Production Of Plant-made Pharmaceuticals Research Articles

Produção de biológicos em plataformas vegetais e seus impactos na segurança alimentar – análise de lacunas na regulação vigente

The use of genetically modified plants to synthesize proteins that are subsequently processed, regulated, and sold as pharmaceuticals is already a global reality. However, the issue of technological development and industrial production of biological products being expressed in transgenic plants and their impact on the safety of the food chain needs to be discussed more deeply. In this paper, we present an extensive literature review on the standards and regulatory guidelines as well as on biosafety in Brazil with regard to the production of plant-made pharmaceuticals; compare them with the regulatory guidelines of other countries/regions such as USA, Canada, European Union, Cuba, and Argentina; and analyze gaps in the Brazilian regulation. We suggest that some regulations must be reviewed and conclude that the Brazilian regulatory authorities should establish an interrelated regulatory framework together, which would enable industrial production while protecting human and animal health as well as the environment.

Assessment of long-term cryopreservation for production of hCTLA4Ig in transgenic rice cell suspension cultures

For the commercialization of plant-made pharmaceuticals (PMPs) using transgenic plant cell cultures, the establishment of a cell-banking system has been known to be an essential process. Plant cells are traditionally maintained by repeated subcultures. However, this method has several problems including genetic instability of transformed cell lines, time- and cost-consuming. In this study, long-term cryopreserved rice suspension cells were firstly investigated for the production of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig). The cryopreserved cells for 5 years were regrowed to callus successfully and then suspended into the liquid medium. Consequently, the maximum cell mass and the hCTLA4Ig production were similar levels compared to those of the non-cryopreserved cells (control) even though hCTLA4Ig productivity was 1.7-fold higher than that of control. To further assess the level of improvements in hCTLA4Ig productivity in cryopreserved cells, hCTLA4Ig production profiles were statistically assessed between data of the cryopreserved cells for 5 years and annual data of non-cryopreserved cells maintained by subculture for 5 years. These results also indicate that hCTLA4Ig productivity in cryopreserved cells for 5 years was significantly increased (p-value: <0.001, 95% confidence interval) and it could be related to cell lysis resulting in release of hCTLA4Ig which was confirmed by the measurement of electrolyte leakage. In conclusion, we show that the long-term cryopreservation of transgenic rice cells was possible to support stable cell lines for the production of PMPs.

Production of Plant Made Pharmaceuticals: From Plant Host to Functional Protein

In the last two decades plants have emerged as valuable alternatives to mammalian cells for the production of pharmaceuticals and their potential as expression systems was shown by the commercial availability and acceptance of several plant made therapeuticals in clinical trials. Plants have many advantages over yeast, insect and bacterial expression systems such as the potential to properly fold the expressed proteins and the synthesis of more human-like N-glycans on the proteins. However, several constraints, such as expression yields, downstream processing and structural authenticity, currently limit the widespread use of plant expression systems. In this review, the focus is on the current limitations of plant systems for the production of pharmaceuticals and the possibilities to overcome these obstacles. A comparison is made with insect cell and yeast expression systems. Furthermore, the importance of glycosylation, in particular N-glycosylation for the biological function(s) of therapeutics in the human body will be discussed in detail and an overview of the state of art in the humanization of the N-glycosylation pathway in plants is provided.

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

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.

Improvement of Human lysozyme Expression in Transgenic Rice Grain by Combining Wheat (Triticum aestivum) puroindoline b and Rice (Oryza sativa) Gt1 Promoters and Signal Peptides

Heterologous protein expression levels in transgenic plants are of critical importance in the production of plant-made pharmaceuticals (PMPs). We studied a puroindoline b promoter and signal peptide (Tapur) driving human lysozyme expression in rice endosperm. The results demonstrated that human lysozyme expressed under the control of the Tapur cassette is seed-specific, readily extractable, active, and properly processed. Immuno-electron microscopy indicated that lysozyme expressed from this cassette is localized in protein bodies I and II in rice endosperm cells, demonstrating that this non-storage promoter and signal peptide can be used for targeting human lysozyme to rice protein bodies. We successfully employed a strategy to improve the expression of human lysozyme in transgenic rice grain by combining the Tapur cassette with our well established Gt1 expression system. The results demonstrated that when the two expression cassettes were combined, the expression level of human lysozyme increased from 5.24 +/- 0.34 mg(-1) g flour for the best single cassette line to 9.24 +/- 0.06 mg(-1) g flour in the best double cassette line, indicating an additive effect on expression of human lysozyme in rice grain.

EFFECTS OF BENZYLAMINOPURINE, 6-(δ, δ-DIMETHYLALLYAMINO)-PURINE AND THIDIAZURON ON SHOOT ORGANOGENESIS OF NICOTIANA SPECIES

Abstract Regenerability is an essential trait for plant varieties developed for the production of plant-made pharmaceuticals (PMPs). As part of the effort to develop optimal plant varieties for PMP production, two experiments were conducted in this study to determine and/or improve the shoot organogenic potential of Nicotiana species with important traits for PMP-oriented breeding. Effects of benzylaminopurine (BA) and 6-(δ,δ-Dimethylallyamino)-purine (2iP) on shoot organogenic potential of 46 Nicotiana accessions, representing 15 species, were examined in the first experiment. BA was found to be more effective than 2iP in enhancing shoot organogenesis for 23 Nicotiana accessions from 10 species, while 2iP was more effective than BA for only four accessions, including two from N. paniculata, one from N. obtusifolia and one from N. rustica. Effects of varying concentrations of thidiazuron (TDZ) in combination with indole-3-acetic acid (IAA) on shoot organogenic potential of 12 poorly responsive Nicotiana a...