**2.1 Platforms based on transgenic plants**

Transgenic plants have been the most widely used platforms for recombinant protein production. To obtain stable transgenic lines, the gene encoding the desired protein is cloned into an expression construct, which generally includes a promoter and regulatory elements that ensure efficient RNA processing and protein synthesis [21]. This expression construct is then stably integrated into the plant nuclear genome, resulting in the stable inheritance of the transgene and expression of stable pharmaceutical proteins over generations [22]. Two major transformation strategies have been employed to insert expression constructs into the nuclear genome: *Agrobacterium*mediated transformation in dicotyledonous species (dicots) and particle bombardment of DNA-coated gold or tungsten beads in monocotyledonous species (monocots) [3]. Transgenic plant lines offer several advantages as platforms for molecular pharming: they are suitable for long-term production of recombinant pharmaceutical proteins and are highly scalable, as each line can be used to produce seeds, which increase the number of plants in every generation. Ultimately, the production capacity of recombinant pharmaceutical proteins in transgenic plants is practically unlimited, as it only depends on the number of hectares available for the plant culture. The major drawbacks of transgenic plants are the long development and scale-up timescales, the unreliable production yields, and the potential spread of pharmaceutical crops in the environment and into the food chain by outcrossing and seed dispersal [3].

The development of simple transformation technologies has expanded the number of host plants available for molecular pharming. Currently, the major molecular pharming transgenic platforms are based on leafy crops, seeds, fruits, and vegetable crops. Leafy crops are benefic in terms of biomass yield and high soluble protein levels. Additionally, leaf harvesting does not need flowering and thus considerably reduces contamination through pollen or seed dispersal [23]. One disadvantage of leafy crops is that proteins are synthesized in an aqueous environment, which is more prone to protein degradation, resulting in lower production yields [24]. In fact, the mature leaves possess very large extra cytoplasmic vacuolar compartments containing various active proteolytic enzymes that are involved in the degradation of native and foreign proteins. This is particularly problematic in the case of therapeutic peptide production because short heterologous peptides have an inherent instability in plant cells [25]. In addition to the protein instability, the harvested material has limited shelf life and needs to be processed immediately after harvest.

Tobacco has been the most widely used leafy crop for molecular pharming. The major advantages of using tobacco to express pharmaceutical proteins are its high biomass yield, well-established technology for gene transfer and expression, yearround growth and harvesting, and the existence of large-scale infrastructure for processing [23]. However, the natural production of nicotine and other alkaloids in tobacco poses some safety issues in its use as a host system for heterologous protein production. Therefore, tobacco varieties with low nicotine and alkaloid levels have been produced to diminish the toxicity and overcome those safety issues. Recent studies have led to the approval of the first monoclonal antibody produced in

**53**

*Prospects for the Production of Recombinant Therapeutic Proteins and Peptides in Plants…*

transgenic tobacco plants, in phase I clinical trial [26]. Additionally, a 2018 publication reported the stable expression of adalimumab (a monoclonal antibody against *tumor necrosis factor-alpha (*TNF-α)) in tobacco plants [27]. Other leafy crops commonly used in molecular pharming include alfalfa and clover [19].

As an alternative to leafy crops, plant seeds have proven to be versatile hosts

for recombinant proteins of all types, including peptides or short and long polypeptides as well as complex, noncontiguous proteins like antibodies and other immunoglobulins [28]. The expression of proteins in seeds can overcome the shortcomings of leafy crops in terms of protein stability and storage. Seeds possess specialized storage compartments, such as protein bodies and vacuoles, which provide the appropriate biochemical environment for protein accumulation, thus protecting the proteins expressed in seeds from proteolytic degradation [29]. Reports have demonstrated that antibodies expressed in seeds remain stable for at least 3 years at room temperature without detectable loss of activity [30]. Furthermore, the small size of most seeds permits to achieve a high recombinant protein concentration in a small volume, which facilitates extraction and downstream processing and reduces the costs of the overall manufacturing process [31]. One essential property of seeds is dormancy, which not only permits the stability of recombinant proteins but also allows a complete decoupling of the cycle of cultivation from the processing and purification of the protein [28]. Finally, proteins expressed in the seed do not normally interfere with vegetative plant growth, and this strategy also reduces exposure to herbivores and other nontarget organisms such as microbes in the biosphere [21]. Several crops have been studied for seed-based production, including cereals, such as maize, rice, barley, and wheat; legumes, such as pea and soybean; and oilseeds such as safflower and rapeseed. Maize has several advantages for seed-based expression of proteins; it has the highest biomass yield among food crops, and it is easy to transform, in vitro manipulate, and scale up [24]. These potentialities were explored by Prodigene Inc. for the production of the first commercially available plant-made protein, avidin (a protein with affinity for biotin used in biochemical assays). Other maize-derived protein products developed by this company include β-glucuronidase, aprotinin, laccase, and trypsin [32]. Prodigene was the first company to demonstrate the commercial benefits of plant-based platforms and was also a forerunner in the study of the economic impact of downstream processing in molecular pharming, having developed several successful approaches to recover

intact and functional recombinant seeds from maize [3].

and collagen (pre-clinical stage).

Maize has also been used to produce recombinant pharmaceutical proteins, including enzymes, vaccines, and antibodies [32, 33]. One of the most notable therapeutic proteins produced in maize is Meristem Therapeutics' gastric lipase, an enzyme intended for the treatment of exocrine pancreatic insufficiency—a disease significantly affecting cystic fibrosis sufferers—that has completed phase II clinical trial. In addition to this enzyme, Meristem Therapeutics has developed two other maize-derived products, human lactoferrin (phase I clinical trial), whose intellectual property was later acquired by Ventria Bioscience (http://www.ventria.com/),

Rice is another leading platform for recombinant protein and peptide production. Similar to maize, rice is easy to transform and scale up, but unlike maize, rice is self-pollinating, which reduces the risk of horizontal gene flow. Ventria Bioscience, in its ExpressTec platform, has used rice to produce recombinant pharmaceutical proteins, including human albumin, transferrin, lactoferrin, lysozyme, and vaccines against human rabies and Lyme disease. Its lead therapeutic candidate VEN100, whose active ingredient is lactoferrin, has been shown to reduce significantly antibiotic-associated diarrhea in high-risk patients and recently completed phase

*DOI: http://dx.doi.org/10.5772/intechopen.84419*

### *Prospects for the Production of Recombinant Therapeutic Proteins and Peptides in Plants… DOI: http://dx.doi.org/10.5772/intechopen.84419*

transgenic tobacco plants, in phase I clinical trial [26]. Additionally, a 2018 publication reported the stable expression of adalimumab (a monoclonal antibody against *tumor necrosis factor-alpha (*TNF-α)) in tobacco plants [27]. Other leafy crops commonly used in molecular pharming include alfalfa and clover [19].

As an alternative to leafy crops, plant seeds have proven to be versatile hosts for recombinant proteins of all types, including peptides or short and long polypeptides as well as complex, noncontiguous proteins like antibodies and other immunoglobulins [28]. The expression of proteins in seeds can overcome the shortcomings of leafy crops in terms of protein stability and storage. Seeds possess specialized storage compartments, such as protein bodies and vacuoles, which provide the appropriate biochemical environment for protein accumulation, thus protecting the proteins expressed in seeds from proteolytic degradation [29]. Reports have demonstrated that antibodies expressed in seeds remain stable for at least 3 years at room temperature without detectable loss of activity [30]. Furthermore, the small size of most seeds permits to achieve a high recombinant protein concentration in a small volume, which facilitates extraction and downstream processing and reduces the costs of the overall manufacturing process [31]. One essential property of seeds is dormancy, which not only permits the stability of recombinant proteins but also allows a complete decoupling of the cycle of cultivation from the processing and purification of the protein [28]. Finally, proteins expressed in the seed do not normally interfere with vegetative plant growth, and this strategy also reduces exposure to herbivores and other nontarget organisms such as microbes in the biosphere [21]. Several crops have been studied for seed-based production, including cereals, such as maize, rice, barley, and wheat; legumes, such as pea and soybean; and oilseeds such as safflower and rapeseed. Maize has several advantages for seed-based expression of proteins; it has the highest biomass yield among food crops, and it is easy to transform, in vitro manipulate, and scale up [24]. These potentialities were explored by Prodigene Inc. for the production of the first commercially available plant-made protein, avidin (a protein with affinity for biotin used in biochemical assays). Other maize-derived protein products developed by this company include β-glucuronidase, aprotinin, laccase, and trypsin [32]. Prodigene was the first company to demonstrate the commercial benefits of plant-based platforms and was also a forerunner in the study of the economic impact of downstream processing in molecular pharming, having developed several successful approaches to recover intact and functional recombinant seeds from maize [3].

Maize has also been used to produce recombinant pharmaceutical proteins, including enzymes, vaccines, and antibodies [32, 33]. One of the most notable therapeutic proteins produced in maize is Meristem Therapeutics' gastric lipase, an enzyme intended for the treatment of exocrine pancreatic insufficiency—a disease significantly affecting cystic fibrosis sufferers—that has completed phase II clinical trial. In addition to this enzyme, Meristem Therapeutics has developed two other maize-derived products, human lactoferrin (phase I clinical trial), whose intellectual property was later acquired by Ventria Bioscience (http://www.ventria.com/), and collagen (pre-clinical stage).

Rice is another leading platform for recombinant protein and peptide production. Similar to maize, rice is easy to transform and scale up, but unlike maize, rice is self-pollinating, which reduces the risk of horizontal gene flow. Ventria Bioscience, in its ExpressTec platform, has used rice to produce recombinant pharmaceutical proteins, including human albumin, transferrin, lactoferrin, lysozyme, and vaccines against human rabies and Lyme disease. Its lead therapeutic candidate VEN100, whose active ingredient is lactoferrin, has been shown to reduce significantly antibiotic-associated diarrhea in high-risk patients and recently completed phase

*Genetic Engineering - A Glimpse of Techniques and Applications*

**2.1 Platforms based on transgenic plants**

processed immediately after harvest.

pharming encompasses platforms based on stably transformed whole-plants transgene insertion in the nuclear or plastid genome, transient expression using agroinfiltration, viral and hybrid vectors; microalgae and aquatic plants (e.g., duck-weed) stably transformed; and in vitro culture systems (e.g., cell suspensions, hairy roots, and moss protonema) [19]. Each platform has particular advantages and limitations; therefore its selection is done on a case-by-case basis, depending on economic considerations as well as on the product characteristics and intended use [20].

Transgenic plants have been the most widely used platforms for recombinant protein production. To obtain stable transgenic lines, the gene encoding the desired protein is cloned into an expression construct, which generally includes a promoter and regulatory elements that ensure efficient RNA processing and protein synthesis [21]. This expression construct is then stably integrated into the plant nuclear genome, resulting in the stable inheritance of the transgene and expression of stable pharmaceutical proteins over generations [22]. Two major transformation strategies have been employed to insert expression constructs into the nuclear genome: *Agrobacterium*mediated transformation in dicotyledonous species (dicots) and particle bombardment of DNA-coated gold or tungsten beads in monocotyledonous species (monocots)

[3]. Transgenic plant lines offer several advantages as platforms for molecular pharming: they are suitable for long-term production of recombinant pharmaceutical proteins and are highly scalable, as each line can be used to produce seeds, which increase the number of plants in every generation. Ultimately, the production capacity of recombinant pharmaceutical proteins in transgenic plants is practically unlimited, as it only depends on the number of hectares available for the plant culture. The major drawbacks of transgenic plants are the long development and scale-up timescales, the unreliable production yields, and the potential spread of pharmaceutical crops in the

environment and into the food chain by outcrossing and seed dispersal [3].

The development of simple transformation technologies has expanded the number of host plants available for molecular pharming. Currently, the major molecular pharming transgenic platforms are based on leafy crops, seeds, fruits, and vegetable crops. Leafy crops are benefic in terms of biomass yield and high soluble protein levels. Additionally, leaf harvesting does not need flowering and thus considerably reduces contamination through pollen or seed dispersal [23]. One disadvantage of leafy crops is that proteins are synthesized in an aqueous environment, which is more prone to protein degradation, resulting in lower production yields [24]. In fact, the mature leaves possess very large extra cytoplasmic vacuolar compartments containing various active proteolytic enzymes that are involved in the degradation of native and foreign proteins. This is particularly problematic in the case of therapeutic peptide production because short heterologous peptides have an inherent instability in plant cells [25]. In addition to the protein instability, the harvested material has limited shelf life and needs to be

Tobacco has been the most widely used leafy crop for molecular pharming. The major advantages of using tobacco to express pharmaceutical proteins are its high biomass yield, well-established technology for gene transfer and expression, yearround growth and harvesting, and the existence of large-scale infrastructure for processing [23]. However, the natural production of nicotine and other alkaloids in tobacco poses some safety issues in its use as a host system for heterologous protein production. Therefore, tobacco varieties with low nicotine and alkaloid levels have been produced to diminish the toxicity and overcome those safety issues. Recent studies have led to the approval of the first monoclonal antibody produced in

**52**

II clinical trial [34]. Rice has also been widely used as host for peptide expression, especially for the production of allergen peptides (e.g., pollen and mite allergies) [35, 36]. Recent studies report that rice has the potential to offer an oral delivery system for vaccine antigens and therapeutic proteins and peptides [25, 35, 37].

Barley seeds have also been developed as commercial platforms. In comparison to other cereal crops, barley is less widely grown. However, this fact added to the self-pollinating nature of barley can be viewed as an advantage since the risk of contamination and outcrossing with non-transgenic crops is minimized. Considering this benefit, an Iceland-based company, ORF Genetics (https://orfgenetics.com/), has targeted barley grain as the expression host for several human cytokines and growth factors [19]. Other molecular pharming companies, such as Ventria Bioscience and Maltagen, have also been developing barley-based production platforms. Although barley is still recognized for its recalcitrance to transformation, over the last decade some progress has been made in the development of reliable transformation procedures [38].

The use of legume seeds, such as soybean and pea, for the production of recombinant pharmaceutical proteins, has been less explored than cereal-based platforms, with platforms based on legume seeds having yet to achieve commercial success. However, the fact that legume seeds have exceptionally high protein content (20–40%) can be exploited to achieve high yields of recombinant protein [39]. Soybean seeds have been used to express recombinant growth factors [40, 41], coagulation factors [42], and vaccine peptides [43]. Transgenic pea seeds have been previously used to produce a single-chain Fv fragment (scFV) antibody used in cancer diagnosis and therapy [44]. In another study, pea seeds were used to produce a vaccine that showed high immunogenicity and protection against rabbit hemorrhagic disease virus [45].

Safflower and rapeseed seeds are rich in oil and are, thus, referred as oilseeds. Oilseeds can provide useful recombinant pharmaceutical protein production systems. SemBioSys (http://www.sembiosys.ca/), with its oleosin-fusion platform, has been a pioneer in that field. Oleosins are the principal membrane proteins of oil bodies; oleosins confer peculiar structural properties to the oil bodies that offer simple extraction and purification procedures [46]. In the oleosin-fusion platform the recombinant protein is fused with oleosin and consequently targeted to the oil bodies. The fusion protein is then recovered through simple purification of the oil bodies and separated from oleosin by endoprotease digestion. Commercial production of hirudin in safflower by SemBioSys constituted the first report of an oilseedderived protein [47]. The company has been focusing on safflower as its primary host ever since, with safflower-derived insulin being in phase I clinical trial [32].

Finally, fruit and vegetable crops can also be employed for molecular pharming. A major advantage of protein expression in fruit and vegetable crops is that edible organs can be consumed uncooked, unprocessed, or partially processed, making them particularly suitable for the production of recombinant subunit vaccines, nutraceuticals, and antibodies designed for topical application [29]. The oral delivery of recombinant therapeutics is one of the differentiating factor of molecular pharming in comparison to mainstream biopharmaceutical production systems, with several pharmaceutical products being produced in tomato fruits, potato tubers, and lettuce leaves for this purpose [3]. Tomato fruits are particularly useful for protein expression because the fruits are palatable as raw tissue but can also be lyophilized and stored for a long time [25]. Recently, human coagulation factor IX (hFIX) was expressed specifically in tomato fruits, constituting the first report on the expression of hFIX in plant [48]. Another study described the expression in tomato fruits of a thymosin α1 concatemer [49], an immune booster that plays an important role in the maturation, differentiation, and function of T cells.

**55**

system for oral drug delivery [57].

*Prospects for the Production of Recombinant Therapeutic Proteins and Peptides in Plants…*

The thymosin α1 concatemer derived from transgenic tomatoes exhibited biological activity and was proven to stimulate the proliferation of mice splenic lymphocytes in vitro. Moreover, thymosin α1 specific activity was higher when produced in tomato than in *Escherichia coli*, demonstrating the authenticity of the plant-made product. Other examples of tomato fruit expression include F1-V [50], a candidate subunit vaccine against pneumonic and bubonic plague, and β-secretase [51], to

In conclusion, platforms based on transgenic plants are a promising alternative to the conventional biopharmaceutical production platforms since they provide a stable source of pharmaceutical proteins and are also the most scalable of all molecular pharming platforms. This scalability of transgenic plants ensures the production of recombinant pharmaceutical proteins at levels previously inaccessible, namely, the commodity bulk production of monoclonal antibodies. In the current scenario of growing pharmaceutical demand, especially in developing countries, the use of transgenic plants can be game changing since they provide a highly scal-

Transplastomic plants are a valuable alternative to transgenic plants for the production of recombinant pharmaceutical proteins. Transplastomic plants are obtained by the insertion of expression constructs into the plastid genome by particle bombardment. Since the *Agrobacterium* T-DNA (transfer DNA) complex is targeted to the nucleus, it is unsuitable for gene transfer to chloroplasts [24, 52]. Following the transformation procedure, the bombarded leaf explants are regenerated, and transplastomic plants with homoplastomic transformation (in which every chloroplast carries the transgene) are finally selected, recurring to a selection medium containing spectinomycin or in combination with streptomycin [53].

Plastid transformation can result in high yields of heterologous proteins because multiple copies of the genome are present in each plastid, and photosynthetic cells may contain hundreds or thousands of plastids [54]. As an example, the expression of a proteinaceous antibiotic in tobacco chloroplasts has achieved up to 70% of the total soluble proteins, which is the highest recombinant protein accumulation accomplished so far in plants [55]. Furthermore, chloroplasts provide a natural biocontainment of transgene flow since genes in chloroplast genomes are maternally inherited and consequently not transmitted through pollen, thereby avoiding unwanted escape into the environment. Other advantages of chloroplast engineering include the ability to express several genes as operons, and the accumulation of recombinant proteins in the chloroplast, thus reducing toxicity to the host plant [24]. Finally, transplastomic production platforms offer the possibility of oral delivery

[54, 56]. In fact, it has been demonstrated that chloroplast-derived therapeutic proteins, delivered orally via plant cells, are protected from degradation in the stomach, probably due to the bioencapsulation of the therapeutic protein by the plant cell wall. They are subsequently released into the gut lumen by microbes that digest the plant cell wall, where the large mucosal intestine area offers an ideal

A shortcoming of expressing proteins via the chloroplast genome is that routine

plastid engineering is still limited to tobacco, a crop that is not edible and thus unsuitable for oral delivery of therapeutic proteins. In addition, the synthesis of glycoproteins is not possible in the chloroplast system, as plastids do not carry out glycosylation [24]. Nevertheless, the expression of human somatotropin [58] in tobacco established that chloroplasts are capable of properly folding human proteins with disulfide bonds. In another study, the production of native cholera toxin B subunit

*DOI: http://dx.doi.org/10.5772/intechopen.84419*

serve as a vaccine antigen against Alzheimer's disease.

able and low-cost means of producing medicines.

**2.2 Platforms based on transplastomic plants**

### *Prospects for the Production of Recombinant Therapeutic Proteins and Peptides in Plants… DOI: http://dx.doi.org/10.5772/intechopen.84419*

The thymosin α1 concatemer derived from transgenic tomatoes exhibited biological activity and was proven to stimulate the proliferation of mice splenic lymphocytes in vitro. Moreover, thymosin α1 specific activity was higher when produced in tomato than in *Escherichia coli*, demonstrating the authenticity of the plant-made product. Other examples of tomato fruit expression include F1-V [50], a candidate subunit vaccine against pneumonic and bubonic plague, and β-secretase [51], to serve as a vaccine antigen against Alzheimer's disease.

In conclusion, platforms based on transgenic plants are a promising alternative to the conventional biopharmaceutical production platforms since they provide a stable source of pharmaceutical proteins and are also the most scalable of all molecular pharming platforms. This scalability of transgenic plants ensures the production of recombinant pharmaceutical proteins at levels previously inaccessible, namely, the commodity bulk production of monoclonal antibodies. In the current scenario of growing pharmaceutical demand, especially in developing countries, the use of transgenic plants can be game changing since they provide a highly scalable and low-cost means of producing medicines.
