Dean S. Milbrath

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.

Role of local antibody density effects on immunosorbent efficiency

Abstract This study evaluates the effect of immobilized antibody density on the performance of an immunosorbent. In contrast to previous studies that emphasize the correlation of high volume averaged antibody density with immunosorbent performance, we have studied the effects of locally high antibody density and spatial distribution on the antigen binding efficiency under conditions of dynamic loading and elution. The distribution of an anti-human Protein C monoclonal antibody immobilized on 3M Emphaze AB1 Biosupport Medium was evaluated. The distribution of immobilized antibody was controlled by a two-step sequence of permeation and reaction. Labeled antibody was visualized by immunofluorescence. Conditions of low pH, low temperature, and the presence of a competitor nucleophile sufficiently depressed the Thiele modulus for coupling to enable permeation of the antibody. The adsorption of the permeated antibody was enhanced by the presence of 0.75 M Na2SO4, and then the pH was raised to achieve rapid covalent coupling. Bead-averaged antibody densities of 1–11 mg/ml of hydrogel support were studied. Immunosorbents containing more evenly distributed antibody gave a two- to three-fold greater antigen binding efficiency than those with locally high antibody densities. No appreciable changes in mass transfer characteristics were observed using breakthrough analysis for immunosorbents with distributed versus locally high antibody density.

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).

Reproducibility of physical characteristics, protein immobilization and chromatographic performance of 3M emphaze biosupport medium AB 1

Abstract 3M Emphaze Biosupport Medium AB 1, a polymeric azlactone-functional support, provides for rapid and efficient coupling of proteins for uses such as affinity chromatography. The support is easy to use, with no preactivation or additional reagents required. It rapidly immobilizes proteins from solutions between pH 4 and 11. Materials used in chromatographic processes need to be reproducible, in order to provide uniform performance. The following characteristics have been monitored in order to evaluate the reproducibility of Emphaze Biosupport Medium: particle size distribution, pore size distribution, surface area, bed volume, protein exclusion limits, pressure and flow performance, protein coupling and affinity performance.