Quasi-perfusion method for scaling and optimisation of a suspension stable producer cell line for continuous manufacturing of lentiviral vectors at high cell density

Résumé fourni par la source

Viral vectors are essential tools in cell and gene therapy, enabling efficient delivery of therapeutic genes and long-term expression. Lentiviral vectors (LVs) are particularly important for the manufacture of CAR-T cells; however, their production is limited by short vector half-life and low purification yields. Overcoming these challenges is critical to improving the scalability and efficiency of LV-based therapies. Continuous manufacturing offers a promising solution, as continuous removal of LV from bioreactors can increase recovery, support high cell density (HCD) cultures, and improve volumetric productivity (VP). In this study, a stable producer cell line (SPCL), WinPac-RDpro-GFP (WRH1), was used. WRH1 replaces the cytotoxic VSV-G envelope with the non-cytotoxic RDpro, enabling prolonged continuous vector production. A scale-down method was developed to optimise suspension HEK-293T cultures. Using a 24-well microwell plate (MWP) shaken at 195 rpm, a maximum viable cell density (VCD) exceeding 50 million viable cells per mL was achieved. The Froude number was applied as a novel scaling parameter, enabling successful scale-up between MWPs and shake flasks for HEK quasi-perfusion culture. Adaptation to serum-free suspension culture resulted in a 40% reduction in cell-specific productivity (CSP). Despite this, VP increased 4.6-fold due to the HCD achieved under quasi-perfusion conditions. Overall, this work demonstrates the potential of HCD quasi-perfusion culture using a suspension stable producer cell line to significantly improve LV manufacturing performance.

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