Understanding mixing, scaling and stress-related challenges in transient transfection processes

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Transient gene expression is traditionally used for rapid production of recombinant proteins to support preclinical studies during drug development as well as for virus production. Advances in this technology have led to an increase in titer and these, combined with a well defined robust process, provides an opportunity for transient gene expression to be an alternative tool for rapid production of clinical material. Studies have addressed the physicochemical characterisation of the system including type of transfection reagent, DNA to transfection reagent ratio and transfection conditions e.g. DNA to cell ratio, ratio of transfection solution (and type) to culture volume, flow conditions. Most of the work have been carried out at the small scale and recent results have indicated variability across scales in performance. Very limited publications have focused on the scale-up of a transient transfection process at industrially-relevant scale and there is a lack of understanding of the engineering factors/parameters which affects scalability. Engineering studies aimed at the evaluation of factors underpinning mixing process at the macro and microscale are essential to the mechanistic understanding of the process and will enable definition of optimal scaling strategies. Similarly, techniques able to measure energy dissipation at high resolution are needed to accurately determine the stress along the cell path which might affect the transient transfection process performance. This project builds on a successful UCL-Cytiva CoE project focusing on the macroscale characterisation of the Allegro STR Bioreactor. As part of the latter, two 1L scale prototypes of the Allegro for engineering and biological studies, respectively, are available at UCL. The Pall team will bring direct experience of internal and external transient transfections in both suspension and adherent format. The project objectives will be1. To conduct tailored biological and mixing studies at 1L scale2. To develop a novel technique which could be widely used for stress profiles determination3. To establish optimisation protocols for transient transfection processesProposed timelineYear 1 Biological and mixing studies in the 1L Allegro STR prototype to define design space and verify scalabilityYear 2 Establishment of HEK293 (suspension-adapted) culture, transfection and analyticsYear 3 Novel technique development for quantification of cumulative stress along cell pathlinesYear 4 Process optimisation and verification at scaleProposed timelineYear 1 Biological and mixing studies in the 1L Allegro STR prototype to define design space and verify scalabilityYear 2 Establishment of HEK293 (suspension-adapted) culture, transfection and analyticsYear 3 Novel technique development for quantification of cumulative stress along cell pathlinesYear 4 Process optimisation and verification at scale