Development of an integrated platform for transient production of recombinant protein biopharmaceuticals using disposable processing technology

Recombinant protein and antibody biopharmaceuticals produced predominantly by mammalian cells in culture are a major class of therapeutic drugs, valued at $35bn pa. Transient expression technologies potentially enable the rapid manufacture from gene to protein of candidate recombinant therapeutic proteins by making product available in days rather than months. This supports the initial high-throughput in vitro studies to identify a therapeutic product and the production of larger quantities of recombinant protein for in vivo testing. There is now considerable industrial interest in this approach. Single-use, disposable biomanufacturing components for both upstream and downstream processes are accepted by industry as a viable route to reduce fixed and operating costs (e.g. capital equipment, cleaning) and to reduce space requirements. In broad terms, the objective of this proposal is to enable a student to develop a 'whole process vision' of integrated biomanufacturing using a combination of transient production and disposable bioprocessing. The project is targeted at the development of a rapid, low-cost manufacturing solution using a combination of innovations in upstream gene expression technology (Sheffield) and downstream bioseparation technology (Pall). Our model system will be the production of a monoclonal antibody in mammalian cell culture. We aim to design a novel disposable platform for rapid and low-cost protein production, a 'bioprocess on a bench'. Current technologies for transient production of recombinant proteins generally result in low volumetric product titres (1-20 mg L-1). To be of industrial use, it is necessary to significantly intensify transient production processes (100-500 mg L-1). Based on polyethylenimine-mediated transfection of CHO cells in chemically-defined media, DCJ's group has previously demonstrated that transient rMab production can be significantly improved by control of cell proliferation to maintain transcriptionally active rDNA in the host cell population for extended periods (Galbraith et al., 2006; Tait et al., 2004). Our model system will be based on commercially available CHO-S cells maintained in a chemically defined environment (Invitrogen) transfected with MAb-encoding plasmid vectors available in-house. To date, no study has attempted to improve transient productivity by simultaneous rational optimisation of numerous process parameters with discrete quantification and statistical analysis of their relative influence. The transient production process should be simple to implement, scalable, consume minimal rDNA, reproducible, cost-effective and GMP-compatible. For transient processes that consume plasmid DNA, we will include E.coli-based plasmid preparation as a sub-process in the overall process design and economic analysis. One challenge will be to integrate the upstream process stream with appropriately interfaced disposable processing components to generate a scaleable, robust 'plug and play' downstream process train capable of the necessary unit operations: particulate removal, product capture, concentration and purification. Pall have a large range of pre-packed disposable filtration products including direct or tangential flow, and chromatographic products both sorbents and columns and membrane chromatography devices. In addition Pall has a fully resourced R&D organisation available to this project to support new product developments in these areas. Bioseparation processes will be designed and optimised with respect to scalability (scale-up/down, out), cost-of-goods, speed/throughput, cumulative yield and product purity characteristics (e.g. alternatives to Protein A as an affinity sorbent, removal of gene delivery vehicle, host cell protein assays etc). An overall objective will be to balance process cost (costs-of-goods; labour time, materials etc) against product yield and purity to design a robust production platform.