Plasmid Impurity Sizing Of The nptII Kanamycin Resistance Gene In Recombinant Adeno-Associated Virus By Droplet Digital Polymerase Chain Reaction

By Sabine Geiger, Felicia Thoennissen, Marina Magerl, Sophie Beer, Paul Zanotti, Sonya M. Schermann, and Markus Horer

Recombinant adeno-associated virus (rAAV) production often results in plasmid impurities, which consist of heterogeneous DNA fragments from production plasmids that are undesired byproducts. While regulatory authorities require accurate quantification of DNA impurities, traditional qPCR methods primarily focus on detecting small fragments of the sequence of interest, offering limited insights into the size distribution of mispackaged DNA species. A potential risk associated with these impurities is the horizontal gene transfer of manufacturing plasmid-derived resistance genes, which could go undetected by current analytical methods.

To better understand the impact of these plasmid-derived DNA sequences, we developed a novel ddPCR method to assess the size profile of antibiotic resistance gene impurities in rAAV products, complemented by orthogonal approaches such as LR-NGS and transcriptional profiling.