Optimizing and developing a scalable, chemically defined, animal component-free lentiviral vector production process in a fixed-bed bioreactor

Abstract

Lentiviral vectors (LVVs) play a critical role in gene delivery for exvivo 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 2.5×104 to 5×103 cells/cm2, delaying the transient transfection from 24 to 120h post-seeding, reducing plasmid DNA to 167ng/cm2, and adding 5mM sodium butyrate 6h 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 1.2×106 TU/cm2 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.