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Recombinant adeno-associated virus (rAAV) is one of the main vectors used in gene therapy. An accurate genome titer is not only critical for clinical dosing, but also a prerequisite for many analytical assays for AAV product characterization. AAV genome titer is traditionally determined by qPCR; however, assay precision is not optimal despite extensive efforts. More recently, droplet digital PCR (ddPCR) emerged as a powerful alternative that offers excellent accuracy and precision. However, currently ddPCR is not as widely available as qPCR and operates at a lower throughput and a higher cost. In this paper, we introduce an improved qPCR method with two major optimizations: (1) using an AAV reference material as qPCR standard instead of plasmid DNA and (2) implementing a "digestion-free" method by adding 5% Tween 20 to standard and sample preparations. The new method has been extensively tested with AAV of different serotypes, purification status, and transgenes encapsidated and was found to be highly accurate, precise, and robust. This significantly improved and simplified assay can be easily adopted by researchers in the gene therapy field and further automated for high-throughput applications., (© 2020 The Author(s).)

Recombinant adeno-associated virus (rAAV) is a vector with increasing popularity in the field of gene therapy. Like other drug substances manufactured in cell lines, rAAV vectors are commonly contaminated with host cell DNA, and the levels must be carefully monitored. The current method for residual DNA quantification in rAAV was adapted from protein programs and required sample digestion by proteinase prior to qPCR analysis. While the method worked effectively, it was unclear if proteinase digestion was essential for releasing DNA from rAAV capsids and improving qPCR efficiency. In this study, we systematically investigated the role of each component and treatment with the goal to simplify and streamline the method. It was determined that the proteinase digestion step was dispensable, while the addition of Tween 20 to rAAV samples was essential for accurate quantification of residual DNA. Based on this finding, a digestion-free method has been established that requires only a one-step sample preparation-addition of Tween 20. The method has been tested extensively with an rAAV9-based drug substance and process intermediates and verified with other rAAV serotypes. This significantly simplified and faster assay can be easily automated for high-throughput applications.