Assessing key attributes of adeno-associated viral proteins using UHPLC-FLD-intact accurate mass analysis

Significance of the Topic

Adeno-associated viral (AAV) vectors are central to modern gene therapy applications due to their favorable safety profile and high transduction efficiency. Accurate characterization of AAV capsid proteins (VP1, VP2, VP3), their relative abundance and post-translational modifications (PTMs) is essential for ensuring product consistency, potency and regulatory compliance during development and manufacturing.

Objectives and Study Overview

This work presents a rapid workflow combining ultra-high-performance liquid chromatography with fluorescence detection (UHPLC-FLD) and high-resolution accurate mass spectrometry (HRAM MS) to evaluate critical quality attributes of AAV capsid proteins across multiple serotypes. Key goals included:

• Quantitative assessment of VP1 : VP2 : VP3 ratios by FLD peak integration.
• Identification and mass measurement of intact VPs and their proteoforms by HRAM Orbitrap analysis.
• Comparison of internal and commercial AAV samples to highlight batch-to-batch or serotype-dependent variations.

Methodology and Instrumentation

Sample Preparation:
Buffer exchange of AAV1, AAV6, AAV8 and AAV9 samples into 80 % water/20 % acetonitrile with reducing agent, followed by incubation to denature capsids.

Chromatography and FLD:
Separation on a C4 column (1.0 × 100 mm, 1.7 µm) at 80 °C using a water/acetonitrile gradient with 0.1 % difluoroacetic acid. Fluorescence detection parameters were optimized to resolve VP1–VP3.

Mass Spectrometry:
Thermo Scientific Orbitrap Exploris MX equipped with a BioPharma Option. ESI-HRAM settings delivered mass accuracy better than 10 ppm. Data deconvolution was performed using BioPharma Finder 5.0 to assign intact masses and PTMs.

Main Results and Discussion

The combined UHPLC-FLD-HRAM MS approach enabled:

• Clear separation and quantitation of VP1, VP2 and VP3 with reproducible FLD peak areas.
• Detection of truncated proteoforms and PTMs (deamidation, phosphorylation, acetylation) across serotypes.
• Mass confirmation of each VP with <10 ppm accuracy, including low-abundance VP1 and VP2 species.
• Observation that commercial AAV9 displayed lower VP1 : VP3 and VP2 : VP3 ratios compared to an internally produced AAV9 batch.
• Identification that AAV8 samples had prominent phosphorylation on VP1/VP2 and unique VP3 acetylation patterns.

Benefits and Practical Applications

This workflow provides:

• High-throughput QC monitoring of AAV capsid composition and PTM profile.
• Fast serotype confirmation and batch consistency checks.
• Support for process development, comparability studies and release testing in gene therapy manufacturing.

Future Trends and Opportunities

Emerging directions include:

• Integration of native MS for higher-order structure analysis of intact capsids.
• Implementation of automation and online data processing to accelerate QC turnaround.
• Expansion to other viral vectors and engineered capsids with novel modifications.
• Coupling with advanced PTM mapping tools to elucidate capsid heterogeneity in greater detail.

Conclusion

The UHPLC-FLD-HRAM Orbitrap MS method delivers a powerful platform for rapid, accurate assessment of AAV capsid protein ratios, identities and PTMs. Its sensitivity, speed and mass accuracy make it well suited for supporting large-scale gene therapy manufacturing and ensuring product quality.

Reference

Reiko Kiyonami et al. Assessing key attributes of adeno-associated viral proteins using UHPLC-FLD-intact accurate mass analysis. Thermo Fisher Scientific application note, 2022.