CD19-CAR-T phenotyping during Prodigy manufacturing uncovers critical T cell transition

Advances in cell therapy engineering have driven chimeric antigen receptor T cell manufacturing from manual procedures to highly automated and technically sophisticated production systems. To meet the increasing demand for high-throughput manufacturing, current efforts focus on accelerating production while maintaining/increasing product quality. Yet, to date, the precise daily progression of the T cell states during manufacturing have not been examined in detail. Here we characterize anti-CD19 CAR-T cell manufacturing on a CliniMACS Prodigy platform under current good manufacturing practice (cGMP) conditions, integrating cytotoxic activity profiling with comprehensive phenotyping across a complete production cycle. Using over 130 extra- and intracellular markers related to T cell activation, CAR signaling, metabolism, apoptosis, and hypoxia, we identify a pivotal transition point between process days 5 and 7 marking the shift from early activation to a stable/durable, functionally more specific T cell phenotype. These data shed light on cellular remodeling throughout semi-automated CAR-T manufacturing and provide a mechanistic framework for modulating T cell phenotypes across distinct production phases.