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Cross-linked macroporous beaded polymer matrices, with pendant hydroxyl groups, were synthesized by the copolymerization of 2-hydroxyethyl methacrylate and ethylene glycol dimethacrylate using suspension polymerization methodology. Novel affinity chromatography matrices were synthesized using various diisocyanates as bifunctional reagents to couple the macroporous polymeric supports, of controlled particle size distribution, with alpha and beta-cyclodextrins. The optimal conditions to couple the hydroxyl groups of cyclodextrin (ligand) and the polymeric supports through urethane linkages were established iteratively using various diisocyanates. Efficacy of ligand binding on the matrix and nonspecific interactions of the synthesized affinity matrices were evaluated to establish the best support and spacer arm. 2,4-Tolylene diisocyanate was established as the best spacer arm on the basis of high ligand binding and low nonspecific interactions. The characteristics of the synthesized affinity matrices toward the adsorption of alpha and beta-cyclodextrin glycosyltransferase (CGTase) were investigated. The binding of beta-CGTase was the highest on affinity matrices with the polymeric methylene diisocyanate spacer. The optimal conditions to regenerate the matrices were also established.

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