Senior Director Thermo Fisher Scientific Carlsbad, California, United States
Abstract: Gene-modified cell therapies, such as Chimeric Antigen Receptor T-cell (CAR-T) therapy, have revolutionized cancer treatment and show potential for treating genetic disorders and autoimmune diseases. The manufacturing process involves isolating, activating, and expanding human T cells, for which CD3/CD28 Dynabeads offer a streamlined solution. These beads have been used in over 200 clinical trials; however, since they are non-biodegradable, their removal from the final product is crucial. Newer systems with detachable beads still consider residual beads as contaminants, with the FDA requiring fewer than 100 beads per 3 million cells in the final product. Consequently, a precise and accurate assay for detecting and quantifying residual beads is essential for product release testing. Current methods are either labor-intensive, have low throughput, or require specialized equipment. This study introduces a novel imaging flow cytometry-based assay designed to improve quality control and reliability in product release testing. The study employed the Attune CytPix flow cytometer, which combines traditional cytometry with high-content imaging, making it ideal for detecting rare events like residual beads in cell products. Over 300,000 images per sample were captured and analyzed using the instrument's software to assess object and pixel attributes. Twenty-six image analysis parameters were explored in various combinations to determine the optimal gating and analysis strategy for distinguishing cells and beads. The analytical procedure was validated according to ICHQ2R2 guidelines to ensure consistency, accuracy, and reproducibility. The method proved reproducible across different users, instruments, and labs, with a lower detection limit of 20 beads and a lower quantification limit of 20 beads per million cells. This novel assay offers a reliable alternative to existing residual bead detection methods and can be implemented on a platform commonly available in cell therapy manufacturing facilities.
