Experimental and mass transfer modelling in agarose beads

The typical drawbacks of chromatographic separations are associated to the inability of affinity columns to handle high volumetric flow rates at reasonable ligand utilisation efficiencies. Membrane-based separation processes can be used in order to overcome these limitations. The ligand immobilization can be realized by deposition of a high capacitive gel on the convective pore walls of the membrane. To understand the optimal coating layer design, the mass transfer can be investigated in conventional resins. This work includes characterization of agarose-based stationary phases and mass transfer description for immunoglobulin G and a fragment from its digestion. The enzymatic digestion of IgG was performed using papain and fragments of known molecular weight were isolated. The breakthrough curves obtained were simulated in MATLAB using the general rate model and Langmuir kinetics. The parameters needed were derived from empirical correlations and experimentally: particle diameter and size distribution were determined at the microscope; extra-particle porosity of the packed bed was determined through Blake-Kozeny equation; intra-particle porosity was determined through iSEC. The modelling allowed the determination of binding capacity and pore diffusivity of the resins. The latter decreased gradually with increasing agarose concentration as regards the 2%w, 4%w and 6%w beads. Binding capacity is proportional to pore accessibility, which decreased with increasing agarose concentration, and to ligand density. An abrupt reduction of both parameters was detected in the 8%w beads and was related to the low accessibility of the target molecules into the beads diffusive pores, which were the smallest among all the resins tested. These findings can be used to optimize the pore size of chromatographic stationary phases for antibodies purification, with particular focus on novel membranes adsorbers, which allow lower pressure drop, higher flow rates and higher productivity.