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European Biopharmaceutical Review

Turning a New Leaf

Recent strides in plant expression system development has fuelled new hopes for the field of plant-made pharmaceuticals (PMPs). However, addressing the environmental risks is crucial in order to help this technology make the transition through to commercialisation.

Historically, plants have provided a myriad of products that have been used to improve human health. Recent advances in biotechnology have allowed us to further increase this suite of products. For example, in 1986 a human growth hormone was produced in transgenic tobacco and three years later, monoclonal antibodies (mAbs) were successfully expressed in transgenic plants. The successes of these advances created the potential for plants as bioreactors for pharmaceutical protein production. In the last two decades, a broad range of functionally active vaccines and therapeutic proteins have been produced by a diverse species of plants, and over 100 patent families, 600 publications and more than 180 biotechnology companies have been established (1-3). The future of PMPs in the real world of pharmaceutical protein production is promising, but suffers from critical setbacks in protein expression and purification technologies, the lack of continued interest from Big Pharma companies, and public concerns on genetically modified (GM) plants.

Innovations in Expression Technology

A major advantage of plant production systems is low-cost upstream protein expression. Unlike mammalian cell culture, which requires capital-demanding bioreactors and expensive tissue culture media and operations, pharmaceutical protein production in plants is easily set up and scaled. Plants can produce large volumes of pharmaceutical proteins efficiently and sustainably, which was initially achieved through the generation of stable transgenic plants (1). In this strategy, plant cells are transformed with the gene of interest into a nuclear genome. The stable integration of the target gene in the nuclear genome allows for Mendelian inheritance of the transgene over generations, and in turn, the stable expression of transgenic proteins. The resulting transgenic lines can then be propagated to establish a master seed bank as an inexpensive and permanent genetic source for future large-scale production.

This strategy, however, suffered several technology and regulatory setbacks. Firstly, it requires a relatively long timeframe to create and select the initial transgenic plants. Further to this, the expression level of the therapeutic protein either fluctuates widely, or is unstable not only between individual plants but also between different generations of the same plant line. These uncertainties are caused by the randomness of transgene insertion into the plant genome and by post-transcriptional gene silencing. Secondly, the potential risk of unwanted transgene outflow from GM plants to neighbouring plants or their wild relatives has also raised regulatory and public acceptance issues.

These scientific and regulatory challenges have been addressed by new expression innovations and strategies. For example, in response to concerns for potential transgene escape, transgenic plants are increasingly being grown in containment or in areas with natural geographic barriers to isolate them from their agricultural or wild relatives (1). Novel transient plant expression systems have also been developed to address the accumulation level, consistency and speed of therapeutic protein production. One of the more robust transient expression vectors is the ‘deconstructed’ viral vector system. The MagnICON system is based on replicationcompetent tobacco mosaic virus (TMV) and potato virus X (PVX) genomes under the control of plant promoters. Once delivered to plant cells, the TMV or PVX genome is transcribed and spliced to generate a functionally infective replicon (4).

Another example is the geminivirus-based expression system, which is a DNA replicon system that allows rapid high-yield production of proteins (5). In these systems, Agrobacterium tumefaciens – a bacterium – is used to deliver the deconstructed viral vectors to plant cells to eliminate the need for systemic viral spread within the plant. This approach also prevents transgene loss during systemic spread, and allows the technology to be applied to a diversity of plant species beyond the natural host(s) of the virus. The deletion of viral coat protein genes in these systems also facilitates the high protein yield of a viral system without the concern of generating infectious virions. Thus, the deconstructed viral vector system provides the flexibility of nuclear gene expression with the speed and yield of viral vectors. Furthermore, since non-transgenic plants are used for transient expression vector delivery, public concerns for GM plants are irrelevant for this strategy.

Overall, deconstructed virus-based transient expression systems allow for a high and consistent level of therapeutic protein accumulation within one to two weeks after vector infiltration, while also reducing public concerns and providing the most convenient technology for obtaining the initial research material (milligram to gram level) for preclinical studies.Moreover, these vectors have been modified for expressing therapeutic proteins in stable transgenic plants under specific inducible conditions to facilitate scale-up while retaining consistent and high protein expression levels, as well as transgene stability (1). Therefore, a rapid evaluation of therapeutic candidates and transitioning to a large-scale commercial production platform can now be accomplished by employing the combination of both transient and stable transgenic plant technologies based on deconstructed viral vectors.



Downstream Processing

The importance of downstream processing for extraction and purification of pharmaceutical proteins from plants has been realised and increasing efforts have been applied to address this critical issue.While ‘immunisation-by-eating’ still presents a possibility to deliver plant-derived vaccines, considerations of regulatory issues have pointed to the necessity of developing processing technologies to produce vaccines and therapeutics with defined unit dosage.

It is often stated incorrectly that downstream processing of PMPs is simpler and less expensive than in a mammalian system. In fact, the unique properties of plant tissues present both challenges and opportunities for downstream processing. For example, plants usually produce more solid debris than other organisms and some plant species, such as those in the tobacco family, are rich in phenolics and alkaloids. As a result, direct loading of plant extracts onto chromatography resins may cause resin fouling and poor binding of target proteins. As such, an additional nonchromatographic separation step, such as aqueous twophase partitioning system, is required to remove these plant compounds from plant extract.

On the other hand, plants also provide opportunities to develop innovative extraction and purification strategies. For instance, Oleosins, a class of plant seed oil-body associated small proteins have been explored as Protein A fusion partners to produce a single-use and low-cost Protein A alternative in safflower plants (6). Monoclonal antibodies produced in plants can form complexes with oleosin- Protein A fusion and be readily purified by extracting with oil bodies. Another example is the development of a nanoparticle based on a tobamovirus displaying a Protein A fragment. These nanoparticles can be produced at high levels in plants and used in a simple mAb purification process to achieve more than 90 per cent product purity (7). Since PMPs have low contamination risks by animal or human pathogens, the tedious viral validation step required in purifying mammalian cell-derived therapeutics could be eliminated, providing a potential time and cost saving opportunity.

The promise of PMP production on an agricultural scale demands a processing platform with extraordinarily largescale capabilities. As a result, alternative non columnbased technologies such as precipitation and membrane chromatography have been explored for very large-scale downstream processing, aiming to enhance manufacture scalability, as well as to provide separation power similar or better than that of column chromatography. For example, it was demonstrated that a cation-exchange membrane chromatography was very efficient in purifying tobaccoproduced mAbs due to its high capacity for large-size impurities which are particularly abundant in plant extracts and a major cause for column clotting (1). As these technologies are being optimised and becoming more sophisticated, large-scale downstream processing of PMPs will become routine and attention will be shifted to the regulatory compliance of these processes to FDA’s cGMP regulations.

Biobetters by Plants?

Plants have been mostly proposed as vehicles to produce biosimilars because of their capacity to generate proteins at a low cost, their ability to make appropriate post-translational modifications on recombinant proteins, and the low contamination risks by animal or human pathogens (1,3). More recently, plants also have been recognised as a promising system to make safer and more effective biobetters due to the development of transgenic plant lines that offer specific and unique properties in protein processing and modification (3,8). For example, a Gaucher disease enzyme commercially produced in chinese hamster ovary cells, requires in vitro N-glycan processing to achieve the desired efficacy. In contrast, the carrot cell-produced version of this enzyme already has the required glycoform, therefore, eliminates the cost of N-glycan processing, and may have resulted in better and more consistent efficacy (3).

The minor difference in protein glycosylation between plant and mammalian cells was one of the main issues of PMP technology, since the possibility of inducing plant-glycan specific antibodies could reduce its therapeutic efficacy by accelerating clearance from plasma, or cause potential adverse effects through immune complex formation. Fortunately, glycoengineering has yielded ‘humanised’plant lines that produce PMPs with mammalian glycoforms by knocking out enzyme genes for making plant specific glycans and/or introducing mammalian glycosylation genes into plant cells (8). Remarkably,mAbs produced from these glycoengineered plants not only have mammalian glycoforms, but also with a high degree of glycan uniformity that cannot be produced by mammalian cells or achieved by in vitro treatments.This portfolio of plant lines, therefore, provides a superior system for producing PMPs with defined and uniform carbohydrate constituents.This development has equipped plants with new advantages beyond the traditional cost-saving benefit, and opened a new avenue for producing biobetters. Indeed, several mAbs against human immunodeficiency virus and CD30 produced in these genetically modified plants showed improved virus neutralisation potency or antibody-dependent cytotoxicity (ADCC) respectively, compared to the same mAb produced in mammalian cells (8). With these plant lines, in theory, one can ultimately design custom therapeutic mAbs with a tailor-made glycoform that is best suited either for its efficacy or safety depending on the clinical application. For example, a specific glycoform can be selected to enhance Fc receptor (FcγR) or C1q binding to facilitate ADCC, and/or complement-dependent cytotoxicity (CDC) to increase the efficacy of certain therapeutic mAbs; while other glycoforms can be chosen to avoid unwanted ADCC and CDC-induced inflammation to ensure the safety of other mAb therapeutics that have different clinical applications.

Where Are the Products?

The most severe criticism of the PMP field is the absence of approved human products after two and half decades of active R&D.To date, only a plant-derived poultry vaccine is on the market, but no therapeutics or vaccine has been approved for human use in the US (3). In addition to the technical difficulties described above, the lack of interest from the large pharmaceutical companies, the uncertainty of regulatory hurtles and public concerns on GM plants also contributed to the under achievement in this area.Despite these setbacks, several PMPs are in late-stage clinical trials. Recently, a secretory IgA for the prevention of tooth decay and a human intrinsic factor for treating vitamin B-12 deficiency were approved for human application in Europe and Ukraine (3). One of the ways to deal with the regulatory uncertainties and public biosafety concerns is to produce therapeutics with plant cells in a setting that mimics mammalian cell cultures. For example, carrot cell cultures are being used to make PMPs in simple, cost-effective plastic bags. Also, a Phase 3 clinical trial was completed for glucocerebrosidase for treating Gaucher disease, one of the product candidates made in such cultures (3). Similarly, duckweeds – a small clonal aquatic plant – are grown in plastic containers for producing α interferon to treat hepatitis C, and just passed a Phase 2 human clinical study (3). Several PMPs produced in traditional whole plants grown in fields are also under clinical testing. For example, insulin produced in safflower has been tested in Phase 1 and 2 clinical trials for treating diabetes and was found bioequivalent to Eli Lilly’s Humulin R (3).

Conclusion

The success of these PMPs has slowly warmed up the interests of large pharmaceutical companies in the PMPs themselves or their production technologies. In 2009, Pfizer entered into an agreement with Protalix to license the worldwide rights for commercialising the plant-produced glucocerebrosidase for Gaucher disease treatment. In 2006, Bayer bought Icon Genetics, the company that developed the MagnICON deconstructed virus expression vectors. This has resulted in the construction of a cGMP-compliant PMP manufacture facility and the initialisation of a Phase 1 clinical trial on tobacco-produced idiotypic antibodies for the treatment of non-Hodgkin’s lymphoma in 2010.

Together with the development of innovative expression, glycoengineering and downstream processing technology, the involvement of big drug companies is a promising sign of regrowth and gives new hopes for the PMP field.The remaining hurdles are winning regulatory approval for the first few PMP therapeutics and addressing public concerns for the potential biosafety and environmental risks of PMP technologies.This is an exciting and critical time for the PMP field, as once the first few PMP therapeutics win regulatory approval, plant production technology will finally earn its place in drug production and make significant contributions for cost-effective, highly scalable, and safe production of biobetters and biosimilars.

References
  1. Chen Q, Expression and manufacture of pharmaceutical proteins in genetically engineered horticultural plants, in transgenic horticultural crops: challenges and opportunities, Essays by Experts, Taylor & Francis: pp86-126, 2011
  2. Lai H, Engle M, Fuchs A, Keller T, Johnson S, Gorlatov S, Diamond MS and Chen Q, Monoclonal antibody produced in plants efficiently treats West Nile virus infection in mice, Proc Natl Acad Sci USA 107: pp2,419-2,424, 2010
  3. Faye L and Gomord V, Success stories in molecular farming – a brief overview, Plant Biotechnol J 8: pp525-528, 2010
  4. Giritch A, Marillonnet S, Engler C, van Eldik G, Botterman J, Klimyuk V and Gleba Y, Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors, Proc Natl Acad Sci USA 103: pp14,701-14,706, 2006
  5. Chen Q, He J, Phoolcharoen W and Mason HS, Geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants, Hum Vaccin 7: pp331-338, 2011
  6. Capuano F, Beaudoin F, Napier JA and Shewry PR, Properties and exploitation of oleosins, Biotechnol Adv 25: pp203-206, 2007
  7. Werner S, Marillonnet S, Hause G, Klimyuk V and Gleba Y, Immunoabsorbent nanoparticles based on a tobamovirus displaying protein A, Proc Natl Acad Sci USA 103: pp17,678- 17,683, 2006
  8. Gomord V, Fitchette AC, Menu-Bouaouiche L, Saint-Jore-Dupas C, Plasson C, Michaud D and Faye L, Plant-specific glycosylation patterns in the context of therapeutic protein production, Plant Biotechnol J 8: pp564-587, 2010


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Qiang Shawn Chen is a Professor in the Center for Infectious Diseases at Arizona State University (ASU). Shawn’s lab focuses on optimising the expression and assembly of monoclonal antibodies and designing novel mAb fusion proteins to enhance their targeting and efficacy. He has spent more than 10 years in the biotechnology and pharmaceutical industry directing research in therapeutic protein development in both plant and mammalian cell culture systems. Prior to joining ASU, he was the Associate Director of Protein Chemistry at Cardinal Health and a Senior Scientist at Monsanto. Email: shawn.chen@asu.edu
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