Immobilized metal-ion affinity chromatography of human antibodies and their proteolytic fragments

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 γ-globulines (IgG), Cohn II–III fractions, and their pepsin cleaved fragments: F(ab′)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 F(ab′)2 fragments and could be then separated in one step by IMAC. When chelated Zn(II) or Co(II) are used as ligands, the F(ab′)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 F(ab′)2 fragments.