Impact of Serum/Xeno-Free Medium and Cytokine Supplementation on CAR-T Cell Therapy Manufacturing in Stirred Tank Bioreactors

Abstract

Chimeric antigen receptor T-cell (CAR-T) therapies have demonstrated clinical efficacy in treating haematological malignancies, resulting in multiple regulatory approvals. However, there is a need for robust manufacturing platforms and the use of GMP-aligned reagents to meet the clinical and commercial demands. This study investigates the impact of serum/xeno-free medium (SXFM) and cytokine supplementation on CAR-T cell production in static and agitated culture systems, using 24-well plate G-Rex vessels and 500 mL stirred tank bioreactors (STRs), respectively. Under static conditions, SXFM media supported CAR-T cell expansion with growth kinetics comparable to foetal bovine serum, FBS-based RPMI, irrespective of the cytokine supplementation (IL-2 or the combination of IL-7 and IL-15). In contrast, when the expansion was conducted using STRs, several differences were observed with SXFM. Particularly, when supplemented with IL-2 SXFM, it increased transduction efficiency, supporting accelerated proliferation relative to FBS-containing RPMI. Additionally, SXFM maintained a higher CD4:CD8 ratio at harvest, a feature associated with improved clinical outcomes. No significant differences were observed in the CAR-T cell populations' differentiation status or activation and exhaustion profiles across the conditions. These results suggest that SXFM enables CAR-T cell manufacturing in STRs, improving key quality attributes such as transduction efficiency, growth kinetics, and CD4:CD8 ratio compared to FBS-supplemented medium.