Protein A immobilized poly(methylmethacrylate-co-hydroxyethylmethacrylate)microbeads for IgG adsorption

Poly(methylmethacrylate-co-2-hydroxyethylmethacrylate) microbeads in the si ze range of 1.5-2.0 mu m 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., hexamethyle ne diamine (HDMA) by using-across-linker i.e., glutaraldehyde and protein A . The optimal concentration obtained for modifications are as follows: sodi um periodate concentration: 0.467 x 10(-2) mmol/mL; HMDA concentration: 3.5 x 10-2 mmol/mL; and glutaraldehyde concentration: 0.7 x 10(-6) mmol/mL. Yi elds of immobilization of protein A onto the plain and periodate oxidized m icrobeads were found very close, and were in the range of 0.01-0.02 mg prot ein A/g microbeads. The optimal conditions for immobilization are as follow s: the initial protein A concentration: 0.1 mg/mL; temperature: 25 degrees C; pH:9.5; and immobilization time:120 min. Incorporation of protein A at t hese conditions resulted in 0.825 mg protein Alg microbeads. The HIgG adsor ption onto these protein A incorporated microbeads was 41 mg HIgG/g microbe ads.