Separation of Full and Empty AAV Capsids by Anion-Exchange Chromatography Using Choline-Type Salts

Abstract

Development of clinically desirable AAV vectors with optimal genome design requires rapid and accurate analytical methods to assess AAV quality. Anion-exchange (AEX) chromatography provides a powerful analytical method for full/empty AAV capsid ratio determination. However, the current AEX methodology for separation of empty and full AAV capsids largely relies on the use of the highly toxic tetramethylammonium chloride (TMAC). Here, we describe a novel analytical AEX method for separation of empty and full AAV capsids that uses only non-toxic, choline-type compounds that contain structural similarity to the quaternary ammonium ligand present on the surface of AEX resin. Choline-Cl gradient, combined with sensitive fluorescence detection, allowed a safe and effective separation of empty and full AAV capsids with reproducible empty/full ratio determination. The choline-based assay was suitable for commonly used serotypes, AAV2, AAV5, AAV6, and AAV8. The limit of detection was 3.9 × 108 virus particles in the assay. A gradient-hold step-gradient elution with choline-Cl resulted in enhanced baseline separation of empty and full AAV8 capsids. In summary, the use of choline-Cl in the AEX assay is recommended for empty/full capsid ratio determination and other applications in AAV production, and it eliminates the necessity of using toxic TMAC.

Keywords: Adeno-Associated Virus, Anion Exchange Chromatography, Choline, Tetramethylammonium Chloride