Abstract
Molecular pharming is a cost-effective platform for the production of recombinant proteins in plants. Although the biopharmaceutical industry still relies on a small number of standardized fermentation-based technologies for the production of recombinant proteins there is now a greater awareness of the advantages of molecular pharming particularly in niche markets. Here we discuss some of the technical, economic and regulatory barriers that constrain the clinical development and commercialization of plant-derived pharmaceutical proteins. We also discuss strategies to increase productivity and product quality/homogeneity. The advantages of whole plants should be welcomed by the industry because this will help to reduce the cost of goods and therefore expand the biopharmaceutical market into untapped sectors.
This is a preview of subscription content, access via your institution.

References
Allen GC, Hall GE Jr, Childs LC, Weissinger AK, Spiker S, Thompson WF (1993) Scaffold attachment regions increase reporter gene expression in stably transformed plant cells. Plant Cell 5:603–613
Allen GC, Hall G Jr, Michalowski S, Newman W, Spiker S, Weissinger AK, Thompson WF (1996) High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco. Plant Cell 8:899–913
Arakawa T, Yu J, Chong DK, Hough J, Engen PC, Langridge WH (1998) A plant-based cholera toxin B subunit-insulin fusion protein protects against the development of autoimmune diabetes. Nat Biotechnol 16:934–938
Aspelund MT, Glatz CE (2010) Purification of recombinant plant-made proteins from corn extracts by ultrafiltration. J Membr Sci 353:103–110
Aviezer D, Brill-Almon E, Shaaltiel Y, Hashmueli S, Bartfeld D, Mizrachi S, Liberman Y, Freeman A, Zimran A, Galun E (2009a) Novel enzyme replacement therapy for Gaucher disease: ongoing Phase III clinical trial with recombinant human glucocerebrosidase expressed in plant cells. Mol Genet Metab 96:S13–S14
Aviezer D, Brill-Almon E, Shaaltiel Y, Hashmueli S, Bartfeld D, Mizrachi S, Liberman Y, Freeman A, Zimran A, Galun E (2009b) A plant-derived recombinant human glucocerebrosidase enzyme—a preclinical and phase I investigation. PLoS ONE 4:e4792
Bardor M, Faveeuw C, Fitchette AC, Gilbert D, Galas L, Trottein F, Faye L, Lerouge P (2003) Immunoreactivity in mammals of two typical plant glyco-epitopes, core alpha (1,3)-fucose and core xylose. Glycobiology 13:427–434
Boothe J, Nykiforuk C, Shen Y, Zaplachinski S, Szarka S, Kuhlman P, Murray E, Morck D, Moloney MM (2010) Seed-based expression systems for plant molecular pharming. Plant Biotechnol J 8:588–606
Buyel JF, Fischer R (2012) Predictive models for transient protein expression in tobacco (Nicotiana tabacum L.) can optimize process time, yield, and downstream costs. Biotechnol Bioeng 109:2575–2588
Cardineau GA (2008) Vectors and cells for preparing immunoprotective compositions derived from transgenic plants. US Patent Application (Boyce Thompson Institute and Dow Agrosciences LLC)
Castilho A, Neumann L, Gattinger P, Strasser R, Vorauer-Uhl K, Sterovsky T, Altmann F, Steinkellner H (2013) Generation of biologically active multi-sialylated recombinant human EPOFc in plants. PLoS ONE 8:e54836
Claparols MI, Bassie L, Miro B, Del Duca S, Rodriguez-Montesinos J, Christou P, Serafini-Fracassini D, Capell T (2004) Transgenic rice as a vehicle for the production of the industrial enzyme transglutaminase. Transgen Res 13:195–199
Conley AJ, Joensuu JJ, Richman A, Menassa R (2011) Protein body-inducing fusions for high-level production and purification of recombinant proteins in plants. Plant Biotechnol J 9:419–433
Conrad U, Fiedler U (1998) Compartment-specific accumulation of recombinant immunoglobulins in plant cells: an essential tool for antibody production and immunomodulation of physiological functions and pathogen activity. Plant Mol Biol 38:101–109
CPMP (Committee for Proprietary Medicinal Products) (2002) Points to consider on quality aspects of medicinal products containing active substances produced by stable transgene expression in higher plants (EMEA/CPMP/BWP/764/02). EMEA, London UK
CPMP (Committee for Proprietary Medicinal Products) (2009) Guideline on the quality of biological active substances produced by stable transgene expression in higher plants (EMEA/CHMP/BWP/48316/2006). EMEA, London
Datta K, Baisakh N, Oliva N, Torrizo L, Abrigo E, Tan J, Rai M, Rehana S, Al-Babili S, Beyer P, Potrykus I, Datta SK (2003) Bioengineered ‘golden’ indica rice cultivars with β-carotene metabolism in the endosperm with hygromycin and mannose selection systems. Plant Biotechnol J 1:81–90
Dow AgroSciences (2006) Press release: Dow AgroSciences achieves world’s first registration for plant-made vaccines (http://www.eurekalert.org/pub_releases/2006-01/i-daa013106.php. Accessed 5th December 2013)
Farre G, Twyman RM, Zhu C, Capell T, Christou P (2011) Nutritionally enhanced crops and food security: scientific achievements versus political expediency. Curr Opin Biotechnol 22:245–251
Faye L, Boulaflous A, Benchabane M, Gomord V, Michaud D (2005) Protein modifications in the plant secretory pathway: current status and practical implications in molecular pharming. Vaccine 23:1770–1778
FDA, USDA (2002) Draft guidance: drugs, biologics, and medical devices derived from bioengineered plants for use in humans and animals. FDA, Rockville
Ferrer-Miralles N, Domingo-Espín J, Corchero JL, Vázquez E, Villaverde A (2009) Microbial factories for recombinant pharmaceuticals. Microb Cell Fact 8:17
Fischer R, Emans NJ, Twyman RM, Schillberg S (2003) Molecular pharming of industrial proteins in plants. In: Fingerman M, Nagabhusmanam R (eds) Recent advances in marine biotechnology. Science Publishers Inc., Enfield NH, pp 279–313
Fischer R, Stoger E, Schillberg S, Christou P, Twyman RM (2004) Plant-based production of biophramaceuticals. Curr Opin Plant Biotechnol 7:152–158
Fischer R, Schillberg S, Hellwig S, Twyman RM, Drossard J (2012) GMP issues for recombinant plant-derived pharmaceutical proteins. Biotechnol Adv 30:434–439
Fischer R, Schillberg S, Buyel JF, Twyman RM (2013) Commercial aspects of pharmaceutical protein production in plants. Curr Pharm Des 19:5471–5477
Garcia-Casado G, Sanchez-Monge R, Chrispeels MJ, Armentia A, Salcedo G, Gomez L (1996) Role of complex asparagine-linked glycans in the allergenicity of plant glycoproteins. Glycobiology 6:471–477
Gomord V, Faye L (2004) Post-translational modification of therapeutic proteins in plants. Curr Opin Biotechnol 7:171–181
Gomord V, Chamberlain P, Jefferis R, Faye L (2005) Biopharmaceutical production in plants: problems, solutions and opportunities. Trends Biotechnol 23:559–565
Gomord V, Fitchette AC, Menu-Bouaouiche L, Saint-Jore-Dupas C, Plasson C, Michaud D, Faye L (2010) Plant-specific glycosylation patterns in the context of therapeutic protein production. Plant Biotechnol J 8:564–587
Halweg C, Thompson WF, Spiker S (2005) The rb7 matrix attachment region increases the likelihood and magnitude of transgene expression in tobacco cells: a flow cytometric study. Plant Cell 17:418–429
Heinrichs A (2010) Small RNAs: Dispensable dicer. Nat Rev Mol Cell Biol 10:461
Hiatt AH, Caffertey R, Bowdish K (1989) Production of antibodies in transgenic plants. Nature 342:76–78
Hofbauer A, Stoger E (2013) Subcellular accumulation and modification of pharmaceutical proteins in different plant tissues. Curr Pharm Des 19:5495–5502
Hollak CE, vom Dahl S, Aerts JM, Belmatoug N, Bembi B, Cohen Y, Collin-Histed T, Deegan P, van Dussen L, Giraldo P, Mengel E, Michelakakis H, Manuel J, Hrebicek M, Parini R, Reinke J, di Rocco M, Pocovi M, Sa Miranda MC, Tylki-Szymanska A, Zimran A, Cox TM (2010) Force majeure: therapeutic measures in response to restricted supply of imiglucerase (Cerezyme) for patients with Gaucher disease. Blood Cells Mol Dis 44:41–47
Hood EE (2002) From green plants to industrial enzymes. Enzyme Microbial Technol 30:279–283
Hood EE, Witcher DR, Maddock S, Meyer T, Baszczynski C, Bailey M, Flynn P, Register J, Marshall L, Bond D, Kulisek E, Kusnadi A, Evangelista R, Nikolov Z, Wooge C, Mehigh RJ, Hernan R, Kappel WK, Ritland D, Li CP, Howard JA (1997) Commercial production of avidin from transgenic maize: characterization of transformant, production, processing, extraction and purification. Mol Breed 3:291–306
Jurgens G (2004) Membrane trafficking in plants. Annu Rev Cell Dev Biol 20:481–504
Kawaguchi R, Bailey-Serres J (2002) Regulation of translational initiation in plants. Curr Opin Plant Biol 5:460–465
Kim CS, Woo YM, Clore AM, Burnett RJ, Carneiro NP, Larkins BA (2002) Zein protein interactions, rather than the asymmetric distribution of zein mRNAs on endoplasmic reticulum membranes, influence protein body formation in maize endosperm. Plant Cell 14:655–672
Knäblein J (2005) Plant-based expression of biopharmaceuticals. In: Meyers RA (ed) Encyclopedia of molecular cell biology and molecular medicine, 2nd edn. Wiley & Sons, NY, pp 385–410
Ko K, Tekoah Y, Rudd PM, Harvey DJ, Dwek RA, Spitsin S, Hanlon CA, Rupprecht C, Dietzschold B, Golovkin M, Koprowski H (2003) Function and glycosylation of plant-derived antiviral monoclonal antibody. Proc Natl Acad Sci USA 100:8013–8018
Kohli A, Twyman RM, Abranches R, Wegel E, Stoger E, Christou P (2003) Transgene integration, organization and interaction in plants. Plant Mol Biol 52:247–258
Kohli A, González-Melendi P, Abranches R, Capell T, Stoger E, Christou P (2006) The quest to understand the basis and mechanisms that control expression of introduced transgenes in crop plants. Plant Signal Behav 1:185–195
Kunka K, Nabil A, Kostadin G, Atanas A (2005) Molecular pharming in plants: an approach of agricultural biotechnology. J Cell Mol Biol 4:77–86
Lico C, Twyman RM, Pezzotti M, Avesani L (2012) The use of plants for the production of therapeutic human peptides. Plant Cell Rep 31:439–451
Lu J, Sivamani E, Azhakanandam K, Samadder P, Li X, Qu R (2008) Gene expression enhancement mediated by the 5′ UTR intron of the rice rubi3 gene varied remarkably among tissues in transgenic rice plants. Mol Genet Genomics 279:563–572
Ma JKC, Lehner T, Stabila P, Fux CI, Hiatt A (1994) Assembly of monoclonal antibodies with IgG1 and IgA heavy-chain domains in transgenic tobacco plants. Eur J Immunol 24:131–138
Ma JKC, Drake PMW, Christou P (2003) The production of recombinant pharmaceutical proteins in plants. Nat Rev Genet 4:794–805
Maqbool SB, Christou P (1999) Multiple traits of agronomic importance in transgenic indica rice plants: analysis of transgene integration patterns, expression levels and stability. Mol Breed 5:471–480
Maqbool SB, Riazuddin S, Nguyen TL, Gatehouse AMR, Gatehouse JA, Christou P (2001) Expression of multiple insecticidal genes confers broad resistance against a range of different rice pests. Mol Breed 7:85–93
Masip G, Sabalza M, Pérez-Massot E, Banakar R, Cebrian D, Twyman RM, Capell T, Albajes R, Christou P (2013) Paradoxical EU agricultural policies on genetically engineered crops. Trends Plant Sci 18:312–324
Mitsuhara I, Ugaki M, Hirochika H, Ohshima M, Murakami T, Gotoh Y, Katayose Y, Nakamura S, Honkura R, Nishimiya S, Ueno K, Mochizuki A, Tanimoto H, Tsugawa H, Otsuki Y, Ohashi Y (1996) Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants. Plant Cell Physiol 37:49–59
Morrow T (2012) Gaucher’s disease treatment option rides on carrot cells’ biologic power. Managed Care 21:45–46
Naqvi S, Zhu C, Farre G, Ramessar K, Bassie L, Breitenbach J, Perez Conesa D, Ros G, Sandmann G, Capell T, Christou P (2009) Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways. Proc Natl Acad Sci USA 106:7762–7767
Naqvi S, Farré G, Sanhauja G, Capell T, Zhu C, Christou P (2010) When more is better: multigene engineering in plants. Trend Plant Sci 15:48–56
Nicholson L, Gonzalez-Melendi P, Van Dolleweerd C, Tuck H, Ma JKC, Fischer R, Christou P, Stoger E (2005) A recombinant multimeric immunoglobulin expressed in rice shows assembly-dependent subcellular localization in endosperm cells. Plant Biotechnol J 3:115–127
Nykiforuk CL, Shen Y, Murray EW, Boothe JG, Busseuil D, Rhéaume E, Tardif JC, Reid A, Moloney MM (2011) Expression and recovery of biologically active recombinant apolipoprotein AI (Milano) from transgenic safflower (Carthamus tinctorius) seeds. Plant Biotechnol J 9:250–263
O’Keefe BR, Vojdani F, Buffa V, Shattock RJ, Montefiori DC, Bakke J, Mirsalis J, D‘Andrea A, Hume SD, Bratcher B, Saucedo CJ, McMahon JB, Pogue GP, Palmer KE (2009) Scaleable manufacture of HIV-1 entry inhibitor griffithsin and validation of its safety and efficacy as a topical microbicide component. Proc Natl Acad Sci USA 106:6099–6104
Parra G, Bradnam K, Rose AB, Korf I (2011) Comparative and functional analysis of intron-mediated enhancement signals reveals conserved features among plants. Nucleic Acids Res 39:5328–5333
Paul M, Ma JKC (2011) Plant-made pharmaceuticals: leading products and production platforms. Biotechnol Appl Biochem 58:58–67
Paul MJ, Teh AYH, Twyman RM, Ma JKC (2013) Target product selection—where can molecular pharming make the difference? Curr Pharm Des 19:5478–5485
Peremarti A, Twyman RM, Gómez-Galera S, Naqvi S, Farré G, Sabalza M, Miralpeix B, Dashevskaya S, Yuan D, Ramessar K, Christou P, Zhu C, Bassie L, Capell T (2010) Promoter diversity in multigene transformation. Plant Mol Biol 73:363–378
Peters J, Stoger E (2011) Transgenic crops for the production of recombinant vaccines and anti-microbial antibodies. Human Vaccines 7:367–374
Ramessar K, Capell T, Christou P (2008a) Molecular pharming in cereal crops. Phytochem Rev 7:579–592
Ramessar K, Capell T, Twyman RM, Quemada H, Christou P (2008b) Trace and traceability—a call for regulatory harmony. Nat Biotechnol 26(9):975–978
Ramessar K, Rademacher T, Sack M, Stadlmann J, Platis D, Stiegler G, Labrou N, Altmann F, Ma JKC, Stoger E, Capell T, Christou P (2008c) Cost-effective production of a vaginal protein microbicide to prevent HIV transmission. Proc Natl Acad Sci USA 105:3727–3732
Ramessar K, Capell T, Twyman RM, Christou P (2010) Going to ridiculous lengths: european coexistence regulations for GM crops. Nat Biotechnol 28:133–136
Sabalza M, Miralpeix B, Twyman RM, Capell T, Christou P (2011) EU legitimizes GM crop exclusion zones. Nat Biotechnol 29:315–317
Sabalza M, Madeira L, Van Dolleweerd C, Ma JKC, Capell T, Christou P (2012) Functional characterization of the recombinant HIV-neutralizing monoclonal antibody 2F5 produced in maize seeds. Plant Mol Biol 80:477–488
Sabalza M, Vamvaka E, Christou P, Capell T (2013) Seeds as a production system for molecular pharming applications: status and prospects. Curr Pharm Des 19:5543–5552
Schillberg S, Zimmermann S, Voss A, Fischer R (1999) Apoplastic and cytosolic expression of full-size antibodies and antibody fragments in Nicotiana tabacum. Transgen Res 8:255–263
Schillberg S, Zimmermann S, Findlay K, Fischer R (2000) Plasma membrane display of antiviral single chain Fv fragments confers resistance to tobacco mosaic virus. Mol Breed 6:317–326
Schillberg S, Raven N, Fischer R, Twyman RM, Schiermeyer A (2013) Molecular farming of pharmaceutical proteins using plant suspension cell and tissue cultures. Curr Pharm Des 19:5531–5542
Sexton A, Harman S, Shattock RJ, Ma JKC (2009) Design, expression, and characterization of a multivalent, combination HIV microbicide. FASEB J 23:3590–3600
Shaaltiel Y, Bartfeld D, Hashmueli S, Baum G, Brill-Almon E, Galili G, Dym O, Boldin-Adamsky SA, Silman I, Sussman JL, Futerman AH, Aviezer D (2007) Production of glucocerebrosidase with terminal mannose glycans for enzyme replacement therapy of Gaucher’s disease using a plant cell system. Plant Biotechnol J 5:579–590
Sharma AK, Sharma MK (2009) Plants as bioreactors: recent developments and emerging opportunities. Biotechnol Adv 27:811–832
Sharma MK, Singh NK, Jani D, Sisodia R, Thungapathra M, Gautam JK, Meena LS, Singh Y, Ghosh A, Tyagi AK, Sharma AK (2008) Expression of toxin coregulated pilus subunit A (TCPA) of Vibrio cholerae and its immunogenic epitopes fused to choleratoxin B subunit in transgenic tomato (Solanum lycopersicum). Plant Cell Rep 27:307–318
Shattock RJ, Moore JP (2003) Inhibiting sexual transmission of HIV-1 infection. Nat Rev Microbiol 1:25–34
Spök A, Twyman RM, Fischer R, Ma JK, Sparrow PA (2008) Evolution of a regulatory framework for pharmaceuticals derived from genetically modified plants. Trends Biotechnol 26:506–517
Stevens LH, Stoopen GM, Elbers IJ, Molthoff JW, Bakker HA, Lommen A, Bosch D, Jordi W (2000) Effect of climate conditions and plant developmental stage on the stability of antibodies expressed in transgenic tobacco. Plant Physiol 124:173–182
Stoger E, Vaquero C, Torres E, Sack M, Nicholson L, Drossard J, Williams S, Keen D, Perrin Y, Christou P, Fischer R (2000) Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies. Plant Mol Biol 42:583–590
Stoger E, Sack M, Perrin Y, Vaquero C, Torres E, Twyman RM, Christou P, Fischer R (2002a) Practical considerations for pharmaceutical antibody production in different crop systems. Mol Breed 9:149–152
Stoger E, Sack M, Fischer R, Christou P (2002b) Plantibodies: applications, advantages and bottlenecks. Curr Opin Biotechnol 13:161–166
Stoger E, Ma JKC, Fischer R, Christou P (2005) Sowing the seeds of success: pharmaceutical proteins from plants. Curr Opin Biotechnol 16:167–173
Strasser R (2013) Engineering of human-type O-glycosylation in Nicotiana benthamiana plants. Bioeng 4:191–196
Streatfield SJ (2007) Approaches to achieve high-level heterologous protein production in plants. Plant Biotechnol J 5:2–15
Topping JF, Wei W, Lindsey K (1991) Functional tagging of regulatory elements in the plant genome. Development 112:1009–1019
Tremblay R, Wang D, Jevnikar AM, Ma S (2010) Tobacco, a highly efficient green bioreactor for production of therapeutic proteins. Biotechnol Adv 28:214–221
Tu J, Zhang G, Datta K, Xu C, He Y, Zhang Q, Khush GS, Datta SK (2000) Field performance of transgenic elite commercial hybrid rice expressing Bacillus thuringiensis δ-endotoxin. Nat Biotechnol 18:1101–1104
Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R (2003) Molecular pharming in plants: host systems and expresion technology. Trends Biotechnol 21:570–578
Twyman RM, Schillberg S, Fischer R (2005) Transgenic plants in the biopharmaceutical market. Exp Opin Emerg Drugs 10:185–218
Twyman RM, Schillberg S, Fischer R (2013) Optimizing the yield of recombinant pharmaceutical proteins in plants. Curr Pharm Des 19:5486–5494
Vain P, Worland B, Kohli A, Snape JW, Christou P, Allen GC, Thompson WF (1999) Matrix attachment regions increase transgene expression levels and stability in transgenic rice plants and their progeny. Plant J 18:233–242
Van Ree R, Cabanes-Macheteau M, Akkerdaas J, Milazzo JP, Loutelier-Bourhis C, Rayon C, Villalba M, Koppelman S, Aalberse R, Rodriguez R, Faye L, Lerouge P (2000) Beta (1,2)-xylose and alpha (1,3)-fucose residues have a strong contribution in IgE binding to plant glycoallergens. J Biol Chem 275:11451–11458
Vaquero C, Sack M, Schuster F, Finnern R, Drossard J, Schumann D, Reimann A, Fischer R (2002) A carcinoembryonic antigen specific diabody produced in tobacco. FASEB J 16:408–410
Vitale A, Denecke J (1999) The endoplasmic reticulum: gateway of the secretory pathway. Plant Cell 11:615–628
Wakasa Y, Takaiwa F (2013) The use of rice seeds to produce human pharmaceuticals for oral therapy. Biotechnol J 8:1133–1143
Wandelt CI, Khan MR, Craig S, Schroeder HE, Spencer D, Higgins TJ (1992) Vicilin with carboxy-terminal KDEL is retained in the endoplasmic reticulum and accumulates to high levels in the leaves of transgenic plants. Plant J 2:181–192
Wilson C, Bellen HJ, Gehring WJ (1990) Position effects on eukaryotic gene expression. Ann Rev Cell Biol 6:679–714
Yang Z, Bennett EP, Jorgensen B, Drew DP, Arigi E, Mandel U, Ulvskov P, Levery SB, Clausen H, Petersen BL (2012) Toward stable genetic engineering of human O-glycosylation in plants. Plant Physiol 160:450–463
Ye G, Tu J, Hu C, Datta K, Datta SK (2001) Transgenic IR72 with fused Bt gene cry1Ab/cry1Ac from Bacillus thuringiensis is resistant against four lepidopteran species under field conditions. Plant Biotechnol 18:125–133
Zimmermann S, Schillberg S, Liao YC, Fisher R (1998) Intracellular expression of TMV-specific single-chain Fv fragments leads to improved virus resistance in Nicotiana tabacum. Mol Breed 4:369–379
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Sabalza, M., Christou, P. & Capell, T. Recombinant plant-derived pharmaceutical proteins: current technical and economic bottlenecks. Biotechnol Lett 36, 2367–2379 (2014). https://doi.org/10.1007/s10529-014-1621-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-014-1621-3
Keywords
- Molecular pharming
- Plant-made pharmaceuticals
- Plant production systems
- Proteins from recombinant plants
- Recombinant pharmaceutical proteins