AFFINITY CROSS-FLOW FILTRATION - PURIFICATION OF IGG WITH A NOVEL PROTEIN-A AFFINITY MATRIX PREPARED FROM 2-DIMENSIONAL PROTEIN CRYSTALS

In this article, we describe the use of 1- to 2-mu m sized affinity mi croparticles for the isolation and purification of IgG from artificial Igc-human serum albumin mixtures and clarified hybridoma cell culture supernatants by affinity cross-flow filtration. Affinity microparticl es were prepared from cell wall fragments of Clostridium thermohydrosu lfuricum L111-69, in which the peptidoglycan-containing layer was comp letely covered with a hexagonally ordered S-layer lattice. After cross linking the S-layer protein with glutaraldehyde, carboxyl groups from acidic amino acids were activated with carbodiimide and used for immob ilization of Protein A. Quantitative determination confirmed that Prot ein A molecules formed a monomolecular layer on the outermost surface of the S-layer lattice. Affinity microparticles were found to withstan d high centrifugal and shear forces and revealed no Protein A leakage or S-layer protein release under cross-flow conditions between pH 2 to 12. The IgG-binding capacity of affinity microparticles was investiga ted under crossflow conditions and compared with that obtained in batc h adsorption processes. (C) 1994 John Wiley and Sons, Inc.