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Preparation of pure, high titer, pseudoinfectious Flavivirus particles by hollow fiber tangential flow filtration and anion exchange chromatography
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Preparation of pure, high titer, pseudoinfectious Flavivirus particles by hollow fiber tangential flow filtration and anion exchange chromatography
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Journal Article
Preparation of pure, high titer, pseudoinfectious Flavivirus particles by hollow fiber tangential flow filtration and anion exchange chromatography
2015
Overview
•A purification procedure for a replication-deficient flavivirus is described.•Purification is achieved by tangential flow filtration followed by anion exchange chromatography.•The use of a convective monolithic anion exchanger supports recovery of infectious virus.•Purification results in ∼60% recovery of infectious virus titer.•Vaccine virus with titers as high as 1×109FFU/mL have been prepared.
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.
Publisher
Elsevier Ltd,Elsevier Limited
Subject
/ Anion exchange chromatography
/ Anions
/ Chromatography, Ion Exchange - methods
/ Flavivirus - growth & development
/ Flavivirus - isolation & purification
/ Genes
/ Hollow fiber tangential flow filtration
/ Methods
/ Monolith
/ Proteins
/ Vaccines
/ virion
/ Virion - growth & development
/ Virion - isolation & purification
/ Viruses
