PMP, plant-made pharmaceutical(s); cGMP, current good manufacturing practice; ORF, open reading frame; IgG, immunoglobulin G; HIV, human immunodeficiency virus; CHO, Chinese hamster ovary; UTR, untranslated region
The number of approaches to recombinant protein production in plants is greater than ever before. Development of these new and improved technologies as production platforms for plant-made pharmaceuticals has and will continue to create new commercial opportunities in the pharmaceutical sector. However, it is inevitable that no single system will be optimal for the production of all recombinant proteins of interest in plants due to both the physical characteristics and the envisaged therapeutic application of each product. Here, we review a range of promising product/platform pairs emphasizing synergies during production and in clinical trials.
The market for recombinant protein pharmaceuticals is large and growing rapidly, with nearly all large pharmaceutical companies reporting an increasing revenue share from these products rather than small-molecule drugs 1. Typically, these pharmaceuticals are manufactured using mammalian or bacterial cell-based systems, which are complex to operate and inherently vulnerable to contamination with human pathogens. Production facilities compatible with current good manufacturing practice (cGMP) require huge capital expenditure and involve considerable financial risk. Plant biotechnology has the potential to overcome some of these limitations, but several hurdles remain before this new industry can effectively compete in the pharmaceutical sector.
Interest in the potential of plants as biofactories for recombinant proteins of pharmaceutical relevance was first piqued by the report of a tobacco line engineered to accumulate a functional murine monoclonal antibody (mAb) 2. This report built on previous work with transgenic plants and identified the plant endomembrane system as a set of compartments in which complex heterologous glycoproteins could correctly fold and assemble. Other examples of recombinant protein pharmaceuticals in plants followed and early descriptions include human serum albumin 3 and hepatitis B surface antigen (HBsAg) 4. There are now about 20 plant-made pharmaceuticals (PMPs) in development as potential products (Table 1). After 20 years of research and development (R&D), these candidates are now starting to make an appearance in the marketplace.
|Apo-A1Milano||Therapeutic protein||Cardiovascular disease||SemBioSys Genetics, (Calgary, Canada)||Safflower||Preclinical. Still under development as of 03/2010.||http://www.sembiosys.comhttp://www.sembiosys.com/Docs/ACC.10_Abstract.pdf|
|Plantechno srl. (Vicomoscano, Cremona, Italy)||Transgenic rice||Preclinical. Patent protection in the USA (US2010/0168006A1).||http://www.plantechno.com|
|Insulin (SBS-1000)||Therapeutic protein||Diabetes||SemBioSys Genetics||Safflower||Phase I/II completed Q1 2009.||5|
|Glucocerebrosidase (UPLYSO)||Therapeutic enzyme||Gaucher's disease||Protalix (Carmiel, Israel)||Carrot cell culture||Phase III trial complete Sept. 2009. Currently available under the FDA's Expanded Access Program, full licensure being sought.||6,7, http://www.protalix.com/&clinicaltrials.gov identifiers NCT00258778, NCT00376168|
|Alpha-galactosidase (PRX-102)||Therapeutic enzyme||Fabry's disease||Protalix||Carrot cell culture||Preclinical||See website|
|Acetylcholesterase (PRX-105)||Therapeutic enzyme||Biodefense||Protalix||Carrot cell culture||Phase I (March 2010)||See website|
|Antitumor necrosis factor (Pr-anti-TNF)||Antibody||Arthritis||Protalix||Carrot cell culture||Preclinical||See website|
|β-Glucosidase||Therapeutic protein||Gaucher's disease||Plantechno srl||Tobacco seeds||Preclinical||See website|
|TransPharma srl (Trieste, Italy)||Transgenic tobacco||Phase I||See website|
|2G12 IgG||Antibody||HIV prophylactic||Pharma-planta consortium||Transgenic tobacco||Phase I (commencing Q2 2009)||http://www.pharma-planta.org|
|Interferon-alpha modified release (Locteron®)||Cytokine||Hepatitis C||Biolex Therapeutics (Pittsboro, NC, USA)||Lemna (Duckweed)||Phase IIb (April 2009-ongoing)||http://www.biolex.com & 8|
|Recombinant plasmin (BLX-155)||Therapeutic enzyme||Thrombosis prophylaxis||Biolex Therapeutics||Lemna (Duckweed)||Preclinical||See website|
|Anti-CD20 mAb (BLX-301)||Antibody||Non-Hodgkin's lymphomas||Biolex Therapeutics||Lemna (Duckweed)||Preclinical||9|
|Human serum albumin||Therapeutic protein||Maintenance of blood plasma pressure||Agragen (Cincinatti, OH, USA)||Flax||Preclinical||http://www.plantpharma.org/2005/06/state-shouldnt-miss-opportunities-with-agragen/|
|Lactoferrin||Dietary||GI infections in infants||Ventria Bioscience (Fort Collins, CO, USA)||Transgenic rice||FDA GRAS application withdrawn||http://www.ventria.com/ & 10|
|Meristem therapeutics (Clarmont-Ferrand, France)||Transgenic maize||Phase I||See website|
|Lysozyme||Dietary||GI infections in infants||Ventria Bioscience||Transgenic rice||FDA GRAS application withdrawn||11|
|(RhinoRx)||Antibody||Rhinovirus prophylactic||Planet Biotechnology (Hayward, CA, USA)||Transgenic tobacco leaves||Phase II||http://www.planetbiotechnology.com|
|Guy's 13 SIgA (CaroRx)||Antibody||Dental caries||Planet Biotechnology||Transgenic tobacco leaves||Phase II complete. Approved for use in the EU, but not marketed.||http://www.planetbiotechnology.com|
|Collagen||Structural protein||Reconstructive surgery||Meristem therapeutics||Transgenic maize||Preclinical||http://web.archive.org/web/20071012232910/www.meristem-therapeutics.com/rubrique.php3?id_rubrique=64|
|Therapeutic enzyme||Cystic fibrosis, pancreatitis||Meristem therapeutics||Transgenic maize||Phase IIa||http://web.archive.org/web/20071012232733/www.meristem-therapeutics.com/rubrique.php3?id_rubrique=38|
|Human intrinsic factor||Dietary||Vitamin B12 deficiency||Cobento Biotech AS (Aarhus, Denmark)||Transgenic Arabidopsis||Phase II complete. Marketed in the EU.||12,13|
|Pandemic and seasonal influenza vaccines||Vaccine (virus-like particle)||Risk of influenza transmission||Medicago (Quebec City, Canada)||Proficia™ infiltrated N. benthamiana||Phase I complete Dec. 2009. Phase II Q4 2010.||http://www.medicago.com Clinical trial NCT00984945|
|Hepatitis B surface antigen||Vaccine||Hepatitis B||Arntzen group, Arizona State University||Transgenic potato||Phase I||14|
|Thomas Jefferson University/Polish NAS (Posnan, Poland)||Transgenic lettuce||Phase I||15|
|Rabies glycoprotein||Vaccine||Rabies||Yusibov group, Fraunhofer USA||Viral vectors in spinach||Phase I||16|
|Various anti-idiotype IgG antibodies||Vaccine||Non-Hodgkin's lymphomas||Bayer Innovation (Halle, Germany)||MagnICON infiltrated N. benthamiana||Phase I (Dec. 2009)||http://www.bayer-innovation.com17 clinical trial NCT01022255|
|Norwalk virus capsid protein||Vaccine||Norovirus vaccine||Arntzen group, Arizona State University||Transgenic potato||Phase I||18|
Plant biotechnology for recombinant protein expression now encompasses a range of different technologies. The first approaches using transgenic plants have been supplemented by new techniques, leading to dramatically improved yields and product consistency. In combination with work to clarify and mature the regulatory framework surrounding PMPs, these advances constitute potential for current and future commercial enterprise in the field. In this review, we will compare plant production methodologies and discuss the prospects of leading PMPs with regard to regulatory requirements and commercial potential.
Technological development in the field has led to an expansion in the number of well-developed systems available for the production of recombinant pharmaceuticals in plants. Many plant species are now amenable to genetic manipulation and the details of each have been reviewed elsewhere 19. The use of a certain set of genetic elements in combination with the transgene of interest has allowed high-yield expression in both the roots and leaves of transgenic plant lines as well as bursts of transient expression in nontransgenic Nicotiana benthamiana plants. In this section, we will highlight the key features of the main production platforms using products that are in development.
These production platforms are:
So far, correlations between the nature of a target recombinant protein and the best expression system for that protein have only been made on the broadest basis. A deeper understanding of the biochemical nature of proteins that are critical for different expression strategies is still required. In addition, the regulatory framework relevant to the different expression systems is certainly more advanced in some cases than in others. Broadly, the relative strengths and weaknesses of each class of expression system is summarized in Table 2.
|Open-field tobacco||Glasshouse tobacco||Rhizosecretion||N. benthamiana transient expression||Cell culture|
|Typical model PMP or product||Antibodies, antigens||Antibodies, antigens||Small proteins (CV-N), antibodies||Antibodies, antigens||Veterinary vaccines, glucocerebrosidase (UPLYSO)|
|Yield (range)||++ (15–50 mg/kg leaf fresh weight)||++ (15–50 mg/kg leaf fresh weight)||+ (0.25–3 µg/mL/ 24 H)||+++ (0.5–4 g/kg leaf fresh weight)||+++|
|Downstream purification burden||+||+||+++||+||++|
The first recombinant protein products from plant biotechnology to reach the marketplace were not pharmaceuticals but diagnostic enzymes and reagents. The production and purification of egg white avidin and bacterial beta-glucuronidase for commercial purposes were first reported in 1998 20,21, and both have been marketed at some point through collaboration between the former biotech company Prodigene (College Station, TX, USA) and Sigma–Aldrich (St. Louis, MO, USA). However, despite active R&D efforts and venture capital, new nonpharma products entering the marketplace have remained scarce. Both of these forerunner products were expressed in a transgenic food crop (maize) in an open-field setting, in order to take advantage of the high protein yields of this crop and the scalability and economy of conventional agronomic practices 22.
The use of food crops for recombinant protein production has been associated with negative sentiment among consumer groups and the food industry, which has led to the U.S. Food and Drug Administration (FDA) adopting a “zero tolerance” policy on PMPs transgene release and contamination of food crops in current PMPs draft guidance documents to industry (www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm124811.pdf). The high-profile loss of containment experienced by Prodigene has led the United States Department of Agriculture/Animal and Plant Health Inspection Service to impose additional requirements on all future field releases of PMP crops in the USA (a copy of the current draft environmental risk assessment is available at http://www.aphis.usda.gov/brs/pdf/complete_eis.pdf). These factors have led to a noticeable shift away from open-field, transgenic food crops toward fully contained, nonfood crop systems for PMPs manufacturing. Unfortunately, this has left the field outside of the established route to commercialization, defined by these forerunner nonpharma products.
Cobento Biotech AS (Aarhus, Denmark) has developed a production system for recombinant human intrinsic factor (rhIF) based on the thale cress Arabidopsis thaliana12,13. A. thaliana is a hardy annual weed with a short generation time that can be readily transformed and grown at high densities in a glasshouse environment. It is not cultivated for commercial use and has no sexually compatible weedy relatives (as stated in the risk assessment lodged with the European Food Safety Authority, http://www.efsa.europa.eu/EFSA/DocumentSet/02_gmo_partii_summary_gmb12vitamin_en.pdf?ssbinary=true), greatly reducing the potential for dissemination of the transgene. The yield of rhIF reported in Cobento's system (70 mg/kg fresh weight, 13) can be compared favorably to that reported for the production of rhIF using a baculovirus system (1–2 mg/L, 23) and the methylotrophic yeast Pischia pastoris (30–40 mg/L, 24). As a widely used model plant, the molecular biology and development of A. thaliana is extensively characterized and a well-annotated genome is available. The identification of specific promoter sequences has resulted in the development of transgenic lines that restrict the expression of transgenic gene products to specific tissues or developmental phases. As the rosette leaves of these plants are small and proportionally poor sources of biomass, future development of this technology as a competitive production platform may focus on achieving high levels of transgenic protein accumulation in the seeds 25.
A unique approach to downstream purification from seeds has been developed by SemBioSys Genetics (Calgary, Canada). Seeds of many plant species accumulate large quantities of triacylglycerides in oil bodies that are stabilized during seed desiccation by the oleosins, a family of apolipoproteins. Recombinant proteins can be designed to associate with oleosin in vivo either through a direct genetic fusion with an oleosin open reading frame (ORF) or through a fusion with an ORF encoding a single-chain variable fragment (scFv) specific for endogenous oleosin. Oil bodies accumulating recombinant protein can then be readily separated from other protein-rich subcellular compartments by centrifugation. This approach has been used to produce insulin in A. thaliana seeds 5 and production for clinical trial has been scaled-up in safflower (Carthamus tinctorius L.).
A seed-oriented production approach has also been demonstrated for other plants, including cereals ( maize, rice, barley, and wheat 26), legumes (pea and soybean), and the oilseed crop safflower. Ventria Biosciences (Fort Collins, CO, USA) has developed human lysozyme 11,27,28 and lactoferrin 10 as products derived from transgenic rice grains. Through a combination of technologies including codon optimization for rice and the use of highly transcribed tissue-specific promoters, researchers were able to obtain a yield of 5 g/kg dehusked rice grain for lactoferrin. A high level of lysozyme accumulation in rice endosperm was achieved by introducing two transgenes into the plant simultaneously. Each gene was designed to encode lysozyme in the context of two distinct sets of cis-elements and with two separate signal peptides, which have been shown to influence the routing of the protein product through the secretory pathway in rice 28. Higher expression levels in these double transgenic lines may be attributable to greater utilization of endoplasmic reticulum subdomains for translation. This approach may have general relevance to other plant platforms wherein increased yields are required to achieve cost-effective production by reducing postharvesting (downstream) purification costs.
Recombinant antibodies have unlocked the potential of mAbs in immunotherapy by increasing both repertoire and affinity while reducing the immunogenicity of nonhuman antibodies. The high cost of production associated with the production of these drugs in mammalian cell bioreactors combined with high therapeutic doses required frequently limited access to these treatments to a select group. The production of several classes of antibodies and antibody-based molecules has been reported in plants (reviewed recently 29).
Planet Biotechnology (Hayward, CA, USA) has progressed the development of CaroRX™, a secretory immunoglobulin A (SIgA) designed to block colonization of the oral cavity by Streptococcus mutans through binding to the bacterial surface adhesin (an archive of products in development is available at http://web.archive.org/web/20080531015736/http://www.planetbiotechnology.com/products.html). SIgA is a heterodecameric complex consisting of four heavy chains, four light chains, the J chain, and the secretory component of the poly-immunoglobulin receptor. The development of a transgenic tobacco (Nicotiana tabacum) line expressing each of the four component protein chains was achieved through conventional plant breeding techniques. Despite lengthy generation times and the need to screen many progeny, the use of transgenic lines bred in this manner remains a robust approach for the production of heteromultimeric complexes in plants. In this example, antibody was produced from leaf matter grown in an open-field site in Kentucky. The scalability of plant production in such an environment, coupled with the application of conventional agricultural machinery for planting, husbandry, and harvesting, is an attractive economic argument for such an approach. However, the need to address tightening regulatory oversight, even in case of a nonfood crop such as tobacco, may impact both the business case and sustainability of open-field production.
The Pharma-Planta consortium (http://www.pharma-planta.org), a European Union (EU)–funded collaborative research project designed to define procedures and methods for the production of pharmaceutical proteins in plants, is aiming to deliver a second plant-derived mAb to clinical trials. 2G12 immunoglobulin G (IgG) was identified from human sera as a potent, broadly neutralizing antibody against many human immunodeficiency virus (HIV) isolates 30. A contained glasshouse scenario was envisaged for production of 2G12 in transgenic tobacco plants. The cost of biomass produced in such facilities will always exceed that of open-field crops due to high start-up costs, labor, and running costs. However, there are several key advantages. Growing plants in a glasshouse under containment eliminates both potential gene flow through the escape of pollen and release of the product into the environment, facilitating compliance with environmental regulations. Close control of environmental factors can be used to optimize PMP yield in modern greenhouses, and temperature has been shown to affect the yield of a murine IgG and the HIV microbicide cyanovirin-N (CV-N) in transgenic tobacco 31. The glasshouse environment also limits the potential for physical damage to the crop, which has also been linked to yield variability in IgG and CV-N tobacco plants (unpublished data from our group). Both these factors may improve batch-to-batch consistency after downstream processing, which is a core principle of cGMP, an internationally harmonized set of standards for the production of therapeutics.
Glasshouse-grown transgenic plants therefore represent a technically feasible production system for PMP. However, limited yields have hampered the adoption of such an environment for commercial production purposes. Increasing PMP yield decreases both the cost of plant cultivation and the burden on downstream processing. High-level transient expression of PMP transcripts can be achieved through the use of Agrobacterium leaf infiltration technique in combination with optimized expression vectors. Medicago (Quebec City, Quebec, Canada) has based a production methodology for PMPs including antibodies and influenza vaccines (seasonal and pandemic) around its proprietary Proficia™ technologies. Proficia™ incorporates plasmid vectors based on highly transcribed cis-regulatory elements, including a range of promoter sequences isolated from photosynthetic genes. These vectors are designed to include multiple expression cassettes, allowing for the coexpression of separate protein products, which may represent a subunit of a multimeric PMP, an inhibitor of posttranscriptional gene silencing, or an enzyme involved in a glycosylation pathway 32. Using this system, the bioaccumulation of a mAb in N. benthamiana was reported to approach 25% total soluble protein or 1.5 g/kg fresh leaf weight (g/kg flw) 32, a figure that exceeds that of most transgenic plant lines expressing mAbs. Medicago has recently committed to develop production facilities for influenza vaccines using this system in both Canada and France.
Further efforts to improve expression levels after Agrobacterium leaf infiltration have focused on the use of plasmids designed to use sequences derived from plant viruses. These sequences may take the form of simple translational enhancers, such as the cowpea mosaic virus 5′ and 3′ untranslated regions (UTRs) that form part of the pEAQ-HT series of vectors 33, or they may include viral genes or even entire recombinant genomes based within plasmid vectors. A vector system based on bean yellow dwarf virus, a member of the single-stranded DNA virus family Geminiviridae, utilizes two viral ORFs and two viral UTRs to direct the synthesis and activity of a viral replicase in plants 34,35. DNA copies of the expression cassette consisting of a constitutive promoter, transgene ORF, and a terminator produced by this replicase then act to maximize transcription of the desired gene product. This system has been employed on a laboratory scale in N. benthamiana leaves to produce an Ebola-directed mAb (0.5 g/kg flw 35) and virus-like particles consisting of either the hepatitis C core antigen or norovirus capsid protein (0.8 and 0.53 g/kg flw, respectively; 34).
ICON Genetics (Halle, Germany), a wholly owned subsidiary of Bayer Innovation (Dusseldorf, Germany), is developing a separate set of vectors for the commercial production of patient-specific antibodies as idiotype vaccines for the treatment of non-Hodgkin's lymphomas (NHLs) 36. In this system, the leaves of nontransgenic N. benthamiana plants are infiltrated with a mixed Agrobacterium culture capable of directing the expression of an antibody idiotype isolated from the patient's lymphoma in the context of either the tobacco mosaic virus or potato virus X minigenome. Two viral genomes are routinely used to avoid segregation of one transgene as the virus replicates. Both minigenomes include an RNA-dependent RNA polymerase (RDRP) and are thought to boost protein yields through a mechanism based on RDRP-directed transcript amplification as well as virus-directed cell-to-cell movement 37. Yields of antibody (IgG1) in the latest version of this system were reported to range from 0.5 to 4.8 g/kg flw 17. The authors estimate that 225 mg of product would suffice for quality control and therapy of a single patient, and using the proposed system, this quantity could be produced within 2 weeks of the completion of molecular cloning. Other demonstrations of this technology have reported yields of the fluorophore green fluorescent protein in excess of 4 g/kg flw 38.
Given the advantages of speed and scalability inherent to transient expression approaches, these yields can be compared favorably to reported mammalian cell culture IgG yields of 10 g/L using an immortalized human retina cells (PER.C6®; Crucell, Leiden, the Netherlands) or Chinese hamster ovary (CHO) cells 39. Although the high yield of transient expression systems greatly reduces the burden on downstream processing, the concomitant introduction of the Gram-negative bacterium Agrobacterium tumefaciens into the biomass increases the risk of bacterial endotoxin contamination. Optimization of downstream processing procedures may reduce the release of endotoxin from bacterial cell membranes, but costly lipopolysaccharide monitoring and removal is likely to be required for products produced in this fashion to meet cGMP guidelines.
In addition to the insertion of transgenes into the plant nucleus and their transcription from either episomes or as integrated elements, the introduction of transgenes into the plastid genome has also been used to express high levels of recombinant proteins in plant leaf tissue. Typically, this is achieved through the microbombardment of leaf sections with gold particles coated with linear DNA fragments designed to integrate into the plastid genome via homologous recombination (HR). This approach tends to yield high levels of protein accumulation due to the high multiplicity of the transgene after segregation. In addition, transplastomic gene expression is not limited by epigenetic effects often observed at chromosomal loci such as transcriptional gene silencing and the positional effects of random transgene insertion. Plastids lack posttranscriptional modification pathways, characteristic of the eukaryote secretory pathway, principally glycosylation, which may render them unsuitable for the production of PMPs that rely on such modifications for stability or functionality.
Currently, there are no companies actively seeking to exploit plastid-based technologies, despite numerous reports in the scientific literature of chloroplast expression of proteins with pharmaceutical potential, such as antigens and antibiotics (for a recent review, see 40). Chlorogen (St. Louis, MO, USA), now a defunct biotech start-up, acquired or licensed an extensive portfolio of Intellectual Property relating to chloroplast transformation. The sale of this company's assets may allow a new player to attempt to commercialize this process in the future.
The majority of recombinant protein pharmaceuticals currently available are produced in bioreactors using mammalian, insect, or microbial cells. Plant cell bioreactors maintain several key advantages of plant-based production over conventional systems: they do not harbor human-trophic pathogens, and they are typically cheaper to operate and scale-up due to the robust nature and simple growth medium requirements of plant cells 41. These systems are not subject to several perceived disadvantages of whole plant PMPs production as there is a greatly reduced potential for gene flow and contamination to the environment and food chain, and a greater compatibility with cGMP. Importantly, the downstream processing of product may be simplified by the comparative lack of secondary metabolics, fibers, and oils or waxes compared with production in whole plants. Challenges remain over the yield of many products in plant cell bioreactors, with reported yields for recombinant antibodies falling far short of those reported in mammalian (CHO) cell systems. Recent research has suggested that this disparity may be at least partly due to the comparative lack of maturity of plant cell systems with regards to media composition, fermenter design, and cell line engineering 42,43.
Two companies are actively involved in PMPs production from cell culture-based systems. Dow Agrosciences (Indianapolis, IN, USA) have developed a plant cell bioreactor system (Concert™) for the production of veterinary vaccines. The lead product, a subunit vaccine against Newcastle disease in poultry, has been produced using transformed N. tabacum-1 (NT-1) cells cultivated in a traditional nondisposable bioreactor (US patent application US2008/0076177, Cardineau et al.). Despite receiving regulatory approval in 2006 (the corresponding press release is available at http://web.archive.org/web/20080123120809/http://www.dowagro.com/animalhealth/resources/firstlic.htm), no attempt to commercialize this product has yet been made, although a second vaccine employing this technology is still under development (http://www.dowagro.com/animalhealth/resources/news/20070827b.htm). Comparatively low product yield [8 µg/mL in culture medium, (US patent application US2008/0076177, Cardineau et al.)] may be a limiting factor to commercial production with this system.
Protalix Biotherapeutics (Carmiel, Israel) has employed a system using carrot cells and disposable, highly scalable polyethelene bioreactors to produce three candidate products. The production of the lead candidate, human glucocerebrosidase (GCD), has been described 6. Recombinant GCD purified from CHO cells (Genzyme's Cerezyme®; Genzyme, Cambridge, MA, USA) is currently used as an effective treatment for Gaucher's disease, a lysosomal storage disorder primarily affecting macrophages. The use of Cerezyme® is limited by the high cost associated with production and the postproduction enzymatic modification of glycan residues necessary for effective macrophage uptake. Protalix has made significant modifications to the protein to alter its subcellular accumulation within the cells, including the addition of a C-terminal vacuolar sorting signal from tobacco chitinase 44. Subsequent glycoanalysis of plant recombinant GCD (prGCD) revealed the presence of paucimannosidic glycans suitable for macrophage uptake 6, hence eliminating the requirement for further processing to expose these ligands.
Moss is adaptable for growth in culture vessels and has been developed as an expression platform for PMPs by Greenovation GmbH (Heilbronn, Germany; http://www.greenovation.com) 45. Moss protonema cultivated in bioreactors is capable of photosynthesis, further reducing the nutrient requirements in the culture medium. In Greenovation GmbH's system, the recombinant protein product is designed to be secreted from the protonema and harvested from the medium. Moss is an unusual plant as it has been shown to undergo HR at nuclear loci at a significant frequency 46. Combined with excellent genomic resources 47, this trait allows researchers to design transgenes to integrate into specific regions of the moss genome. This approach may reduce variability in expression levels between transgenic lines, thus facilitating a rapid and predictable scale-up of production. Second, knowledge of the integration site of the transgene may be advantageous to any application for IP protection and regulatory approval. Finally, targeted mutation of the moss genome using HR can be used to create moss lines with improved protein processing characteristics, for example, engineered glycosylation patterns 48.
Lemna species, collectively known as duckweed, are protein-rich, clonal, and rapidly growing aquatic plants that are adaptable as PMP production systems 49. Using Lemna as an expression platform, Biolex Therapeutics (Pittsboro, NC, USA) has focused on developing products with superior clinical efficacy than existing related drugs on the market. BLX-301, a CD20-specific antibody with potential to treat NHLs, is produced in the context of a regulated glycosylation pathway achieved through the use of RNA interference. Antibodies produced in this fashion were shown to bear a more homogeneous population of glycans that correlated with improved antibody effector functions 9. A second product, human plasmin (BLX-301), has been under investigation as an anticoagulant for over 50 years. However, owing to difficulties encountered during the purification of active plasmin for blood sera and later from nonplant recombinant sources, and the relative tractability of enzymes upstream in the plasmin pathway, no therapeutic version has yet been developed. Recently, the therapeutic potential of two deleted versions of plasmin expressed in Escherichia coli was demonstrated 50,51. In contrast, full-length human plasmin accumulated in an active conformation and at high yields in the Lemna production system, and results for preclinical trials are available (http://www.biolex.com/pdfs/Biolex%20Press%20Release%20-%20SIR%20Presentation%2031808.pdf). It is proposed that the domains retained in the Lemna product may enhance efficacy by increasing affinity for fibrin, the major component of blood clots. Lemna-derived plasmin may also benefit from an improved safety profile as it is correctly inhibited by endogenous plasmin inhibitors. The development of the company's lead product, interferon-alpha-2b in a novel modified-release formulation (Locteron®), is now supported by significant venture capital (Biolex press release available at http://www.biolex.com/pdfs/Biolex%20Series%20D%20-%20October%206%202008.pdf). This investment, the fourth injection of funds into the company, underlines the confidence of the biotech sector in Biolex's technology and product portfolio.
The collection of recombinant proteins from the space around the tissues of transgenic plants is an attractive modality for PMP production. Two approaches have been described: the collection of apoplastic fluids either as tobacco leaf guttation fluids 52, or via vacuum collection 53, and of root exudates (rhizosecretion) 54,55. Compared with homogenized plant tissue, these fluids are excellent feedstocks for downstream purification as they do not require expensive and lossy initial processing steps such as mechanical disruption and extract clarification. Furthermore, as the plant tissue is not disrupted during harvesting, the feedstock is not contaminated with intracellular proteases or degraded fragments of the product, and continuous production can be envisaged. These approaches also retain many advantages of using whole plants: genetic stability 56, photoautotrophy, and scalability. Despite these advantages, the vacuum collection of alpha-galactosidase A from plant leaves as demonstrated by the former biotech company Biosource Technologies remains the only reported large-scale application of a nondestructive harvesting approach 57.
Because of the porosity of plant cell walls, the accumulation of recombinant proteins in exudates is influenced by the effective molecular radius of the molecule. These systems may therefore be most suited to the production of smaller proteins, or those with compact structures, such as antibodies 54,58. Noncovalent interactions between the PMPs and molecules present in plant tissues may also prevent efficient harvesting through washing with a physiological buffer. High ionic strength buffers have typically been used to disrupt these interactions and release PMPs from suspension cultured plant cells 59, but it remains to be shown whether this approach is viable in rhizosecretion models under continuous production.
Product yield has remained a major drawback for systems relying on plant tissue exudates. Guttation of secreted alkaline phosphatase (SEAP) from transgenic tobacco leaves was quantified as 0.15–1.1 µg/g leaf dry weight per day 52. The authors state that no attempt was made to optimize yield in this system; nevertheless, it is difficult to envisage a cost-effective engineering solution to collect this material. Efforts to increase rhizosecretion yield have included the use of root-specific promoters 60, induction of hairy roots 61, co-expression of secreted protease inhibitors 62, and the addition of plant growth regulators 54. In the last example, the maximum rate of rhizosecretion of a murine IgG and cyanovirin-N (CV-N) was reported as 58 and 766 µg/g root dry weight/24 H, respectively. These yields represent a fivefold increase over previously reported rates 63,64. Establishing a suitable engineering solution for the collection of hydroponic fluid on a large scale, either through the use of recirculating nutrient film technique trays or batch production in sterile containers, is the next step in developing a rhizosecretion-based PMPs production system.
As with all novel drugs, PMPs must pass through a series of clinical trials in order to gain regulatory approval for marketing. The precise path of each PMP through clinical trials is subject to the nature of the drug; for example, plant-produced versions of biologic pharmaceuticals already on the market (“biosimilars”) or drugs designed to treat orphan or rare diseases may enjoy a shorter passage through the approval process. Such drugs may therefore be attractive candidates for development as PMPs. It is clear, however, that the production process of candidate PMPs must be designed from the very beginning to cope with the regulatory requirements for clinical trials. Guidance for the production of recombinant protein pharmaceuticals specifically from transgenic plants has now been adopted in the EU (EMEA/CHMP/BWP/48316/2006). A more general set of guidelines is under draft in the USA (FDA CVM GFI #153, www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm124811.pdf) that also covers some aspects of transient expression systems. In the case of transgenic plants, these guidelines establish require-ments for host plant characterization and seed banking. Specifications for PMPs characterization must be established on a case-by-case basis but should include a suitable comparison with the natural counterpart where feasible and appropriate.
The analysis of plant-specific modifications, such as specific complex glycosylation patterns and other posttranslational modifications, is an important component of PMP specifications. Considerable concern has centered on the induction of inappropriate immune responses to parenterally administered PMPs triggered by plant-specific glycoforms. Production using seed crops, or in systems wherein the transgene product is effectively retained in the early secretory pathway, may be preferred in order to limit the addition of these moieties. However, there is little evidence that these reactions, when they do occur, are clinically significant 65. Indeed, in the results of a phase I clinical trial of prGCD (Protalix Biotherapeutics trial reference NCT00258778 and 7), no specific antibodies were raised to prGCD, which bears a proportion of glycans with plant-specific monosaccharides and sugar linkages. In the case of this drug, the orphan disease status of the indication in combination with the high cost of existing therapies has allowed Protalix to apply for a “fast track” to FDA approval (a description of the process is available here http://www.fda.gov/AboutFDA/CentersOffices/CBER/ucm122932.htm), which includes enhanced regulatory oversight and rolling reviews of the new drug application (NDA). The clinical use of prGCD has also been accelerated by the apparent contamination of the existing CHO cell-derived product (Cerezyme®, Genzyme) with a potentially pathogenic calcivirus (Genzyme press release available at http://www.genzyme.com/corp/media/GENZ%20PR-061609.asp). In contrast, most plant systems offer limited or no exposure to human-trophic viral adventitious agents.
Glycoprotein pharmaceuticals from all production systems are routinely analyzed for the homogeneity and nature of their associated carbohydrates 66, including those produced using mammalian cell systems, as these moieties can influence the potency and pharmacokinetic profile of the drug. Various plant systems including those based on Nicotiana spp. and Lemna have been developed with altered glycosylation pathways 9,67,68, opening up the possibility of glycoengineering PMPs for improved therapeutic effect. This has particular relevance to antibodies, such as Bayer Innovation's anti-idiotype NHL vaccines, which are currently undergoing phase 1 clinical trials (clinicaltrials.gov identifier NCT01022255). It will be interesting to compare the results of this trial against a previous iteration of this technology, which utilized plant-produced scFvs 36.
Insulin produced using the safflower seed oil body system (SBS-1000, Sembiosys Genetics) has progressed through a phase I/II trial involving 23 healthy volunteers. The human arm of the study aimed to establish bioequivalence between SBS-1000 and Humulin-R® (Eli Lilly, Indianapolis, IN, USA), a widely used off-patent insulin product produced in bacterial fermentors. In each group, the total glucose infusion required to maintain a euglycemic clamp and insulin concentration in the serum of healthy volunteers was found to be within an equivalence range. Adverse reactions to the product were also within an acceptable profile for the administration of recombinant human insulin. This demonstration of bioequivalence will likely reduce the requirement for further clinical efficacy data in order to obtain a license from the relevant agencies. Guidance on this process has been issued in the EU (CHMP/437/2004), and in the USA certain biosimilar products have been approved for an abbreviated NDA. However, as biosimilar products such as SBS-1000 lack demonstrable clinical advantages over rival generics, the economic advantages of the plant production system must be sufficient on their own. In contrast, a phase I trial of interferon-alpha from Lemna (Locteron®, Biolex Therapeutics 8) aimed to demonstrate a superior pharmacokinetic profile when compared with Intron A (Merck, Whitehouse Station, NJ, USA) through the use of a modified-release formulation, and will therefore require full approval as a new drug. Nevertheless, formulating PMPs to achieve superior clinical efficacy may be the key to market penetration.
Subunit vaccines delivered as unprocessed or minimally processed plant biomass preparations have also entered phase I clinical trials. These include HBsAg from potato 14 and lettuce 15, rabies glycoprotein from spinach 16, enterotoxigenic E. coli heat-labile toxin B subunit from corn 69 and potato 70, and norovirus nucleocapsid from potato 18. Each preparation was well tolerated. The efficacy of this approach will be largely determined by the relevant stability of the antigen in the gut, the development of suitable adjuvants, and methods to ensure consistent dosing.
In this review, we have covered only some of the approaches described to generate recombinant proteins from plants. The myriad of plant production systems available offers unrivalled flexibility to create financially and technically viable approaches for the production of PMPs, but the lack of focus on a particular technology has doubtlessly delayed the application of plant biotechnology as a whole to this field. Thus, the pathway to commercialization for PMPs remains unclear. It is likely that the first wave of human therapeutics from plants will employ cell culture approaches, by virtue of compatibility with existing regulatory frameworks.
Three broad classes of drug have received particular attention from the PMP community. The first class is therapeutic antibodies and antibody-derived proteins. Antibody PMPs manufacture is now supported by a well-developed set of technologies, which can offer excellent yields and unique technical advantages. Indeed, plants remain the only viable production platform for the manufacture of SIgA molecules. Antibodies produced in plants have been approved for topical use in the EU (CaroRX™, Planet Biotechnology) and are progressing through clinical trials for systemic use in the USA (NHL scFvs). Transgenic tobacco, in either an open-field or contained glasshouse environment, offers an inexpensive manufacturing route that is currently employed in the production of antibodies for topical use (e.g., CaroRX™ and mAb 2G12) as well as an antibody used in downstream processing of a hepatitis B vaccine (anti-HbsAg; CIBR, Cuba; 71). The involvement of the EU Pharma-Planta consortium in bringing mAb 2G12 to the brink of a phase I clinical trial has left clear regulatory guidelines in place for greenhouse production of PMPs from whole plant biomass.
The second group is subunit vaccine antigens. Noninvasive immunization by oral delivery is an appealing route of immunization, and plant expression hosts have linked with oral delivery since the inception of the technology. Several “edible vaccines” have progressed to phase I clinical trials, although important issues regarding dose standardization and contamination of the food chain, as well as the efficacy of oral vaccination, remain to be addressed. Plant-produced oral vaccine antigens may find greatest relevance as the boosting components of heterologous prime-boost strategies, as demonstrated for measles and hepatitis B vaccines 14,72. The rapid production of vaccine antigens in transiently transfected leaves of N. benthamiana provides a further potential advantage of plant production for vaccine antigens. This approach is exemplified by the seasonal and pandemic influenza vaccines currently in clinical trials sponsored by Medicago.
The third class of PMPs is therapeutic enzymes. At least initially, PMPs in this class are likely to be “biosimilars” such as insulin (SBS-100, SemBioSys Genetics), glucocerebroside (UPLYSO, Protalix), and interferon-alpha (Locteron®, Biolex Therapeutics). These products benefit from a potentially abbreviated review process and streamlined product development. The use of PMP technologies may allow the marketing of biosimilar versions of drugs with extant patent protection in other production systems. However, in view of the vast manufacturing capacity already in place in the generics sector, PMPs must also rely on the advantages of a plant production platform to compete effectively.