Optimizing Adeno-Associated Virus (AAV) Capsid Protein Analysis Using UPLC and UPLC-MS

Importance of the Topic

Adeno-associated viruses (AAVs) are leading vectors in gene therapy because of their low immunogenicity, broad tissue tropism, and sustained transgene expression. Precise characterization of AAV capsid proteins—VP1, VP2, and VP3—is critical for understanding vector infectivity, ensuring batch-to-batch consistency, and meeting regulatory quality standards. Traditional techniques (ELISA, Western blot, SDS-PAGE) are often laborious or lack serotype specificity, driving demand for rapid, robust, and sensitive LC-MS and optical methods for intact capsid protein analysis.

Objectives and Study Overview

This application note demonstrates development of optimized UPLC-FLR and UPLC-MS workflows on the Waters BioAccord System for:

• Separation and intact-mass analysis of AAV capsid proteins.
• Quantitative stoichiometry measurement via intrinsic fluorescence.
• Case study on AAV8 serotype and extension to serotypes 1, 2, 5, 6, 8, and 9.

Methodology and Instrumentation

Chemicals and Samples:

• rAAV serotypes sourced from BioReliance and Vigene Bioscience.
• Mobile phases: water and acetonitrile with 0.1% difluoroacetic acid (DFA).

Sample Preparation:

• Dilution to 1×10¹³ GC/mL or as-received.
• Acetic acid denaturation (10% v/v, 15 min), centrifugation, direct injection of ~1 µg (LC-MS) or 0.1 µg (LC-FLR).

Instrumentation:

• BioAccord System: ACQUITY UPLC I-Class PLUS, ACQUITY FLR Detector, ACQUITY RDa Mass Detector under waters_connect informatics with UNIFI intact mass workflow.
• Columns evaluated: BEH C4 (300 Å), BEH C8 (130 Å), Peptide BEH C18 (300 Å).
• UPLC gradient: 80–20% A to B over 20 min at 0.2 mL/min, column at 80 °C.
• MS settings: ESI+, 400–7000 m/z, cone voltage 65 V, desolvation 550 °C, capillary 1.5 kV, Leu-enkephalin lock mass.

Main Results and Discussion

Chromatographic Optimization:

• Formic acid modifier with C8 column co-eluted VP1/VP2/VP3.
• Switch to DFA improved peak resolution; C4 (300 Å) further sharpened peaks and increased MS response.

Intact Mass Analysis:

• Resolved peaks at 6.74, 7.10, and 8.32 min correspond to VP1 (81 668 Da), VP2 (66 518 Da), VP3 (59 805 Da).
• Detected N-terminal acetylation, phosphorylation (+80 Da) on VP1/VP2, N-term Met removal, and VP3 clip (50 592 Da) from Asp-Pro hydrolysis.

Stoichiometry by Optical Detection:

• UV detection yielded VP1:VP2:VP3 ratios close to 1:1:10 but required higher load.
• FLR (Ex280/Em350 nm) enhanced sensitivity ~50× (detection of 50 ng), enabling accurate stoichiometry at sub-microgram levels.

Applicability to Other Serotypes:

• Serotypes 1, 6, 8, and 9 showed similar profiles; phosphorylation and acetylation were conserved.
• AAV2 and AAV5 exhibited co-elution; use of C18 column for AAV5 improved VP1 recovery and resolved altered elution order, reflecting hydrophobicity differences.

Benefits and Practical Applications

The BioAccord-based workflows deliver:

• Robust separation and intact-mass determination of capsid proteins with minimal method development effort.
• Quantitative optical readout for routine QC of capsid stoichiometry and purity.
• Automation and compliance support for gene therapy product development and lot release.

Future Trends and Applications

Advancements may include integration of high-throughput UPLC-MS pipelines for multiple serotypes, expanded analysis of PTM heterogeneity, real-time process monitoring, and adaptation to novel viral vectors (e.g., lentivirus). Machine-learning-driven data analysis within informatics platforms will further streamline characterization and support regulatory submissions.

Conclusion

The optimized UPLC-FLR and UPLC-MS methods on the BioAccord System provide sensitive, reproducible, and generalizable workflows for intact AAV capsid protein analysis. These approaches facilitate detailed mass measurement, PTM characterization, and precise stoichiometry assessment, addressing critical needs in gene therapy vector development and quality control.

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