**1. Introduction**

The term "molecular pharming," blend of pharmaceutical and farming, surfaced in the literature in the 1980s to refer to the production of high-value compounds in

transgenic animals. Nowadays, the expression is mainly employed to the production of recombinant pharmaceutically relevant proteins or secondary products in plants [1–3].

The roots of molecular pharming can be traced back to the mid-1980s when plants started to be genetically engineered to act as bioreactors that produced pharmaceutically relevant proteins. Barta et al. [4] demonstrated that tobacco and sunflower callus tissues were capable of expressing transcripts of a human growth hormone fusion gene. Although no protein was detected, this was the first report of plants expressing human genes and established plants as a potential production system for recombinant therapeutic proteins. Later on, the expression of a full-sized IgG in tobacco [5] was a major breakthrough since it revealed the ability of plants to produce complex functional mammalian proteins of pharmaceutical relevance. In 1990, the "authenticity" of plant-derived recombinant proteins was proved even further with the production of the first human protein (serum albumin) with confirmed native structure in tobacco and potato [6].

After several studies that demonstrated the capacity of various plant species and systems to produce recombinant pharmaceutical proteins and peptides, during the 1990s, the field of molecular pharming gained support and interest from the plant biotechnology community. The scientific attention was followed by commercial interest, with many start-up companies being created to capitalize the advantages of plants in relation to the established platforms. These advantages include being a more costeffective, scalable, and safer means of producing pharmaceutically relevant proteins and peptides. In opposition to the fermentation-based traditional platforms that require a massive investment in bioreactors, plant-based production systems can be established with minimal investment and offer a myriad of different hosts and platforms [7]. However, the expectation that plants could easily compete for the market share of some well-established biopharmaceutical platforms, such as Chinese hamster ovary (CHO) cells, and that they could motivate the mainstream pharmaceutical industry to switch platforms was overinflated. The CHO epithelial cell lines are the most commonly used mammalian hosts for industrial production of therapeutic recombinant proteins. The technical limitations of plants, especially their lower yields compared to mammalian cell lines, allied to the colossal existing investment in fermentation infrastructures, the unfavorable public opinion on OGMs, and regulatory uncertainty, lead the mainstream pharmaceutical industry to be cautious and to a consequent stagnation of the molecular pharming field in the 2000s [8, 9]. This situation induced a change of paradigm concerning molecular pharming: the initial vision of a highly scalable and low-cost production system, while still valid, was replaced by the idea of a production system for certain niche products that are not easily manufactured by conventional systems [8, 9].

Molecular pharming embraces several platforms and technologies with different advantages and limitations, related by their use of plant tissues. Conversely to conventional biopharmaceutical production systems that are based on few selected platforms, particularly the bacterium *Escherichia coli*, yeasts such as *Pichia pastoris*, and mammalian cell lines such as Chinese hamster ovary (CHO) cells [3], pharming platforms range from plant cells or unicellular plants growing in bioreactors to whole plants growing in soil or hydroponic environments. Further, the technologies include stable integration of DNA into the nuclear genome or plastid genome and transient expression by infiltrating leaves with expression vectors based on *Agrobacterium tumefaciens*, plant viruses, or hybrids [3, 8]. This great diversity of molecular pharming confers adaptability and flexibility, allowing the selection of the most suitable platform for each product, but has also conduced to fragmentation. This fragmentation meant that in the early days of molecular pharming there was no driving force to establish molecular pharming as a single competitive platform. Consequently, no actions were made to match the industry requirements for high yields, standardized procedures, and good manufacturing practices (GMP) [7, 9]. More recently, efforts have been made to mimic the mainstream

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**peptides**

pharmaceuticals.

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

biopharmaceutical industry and place a focus only on a small number of platforms, namely, plant cell cultures, nuclear transgenic plants, and leafy plants transiently transformed [3, 10]. Since 2010 the attention of the biopharmaceutical industry to molecular pharming has been renewed as result of its consolidation on a small number

In 2012, the FDA approval of the first recombinant plant-derived therapeutic for human use, Protalix Biotherapeutics' taliglucerase alfa (Elelyso™), was an important breakthrough for molecular pharming. The enzyme taliglucerase alfa is a carrot cellexpressed human recombinant β-glucocerebrosidase and is prescribed for the treatment of Gaucher's disease, a lysosomal storage disorder [11]. Imiglucerase, a recombinant form of glucocerebrosidase commercialized under the name Cerezyme®, was already produced in CHO cells. In this production platform, the enzyme required subsequent in vitro exposure to mannose residues in order to have biological activity, resulting in a time-consuming and expensive manufacturing process. Besides, this platform also has potential safety problems, namely, the risk of viral contamination, allergies, and other adverse reactions. In comparison, the plant-based platform is safer and less timeconsuming and has reduced production costs, since the mannose units are posttranslationally added in vivo [11]*.* Glucocerebrosidase is a clear example of a target product in which safety, cost, and downstream processing issues were solved by switching from a traditional platform to molecular pharming. Another example that gathered mediatic exposure was ZMapp, a cocktail of three chimeric monoclonal antibodies targeting the Ebola virus surface glycoprotein produced in *Nicotiana benthamiana* using a hybrid transient expression system, the magnICON system. ZMapp was developed during the Ebola outbreak of 2014 by Mapp Biopharmaceutical Inc. (San Diego, USA), following initial studies on nonhuman primates [12]. ZMapp has since been used in humans under emergency compassionate protocols [13] and randomized controlled trials [14]. Following these examples of success, there has been a continuous increase in clinical trial applications and manufacturing capacity, which has also been correlated with the conception of more tangible regulations concerning plant-derived

Although plants are still unlikely to substitute the established platforms [8], the recent promising developments in the field of molecular pharming demonstrate that glucocerebrosidase was not a lone case of success and that plant-based platforms could provide countless opportunities for the biopharmaceutical market. Plants combine the advantage of a full posttranslational modification potential with simple growth requirements and theoretically unlimited scalability in the case of field-grown whole plants. Plant-based platforms are versatile and allow the targeting of recombinant proteins and peptides produced to different organs or subcellular compartments, which provides an additional protection against proteolysis. Finally, plants are a safe host for therapeutic protein and peptide production since they do not harbor human or animal pathogens [15]. Therefore, instead of facing the red ocean of established pharmaceutical industries [16], molecular pharming is now evolving as a disruptive technology that creates its own marketplace by offering rapid drug development and production, unparalleled scalability, unique quality attributes such as tailored glycan structures, individualized therapies, and oral or topical applications

of minimally processed plant tissues, thus reducing downstream costs [17].

**2. Plant platforms for the production of therapeutic proteins and** 

The continuous development of genetic engineering technologies for plants has resulted in an expansion of well-established plant-based platforms [18]. Molecular

of platforms and some target products that meetindustry demands [8, 9].

*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*

biopharmaceutical industry and place a focus only on a small number of platforms, namely, plant cell cultures, nuclear transgenic plants, and leafy plants transiently transformed [3, 10]. Since 2010 the attention of the biopharmaceutical industry to molecular pharming has been renewed as result of its consolidation on a small number of platforms and some target products that meetindustry demands [8, 9].

In 2012, the FDA approval of the first recombinant plant-derived therapeutic for human use, Protalix Biotherapeutics' taliglucerase alfa (Elelyso™), was an important breakthrough for molecular pharming. The enzyme taliglucerase alfa is a carrot cellexpressed human recombinant β-glucocerebrosidase and is prescribed for the treatment of Gaucher's disease, a lysosomal storage disorder [11]. Imiglucerase, a recombinant form of glucocerebrosidase commercialized under the name Cerezyme®, was already produced in CHO cells. In this production platform, the enzyme required subsequent in vitro exposure to mannose residues in order to have biological activity, resulting in a time-consuming and expensive manufacturing process. Besides, this platform also has potential safety problems, namely, the risk of viral contamination, allergies, and other adverse reactions. In comparison, the plant-based platform is safer and less timeconsuming and has reduced production costs, since the mannose units are posttranslationally added in vivo [11]*.* Glucocerebrosidase is a clear example of a target product in which safety, cost, and downstream processing issues were solved by switching from a traditional platform to molecular pharming. Another example that gathered mediatic exposure was ZMapp, a cocktail of three chimeric monoclonal antibodies targeting the Ebola virus surface glycoprotein produced in *Nicotiana benthamiana* using a hybrid transient expression system, the magnICON system. ZMapp was developed during the Ebola outbreak of 2014 by Mapp Biopharmaceutical Inc. (San Diego, USA), following initial studies on nonhuman primates [12]. ZMapp has since been used in humans under emergency compassionate protocols [13] and randomized controlled trials [14].

Following these examples of success, there has been a continuous increase in clinical trial applications and manufacturing capacity, which has also been correlated with the conception of more tangible regulations concerning plant-derived pharmaceuticals.

Although plants are still unlikely to substitute the established platforms [8], the recent promising developments in the field of molecular pharming demonstrate that glucocerebrosidase was not a lone case of success and that plant-based platforms could provide countless opportunities for the biopharmaceutical market. Plants combine the advantage of a full posttranslational modification potential with simple growth requirements and theoretically unlimited scalability in the case of field-grown whole plants. Plant-based platforms are versatile and allow the targeting of recombinant proteins and peptides produced to different organs or subcellular compartments, which provides an additional protection against proteolysis. Finally, plants are a safe host for therapeutic protein and peptide production since they do not harbor human or animal pathogens [15]. Therefore, instead of facing the red ocean of established pharmaceutical industries [16], molecular pharming is now evolving as a disruptive technology that creates its own marketplace by offering rapid drug development and production, unparalleled scalability, unique quality attributes such as tailored glycan structures, individualized therapies, and oral or topical applications of minimally processed plant tissues, thus reducing downstream costs [17].
