Hydrophobic microbeads as an alternative pseudo-affinity adsorbent for recombinant human interferon-alpha via hydrophobic interactions

Abstract

Hydrophobic interaction chromatography (HIC) is increasingly used for protein purification, separation and other biochemical applications. The aim of this study was to prepare hydrophobic microbeads and to investigate their recombinant human interferon-alpha (rHuIFN-alpha) adsorption capability. For this purpose, we had synthesized functional monomer, N-methacryloyl-L-phenylalanine (MAPA), to provide a hydrophobic functionality to the adsorbent. The poly(2-hydroxyethyl methacrylate-N-methacryloyl-L-phenylalanine) [poly(HEMA-MAPA)] microbeads were prepared by suspension copolymerization. microbeads were characterized using FTIR, swelling behavior, and SEM micrographs. Equilibrium swelling ratio of poly(HEMA-MAPA) and poly(HEMA) microbeads were 53.3% and 69.3%, respectively. The specific surface area and average pore diameters determined by BET apparatus were 17.4 m(2)/g and 47.3 angstrom for poly(HEMA) microbeads and 18.7 m(2)/g and 49.8 angstrom for poly(HEMA-MAPA) microbeads. Adsorption experiments were performed under different conditions. Maximum rHuIFN-alpha adsorption capacity was found to be 137.6 +/- 6.7 mg/g by using poly(HEMA-MAPA) microbeads with a size range of 150-250 mu m and containing 327 mu mol MAPA/g microbeads. Compared with poly(HEMA-MAPA) microbeads, nonspecific rHuIFN-alpha adsorption onto plain poly(HEMA) microbeads was very low, about 4.2 +/- 2.3 mg/g. To determine the effects of adsorption conditions on possible conformational changes of rHuIFN-alpha structure, fluorescence spectrophotometry was employed. Repeated adsorption-elution processes showed that these microbeads are suitable for repeatable rHuIFN-alpha adsorption