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Empty and Full Separation of Adeno-Associated Virus Vectors by Anion Exchange Membrane Chromatography

A Hejmowski(1) K Boenning(1) A Kavara(1) J Huato(1) M Schofield()

1:Pall Life Sciences

Pall Corporation, 20 Walkup Drive, Westborough, MA 01581, USA

The approval of adeno-associated virus (AAV) gene therapies has led to increased research into AAV production and the burgeoning of promising clinical trials. However, significant challenges persist in AAV purification as AAV harvests typically contain a majority population of empty capsids that generate an immune response without delivering the therapeutic payload. In addition, subtle differences between therapies restrict platformability.

Here we assess the performance of anion exchange (AEX) membrane chromatography as the polishing stage of an AAV platform process following affinity purification. Utilizing a novel 1 mS/cm step gradient approach, we are able to demonstrate separation of empty and full AAV capsids of serotypes 5, 8, and 9 with the Mustang® Q XT membrane. This process maximizes the high flow rate benefits of membrane chromatography relative to traditional column chromatography, while providing improved separation. Distinct populations in the UV 260/280 chromatogram, analytical trends with PCR and ELISA tests, and capsid standards prepared by ultracentrifugation reaffirm separation. This technique is scalable between the 0.86 mL Mustang Q XT Acrodisc® unit and 5 mL Mustang Q XT capsule, and effectively clears residual host cell protein and DNA contaminants.

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