Fast Affinity Chromatography Using Small Particle Silica-Based Packing Materials

Abstract

Affinity chromatography is one of the most powerful techniques for the purification of biologically active proteins available (for review see [1]). The ability of this method to purify proteins is based on highly specific, selective or characteristic interactions with immobilized ligands. Several advantages over traditional soft gel affinity supports have been observed with the use of small particle silica based materials for high performance affinity chromatography. These include greatly improved mass transfer properties which allow separations that are not always practical in the low performance mode, greatly reduced equilibration and isolation times, high available ligand densities, small elution volumes, excellent recovery of very small quantities of protein and high dynamic capacities. The criteria for developing a general, derivatizable, high performance support for high performance affinity chromatography are discussed. The step-by-step examination of these criteria and experimental evidence for determining parameters such as ligand density, non-specific adsorption and column life time for such a system are described. Chromatographic results are shown for preparative separations of (i) receptor proteins, (ii) antibodies and (iii) active enzymes.