Preparation of protein A-attached poly(HEMA) cryogels for rheumatoid arthritis treatment

Rheumatoid arthritis is an autoimmune disorder in which the immune system of the body attacks healthy cells. RA prevails among 1-3 % of the world population. In recent years, the RA treatment methods have advanced greatly. One of the methods involving the removal of only pathogenic antibodies out of the plasma is immunoadsorption. Affinity chromatography applications utilizing Staphyloccal Protein A as the ligand have been widely used in antibody purification. Protein A is a protein which binds specifically to Fc domain of the antibodies.In the present study, protein A immobilized poly(2-hydroxyethyl methacrylate) cryogel was prepared. Firstly, poly(HEMA) cryogel was prepared by bulk polymerization which involves freezing of the monomer in aqueous solution in a plastic syringe. After thawing, adjacent pores of 10-200 µm in size formed in the matrix of poly(HEMA) cryogel. The cryogel was then activated by cyanogen bromide (CNBr) followed by immobilization of protein A. The protein A immobilized poly(HEMA) cryogel was characterized by surface area measurements, swelling tests, scanning electron microscopy (SEM) and FTIR. The maximum IgG adsorption by the immobilized cryogel was found to be 83.2 mg/g at pH 7.4. Higher adsorption capacity of IgG (as high as 98.7 mg/g) was reached by the cryogel when human plasma was used. The non-specific IgG adsorption onto the poly(HEMA) cryogel was about 0.38 mg/g. The IgG adsorbed was eluted using 0.1 M glycine-HCl buffer (pH 3.5) with a purity of 85 %. Maximum IgG adsorption capacity was determined to be 98.8 mg/g at 37 oC in temperature experiments.It was observed that the rheumatoid factor increased as the immobilized protein A in the cryogel was increased. The computed Rs values for a mixture of Human Serum Albumin (HSA) and Immunoglobulin G (IgG) proteins and human plasma were 2.28 and 2.63, respectively.It was observed that the factors of Clotting Time (CT), Active Partial Tromboplastine Time (APTT), Protrombine Time (PT) and Fibrinogen Time (FT) increased with protein A immobilized poly(HEMA) cryogel as compared to poly(HEMA) cryogel.Protein A immobilized poly(HEMA) cryogel used for repetitive adsorption/desorption of IgG without noticeable decrease in IgG adsorption capacity after ten cycles.