Plant-made Pharmaceuticals Research Articles

Plant‐made pharmaceuticals: Leading products and production platforms

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.

Variation of Total Soluble Protein Content in Fruit among Six Greenhouse Tomato Cultivars

Tomato has been considered as a potential biological production platform for plant-made pharmaceuticals (PMPs). However, information is limited for protein productivity and dynamics in tomato fruit. As part of our PMP production project, total soluble protein (TSP) content of green and red fruits was analyzed for selected six greenhouse cultivars. The six cultivars consisted of various fruit types (including cluster, cherry, mini plum, and grape types) with sizes ranging from 10 to 150 and from 14 to 188 g fresh weight for green and red fruits, respectively. In fruit TSP content per unit dry weight (DW), approximately seven- to eightfold variation was observed among cultivars for both green and red fruits. There was no significant correlation between fruit TSP content per DW and fruit DW or fruit type over the cultivars irrespective of fruit ripening stage, indicating that fruit weight or fruit type of a cultivar cannot be an indicator of the TSP content per DW. There was also an inconsistent trend in differences in fruit TSP content per whole fruit between green and red fruit among cultivars. Our results suggest that a low-yielding cultivar can produce high TSP content per unit ground area resulting from high TSP content per DW in fruit. A background tomato cultivar or genotype candidate for commercial PMP production should thus be selected from among various fruit types considering both yield potential and fruit TSP content.

Review article: Commercialization of whole-plant systems for biomanufacturing of protein products: evolution and prospects

Technology for enabling plants to biomanufacture nonnative proteins in commercially significant quantities has been available for just over 20 years. During that time, the agricultural world has witnessed rapid commercialization and widespread adoption of transgenic crops enhanced for agronomic performance (herbicide-tolerance, insect-resistance), while plant-made pharmaceuticals (PMPs) and plant-made industrial products (PMIPs) have been limited to experimental and small-scale commercial production. This difference in the rate of commercial implementation likely reflects the very different business-development challenges associated with 'product' technologies compared with 'enabling' ('platform') technologies. However, considerable progress has been made in advancing and refining plant-based production of proteins, both technologically and in regard to identifying optimal business prospects. This review summarizes these developments, contrasting today's technologies and prospective applications with those of the industry's formative years, and suggesting how the PM(I)P industry's evolution has generated a very positive outlook for the 'plant-made' paradigm.

Molecular farming, patents and access to medicines

Transgenic plants have several advantages over other expression systems for the production of recombinant medicines, including low costs, large-scale production and the ability to produce complex multimeric proteins with appropriate post-translational modifications. Several plant-made pharmaceuticals, including the enzyme glucocerebrosidase, insulin and IFN-α2b, are approaching commercialization and these developments have been accompanied by considerable patenting activity. In the present article, we investigated plant-made pharmaceutical patents between the years 2002 and 2008. There was a clear downward trend in the number of patents filed between these years and a greater number of patents were filed by public sector institutions or inventors than by the private sector. The USA dominated patenting activity providing nearly 30% of inventors. The majority of patents were for vaccine candidates (55%), followed by therapeutics (38%) and antibodies (7%). The relationship of patenting to innovation and access to medicines, particularly in the developing world, will be discussed.

Plant-made pharmaceuticals for the prevention and treatment of autoimmune diseases: where are we?

Molecular farming in plants or plant cell cultures represents a viable alternative technology that holds great promise for the low-cost and large-scale production of recombinant proteins. The particular case of plant-based vaccines for the prevention of autoimmune diseases is addressed here, presenting a comprehensive overview of the different molecules and expression technologies that have been investigated so far in both academia and industry. The potential of plants not only as bioreactors but also as delivery systems for pharmaceuticals is discussed, and the advantages of oral delivery of autoantigens for the induction of immune tolerance are highlighted.

Success stories in molecular farming—a brief overview

Success stories in molecular farming—a brief overview

Evaluating biological containment strategies for pollen-mediated gene flow

Several biological containment methods have been developed to reduce pollen dispersal; many of them only have a proof of concept in a model plant species. This review focuses on biological containment measures which were tested for their long-term efficiency at the greenhouse or field scale level, i.e. plastid transformation, transgene excission, cleistogamy and cytoplasmic male sterility (CMS). Pollen-mediated gene transfer in transplastomic tobacco could occur at very low frequencies if the predominant mode of inheritance is maternal. Transgene excision from tobacco pollen can be made highly efficient by coexpression of two recombinases. For cleistogamous oilseed rape it was shown that some flowers were partially open depending on genotypes, environment and recording dates. Reports on the stability of CMS in maize and sunflower indicated that there is a high variability for different genotypes under different environmental conditions and over successive years. But for both crop types some stable lines could be selected. These data demonstrate that the biological containment methods discussed are very promising for reducing gene flow but that no single containment strategy provides 100% reduction. However, the necessary efficiency of biological containment methods depends on the level of containment required. The containment level may need to be higher for safety purposes (e.g. production of special plant-made pharmaceuticals), while much lower containment levels may already be sufficient to reach coexistence goals. It is concluded that where pollen-mediated gene flow must be prevented altogether, combinations of complementary containment systems will be required.

Development of Plant-made Pharmaceuticals

Development of Plant-made Pharmaceuticals

형질전환 식물세포배양을 이용한 바이오의약품 생산

Transgenic plant cell cultures for the production of biopharmaceuticals including monoclonal antibodies, recombinant proteins have been regarded as an alternative platform in addition to traditional microbial fermentation and mammalian cell cultures. Plant-made pharmaceuticals (PMPs) have several advantages such as safety, cost-effectiveness, scalability and possibility of complex post-translational modifications. Increasing demand for the quantity and diversity of pharmaceutical proteins may accelerate the industrialization of PMP technology. Up to date, there is no plant-made recombinant protein approved by USFDA (Food and Drug Administration) for human therapeutic uses due to the technological bottlenecks of low expression level and slight differences in glycosylation. Regarding expression levels, it is possible to improve the productivity by using stronger promoter and optimizing culture processes. In terms of glycosylation, humanization has been attempted in many ways to reduce immune responses and to enhance the efficacy as well as stability. In this review article, all these respects of transgenic plant cell cultures were summarized. In addition, we also discuss the general characteristics of plant cell suspension cultures related with bioreactor design and operation to achieve high productivity in large scale which could be a key to successful commercialization of PMPs.

Biosafety considerations associated with molecular farming in genetically modified plants.

The production in genetically modified plants of recombinant proteins for pharmaceutical or industrial use, also referred to as molecular deserves increasing interest due to its potential advantages. However, this type of application of genetic engineering also raises some biosafety concerns, in particular regarding aspects such as transgene spread in the environment or accidental contamination of the food and feed chains. This review presents the current state of the art of this sector, discusses some relevant regulatory issues and outlines important scientific aspects that should be considered during the safety assessment of genetically modified plants grown for this purpose. In particular, it addresses general strategies as well as specific potential containment measures that could be applied to limit the potential environmental and human health impacts linked to plant molecular farming. Key words: Biopharming, biosafety, confinement, containment, genetically modified organism, plant molecular farming, plant-made industrials, plant-made pharmaceuticals.

Production and characterization of an orally immunogenic Plasmodium antigen in plants using a virus‐based expression system

Increasing numbers of plant-made vaccines and pharmaceuticals are entering the late stage of product development and commercialization. Despite the theoretical benefits of such production, expression of parasite antigens in plants, particularly those from Plasmodium, the causative parasites for malaria, have achieved only limited success. We have previously shown that stable transformation of tobacco plants with a plant-codon optimized form of the Plasmodium yoelii merozoite surface protein 4/5 (PyMSP4/5) gene resulted in PyMSP4/5 expression of up to approximately 0.25% of total soluble protein. In this report, we describe the rapid expression of PyMSP4/5 in Nicotiana benthamiana leaves using the deconstructed tobacco mosaic virus-based magnICON expression system. PyMSP4/5 yields of up to 10% TSP or 1-2 mg/g of fresh weight were consistently achieved. Characterization of the recombinant plant-made PyMSP4/5 indicates that it is structurally similar to PyMSP4/5 expressed by Escherichia coli. It is notable that the plant-made PyMSP4/5 protein retained its immunogenicity following long-term storage at ambient temperature within freeze-dried leaves. With assistance from a mucosal adjuvant the PyMSP4/5-containing leaves induced PyMSP4/5-specific antibodies when delivered orally to naïve mice or mice primed by a DNA vaccine. This study provides evidence that immunogenic Plasmodium antigens can be produced in large quantities in plants using the magnICON viral vector system.

Dynamics of global disclosure through patent and journal publications for biopharmaceutical products

Examining the relationships between invention disclosure type in industry and academia and geographical disclosure trends, using plant-made pharmaceuticals as a single-sector model for the biopharmaceutical industry.

Optimization of growth performance of freshly induced carrot suspensions concerning PMP production

Carrot suspension cultures are efficient production systems for plant-made pharmaceuticals (PMP); however, frequently, the reduction of transgene expression in long-running transgenic cell lines limits the productivity. Freshly induced carrot suspensions have been identified for a constant and high biomass production with optimization of culture conditions and cultivars. A small-volume monitoring method was adapted to freshly induced carrot suspension that minimized the heterogeneity of freshly induced meristematic carrot cell lines. Using this system combined with the adaptation of growth conditions, we identified the cultivar Rote Riesen as having the highest increase in dry biomass with an average of 6.5 g/L weekly, stable and consistent after 8 wk. As a model PMP, VP60—the capsid protein from the rabbit hemorrhagic disease virus—was produced in a carrot suspension with a maximal accumulation of 2.4 μg/g dry mass. The developed method guarantees comparable investigations of different transgene expression in various freshly induced small-volume carrot cell lines.

Investigating recombinant protein exudation from roots of transgenic tobacco

It is widely acknowledged that plant-made pharmaceuticals (PMPs) offer numerous benefits, including inexpensive production, biological safety and the facility for production at agricultural scale. At the same time, it is important to minimize any potential risk associated with this new technology, including the potential release of bioactive proteins into the environment. To address this issue, we studied transgenic Nicotiana benthamiana and Nicotiana tabacum plants expressing two recombinant single-chain variable fragment (scFv) antibodies, respectively scFvB9 and scFvH10. ScFvB9 was raised against glycoprotein G1 of Tomato spotted wilt virus (TSWV), and scFvH10 was raised against human tumor-associated antigen tenascin-C. Both antibodies were targeted to the secretory pathway using the N-terminal signal peptide from Phaseolus vulgaris polygalacturonase-inhibiting protein (PGIP), and scFvH10 carried in addition a C-terminal KDEL tetrapeptide for retention in the endoplasmic reticulum (ER). Sterile hydroponic cultures were established, allowing us to investigate whether scFvB9 and scFvH10 were present in root exudates. Intercellular fluids extracted from different plant tissues were analyzed by western blotting revealing the presence of scFvB9. Successful secretion of scFvB9 in hydroponic medium was also demonstrated, whereas no scFvH10 could be detected in the leaf, stem or root apoplast, nor secreted into the hydroponic medium. Our results show that scFvH10 release or diffusion from the roots of transgenic plants was not occurring, suggesting that the KDEL signal might contribute to the environmental biosafety of crops producing PMPs.

Evolution of a regulatory framework for pharmaceuticals derived from genetically modified plants

The use of genetically modified (GM) plants to synthesize proteins that are subsequently processed, regulated and sold as pharmaceuticals challenges two very different established regulatory frameworks, one concerning GM plants and the other covering the development of biotechnology-derived drugs. Within these regulatory systems, specific regulations and guidelines for plant-made pharmaceuticals (PMPs)--also referred to as plant-derived pharmaceuticals (PDPs)--are still evolving. The products nearing commercial viability will ultimately help to road test and fine-tune these regulations, and might help to reduce regulatory uncertainties. In this review, we summarize the current state of regulations in different countries, discuss recent changes and highlight the need for further regulatory development in this burgeoning, new industry. We also make the case for the harmonization of international regulations.

Molecular Farming – Plant Biologicals, EuroSciCon (http://www.euroscicon.com). BioPark Hertfordshire, Welwyn Garden City, 29 January 2008

Molecular Farming – Plant Biologicals, EuroSciCon (http://www.euroscicon.com). BioPark Hertfordshire, Welwyn Garden City, 29 January 2008

Molecular pharming in cereal crops

There are many different agricultural expression systems that can be used for the large-scale production of recombinant proteins, but field-grown cereal crops are among the most attractive because recombinant proteins can be targeted to accumulate in the seed, and specifically in the endosperm, which has evolved naturally as a protein storage tissue. Within the developing endosperm, proteins are supplied with molecular chaperones and disulfide isomerases to facilitate folding and assembly, while the mature tissue is desiccated to prevent proteolytic degradation. Proteins expressed in cereal seeds can therefore remain stable for years in ambient conditions. Recent basic research has revealed a surprising diversity of protein targeting mechanisms in the endosperm, which can help to control post-translational modification and accumulation. Applied research and commercial development has seen several pharmaceutical proteins produced in cereals reach late stage preclinical development and the first clinical trials, with a number of companies now dedicated to developing cereal-based production platforms. In this review we discuss the basic science of molecular pharming in cereals, some of the lead product candidates, and challenges that remain to be addressed including the emerging regulatory framework for plant-made pharmaceuticals.

PERSISTENCE AND DEGRADATION OF MAIZE-EXPRESSED VACCINE PROTEIN, ESCHERICHIA COLI HEAT-LABILE ENTEROTOXIN SUBUNIT B, IN SOIL AND WATER

Transgenic plants represent an innovative platform for the cost-effective large-scale production of various pharmaceutical proteins. The eventual open-field production of plant-made pharmaceuticals (PMPs) requires risk assessment to determine the potential for harm to the surrounding ecosystem. In the present study, the environmental persistence of a transgenic maize-expressed antigen, Escherichia coli heat-labile enterotoxin subunit B (LTB), was studied under laboratory conditions. To semiquantitatively monitor the persistence of LTB in soil, extraction with a high-salt, high-pH extraction buffer was optimized using the closely homologous Vibrio cholerae enterotoxin subunit B (CTB) as a test substance. The time to dissipation of 50% (DT50) of the extractable fraction of maize-expressed LTB was 4 to 15 d in pond water and 35 to 90 d in soils. Both extraction efficacy and persistence were strongly affected by the matrix type and incubation conditions. In contrast with maize-expressed LTB, the DT50 for bacterially produced LTB and CTB was less than 4 d both in pond water and soil. Although maize-expressed LTB was more stable than bacterially produced analogue, its dissipation was governed by an initial lag, which could be attributed to release from the plant material, followed by rapid decline.

Pharma-Planta: road testing the developing regulatory guidelines for plant-made pharmaceuticals

Significant advances over the last few years have seen plant-made pharmaceuticals (PMPs) move from the exploratory research phase towards clinical trials, with the first commercial products for human use expected to reach the market by 2009. Europe has yet to witness the commercial application of PMP technology, although at least one product has begun phase II clinical trials with others following close behind. These emerging products are set to challenge the complex and overlapping regulations that currently govern GM plants and 'conventional' pharmaceutical production. The areas of responsibility are being mapped out between the different EU regulatory agencies, with specific guidelines currently being drawn up for the regulation of PMPs. This article discusses issues surrounding the development of robust risk-assessment and risk-management practices based on health and environmental impact, while working with EU regulatory authorities to ensure appropriate regulatory oversight.

From Farming to "Pharming". Risks and Policy Challenges of Third Generation GM Crops

Commercial and academic activities in the production of pharmaceuticals or other substances of industrial interests from genetically modified plants, i.e. molecular farming, have so far centred in the USA and Canada. Recent increases in EU activities and the proximity to market stage of the first plant-made pharmaceuticals, some of which from EU based companies, represent a call to action for EU regulators. Drawing on the North American debate on molecular farming it will be argued that both the rationale of and the risk issues associated with molecular farming will differ significantly from those of first generation GM crops. Based on these differences, the suitability of the existing regulatory framework, which essentially was developed in response to the arrival of insecticide and herbicide tolerant crops for food and feed use, is discussed. Possible options for adapting the already complex EU regulatory system to cater for molecular farming are examined. It will be argued that the policy challenges posed will inevitably spark a broader public debate. As an issue for debate, molecular farming is located at two crossroads: of the risk debate on agricultural biotechnology and the sustainability debate on renewables and greening of industry and of red and green biotechnology. Complex scientific, technical and legal issues, new issues at stake and a new pattern of actors are likely to give EU regulators a difficult time.