PREPARATIVE AFFINITY-CHROMATOGRAPHY OF PROTEINS - INFLUENCE OF THE PHYSICAL-PROPERTIES OF THE BASE MATRIX

Several silica-based high-performance affinity media differing in pore size and surface area were synthesized for the immobilization of proteins containing primary amino groups. Surface characterization of these silica-based media was carried out by mercury intrusion porosimetry and the pore surface area and pore diameters were compared. The intrinsic binding capacities for proteins of different molecular weights were determined by static binding studies. Concanavalin A was covalently immobilized on these media and its dynamic binding capacity was determined in the affinity mode by frontal uptake studies. The studies show that binding capacity increases with increasing pore size, then decreases as the pore size becomes sufficiently large to significantly decrease the surface are. Thus, an ideal affinity chromatographic support would have sufficiently large pores accessible for proteins to penetrate, yet would preserve the maximum surface area for binding. These studies suggest that a final bonded phase pore diameter of at least 200 Å is desirable for the preparative affinity chromatography of proteins of molecular weight in excess of 150 000 daltons. © 1990.