Hydroxyapatite is a well-known multimodal chromatographic adsorbent, which combines cation exchange interactions with phosphoryl groups together with calcium metal affinity. Hydroxyapatite has been used extensively to remove aggregates from monoclonal antibodies (mAb). Despite widespread use, however, little has been published regarding the kinetics of adsorption, especially for multicomponent systems. In this work, we examine the adsorption and separation of a mAb monomer and its dimer on two types of ceramic hydroxyapatite, CHT Type I and CHT Type II, which differ in pore size and surface area. The internal structure of both adsorbents was first evaluated using transmission electron microscopy (TEM) and inverse size exclusion chromatography (iSEC). Adsorption equilibrium and kinetics of mAb monomer and dime, individually and in mixtures, were then characterized based on batch measurements and by confocal laser scanning microscopy (CLSM). The isotherm data under various NaCl and Na<sub>2</sub>HPO<sub>4</sub> concentrations indicate that mAb binding follows a cation exchange mechanism which is selective for the dimer compared to the monomer while the kinetics is dominated by pore diffusion for both, but with effective diffusivities that are strongly influenced by the molecular size and CHT type. Displacement of the monomer by the dimer was found to occur relatively quickly for mixture adsorption and was found to be largely mass transfer controlled, especially at higher salt concentrations. Separation by frontal analysis, where the mixture is continuously fed to the column under the binding condition, was demonstrated over a range of residence times and with different monomer-dimer feed ratios. Finally, a mechanistic model was developed to describe competitive binding and the frontal analysis process assuming pore diffusion control with adsorption described by multicomponent Langmuir isotherms. The model can be used to optimize protein load to maximize productivity curtailing much of the pure empiricism that has been traditionally associated with the application of CHT for protein purification.

Competitive binding of monomer-dimer mAb mixtures and their separation by frontal analysis using ceramic hydroxyapatite

2019 Spring National Meeting

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