Intensification of rAAV Production Based on HEK293 Cell Transient Transfection

Recombinant adeno-associated virus (rAAV) vectors are widely used in gene therapies, but the rapidly increasing global demand has created a significant challenge for rAAV manufacturing, where production capacity remains a critical bottleneck. To address this, strategies to enhance production yields are urgently needed. This study presents an innovative approach to rAAV production using high cell density (HCD) stirred tank perfusion culture. rAAV1 and rAAV9 vectors carrying GFP cargo were used as models, with triple-plasmid transfection performed in suspension HEK293 cells at a high viable cell density of 50 million cells/mL in culture then maintained at ≥ 30 million cells/mL throughout production. Transfection and production parameters were first optimized in a 5 mL pseudo-perfusion spin tube screening system at HCD. A proof-of-concept was then demonstrated by scaling up to a 200 mL stirred tank bioreactor (STR) in perfusion mode. This intensified process achieved rAAV9 production levels per cell comparable to those observed in reference shake flask cultures at 1 million cells/mL. By implementing transfection at very HCD in a perfusion-based STR, this approach has the potential to significantly enhance rAAV volumetric production capacity, providing a promising solution to meet the growing demand for gene therapies.