Protein A Immobilized Poly(Methylmethacrylate-Co-Hydroxyethylmethacrylate) Microbeads for IgG Adsorption

Abstract

Poly(methylmethacrylate-co-2-hydroxyethylmethacrylate) microbeads in the size range of 1.5-2.0μm were prepared by a phase inversion polymerization. The hydroxyl groups were activated by periodate oxidation, and the active ligand, i.e., protein A was immobilized via a spacer-arm, i.e., hexamethylene diamine (HDMA) by using a cross-linker, i.e., glutaraldehyde and protein A. The optimal concentration obtained for modifications are as follows: sodium periodate concentration: 0.467 × 10⁻²mmol/mL; HMDA concentration: 3.5 × 10⁻²mmol/mL; and glutaraldehyde concentration: 0.7 × 10⁻⁶mmol/mL. Yields of immobilization of protein A onto the plain and periodate oxidized microbeads were found very close, and were in the range of 0.01-0.02 mg protein A/g microbeads. The optimal conditions for immobilization are as follows: the initial protein A concentration: 0.1 mg/mL; temperature: 25°C; pH: 9.5; and immobilization time:120 min. Incorporation of protein A at these conditions resulted in 0.825 mg protein A/g microbeads. The HIgG adsorption onto these protein A incorporated microbeads was 41 mg HIgG/g microbeads.