Separation and analysis of adeno-associated virus vectors using a 3 μm monodisperse strong anion exchange chromatography column

Importance of the Topic

Adeno associated viruses (AAVs) are widely used as gene therapy vectors due to their low immunogenicity and safety profile. Accurate separation and quantitation of empty and full capsids is critical for ensuring product efficacy and safety in biopharmaceutical applications.

Objectives and Study Overview

This study demonstrates the development and optimization of strong anion exchange chromatography methods for separating empty and full AAV capsids. Using a 3 µm monodisperse ProPac 3R SAX column, two strategies are explored: a simple linear salt gradient for AAV1, AAV6, and AAV8, and a variant with an isocratic hold to enhance resolution for AAV6.

Methodology

Samples of AAV1, AAV6, and AAV8 were provided at 2 × 10^13 viral genomes per mL and spiked to a 1:10 empty to full capsid ratio for linear gradient experiments. AAV6 full capsid samples were analyzed directly for isocratic hold studies. Separations were performed at pH 9.0 above the AAV isoelectric point, with a flow rate of 0.2 mL/min, injection volume of 0.2 µL, and column temperature of 20 °C.

Used Instrumentation

• UHPLC system: Thermo Scientific Vanquish Flex
• Detector: Vanquish fluorescence detector (Ex 280 nm, Em 330 nm)
• Column: ProPac 3R SAX, 3 µm, 2 × 50 mm
• Software: Chromeleon 7.2.10 for data acquisition and analysis

Main Results and Discussion

The simple linear salt gradient achieved baseline separation of empty and full capsids for AAV1, AAV6, and AAV8, with an additional impurity peak observed for AAV6 and AAV8. Lot to lot reproducibility was confirmed across three ProPac 3R SAX columns. Incorporating an isocratic hold at 17% mobile phase B improved resolution between empty and full capsids. However, extending the hold time beyond 4 minutes led to broadening and reduced peak intensity of the full capsid due to delayed elution. Orthogonal techniques such as analytical ultracentrifugation or cryo electron microscopy are recommended to validate empty/full ratios and optimize hold times.

Benefits and Practical Applications

• High resolution separation of AAV empty and full capsids suitable for QC and R&D
• Robust performance with minimal method complexity
• Reproducible lot to lot results enable consistent analytical workflows
• Method flexibility allows tuning of gradient and isocratic holds for challenging samples

Future Trends and Potential Applications

Continued development may include calibration with purified capsid standards, integration of multi attribute chromatographic methods, and application to a broader range of AAV serotypes. Combining AEX with orthogonal technologies will enhance characterization depth. Scale down and scale up workflows can support both academic research and commercial manufacturing environments.

Conclusion

The ProPac 3R 3 µm SAX column enables efficient and reproducible separation of AAV empty and full capsids. A linear salt gradient provides baseline resolution for multiple serotypes, and an isocratic hold at optimized salt concentrations further improves separation for AAV6. This approach supports accurate characterization essential for gene therapy product development and quality control.

References

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2. Aebischer MK, Gizardin-Fredon H, Lardeux H, et al. Anion exchange chromatography at the service of gene therapy: Baseline separation of full/empty AAV capsid by screening conditions and step gradient elution mode. Int J Mol Sci. 2022;23(20):12332.
3. Khatwani SL, Pavlova A, Pirot Z, et al. Anion exchange HPLC assay for separation and quantification of empty and full capsid in multiple AAV serotypes. Mol Ther Methods Clin Dev. 2021;21:548–558.
4. Ma K, Bechler S. Salt gradient separation and analysis of AAV samples using a 3 µm monodisperse SAX chromatography column. Thermo Fisher Scientific AN001816-EN. 2023.
5. Mietzsch M, Liu W, Ma K, et al. Production and characterization of an AAV1-VP3 only capsid: An analytical benchmark standard. Mol Ther Methods Clin Dev. Published May 08, 2023.