Elena Cristea

Introduction

Bioengineered plants are use to produce protein therapeutics from plants for more than two decades. Until recent time, pharmaceuticals were produced from small organic molecules, synthesized by microorganisms or using the chemical pathways. Usually short peptide chains (containing less than 30 amino acids) are synthesized chemically, but large proteins are best produced by living cells (Joshi & Lopez, 2005), (Thomas, 2002). These days the attention on larger and more complicated therapeutic molecules is focused. Considering the fact that proteins play a very important role in cell biology, they have large uses as therapeutics, both for preventing and curing diseases (Thomas, 2002). The increasing demand for pharmaceuticals all around the world led to the development of bioengineered plants for their production, since production in plants systems has some advantages such as:

• Lower production costs compared to protein production in mammalian cells, larger proteins compared to the ones produced in bacteria or yeast
• Production units that can be economically expanded or reduced on the demand of the specific pharmaceutical, given the possibility to produce high amounts of biomass and a fast gene-to-protein time
• Elimination of the risk of human or animal virus and prion transmission or of contamination by harmful substances of chemical synthesis
• Facilitated storage (especially in case of seeds)
• Suitable for production of large proteins

Until today, more than 95 therapeutic proteins or peptides have been licensed. Bacterial, fungal and mammalian cells are used for that purpose. The technologies using bacterial and fungal cells, being improved, have proven their efficiency, but at the same time, their limitations to produce larger proteins have been revealed. Systems using eukaryotic cells, such as mammalian cells, have shown their capacity to produce human, complex proteins. These systems are developing fast, but also have their limitations like difficult production in a bioreactor, limitations to production volumes, a high price for investments, a high cost/volume ratio (Landry & Vézina, 2001)(Huang & McDonald, 2009).

Since almost all proteins produced by other existing systems (antibodies, vaccines, plasma proteins) have already been manufactured in plants, these are considered a good alternative system in order to satisfy the growing need for therapeutic proteins (Joshi & Lopez, 2005), (Davies & Ph, 2008).

Discussion

Different recombinant plant protein production systems

The production of recombinant proteins is done by transgenesis. The methods used for the genetic transformation of the plants are very similar to those used for yeasts and mammalian cells (Davies & Ph, 2008). There are a few ways to induce a genetic modification in plant cells:

• Microinjection of genetic material directly into the cell
• Biolistic DNA delivery
• Electroporation
• Treatment with polyethylene glycol in the presence of divalent cations
• Agrobecterium-mediated infiltration
• Use of viruses (Tobacco mosaic virus, Cucumber mosaic virus, Potato virus, Tomato mosaic virus) (Schillberg, Twyman, & Fischer, 2005), (Egelkrout, Rajan, & Howard, 2012).

Each of these methods has advantages and disadvantages, but the most used in plant transgenesis are the Agrobacterium-mediated infiltration and the use of viruses (Egelkrout, Rajan, & Howard, 2012), (Hefferon, 2012).

Two major production approaches were identified:

• Production in whole mature plants
• Production using cell lines (in liquid medium to form cell suspensions or in solid medium)

In most plant systems used for a high production of recombinant proteins, the plant cells are not multiplied in vitro in bioreactors, but grown in conditions which allow the regeneration of mature plants. It’s the mature plants that are used in most cases for production and this is one of the differences from other protein production systems, although  studies on bioreactors for plant cell lines have been undertaken, and some authors consider growth in bioreactor as being easier and more efficient. In vitro aseptic suspension culture of plant cells, tissues or organs under controlled environment has been developed for the production of medicinal secondary metabolites e.g. shikonin and paclitaxel (Huang & McDonald, 2009).

Some other advantages of plant cell cultures over whole plants are:

• Simplified purification
• Consistency in product quality and homogeneity under controlled environmental conditions
• Facility to meet GMP requirements
• Elimination of the need of greenhouse or field grown plants
• Ability to use inducible promoter systems
• Lower probability of endotoxin and mycotoxin contamination derived from the plant and soil source
• Reduced risks related to transgene migration (Huang & McDonald, 2009)

The systems used for mature plants have been adapted to their anatomical and physiological requirements.  Within this class two large groups of plant systems are developed in order to benefit from the most accessible sources of biomass. The most common are foliage and seeds (Spök, Twyman, Fischer, Ma, & Sparrow, 2008). In the case of tobacco, alfalfa and other species with abundant foliage, the genes responsible for those parts are used as target genes. Transplastomic technology, when heterologous proteins are expressed in the chloroplast, offers the benefit of higher protein expression levels (Fischer, Twyman, & Schillberg, 2003). For corn, rapeseed, safflower, rice, soybeans and other legumes, the expression vectors stimulate the production and accumulation of recombinant proteins in the seed. Each of these strategies has its own advantages and disadvantages (Huang & McDonald, 2009), (Landry & Vézina, 2001), (Ramessar, Sabalza, Capell, & Christou, 2008). The leaves are metabolically active and complex which offers many possibilities, but they also have important protease activity that limits the accumulation of certain proteins. The seeds have the advantage of having lower water content therefore offer a more stable storage medium. However, they are not suitable for the synthesis of certain protein complexes, and the need to reach flowering may represent an increased risk of dispersion the transgene (Landry & Vézina, 2001)(Stoger, Ma, Fischer, & Christou, 2005), (Stoger et al., 2005). Tobacco is one of the favourite plants that are used in a whole plant production. Proteins that are engineered in the seeds of plants such as legumes, maize or barley have a high accumulation level and facilitated product storage. Proteins that are engineered in plants that have high oil content in the seeds (peanut, safflower) offer additional advantages – in complex with the seed oil they benefit from opportunities for efficient extraction, formulation and delivery systems (Joshi & Lopez, 2005) (Mascia & Flavell, 2004).

Purification

In any heterologous production system, the recombinant molecule must be extracted and purified from the set of proteins of the endogenous body. In the process of separation, the molecule can be purified by conventional methods: chromatography or electrophoresis (Schillberg, Twyman, & Fischer, 2005). These are the initial phases of extraction and purification, which are problematic in most cases, especially because of the rapid proteolysis that occurs during the homogenization of tissue.  Some production strategies have been developed to perform the first steps of purification in a simplified manner. For example, in some oil seeds, the recombinant protein is fused with a protein that is part of the lipid globules accumulating during seed maturation. Another novel approach is the utilization of GVGVP, a protein-based polymer, which is coded by synthetic genes. GVGVP will act as a fusion protein, facilitating purification in a way that chromatography is no longer needed (Daniell, Streatfield, Wycoff, & Daniell, 2001).

Challenges in plant proteins production

Complete realization of the plant-derived engineered proteins requires solutions for problems such as bioequivalence and product consistency. The most important issues related to the production of proteins in plants are related to:

• Post-translational modifications (PTMs) such as N-glycosylation, O-glycosylation, phosphorylation, hydroxylation, oxidation, deamidation, glycation, gamma-carboxylation (Gomord & Faye, 2004)
• Proteolysis

PTMs are key factors in the structure and function of eukaryotic proteins. Among the protein PTMs, glycosylation is the most important in terms of bioactivity and acceptance by the pharmaceutical and biotechnology industries and regulatory agencies (Joshi & Lopez, 2005).

Under the present form the plants are not yet ideal for the production of proteins because they produce molecules with a glycosylation that is not always compatible with therapeutic application in humans. This way, the change of glycosylation capacity in plants, so their expression system is better suited for the production of human therapeutic glycoproteins, is the subject of many studies (Landry & Vézina, 2001) (Doran, 2000). PMTs are the processes happening in endoplasmic reticulum and Golgi apparatus after translation. The major difference in the structure of plant and mammalian proteins is made during the late processes in the Golgi apparatus.

One of the most studied processes is the N-glycosylation (attachment of glycosyl to the nitrogen on arginine and asparagine side chains). Although protein folding, assembly and glycosylation have similar mechanisms for proteins entering the secretory pathways: N-linked glycosylation in plants occurs in the ER and Golgi apparatus, O-linked glycosylation only occurs in the Golgi apparatus, and molecular chaperones in the ER help to fold the protein), certain differences in the capacity for post-translational modification have been observed between plant and animal cells. Plant cells tend to attach alpha-(1,3)-fucose and beta-(1,2)-xylose in the glycan of plant-made recombinant human glycoprotein, that are absent in animal cells. Furthermore, plant-made recombinant human glycoprotein generally lacks the terminal galactose and sialic acid residues, which have been found on many human glycoproteins. These small differences in the glycan structures of plant-derived recombinant human glycoproteins tend to impact the product stability, biological activity and immunogenicity to humans (Huang & McDonald, 2009).

Another form of post-translational modification is O-glycosylation – it is less studied, but recent discoveries reveal that it is important in the production of more complex proteins. O-glycosylation patterns of therapeutic proteins have never yet been elucidated, although O-linked glycans are found on several proteins of clinical interest (Pujol et al., 2007), (Gomord & Faye, 2004).

Phosphorylation is also an important issue which appeared with the development of the demand for more complex pharmaceuticals. It is known that the enzymes responsible for phoshporylation are different in plant cells and mammalian cells. Tyrosine phosphatase is responsible in mammalian cells, while in plant cells it is serine-threonine phosphatase, although recent studies suggest that tyrosine-kinase pathway is also possible in plants and that they would be capable to phosphorilate in a mammalian manner (Egelkrout, Rajan, & Howard, 2012).

Proteolysis is another process, influencing, especially, the stability of plant-made proteins, which is a very important factor, crucial in the case of edible vaccines. Proteases found in the secretory pathway, as well as in the rest of the cell represent a major hurdle for the efficient production of recombinant proteins. For plants, yields in recombinant protein not only depend on the efficient expression rate of the transgene, but also on the stability of the resulting protein during the whole expression/ recovery process. Proteases which are present in the different compartments of plant cells may alter the stability of foreign proteins in a dramatic way, either in vivo, or in vitro in the process of their recovery from plant tissues. Vacuolar proteases, active in mildly-acidic conditions, in particular, were found as potentially damaging for the integrity of recombinant proteins expressed in vegetative organs of transgenic plants. In order to avoid proteolysis, strategies based on the fusion of appropriate peptidic signals to the expressed proteins were proposed. The main purpose of this is to direct proteins’ accumulation in alternative compartments such as the ER or the chloroplast (Pujol et al., 2007), (Doran, 2000), (Ramessar, Sabalza, Capell, & Christou, 2008).

“Humanization” of plant proteins Different strategies

The differences in the post-translational modification pathways limit the possibilities of application of plant proteins. Different strategies are used for the “humanization” of proteins produced in plants. Considering the fact that the major difference between plant and mammalian protein is due to the maturation processes occurring in the Golgi apparatus, one approach is to block the proteins to be transferred from the endoplasmic reticulum.  Analyzing different studies on the “humanization” of plant proteins, three main approaches were revealed.

• Inhibition of glycosyltransferases– enzymes responsible for glycosylation
• Retention in the endoplasmic reticulum
• Sialylation

One of the important advances made in the recent years for the “humanization” of plant proteins was the discovery of terminal sialic acids. Most mammalian glycoproteins have glycan chains that terminate with sialic acid residues, which are important for intermolecular and cellular interactions. In the absence of terminal sialic acids, glycoconjugates are detected by hepatic asialoglycoprotein receptors and removed from the serum, making these proteins biologically ineffective. It was determined that plants are also able of sialylation, although the levels are low compared to the ones from mammalian cells. Experiments suggest that plants can be programmed to glycosylate in mammalian manner and, for that purpose, their capacity of sialylation should be increased. At least five more genes are necessary for that, which should be expressed in the appropriate manner. Also, the addition of b1-4 galactose and the removal of a1-3 fucose and b1-2xylose are other major steps towards humanizing plant glycans (Joshi & Lopez, 2005), (Landry & Vézina, 2001).

Plant-derived biopharmaceuticals

Blood and plasma proteins

Blood and plasma proteins are currently produced by extraction from samples of blood which was not retained for transfusion purposes. This method has certain limits which have impact on the industry: the supplies can fluctuate and the source is considered by the public as dangerous. Also possible contamination by viruses and prions remains a major concern. Many plasma proteins have already been produced in plants. The human albumin, a plasma protein used in the control of hypovolemia and hypoalbuminemia occurring during some surgeries and excipient of several drugs, was produced successfully in potato and tobacco. The plant production system is ideal to meet the requirements for large quantities. Other proteins of the same group such as aprotinin, enkephalin and hemoglobin have been produced in various plant systems. I collagen, a triple helix molecule assembly involved in several complex mechanisms such as organogenesis, stowage and cell proliferation, hemostasis and tissue regeneration was also produced in tobacco plants for therapeutic use, but also in the cosmetic industry (Landry & Vézina, 2001).

Table 1

Blood and plasma proteins produced in plants (Landry & Vézina, 2001) (Daniell, Streatfield, Wycoff, & Daniell, 2001)

Protein	Application and specificity	Plant
Albumin	Control of blood volume	Potato,  tobacco
Aprotinin	Antifibrinolytic	Maize
Homotrimericcollagen I	Homeostatic agent, tissue sealant and other	tobacco
Enkephalins	Analgesic	Tobacco, arabidopsis
Hemoglobin	Blood substitute	Tobacco
Hirudin	Thrombin inhibitor	Canola
Granulocyte-macrophage colony stimulating factor	Neutropenia	Tobacco
Serum albumin	Liver cirrhosis, burns, surgery	Tobacco

Antibodies

Antibodies are a prominent category of plant-derived products; examples include antibodies that recognize serum glycoproteins and have important and diverse roles in the diagnosis and treatment of cancers, microbial infections and other diseases. The first demonstration of antibodies production in plants was conducted in 1989 by Hiatt and al. Since then, other antibodies or antibody fragments for therapeutic use were produced in various plant systems, including antibodies directed against human immunoglobulins, creatine kinase and the tumour antigen of cancer colon. Antibody fragment (calledscFv) was also produced against carcinoembryonic antigen, a marker of tumour growth (Landry & Vézina, 2001).

Monoclonal antibodies are the fastest growing sector of these new biotech-derived products and the closest to reaching the market. These pharmaceuticals represent more than 25% of the total biotech-derived protein market and, at present, are produced mainly in mammalian cell culture systems. The production of antibodies and antibody fragments in plants and their successful storage in dried leaves, seeds and tubers demonstrates the potential for long-term storage of these proteins and validates the plant-based production system (Joshi & Lopez, 2005).

Some successes in this field are the production of production of various types of antibodies such as recombinant IgG or secretory IgA, antibodies used as therapeutic agents. Antibodies are complex molecules. As an example, the immunoglobulin G class are tetramers consisting of two identical polypeptides of 450 amino (heavy chains) and two identical polypeptides of 250 amino (light chains). These four polypeptides constituting a molecule IgG are interconnected by several disulfide bonds. The complexity of a secretory IgA is even greater since these immunoglobulins are made of four heavy chains and four light chains connected together by two polypeptides. The assembly of a molecule of secretory IgA requires the successive intervention of two distinct cell types in mammals (Fischer, Stoger, Schillberg, Christou, & Twyman, 2004). These two types of antibodies were successfully produced in biologically active form in transgenic plants, which illustrates the capacity of the plant cellular machinery of assembling mammalian protein, even when they are extremely complex. Over a hundred clinical trials using antibodies are currently studied for the treatment of various diseases such as malfunctions of the immune system, inflammatory diseases, certain cancers, disorders in central nervous system and infectious diseases. Most proposed applications require the use of whole antibodies. Exception of hybridomas, only cells in mammals or transgenic plants are able to associate the heavy and light chains component of the antibody by disulfide bridges. (Landry & Vézina, 2001), (Fischer, Stoger, Schillberg, Christou, & Twyman, 2004).

 Table 2

Antibodies produced in plants (Landry & Vézina, 2001), (Daniell, Streatfield, Wycoff, & Daniell, 2001)

Protein	Application and specificity	Plant
IgGC5-1	Anti-IgGdiagnostic	Alfalfa
IgAagainst S.mutans	Preventionof dental caries	Tobacco
IgGagainstcreatinekinase	Antibodies diagnostic	Tobacco
IgGagainst antigen tumorCO17-1A	treatmentof colon cancer	Tobacco
ScFvagainstantigen carcinoembryonic(CEA)	cancerantigen	Grains

Vaccines

Oral and topically applied subunit vaccines are another intense area of research in plant biopharming. Plant produced vaccines for a wide range of human and veterinary applications, including protection from infectious diseases, cancers, and potential biowarfare pathogens, are in development (Thomas, 2002). Indeed, it is possible to trigger an immune response by oral administration of an antigen. Thus, in the case of Hepatitis B, virus which affects more than 2 billion individuals – a vaccine that can be easily distributed and administered in developing countries is highly desirable. Studies in mice demonstrated that the ingestion of potato expressing an antigen of hepatitis B triggers an immune response. Several other types of antigens for oral or parenteral administration products were also produced in plants with the aim of vaccination against different human pathogens, including a potential vaccine against AIDS. The development of a vaccine for oral administration requires precise metering of the quantity of used antigens (Landry & Vézina, 2001), (Karasev et al., 2005), (Giddings, 2001). 

Table 3

Vaccines produced in plants (Landry & Vézina, 2001), (Daniell, Streatfield, Wycoff, & Daniell, 2001), (Ma et al., 2005)

Protein	Application and specificity	Plant
Bet v1	Treatment of type I allergies	Tobacco
Subunit ofcholeratoxinB	Cholera treatment	Potato
GlycoproteinB ofCMV	Treatment of an infection with cytomegalovirus	Tobacco
D2peptideof the proteinB	Autoimmune diabetes treatment	Black bean
VP1	Mucosal vaccine that does not require binding adjuvant	Alfalfa, Black bean
Hemagglutinin	Treatment of influenza	Tobacco
Hepatitis antigen	Treatment of hepatitis B	Tobacco, potato
Enterotoxin B of E.coli	Treatment of diarrhea	Tobacco, potato
Epitope ofP.falciparum	Treatment of malaria	Tobacco
Capsid protein of Norwalk virus	Treatment of diarrhea caused by the Norwalk virus	Tobacco
Protein G of rabies virus	Vaccination against rabies	Tobacco, spinach, tomato

Growth factors, hormones and cytokines

Growth factors, hormones and cytokines represent have a great share in the market of biopharmaceuticals. The products occupying the largest market share in this category are the growth factors of the hematopoietic system such as erythropoietin, G-CSF (granulocyte-colony stimulating factor), GMCSF (granulocyte-macrophage colony stimulating factor), insulin. Several hormones and growth factors were expressed in tobacco: GM-CSF, interferon α and β, erythropoietin and EGF (epidermal growth factor). Also, interleukin-2 that was produced in alfalfa with bioactivity comparable to that of original recombinant protein commonly used in bacterial therapy (Landry & Vézina, 2001).

Table 4

Growth factors, hormones and cytokines produced in plants (Landry & Vézina, 2001)

Protein	Application and specificity	Plant
Somatropin	Growth hormone	Tobacco
Interferon-α	Hepatitis B and C	Rice, turnip
Interferon-β	Hepatitis B and C	Tobacco
Epidermal growth factor (EGF)	Control of cell proliferation	Tobacco
GM-CSF	Growth factor and used hematopoietic factor	tobacco
Homotrimeric collagen	Collagen	Tobacco
α-1 antitrypsin	Cystic fibrosis, liver disease	Rice
Lectoferrin	Antimicrobial	Potato

Enzymes and other products

Enzymes are a group of biopharmaceutical products of special interest for the treatment of certain diseases such as thrombosis (urokinase), Gaucher’s disease or CF (alteplase). The tests made in plants for the production of therapeutic enzymes are conclusive in terms of level of activity, safety, cost and capacity. Glucocerebrosidase can be one example, which is presently produced commercially from extracts of human placentas. About 2000 placentas are required to provide a standard dose without taking into account the risk of contamination with human pathogens. When the gene encoding the glucocerebrosidase is expressed in the tobacco, the content of a single sheet is sufficient to provide the required single dose.
The main problem in this case is the discussed above glycosylation of the mature protein which delays so far the start of clinical tests with this enzyme produced in tobacco leaves (Landry & Vézina, 2001).

 Table 5

Enzymes produced in plants (Landry & Vézina, 2001)

 

Protein	Application and specificity	Plant
Converting enzyme of angiotensin	Hypertension	Tobacco and tomato
Cprotein (serine protease)	Anti-coagulant	Tobacco
Glucocerebrosidase	Gaucher’s disease	Tobacco
α–trichosantine	Inhibits HIV replication	Tobacco

Non-human proteins

Protein	Application and specificity	Plant
Angiotensin-converting  enzyme	Hypertension	Tobacco, tomato
α-tricosanthin from TMV-U1 subgenomic coat protein	HIV therapies	Tobacco
Glucocerebrosidase	Gaucher’s disease	Tobacco
Ricin		Tobacco
Bryodin 1	Potent ribosome-inactivating protein	Tobacco
Chlorogen	Ovarian cancer	Tobacco

Conclusions

Plant proteins production systems are efficient for the fabrication of biopharmaceuticals, in terms of rapidity, costs, quality of proteins, offering the chance to satisfy the increasing demand for protein pharmaceuticals. At the moment, most plant proteins are produced in mature plants, but the production using plant cell lines in bioreactors reveals several advantages and offers new opportunities. Also, challenges such as post-translational modifications and proteolysis, have to be overcome. Post-translational modifications make the major difference between plant and human proteins, N-glycosylation being the most studied. Furthermore, different strategies are researched for the “humanization” of plant proteins, the most important recent advance of this process being the discovery of sialylation pathways in plants. Regardless of problems related to bioequivalence, different pharmaceuticals such as vaccines, blood proteins and enzymes, have already been obtained from plants and used with success.

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