Recombinant adeno-associated viral (rAAV) vectors have emerged as one of the most popular delivery vehicles for gene therapy applications.  During the cell culture process to manufacture, AAV vectors, undesired empty particles are formed.  These particles are capsid shells that are similar to the desired product, but lack the therapeutic gene inside them.  The presence of empty particles at high levels could reduce efficient gene transduction in the targeted cells and are believed to cause immunological responses.  To ensure high therapeutic potency of AAV vector products and enhance their safety profile, it is critical that the manufacturing process can consistently produce viral vector products that contain low levels of empty particles.<br/> <br/>Gradient ultracentrifugation using cesium chloride or iodixanol has proven to be effective in removing empty particles, but the process scale-up for large-scale AAV manufacturing remains a significant challenge.  In this work, we present a scalable anion exchange chromatography method to separate full and empty particles.  The presence of DNA inside the full AAV particles reduces the isoelectric point of the capsid by approximately 0.4 units, and that minor surface charge difference was exploited to achieve the desired separation.  Very shallow linear gradient elutions using chlorine salts was applied to an anion exchange monolith column to achieve the high degree of resolution required for separation of these similarly charged particles.  There are several challenges in implementing a shallow gradient on a monolith column at large-scale manufacturing, including consistent generation of gradients using disposable flow-path chromatography skids, effective fractionation and pooling, and challenging column scale-up.  To overcome these limitations, a novel column process with isocratic wash and elution was developed which enabled separation of empty particles with a higher degree of resolution while maintaining high product recovery.

Development of a scalable chromatography process to separate empty and full adeno-associated vector (AAV) particles

255th National Meeting (2018)

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