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

Significance of the Topic

The development of recombinant adeno-associated viral (rAAV) vectors has revolutionized gene therapy due to their high transduction efficiency and favorable safety profile. Accurate characterization of AAV capsid proteins (VP1, VP2, VP3) and their post-translational modifications (PTMs) is essential to ensure potency, consistency across batches, and compliance with regulatory standards.

Goals and Study Overview

This study presents a robust UHPLC-FLD-HRAM MS workflow employing an Orbitrap Exploris MX system to rapidly determine VP identity, relative abundance, and PTM profiles in multiple AAV serotypes (AAV1, AAV6, AAV8, AAV9). Both in-house and commercial viral preparations were analyzed to demonstrate method applicability and serotype confirmation.

Methodology and Instrumentation

AAV samples were buffer-exchanged into 80% water/20% acetonitrile with a reducing agent and formic acid, then denatured prior to analysis. Separation was performed on a C4 reversed-phase column at elevated temperature using a gradient of 0.1% difluoroacetic acid in water and acetonitrile. Fluorescence detection (excitation 280 nm, emission 350 nm) provided sensitive VP ratio assessment, while full-scan HRAM MS (m/z 700–2800, resolution 30000) delivered intact mass measurements.

Used Instrumentation:

• Vanquish Horizon UHPLC system with Fluorescence Detector F
• Orbitrap Exploris MX mass spectrometer with BioPharma Option
• C4 reversed-phase column and nanoViper fluidic fittings

Main Results and Discussion

The UHPLC-FLD method achieved baseline separation of VP1, VP2, VP3, and associated truncated or modified proteoforms. Deconvoluted mass spectra yielded mass errors below 5 ppm for major and low-abundance species. Key observations included:

• Universal detection of N-terminal acetylation on VP1 and VP3 across serotypes
• Serotype-specific phosphorylation on VP1 and VP2 in AAV8
• Reproducible truncated VP species in internal versus commercial AAV9 samples, reflecting production differences
• Relative quantitation via FLD peak areas revealed batch-dependent VP1:VP3 and VP2:VP3 ratios

Benefits and Practical Applications

• Rapid serotype confirmation and batch comparison with precise intact mass data
• High sensitivity enabling detection of low-abundance PTMs for stringent quality control
• Adaptable platform for diverse AAV serotypes and manufacturing workflows
• Enhanced monitoring of critical quality attributes in gene therapy production

Future Trends and Possibilities

Ongoing advances in mass spectrometry and chromatographic methods will further optimize AAV protein characterization. Automated sample processing, real-time data analysis, and integration with AI-based interpretation promise higher throughput and deeper insights. Emerging techniques such as native MS and middle-down analysis may offer detailed topological information on capsid integrity and PTM distribution, accelerating process development and regulatory approval.

Conclusion

The UHPLC-FLD-HRAM Orbitrap Exploris MX workflow delivers a rapid, sensitive, and accurate solution for comprehensive analysis of AAV capsid proteins and proteoforms. It reliably measures protein identity, relative ratios, and PTMs across multiple serotypes, supporting robust quality control in gene therapy manufacturing.

References

1. Samulski R J, Muzyczka N AAV-Mediated Gene Therapy for Research and Therapeutic Purposes Annu Rev Virol 2014 1 427–451
2. Naso M F, Tomkowicz B, Perry W L, Stroh W R Adeno-Associated Virus as a Vector for Gene Therapy BioDrugs 2017 31 317–334