Characterization of Adeno-Associated Viral assemblies on an Ultra-High Mass Range Hybrid Quadrupole-Orbitrap Mass Spectrometer

Significance of the Topic

Advancements in gene therapy hinge on precise characterization of viral delivery vehicles. Adeno-associated viruses (AAVs) are widely used capsid systems whose therapeutic efficacy and safety depend on detailed insights into capsid assembly, subunit composition, and post-translational modifications.

Study Objectives and Overview

This work assesses the Thermo Scientific Q Exactive UHMR hybrid quadrupole-Orbitrap mass spectrometer for two key applications:

• Native MS of intact AAV capsids to determine empty versus genome-loaded particle ratios.
• LC-MS/MS top-down analysis of denatured AAV capsid proteins (VP1, VP2, VP3) to characterize stoichiometry and proteoforms.

Methodology

Sample Preparation:

• AAV serotypes 2 and 6 were expressed in HEK293 cells and buffer-exchanged into 100 mM ammonium acetate for native analysis.
• AAV6 capsids were acid-treated to dissociate subunits for top-down investigation.

Chromatography and Mass Spectrometry:

• Native MS: Static nanospray via coated emitters and NanoFlex source on the Q Exactive UHMR.
• IEX-FLD: Ion-exchange separation with fluorescence detection to quantify empty versus full capsids.
• RP LC-MS/MS: Denaturing reverse-phase UHPLC (Vanquish Horizon) coupled to UHMR for top-down fragmentation using All Ion Fragmentation (AIF) and higher-energy collisional dissociation (HCD).

Results and Discussion

1. Native Intact Capsid Analysis:

• AAV6 empty : full ratio of approximately 64 : 36 was determined by IEX-FLD.
• AAV2 displayed three distinct VP1 : VP2 : VP3 stoichiometries—5 : 5 : 50, 5 : 6 : 49, and 5 : 7 : 48—resolved in the m/z domain under native conditions.

2. Top-Down Subunit Characterization:

• High-resolution MS confirmed N-terminal acetylation of VP1 and VP3 with mass accuracy < 0.1 Da.
• Phosphorylation sites on VP2 at Ser-11 and Thr-56 were localized via HCD fragment ion series with < 10 ppm tolerance.
• Relative quantitation based on integrated LC-MS peak areas yielded a VP1 : VP2 : VP3 ratio near 7 : 5 : 48 for AAV6.

Practical Benefits and Applications

• Enables rapid quality control of AAV vector preparations by determining packaging efficiency and stoichiometry.
• Provides detailed proteoform mapping to monitor critical PTMs that may affect capsid stability or immunogenicity.
• Facilitates batch-to-batch comparability and compliance with regulatory requirements in gene therapy manufacturing.

Future Trends and Applications

Integration of UHMR-MS with automation and high-throughput workflows will accelerate vector development. Coupling native MS with ion mobility may further resolve assembly variants. Advanced top-down approaches could uncover new PTMs and proteoforms, supporting personalized gene therapy design.

Conclusion

The Orbitrap-based Q Exactive UHMR instrument demonstrated robust performance for both intact native analysis and top-down subunit characterization of AAV capsids, delivering high mass accuracy, sensitivity, and detailed proteoform insights essential for gene therapy vector development.

Instrumentation Used

• Q Exactive UHMR hybrid quadrupole-Orbitrap mass spectrometer
• Vanquish Horizon and Flex UHPLC systems
• Static NanoFlex nanospray source with coated emitters
• Ion-exchange fluorescence detector setup

References

No additional references cited.