Highly sensitive intact mass analysis of AAV capsid proteins using a UHPLC-FLD- HRAM MS platform

Importance of the Topic

Recombinant adeno-associated virus (rAAV) vectors have become essential tools for gene therapy due to their high transduction efficiency, favorable safety profile, and limited immunogenicity.
The AAV capsid, composed of three overlapping structural proteins (VP1, VP2, VP3), governs cell targeting, internalization, and trafficking.
Complete molecular characterization of these capsid proteins, including low-abundance proteoforms, is critical to ensure product safety, consistency, and efficacy.

Objectives and Study Overview

• Demonstrate the performance advantages of the Thermo Scientific™ Orbitrap Exploris™ 480 mass spectrometer for intact mass analysis of AAV capsid proteins.
• Evaluate high-resolution fluorescence detection for rapid VP1:VP2:VP3 ratio assessment.
• Apply a top-down approach to confirm sequences of low-abundance and truncated proteoforms.

Methodology and Instrumentation

AAV6 particles were produced by transient transfection in HEK293 cells, purified, and buffer-exchanged into 80% water/20% acetonitrile with 5 mM TCEP and 0.1% formic acid.
Analyses were performed on a Thermo Scientific™ Vanquish™ Horizon UHPLC system coupled to an Orbitrap Exploris™ 480 with BioPharma Option and a fluorescence detector.

• C4 column (1.0 × 100 mm, 300 Å, 1.7 µm) at 80 °C
• Fluorescence detection at 280 nm excitation and 350 nm emission
• Mobile phases: 0.1% difluoroacetic acid in water (A) and acetonitrile (B)
• Flow rate 0.05 mL/min with a gradient from 20% to 80% B
• Orbitrap settings: 15,000 resolution for intact MS, AGC target 7.5e4, HCD for top-down at 120,000 resolution

Results and Discussion

Intact mass analysis provided clear average mass measurements for VP1, VP2, and VP3 with mass accuracy better than 3 ppm, even at low sample loads (down to 28 ng total protein).
Fluorescence detection enabled accurate quantification of the VP1:VP2:VP3 ratio (approximately 1.3:1:10).
Top-down HCD fragmentation with wide isolation windows confirmed the sequences of two truncated VP3 proteoforms (residues 1–387 and C-terminal fragment), supported by comprehensive fragment ion assignments.

• High-resolution intact spectra sustained for low-abundance species (VP3 clip and truncated VP3) with <10 ppm accuracy
• Top-down MS² identified N-terminal acetylated forms and localized truncation sites with unambiguous fragment coverage
• Sensitivity and spectral clarity enabled by the Orbitrap Exploris 480 support detailed proteoform analysis on an LC timescale

Benefits and Practical Applications

• Robust and sensitive platform for AAV vector characterization in research and QC laboratories
• Rapid intact mass confirmation accelerates batch release and stability studies
• Top-down proteomics ensures detection of processing variants that may affect safety or efficacy

Future Trends and Opportunities

Advances in hardware, novel fragmentation methods, and dedicated software tools will further improve throughput and depth of proteoform analysis.
Integration of multi-omics data and automation may enable real-time monitoring of viral vector production and streamlined compliance with regulatory requirements.

Conclusion

The UHPLC-FLD-HRAM MS platform combining fluorescence detection with Orbitrap Exploris 480 mass spectrometry offers high sensitivity, mass accuracy, and confidence in both intact and top-down analyses of AAV capsid proteins, supporting rigorous characterization and quality control of gene therapy vectors.

Reference

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