Removal of Empty AAV Capsids to Undetectable Levels Using Orthogonal Purification Steps.

Typical AAV Gene Therapy production systems generate cell culture harvests with a high abundance of empty capsids lacking therapeutic DNA compared to full capsids containing the therapeutic DNA. Removal of empty capsids during AAV purification is a central production challenge since they possess similar surface charge properties as full capsids. A robust and scalable purification process capable of completely removing empty capsids from the product is presented in this work. This process employs Anion Exchange Chromatography (AEX) and Zonal Ultracentrifugation (ZUC) as orthogonal purification steps. While AEX differentiates the surface charge between empty and full capsids, ZUC separates via differences in density of the capsids. Heterogeneity of empty capsids was first studied by evaluating empty capsid elution profiles during AEX gradient chromatography and by comparing sedimentation profiles by Analytical Ultracentrifugation (AUC) of empty capsids separated by AEX and ZUC. The results suggest a heterogenous profile of empty capsids in AAV. Separation of AAV by AEX step elution was then studied, demonstrating that there is a distinct sub-population of empty capsids that co-elutes with full capsids, making it difficult to separate them using AEX alone. AAV separation on ZUC results in near-baseline separation of empty capsids from full capsids in addition to removal of aggregates and high molecular weight (HMW) impurities. Finally, three purification trains using the AEX-ZUC combination process were performed to study the robustness of this process in removing empty capsids and other impurities. Results demonstrated that the AEX-ZUC combination process was able to remove empty capsids down to undetectable levels when analyzed by AUC. This process also resulted in reduction of other impurities such as aggregates and HMW species. In addition, Rep protein impurity was also reduced by AEX and then ZUC down to very low levels suggesting that this process can control trace impurities potentially bound to empty capsids. This work shows that the AEX-ZUC combination is a robust and scalable process capable of producing high purity AAV for Gene Therapy applications.