Experimental evaluation and theoretical analysis of convective affinity adsorbers for chromatographic applications

Affinity chromatography represents one of the most important and widely used unit operations in the biotechnology industry. However, traditional packed bead columns suffer from several limitations such as high pressure drop, slow mass transfer through the diffusive pores and strong dependence of the binding capacity on flow rate. One possible alternative to overcome these drawbacks is represented by convective media columns packed with affinity membrane or monoliths. This work presents the purification of immunoglobulin G in columns packed with convective media, and discusses the enhanced performance versus packed bead columns. The stationary phases studied derive from the functionalization of membranes and monoliths with natural and synthetic affinity ligands that show high specificity towards IgG. The affinity materials are completely characterized through adsorption, washing and elution cycles. The separation performance of the affinity supports has been determined by feeding both pure IgG solutions and a cell culture supernatant. Relevant process parameters, such as maximum adsorption capacity, affinity equilibrium constant and selectivity, are evaluated and carefully compared among the different affinity supports. The scale-up of the improved affinity materials for industrial applications is also addressed. The experimental data collected have been used for the validation of a proposed simulation model. Such chromatographic model is based on species mass balance equation over the convective medium, coupled with a suitable kinetic equation which represents the interaction between the IgG target molecule and the ligand immobilized on the porous support. Model simulations are in good agreement with all of the experimental affinity cycles, demonstrating the accuracy of the model to describe the transport phenomena in the column and the adsorption binding mechanism.