Lentiviral vectors (LVVs) play a critical role in gene delivery for ex vivo gene-modified cell therapies. However, the lack of scalable LVV production methods and the high cost associated with them may limit their use. In this work, we demonstrate the optimization and development of a scalable, chemically defined, animal component-free LVV production process using adherent human embryonic kidney 293T cells in a fixed-bed bioreactor. The initial studies focused on the optimization of the culture process in 2D static cultures. Process changes such as decreasing cell seeding density on day 0 from 2.5 × 10 4 to 5 × 10 3 cells/cm 2, delaying the transient transfection from 24 to 120 h post-seeding, reducing plasmid DNA to 167 ng/cm 2, and adding 5 mM sodium butyrate 6 h post-transfection improved functional LVV titers by 26.9-fold. The optimized animal component-free production process was then transferred to the iCELLis Nano bioreactor, a fixed-bed bioreactor, where titers of 1.2 × 10 6 TU/cm 2 were achieved when it was operated in perfusion. In this work, comparable functional LVV titers were obtained with FreeStyle 293 Expression medium and the conventional Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum both at small and large scale.
Rafiq and colleagues have demonstrated the optimization and development of a scalable, chemically defined animal component-free manufacturing platform for the production of functional lentiviral vectors (LVVs) using adherent HEK293T cells in a fixed-bed bioreactor. LVVs play a critical role for both gene therapy and viral vaccine applications.