title: Engineering of a two-step purification strategy for a panel of monoclonal immunoglobulin M directed against undifferentiated human embryonic stem cells.
abstract: A two-step purification strategy comprising of polyethylene glycol (PEG) precipitation and anion-exchange chromatography was developed for a panel of monoclonal immunoglobulin M (IgM) (pI 5.5-7.7) produced from hybridoma cultures. PEG precipitation was optimized with regards to concentration, pH and mixing. For anion-exchange chromatography, different resins were screened of which Fractogel EMD, a polymer grafted porous resin had the highest capacity. Despite its significantly slower mass transfer, the binding capacity was still higher compared to a convection driven resin (monolith). This purification strategy was successfully demonstrated for all 9 IgMs in the panel. In small scale most antibodies could be purified to >95% purity with the exception of two which gave a lower final purity (46% and 85%). The yield was dependent on the different antibodies ranging from 28% to 84%. Further improvement of recovery and purity was obtained by the digestion of DNA present in the hybridoma supernatant using an endonuclease, benzonase. So far this strategy has been applied for the purification of up to 2l hybridoma supernatants.
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, No. 06-01 Centros, Singapore. anne.tscheliessnig@gmx.at
authors.forename: Anne
authors.initials: A
authors.lastname: Tscheliessnig
authors.forename: Danny
authors.initials: D
authors.lastname: Ong
authors.forename: Jeremy
authors.initials: J
authors.lastname: Lee
authors.forename: Siqi
authors.initials: S
authors.lastname: Pan
authors.forename: Gernalia
authors.initials: G
authors.lastname: Satianegara
authors.forename: Kornelia
authors.initials: K
authors.lastname: Schriebl
authors.forename: Andre
authors.initials: A
authors.lastname: Choo
authors.forename: Alois
authors.initials: A
authors.lastname: Jungbauer
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification* / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003601:Cytotoxicity Tests, Immunologic; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects; D005590:Fractional Precipitation / Q000379:methods*; D006801:Humans; D006825:Hybridomas / Q000737:chemistry; D007075:Immunoglobulin M / Q000276:immunology / Q000302:isolation & purification* / Q000494:pharmacology; D051379:Mice; D011092:Polyethylene Glycols / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D007075:Immunoglobulin M; D011092:Polyethylene Glycols