Journal of Chromatography B, 936 (2013) 1–9 Contents lists available at ScienceDirect Journal of Chromatography B j ourna l h om epage: www.elsevier.com/locate/chromb Adsorption and separation of immunoglobulins by novel affinity core–shell beads decorated with Protein L and l-histidine Gulay Bayramoglu a,b , V. Cengiz Ozalp c , M. Yakup Arica a,∗ a Biochemical Processing and Biomaterial Research Laboratory, Gazi University, 06500 Teknikokullar, Ankara, Turkey b Department of Chemistry, Gazi University, 06500 Teknikokullar, Ankara, Turkey c School of Medicine, Istanbul Kemerburgaz University, 34217 Istanbul, Turkey a r t i c l e i n f o Article history: Received 16 May 2013 Accepted 23 July 2013 Available online 31 July 2013 Keywords: Affinity beads Protein L l-Histidine Adsorption Separation Immunoglobulins a b s t r a c t A novel core shell beaded chromatographic materials was prepared by grafting of glycidyl methacry- late (GMA) on to the surface of poly(hydoxypropyl methacrylate/ethyleneglycol dimethacrylate), p(HPMA/EGDMA) beads via surface-initiated atom transfer radical polymerization (SI-ATRP). For grafting GMA, p(HPMA/EGDMA) beads were first modified with an ATRP initiator. A reaction with 2-bromo-2- methylpropionyl bromide of the hydroxyl groups of the beads led to ATRP initiator-covered surfaces. The grafted p(GMA) fibrous chains on the beads were decorated with two different ligands (i.e., Pro- tein L and l-histidine) for separation of Immunoglobulin’s (Igs) from aqueous solution in batch system. The maximum Igs adsorptions on the p(HPMA/EGDMA)-g-p(GMA)-Protein L and p(HPMA/EGDMA)-g- p(GMA)-l-histidine affinity beads were found to be 81.8 and 112.3 mg/g at pH 7.5 and 5.5, respectively. The purity of Igs from human serum was analyzed by HPLC. The Protein L immobilized affinity beads provided purity about 98%. The novel core shell polymeric beads decorated with Protein L showed a good selectivity for Igs molecules from diluted human serum. Adsorption studies of Igs onto Protein L and l-histidine immobilized affinity beads were also carried out in a continuous system. © 2013 Elsevier B.V. All rights reserved. 1. Introduction The selection of chromatographic materials, are dominant factors affecting the chromatographic performance. Among the chromatographic materials, acrylate based polymers have been received more interests on the chromatographic applications due to their high water content, mechanical and biological properties such as mechanical strength, toughness, elasticity, biocompatibility, and easy chemical modification [1–6]. Additionally, these materials surface can be modified with high-density polymer brushes to enhance adsorptive properties via surface-initiated atom transfer radical polymerization (SI-ATRP) technique [7–10]. Controlled rad- ical polymerization strategies, especially ATRP, seem to be the most promising route for decorating particles with polymer brushes, and the method leads to polymers precisely defined in their con- stitution and molar masses [11–15]. Moreover, the reactions can be carried out in the presence of water and have a large tol- erance toward many functional groups. The grafting of supports with epoxy group carrying monomers such as p(GMA) is attrac- tive, and ligands molecules can be readily attached by the epoxy ring opening reaction. Thus, polymers with epoxy groups offer ∗ Corresponding author. Tel.: +90 506 234 6909; fax: +90 312 2213202. E-mail address: yakuparica@tnn.net (M.Y. Arica). numerous functionalization possibilities in mild reaction condi- tions [16–23]. Immuno-affinity chromatography is based on the ability of an antigen to bind specifically and reversibly to its complemen- tary molecule “antibodies”. Protein G and Protein A are the most commonly used biospecific ligands for isolation of immunoglo- bilins G, (IgG), and both proteins are expressed on the surface of gram-positive bacteria, Staphylococcus aureus and  – hemolytic streptococcus, respectively. Both have a strong affinity for the Fc portion of the IgG molecules. Some strains of the anaerobic bacterial species Peptostreptococcus magnus express an Igs-binding surface molecule called Protein L [24], which is known to bind to Igs cappa light chains of a wide range of animal species [25]. Protein L appears to be a virulence determinant [26], and it is a useful immuno- chemical reagent. Since no part of the heavy chain is involved in the binding interaction, Protein L binds a wider range of antibody classes than Protein A or G [27]. Protein L binds to representatives of all antibody classes, including IgG, IgM, IgA, IgE and IgD. Sin- gle chain variable fragments (ScFv) and Fab fragments also bind to Protein L. In this paper, Protein L and l-histidine affinity ligands were immobilized on the p(GMA) grafted p(HPMA/EGDMA) beads. Both affinity beads were well characterized and tested separately in batch, and continuous systems for adsorption of Igs from solutions. Finally, Protein L and l-histidine ligands immobilized beads were 1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.07.025