Fibronectin purification from human plasma in a packed-bed column system with gelatin immobilized PHEMA microspheres

Abstract

Bioaffinity chromatography has a unique and powerful role that is used as a purification tool in the production of therapeutic plasma protein derivatives. In this study, a bioaffinity-ligand, i.e. gelatin, was covalently immobilized with PHEMA microspheres (150-200 μm in diameter). The affinity sorbent carrying 7.5 mg gelatin g-1 polymer was then used to separate fibronectin from human plasma in a packed-bed column system. Fibronectin separation from human plasma on unmodified PHEMA microspheres was 0.45 mg g-1, while much higher adsorption values, up to 21.8 mg g-1, were obtained with gelatin-immobilized microspheres. The fibronectin adsorption capacity of the microspheres decreased with an increase in the recirculation rate of plasma. Fibronectin adsorption increased with decreasing temperature, and the maximum adsorption achieved at 4°C (26.3 mg fibronectin g-1). Up to 94.7% of the adsorbed fibronectin was desorbed by using 2 M urea in the presence of 1 M sodium chloride as elution agent. The adsorption-desorption cycle was repeated ten times using the same affinity column. There was no remarkable reduction in the adsorption capacity of the gelatin-immobilized PHEMA microspheres.