Kinetic studies on the immobilization of antibodies to high-performance liquid chromatographic supports

Several factors can potentially affect the rate of immobilization of proteins onto solid supports, such as those used in affinity-based high-performance liquid chromatography. This study examined several of these factors and their influence on the coupling of periodate-treated rabbit immunoglobulin G antibodies to dihydrazide-activated silica. Items considered included the number of potential coupling sites on the antibodies, the density of activated sites on the support, the relative amount of antibody combined with the support, and the density of the overall reaction slurry. In each case, the rate of change in the solution-phase antibody concentration gave biphasic behavior which could be described by two competing pseudo-first-order reactions. The overall immobilization rate was essentially independent of the density of the support's activated sites (when present at a coverage of 0.1-0.4 μmol/m²) but was strongly influenced by the number of available coupling groups on the antibodies. Increasing the slurry density had no appreciable effect on the immobilization rate, and the reaction rate showed only a small change when using different types of reagents for support activation (e.g., adipic vs oxalic dihydrazide). These results are consistent with a mechanism in which the rate-limiting step during immobilization is the covalent attachment of antibodies to the support and not mass transfer of antibodies to the support's surface.