Performance of in-house optimized orthogonal methods for titration of in-process and purified rAAV

To be able to accurately characterize gene therapy products it is crucial to develop robust analytical strategies. For this we developed and optimized methods for quantification of rAAV genome (vg/mL) and capsid (vp/mL) titers, measured by qPCR/ddPCR and rAAV capsid ELISA assays, respectively. Our focus was on optimizing sample preparation steps and sample pre-dilution conditions into standardized protocols to improve on the assay precision and robustness. Using orthogonal methods, we confirmed the optimized conditions and found ddPCR to have superior performance over qPCR; particularly for in-process material. Overall our data emphasize the importance of thorough optimization and standardization of in-house developed analytical methods for advanced characterization of purified and in-process derived rAAV productions. Furthermore, we have established HPLC based protocols which correlate with viral titers observed by qPCR/ddPCR and ELISA and hence can significantly improve throughput and sample turnaround time.

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