title: Immobilized metal-ion affinity chromatography of human antibodies and their proteolytic fragments.
abstract: Immobilized metal-ion affinity chromatography (IMAC) performed with four different transition metal ions: copper(II), nickel(II), zinc(II) and cobalt(II), was used to study the adsorption properties of human polyclonal gamma-globulines (IgG), Cohn II-III fractions, and their pepsin cleaved fragments: Fab'2 and F'c. In each case, digested products showed lower affinity for metal ions, as well by decreasing pH elution as by competition with imidazole. An explanation was proposed by the presence of a histidine (His) cluster in the F'c domain of IgGs, identified by computer calculation (accessible surface area (ASA) determination) as the more probable His 433-x-His 435 sequence presented in the CH3 domain of human IgG heavy chain. As shown by IMAC and electrophoresis, F'c and undigested IgG have higher affinity for transition metal ions than Fab'2 fragments and could be then separated in one step by IMAC. When chelated Zn(II) or Co(II) are used as ligands, the Fab'2 fragment could be easily recovered under mild conditions (pH 7) in the non-retained fraction. This approach could be used as a powerful alternative to conventional protein A/G methods for the commercial preparation of non immunogen active Fab'2 fragments.
authors.affiliation: Laboratoire d'Interactions Moléculaires et de Technologies de Séparation (LIMTechS), Centre de Recherche de Royallieu, BP 20 529, Université de Technologie de Compiègne, Compiègne, France.
authors.forename: Daniela
authors.initials: D
authors.lastname: Todorova-Balvay
authors.forename: Olivier
authors.initials: O
authors.lastname: Pitiot
authors.forename: Mustapha
authors.initials: M
authors.lastname: Bourhim
authors.forename: Thamarapu
authors.initials: T
authors.lastname: Srikrishnan
authors.forename: Mookambeswaran
authors.initials: M
authors.lastname: Vijayalakshmi
mesh_terms: D000906:Antibodies / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006868:Hydrolysis; D007128:Immunoglobulin Fragments / Q000302:isolation & purification*; D008670:Metals / Q000737:chemistry*; D008958:Models, Molecular
chemical_list: D000906:Antibodies; D007128:Immunoglobulin Fragments; D008670:Metals