In the application note, the anion-exchange based method was evaluated using the TSKgel Q-STAT column in combination with fluorescence detection and a linear choline-Cl gradient. The sensitivity of the assay was determined by a limit of detection (LOD) of ~3.9 ×10⁸ viral capsids (VC) for both empty and full AAV5. In addition, the method was applied to four AAV serotypes (AAV2, AAV5, AAV6 and AAV8), resulting in full-empty separation of each serotype tested, although the linear gradient did not achieve baseline separation for all serotypes. The new method was further optimized by introducing an isocratic hold step, which led to baseline separation even for samples that were difficult to analyze.

This novel AEX method using a non-toxic choline chloride based mobile phase eluent separates empty and full AAV capsids for several serotypes. It enhances the safety for AAV critical quality attribute analyses while preserving the desired separation performance exhibited by its toxic counterpart, TMAC.