Dynamics of competitive binding and separation of monoclonal antibody monomer-dimer mixtures in ceramic hydroxyapatite columns

Abstract

This work examines the separation dynamics of monoclonal antibody monomer/dimer mixtures by frontal analysis using ceramic hydroxyapatite CHT Type I and Type II columns. The binding capacity and selectivity are dependent on the CHT type and salt concentration. While the rate of protein adsorption on CHT Type I is slow and controlled largely by pore diffusion resulting in relatively poor separation, adsorption on CHT Type II is much faster and better separation is obtained than with Type I. However, comparison with predictions based on pore diffusion alone, reveals the presence of additional resistances associated with adsorption and displacement kinetics. A spreading kinetics model assuming multiple binding configurations coupled with pore diffusion was developed to describe these effects and found to be in quantitative agreement with the frontal analysis results and able to predict the separation achieved for conditions outside the range of the experiments. To help validate the assumed mechanism, isocratic elution experiments were also conducted at low protein loads. The chromatograms could be described by the solution of the spreading model coupled with pore diffusion in the linear region of the isotherm with parameters determined from the analytical expressions for the peak moments. This confirms that there is an increasing tendency to spread with slower kinetics as the salt concentration is decreased and binding strength is increased.