Enhanced Separation of AAV Capsids Using Analytical Anion Exchange Monolithic Columns: Emphasis on Organic Modifiers

T. Žvanut, S. Peljhan, S. Lebar, A. Štrancar, A. Gramc Livk

Analytica Chimica Acta, Volume 1376, 2025.

Abstract:

Adeno-associated virus (AAV) vectors remain central to gene therapy; yet, their therapeutic performance and safety critically depend on the precise resolution of empty (E), full (F), and partially filled (P) capsids. Conventional anion exchange chromatography (AEX) often fails to achieve baseline separation between different subpopulations, particularly in complex process samples. This study introduces a significant advancement: high-resolution separation of AAV capsids using an analytical anion exchange monolithic column, CIMac QA HR. By carefully optimizing organic modifiers, Mg²⁺ counterion selection, buffer pH, and modifier concentrations, the method significantly enhances baseline separation between E and F capsids.

The method was validated across multiple serotypes, including AAV2/8, AAV5, AAV8, and AAV9, confirming its broad applicability. Development was conducted on the PATfix biochromatography system, equipped with multiple detectors, including UV-Vis, fluorescence, and multi-angle light scattering (MALS). Notably, the MALS detector enables direct monitoring of capsids in crude samples that are rich in impurities, where alternative techniques often prove insufficient. Further orthogonal validation using techniques like dPCR, ELISA, mass photometry, and CDMS confirmed the method’s accuracy and robustness.

Overall, this approach provides a robust platform for precise AAV capsid characterization, enabling more reliable process development and contributing to the development of safer and more effective gene therapies.

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