Ultrafiltration behavior of recombinant adeno associated viral vectors used in gene therapy

Abstract

Recombinant Adeno-Associated Viruses (AAVs) are increasingly being used as the choice of vectors for gene therapy applications. While ultrafiltration is the unit operation in place to concentrate the AAV and place vector in the final formulation buffer, not enough data, and fundamental understanding of ultrafiltration of AAV vectors exists to date. This work examines the effect of membrane molecular weight cut-off and operating conditions on the flux and transmission of AAV2 and AAV9 serotypes through composite regenerated cellulose (CRC) ultrafiltration membranes. The transmission of AAV through more permeable ultrafiltration membranes was observed to vary as a function of the operating conditions and membrane properties. The effect of operating conditions, membrane permeability, and scale were examined for a completely retentive 30 kDa ultrafiltration membrane. It was found that a 100 kDa ultrafiltration membrane examined in this work could perform as a completely retentive membrane or, conversely, as a membrane significantly permeable to both AAV serotypes. It was found that operating the 30 kDa membrane closer to the pressure independent regime maximized flux and reduced process variability. This study also demonstrated that a flat sheet cassette may be successfully used as a scale-down model for a holderless spiral wound device, enabling users to choose those devices for small scale process development. This is the first study to provide a quantitative understanding of the ultrafiltration behavior of AAVs for gene therapy to guide process development.