CC BY 4.0Couto, Pedro SilvaPedro SilvaCoutoStibbs, Dale J.Dale J.StibbsSpringuel, PierrePierreSpringuelSchultz, UrsulaUrsulaSchultzEffenberger, ManuelManuelEffenbergerGoldrick, StephenStephenGoldrickNavarro‐Velázquez, SergioSergioNavarro‐VelázquezJuan, ManelManelJuanHerbst, LauraLauraHerbstNießing, BastianBastianNießingMestermann, KatrinKatrinMestermannSanges, CarmenCarmenSangesHudecek, MichaelMichaelHudecekRafiq, Qasim A.Qasim A.Rafiq2025-09-252025-09-252025-09https://publica.fraunhofer.de/handle/publica/496250https://doi.org/10.24406/publica-554110.1002/biot.7011410.24406/publica-5541Chimeric 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.enATMPCAR-TGMPBiomanufacturingSerum freeStirred-tank bioreactorXeno-freejournal article