Characterizing the Content and Structure of AAV Capsids by Size Exclusion Chromatography and Orbitrap-Based 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, oligomers, 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 multiangle light scattering (MALS) and a diode-array detector (DAD), followed by orbitrap-based charge detection-mass spectrometry (CD-MS). The SEC step was used to separate AAV capsids (non-aggregates) from oligomers, 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, sensitive, and robust CD-MS determination of empty-filled capsids, capsid oligomers, and encapsidated genomes. Importantly, the empty-to-filled capsid ratio was inaccurate without the SEC step. Together, our analytical platform offers a reliable and comprehensive approach for assessing the rAAV purity and characterizing key quality attributes, including capsid aggregation, capsid oligomerization, and genome packaging.