title: Application of phase partitioning and thiophilic adsorption chromatography to the purification of monoclonal antibodies from cell culture fluid.
abstract: A two-step method for the isolation of an IgG1 monoclonal antibody against horseradish peroxidase from hybridoma cell culture supernatant is described. Purification was achieved using an aqueous two-phase extraction procedure in conjunction with thiophilic adsorption chromatography. In an aqueous two-phase system composed of 5% PEG 1540 and 22% phosphate the monoclonal antibody preferentially associates with the PEG-rich top phase whereas proteins such as albumin and transferrin partition into the salt-rich bottom phase. Final purification of the monoclonal antibody was achieved by subjecting the PEG-rich top phase to thiophilic adsorption chromatography. The monoclonal antibody purified to homogeneity retained its specificity for horseradish peroxidase as revealed by polyacrylamide gel electrophoresis and an enzyme-linked immunosorbent assay. The potential of this purification protocol for large scale applications is discussed.
authors.affiliation: Institute of Biochemistry, University of Leipzig, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Sulk
authors.forename: G
authors.initials: G
authors.lastname: Birkenmeier
authors.forename: G
authors.initials: G
authors.lastname: Kopperschläger
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D002478:Cells, Cultured; D002845:Chromatography / Q000379:methods*; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D006735:Horseradish Peroxidase / Q000276:immunology; D006825:Hybridomas / Q000276:immunology; D007074:Immunoglobulin G / Q000302:isolation & purification*; D051379:Mice; D010710:Phosphates; D011092:Polyethylene Glycols
chemical_list: D000911:Antibodies, Monoclonal; D003470:Culture Media; D007074:Immunoglobulin G; D010710:Phosphates; D011092:Polyethylene Glycols; D006735:Horseradish Peroxidase