Combination of hydrophilic interaction liquid chromatography and top-down mass spectrometry for characterisation of adeno-associated virus capsid proteins.

Adeno-associated virus (AAV) viral vector-based gene therapy is advancing rapidly, offering potential treatments for rare and severe diseases. The AAV capsid consists of a combination of three viral proteins (VPs), VP1, VP2, and VP3, ranging from 59 to 81 kDa and present at a theoretical bulk ratio of 1:1:10. This study employed hydrophilic interaction liquid chromatography (HILIC) and mass spectrometry (MS) to achieve robust separation and detailed characterisation of AAV9 capsid proteins. Advanced top-down MS approaches combining multiple fragmentation techniques (HCD, ETD, EThcD, and UVPD) were successfully applied, increasing the sequence coverage up to 40% for VP3 and confirming N-terminal acetylation on VP1 and VP3. The workflow demonstrated high reproducibility between injection duplicates and was subsequently applied to the characterisation of in-house produced biological replicates of AAV9 samples from HEK293 cells, showing consistent results across them. Analysis of AAV9 derived from Sf9 insect cells, a more complex sample due to higher levels of modification of the capsid VPs, further evidenced method versatility. Overall, this study highlights the potential of HILIC-MS and advanced top-down MS approaches for detailed characterisation of AAV capsid proteins.