ADDRESSING CHALLENGES IN POTENCY ASSAY DEVELOPMENT FOR CAR-T PRODUCTS IN DECENTRALIZED MANUFACTURING: A CASE STUDY FROM ORGENESIS MD.

Introduction: In the rapidly evolving landscape of cellular and gene therapy (CGT) products, ensuring potency is paramount for efficacy and patient safety. Decentralized CGT manufacturing requires simplicity to enable analysts with basic training, brevity due to the short shelf life of fresh products, and automation to facilitate efficient reporting with QC/QA management. Here, we present a case study where Orgenesis MD addresses these challenges through the development of a cytotoxicity assay as a potency assay for CAR-T products. Methods: The Axion Biosystems Maestro Z instrument was utilized for real-time monitoring of impedance values within a controlled environment of temperature and CO2. Impedance values served as indicators of target cell coverage and attachment, with decreases indicating target cell death. Target cells were dosed with CAR-T effectors derived from 3 independent donors. Data was processed in the Axion Axis-Z analysis software to obtain impedance values, cytolysis percentage, and dose-response curves. To determine the potency of our CAR-T products we validated specificity, linearity, and accuracy using various batches produced from different viral vectors. The studies identified critical points in the potency assay including defined culture parameters and E:T ratios. The assay demonstrated sensitivity in differentiating between CAR-T batches and revealed notable differences between various vectors used. Unlike conventional endpoint assays, the Axion enables retrospective data analysis and comparison with clinical outcomes, potentially offering valuable insights into CAR-T efficacy over extended co-culture durations. Cytotoxicity assay development addressed critical parameters and demonstrated the sensitivity of the potency assay to discern between the relative potency of CAR-T batches. These findings represent significant progress towards establishing a fully validated potency assay for CAR-T products that addresses the specific needs of decentralized CGT manufacturing. Further qualification and validation studies will refine assay parameters, ensuring robustness across production batches and product constructs, ultimately enhancing the quality and consistency of CGT products and improving patient outcomes. [ABSTRACT FROM AUTHOR]