Methacryloylamidocysteine functionalized poly(2-hydroxyethyl methacrylate) beads and its design as a metal-chelate affinity support for human serum albumin adsorption

Metal chelate affinity chromatography is a widely used technique for separation of proteins. An expensive and time consuming step in this process is coupling of a chelating ligand to the chromatographic support. This chelating ligand coordinates metal ions, which in turn bind proteins. The toxicity of chemicals required for coupling and their slow release during the separation process are of considerable concern. This is an important issue in the context of purification of proteins which are used in pharmaceutical purposes. In this study, a simpler metal-chelate support design was described. It was shown that poly(hydroxyethyl methacrylate-methacryloylamidocysteine) [poly(HEMA-MAC)] beads (formed by chelating MAC with Cu2+ directly) can be used for metal-chelate affinity separation. MAC was synthesized by using methacryloyl chloride and cysteine. Spherical beads with an average size of 140-180 mum were obtained by the radical suspension polymerization of HEMA and MAC conducted in an aqueous dispersion medium. poly(HEMA-MAC) beads have a specific surface area of 18.9 m(2)/g. poly(HEMA-MAC) beads were characterized by swelling studies, FTIR, SEM and elemental analysis. The poly(HEMA-MAC) beads with a swelling ratio of 72%, and containing 3.9 mmol MAC/g were used in the adsorption of human serum albumin (HSA) from aqueous solutions. The adsorption of HSA was 22.8 mg/g. Cu2+ chelation increased the HSA adsorption (46.6 mg/g). The maximum HSA adsorption was observed at pH 6.0. The adsorption phenomena appeared to follow a typical Langmuir isotherm. The maximum capacity (q(m)) of the Cu2+-chelated beads for HSA adsorption (193 mg/g) was greater than that of the poly(HEMA-MAC) beads (37 mg/g). The dissociation constant (k(d)) values was found to be 4.6 x 10(-5) M with poly(HEMA-MAC) beads, and was found to be 0.22 x 10(-5) M with Cu2+-chelated beads. Elution of HSA was obtained using 0.1 M Tris/HCl buffer containing 0.5 M NaSCN. High elution efficiencies (up to 100% of the adsorbed HSA) were observed. It was possible to reuse Cu2+-chelated beads without significant decreases in the adsorption capacities. (C) 2004 Elsevier B.V. All rights reserved.