Comprehensive Adeno-Associated Virus Critical Quality Attribute Analysis with Liquid Chromatography-Mass Spectrometry

Importance of the Topic

Adeno-associated viruses (AAV) are widely used gene therapy vectors due to their ability to deliver genetic material safely and efficiently. Rigorous monitoring of their critical quality attributes (CQAs) such as post-translational modifications, host cell protein (HCP) impurities, and sequence variants is essential to ensure product safety, potency, and regulatory compliance.

Objectives and Study Overview

This study aims to establish a comprehensive liquid chromatography–mass spectrometry (LC–MS) workflow for AAV CQA analysis. It integrates intact mass analysis, subunit and peptide mapping, host cell protein profiling, and sequence variant analysis (SVA) to provide a unified platform that improves upon traditional ELISA and sequencing methods.

Methods and Instrumentation

Sample Preparation:

• Intact AAVs underwent buffer exchange into reducing conditions (10 kDa filter, tris(2-carboxyethyl)phosphine, 80% water/20% acetonitrile, 0.1% formic acid).
• For HCP and SVA, capsids were denatured, reduced, alkylated, and digested with trypsin and rAsp-N.

Chromatography and Mass Spectrometry:

• Agilent 1290 Infinity II LC coupled to a 6545XT AdvanceBio LC/Q-TOF with dual Agilent Jet Stream source.
• Columns: Zorbax Diphenyl RRHD 300 Å (2.1×150 mm, 2.7 µm) for intact analysis; AdvanceBio Peptide Mapping (2.1×150 mm, 1.8 µm) for peptide workflows.
• Flow rate: 0.4 mL/min; Column temperature: 60 °C.
• Data acquisition: MassHunter Acquisition B.09.00 with SWARM autotune; iterative MS/MS for mapping, HCP, and SVA.
• Data processing: Protein Metrics Byos with SVA Validator algorithm.

Main Results and Discussion

Intact vs. Peptide Reconstruction:
Reconstructed theoretical intact spectra from peptide mapping accurately highlighted low-abundance PTMs responsible for shoulder peaks in experimental mass profiles of VP1, VP2, and VP3 capsid proteins.

Host Cell Protein Profiling:
LC–MS/MS enabled identification and relative quantitation of residual HCPs post AAV8 purification. AAV8 proteins dominated the profile, but several enzyme and uncharacterized host proteins were detected at varying abundances.

Sequence Variant Analysis:
Multiple amino acid variants (e.g., Gly→Asp, Met→Ile/Leu, Ser→Asn) were identified with low relative abundances (0.03–4.47%) and confirmed via MS/MS fragmentation patterns.

Benefits and Practical Applications

The integrated LC–MS workflow offers high specificity and sensitivity for simultaneous assessment of multiple AAV CQAs. It streamlines quality control by replacing separate ELISA and sequencing assays with a single platform, enhancing throughput and depth of molecular characterization.

Future Trends and Opportunities

Advances in mass spectrometry hardware and software will further increase acquisition speed and sensitivity, enabling deeper proteoform analysis of viral vectors. Automation and data analysis algorithms will facilitate real-time monitoring, and expansion into native MS and single-particle techniques may provide new insights into capsid heterogeneity and genome packaging.

Conclusion

A comprehensive LC–MS approach for AAV characterization effectively monitors critical quality attributes including PTMs, HCPs, and sequence variants. This methodology enhances analytical rigor for gene therapy development and supports robust quality control frameworks.

References

1. Huang Y. et al. Journal of Pharmaceutical and Biomedical Analysis. 2021;200:114069.
2. Valliere-Douglass J. et al. PDA Journal of Pharmaceutical Science and Technology. 2019;73:622–634.