Hydrophobic interaction chromatography of proteins III. Transport and kinetic parameters in isocratic elution.

The effective pore diffusivities, D(e), of five model proteins (ribonuclease A, lysozyme, alpha-lactalbumin, ovalbumin, and BSA) in eight commercial phenyl hydrophobic interaction chromatography (HIC) media were determined by analyzing the plate height data from isocratic elution using the first two moments of the general linear rate model. The adsorbents represent a diverse set of HIC media that are widely used for protein purification. The estimated pore diffusivities were used to calculate the elution profiles of proteins in these adsorbents and were compared with the elution profiles obtained experimentally. High protein loading and sample protein concentration led to the underestimation of the pore diffusivity by the linear rate model. Comparisons between the calculated and the experimental profiles suggest that the pore diffusivities obtained from the linear rate model are generally accurate for proteins with low structural flexibility but not for more flexible ones, presumably because conformational change effects contribute significantly to the overall HETP. The general linear rate model was modified to account for the protein folding/unfolding kinetics, and parameter values could be estimated by fitting the experimental elution profiles to the modified model. In addition to conformational change, adsorbent type also had a significant effect on the accuracies of the pore diffusivities estimated by the linear rate model. The results also show that pore diffusion was the rate-limiting step in all absorbents for rigid proteins such as ribonuclease A and lysozyme. For structurally flexible proteins, conformational change contributed significantly to the overall reduced plate heights of the isocratic elution peaks. The physical properties of adsorbents, such as protein accessible porosity, pore size distribution, pore radius and pore connectivity, play important roles in determining the effective protein pore diffusivities.