Immobilization of Protein A at high density on azlactone-functional polymeric beads and their use in affinity chromatography

Abstract

This paper presents the results of the use of highly cross-linked, porous, hydrophilic copolymer beads with protein immobilized on their surface for affinity chromatography. Copolymer beads composed of vinyldimethyl azlactone (oxazolone) and methylene-bis-acrylamide in various ratios, with up to 3/5 mequiv./g azlactone functionality, will undergo nucleophilic attack by amines, as well as by thiols and alcohols. The ring-opening reaction of a nucleophile-containing ligand ( e.g., a protein) resulted in covalent attachment to the support. The reaction was rapid, half-complete in about 5 min, yielding proteins immobilized at very high densities, recombinant Protein A at 397 mg/g, and human immunoglobulin G at 225 mg/g. The reaction proceeded at significant levels from pH 4 to 9. There was a marked enhancement in the amount of protein coupled, its rate of reaction, and its biological activity when Protein A was made to react in the presence of high concentrations of sodium sulfate. Evaluatioin of affinity columns, prepared with Protein A immobilized at over 200 mg/g, gave molar ratios f immunoglobulin G to immobilized Protein A of 1:1 or greater. Up to 56 mg of immunoglobulin G was recovered per ml of column bed volume. The support combined high flow-rates with low back-pressures and nobed-volume changes upon changing mobile phases, including highly ionic aqueous solvents and ethanol.