Large Scale Purification of AAV with Continuous Flow Ultracentrifugation

Since its first approval in Europe as gene therapy drug in human use in 2012, adeno-associated viral (AAV) vectors have gained more and more attentions in the field for gene therapy research. So far AAV vectors have usually been purified through either density gradient ultracentrifugation in small volume centrifuge tubes or column chromatography. Though these purification methods have their unique benefits, there is still a need for technology that can process large volume of lysate with high AAV recovery rate. We reasoned that continuous flow ultracentrifugation could meet these requirements. We tested the Alfa Wasserman's AW Promatix 1000™, a research scale continuous flow ultracentrifuge, as a prove of concept for AAV vector purification. In the initial experiments, we tested cesium chloride (CsCl) solution as density gradient media for AAV vector purification but found out that CsCl solution was not stable enough to form a linear gradient even when sucrose was added to increase its viscosity for AAV purification. We then tested iodixanol solution as density gradient media and got satisfactory purification of AAV vectors. Our results indicate that we can obtain near-purified AAV vectors in a single-step of centrifugation with AAV recovery rate exceeding 50%. Further experiments indicate that minor impurities associated with the purified AAV vectors could be removed by adding salts to the iodixanol solution such as CsCl to increase the ionic strength of the density gradient. The data presented here indicate that continuous flow ultracentrifugation can be used for large scale purification of AAV vectors and it should provide an additional tool to facilitate the translation from research to the clinic.