title: A membrane-based purification process for cell culture-derived influenza A virus.
abstract: A simple membrane-based purification process for cell culture-derived influenza virus was established that relies on only two chromatographic unit operations to achieve the contamination limits required according to regulatory authorities. After clarification and concentration, a pseudo-affinity membrane adsorber (sulfated cellulose, SCMA) was applied for virus capture. The subsequent polishing step consisted of a salt-tolerant anion exchange membrane adsorber (STMA) to bind residual DNA. For the presented process neither a buffer exchange step nor a nuclease step for further DNA digestion were required. As a starting point, a two-salt strategy (including a polyvalent ion) was employed to screen STMA conditions in a 96-well plate format. After optimization on chromatographic laboratory scale, the virus recovery was up to 97% with a residual DNA level below 0.82%. In addition, the STMA was characterized regarding its dynamic binding capacity and the impact of flow rate on yields and contamination levels. Overall, the total virus yield for influenza virus A/PR/8/34 (H1/N1) of this two-step membrane process was 75%, while the protein and the DNA contamination level could be reduced to 24% and at least 0.5%, respectively. With 19.8μg protein and 1.2ng DNA per monovalent dose, this purity level complies with the limits of the European Pharmacopeia for cell culture-derived vaccines for human use. Overall, the presented downstream process might serve as a generic and economic platform technology for production of cell culture-derived viruses and viral vectors.
authors.affiliation: Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, 39106 Magdeburg, Germany. Electronic address: weigel@mpi-magdeburg.mpg.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Weigel
authors.affiliation: Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, 39106 Magdeburg, Germany; Faculty of Pharmaceutical Biotechnology, Biberach University of Applied Sciences, 88400 Biberach, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Solomaier
authors.affiliation: Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, 39106 Magdeburg, Germany; Department of Biotechnology, Bielefeld University of Applied Sciences, 33615 Bielefeld, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Wehmeyer
authors.affiliation: Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, 39106 Magdeburg, Germany; Biotechnology, Brandenburg University of Technology Cottbus-Senftenberg, 01968 Senftenberg, Germany.
authors.forename: Alessa
authors.initials: A
authors.lastname: Peuker
authors.affiliation: Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, 39106 Magdeburg, Germany; Chair of Bioprocess Engineering, Otto-von-Guericke University, 39106 Magdeburg, Germany.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Wolff
authors.affiliation: Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, 39106 Magdeburg, Germany; Chair of Bioprocess Engineering, Otto-von-Guericke University, 39106 Magdeburg, Germany.
authors.forename: Udo
authors.initials: U
authors.lastname: Reichl
mesh_terms: D000327:Adsorption; D000818:Animals; D000838:Anions / Q000737:chemistry; D001709:Biotechnology / Q000379:methods; D018929:Cell Culture Techniques / Q000379:methods*; D002482:Cellulose / Q000031:analogs & derivatives; D002853:Chromatography, Liquid / Q000191:economics / Q000379:methods*; D059018:DNA Contamination; D004279:DNA, Viral / Q000302:isolation & purification; D004285:Dogs; D006801:Humans; D009980:Influenza A virus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D007252:Influenza Vaccines / Q000737:chemistry / Q000494:pharmacology; D061985:Madin Darby Canine Kidney Cells; D008566:Membranes / Q000737:chemistry / Q000821:virology; D014764:Viral Proteins / Q000302:isolation & purification; D014773:Virology / Q000379:methods*
chemical_list: D000838:Anions; D004279:DNA, Viral; D007252:Influenza Vaccines; D014764:Viral Proteins; D002482:Cellulose; C020254:cellulose sulfate
keywords: Downstream process; Influenza virus vaccine purification process; Primary amine anion exchange membrane; Pseudo-affinity membrane chromatography; Salt-tolerant membrane chromatography; Sulfated cellulose membrane adsorber