Development and qualification of quantitative assays for detecting process-related nucleic acid impurities in recombinant AAV preparations

Residual DNA impurities in recombinant adeno-associated virus (rAAV) vector preparations pose an important analytical and regulatory challenge, yet current methods provide limited information on their integrity. Here, we developed multiplex digital PCR (dPCR) workflows that quantitatively distinguish full-length from fragmented DNA impurities originating from plasmid backbones (ampicillin and kanamycin resistance genes) and host-cell DNA (adenoviral E1A and SV40 large T antigen). By strategically positioning terminal and internal assays across each target, the method enables integrity assessment beyond the capabilities of other methods. In our CsCl-purified scAAV9 preparations, plasmid-derived sequences were abundant and largely DNase I resistant, confirming their encapsidation, whereas host-cell-derived impurities were present at low levels and mostly removed by nuclease treatment. Obtained dPCR results were further confirmed using countable PCR, a recently introduced single-molecule quantification technology, and with nanopore sequencing. Moreover, countable PCR platform was shown to be less sensitive to the highly concentrated AAV background, when quantifying rare targets. Overall, our results demonstrate that multiplex dPCR is an informative tool for integrity profiling of impurities in rAAV manufacturing.