A versatile and GMP-compliant bead-based IFN-γ potency assay for standardized quality control and release testing of CAR-T cell drug products.

Potency is a critical specification for CAR-T cells and is typically determined by co-culture of CAR-T cells with target cell lines. However, cell line heterogeneity hampers standardization and can lead to analytical variation impacting potency test results. Further, both the assay and cell line maintenance are labor, time, and resource intensive. Here, we developed and validated a versatile, fully standardized, and GMP-compliant CAR-T cell potency assay. This assay utilizes antigen-loaded beads instead of target cell lines and was fully validated for CD19 and qualified for CD7 and HER2 CAR-T cells. Incubation of streptavidin beads with recombinant biotinylated antigen yielded a dose-dependent bead loading. Subsequent incubation of CAR-T cells with increasing amounts of antigen-loaded beads yielded a dose-dependent secretion of IFN-γ, whereas non-loaded or MOCK antigen-loaded beads did not significantly trigger IFN-γ secretion. Notably, as assessed for CD19 CAR-T cells, cryopreserved CAR-T cells yielded lower potency than fresh CAR-T cells and potency results correlated with the total amount of CAR-T cells in the test sample. Therefore, the assay was fully standardized using a fixed amount of 50 k CAR-T cells per test, a fixed amount of antigen loaded onto the beads (1 pg/bead), and a fixed amount of 500 k antigen-loaded beads. A quantitative and statistically substantiated potency threshold for batch release was established for fresh as well as cryopreserved CAR-T cell drug products This fully standardized assay protocol and validation strategy provides a facile potency assay for CAR-T cell drug products that can be implemented for essentially any antigen of interest.