Universal ddPCR-based assay for the determination of lentivirus infectious titer and lenti-modified cell vector copy number.

The translation of cell-based therapies from research to clinical setting requires robust analytical methods that successfully adhere to current good manufacturing practices and regulatory guidelines. Lentiviral vectors are commonly used for gene delivery to generate genetically modified therapeutic cell products. For some cell therapy products, standardized characterization assays for potency and safety have gained momentum. Translational applications benefit from assays that can be deployed broadly, such as for lentiviral vectors with various transgenes of interest. Development of a universal method to determine lentivirus infectious titer and vector copy number (VCN) of lenti-modified cells was performed using droplet digital PCR (ddPCR). Established methods relied on a ubiquitous lenti-specific target and a housekeeping gene that demonstrated comparability among flow cytometry-based methods. A linearized plasmid control was used to determine assay linearity/range, sensitivity, accuracy, and limits of quantification. Implementing this assay, infectious titer was assessed for various production runs that demonstrated comparability to the flow cytometry titer. The ddPCR assay described here also indicates suitability in the determination of VCN for genetically modified CAR-T cell products. Overall, the development of these universal assays supports the implementation of standardized characterization methods for quality control.