Plant Molecular Farming: A Viable Platform for Recombinant Biopharmaceutical Production

The demand for recombinant proteins in terms of quality, quantity, and diversity is increasing steadily, which is attracting global attention for the development of new recombinant protein production technologies and the engineering of conventional established expression systems based on bacteria or mammalian cell cultures. Since the advancements of plant genetic engineering in the 1980s, plants have been used for the production of economically valuable, biologically active non-native proteins or biopharmaceuticals, the concept termed as plant molecular farming (PMF). PMF is considered as a cost-effective technology that has grown and advanced tremendously over the past two decades. The development and improvement of the transient expression system has significantly reduced the protein production timeline and greatly improved the protein yield in plants. The major factors that drive the plant-based platform towards potential competitors for the conventional expression system are cost-effectiveness, scalability, flexibility, versatility, and robustness of the system. Many biopharmaceuticals including recombinant vaccine antigens, monoclonal antibodies, and other commercially viable proteins are produced in plants, some of which are in the pre-clinical and clinical pipeline. In this review, we consider the importance of a plant- based production system for recombinant protein production, and its potential to produce biopharmaceuticals is discussed.


Introduction
Recombinant proteins are complex exogenous ("foreign") proteins that are produced in expression hosts, and mainly used as medical diagnostic reagents and in human healthcare as vaccines, drugs, or monoclonal antibodies [1]. The prominent role and increasing market demand for high-value recombinant proteins in novel drug discovery creates an opportunity for the development of various protein expression hosts to manufacture proteins by following the existing rigid standards laid down for veterinary and human applications. The industry is focusing mainly on already established production platforms using prokaryotic and eukaryotic expression host systems such as Escherichia coli, a selection of yeast, insect, and mammalian cell cultures, due to their well-defined processes in-line with current good manufacturing practice (cGMP) [2]. Moreover, industrially established mammalian and other cell cultures have stringent regulatory approval in place, which hinders the industrial acceptance of the new technology or production system. Bacterial expression systems offer rapid production with high product yield, whereas Saccharomyces cerevisiae and Pichia pastoris (yeast) offer post-translational modifications (PTMs) which are essential for functional activity of the recombinant proteins [3]. The majority of the Table 1. Available expression platforms for recombinant protein production with potential advantages and disadvantages (adapted from Shanmugaraj et al., 2020) [25].

Plant-Derived Recombinant Proteins
The concept of using plants for the production of foreign proteins including pharmaceutical and non-pharmaceutical proteins has been well explored and documented. Many reports proved the ability of in vivo and in vitro plant systems to produce vaccine candidates both for veterinary and human applications and showed that plant-produced antigens elicit potential immune responses in animal models and even confer protection in animal challenge experiments. Examples of the variety of pharmaceutical and non-pharmaceutical proteins expressed in plant systems are illustrated in Tables 2 and 3.
Tobacco has been engineered to express a variety of antigens in the nucleus and chloroplast including, but not limited to, chikungunya, dengue, Ebola, influenza, and Zika. The transformation protocols for recombinant protein production are also established for fruits and vegetables such as tomatoes and potatoes. Transgenic potatoes expressing the S1 glycoprotein of the infectious bronchitis virus confers protection to chickens upon virus challenge [26]. Leafy crops such as lettuce, alfalfa, and clover have been investigated for molecular farming to obtain the oral delivery of vaccine antigens eliminating purification and injections. The lettuce chloroplast-derived booster vaccine using lyophilized plant cells expressing the poliovirus capsid protein induced neutralization antibodies in mice primed with inactivated poliovirus vaccine (IPV) and conferred protection against all polio serotypes [27]. Plant systems have also been evaluated for the expression of virus-like particles (VLP) of many viruses including norovirus, poliovirus, foot-and-mouth disease virus, influenza, [28][29][30][31], and the potential for plant-derived VLPs to be used as candidate vaccines and reagents has been reviewed in detail elsewhere [18,32,33]. Apart from expressing antigens for human diseases, several antigens for veterinary applications and non-pharmaceutical proteins have also been well tested for expression in plants, and are particularly gaining attention due to the fact that these products can quickly reach the market due to lower regulatory burden [9]. This was clearly evidenced by the commercialization of avidin [34], β-glucuronidase [35], and trypsin [36] by the US-based biotechnology company ProdiGene, Inc. The vaccine against Newcastle disease virus (NDV) was the first plant-based poultry vaccine (Dow Agrosciences) that obtained regulatory approval from the United States Department of Agriculture in 2006, opening a new avenue for the commercialization of plant-derived vaccines. Currently, many plant-derived non-pharmaceutical and pharmaceutical proteins are in clinical development.
Although proof-of-concept and efficacy of many vaccine candidates proved the feasibility and scalability of the robust plant system, it is high time to compete with the established expression systems. Now the plant-based good manufacturing practices (GMP) complaint production facilities such as Fraunhofer (Germany), Kentucky BioProcessing (USA), Medicago (Canada), and Protalix Biotherapeutics (Israel) are available to manufacture GMP materials for human clinical trials. Fraunhofer IME received a GMP license for the production of neutralizing anti-HIV antibody 2G12 in tobacco for phase I clinical testing [37]. The plant molecular farming research community continuously thrives to set up a regulatory framework for plant-derived products.

Strategies Used for Recombinant Protein Production in Plants
The expression methods used for the recombinant protein production in plants can be either stable or transient expression. PMF relies on following approaches for the expression of vaccine candidates, i.e., stable nuclear transformation, stable chloroplast transformation, or transient expression, by using plant viral vectors and stable transformation of hydroponically grown plants in which recombinant proteins are recovered from the medium [119] (Figure 1). Stable nuclear transformation is the traditional strategy of genetic manipulation in plants for recombinant protein production. The transgene in the plant expression vector can be introduced into the in vitro grown plantlets either with Agrobacterium tumefaciens-mediated transformation or particle bombardment, and stable transgenic lines can be developed. The best transgenic line for protein production will be subsequently screened from the pool of transgenic lines. By this method, recombinant proteins can be produced in successive generations, as the transgene has been stably integrated into the plant genome. The model plants such as Arabidopsis thaliana and tobacco were more commonly used during the early stages of genetic transformation to develop stable transformants [79]. Stable transformation in plants requires substantial time and is a labor-intensive process, and the protein expression is insufficient to meet the industrial-level protein production. However, the antigen expression in stable transgenic line could be used for developing oral vaccines that could reduce the cost associated with protein purification [120,121].
Alternatively, transient expression based on agroinfiltration or virus-based vectors have been developed to complement transgenic plants that offer rapid and high-level protein expression within a few days. The drawbacks and challenges associated with stable expression, such as Stable nuclear transformation is the traditional strategy of genetic manipulation in plants for recombinant protein production. The transgene in the plant expression vector can be introduced into the in vitro grown plantlets either with Agrobacterium tumefaciens-mediated transformation or particle bombardment, and stable transgenic lines can be developed. The best transgenic line for protein production will be subsequently screened from the pool of transgenic lines. By this method, recombinant proteins can be produced in successive generations, as the transgene has been stably integrated into the plant genome. The model plants such as Arabidopsis thaliana and tobacco were more commonly used during the early stages of genetic transformation to develop stable transformants [79].
Stable transformation in plants requires substantial time and is a labor-intensive process, and the protein expression is insufficient to meet the industrial-level protein production. However, the antigen expression in stable transgenic line could be used for developing oral vaccines that could reduce the cost associated with protein purification [120,121].
Alternatively, transient expression based on agroinfiltration or virus-based vectors have been developed to complement transgenic plants that offer rapid and high-level protein expression within a few days. The drawbacks and challenges associated with stable expression, such as insufficient protein expression, time, and consistency, have been overcome by the development of novel strategies involving deconstructed viral vector systems such as MagnICON ® technology, geminiviral, and pEAQ, which allows rapid accumulation of recombinant proteins in a short time [12]. Hence, it is considered as a suitable convenient platform, especially for the production of vaccine antigens or monoclonal antibodies against infectious diseases (Figure 2). Gleba et al. (2007) summarized the application of plant viral vectors for the transient expression of heterologous proteins in plants [122]. Plant transient expression holds tremendous potential to produce rapid-response proteins, emergency vaccines, or biologics, which was impressively shown during the Ebola outbreak in 2014. Mapp Biopharmaceutical Inc., USA produced an experimental drug ZMapp, an anti-Ebola antibody cocktail of three chimeric monoclonal antibodies manufactured in tobacco plants (Nicotiana benthamiana) to treat humans during the recent Ebola outbreak [123]. During a pandemic situation, in order to cope with a rapidly spreading infectious disease, production methods should meet the demand for production targets of strategic vaccines to control the disease. One of the recent examples is the pandemic, corona virus disease (COVID-19). The virus has spread rapidly, and millions of people have been affected across 6 continents in few months, posing a constant threat to global health. This infection has created a massive demand for diagnostic reagents, vaccines, and therapeutic development. Given the speed advantages, and proven viability of the plant production platform, the transient expression system in particular could be employed to produce recombinant proteins at high levels to meet the sudden demand for production of viral antigens or antiviral proteins that could be used as research reagents, emergency vaccines (SARS-CoV-2 subunit and virus-like particle vaccines), or other biopharmaceuticals to fight against COVID-19 [25,124]. The neutralizing monoclonal antibodies against SARS-CoV-2 could also be produced in plants with minimal investment, which could be used for passive immunotherapy [125]. Recently, Medicago (Quebec, Canada), Kentucky BioProcessing (Owensboro, KT, USA), and iBio (Bryan, TX, USA) joined the global race for developing potential plant-based vaccines for COVID-19 [126]. By using the transient expression platform, recombinant protein production in plants could be scaled up rapidly, and milligram quantities of proteins could be produced in a timeframe of less than 4 weeks after receiving the corresponding gene construct [5,59,127].
Plants 2020, 9, x FOR PEER REVIEW 12 of 21 has spread rapidly, and millions of people have been affected across 6 continents in few months, posing a constant threat to global health. This infection has created a massive demand for diagnostic reagents, vaccines, and therapeutic development. Given the speed advantages, and proven viability of the plant production platform, the transient expression system in particular could be employed to produce recombinant proteins at high levels to meet the sudden demand for production of viral antigens or antiviral proteins that could be used as research reagents, emergency vaccines (SARS-CoV-2 subunit and virus-like particle vaccines), or other biopharmaceuticals to fight against COVID-19 [25,124]. The neutralizing monoclonal antibodies against SARS-CoV-2 could also be produced in plants with minimal investment, which could be used for passive immunotherapy [125]. Recently, Medicago (Quebec, Canada), Kentucky BioProcessing (Owensboro, KT, USA), and iBio (Bryan, TX, USA) joined the global race for developing potential plant-based vaccines for COVID-19 [126]. By using the transient expression platform, recombinant protein production in plants could be scaled up rapidly, and milligram quantities of proteins could be produced in a timeframe of less than 4 weeks after receiving the corresponding gene construct [5,59,127].  Alternately, chloroplast expression focuses on expressing the transgenes in chloroplast by the precise insertion of foreign DNA by homologous recombination into the chloroplast genome. Much progress has been made in chloroplast engineering in recent years. The transformation of the chloroplast genome has many advantages over nuclear transformation which includes higher protein production, lack of gene silencing and position effect, polycistronic mRNA expression, and prevention of transmission of foreign DNA through pollen by uniparental plastid gene inheritance (maternal inheritance) in crop plants [128][129][130][131].
Similar to bacterial and mammalian cells, heterologous protein production can be achieved by using individual suspension of plant cells rather than whole plants. The cell suspension derived from undifferentiated callus grown in liquid medium can be scaled up in bioreactors for large-scale protein production under an aseptic environment. The first USDA-approved poultry vaccine and the first FDA-approved recombinant plant-produced pharmaceutical protein "Elelyso" were produced in tobacco and carrot cell suspension cultures, respectively, which proved the importance and competitiveness of plant suspension culture in high-value protein production in the biopharmaceutical industry [121,[132][133][134]. Hairy root cultures are also being explored as an alternative recombinant protein production system due to their ease in protein recovery and low costs. The recombinant proteins are secreted from the transgenic plant roots into the culture medium viz., rhizosecretion; hence, this allows continuous protein production and recovery from the culture medium without the requirement of cell lysis during extraction. Moreover, recombinant proteins produced from root cultures attribute to the improved protein quality and quantity without complex downstream processing that could eventually reduce production costs as well [135]. A recent review on the applications of hairy root cultures for protein production has been extensively discussed by Gutierrez-Valdes et al. (2020) [136].

Perspectives
Although plants are attractive with several unique advantages, they are unable to compete with the existing microbial and mammalian systems, as both are well established and characterized, especially in terms of GMP manufacturing and regulatory approval in an industrial setting. Even after many years of research, which has shown the proof-of-concept of expressing many therapeutic proteins in plants, the process of producing therapeutic proteins from the lab bench to commercialization is slow. Hence, in order to move forward, the commercial potential and economic sustainability of technology needs to be exploited by developing veterinary vaccines, non-pharmaceutical diagnostic, cosmetic products, and industrial enzymes in plants, as they have a low regulatory burden compared to therapeutic proteins [2,9]. This technology can also be employed to reproduce rapid response vaccines or diagnostic reagents against emerging infections. For the past few years, extensive research has been carried out to combat the several emerging diseases including Zika, chikungunya, Nipah, SARS-CoV, MERS-CoV, and more recently SARS-CoV-2. Even though several efforts have been made for many years to develop effective vaccine candidates for many of those emerging and zoonotic diseases, still, there are no vaccine candidates or therapeutic measures available commercially. Even if a successful vaccine or drug developed against such diseases, it is unlikely that it would have a significant impact on developing and under-developed countries, due to the high cost associated with it, and scalability concern. In such a scenario, a plant-derived vaccine or diagnostic reagent would be a feasible approach to rapidly respond to the demand and need for recombinant proteins. However, harnessing the full potential of this plant molecular farming technology for cost-effective vaccines or drug development will be evident in the upcoming years.

Conclusions
Plants have both economic and technical advantages over conventional expression systems for the production of pharmaceutical and non-pharmaceutical products. The different PMF technologies such as nuclear, chloroplast expression, viral transfection, and transient expression systems have their unique features, enabling them to address a production of diversified product "targets" with less production constraints in a short time. Many scientific and technical challenges associated with the plant platform were met in recent years. However, the regulatory burden associated with therapeutic protein production is a major barrier that hinders the widespread acceptance of the plant system. Considering the low costs and greater scalability of plant production systems, the commercialization of non-pharmaceutical proteins is straightforward and faster due to lower regulatory challenges. Hence, the universal acceptance of the technology will be strongly influenced by the regulatory framework and restrictions applied to plant-derived products worldwide. The demand for industrially or pharmaceutically useful recombinant proteins, together with demonstrated production capability and economic feasibility of the plant system, suggests a bright future for the plant-made biologics.

Conflicts of Interest:
The authors declare no conflict of interest.