Monosize microbeads for pseudo-affinity adsorption of human insulin.

Affinity adsorption technique is increasingly used for protein purification, separation and other biochemical applications. Therapeutic molecules such as antibodies, cytokines, therapeutic DNA and plasma proteins must be purified before characterization and utilization. The aim of this study was to prepare micronsized spherical polymeric beads and to investigate the extent of their human insulin adsorption capability. Monosize poly(ethylene glycol dimethacrylate-N-methacryloyl-(L)-histidine) [poly(EDMA-MAH)] beads were prepared by modified suspension copolymerization. Functional monomer (MAH) was synthesized using methacryloyl chloride and L-histidine. The beads were characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, swelling test and elemental analysis. MAH incorporation into monosize polymeric beads, having an average size around 2-3 μm, was estimated as 55.3 μmol MAH/g bead. Equilibrium swelling ratios of poly(EDMA-MAH) and poly(EDMA) beads were 65% and 55%, respectively. Adsorption experiments were performed under different conditions (i.e., pH, temperature, protein concentration and ionic strength). It was found that adsorption characteristics are strongly depend on these conditions. Maximum insulin adsorption capacity was achieved as 24.7 mg insulin/g poly(EDMA-MAH) beads. Results were well fitted to the Langmuir isotherm model. Compared with poly(EDMA-MAH), nonspecific insulin adsorption onto poly(EDMA) beads was very low (0.61 mg insulin/g bead) and can be negligible. It was observed that insulin could be repeatedly adsorbed and desorbed (at least 10 times) without significant loss in adsorption capacity.