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

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-α (rHuIFN-α) 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 m2/g and 47.3 Å for poly(HEMA) microbeads and 18.7 m2/g and 49.8 Å for poly(HEMA–MAPA) microbeads. Adsorption experiments were performed under different conditions. Maximum rHuIFN-α 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 μm and containing 327 μmol MAPA/g microbeads. Compared with poly(HEMA–MAPA) microbeads, nonspecific rHuIFN-α 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-α structure, fluorescence spectrophotometry was employed. Repeated adsorption–elution processes showed that these microbeads are suitable for repeatable rHuIFN-α adsorption.