Analysis of AAV-Extracted DNA by Charge Detection Mass Spectrometry Reveals Genome Truncations

Anal Chem. 2023 Mar 7;95(9):4310-4316. doi: 10.1021/acs.analchem.2c04234. Epub 2023 Feb 22.

Abstract

Adeno-associated virus (AAV) is a widely used gene therapy vector. The intact packaged genome is a critical quality attribute and necessary for an effective therapeutic. In this work, charge detection mass spectrometry (CDMS) was used to measure the molecular weight (MW) distribution for the genome of interest (GOI) extracted from recombinant AAV (rAAV) vectors. The measured MWs were compared to sequence masses for a range of rAAV vectors with different GOIs, serotypes, and production methods (Sf9 and HEK293 cell lines). In most cases, the measured MWs were slightly larger than the sequence masses, a result attributed to counterions. However, in a few cases, the measured MWs were significantly smaller than the sequence masses. In these cases, genome truncation is the only reasonable explanation for the discrepancy. These results suggest that direct analysis of the extracted GOI by CDMS provides a rapid and powerful tool to evaluate genome integrity in gene therapy products.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA* / genetics
  • Dependovirus* / genetics
  • Genetic Vectors
  • HEK293 Cells
  • Humans
  • Mass Spectrometry
  • RNA

Substances

  • DNA
  • RNA