A Novel and Simplified Anion Exchange Flow‐Through Polishing Approach for the Separation of Full From Empty Adeno‐Associated Virus Capsids

ABSTRACT Adeno‐associated viruses (AAV) are widely used viral vectors for in vivo gene therapy. The purification of AAV, particularly the separation of genome‐containing from empty AAV capsids, is usually time‐consuming and requires expensive equipment. In this study, we present a novel laboratory scale anion exchange flow‐through polishing method designed to separate full and empty AAV. Once the appropriate conditions are defined, this method eliminates the need for a chromatography system. Determination of optimal polishing conditions using a chromatography system revealed that the divalent salt MgCl 2 resulted in better separation of full and empty AAV than the monovalent salt NaCl. The efficacy of the method was demonstrated for three distinct AAV serotypes (AAV8, AAV5, and AAV2) on two different stationary phases: a membrane adsorber and a monolith, resulting in a 4‐ to 7.5‐fold enrichment of full AAV particles. Moreover, the method was shown to preserve the AAV capsids’ functional potency and structural integrity. Following the successful establishment of the flow‐through polishing approach, it was adapted to a manual syringe‐based system. Manual flow‐through polishing using the monolith or membrane adsorber achieved 3.6‐ and 5.4‐fold enrichment of full AAV, respectively. This study demonstrates the feasibility of separating full and empty AAV without complex linear or step gradient elution and the necessity of specialized equipment. Flow‐through polishing provides a rapid and easy‐to‐perform platform for polishing multiple vector preparations, addressing a critical aspect in the research and development of novel gene therapies.