Evaluation of factors which affect column performance with immobilized monoclonal antibodies. Model studies with a...

Methods are described for the automated evaluation of affinity columns by frontal boundary analysis. These methods were used to evaluate the performance of immunoaffinity columns based on antilysozyme monoclonal antibody-lysozyme immunoaffinity system. This model system enabled the effects of (i) matrix activation and (ii) the density of immobilized antibody on the change in specific activity of immobilized antibody to be quantitatively assessed. Experimental data were accumulated with carbonyldiimidazole-activated Fractogel HW65F and Trisacryl GF2000 resins and cyanogen bromide-activated Sepharose 4B. An increase in the molar ratio between the concentration of the active groups on the activated matrix and the concentration of immobilized antibody ligands did not result in significant change in the specific activity of the immobilized antibody in the immunochromatographic system. However, increased antibody density with the Fractogel HW65F resin resulted in an increase in the apparent heterogeneity of antibody binding sites for lysozyme and a significant decrease in the specific activity of the immobilized antibody. Furthermore, data from size-exclusion studies with these immunoaffinity matrices demonstrated that at high antibody densities, the accessibility of the immobilized antibody was further decreased due to steric resistance as the antigen size increased.