Competitive binding of monoclonal antibody monomer-dimer mixtures on ceramic hydroxyapatite

Abstract

The internal structure of ceramic hydroxyapatite CHT Type I and Type II and the adsorption behavior of a monoclonal antibody in monomeric and dimeric forms are determined. Both CHT types contain elongated nanocrystals with size about 20 x 100 nm for Type I and about 50 x 200 nm for Type II. Internal porosities and apparent pore radii based on inverse size exclusion chromatography are 0.73 and 30 nm for Type I and 0.70 and 49 nm for Type II. Adsorption isotherms show maximum capacities of 95 and 110 mg/mL of particle for monomer and dimer on Type I and of 55 and 67 mg/mL of particle on Type II, in approximate agreement with the ratio of surface areas. The isotherms are dependent on the Na+ concentration consistent with an electrostatically driven mechanism. Mixture adsorption shows selectivity toward the dimer, with mean α-values of 4.3 and 5.8 for Type I and II, respectively. Effective pore diffusivities for non-binding conditions are 0.54 × 10−7 and 0.33 × 10−7 cm2/s for monomer and dimer in CHT Type I increasing to 0.94 × 10−7 and 0.66 × 10−7 cm2/s as a result of the larger pore size of Type II. Effective pore diffusivities for strong binding conditions, obtained by confocal microscopy, are much smaller than the non-binding values for Type I but essentially the same for Type II, indicating that diffusional hindrance by the bound protein is greater in the smaller pores of Type I. Mixture confocal adsorption experiments show that the competitive binding kinetics is largely controlled by pore diffusion in both CHT types with the dimer readily displacing the monomer even for strong binging conditions, but is much faster on Type II.