Streamlined Characterization of Recombinant AAVs via Charge Detection Mass Spectrometry: Insights from the USP AAV8 Reference Standards

Importance of the Topic

Recombinant adeno‐associated viruses (rAAVs) are critical vectors for gene therapy, offering low immunogenicity and long‐term expression. Comprehensive characterization of rAAV preparations is vital to ensure safety, efficacy, and consistent dosing. However, the inherent heterogeneity of viral capsids—empty, partially filled, and full particles—presents analytical challenges that conventional mass spectrometry and chromatography cannot fully resolve.

Objectives and Study Overview

This application note demonstrates the use of Charge Detection Mass Spectrometry (CDMS) on a commercial Xevo CDMS system to characterize USP rAAV8 empty and full reference standards. The goals were to measure individual particle mass, calculate empty/full capsid ratios, identify impurities, and compare results with orthogonal methods such as sedimentation velocity analytical ultracentrifugation (sv-AUC), mass photometry (MP), and SEC-MALS.

Methodology and Instrumentation

Samples of USP rAAV8 empty and full capsid standards were buffer‐exchanged into 200 mM ammonium acetate with 0.01 % Pluronic F‐68 and analyzed by nano‐electrospray ionization in positive mode. Ions were trapped in an electrostatic linear ion trap for 100 ms, and data were acquired over 15–20 min. The waters_connect™ CDMS Toolkit software managed data acquisition, method definition, and automated analysis.

Key Results and Discussion

CDMS resolved distinct mass peaks at ~3.8 MDa for empty capsids and ~4.8 MDa for full capsids, with full width at half maximum below 0.2 MDa. Charge distributions averaged ~155e for both species. Analysis of the empty standard yielded 98.5 % empty, 0.9 % partially filled, and 0.6 % full capsids. The full standard contained 4 % empty, 16 % partially filled, 77 % full, and 3 % overfull particles. Comparison with sv-AUC, MP, and SEC-MALS highlighted consistency in overall full-capsid content when partial species were grouped, while underscoring CDMS’s superior resolution of heterogeneous subpopulations.

Benefits and Practical Applications

• High‐resolution, single‐particle mass measurement enables precise empty/full quantitation.
• Minimal sample preparation accelerates throughput.
• Enhanced sensitivity detects low‐abundance impurities, supporting quality control in gene therapy pipelines.

Future Trends and Opportunities

Advances in CDMS hardware and software promise higher throughput and broader mass range capabilities. Integration with automated sample handling and bioinformatics will facilitate routine characterization of diverse viral vectors, protein complexes, and nanoparticle formulations. These developments will support regulatory compliance and accelerate biopharmaceutical development.

Conclusion

The Xevo CDMS platform, coupled with waters_connect CDMS Toolkit, offers a streamlined workflow for high‐resolution rAAV characterization. Its ability to resolve empty, partial, full, and overfull capsids with minimal preparation makes it a powerful tool for rigorous quality assessment in gene therapy research and manufacturing.

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