Developing A Scalable Full Empty Separation Step For An AAV Capsid By Anion Exchange Chromatography In Step Gradient Elution Mode

By R. Staffler, E. Tsiaousi, J. Weizenegger, J. Babic, B. Alkharrat, C. Weiss, M. Boscher, J. Wagner, S. Petrik, S. Ritter, C. Zach, M. Lott, T. Klotzler, A. Heinlein, M. Gora, A. Youssef, A. Schobert, and M. Langhauser

Packaging heterogeneity in recombinant adeno-associated virus (rAAV) manufacturing can result in product-related impurities, such as ‘empty’ capsids, capsids with partial vector genomes, and those containing various DNA impurities. Reducing or eliminating ‘empty’ capsids in downstream processing, particularly at a scalable level, remains a technical challenge. In this study, we present a fully scalable method that efficiently enriches the percentage of full capsids through anion exchange chromatography.

We developed an anion exchange (AEX) chromatography step using isocratic elution, which was optimized through design of experiment (DoE) studies. After screening different resins, we selected a strong anion exchanger for further optimization in a step gradient mode. The data from DoE experiments were analyzed using UV and Stunner (Unchained Labs) chromatography, with results validated by qPCR for recovery data and mass photometry for enrichment and full capsid percentages.

Our studies revealed that the capsid load and salt concentration during the empty particle elution phase are critical parameters for achieving a high percentage of full particles with a satisfactory yield. The scalability of the AEX step was confirmed by processing material from a 50-liter scale run, applying the optimized conditions. We achieved up to 3-fold enrichment of full capsids, with about 75% full capsids as determined by mass photometry, and a vector genome recovery of approximately 70%.

This AEX chromatography process can be incorporated into downstream processing as needed, depending on product specifications. It is fully scalable and adaptable to different expression cassettes or capsids, providing a versatile tool for rAAV production.