Harvesting AAV by tangential flow filtration using reverse asymmetric membranes.

Efficient bioreactor clarification for harvesting virus particles is often challenging. Tangential flow filtration is attractive as it can be easily adapted for batch and perfusion operations. Here the feasibility of using reverse asymmetric hollow fiber membranes, where the more open support structure faces the feed stream, has been investigated for harvesting adeno associated virus serotype 2. The open support structure of these membranes stabilizes a secondary membrane consisting of rejected particulate matter. It is essential that the stabilized secondary membrane remains highly permeable. Flux stepping experiments were conducted in total recycle mode in order to determine the critical flux. The critical flux is the maximum stable flux. Higher fluxes lead to a rapid increase in transmembrane pressure under constant flux operation. The critical flux is shown to increase with increasing wall shear rate (feed flow rate). The reduction in turbidity of the permeate relative to the feed decreases with increasing wall shear rate. Harvesting adeno associated virus was conducted at a wall shear rate of 2000 s^-1. The permeate flux was set at 15 Lm^-2 h^-1. The feed was concentrated till the transmembrane pressure reached 3.5 kPa. Diafiltration then commenced using 3 diavolumes. While commencing diafiltration with a smaller feed volume will reduce diluent usage and dilution of the product, it is essential that the transmembrane pressure is not too high to create a compacted low permeability secondary membrane. Here the transmembrane pressure was almost constant at 3.5 kPa during diafiltration. Virus recovery was 94%.