Comprehensive analysis of adeno-associated virus quality using 3 μm monodisperse Strong Anion Exchange and Size Exclusion chromatography columns

Significance of the Topic

Adeno-associated viruses (AAVs) serve as critical vectors in gene therapy due to their favorable safety profile and long-term expression. Reliable separation and quantification of empty and full capsids, along with aggregate measurement, are essential for ensuring product efficacy and regulatory compliance.

Objectives and Study Overview

This study evaluates novel 3 µm monodisperse Strong Anion Exchange (SAX) and Size Exclusion Chromatography (SEC) columns for robust, high-resolution analysis of AAV fill state, titer, and aggregation. Comparisons with conventional columns assess performance gains in speed, reproducibility, and resolution.

Methodology and Instrumentation

A Thermo Scientific Vanquish Flex UHPLC system with fluorescence detection was used for SAX analysis on a ProPac 3R SAX 3 µm column (2 × 50 mm), employing linear salt gradients and gradients with isocratic holds. SEC-MALS characterization of AAV5 was performed on a SurePac Bio 550 SEC MDi 3 µm column (4.6 × 150 mm) with Wyatt Technology ASTRA software. Data acquisition and analysis utilized Chromeleon CDS and ASTRA.

Used Instrumentation

• Thermo Scientific Vanquish Flex UHPLC system with fluorescence detector
• ProPac 3R SAX 3 µm column (2 × 50 mm)
• SurePac Bio 550 SEC MDi 3 µm column (4.6 × 150 mm)
• Conventional SEC column (5 µm, 4.6 × 300 mm)
• Chromeleon 7.2.10 Chromatography Data System
• Wyatt Technology ASTRA 8.1.2 for SEC-MALS

Main Results and Discussion

The ProPac 3R SAX column achieved baseline separation of empty and full capsids across AAV1, AAV6, and AAV8 using a simple linear gradient. Incorporating an isocratic hold at 17 % mobile phase B further enhanced resolution, delivering asymmetry close to unity for full capsids. SEC-MALS with the SurePac column halved analysis time and sample consumption compared to conventional columns, while doubling plate counts and improving aggregate resolution, particularly for dimer and trimer species. Repeatability tests showed coefficients of variation below 5 % for monomer peaks, with aggregate quantification consistent across runs.

Benefits and Practical Applications

• Faster throughput and reduced solvent and sample use
• High-resolution separation for precise empty/full capsid quantification
• Improved aggregate detection and molecular weight determination
• Robust column-to-column reproducibility supports QC and regulatory workflows

Future Trends and Opportunities

Future developments may include integration of orthogonal techniques such as analytical ultracentrifugation and cryo-EM, optimization of isocratic conditions for diverse serotypes, miniaturized high-throughput formats, real-time monitoring, and application of machine learning for data interpretation in viral vector analytics.

Conclusion

Monodisperse 3 µm SAX and SEC columns offer a reliable, rapid, and high-resolution platform for comprehensive AAV characterization, enhancing QC capabilities while reducing operational demands. SEC-MALS remains the preferred approach for simultaneous titer and aggregate analysis.

References

1. Wang C; Mulagapati SHS; Chen Z; Du J; Zhao X; Xi G; Chen L; Linke T; Gao C; Schmelzer AE; Liu D. Developing an Anion Exchange Chromatography Assay for Determining Empty and Full Capsid Contents in AAV6. Molecular Therapy – Methods & Clinical Development 2019;15:257–263.
2. Aebischer MK; Gizardin-Fredon H; Lardeux H; Kochardt D; Elger C; Haindl M; Ruppert R; Guillarme D; D’Atri V. Anion-Exchange Chromatography at the Service of Gene Therapy: Baseline Separation of Full/Empty Adeno-Associated Virus Capsid by Screening of Conditions and Step Gradient Elution Mode. International Journal of Molecular Sciences 2022;23(20):12332.
3. Khatwani SL; Pavlova A; Pirot J; Zhu X. Anion-exchange HPLC assay for separation and quantification of empty and full capsid in multiple adeno-associated virus serotypes. Molecular Therapy – Methods & Clinical Development 2021;21:548–558.