Breakthrough performance of high-capacity membrane adsorbers in protein chromatography

Abstract

The breakthrough of lysozyme (HEWL) and bovine serum albumin (BSA) on high-capacity cation exchange membranes was studied (Sartobind-S; Sartorius AG, Germany). The ligand density of these membranes is increased significantly by anchoring sulfonic acid groups (SO 3H) to cross-linked polymer chains grafted onto the basic polymer resulting in a three-dimensional adsorbing layer. The static and dynamic protein binding behavior was characterized for two types of membranes with a different degree of modification. Static capacities were found to be increased up to 100-fold compared to a conventional modification with a direct coupling of the ligands to the stationary phase. A dimensionless treatment of the breakthrough zone in frontal analysis led to the identification of a rate-limiting diffusive mechanism resulting from slow transport of proteins into the three-dimensional adsorbing layer. A combination of pore diffusion and surface diffusion is well suited to explain the effect of the feed concentration upon the sharpness of the breakthrough curves. These results reveal that the common assumption of an ideal interaction between protein and ligand in membrane adsorption chromatography is not realistic for a theoretical description of these high-capacity membranes. The analysis of breakthrough performance forms the basis for an optimization of the membrane modification leading to a ‘useful’ ligand density with high capacities at enhanced mass transfer. The dimensionless analysis of fixed-bed adsorption is outlined for different rate limitations resulting from various mass transfer mechanisms and binding kinetics. This frame-work enables a comparison between the performance of both, membrane adsorbers and conventional gel beads, and an appropriate choice of operating conditions.