomas (SSAs) in proximal colon which accounts for 35% in CRC. Joshi and team used phage display to identify a peptide that binds specifically to SSAs. Many SSA cells have the V600E mutation in BRAF. Therefore, peptide selection was performed with an M13 Ph.D.-7 phage display library using a biopanning strategy with subtractive hybridization with HT29 colorectal cancer cells containing the V600E mutation in BRAF [45]. Binding of fluorescently labeled peptide, KCCFPAQ, to colorectal cancer cells was evaluated with confocal fluorescence

*WL and FL images of representative lesions. (a–c) The lesions that are visible in WL show increased fluorescence. (d) A lesion that is visible in WL has enhanced visibility in FL. (e, f) Flat lesions that were only* 

*DOI: http://dx.doi.org/10.5772/intechopen.84281*

and patients [48].

#### *Targeting Peptides Derived from Phage Display for Clinical Imaging DOI: http://dx.doi.org/10.5772/intechopen.84281*

*Bacteriophages - Perspectives and Future*

mouse negative (**Figure 3B**).

from phage display for early detection of CRC [45].

to account for approximately 60% of CRCs, and up to 35% are now attributed to the serrated pathway [44]. White-light endoscopy (WLE) that is normally used in colonoscopy is sensitive to gross morphologic abnormalities, such as polyps. Dysplasia that is flat in morphology, focal in size, and patchy in distribution appears "invisible" on conventional wide-field endoscopy. Therefore, imaging methods with improved contrast and sensitivity to molecular rather than morphological properties that could improve early detection and prevention of CRC are in needed. There are several reports on discovery and validation of targeted peptides derived

One group used phage display to identify a peptide that binds to dysplastic colonic mucosa in vivo in a genetically engineered mouse model of colorectal tumorigenesis, *CPC;Apc* [46, 47]. A peptide, QPIHPNNM, was isolated after several rounds of in vivo T7 library biopanning. The peptide was synthesized, fluorescently labeled, and purified. The peptide was sprayed topically in mouse distal colon. The wide-field fluorescent videos were recorded. After quantitative image analysis, the fluorescent-labeled peptide was significantly bound twofold greater to the colonic adenomas when compared to the control peptide. The target peptide also showed minimal binding to an activated KrasG12D mutant mouse model that demonstrates hyperplastic polyp-like features used as a control hyperplastic model that does not progress to carcinoma (**Figure 3A**) and the lumen of a *CPC;Apc* bred

c-Met overexpression has been shown to occur as an early event in colorectal adenocarcinoma. A peptide that has high affinity for the extracellular domain of human c-Met was discovered using an M-13 phage display library. GE-137 is a water-soluble cyclic peptide (AGSCYCSGPPRFECWCYETEGT) labeled with a cyanine dye with a high affinity for human c-Met. The quantitative biodistribution, pharmacokinetics, binding specificity, and qualitative fluorescence of GE-137 were assessed in a CRC xenograft mouse model using subcutaneous injection of the c-Met-expressing human CRC cell line HT29. Intravenously administered GE-137 accumulated in the c-Met-expressing tumor xenografts and left a fluorescent signal in the tumors and kidneys 120 and 240 minutes

*Images from wide-field endoscopy videos after topical application of fluorescence-labeled peptides. The top and bottom panels represent frames from white light and fluorescence, respectively. (A) The hyperplastic epithelium after QPI peptide application, (B) the lumen of a CPC;Apc bred mouse negative for Cre recombinase (control litter mate), (C) single adenoma after control peptide application, (D) single adenoma, and (E) multiple* 

*adenomas in a CPC;Apc mouse after QPI peptide application. Used with permission [47].*

**120**

**Figure 3.**

after injection (**Figure 4**). In a pilot study in 15 patients at high risk of colorectal neoplasia, a total of 101 lesions were detected during first inspection with white light (WL), and an additional 22 were detected during second inspection with dual WL/fluorescence (FL). After immunohistochemical analysis, 36 hyperplastic lesions and 8 serrated polyps were identified. Most of these were visible in fluorescent mode (94 and 100%, respectively), and the majority (78 and 87%, respectively) showed increased fluorescence. From the data, GE-137 peptide showed some specificity to hyperplasia and serrated lesions in CRC mouse model and patients [48].

Overexpression of EGFR has been reported in as high as 97% of colonic adenocarcinomas, and it is a validated biomarker for CRC. In one study, the extracellular domain of EGFR (EGFR-ECD) was expressed and purified [49]. A library of M13 bacteriophage was used to select peptide candidates that bind specifically to EGFR-ECD. A peptide, QRHKPRE, that is specific for EGFR was developed and validated. Peptide binding to cells occurred within 2.46 minutes and had an affinity of 50 nM. A NIR fluorescence endoscope was used to perform *in vivo* imaging to validate peptide binding to spontaneous colonic adenomas in a *CPC;Apc* mouse model via topical administration (**Figure 5**). T/B ratios of polyps and flat lesions were 4.0 ± 1.7 and 2.7 ± 0.7, respectively. Subsequently, specific peptide binding to human colonic adenomas was assessed on immunohistochemistry and immunofluorescence. On human colonic specimens, greater intensity from peptide binding to dysplasia than normal was found with a 19.4-fold difference.

In another study, a phage-derived peptide was tested for specific binding to sessile serrated adenomas (SSAs) in proximal colon which accounts for 35% in CRC. Joshi and team used phage display to identify a peptide that binds specifically to SSAs. Many SSA cells have the V600E mutation in BRAF. Therefore, peptide selection was performed with an M13 Ph.D.-7 phage display library using a biopanning strategy with subtractive hybridization with HT29 colorectal cancer cells containing the V600E mutation in BRAF [45]. Binding of fluorescently labeled peptide, KCCFPAQ, to colorectal cancer cells was evaluated with confocal fluorescence

#### **Figure 4.**

*WL and FL images of representative lesions. (a–c) The lesions that are visible in WL show increased fluorescence. (d) A lesion that is visible in WL has enhanced visibility in FL. (e, f) Flat lesions that were only visible in FL. Used with permission [48].*

#### **Figure 5.**

In vivo imaging of colon in CPC;Apc mouse. *(a) WL image of colon in CPC;Apc mouse shows the presence of polyp (arrow). (b) NIR fluorescence image after topical administration of QRH\*-Cy5.5 shows increased intensity from polyp (arrow) and several flat lesions (arrowheads). (c) Image with Cy5.5-control peptide (PEH\*-Cy5.5) shows minimal signal. (d) WL image shows no grossly visible lesions (polyps). (e) NIR fluorescence image with QRH\*-Cy5.5 shows the presence of flat lesions (arrowheads). (f) Image with Cy5.5 control peptide shows minimal signal. Used with permission [49].*

microscopy. The peptide showed an apparent dissociation constant of Kd = 72 nM and an apparent association time constant of K = 0.174/minute. Toxicity was also assessed in rats. In the clinical safety study, fluorescently labeled peptide was topically administered, using a spray catheter, to the proximal colon of 25 subjects undergoing routine outpatient colonoscopies. Subsequently, endoscopists resected identified lesions, which were analyzed histologically by gastrointestinal pathologists. Fluorescence intensities of SSAs were compared with those of normal colonic mucosa. During fluorescence imaging of patients during endoscopy, regions of SSA had 2.43-fold higher mean fluorescence intensity than that for normal colonic mucosa. Fluorescence labeling distinguished SSAs from normal colonic mucosa with 89% sensitivity and 92% specificity.

#### *5.4.2 Confocal laser endomicroscopy*

Confocal laser endomicroscopy (CLE) is an endoscopic modality that based on tissue illumination using a low power laser and the subsequent detection of fluorescent light that is reflected back from the tissue through a pinhole. The term "confocal" refers to the alignment of both illumination and collection systems in the same plane. This alignment dramatically increases the spatial resolution of CLE and enables cellular imaging and evaluation of tissue architecture at the focal plane. CLE can be used to guide biopsies and has been demonstrated in a number of clinical studies to detect cancer in the digestive tract, bladder, cervix, ovary, oral cavity, and lungs. CLE requires the use of a fluorescent contrast agent to enhance visualization of cells. Contrast agents can be administered intravenously or topically. Intravenous fluorescein sodium and acriflavine are widely used contrast agents.

**123**

**Figure 6.**

*200 μm. Used with permission [50].*

*Targeting Peptides Derived from Phage Display for Clinical Imaging*

derived from phage display are summarized below.

CLE can be performed shortly following injection with its fluorescence lasting approximately 30 minutes. However, due to the lack of specificity of conventional contrast agents, there is an increasing use of tissue-specific binding molecular probes in CLE. The studies that used CLE in combination with a targeting peptide

In another study, Qiu and team demonstrated vertical cross-sectional (XZ-plane) images of NIR fluorescence with a handheld dual-axis confocal endomicroscope that revealed a specific binding of a Cy5.5-labeled peptide (LTTHYKLGGGSK-Cy5.5) to premalignant colonic mucosa in mice [50]. This targeting peptide was selected using in vivo phage display technology in a *CPC;Apc* mouse model which developed adenomas spontaneously in the distal colon. NIR vertical cross-sectional fluorescence images of fresh mouse colonic mucosa demonstrate histology-like imaging performance as shown in **Figure 6**. The peptide showed specific binding and distinguished premalignant colonic mucosa from

Gastric cancer vessels may have many differentiating characteristics compared to normal vessels. However, identification of gastric cancer vascular endothelial cells is difficult. Co-culture of gastric cancer cells and vascular endothelial cells was suggested to simulate gastric solid tumor mass. In a study, GEBP11, a nine amino

*Vertical cross-sectional image of colonic dysplasia. (a) Chemical structure of LTTHYKL peptide with GGGSK linker and Cy5.5 fluorophore. (b) NIR fluorescence image from CPC; Apc mouse colon ex vivo shows vertically oriented dysplastic crypts. (c) The border between normal colonic mucosa and dysplasia shows increased contrast from specific binding of the LTT\*-Cy5.5 peptide. (d) Corresponding histology (H&E), scale bar* 

Hsiung et al. used an M13 phage library to identify peptides that would specifically bind dysplastic colonic mucosa from fresh human colonic biopsies [39]. A peptide (VRPMPLQ ) that bound to colonic dysplasia was identified, synthesized, and conjugated with fluorescein for in vivo testing in a pilot study in patients undergoing routine colonoscopy using a flexible-fibered confocal microscope. Fluorescence images and videos of bound topically administered peptide collected in vivo showed that the selected peptide bound more strongly to dysplastic colonocytes than to the adjacent normal mucosa in the same subject with 81% sensitivity

*DOI: http://dx.doi.org/10.5772/intechopen.84281*

and 82% specificity.

normal mucosa.

#### *Targeting Peptides Derived from Phage Display for Clinical Imaging DOI: http://dx.doi.org/10.5772/intechopen.84281*

*Bacteriophages - Perspectives and Future*

microscopy. The peptide showed an apparent dissociation constant of Kd = 72 nM and an apparent association time constant of K = 0.174/minute. Toxicity was also assessed in rats. In the clinical safety study, fluorescently labeled peptide was topically administered, using a spray catheter, to the proximal colon of 25 subjects undergoing routine outpatient colonoscopies. Subsequently, endoscopists resected identified lesions, which were analyzed histologically by gastrointestinal pathologists. Fluorescence intensities of SSAs were compared with those of normal colonic mucosa. During fluorescence imaging of patients during endoscopy, regions of SSA had 2.43-fold higher mean fluorescence intensity than that for normal colonic mucosa. Fluorescence labeling distinguished SSAs from normal colonic mucosa

In vivo imaging of colon in CPC;Apc mouse. *(a) WL image of colon in CPC;Apc mouse shows the presence of polyp (arrow). (b) NIR fluorescence image after topical administration of QRH\*-Cy5.5 shows increased intensity from polyp (arrow) and several flat lesions (arrowheads). (c) Image with Cy5.5-control peptide (PEH\*-Cy5.5) shows minimal signal. (d) WL image shows no grossly visible lesions (polyps). (e) NIR fluorescence image with QRH\*-Cy5.5 shows the presence of flat lesions (arrowheads). (f) Image with Cy5.5-*

Confocal laser endomicroscopy (CLE) is an endoscopic modality that based on tissue illumination using a low power laser and the subsequent detection of fluorescent light that is reflected back from the tissue through a pinhole. The term "confocal" refers to the alignment of both illumination and collection systems in the same plane. This alignment dramatically increases the spatial resolution of CLE and enables cellular imaging and evaluation of tissue architecture at the focal plane. CLE can be used to guide biopsies and has been demonstrated in a number of clinical studies to detect cancer in the digestive tract, bladder, cervix, ovary, oral cavity, and lungs. CLE requires the use of a fluorescent contrast agent to enhance visualization of cells. Contrast agents can be administered intravenously or topically. Intravenous fluorescein sodium and acriflavine are widely used contrast agents.

with 89% sensitivity and 92% specificity.

*control peptide shows minimal signal. Used with permission [49].*

*5.4.2 Confocal laser endomicroscopy*

**122**

**Figure 5.**

CLE can be performed shortly following injection with its fluorescence lasting approximately 30 minutes. However, due to the lack of specificity of conventional contrast agents, there is an increasing use of tissue-specific binding molecular probes in CLE. The studies that used CLE in combination with a targeting peptide derived from phage display are summarized below.

Hsiung et al. used an M13 phage library to identify peptides that would specifically bind dysplastic colonic mucosa from fresh human colonic biopsies [39]. A peptide (VRPMPLQ ) that bound to colonic dysplasia was identified, synthesized, and conjugated with fluorescein for in vivo testing in a pilot study in patients undergoing routine colonoscopy using a flexible-fibered confocal microscope. Fluorescence images and videos of bound topically administered peptide collected in vivo showed that the selected peptide bound more strongly to dysplastic colonocytes than to the adjacent normal mucosa in the same subject with 81% sensitivity and 82% specificity.

In another study, Qiu and team demonstrated vertical cross-sectional (XZ-plane) images of NIR fluorescence with a handheld dual-axis confocal endomicroscope that revealed a specific binding of a Cy5.5-labeled peptide (LTTHYKLGGGSK-Cy5.5) to premalignant colonic mucosa in mice [50]. This targeting peptide was selected using in vivo phage display technology in a *CPC;Apc* mouse model which developed adenomas spontaneously in the distal colon. NIR vertical cross-sectional fluorescence images of fresh mouse colonic mucosa demonstrate histology-like imaging performance as shown in **Figure 6**. The peptide showed specific binding and distinguished premalignant colonic mucosa from normal mucosa.

Gastric cancer vessels may have many differentiating characteristics compared to normal vessels. However, identification of gastric cancer vascular endothelial cells is difficult. Co-culture of gastric cancer cells and vascular endothelial cells was suggested to simulate gastric solid tumor mass. In a study, GEBP11, a nine amino

#### **Figure 6.**

*Vertical cross-sectional image of colonic dysplasia. (a) Chemical structure of LTTHYKL peptide with GGGSK linker and Cy5.5 fluorophore. (b) NIR fluorescence image from CPC; Apc mouse colon ex vivo shows vertically oriented dysplastic crypts. (c) The border between normal colonic mucosa and dysplasia shows increased contrast from specific binding of the LTT\*-Cy5.5 peptide. (d) Corresponding histology (H&E), scale bar 200 μm. Used with permission [50].*

acid vascular homing peptide, was screened and identified using Ph.D.C7C phage display peptide library kit panning against Co-HUVECS cells [51]. Liu et al. used FITC-GEBP11 to identify gastric cancer in mouse model [52]. A whole-body fluorescent imaging was first used to screen for the strongest specific fluorescent signal in xenograft models after tail vein injection of FITC-GEBP11. A specific signal was observed in both subcutaneous and orthotopic xenograft models in vivo, whereas the group injected with FITC-URP, a control peptide, showed no fluorescent signals. In addition, neoplastic and nonneoplastic gastric mucosae obtained from the patients were incubated with FITC-GEBP11 or FITC-URP for 30 minutes and were scanned with CLE. A specific signal of GEBP11 was observed in 26/28 neoplastic human specimens and in 8/28 samples of nonneoplastic specimens (p < 0.01).

Sturm and team developed a peptide (ASYNYDA) that binds specifically to high-grade esophageal dysplasia and adenocarcinoma using phage display technology [53]. After peptide specific binding validation in human esophageal cancer specimens, they applied peptide topically and performed confocal endomicroscopy in 25 patients. The targeting peptide showed 3.8-fold greater fluorescence intensity for esophageal neoplasia compared with Barrett's esophagus and squamous epithelium with high sensitivity and specificity. The peptide revealed no toxicity in animals or patients.

In a pilot study, Palma and team identified dysplasia lesions in ulcerative colitis (UC) patients using CLE and a fluorescently labeled peptide [54]. A phage-derived peptide (VRPMPLQ ) was synthesized and conjugated with fluorescein. Eleven suspected dysplasia specimens were collected from nine UC patients. Specimens were stained with the peptide and subsequently inspected by CLE. The CLE images were correlated to histological results from specialists. The peptide showed a different pattern on dysplastic mucosa compared to nondysplastic lesions. However, due to several restrictions of this study, further studies on larger UC patients are required for systematic validation.

ErbB2 expression in early breast cancer can predict tumor aggressiveness and clinical outcomes in patients. Up to 30% of all breast cancers express ErbB2, also known as HER2. Immunohistochemistry is commonly used to evaluate ErbB2 expression, but it has limitations due to tumor heterogeneity. Therefore, the use of a specific biomarker for ErbB2 is increasingly popular. One study used a NIR-labeled ErbB2 peptide and a handheld dual-axis CLE to detect in human xenograft breast tumors and human specimens [55]. As a result, they found significantly greater peptide binding to xenograft breast cancer in vivo and to human specimens of invasive ductal carcinoma that express ErbB2 ex vivo. From the data, a miniature dual-axis confocal fluorescence endomicroscope with ErbB2-specific peptide could be implemented to support future image-guided surgery.

#### **5.5 Multimodality imaging**

Radiation-induced pulmonary fibrosis (RIPF) is a serious side effect of radiation therapy, especially in lung and breast cancers. Computed tomography (CT) imaging is currently utilized to identify and monitor RIPF. However, anatomical change interference is a major limitation of CT. Therefore, RIPF detection and observing techniques need to be improved. Collagen accumulation is common in fibrosis. In one study, a collagen-targeting peptide was fabricated to maximize the visualization of fibrosis using fluorescence endomicroscope imaging [37]. The probe showed moderate binding ability to collagen in a fibrosis in vitro binding assay and on lung tissue specimens. The probe showed a similar binding pattern on lung specimens compared to antibody. But its sensitivity was not as good as the collagen-binding antibody.

**125**

*Targeting Peptides Derived from Phage Display for Clinical Imaging*

In another study, Zhang and team evaluated the potential applicability of GEBP11 peptides in gastric cancer diagnosis and radiotherapy. They developed iodine 131-labeled GEBP11 peptides and derivatives [56]. The clinical potential of GEBP11 peptides was determined with multimodality imaging methods. Cerenkov and SPECT imaging showed significantly higher tumor uptake for 131I-2PEG- (GEBP11)3 trimer compared to monomer. Higher tumor accumulation and better T/B ratio of 131I-2PEG-(GEBP11)3 trimer were observed. Treating with 131I-2PEG- (GEBP11)3 trimer exhibited a significant tumor growth suppression compared to control and monomer groups. The tumor volume and vasculature decreased significantly after treatment with 131I-2PEG-(GEBP11)3 trimer, resulting in prolonged survival time. In addition, 131I-2PEG-(GEBP11)3 trimer showed no significant hepatic or renal toxicity. In conclusion, 131I-2PEG-(GEBP11)3 trimer could be a potential ligand used to identify gastric cancer and in antiangiogenic therapy.

The discovery and development of therapeutic drugs and diagnostic probes are a time-consuming, expensive, and complex process. The processes involve experts from a wide range of disciplines such as medicinal chemistry, biochemistry, molecular biology, medicine, and pharmacology. It has been estimated that from about 10,000 new chemical entities identified, only one will reach the market in an average time of 16 years. Phage display, and particularly peptide phage display, has played a major role in the development pipeline for bringing peptide therapeutics into the clinic. Phage-derived peptides play an important role in disease detection and therapy, including in clinical imaging. The potential of peptides in preclinical and clinical molecular imaging is tremendous. Molecular imaging offers invaluable opportunities to explore complex disease-related biological processes at the molecular level in vivo. The emergence of current molecular imaging technologies is dependent not only on the progress of imaging systems but, more importantly, also on molecular imaging probes. Peptide-based imaging has now become an established approach in nuclear imaging, and its application is expanding to other imaging modalities. Considering the emergence of novel library designs and innovative selection strategies, we are confident that phage-derived peptides will continue to be promising biomarkers for early cancer detection, in metabolic abnormalities,

*DOI: http://dx.doi.org/10.5772/intechopen.84281*

**6. Future prospects**

and in personalized medicine.

*Targeting Peptides Derived from Phage Display for Clinical Imaging DOI: http://dx.doi.org/10.5772/intechopen.84281*

In another study, Zhang and team evaluated the potential applicability of GEBP11 peptides in gastric cancer diagnosis and radiotherapy. They developed iodine 131-labeled GEBP11 peptides and derivatives [56]. The clinical potential of GEBP11 peptides was determined with multimodality imaging methods. Cerenkov and SPECT imaging showed significantly higher tumor uptake for 131I-2PEG- (GEBP11)3 trimer compared to monomer. Higher tumor accumulation and better T/B ratio of 131I-2PEG-(GEBP11)3 trimer were observed. Treating with 131I-2PEG- (GEBP11)3 trimer exhibited a significant tumor growth suppression compared to control and monomer groups. The tumor volume and vasculature decreased significantly after treatment with 131I-2PEG-(GEBP11)3 trimer, resulting in prolonged survival time. In addition, 131I-2PEG-(GEBP11)3 trimer showed no significant hepatic or renal toxicity. In conclusion, 131I-2PEG-(GEBP11)3 trimer could be a potential ligand used to identify gastric cancer and in antiangiogenic therapy.

#### **6. Future prospects**

*Bacteriophages - Perspectives and Future*

animals or patients.

for systematic validation.

**5.5 Multimodality imaging**

acid vascular homing peptide, was screened and identified using Ph.D.C7C phage display peptide library kit panning against Co-HUVECS cells [51]. Liu et al. used FITC-GEBP11 to identify gastric cancer in mouse model [52]. A whole-body fluorescent imaging was first used to screen for the strongest specific fluorescent signal in xenograft models after tail vein injection of FITC-GEBP11. A specific signal was observed in both subcutaneous and orthotopic xenograft models in vivo, whereas the group injected with FITC-URP, a control peptide, showed no fluorescent signals. In addition, neoplastic and nonneoplastic gastric mucosae obtained from the patients were incubated with FITC-GEBP11 or FITC-URP for 30 minutes and were scanned with CLE. A specific signal of GEBP11 was observed in 26/28 neoplastic human specimens and in 8/28 samples of nonneoplastic specimens (p < 0.01). Sturm and team developed a peptide (ASYNYDA) that binds specifically to high-grade esophageal dysplasia and adenocarcinoma using phage display technology [53]. After peptide specific binding validation in human esophageal cancer specimens, they applied peptide topically and performed confocal endomicroscopy in 25 patients. The targeting peptide showed 3.8-fold greater fluorescence intensity for esophageal neoplasia compared with Barrett's esophagus and squamous epithelium with high sensitivity and specificity. The peptide revealed no toxicity in

In a pilot study, Palma and team identified dysplasia lesions in ulcerative colitis (UC) patients using CLE and a fluorescently labeled peptide [54]. A phage-derived peptide (VRPMPLQ ) was synthesized and conjugated with fluorescein. Eleven suspected dysplasia specimens were collected from nine UC patients. Specimens were stained with the peptide and subsequently inspected by CLE. The CLE images were correlated to histological results from specialists. The peptide showed a different pattern on dysplastic mucosa compared to nondysplastic lesions. However, due to several restrictions of this study, further studies on larger UC patients are required

ErbB2 expression in early breast cancer can predict tumor aggressiveness and clinical outcomes in patients. Up to 30% of all breast cancers express ErbB2, also known as HER2. Immunohistochemistry is commonly used to evaluate ErbB2 expression, but it has limitations due to tumor heterogeneity. Therefore, the use of a specific biomarker for ErbB2 is increasingly popular. One study used a NIR-labeled ErbB2 peptide and a handheld dual-axis CLE to detect in human xenograft breast tumors and human specimens [55]. As a result, they found significantly greater peptide binding to xenograft breast cancer in vivo and to human specimens of invasive ductal carcinoma that express ErbB2 ex vivo. From the data, a miniature dual-axis confocal fluorescence endomicroscope with ErbB2-specific peptide could

Radiation-induced pulmonary fibrosis (RIPF) is a serious side effect of radiation therapy, especially in lung and breast cancers. Computed tomography (CT) imaging is currently utilized to identify and monitor RIPF. However, anatomical change interference is a major limitation of CT. Therefore, RIPF detection and observing techniques need to be improved. Collagen accumulation is common in fibrosis. In one study, a collagen-targeting peptide was fabricated to maximize the visualization of fibrosis using fluorescence endomicroscope imaging [37]. The probe showed moderate binding ability to collagen in a fibrosis in vitro binding assay and on lung tissue specimens. The probe showed a similar binding pattern on lung specimens compared to antibody. But its sensitivity was not as good as the collagen-binding

be implemented to support future image-guided surgery.

**124**

antibody.

The discovery and development of therapeutic drugs and diagnostic probes are a time-consuming, expensive, and complex process. The processes involve experts from a wide range of disciplines such as medicinal chemistry, biochemistry, molecular biology, medicine, and pharmacology. It has been estimated that from about 10,000 new chemical entities identified, only one will reach the market in an average time of 16 years. Phage display, and particularly peptide phage display, has played a major role in the development pipeline for bringing peptide therapeutics into the clinic. Phage-derived peptides play an important role in disease detection and therapy, including in clinical imaging. The potential of peptides in preclinical and clinical molecular imaging is tremendous. Molecular imaging offers invaluable opportunities to explore complex disease-related biological processes at the molecular level in vivo. The emergence of current molecular imaging technologies is dependent not only on the progress of imaging systems but, more importantly, also on molecular imaging probes. Peptide-based imaging has now become an established approach in nuclear imaging, and its application is expanding to other imaging modalities. Considering the emergence of novel library designs and innovative selection strategies, we are confident that phage-derived peptides will continue to be promising biomarkers for early cancer detection, in metabolic abnormalities, and in personalized medicine.

#### **Author details**

Supang Khondee1 and Wibool Piyawattanametha2,3\*

1 School of Pharmaceutical Sciences, University of Phayao, Phayao, Thailand

2 Faculty of Engineering, Department of Biomedical Engineering, King Mongkut's Institute of Technology Ladkrabang (KMITL), Bangkok, Thailand

3 Institute for Quantitative Health Science and Engineering (IQ ), Michigan State University, Michigan, USA

\*Address all correspondence to: wibool@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**127**

*Targeting Peptides Derived from Phage Display for Clinical Imaging*

multimeric RGD peptides for targeting tumors. Journal of Medicinal Chemistry.

[11] Comeau AM et al. Exploring the prokaryotic virosphere. Research in Microbiology. 2008;**159**(5):306-313

[12] Ofir G, Sorek R. Contemporary

[13] Drulis-Kawa Z, Majkowska-Skrobek G, Maciejewska

B. Bacteriophages and phage-derived proteins, application approaches. Current Medicinal Chemistry.

[14] Murphy FA et al. Virus Taxonomy: Classification and Nomenclature of Viruses. Vol. 10. New York: Springer Science & Business Media; 2012

[15] Russel M, Lowman HB, Clackson T. Introduction to phage biology and phage display. In: Phage Display: A Practical Approach. New York: Oxford University

[16] Barbas CF, et al. Phage Display: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press; 2001.

[17] Pal G, Fellouse FA. Methods for the construction of phage-displayed libraries. In: Phage Display in Biotechnology and Drug Discovery. Florida: CRC Press; 2005. pp. 131-162

[18] Zeri AC et al. Structure of the coat protein in fd filamentous bacteriophage particles determined by solid-state NMR spectroscopy. Proceedings of the National Academy of Sciences.

2003;**100**(11):6458-6463

[19] Bratkovic T. Progress in phage display: Evolution of the technique and

phage biology: From classic models to new insights. Cell. 2018;**172**(6):1260-1270

2015;**22**(14):1757-1773

Press; 2004. pp. 1-26

pp. 1-24

2006;**49**(7):2268-2275

*DOI: http://dx.doi.org/10.5772/intechopen.84281*

[1] Lee S, Xie J, Chen X. Peptidebased probes for targeted

molecular imaging. Biochemistry.

[3] Wu AM, Olafsen T. Antibodies for molecular imaging of cancer. The Cancer Journal. 2008;**14**(3):191-197

[4] Derda R et al. Diversity of phagedisplayed libraries of peptides during panning and amplification. Molecules.

[5] Smith GP. Filamentous fusion phage: Novel expression vectors that display cloned antigens on the virion surface. Science. 1985;**228**(4705):1315-1317

[6] Zhang ZF et al. Screening and selection of peptides specific for esophageal cancer cells from a phage display peptide library. Journal of Cardiothoracic Surgery. 2014;**9**(1):76

[7] Witt H et al. Identification of a rhabdomyosarcoma targeting peptide by phage display with sequence similarities to the tumour lymphatic-homing peptide LyP-1. International Journal of Cancer.

[8] Hui X et al. Specific targeting of the vasculature of gastric cancer by a new tumor-homing peptide CGNSNPKSC. Journal of Controlled

[9] Wang W et al. Near-infrared optical imaging of integrin αvβ3 in human tumor xenografts. Molecular Imaging.

[10] Ye Y et al. Design, synthesis, and evaluation of near infrared fluorescent

2009;**124**(9):2026-2032

Release. 2008;**131**(2):86-93

2004;**3**(4):15353500200404148

[2] De Jong M et al. Tumor imaging and therapy using radiolabeled somatostatin analogues. Accounts of Chemical Research. 2009;**42**(7):873-880

2010;**49**(7):1364-1376

**References**

2011;**16**(2):1776-1803

*Targeting Peptides Derived from Phage Display for Clinical Imaging DOI: http://dx.doi.org/10.5772/intechopen.84281*

#### **References**

*Bacteriophages - Perspectives and Future*

**126**

**Author details**

Supang Khondee1

University, Michigan, USA

provided the original work is properly cited.

\*Address all correspondence to: wibool@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

and Wibool Piyawattanametha2,3\*

Institute of Technology Ladkrabang (KMITL), Bangkok, Thailand

1 School of Pharmaceutical Sciences, University of Phayao, Phayao, Thailand

2 Faculty of Engineering, Department of Biomedical Engineering, King Mongkut's

3 Institute for Quantitative Health Science and Engineering (IQ ), Michigan State

[1] Lee S, Xie J, Chen X. Peptidebased probes for targeted molecular imaging. Biochemistry. 2010;**49**(7):1364-1376

[2] De Jong M et al. Tumor imaging and therapy using radiolabeled somatostatin analogues. Accounts of Chemical Research. 2009;**42**(7):873-880

[3] Wu AM, Olafsen T. Antibodies for molecular imaging of cancer. The Cancer Journal. 2008;**14**(3):191-197

[4] Derda R et al. Diversity of phagedisplayed libraries of peptides during panning and amplification. Molecules. 2011;**16**(2):1776-1803

[5] Smith GP. Filamentous fusion phage: Novel expression vectors that display cloned antigens on the virion surface. Science. 1985;**228**(4705):1315-1317

[6] Zhang ZF et al. Screening and selection of peptides specific for esophageal cancer cells from a phage display peptide library. Journal of Cardiothoracic Surgery. 2014;**9**(1):76

[7] Witt H et al. Identification of a rhabdomyosarcoma targeting peptide by phage display with sequence similarities to the tumour lymphatic-homing peptide LyP-1. International Journal of Cancer. 2009;**124**(9):2026-2032

[8] Hui X et al. Specific targeting of the vasculature of gastric cancer by a new tumor-homing peptide CGNSNPKSC. Journal of Controlled Release. 2008;**131**(2):86-93

[9] Wang W et al. Near-infrared optical imaging of integrin αvβ3 in human tumor xenografts. Molecular Imaging. 2004;**3**(4):15353500200404148

[10] Ye Y et al. Design, synthesis, and evaluation of near infrared fluorescent multimeric RGD peptides for targeting tumors. Journal of Medicinal Chemistry. 2006;**49**(7):2268-2275

[11] Comeau AM et al. Exploring the prokaryotic virosphere. Research in Microbiology. 2008;**159**(5):306-313

[12] Ofir G, Sorek R. Contemporary phage biology: From classic models to new insights. Cell. 2018;**172**(6):1260-1270

[13] Drulis-Kawa Z, Majkowska-Skrobek G, Maciejewska B. Bacteriophages and phage-derived proteins, application approaches. Current Medicinal Chemistry. 2015;**22**(14):1757-1773

[14] Murphy FA et al. Virus Taxonomy: Classification and Nomenclature of Viruses. Vol. 10. New York: Springer Science & Business Media; 2012

[15] Russel M, Lowman HB, Clackson T. Introduction to phage biology and phage display. In: Phage Display: A Practical Approach. New York: Oxford University Press; 2004. pp. 1-26

[16] Barbas CF, et al. Phage Display: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press; 2001. pp. 1-24

[17] Pal G, Fellouse FA. Methods for the construction of phage-displayed libraries. In: Phage Display in Biotechnology and Drug Discovery. Florida: CRC Press; 2005. pp. 131-162

[18] Zeri AC et al. Structure of the coat protein in fd filamentous bacteriophage particles determined by solid-state NMR spectroscopy. Proceedings of the National Academy of Sciences. 2003;**100**(11):6458-6463

[19] Bratkovic T. Progress in phage display: Evolution of the technique and its applications. Cellular and Molecular Life Sciences. 2010;**67**(5):749-767

[20] Pande J, Szewczyk MM, Grover AK. Phage display: Concept, innovations, applications and future. Biotechnology Advances. 2010;**28**(6):849-858

[21] Hamzeh-Mivehroud M, Mahmoudpour A, Dastmalchi S. Identification of new peptide ligands for epidermal growth factor receptor using phage display and computationally modeling their mode of binding. Chemical Biology & Drug Design. 2012;**79**(3):246-259

[22] Matz J, Chames P. Phage display and selections on purified antigens. In: Antibody Engineering. Heidelberg: Springer; 2012. pp. 213-224

[23] Smolarek D, Bertrand O, Czerwinski M. Variable fragments of heavy chain antibodies (VHHs): A new magic bullet molecule of medicine? Advances in Hygiene & Experimental Medicine. 2012;**66**:348-358

[24] Kolonin MG et al. Synchronous selection of homing peptides for multiple tissues by in vivo phage display. The FASEB Journal. 2006;**20**(7):979-981

[25] Ludtke JJ et al. In vivo selection and validation of liver-specific ligands using a new T7 phage peptide display system. Drug Delivery. 2007;**14**(6):357-369

[26] Molenaar TJ et al. Uptake and processing of modified bacteriophage M13 in mice: Implications for phage display. Virology. 2002;**293**(1):182-191

[27] Wu M et al. Mapping alveolar binding sites in vivo using phage peptide libraries. Gene Therapy. 2003;**10**(17):1429

[28] Arinaminpathy Y et al. Computational analysis of membrane proteins: The largest class of drug

targets. Drug Discovery Today. 2009;**14**(23-24):1130-1135

[29] Jones ML et al. Targeting membrane proteins for antibody discovery using phage display. Scientific Reports. 2016;**6**:26240

[30] Alfaleh MA et al. Strategies for selecting membrane protein-specific antibodies using phage display with cell-based panning. Antibodies. 2017;**6**(3):10

[31] Schooltink H, Rose-John S. Designing cytokine variants by phagedisplay. Combinatorial Chemistry & High Throughput Screening. 2005;**8**(2):173-179

[32] Westwater C et al. Use of genetically engineered phage to deliver antimicrobial agents to bacteria: An alternative therapy for treatment of bacterial infections. Antimicrobial Agents and Chemotherapy. 2003;**47**(4):1301-1307

[33] Lau JL, Dunn MK. Therapeutic peptides: Historical perspectives, current development trends, and future directions. Bioorganic & Medicinal Chemistry. 2017;**26**:2700-2707

[34] Rothe A, Hosse RJ, Power BE. In vitro display technologies reveal novel biopharmaceutics. The FASEB Journal. 2006;**20**(10):1599-1610

[35] Zuraw, B, Yasothan U, Kirkpatrick P. Ecallantide. Nature Reviews Drug Discovery. 2010;**9**:189-190

[36] Froidevaux S, Eberle AN. Somatostatin analogs and radiopeptides in cancer therapy. Peptide Science. 2002;**66**(3):161-183

[37] Caravan P et al. Collagen-targeted MRI contrast agent for molecular imaging of fibrosis. Angewandte Chemie International Edition. 2007;**46**(43):8171-8173

**129**

*Targeting Peptides Derived from Phage Display for Clinical Imaging*

[47] Miller SJ et al. In vivo fluorescence-

based endoscopic detection of colon dysplasia in the mouse using a novel peptide probe. PLoS One.

[48] Burggraaf J et al. Detection of colorectal polyps in humans using an intravenously administered fluorescent peptide targeted against c-Met. Nature

[49] Zhou J et al. EGFR overexpressed in colonic neoplasia can be detected on wide-field endoscopic imaging. Clinical and Translational Gastroenterology.

[50] Qiu Z et al. Targeted vertical crosssectional imaging with handheld nearinfrared dual axes confocal fluorescence endomicroscope. Biomedical Optics

Medicine. 2015;**21**(8):955

Express. 2013;**4**(2):322-330

2006;**84**(9):764-773

[51] Liang S et al. Screening and identification of vascular-endothelialcell-specific binding peptide in gastric cancer. Journal of Molecular Medicine.

[52] Liu L et al. In vivo molecular imaging of gastric cancer in humanmurine xenograft models with confocal laser endomicroscopy using a tumor vascular homing peptide. Cancer Letters. 2015;**356**(2):891-898

[53] Sturm MB et al. Targeted imaging of esophageal neoplasia with a fluorescently labeled peptide: First-in-human results. Science Translational Medicine. 2013;**5**(184):184ra61-184ra61

[54] De Palma GD et al. Detection of colonic dysplasia in patients with ulcerative colitis using a targeted fluorescent peptide and confocal laser endomicroscopy: A pilot study. PLoS

[55] Gao Z et al. In vivo near-infrared

One. 2017;**12**(6):e018050e9

imaging of ErbB2 expressing

2011;**6**(3):e17384

2015;**6**(7):e101

*DOI: http://dx.doi.org/10.5772/intechopen.84281*

[38] Rossez Y et al. Early detection of colonic dysplasia by magnetic resonance molecular imaging with a contrast agent raised against the colon cancer marker MUC5AC. Contrast Media & Molecular Imaging.

[39] Hsiung PL et al. Detection of colonic dysplasia in vivo using a targeted heptapeptide and confocal microendoscopy. Nature Medicine.

2013;**12**(5):7290.2012. 00047

[41] Li ZJ et al. A novel peptide specifically targeting the vasculature of orthotopic colorectal cancer for imaging detection and drug delivery. Journal of Controlled Release.

[42] Liu Z et al. Characterization of TCP-1 probes for molecular imaging of colon cancer. Journal of Controlled

[43] Zhou Q et al. In vivo photoacoustic tomography of EGFR overexpressed

[44] Snover DC. Update on the serrated pathway to colorectal carcinoma. Human Pathology. 2011;**42**(1):1-10

[45] Joshi BP et al. Detection of sessile serrated adenomas in the proximal colon using wide-field fluorescence endoscopy. Gastroenterology. 2017;**152**(5):1002-1013. e9

[46] Elahi SF et al. Targeted imaging of colorectal dysplasia in living mice with fluorescence microendoscopy.

Biomedical Optics Express.

2011;**2**(4):981-986

2010;**148**(3):292-302

Release. 2016;**239**:223-230

in hepatocellular carcinoma mouse xenograft. Photoacoustics.

2016;**4**(2):43-54

[40] Shi J et al. Technetium 99m–labeled VQ peptide: A new imaging agent for the early detection of tumors or premalignancies. Molecular Imaging.

2016;**11**(3):211-221

2008;**14**(4):454

*Targeting Peptides Derived from Phage Display for Clinical Imaging DOI: http://dx.doi.org/10.5772/intechopen.84281*

[38] Rossez Y et al. Early detection of colonic dysplasia by magnetic resonance molecular imaging with a contrast agent raised against the colon cancer marker MUC5AC. Contrast Media & Molecular Imaging. 2016;**11**(3):211-221

*Bacteriophages - Perspectives and Future*

its applications. Cellular and Molecular Life Sciences. 2010;**67**(5):749-767

targets. Drug Discovery Today. 2009;**14**(23-24):1130-1135

2016;**6**:26240

2017;**6**(3):10

2005;**8**(2):173-179

[29] Jones ML et al. Targeting membrane proteins for antibody discovery using phage display. Scientific Reports.

[30] Alfaleh MA et al. Strategies for selecting membrane protein-specific antibodies using phage display with cell-based panning. Antibodies.

[31] Schooltink H, Rose-John S. Designing cytokine variants by phagedisplay. Combinatorial Chemistry & High Throughput Screening.

[32] Westwater C et al. Use of

genetically engineered phage to deliver antimicrobial agents to bacteria: An alternative therapy for treatment of bacterial infections. Antimicrobial Agents and Chemotherapy. 2003;**47**(4):1301-1307

[33] Lau JL, Dunn MK. Therapeutic peptides: Historical perspectives, current development trends, and future directions. Bioorganic & Medicinal Chemistry. 2017;**26**:2700-2707

[34] Rothe A, Hosse RJ, Power BE. In vitro display technologies reveal novel biopharmaceutics. The FASEB Journal.

[35] Zuraw, B, Yasothan U, Kirkpatrick P. Ecallantide. Nature Reviews Drug

Somatostatin analogs and radiopeptides in cancer therapy. Peptide Science.

[37] Caravan P et al. Collagen-targeted MRI contrast agent for molecular imaging of fibrosis. Angewandte Chemie International Edition.

2006;**20**(10):1599-1610

Discovery. 2010;**9**:189-190

2002;**66**(3):161-183

2007;**46**(43):8171-8173

[36] Froidevaux S, Eberle AN.

Identification of new peptide ligands for epidermal growth factor receptor using phage display and computationally modeling their mode of binding. Chemical Biology & Drug Design.

[22] Matz J, Chames P. Phage display and selections on purified antigens. In: Antibody Engineering. Heidelberg:

Czerwinski M. Variable fragments of heavy chain antibodies (VHHs): A new magic bullet molecule of medicine? Advances in Hygiene & Experimental

[24] Kolonin MG et al. Synchronous selection of homing peptides for

multiple tissues by in vivo phage display. The FASEB Journal. 2006;**20**(7):979-981

[25] Ludtke JJ et al. In vivo selection and validation of liver-specific ligands using a new T7 phage peptide display system. Drug Delivery. 2007;**14**(6):357-369

[26] Molenaar TJ et al. Uptake and processing of modified bacteriophage M13 in mice: Implications for phage display. Virology. 2002;**293**(1):182-191

[27] Wu M et al. Mapping alveolar binding sites in vivo using phage peptide libraries. Gene Therapy.

Computational analysis of membrane proteins: The largest class of drug

[28] Arinaminpathy Y et al.

2003;**10**(17):1429

Springer; 2012. pp. 213-224

[23] Smolarek D, Bertrand O,

Medicine. 2012;**66**:348-358

[20] Pande J, Szewczyk MM, Grover AK. Phage display: Concept, innovations, applications and future. Biotechnology Advances.

2010;**28**(6):849-858

2012;**79**(3):246-259

[21] Hamzeh-Mivehroud M, Mahmoudpour A, Dastmalchi S.

**128**

[39] Hsiung PL et al. Detection of colonic dysplasia in vivo using a targeted heptapeptide and confocal microendoscopy. Nature Medicine. 2008;**14**(4):454

[40] Shi J et al. Technetium 99m–labeled VQ peptide: A new imaging agent for the early detection of tumors or premalignancies. Molecular Imaging. 2013;**12**(5):7290.2012. 00047

[41] Li ZJ et al. A novel peptide specifically targeting the vasculature of orthotopic colorectal cancer for imaging detection and drug delivery. Journal of Controlled Release. 2010;**148**(3):292-302

[42] Liu Z et al. Characterization of TCP-1 probes for molecular imaging of colon cancer. Journal of Controlled Release. 2016;**239**:223-230

[43] Zhou Q et al. In vivo photoacoustic tomography of EGFR overexpressed in hepatocellular carcinoma mouse xenograft. Photoacoustics. 2016;**4**(2):43-54

[44] Snover DC. Update on the serrated pathway to colorectal carcinoma. Human Pathology. 2011;**42**(1):1-10

[45] Joshi BP et al. Detection of sessile serrated adenomas in the proximal colon using wide-field fluorescence endoscopy. Gastroenterology. 2017;**152**(5):1002-1013. e9

[46] Elahi SF et al. Targeted imaging of colorectal dysplasia in living mice with fluorescence microendoscopy. Biomedical Optics Express. 2011;**2**(4):981-986

[47] Miller SJ et al. In vivo fluorescencebased endoscopic detection of colon dysplasia in the mouse using a novel peptide probe. PLoS One. 2011;**6**(3):e17384

[48] Burggraaf J et al. Detection of colorectal polyps in humans using an intravenously administered fluorescent peptide targeted against c-Met. Nature Medicine. 2015;**21**(8):955

[49] Zhou J et al. EGFR overexpressed in colonic neoplasia can be detected on wide-field endoscopic imaging. Clinical and Translational Gastroenterology. 2015;**6**(7):e101

[50] Qiu Z et al. Targeted vertical crosssectional imaging with handheld nearinfrared dual axes confocal fluorescence endomicroscope. Biomedical Optics Express. 2013;**4**(2):322-330

[51] Liang S et al. Screening and identification of vascular-endothelialcell-specific binding peptide in gastric cancer. Journal of Molecular Medicine. 2006;**84**(9):764-773

[52] Liu L et al. In vivo molecular imaging of gastric cancer in humanmurine xenograft models with confocal laser endomicroscopy using a tumor vascular homing peptide. Cancer Letters. 2015;**356**(2):891-898

[53] Sturm MB et al. Targeted imaging of esophageal neoplasia with a fluorescently labeled peptide: First-in-human results. Science Translational Medicine. 2013;**5**(184):184ra61-184ra61

[54] De Palma GD et al. Detection of colonic dysplasia in patients with ulcerative colitis using a targeted fluorescent peptide and confocal laser endomicroscopy: A pilot study. PLoS One. 2017;**12**(6):e018050e9

[55] Gao Z et al. In vivo near-infrared imaging of ErbB2 expressing

breast tumors with dual-axes confocal endomicroscopy using a targeted peptide. Scientific Reports. 2017;**7**(1):14404

[56] Zhang J et al. Targeted radiotherapy with tumor vascular homing trimeric GEBP11 peptide evaluated by multimodality imaging for gastric cancer. Journal of Controlled Release. 2013;**172**(1):322-329

*Bacteriophages - Perspectives and Future*

[56] Zhang J et al. Targeted radiotherapy with tumor vascular homing trimeric

breast tumors with dual-axes confocal endomicroscopy using a targeted peptide. Scientific Reports.

GEBP11 peptide evaluated by multimodality imaging for gastric cancer. Journal of Controlled Release.

2017;**7**(1):14404

2013;**172**(1):322-329

**130**

## *Edited by Renos Savva*

Bacteriophages are viruses that utilise bacterial cells as factories for their own propagation and as safe havens for their genomic material. They are capable of equipping bacteria with properties that bestow environmental advantages. They are also capable of specifically and efficiently killing bacteria.Bacteriophages are resilient in a wide diversity of environments, presumed to be as ancient as life itself, and are estimated to be the most numerous biological entities on the planet. Their overarching capacity to survive via molecular adaptation is supported by an arsenal of encoded enzymatic tools, which also enabled biotechnology.This volume includes contributions that describe bacteriophages as nanomachines, genetic engineers, and also as medicines and technologies of the future, including relevant production and process issues.

Published in London, UK © 2020 IntechOpen © iunewind / iStock

Bacteriophages - Perspectives and Future

Bacteriophages

Perspectives and Future

*Edited by Renos Savva*