Direct Liquid Chromatography/Mass Spectrometry Analysis for Complete Characterization of Recombinant Adeno-Associated...

The requirement for robust analytical methods to characterize adeno-associated virus (AAV) vectors is immediate, as the field advances more AAV gene therapies into the clinic and onto commercialization. AAV capsid proteins (VPs) are critical for viral infectivity and vector potency. Thus, complete characterization of the constituent viral capsid proteins of AAV vectors, including their sequences and post-translational modifications (PTMs), is highly recommended to ensure AAV product quality and consistency. Typically, SDS-PAGE analysis followed by in-gel enzymatic digestion and liquid chromatography/tandem mass spectrometry (LC/MS/MS) is used for the characterization of viral capsid proteins. However, due to the limited recovery of digested peptides from the gel, determination of N-terminal sequences of VPs has not been reported to date. In this study, a direct liquid chromatography/mass spectrometry (LC/MS) intact protein analysis was developed to characterize viral capsid proteins in a variety of AAV serotypes. Both N- and C-terminal sequences of six AAV serotypes have been identified based on accurate mass measurement. This method can be used to confirm the identity of AAV serotype and monitor potential capsid protein heterogeneity. Complete sequence confirmation of AAV2 VPs was achieved through LC/MS/MS analysis of peptides generated using multiple enzymatic digestions. LC/MS/MS analysis confirmed the sequences for both N- and C-termini of capsid VPs and revealed acetylation on the N-termini of VP1 and VP3, consistent with LC/MS intact protein analysis.