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

Anion exchange (AEX) chromatography is a widely used analytical technique for separating empty (E) and full (F) adeno-associated virus (AAV) capsids. However, it sometimes fails to achieve baseline separation between E and F capsids in purified samples, and this challenge is even more pronounced in complex process samples. Moreover, AEX is typically serotype dependent, indicating that a specific method must be developed for each serotype. We propose several strategies that significantly enhance the separation of E and F, and in some instances, facilitate baseline separation while providing insights into additional AAV capsid subpopulations, including partially filled capsids, overfilled capsids, and aggregates.

By using an AEX analytical Convective Interaction Media (CIM) monolithic column, we demonstrate how various organic solvents, their concentrations, the choice of Mg2+ counterions, and the pH of buffers affect the separation of E and F AAV capsids. These findings align with other orthogonal techniques and are incorporated into an enhanced chromatographic AEX method.

The added value of this method lies in its rapid and enhanced characterization of a broad range of samples from various AAV serotypes and purities. It ultimately promotes the development of safer and more effective gene therapies.