Margaret M. Walker

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.

Crosslinked, hydrophilic, azlactone-functional polymeric beads : a two-step approach

Abstract Crosslinked, hydrophilic, azlactone-functional polymeric beads are readily prepared via a two-step approach involving: (1) reverse phase suspension copolymerization of N-acryloyl-amino acids with water-soluble crosslinkers and, optionally, dimethylacrylamide, followed by (2) cyclodehydration of pendant acylamino acid groups to azlactones using acetic anhydride. Azlactone functionalities of 0.3 to 3.0 meq/g (typically greater than 70% of the theoretical value) were achieved by this procedure. The azlactone-functional group in these beads was found to be quite reactive towards amine nucleophiles, even in aqueous solution where little competition from hydrolysis was observed. Rapid, covalent coupling of protein could be accomplished from aqueous media under mild conditions, and indicated a potential for extremely high coupling densities (up to 245 mg protein/g of beads).