14A Real Success Story: Plantibodies for Human Therapeutic UseJorg Knablein, Merardo Pujol, and Carlos Borroto
About 80% of the currently more than 200 biopharmaceuticals were approved after2000, and more than 10 of those reached blockbuster status (>US$ 1 billion perannum) in 2004 already. All together, with the highest growth rates (compoundannual growth rate of 20%) within the entire pharma market, biopharmaceuticalsales in 2006 were about US$ 60 billion and have almost reached more thanUS$ 100 billion revenues in the year 2011 . Over the last couple of years, though itbecame obvious that production capacities for biopharmaceuticals with conven-tional bioreactors would be a bottleneck and that worldwide fermentation capacitiesare limited keeping in mind that the annual demand for a rst-generationbiopharmaceutical such as Bayers interferon beta is 2 kg, vs 300 kg, for example,a second-generation antibody such as Genentechs rituximab. One promisingsolution to these capacity crunches is the use of transgenic plants to producebiopharmaceuticals [2,3].
14.2SWOT Analysis Reveals a Ripe Market for Plant Expression Systems
When analyzing different expression systems regarding their SWOT, that is,strengths, weaknesses, opportunities, and threats in 2002, the advantages of plantsand their potential to circumvent the worldwide capacity limitations for bio-pharmaceutical production became quite obvious already (Figure 14.1a) .Comparison of transgenic animals , mammalian cell culture [6,7], plant expres-sion systems , insect cells , yeast , and bacteria shows certain advan-tages for each of the systems. In this order, one can compare these systems in termsof, for example, their development time or speed. Transgenic animals have thelongest cycle time (18months to develop a goat), followed bymammalian cell culture,plants, yeast, and bacteria (1 day to transform Escherichia coli). If one looks atoperating and capital costs, safety and scalability, the data show that plants
Modern Biopharmaceuticals: Recent Success Stories, First Edition. Edited by Jrg Knblein.# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.
Figure 14.1 (a) SWOT analysis in 2002. (b) SWOT analysis in 2012. Source: (a, b) Adopted fromRef. .
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are benecial: therefore, in the comparison they are shown on the right-hand side. But even for glycosylation, multimeric assembly and folding (whereplants were not shown on the right-hand side, meaning other systems are bene-cial), some plant expression systems have moved in that direction in the meantime(see Figure 14.1b) (see also Chapter 20).Examples are the moss system from Greenovation Biotech, Germany . This
system performs proper folding and assembly of even such complex proteins suchas the homodimeric VEGF. For this molecule also the sugar pattern could success-fully be reengineered from plant- to humanlike glycosylation. Other plants as wellare successfully used as expression systems: Medicago CA (alfalfa), MERISTEMTherapeutics, France (corn), ICON Genetics, Germany (tobacco). By the use of twononcompeting vectors ICON was able to coexpress the heavy and light chains of anantibody, resulting in yields of up to 0.5 g of assembled full-size IgG1 per kilogram offresh-leaf biomass (see also Ref. ).In addition to such high yields and the potential of performing human glycosyla-
tion, plants also enjoy the distinct advantage of not harboring any pathogens, whichare known to harm animal cells, nor do the products contain any microbial toxins(e.g., endotoxins), TSE (Transmissible Spongiform Encephalopathies), prions, or onco-genic sequences. In fact, humans are exposed to a large, constant dose of living plantviruses in the diet without any known effects/illnesses. Plant production of bio-pharmaceuticals also has advantages with regard to their scale and speed ofproduction. Plants can be grown in ton quantities (using existing plant/croptechnology, such as commercial greenhouses), be extracted with industrial-scaleequipment, and produce kilogram-size yields from a single plot of cultivation[18,21]. These economies of scale are expected to reduce the cost of productionof pure pharmaceutical-grade therapeutics by more than two orders of magnitudeversus current bacterial fermentation or cell culture reactor systems , (plus rawmaterial cost of goods sold, COGS are estimated to be as low as 10% of conventionalcell culture expenses).
14.3Current Status of Plant-Made Biopharmaceuticals
Although a growing list of heterologous proteins were successfully produced in anumber of plant expression systems (Figure 14.2) with their manifold advantages,there are also obvious downsides [18,21].Nonal regulatory guidelines exist, althoughregulatory authorities [Food and Drug Administration (FDA), European MedicineEvaluation Agency (EMA), Biotechnology Regulatory Service (BRS), and the Bio-technology Industry Organization (BIO)] have drafted guidelines on plant-derivedbiopharmaceuticals. The FDA has also issued several PTC (points to consider)guidelines about plant-based biologics, and review of the July 2002 PTC conrmsthat the FDA supports this eld and highlights the benets of plant expressionsystems including the absence of any pathogens to man from plant extracts [16,17].But as of today, none of the major authorities have nalized their guidelines.
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The main concerns of using plant expression systems are societal ones aboutenvironmental impacts, segregation risk, and contamination of the food chain.These threats, however, can easily be dealt with using nonedible plants (nonfood,non-feed), applying advanced containment technologies (GMP greenhouses, bio-reactors), and avoiding open-eld production (see also ).Owing to the obvious strengths of plant expression systems, there has been
explosive growth in the number of start-up companies. Since 1990s a number ofpromising plant expression systems have been developed, and even big pharma-ceutical companies have become more interested (see Figure 14.2). Recent yearshave seen several major biotech companies entering partnerships; which clearlydemonstrates that, in general, there has been sufcient basic research and exper-imentation with various crops to provide the overall proof of concept that transgenicplants can produce biopharmaceuticals for human use. Indeed, Cuba has promotedthis technology and, on April 11, 2006, the Cuban Ministry of Public Healthapproved what became the worlds rst plantibody . Owing to their impor-tance, the development of plant-based antibodies has been a major eld since 1980salready (Figure 14.3).
Figure 14.2 List of plant-made biopharmaceuticals (approved and in the pipeline).
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14.4The CB Hep1 Case Story
One of the lead products of the Cuban biotechnology industry has been the hepatitisB vaccine (Tradename: Heberbiovac1) produced by the Center for Genetic Engineer-ing and Biotechnology of Havana (Centro de Ingeniera Genetica y Biotecnologa,CIGB). The active pharmaceutical ingredient (API) of Heberbiovac is the hepatitis Bsurface antigen (HBsAg) recombinantly produced in yeast. A fundamental step inthe purication process of this API from yeast is the afnity chromatography usingthe mice-derived monoclonal antibody CB Hep1.The international commercial success of Heberbiovac entails expansion of
vaccine production up to several millions of doses per year, which concurrentlydemands for increasing needs of the CBHep1monoclonal antibody (catch antibody)for HBsAg immunopurication.Besides that, the international trend of regulatory, biosafety, and ethical require-
ments regarding proteins produced in animals (or including animal-derived com-ponents) has greatly increased over the last few years, boosting the strengths ofexpression systems lacking animal components. One exception is the FDA approvalof ATryn1 on February 6, 2009. ATryn is the brand name of the anticoagulant
Figure 14.3 List of plantibodies since 1980s.
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antithrombin manufactured by the Massachusetts-based US company GTC Bio-therapeutics . It is made from the milk of goats that have been geneticallymodied to produce human antithrombin, a plasma protein with anticoagulantproperties. Microinjection was used to insert human antithrombin genes into thecell nucleus of their embryos. ATryn is the rst biopharmaceutical produced usinggenetically engineered animals . GTC states that one genetically modied goat canproduce the same amount of antithrombin in a year as 90 000 blood donations .GTC chose goats for the process, because they reproduce more rapidly than cattleand produce more protein than rabbits or mice.
A joint team from the Recombinant Antibody Department and the Plant Divisionof the CIGB obtained stable transgenic Nicotiana lines through Agrobacteriumtumefaciens-mediated genetic transformation with a binary vector harboring thelight and heavy chain genes for the full CB Hep1 antibody. Clones with high-levelexpression of this plantibody were selected. Processed leaf extracts from transgenicplant clones provided the starting material for the development of a laboratory scalepurication procedure comprising protein A-sepharose chromatography separationof the plantibody. Consistent purity above 90% was achieved using this procedure,allowing biochemical characterization of the plant-derived antibody. Comparisonexperiments involving the plantibody and the ascites-derived antibody resulted insimilar purity levels of the puried HBsAg obtained at laboratory scale with bothantibody sources, suggesting the real potential of this alternative for its practical usein the CB Hep1 vaccine production. The ow chart for production of this plantibodyis shown in Figure 14.4.
14.4.2Development of Large-Scale Downstream Purification Procedures
Hundreds of kilograms of Nicotiana leaves were disrupted, plant extracts wereclaried, and protein A-sepharose afnity chromatography was applied to antibody-containing extracts in several scales. From batches of 500 kg of plant leaves,consistent 4050% recovery and higher than 90% purity was routinely achieved. Upscaled CB Hep1 was tested for its ability to immunopurify the HBsAg fromyeast cultures, in comparison with the antibody obtained from mice ascites. Theplant-made antibody behaved similarly to the mice antibody regarding couplingefciency to sepharose CL-4B.The characterization of CB Hep1 showed that in comparison with the antigenic
determinant recognized in relation to the mice-derived antibody, the plant-madeantibody recognized the same peptides of the HBsAg similar to the murineantibody. The study of glycosylation pattern showed that, coincidently with reportsfrom other research groups, attached sugars varied from complex N-glycans tomannose. However, as expected for this case, the ability of the plant-made antibody
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to selectively immunopurify the HBsAg remained unaffected in spite of the plant-type glycosylation pattern.Good manufacturing practices (GMP) were established for biomass production.
In case of the CBHep1 plantibody, these procedures combined good pharmaceuticalpractices and good agricultural practices in totally conned greenhouse facilities(Figure 14.5), starting from certied seed banks, full traceability of batches (pro-duced in soilless substrate) over plantlet production to harvest. Standard operationprocedures (SOP) put in place, as well as the ne production technologies chosen forbiomass in automated greenhouses (Figure 14.6), guarantee consistent year roundobtainment of biomass batches for downstream purication .
Figure 14.5 Area of greenhouses for plant-made pharmaceuticals biomass production at CIGB,Havana, Cuba.
Working seed bank
Quality clearancefor industrial use
Chart flow for production of plant-derived CB Hep1 antibody
Figure 14.4 Chart flow for production of plantibody CB Hep1.
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Production of recombinant pharmaceuticals from plants requires speci c PTCs,which are very particular for tobacco as expression and production system. For thelatter, a specic regulatory document and procedure concerning recombinant plant-derived pharmaceuticals was established by the Centro para el Control Estatal de laCalidad de los Medicamentos (CECMED), the Cuban regulatory agency. Taking intoaccount two draft regulations previously released by EMA and FDA, the documentwas nally issued by CECMED . After considering the application dossier, in situevaluations of facilities, procedures and processes, and inspection of productionareas, an of cial permission for the industrial scale use of the plantibody CB Hep1was granted by CECMED in April 2006 .In addition to authorizations required for manufacturing, the National Biosafety
Agency (Centro Nacional para la Seguridad Biol ogica, CNBS http://www.medioambiente.cu/oregulatoria/cnsb/index.htm) is controlling all stages fromresearch to production of CB Hep1.
14.5Conclusion and Outlook
While in Western countries much research and development is ongoing in the eldof plant-made pharmaceuticals, up to now no such drug has reached the market.
Figure 14.6 Greenhouse contained production of plant material facilitates consistency and goodmanufacturing practices for plant-made pharmaceuticals.
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Advantages of these production systems are low cost of cultivation, high biomassproduction, relatively fast gene-to-protein time, low capital and operating costs,excellent scalability, eukaryotic posttranslational modications, low risk of humanpathogens, lack of endotoxins, as well as high protein yields.Plants are by far the most abundant and cost-effective renewable resource
uniquely adapted to complex biochemical synthesis. The increasing cost of energyand chemical raw materials, combined with the environmental concerns associatedwith conventional pharmaceutical manufacturing, will make plants even morecompatible in the future .Taking advantage of plant expression systems, the availability of other cheap
protein-based vaccines is rapidly progressing at the periphery of well-establishedWestern pharma market. Drivers of this development are health systems lackingresources  and easy transfer of biotechnological know how. Western healthsystems still rich in resources also might benet from original drugs. The cost ofvery expensive hormone therapies (erythropoietin, human growth hormone, etc.) could fall dramatically within the next decade due to the use of plant expressionsystems....