Development of a high-yield cGMP lentiviral vector manufacturing process using suspension culture, a syenex lvv enhancer, and a closed aseptic system.

Abstract Background Lentiviral vectors (LVV) are widely used to genetically engineer T cells for cancer immunotherapies, including CAR-T and TCR-T. Cost-effective production of large, cGMP-compliant batches of LVVs is essential to enhancing the accessibility of these therapies. A critical limitation of adherent 293T-based LVV production in multi-layered cell factories is its labor-intensive process and inherent lack of scalability. Furthermore, end-point sterilization filtration can lead to over 50% titer losses due to the large size of LVV particles. To address these challenges, we developed a scalable, cost-effective cGMP process featuring: (1) serum-free HEK293TH suspension cultures, (2) Syenex LVV enhancer, and (3) a fully closed aseptic processing system. Methods (1) More than 100 HEK293T cell clones were screened for high LVV packaging efficiency following transient plasmid transfection. Selected clones were adapted to serum-free suspension culture, achieving densities up to 1.4 × 10^7 cells/mL. A single high-performing clone was expanded, banked, and designated 293TH cells. (2) 293TH cells were cultured in suspension to 4E6 cells/mL (viability > 90%) and transfected with a 4-plasmid system + Syenex enhancer plasmid. After 48–72 hours, the cell suspension was clarified by filtration, and LVVs were harvested from the supernatant. (3) A closed downstream system was developed, comprising cell harvest, tangential flow filtration, and chromatographic purification. This closed downstream production system eliminates the need of terminal sterilizing filtration. The system sterility was validated via aseptic process simulation. Results (1) Suspension culture achieved titers of 1E7 - 2E8 TU/mL (measured by flow cytometry using Jurkat cells) with superior scalability compared to adherent cultures. (2) The Syenex enhancer consistently increased yields 3 to 5 folds across the scale-up process (from 30 mL shaker flasks to 20 L bioreactors) and with multiple transgenes. (3) The closed processing system improved LVV recovery up to 80%. Conclusion Our integrated LVV manufacturing platform combines suspension 293TH cells, Syenex enhancer technology, and a closed processing system to enable high-yield, serum-free, cGMP-compliant LVV production optimized for cell therapy applications.