COMPREHENSIVE AAV CHARACTERIZATIONS USING HRMS: CAPSID PROTEINS, EMPTY/FULL CAPSID RATIO AND, SSDNA ANALYSIS

Importance of the topic

Adeno associated virus (AAV) vectors are critical tools in gene therapy development requiring rigorous analysis of capsid protein composition, genome packaging efficiency and vector integrity. High resolution mass spectrometry driven workflows provide detailed insight into capsid protein heterogeneity, empty and full particle ratios and single stranded DNA content essential for quality control and regulatory compliance.

Objectives and study overview

This study integrates orthogonal high resolution MS approaches to characterize AAV capsid proteins across six serotypes. Key aims include intact mass determination of VP1, VP2 and VP3 subunits, peptide mapping for sequence coverage and post translational modifications identification, quantification of empty versus full capsids by combined chromatographic and charge detection MS methods and analysis of released single stranded DNA from AAV9.

Methodology and instrumentation

The analytical scheme combines ultra performance liquid chromatography coupled to time of flight MS for both intact capsid proteins and enzymatic digests, charge detection mass spectrometry for direct particle mass measurement, anion exchange and size exclusion chromatography for empty/full separation and proteinase K digestion to release encapsidated ssDNA. Data processing utilized dedicated bioinformatics platforms for mass deconvolution, sequence mapping and fractional analysis.

Main results and discussion

Intact mass analyses of AAV1, 2, 5, 6, 8 and 9 confirmed theoretical values for VP1, VP2 and VP3 with sub 5 ppm mass error. Peptide mapping achieved over 98 percent sequence coverage of VP1 and detected low abundance PTMs such as deamidation, oxidation and glycine clipping. Chromatographic empty/full ratios from AEC and SEC showed excellent correlation with charge detection MS quantifications across a range of sample dilutions. Single stranded DNA released from AAV9 was fractionated and revealed intact genome mass around 1.0 MDa alongside degraded fragments and dimerized dsDNA species.

Benefits and practical applications of the method

• Comprehensive capsid protein profiling supports lot to lot consistency and regulatory submissions
• High confidence empty/full quantifications enhance vector potency and safety assessments
• PTM detection and mapping informs vector stability and manufacturing process optimization
• ssDNA integrity analysis verifies genome packaging and informs formulation development

Future trends and potential applications

Advances in high mass charge detection instrumentation promise improved resolution for heterogeneous viral particles. Automation of MS based AAV analytics will accelerate process development and release testing. Integration with machine learning models may enable predictive quality monitoring and adaptive control in viral vector manufacturing.

Conclusion

This integrated HRMS framework delivers a robust and orthogonal toolkit for detailed AAV characterization spanning capsid protein mass and modifications, empty/full particle ratio and genome integrity. Adoption of these methods can streamline gene therapy vector development and ensure high quality and efficacy of clinical products.

Instrumentation used

• BioAccord LC MS System with UPLC TOF MS for intact protein and peptide mapping
• Charge Detection Mass Spectrometry (CDMS) for empty/full capsid analysis
• Anion Exchange Chromatography (AEC) for empty/full separation
• Size Exclusion Chromatography (SEC) with UV detection
• Protein Pak Hi Res Q and Protein Pak columns for ssDNA fractionation

References

1. J Mol Biol 2016 January 29 428(2 Pt A) 292–300 doi:10.1016/j.jmb.2015.06.019
2. Optimized reversed phase LC MS methods for intact protein analysis and peptide mapping of adeno associated virus proteins Hum Gene Ther Methods 2021 doi:10.1089/hum.2021.046