Ligand efficiency in axial and radial flow immunoaffinity chromatography of factor IX

Abstract

Radial flow (RF) columns are attractive for process chromatography primarily because larger throughputs and lower pressure drops are achievable in such columns. Large scale immunoaffinity processes using soft resins can benefit most from this configuration. In this study, we compared immobilized ligand efficiency in axial flow (AF) and RF columns using monoclonal antibody against factor IX as the immobilized ligand and a coagulation factor IX complex as the source material. We examined the effects of flow-rate, total protein loading, feed antigen concentration and direction of flow (centrifugal or centripetal for RF, and downward or upward for AF) on immobilized antibody capacity (measured as mg antigen bound per mg antibody). Our results corroborate earlier work, and suggest that none of the factors, in the ranges examined, significantly altered the efficiency of the monoclonal antibody (MAb) in binding factor IX. We also investigated the efficiency of the immobilized antibody upon reuse and found that, over twenty cycles, there was no significant decrease in antibody efficiency. Our results demonstrated that efficiencies obtainable in AF columns can be achieved in RF columns with the same bed thickness, suggesting that radial dispersion, mass transfer and intraparticular diffusion may not have a significant influence on immunoaffinity chromatography efficiency in RF and AF columns.