Preparation of pure, high titer, pseudoinfectious Flavivirus particles by hollow fiber tangential flow filtration and anion exchange chromatography.

Purification of enveloped viruses such as live flavivirus vaccine candidates poses a challenge as one must retain viral infectivity to preserve immunogenicity. Here we describe a laboratory-scale purification procedure for two replication defective (single-cycle) flavivirus variants for use in a pre-clinical setting. The two step purification scheme based on hollow fiber tangential flow filtration (TFF) followed by anion exchange chromatography using convective interaction media (CIM(®)) monoliths results in a ∼60% recovery of infectious virus titer and can be used to prepare nearly homogenous, highly purified vaccine viruses with titers as high as 1×10(9) focus forming units per mL. Flavivirus virions prepared by this method are 2 and 3 orders of magnitude more pure with respect to dsDNA and BHK host cell proteins, respectively, as compared to the raw feed stream.