Impact Of Sample Treatment On DNA Length Distribution Analysis By Duplex

By M. Magerl, F. Thoennissen, S. Beer, RC. Feiner, M. Hoerer, SM. Schermann, and S. Geiger

Recombinant adeno-associated virus (rAAV)-encapsidated plasmid impurities are unwanted byproducts of vector manufacturing, consisting of heterogeneous DNA fragments from the production plasmids. Regulatory authorities require a detailed characterization of these impurities.

To address this, we developed a novel duplex ddPCR method to assess the length distribution of encapsidated nptII (kanamycin resistance) gene fragments. This method also evaluates how common sample treatments used to disrupt virus capsids before droplet formation can affect the analysis of fragment length by duplex ddPCR. Additionally, the potential risk of horizontal gene transfer of plasmid-derived resistance genes, particularly when packaged as full-length genes, underscores the need for thorough impurity characterization.