Novel Anion Exchange Membrane Chromatography Method for the Separation of Empty and Full Adeno‐Associated Virus

A challenge in the production of recombinant Adeno-Associated Virus (AAV) for gene therapies is the presence of capsids that lack the required gene of interest. The impact of these empty vectors in therapies is not fully understood, however the ability to control the ratio of empty to full particles, which contain the genetic payload, is a necessary step in the purification of these viruses. In this study we demonstrate a novel anion exchange chromatography elution method for enrichment of full AAV particles. A step gradient with small conductivity increases of around 1 mS/cm provides more efficient separation of empty and full AAV serotype 5 across membrane media as compared to conventional linear gradient method. We further explored the use of this approach in optimizing a simpler method for manufacturing processes and scalability to a larger chromatographic volume. With this approach we achieved greater than 4-fold enrichment of full capsids, to give a total of approximately 50–60% full capsids, using a 25 mM Bis-Tris Propane pH 9.0 buffer system with NaCl as the eluting salt. Our results suggest this elution method can be implemented into a scalable process and can provide insight into development of elution methods for other AAV serotypes.

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