Characterization and quantitation of baculoviral DNA in rAAV vectors produced in Sf9 cells

Recombinant adeno-associated virus (rAAV) vectors are widely used in gene therapy due to their ability to transduce various cell types and tissues, sustained gene expression, and relatively safe profile. However, the production of rAAV vectors using the baculovirus/Sf9 system can result in the mispackaging of the baculoviral (recombinant baculoviral [rBV]) DNA. This study aims to characterize and quantify these rBV DNA impurities in rAAV products. We developed a multiplex digital PCR method to model, characterize, and accurately determine rBV DNA impurities. Our findings indicate that rBV DNA within 5 kb of the inverted terminal repeat (ITR) and the mini-F region has a higher probability of being mispackaged into rAAV particles than other regions. Additionally, using regular PCR plus agarose gel analysis and digital PCR, full-length kanamycin and gentamicin antibiotic genes were detected and quantitated in the rAAV. The study also revealed a strand-selective mispackaging of rBV DNA, with no correlation between the amount of rBV DNA impurity and the vector's size conflicting with the prevalent belief that smaller vectors will contain more rBV impurities. These results provide insights into the mechanisms of rBV DNA impurity formation and suggest strategies to reduce these impurities, thereby enhancing the safety and efficacy of rAAV-based gene therapies.