A Streamlined Workflow for the Characterisation and Relative Quantification of Recombinant Adeno-Associated Viruses using Charge Detection Mass Spectrometry

Significance of the Topic

Recombinant adeno-associated viruses are central to gene therapy, requiring precise characterization of capsid integrity and genome packaging to ensure safety and efficacy.

Objectives and Study Overview

This study presents a streamlined charge detection mass spectrometry workflow for assigning and quantifying empty, partial, full, and overfull rAAV capsids and compares results against mass photometry, multi-angle light scattering, and analytical ultracentrifugation.

Methods and Instrumentation

Samples from two manufacturers were buffer-exchanged into ammonium acetate with Pluronic F-68. Ions were generated by nano-electrospray and analyzed using a prototype electrostatic linear ion trap-based CDMS. Custom waters_connect software processed time-domain signals via fast Fourier transform to extract mass-to-charge and charge information for individual ions. Additional orthogonal techniques included:

• Mass photometry (MP)
• Size-exclusion chromatography with multi-angle light scattering (SEC-MALS)
• Analytical ultracentrifugation (AUC)

Main Results and Discussion

• CDMS accurately resolved empty, intermediate, full, and overfull capsid populations and visualized them in two-dimensional heat maps.
• Relative quantification of rAAV8 empty/full ratios by CDMS agreed within 1% of combined orthogonal methods.
• CDMS demonstrated enhanced sensitivity to impurity species not readily distinguished by other techniques.

Benefits and Practical Applications

This workflow delivers rapid, high-resolution characterization of viral capsids, supports process development and quality control, and can be adapted to other biotherapeutic analytes.

Future Trends and Opportunities

Advances may include increased automation, integration with cloud-based data platforms, application to diverse viral vectors, enhanced mass resolution, and tighter coupling with orthogonal analytical methods.

Conclusion

The dedicated CDMS waters_connect application enables a robust, efficient, and versatile platform for detailed rAAV capsid analysis, complementing existing biophysical techniques.

References

No explicit literature references were provided in the source material.