Analytical solutions to accelerate your AAV gene therapy

Importance of the topic

Adeno-associated virus (AAV) vectors are central to modern gene therapy due to their low immunogenicity, safety profile and ability to sustain long-term gene expression. Because AAV products are more complex than traditional biologics—comprising full and empty capsids, host cell proteins, DNA and potential aggregates—precise analytical characterization is critical to ensure both efficacy and safety of gene therapy vectors.

Objectives and overview of the article

This application note outlines a suite of analytical workflows for comprehensive AAV vector characterization. It demonstrates methods to resolve empty versus full capsids, confirm intact capsid mass, analyze capsid proteins and post-translational modifications, and quantify process-related impurities such as host cell proteins and DNA.

Methodology and instrumentation

• Ion-exchange chromatography: Thermo Scientific ProPac SAX-10 columns with fluorescence detection at 280 nm/330 nm to separate empty and full AAV capsids in under 10 minutes.
• Native mass spectrometry: Q Exactive UHMR mass spectrometer operating under charge-reducing conditions to resolve 3 MDa and 4 MDa viral particles across a 27 000–36 000 m/z range.
• LC-HRMS intact protein analysis: Top-down sequencing for accurate molecular weight determination (< 10 ppm) of VP1–VP3 proteins and detection of low-level degradants.
• Peptide mapping by LC-HRMS: Sensitive identification of post-translational modifications (e.g., phosphorylation, oxidation) on capsid proteins.
• Host cell protein analysis: Universal LC-HRMS workflow with SMART Digest and ImmunoAffinity kits achieving LOQ < 0.5 ppm.
• Host cell DNA quantification: GMP-compliant real-time PCR (resDNASEQ) with LOQ of 1.5 pg/mL on QuantStudio 3/5 systems.

Main results and discussion

• Ion-exchange chromatography achieved baseline resolution of empty/full capsids for AAV6 and AAV8, enabling precise quantitation of packaging ratio.
• Native MS spectra displayed well-resolved charge states for both 3 MDa and 4 MDa capsids without bias toward smaller particles, confirming mass heterogeneity.
• Top-down LC-HRMS accurately measured VP1, VP2 and VP3 masses and identified trace degradants at nanogram levels through sequencing.
• Peptide mapping resolved phosphorylation and oxidation variants of VP3 across retention time, supporting detailed PTM profiling.
• Process impurity workflows detected host cell proteins at sub-ppm levels and quantified residual host cell DNA in compliance with regulatory thresholds.

Benefits and practical applications of the method

• Rapid chromatographic separation streamlines QC monitoring of capsid integrity.
• High-resolution native MS enhances understanding of capsid heterogeneity and assembly.
• Top-down and bottom-up MS deliver comprehensive proteoform characterization, including degradants and PTMs.
• Integrated impurity profiling ensures product safety and supports regulatory submissions.
• Fully validated PCR workflow provides reliable quantification of residual DNA contaminants.

Future trends and potential applications

• Development of multi-attribute methods combining chromatography and MS for simultaneous assessment of capsids, proteins and impurities.
• Automation and high-throughput platforms to accelerate process development and manufacturing QC.
• Advancements in MS instrumentation for deeper proteoform coverage and sensitivity toward smaller viral vectors.
• Application of machine learning to interpret complex AAV analytical datasets and detect subtle quality variations.
• Global alignment of regulatory guidelines driving standardization of analytical benchmarks for gene therapy products.

Conclusion

The combination of ion-exchange chromatography, native and high-resolution MS techniques, and real-time PCR offers a robust analytical toolkit for comprehensive characterization of AAV gene therapy vectors. These methods fulfill critical QC requirements, enhance understanding of product heterogeneity and support regulatory compliance for safe, effective gene therapies.

Reference

• Thermo Fisher Scientific. Analytical solutions to accelerate your AAV gene therapy. Application note IN74105-EN, 2022.