Abstract 93: Validation of an impedance-based in vitro potency assay for repeatability and precision

Abstract Cell-based immunotherapies, such as CAR T cells, are emerging as a promising approach to therapeutic intervention against cancer. Developing cell and gene therapy products requires reliable and reproducible methods of quantifying critical quality attributes, such as potency, to ensure product strength and consistency. A variety of assays can be used to assess potency; however, it is crucial that these tests are qualified for assay performance to ensure intra- and inter-assay precision. Although inherently variable, biological assays are often the best methods to investigate a product’s mechanism of action and predict clinical outcomes. Previous work has shown that impedance-based potency assays provide a non-invasive, real-time measure of effector cell-mediated cytotoxicity, thus reducing cell manipulation that impacts data reliability. Here, we describe an in vitro potency assay that demonstrates low variability and high precision across replicates, analysts, and days. Effector cell-mediated cytotoxicity was performed using a CD19-positive liquid tumor cell line (Raji) tethered to the well surface using an anti-CD40 antibody. CD19-specific CAR T effector cells were added 24 hours after the target cells and co-cultured with the target cells across a range of E: T ratios, and cytolysis was monitored for three days. Real-time measurement of cell-mediated cytotoxicity also allowed for the calculation of kill time 50 (KT50), defined as the time required for 50% cytolysis of the target cells. Repeatability was calculated across three replicates per E: T ratio. The co-culture experiment was performed in duplicate by a second analyst and on two subsequent days for a comparison of f three plates as well as two operators to evaluate inter-assay precision. In addition to this, CAR T-mediated cell killing in a multiplate impedance system was also investigated for plate-to-plate variability. The percent coefficient of variation (% CV), a statistical measure that describes the precision and repeatability of an assay, was calculated to assess variability between assay replicates, operators, and plate replicates. In all conditions tested, the percent CV for all E: T ratios was below 20%, illustrating the repeatability and precision of the assay. These data support the use of an impedance-based potency assay for evaluating and characterizing immunotherapy products. Citation Format: Danielle Califano, Jianjie Jiang, Dirk Windgassen, Caitlyn De La Flor, Stacie Chvatal, Denise Sullivan, Daniel Millard. Validation of an impedance-based in vitro potency assay for repeatability and precision abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 93.