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

Bioaffinity chromatography has a unique and powerful role that is used as a purification tool in the production of therapeutic plasma protein derivati ves. In this study, a bioaffinity-ligand, i.e. gelatin, was covalently immo bilized with PHEMA microspheres (150-200 tm in diameter). The affinity sorb ent carrying 7.5 mg gelatin g(-1) polymer was then used to separate fibrone ctin from human plasma in a packed-bed column system. Fibronectin separatio n from human plasma on unmodified PHEMA microspheres was 0.45 mg g(-1), whi le much higher adsorption values, up to 21.8 mg g(-1), were obtained with g elatin-immobilized microspheres. The fibronectin adsorption capacity of the microspheres decreased with an increase in the recirculation rate of plasm a. Fibronectin adsorption increased with decreasing temperature, and the ma ximum adsorption achieved at 4 degreesC (26.3 mg fibronecting(-1)). Up to 9 4.7% of the adsorbed fibronectin was desorbed by using 2 M urea in the pres ence of I Ni sodium chloride as elution agent. The adsorption-desorption cy cle was repeated ten times using the same affinity column. There was no rem arkable reduction in the adsorption capacity of the gelatin-immobilized PHE MA microspheres.