PHARMSCI: Comprehensive AAV Characterizations Using HRMS: Peptide Mapping, Capsid Proteins, Intact ssDNA, and Intact Virus

Significance of the topic

Adeno-associated virus (AAV) vectors are leading platforms for gene therapy applications, yet standardized analytical methods to ensure product quality and safety are not fully established
The ability to characterize protein composition, post-translational modifications, and genome packaging is critical for regulatory compliance, process development, and accurate dosing
High-resolution mass spectrometry approaches combined with orthogonal separation techniques enable detailed profiling of viral capsid proteins, intact single-stranded DNA (ssDNA), and particle integrity

Objectives and overview of the study

This study aimed to develop a comprehensive workflow for AAV characterization addressing peptide mapping, intact capsid protein analysis, ssDNA isolation, and empty/full capsid quantification
Multiple AAV serotypes (1, 2, 5, 6, 8, 9) were analyzed to demonstrate method robustness and applicability across vector variants
Key goals included achieving high sequence coverage of capsid proteins, accurate mass measurements of viral components, and correlation of empty/full particle ratios using complementary platforms

Methods and instrumentation

The analytical workflow combined the following techniques and instruments

• Peptide mapping: Tryptic digestion of AAV5 capsid proteins after surfactant removal by denaturing size-exclusion chromatography (SEC); reversed-phase UHPLC-TOF-MS on a C18 column for peptide analysis
• Intact capsid proteins: RP-LC-MS with intrinsic fluorescence detection using C4 column and difluoroacetic acid modifier to quantify VP1, VP2, VP3 proteins across serotypes
• ssDNA isolation: Proteinase K digestion followed by anion-exchange chromatography (Protein-Pak Hi Res Q) to separate viral genome; buffer exchange into 20 mM ammonium acetate prior to charge detection mass spectrometry (CDMS)
• Empty/full capsid analysis: AEC with fluorescence detection and SEC with UV absorbance at 260 nm and 280 nm to determine particle loading
• Charge-detection MS: MegaDalton Solutions CDMS system for direct measurement of intact viral particles and isolated genome masses
• Data processing: UNIFI 1.9.4 and Empower 3.0 software for spectral deconvolution, peptide identification, and quantitative analysis

Main results and discussion

Peptide mapping achieved 98 percent sequence coverage of the AAV5 VP1 protein and identified low-level post-translational modifications (deamidation, oxidation) above 0.5 percent relative abundance
Intact protein measurements of VP1, VP2, and VP3 from six serotypes matched theoretical masses within one to three daltons, illustrating high mass accuracy of the RPLC-MS method
Empty/full capsid percentages measured by CDMS correlated linearly (R² = 0.9959) with values from AEC and SEC, confirming method agreement across platforms
CDMS analysis of isolated ssDNA from AAV9 revealed monomeric genome mass around 0.9 MDa and a dimeric species at ~1.8 MDa due to base pairing in solution
Chromatographic fractionation separated degraded DNA fragments, intact ssDNA, and residual proteins, supporting detailed characterization of genome integrity
The release of ssDNA from capsids led to partial degradation or pairing with complementary strands, an important consideration for genome quality assessment

Benefits and practical applications

The multi-modality approach enables comprehensive quality control for AAV vectors, addressing regulatory needs for gene therapy products
Small sample requirements (microgram level) and rapid measurement cycles support process development workflows and comparability studies
High resolution and orthogonal validation enhance confidence in critical attributes such as capsid composition, genome loading, and particle integrity
This workflow can be adopted in research, quality assurance/control laboratories, and biomanufacturing to streamline vector characterization

Future trends and opportunities

Integration of automated sample preparation and high-throughput MS platforms will accelerate AAV analytics in industrial settings
Advances in single-particle mass spectrometry may allow direct measurement of individual virus particles for ultra-detailed heterogeneity profiling
Improved informatics and artificial intelligence-driven data analysis could enhance detection of low-abundance variants and novel modifications
Expansion of the method to other viral vectors and gene therapy modalities will broaden its impact on biopharmaceutical development

Conclusion

A comprehensive MS-based analytical strategy was established for peptide mapping, intact capsid protein quantification, ssDNA characterization, and empty/full particle evaluation
High sequence coverage, accurate mass measurement, and consistent empty/full ratios across orthogonal techniques demonstrate method reliability
This robust workflow supports regulatory compliance and accelerates AAV vector development for clinical and commercial applications

References

1. J Mol Biol. 2016 Jan 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 (AAV) proteins. doi:10.1089/hum.2021.046