Role of local antibody density effects on immunosorbent efficiency
Subramanian, A.; Van Cott, K.E.; Milbrath, D.S.; Velander, W.H.
Journal of Chromatography A 672(1-2): 11-24
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.