Development and qualification of a sensitive method for residual feeder cell detection in NK cell therapy products.

Abstract

Cellular therapies must meet stringent regulatory standards for safety and quality. These requirements include the thorough evaluation of all manufacturing components, particularly residual cellular materials, and the potential impact of their presence in the final product. In this study we describe the development and qualification of a safety test method that supports the release of a Natural Killer (NK) cells drug product that is expanded by activation through a combination of cytokines and interaction with feeder cells, derived from a monoclonal K-562 cell line, modified to express 4-1BBL and mbIL-21 genes. In response to the safety and regulatory requirements with the use of feeder cells in NK cell manufacturing for clinical applications, and the limitation of current methodologies, we developed a residual test method enhanced by whole-genome sequencing and copy number analysis via droplet digital PCR (ddPCR). The method guarantees accurate identification of target cells via copy number with high specificity and precision with a coefficient of variation of <10%, linearity (R² = 0.999), and accuracy (72-115% recovery). The linear range reached a lower limit of quantification (LLOQ) of 0.1% and a lower limit of detection (LLOD) of 0.02%. These results support the applicability of this method for residual cell detection and release testing of cellular immunotherapies. Importantly, the methodological framework described here is broadly applicable and can be adapted for other cell therapy products where residual unwanted cells pose a risk of contamination in the final product, offering an adaptable, sensitive, and regulatory-compliant solution for safety testing.