title: Steric exclusion chromatography for purification of cell culture-derived influenza A virus using regenerated cellulose membranes and polyethylene glycol.
abstract: Steric exclusion chromatography has been used for the purification of proteins and bacteriophages using monoliths. The operation is carried out by mixing a crude sample containing the target species with a predetermined concentration and molecular weight of polyethylene glycol (PEG) and loading it onto a non-reactive hydrophilic surface. Product capture occurs by the mutual steric exclusion of PEG between the product and the matrix. Selectivity is significantly influenced by target product size. Product elution is achieved by decreasing the PEG concentration. In this study, a 75cm2cellulose membrane adsorber was used for the purification of a clarified and inactivated influenza A virus broth produced in a 5L bioreactor using suspension Madin Darby canine kidney cells. Product recovery was above 95% based on hemagglutination activity and single radial immunodiffusion assays. Maximum depletion of double stranded host cell DNA and total protein was 99.7% and 92.4%, respectively. Purified virus particles showed no aggregation with a monodisperse peak around 84nm. 250mL of the clarified inactivated virus broth was purified within 40min. The surface area productivity based on the recovery of the viral hemagglutinin antigen was 28-50mgm-2h-1depending on the feed and loading conditions.
authors.affiliation: Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany. Electronic address: marichal-gallardo@mpi-magdeburg.mpg.de.
authors.forename: Pavel
authors.initials: P
authors.lastname: Marichal-Gallardo
authors.affiliation: Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany.
authors.forename: Michael M
authors.initials: MM
authors.lastname: Pieler
authors.affiliation: Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany; Institute of Bioprocess Engineering and Pharmaceutical Technology, Technische Hochschule Mittelhessen, Wiesenstrasse 14, 35390 Gießen, Germany.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Wolff
authors.affiliation: Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany; Chair of Bioprocess Engineering, Otto-von-Guericke University Magdeburg, Universitätsplatz 2, 39106 Magdeburg, Germany.
authors.forename: Udo
authors.initials: U
authors.lastname: Reichl
mesh_terms: D000327:Adsorption; D000818:Animals; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000379:methods*; D002482:Cellulose / Q000737:chemistry*; D002850:Chromatography, Gel / Q000379:methods*; D004285:Dogs; D006386:Hemagglutination Tests; D009980:Influenza A virus / Q000302:isolation & purification*; D061985:Madin Darby Canine Kidney Cells; D008567:Membranes, Artificial; D008970:Molecular Weight; D010316:Particle Size; D011092:Polyethylene Glycols / Q000737:chemistry*
chemical_list: D008567:Membranes, Artificial; D011092:Polyethylene Glycols; D002482:Cellulose
keywords: Influenza A virus purification; Membrane chromatography; Polyethylene glycol; Regenerated cellulose; Steric exclusion chromatography