title: Preparation of pure, high titer, pseudoinfectious Flavivirus particles by hollow fiber tangential flow filtration and anion exchange chromatography.
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 1×10(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.
authors.affiliation: Sanofi Pasteur, Discovery North America, 38 Sidney St., Cambridge, MA 02139, USA. Electronic address: sophia.mundle@sanofipasteur.com.
authors.forename: Sophia T
authors.initials: ST
authors.lastname: Mundle
authors.affiliation: Sanofi Pasteur, Discovery North America, 38 Sidney St., Cambridge, MA 02139, USA.
authors.forename: Maryann
authors.initials: M
authors.lastname: Giel-Moloney
authors.affiliation: Sanofi Pasteur, Discovery North America, 38 Sidney St., Cambridge, MA 02139, USA.
authors.forename: Harry
authors.initials: H
authors.lastname: Kleanthous
authors.affiliation: Sanofi Pasteur, Discovery North America, 38 Sidney St., Cambridge, MA 02139, USA.
authors.forename: Konstantin V
authors.initials: KV
authors.lastname: Pugachev
authors.affiliation: Sanofi Pasteur, Discovery North America, 38 Sidney St., Cambridge, MA 02139, USA.
authors.forename: Stephen F
authors.initials: SF
authors.lastname: Anderson
mesh_terms: D000838:Anions; D002852:Chromatography, Ion Exchange / Q000379:methods; D005374:Filtration; D005416:Flavivirus / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D019562:Viral Load; D014771:Virion / Q000254:growth & development / Q000302:isolation & purification*
chemical_list: D000838:Anions
keywords: Anion exchange chromatography; Flavivirus purification; Hollow fiber tangential flow filtration; Monolith