Characterizing Adeno-Associated Virus Capsids with both Denaturing and Intact Analysis Methods

Adeno-associated virus (AAV) capsids are among the leading gene delivery platforms used to treat a vast array of human diseases and conditions. AAVs exist in a variety of serotypes due to differences in viral protein (VP) sequences, with distinct serotypes targeting specific cells and tissues. As the utility of AAVs in gene therapy increases, ensuring their specific composition is imperative for correct targeting and gene delivery. From a quality control perspective, current analytical tools are limited in their selectivity for viral protein (VP) subunits due to their sequence similiaries, instrumental difficulties in assessing the large molecular weights of intact capsids, and the uncertainity in distinguishing empty and filled capsids. To address these challenges, we combine two distinct analytical workflows that assess the intact capsids and VP subunits separately. First, charge detection-mass spectrometry (CD-MS) was applied for characterization of the intact capsids and then liquid chromatography, ion mobility spectrometry, and mass spectrometry (LC-IMS-MS) separations were used for capsid denaturing measurements. This multi-method combination was applied to 3 AAV serotypes (AAV2, AAV6, and AAV8) to evaluate their intact empty and filled capsid ratios and then examine the distinct VP sequences and modifications present.