Characterizing the content and structure of AAV capsids by size exclusion chromatography and charge detection mass-spectrometry

Adeno-Associated Virus (AAV) is currently the most widely used vector in gene therapy applications. However, a significant challenge in the manufacturing process of recombinant AAV (rAAV) is the presence of empty capsids, AAV aggregates, and partially filled capsids. These components do not provide any therapeutic benefit but add to the overall viral load, which could increase immunogenicity and reduce transduction efficiency. Here, we present a strategy that utilizes size exclusion chromatography (SEC) equipped with multi-angle light scattering (MALS) and a diode-array detector (DAD), followed by charge detection-mass spectrometry (CD-MS). The SEC step was used to separate AAV from aggregates and low molecular weight contaminants. In the second step, we employed direct CD-MS infusion using capillary electrophoresis with a sheath liquid (MS) interface. This approach facilitated automated, reproducible, and robust CD-MS determination of empty-filled capsids. Together, our analytical platform offers a reliable and comprehensive approach for assessing the rAAV purity and characterizing key quality attributes, including capsid aggregation and genome packaging.