title: Simultaneous metal chelate affinity purification and endotoxin clearance of recombinant antibody fragments.
abstract: Endotoxins are frequent contaminants of recombinant proteins produced in Escherichia coli. Due to their adverse effects, endotoxins have to be removed from recombinant proteins prior their use in cell-based assays or parenteral application. Reduction of endotoxin to less than 10 EU mg(-1) is, however, one of the most problematic steps during protein purification from E. coli and often associated with substantial loss of biological materials. The present paper describes the use of a single step procedure enabling metal chelate affinity purification and endotoxin clearance from antibody fragments produced in E. coli using a non-ionic detergent. Endotoxin content was as low as 5 to 9 EU mg(-1) with a recovery of antibody fragments of over 90%. Non-ionic detergent treatment did not compromise integrity and functionality of these multimeric molecules. Furthermore, recombinant antibody fragments did not stimulate endotoxin-sensitive cell lines confirming the low endotoxin content. In conclusion, this one-step protocol is a rapid, cost effective and automation-compatible procedure suitable for recombinant antibody fragments.
authors.affiliation: MorphoSys AG, Lena-Christ-Str. 48, D-82152 Martinsried, Germany.
authors.forename: Timo
authors.initials: T
authors.lastname: Zimmerman
authors.forename: Corinne
authors.initials: C
authors.lastname: Petit Frère
authors.forename: Marion
authors.initials: M
authors.lastname: Satzger
authors.forename: Monika
authors.initials: M
authors.lastname: Raba
authors.forename: Manuela
authors.initials: M
authors.lastname: Weisbach
authors.forename: Katrin
authors.initials: K
authors.lastname: Döhn
authors.forename: Andreas
authors.initials: A
authors.lastname: Popp
authors.forename: Mariel
authors.initials: M
authors.lastname: Donzeau
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry; D001666:Binding Sites, Antibody; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity / Q000379:methods*; D003902:Detergents / Q000737:chemistry; D019281:Dimerization; D042783:Endothelial Cells / Q000502:physiology; D004731:Endotoxins / Q000737:chemistry* / Q000302:isolation & purification; D004926:Escherichia coli; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D018799:Intercellular Adhesion Molecule-1 / Q000276:immunology; D008670:Metals; D008958:Models, Molecular; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D003902:Detergents; D004731:Endotoxins; D007128:Immunoglobulin Fragments; D008670:Metals; D018799:Intercellular Adhesion Molecule-1; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40