Effect of feed flow-rate, antigen concentration and antibody density on immunoaffinity purification of coagulation factor IX.
A simple physical model of immunoaffinity chromatography (IAC) demonstrates that immobilized monoclonal antibody (MAb) capacity in IAC purification will be a function of many parameters, including feed flow-rate and antigen concentration, and MAb density (mg MAb immobilized/ml resin). We studied IAC of factor IX, and examined the effect of parameter variation on MAb capacity. MAb capacity (1) was not affected by feed flow-rate or antigen concentration, and (2) decreased as MAb density increased. (1) Suggested that diffusion of factor IX into the resin bead was not limiting. Characteristic diffusion, convection and reaction times were calculated and used in dimensional analysis to compare their relative magnitudes. If MAb was assumed to be localized to the outer 10% of the bead volume, this analysis concluded that diffusion was not limiting, consistent with the suggestions of our experimental data. (2) Suggests that high MAb densities make MAb less accessible.