Modular, Scalable AAV Purification Process For Safe Vectors And Recoveries Of Up To 50%

By M. Langhauser, J. Weizenegger, E. Tsiaousi, C. Mantzoros, R. Staffler, J. Trommer, M. Boscher, J. Wagner, J. Babic, B. Larena Carnio, A. Heinlein, C. Zach. S. Ritter, M. Gora, T. Kloetzler, B. Voland, N. Spada, A. Schoberth, A. Youssef, and K. Heller

The development of a scalable AAV purification process with high product yield and vector quality is a critical goal in the gene therapy field. In this study, we present a modular and scalable downstream process (DSP) for AAV9 material produced using our proprietary suspension cell process. While chromatography and filtration steps offer scalable DSP options, challenges arise with the scaling of preparative ultracentrifugation, leading us to focus on optimizing chromatography and filtration. To create a suitable scale-down model for process robustness studies, we adjusted column and filter sizes to match production scale, which may impact yield and quality. We also tested several downstream steps to remove adventitious viruses, ensuring product stability and recovery while maintaining high quality and quantity.

Our 2-split plasmid system produces AAV9 capsids with up to 60% full capsid content (mass photometry) in upstream processing, contributing to high product quality from the outset. A full-empty separation step, such as anion exchange chromatography (AEX), may be unnecessary for certain applications, depending on the intended dose, administration route, and risk/benefit profile. By avoiding full capsid enrichment, we achieve higher vector recovery. Furthermore, a high-salt AEX chromatography in flow-through mode was developed as a polishing step to reduce non-packaged impurities, often down to levels at or below the limit of quantification of our sensitive in-house assays.

In conclusion, we have successfully developed a fully scalable DSP platform that delivers high-quality vectors with greater than 50% recovery and integrated adventitious virus removal, positioning the process to meet future regulatory requirements. We encourage further exploration and adoption of this scalable DSP platform to advance gene therapy applications while ensuring high product quality and regulatory compliance.