Preparation of pure,high titer,pseudoinfectious Flavivirus particles by hollow fiber tangential flow filtration and anion exchange chromatography |
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Institution: | 1. Department of Chemical Engineering, Michigan Technological University, USA;2. Wayne County Community College District, USA |
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Abstract: | 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 × 109 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. |
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Keywords: | Hollow fiber tangential flow filtration Anion exchange chromatography Monolith |
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