Characterizing AAVs

Importance of the Topic

AAV-based gene therapies are transforming treatment strategies for inherited and acquired diseases. Due to their complex structure—a ~5 MDa non-enveloped capsid encapsulating a 4.8 kb single-stranded genome—AAV vectors demand rigorous analytical characterization to guarantee safety, potency, and batch-to-batch consistency.

Objectives and Overview

This guide summarizes chromatographic strategies for measuring critical quality attributes of AAV vectors. It outlines methods to assess capsid protein composition, genome encapsidation, aggregate content, and overall vector potency relevant for development, IND/ BLA filings, and lot release.

Methodology and Instrumentation

Comprehensive AAV analysis employs multiple chromatographic modes coupled with advanced detection:

• Size Exclusion Chromatography (SEC) with multi-angle light scattering (MALS) for size distribution, absolute molar mass, aggregate quantitation, and particle titer.
• Anion Exchange Chromatography (AEX) to distinguish empty and full capsids via charge differences of encapsidated DNA.
• Reversed-Phase Liquid Chromatography (RPLC) on BEH C4 columns with DFA/TFA ion pairing to resolve the VP1:VP2:VP3 ratio and detect post-translational modifications.
• Hydrophilic Interaction Chromatography (HILIC) using BEH Amide or MaxPeak HPS columns for enhanced separation of phosphorylated and other polar VP isoforms.
• Peptide mapping workflows combining high-purity RapiZyme Trypsin digestions and BEH/CSH particle columns to confirm sequence integrity and identify deamidation or truncations.

Key consumables include XBridge Premier GTx SEC 450 Å, Protein-Pak Hi Res Q, ACQUITY Premier Protein BEH C4 300 Å, Peptide CSH C18 130 Å, and high-performance hardware (MaxPeak surface technology).

Key Results and Discussion

SEC-MALS methods achieved a twofold acceleration and 50 % higher resolution for aggregate detection while reducing sample consumption threefold.
AEX assays provided clear empty/full capsid separation, enabling precise quantification of encapsidation efficiency.
RPLC and HILIC separations reliably measured the canonical 1:1:10 VP ratio and resolved phosphorylated forms, correlating capsid composition with transduction potency.
Peptide mapping validated capsid protein sequences, identified critical PTMs, and supported stability studies.

Benefits and Practical Applications

• Regulatory-compliant assessment of AAV identity, purity, encapsidation, and potency.
• High-throughput QC workflows reducing analysis time and reagent use.
• Multi-attribute methods enabling simultaneous monitoring of several CQAs.
• Flexible platforms applicable to diverse serotypes and engineered AAV variants.

Future Trends and Possibilities

Ongoing improvements in column chemistries, detector sensitivity, and automation will further streamline AAV analytics.
Integration of orthogonal techniques such as capillary electrophoresis, mass photometry, and native MS will enrich CQA profiling.
Machine learning tools may accelerate method development, data interpretation, and predictive quality control for viral vectors.

Conclusion

Chromatography combined with light scattering and mass spectrometry offers a robust, multi-attribute toolkit for AAV characterization. Optimized columns, reagents, and detection workflows support critical assessments of vector quality, safety, and efficacy across development and manufacturing phases.

References

1. Human Gene Therapy 2021, 32(12), 1501–1511
2. Journal of Pharmaceutical and Biomedical Analysis 2020, 189, 113481
3. Waters Application Note 720007969
4. Waters Application Note 720006825