A Comprehensive ddPCR Portfolio For In-Depth DNA Impurity Profiling

By Sonya M. Schermann, Sabine Geiger, Renée Kober, Felicia Thoennissen, Marina Magerl, Sophie Beer, and Markus Hoerer

DNA impurities in recombinant adeno-associated virus (rAAV) products pose potential risks to patients and must be thoroughly understood. Effective methods for quality control (QC) of the final product, as well as for extended product characterization, are essential. Here, we discuss how multiplex digital droplet polymerase chain reaction (ddPCR) can be applied in both of these areas.

For QC release, multiplexing allows monitoring of multiple DNA impurities in a single assay, reducing the number of required assays and thereby minimizing both time and cost for batch release. We present a duplex method for monitoring residual DNA impurities of different origins and discuss key aspects of method development and the qualification/validation of such assays.

For extended product characterization, we demonstrate that multiplex ddPCR can be used in various applications, such as profiling the length distribution of encapsidated plasmid and host cell DNA—critical attributes when evaluating the potential risk to patients from such sequences. Long-read NGS sequencing, developed in-house, is used as an orthogonal method to validate these results. We also show how multiplex ddPCR can assess whether two different sequences, such as residual rep and cap DNA, are present in the same capsid—an important application for predicting the potential presence of replication-competent AAV (rcAAV) in batches.

Overall, multiplex ddPCR methods provide powerful tools to determine not only the presence but also the quantity, size, and identity of encapsidated residual DNA, making them valuable for both QC and extended characterization of rAAV products.