LC/MS of Intact Adeno-Associated Virus Capsid Proteins for Rapid Confirmation of Product Identity

Importance of the Topic

Adeno-associated viruses (AAVs) are key vectors in gene therapy for rare genetic disorders. Ensuring the correct identity and stoichiometry of their capsid proteins is vital for safety, efficacy, and regulatory compliance. Traditional antibody-based assays are laborious and may not distinguish highly homologous serotypes, motivating the development of rapid, specific analytical workflows.

Study Objectives and Overview

This study presents an optimized liquid chromatography–fluorescence detection–mass spectrometry (LC/FLD-MS) method for intact AAV capsid protein analysis. The goals were:

• To achieve baseline separation of VP1, VP2, and VP3 proteins across seven AAV serotypes (2, 7, 9, 7m8, DJ, rh10, Anc80).
• To confirm primary sequences and detect post-translational or sequence variants without reliance on antibodies.
• To assess relative protein abundance (stoichiometry) and mass accuracy.

Methodology and Instrumentation

Sample Preparation:

• Mixed AAV samples with denaturation buffer (3 M guanidine HCl, 40 mM DTT, 200 mM ammonium bicarbonate, pH 8), heated at 70 °C for 15 min.
• Injected ~1.5×10¹¹ viral genomes per run; no buffer exchange required despite salts and 0.001% Pluronic F-68.

Chromatography and Detection:

• Agilent 1290 Infinity II LC with FLD detection (280 nm excitation, 360 nm emission, 8 µL cell).
• ZORBAX RRHD 300 Å StableBond C18 (2.1 × 100 mm, 1.8 µm) column with high-strength isopropanol gradient (0.1% TFA/FA in water and 90% IPA with 0.1% TFA/FA) at 75 °C, 0.4 mL/min.
• Alternative SB-Diphenyl phase used for serotypes with coelution issues.

Mass Spectrometry:

• Agilent 6545XT AdvanceBio LC/Q-TOF with Dual Jet Stream source; gas temps 350 °C, flow 11–12 L/min; Vcap 4 kV, fragmentor 180 V.
• Acquisition range m/z 900–3,200 at 1 s/s; deconvolution by maximum entropy over 55–85 kDa.

Results and Discussion

The C18 column provided complete resolution of VP1, VP2, and VP3, overcoming coelution seen on C3 and diphenyl phases. For AAV-2, deconvoluted masses matched theoretical VP2/V P3 (≤25 ppm) but revealed a +29.6 Da shift in VP1. Peptide mapping and MS/MS confirmed a single alanine→threonine substitution at residue 77. Application to other serotypes showed accurate mass measurements (≤32 ppm) and required diphenyl selectivity for AAV-9, rh10, and Anc80. Fluorescence peak integration demonstrated VP1:VP2:VP3 ratios close to the expected 1:1:10, with variability in VP1 abundance likely influencing infectivity. Minor deamidations were noted in DJ and rh10, possibly due to culture or sample processing.

Benefits and Practical Applications

• Rapid, antibody-free confirmation of AAV capsid identity and sequence integrity.
• High specificity allows discrimination of closely related serotypes.
• Quantitative insight into capsid stoichiometry aids infectivity assessment.
• Robustness to common buffers and surfactants simplifies workflow in QC and development labs.

Future Trends and Opportunities

• Integration with high-throughput screening of engineered AAV libraries.
• Extension to other viral vectors or large protein complexes.
• Automated data analysis pipelines for routine QC in biomanufacturing.
• Combination with advanced separation media to further enhance resolution.

Conclusion

The optimized LC/FLD-MS workflow using Agilent Infinity II systems and ZORBAX RRHD columns delivers rapid, accurate, and antibody-independent analysis of intact AAV capsid proteins. It enables confirmation of product identity, detection of sequence variants, and reliable stoichiometry assessment across multiple serotypes.

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