Purity and Identity Characterization of Adeno-Associated Virus Capsid Particles by Intact and Bottom-Up Based Liquid Chromatography-Mass Spectrometry Methods

Importance of the Topic

Adeno associated viruses serve as critical delivery vehicles in gene therapy, and detailed characterization of their protein capsids is essential to confirm their integrity, composition and functionality.

Study Objectives and Overview

This work evaluates intact and bottom up liquid chromatography–mass spectrometry workflows for detailed analysis of AAV8 capsid proteins. It demonstrates simultaneous detection of VP1, VP2 and VP3 proteoforms, accurate mass measurement of post translational modifications and comprehensive sequence coverage through peptide mapping.

Methodology and Instrumentation

Sample preparation for intact analysis included buffer exchange with TCEP reduction and high organic mobile phase. Peptide mapping involved denaturation, reduction, alkylation and dual enzyme digestion using trypsin and rAsp N. Chromatographic separation and MS detection were performed on an Agilent 1290 Infinity II LC coupled to a 6545XT AdvanceBio LC/Q TOF with Agilent Jet Stream source. Columns used were Zorbax Diphenyl RRHD for intact analysis and AdvanceBio Peptide Mapping for bottom up. Standard flow rates (0.4 mL/min), injection volumes (20 µL intact, 40 µL digest) and column temperatures (60 °C) were applied. Data acquisition employed the SWARM autotune feature and iterative MS/MS for peptide mapping, with processing in MassHunter BioConfirm 10.0.

Key Results and Discussion

• Intact analysis resolved all three capsid proteins with <10 ppm mass accuracy and chromatographic separation of low‐abundance VP1 and VP2 proteoforms. VP1 exhibited three phosphorylation sites and N-terminal acetylation; VP2 carried at least two phosphosites; VP3 showed ~70 % acetylation.
• Peptide mapping delivered 97.7–100 % combined sequence coverage across VP1, VP2 and VP3 using iterative MS/MS and multiple enzyme digests. Site-specific localization of PTMs was achieved, and low levels of deamidation and oxidation confirmed protein integrity.

Benefits and Practical Applications

This combined intact and bottom up LC-MS approach provides a robust platform for comprehensive assessment of critical quality attributes in AAV vectors. It supports regulatory requirements by delivering precise PTM profiling, proteoform quantitation and sequence verification, enhancing confidence in therapeutic preparations.

Future Trends and Opportunities

Advancements may include expanded multispecific enzyme digestions, automated PTM quantitation algorithms, extension to diverse AAV serotypes and real-time monitoring in manufacturing workflows to accelerate gene therapy development.

Conclusion

The described intact and peptide mapping LC-MS workflows enable high-resolution characterization of AAV capsid proteins, achieving deep sequence coverage, accurate PTM identification and quantitation, thereby strengthening quality control and supporting regulatory submissions.

Reference

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