Porous Magnetic Chelator Support for Albumin Adsorption by Immobilized Metal Affinity Separation Mehmet Odabas  ı, Lokman Uzun, Adil Denizli Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara, Turkey Received 5 March 2003; accepted 16 April 2004 DOI 10.1002/app.20826 Published online in Wiley InterScience (www.interscience.wiley.com). ABSTRACT: Magnetic poly(2-hydroxyethylmethacrylate) (mPHEMA) beads are modified by iminodiacetic acid (IDA) to implify the reactive groups and subsequent binding of Cu 2+ ions to form metal chelate. mPHEMA beads, in the size range of 80 –120 m, were produced by a modified suspen- sion polymerization technique. mPHEMA beads were char- acterized by swelling tests, electron spin resonance (ESR), FTIR, and scanning electron microscopy (SEM). Important results obtained in this study are as follows. The swelling ratio of mPHEMA beads was 34%. The presence of magne- tite particles in the polymeric structure was confirmed by ESR. FTIR data confirmed that the magnetic polymer beads were modified with functional groups IDA. The mPHEMA beads have a spherical shape and porous structure. The effect of pH and concentration of human serum albumin (HSA), on the adsorption of HSA to the metal-chelated magnetic beads, were examined in a batch reactor. Most importantly, the magnetic beads had little nonspecific ad- sorption for HSA (0.5 mg/g) before introducing IDA groups. Cu 2+ chelation increased the HSA adsorption up to 28.4 mg/g. Adsorption behavior can be described at least ap- proximately with the Langmuir equation. Regeneration of the metal-chelated magnetic beads was easily performed with 1.0M NaSCN, pH 8.0, followed by washing with dis- tilled water and reloading with Cu 2+ . © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 93: 2501–2510, 2004 Key words: magnetic polymers; separation techniques; chromatography; proteins INTRODUCTION Human serum albumin (HSA) is the most abundant protein in blood plasma. 1 Research on albumin sepa- ration has attracted considerable attention for its great potential in blood protein manufacture. HSA is iso- lated from human plasma by Cohn’s method. 2 Cohn’s method concerns precipitation of proteins by using ethanol with varying pHs, ionic strengths, and tem- peratures. This technique, however, which is the old- est method of industrial fractionation of proteins, is not highly specific and can give partially denatured proteins. 3 Recently, immobilized metal ion affinity chromatography has become a widespread analytical and preparative separation method for therapeutic proteins, peptides, nucleic acids, hormones, and en- zymes. 4 –14 Many transition metals can form stable complexes with electron-rich compounds (aromatic molecules and olefins) and may coordinate molecules containing O, N, and S by ion dipole interactions. Metal ion ligands are first-row transition metal ions (Zn 2+ , Ni 2+ , Cu 2+ , and Fe 3+ ) incorporated by imino- diacetic acid, nitrilotriacetic acid, amino salicylic acid, and carboxymethylated amino acids. 15–19 The number of histidine residues in the protein is of primary im- portance in the overall affinity for attached metal ions. In addition, factors such as the accessibility, microen- vironment of the binding residue (i.e., the imidazole group of histidine, the thiol group of cysteine, and the indoyl group of tryptophan), cooperation between neighboring amino acid side chains, and local confor- mations play important roles in biomolecule binding. Aromatic amino acids and the amino-terminus of the peptides also have some contributions. 20 The low cost of metals and the ease of regeneration of adsorbents are attractive features of metal-chelate affinity separa- tion. The development of the magnetic adsorbents prom- ises to overcome many of the problems associated with chromatographic separations in packed bed and conventional fluidized bed systems. 21 Magnetic adsor- bents combine some of the best characteristics of flu- idized beds (low pressure drop) and fixed beds (high mass-transfer rates and good fluid–solid contact). 22 Recently, there has been increased interest in the use of magnetic affinity adsorbents in biomolecule cou- pling and protein purification. 23–26 Magnetic adsor- bents can be produced by using both inorganic mate- rials or a number of synthetic and natural polymers. High mechanical resistance, insolubility, and excellent shelf life make inorganic materials ideal as adsorbents. The main disadvantage of inorganic supports is their Correspondence to: A. Denizli (denizli@hacettepe.edu.tr). Journal of Applied Polymer Science, Vol. 93, 2501–2510 (2004) © 2004 Wiley Periodicals, Inc.