Recombinant adeno-associated virus (rAAV) has emerged as an important gene therapy vector with many clinical trials currently in progress. Analytical characterization and quantitation of particle content remain challenges in both the development and production of rAAV vectors. In this study, charge detection mass spectrometry (CDMS) and gel electrophoresis are used to characterize the DNA content of recombinant AAV8 (rAAV8) vectors with a wide range of target genome sizes. We show that the differences between the masses of empty particles and particles with the genome of interest (GOI) are correlated with the expected genome mass. A small systematic deviation (around 2%) is attributed to the packaging of counterions along with the DNA. In addition to the GOI, a broad distribution of heterogeneous DNA is packaged. The distribution peaks are close to the packaging capacity of the rAAV8 vectors. There is also evidence for the co-packaging of small DNA fragments along with the GOI. Finally, we present evidence that incubation at an elevated temperature can reduce the heterogeneity of the packaged DNA. Taken together, these results show that CDMS is a viable tool for characterization of the packaged genome.
Keywords: AAV; CDMS; GOI; adeno-associated virus; charge detection mass spectrometry; genome; mass spectrometry; rAAV.
© 2021 The Authors.