From gene to harvest: insights into upstream process development for the GMP production of a monoclonal antibody in transgenic tobacco plants

Abstract

SummaryThe EU Sixth Framework Programme Integrated Project 'Pharma-Planta' developed an approved manufacturing process for recombinant plant-made pharmaceutical proteins (PMPs) using the human HIV-neutralizing monoclonal antibody 2G12 as a case study. In contrast to the wellestablished Chinese hamster ovary platform, which has been used for the production of therapeutic antibodies for nearly 30 years, only draft regulations were initially available covering the production of recombinant proteins in transgenic tobacco plants. Whereas recombinant proteins produced in animal cells are secreted into the culture medium during fermentation in bioreactors, intact plants grown under nonsterile conditions in a glasshouse environment provide various 'plant-specific' regulatory and technical challenges for the development of a process suitable for the acquisition of a manufacturing licence for clinical phase I trials. During upstream process development, several generic steps were addressed (e.g. plant transformation and screening, seed bank generation, genetic stability, host plant uniformity) as well as productspecific aspects (e.g. product quantity). This report summarizes the efforts undertaken to analyse and define the procedures for the GMP/GACP-compliant upstream production of 2G12 in transgenic tobacco plants from gene to harvest, including the design of expression constructs, plant transformation, the generation of production lines, master and working seed banks and the detailed investigation of cultivation and harvesting parameters and their impact on biomass, product yield and intra/interbatch variability. The resulting procedures were successfully translated into a prototypic manufacturing process that has been approved by the German competent authority.

In vitro plant tissue culture: means for production of biological active compounds

“…Once the plant has been engineered (either by gene editing by CRISPR/Cas9 or by traditional methods), and has passed through in vitro culture, the genetic material can be stored using master and working seed banks (Sack et al 2015). The plants can then be used for the large-scale production of the desired metabolites, including plant-made pharmaceuticals.…”

Expression of recombinant human anti-TNF-α scFv-Fc in Arabidopsis thaliana seeds

“…As the demand for safe and inexpensive means for mass production of recombinant pharmaceutical proteins increases, the production of complex mammalian proteins in transformed plants or plant suspension cells, such as HIV-neutralizing antibody 2G12B expressed in tobacco and rice Sack et al, 2015;Vamvaka et al, 2016), human interleukin-10 fusions expressed in tobacco cell suspension cultures (Kaldis et al, 2013), is desirable. Because plants are suitable for the large scale production of pharmaceutical proteins, and the expressed proteins are functional and almost indistinguishable from their mammalian counterparts, they will be available in significantly larger amounts, and a cost-effective basis (Sharma and Sharma, 2009).…”

Biomanufacturing of protective antibodies and other therapeutics in edible plant tissues for oral applications

“…batch-to-batch consistency) of the recombinant pharmaceutical products according to GMP compliance. The procedures for the GMP/GACP-compliant upstream production of 2G12 in transgenic tobacco plants have been recently summarized by Sack et al (2015b). In general, the manufacturing of recombinant biologically active substances in plants is covered at the EU level by the guideline EMEA/CHMP/ BWP/48316/2006 (http://www.ema.europa.eu/docs/en_GB/doc-ument_library/Scientific_guideline/2009/09/WC500003154.pdf), which basically adapts aspects of the quality guidance already in place for other production systems to the special case of transgenic plants.…”