title: Separation of human IgG fragments using copper, nickel, zinc, and cobalt chelated to CM-Asp-agarose by positive and negative chromatography.
abstract: This study evaluated the feasibility of using immobilized metal-ion affinity chromatography (IMAC) for separation of human Fab fragments using four different transition metal ions copper, nickel, zinc, and cobalt chelated to CM-Asp (carboxymethylaspartate) immobilized on the agarose gel. The Fab and Fc fragments (from human IgG digested with papain) interacted differently with the chelates studied, depending on the adsorption buffer system. The interaction between chelate and Fc fragment is predominantly based on the coordination bonds using adsorption buffer containing NaCl. Negative chromatography was performed on Cu(II)-CM-Asp-agarose obtaining 2.9mg of Fab per mL of adsorbent in nonretained fractions (Fc fragment-free without uncleaved IgG). The adsorption of Fab fragments is governed by electrostatic forces in the absence of NaCl in the adsorption buffer. High selectivity was achieved on Co(II)-CM-Asp-agarose and 5.7mg of Fab per mL of adsorbent was obtained in eluted fractions without Fc fragments, although having uncleaved IgG. The results showed that chromatography on transition metal ions chetated to CM-Asp-agarose is a promising approach to separation of Fab fragments from papain-digested human IgG solution.
authors.affiliation: School of Chemical Engineering, University of Campinas, UNICAMP, 13083-970 Campinas, SP, Brazil.
authors.forename: Cecília Alves
authors.initials: CA
authors.lastname: Mourão
authors.affiliation: School of Chemical Engineering, University of Campinas, UNICAMP, 13083-970 Campinas, SP, Brazil.
authors.forename: Gabriela Pannunzio
authors.initials: GP
authors.lastname: Carmignotto
authors.affiliation: School of Chemical Engineering, University of Campinas, UNICAMP, 13083-970 Campinas, SP, Brazil. Electronic address: sonia@feq.unicamp.br.
authors.forename: Sonia Maria Alves
authors.initials: SMA
authors.lastname: Bueno
mesh_terms: D000327:Adsorption; D002614:Chelating Agents / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000379:methods*; D004587:Electrophoresis, Agar Gel; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification*; D008670:Metals / Q000737:chemistry*
chemical_list: D002614:Chelating Agents; D007074:Immunoglobulin G; D008670:Metals
keywords: Adsorption; CM-Asp (carboxymethylaspartate); Human Fab fragments; IMAC; Negative chromatography