Comparison of antibody binding to immobilized group specific affinity ligands in high performance monolith affinity chromatography

A novel biochromatographic principle is introduced taking the quantitative analysis of affinity interactions between antibodies and immobilized group specific ligands (protein A, G, and L) as example. The name high performanc e monolith affinity chromatography (HPMAC) is proposed for this technique. HPMAC uses rigid, macroporous monoliths, so-called convective interaction m edia (CIM(TM))-disks, as stationary phase. An optimized procedure is descri bed for the covalent immobilization of the group specific affinity ligands to such disks. The binding of polyclonal bovine Ige and a recombinant human antibody (type IgG1-kappa) to all affinity disks is discussed. An essentia l feature of HPMAC is its compatibility to unusually high mobile phase flow rates (> 4 ml/min). Chromatographic experiments are thus completed within seconds without significant loss in binding capacity and retentive power. T his makes HPMAC a promising tool for applications in fast process monitorin g or screening. As an example for the former, the direct quantitative isola tion of recombinant antibodies from serum-free culture supernatant is demon strated. (C) 2000 Elsevier Science B.V. All rights reserved.