Auto‐transduction in lentiviral vector bioprocessing: A quantitative assessment and a novel inhibition strategy

Abstract Lentiviral vectors are highly efficient gene delivery vehicles used extensively in the rapidly growing field of cell and gene therapy. Demand for efficient, large‐scale, lentiviral vector bioprocessing is growing as more therapies reach late‐stage clinical trials and are commercialized. However, despite substantial progress, several process inefficiencies remain. The unintended auto‐transduction of viral vector‐producing cells by newly synthesized lentiviral vector particles during manufacturing processes constitutes one such inefficiency which remains largely unaddressed. In this study, we determined that over 60% of functional lentiviral vector particles produced during an upstream production process were lost to auto‐transduction, highlighting a major process inefficiency likely widespread within the industry. Auto‐transduction of cells by particles pseudotyped with the widely used vesicular stomatitis virus G protein was inhibited via the adoption of a reduced extracellular pH during vector production, impairing the ability of the vector to interact with its target receptor. Employing a posttransfection pH shift to pH 6.7–6.8 resulted in a sevenfold reduction in vector genome integration events, arising from lentiviral vector‐mediated transduction, within viral vector‐producing cell populations and ultimately resulted in improved lentiviral vector production kinetics. The proposed strategy is scalable and cost‐effective, providing an industrially relevant approach to improve lentiviral vector production efficiencies.