The Influence of Protein Size on Adsorption Kinetics and Equilibria in Ion-Exchange Chromatography

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Abstract
The adsorption behavior of lysozyme and γ-globulin on the strong cation-exchanger Streamline SP was studied by determining the equilibrium isotherms with batch experiments. Adsorption isotherms of the binary mixture of both proteins were modeled using the Langmuir parameters derived from single-component systems. Comparison of experimental results with the competitive Langmuir model displayed significant deviations for the adsorption of lysozyme in the mixture. An extended Langmuir model, accounting for the distinct accessibility of the sorbent's surface for the competing proteins, revealed much better consistency with experimental data. Assuming the rate-limiting step of protein uptake is due to mass transport effects, the kinetics were modeled with a rate model. Taking into account diffusion across the li-quid film and within the adsorbent pores, effective pore diffusion coefficients were determined. It was shown that the effective pore diffusion coefficient of both model proteins depends on the total protein concentration and the concentration ratio of the competing proteins in the liquid phase. In the binary mixture the diffusion rate of the faster diffusing protein lysozyme was slowed down by the lower diffusion rate of the larger protein γ-globulin. In packed bed experiments the weaker adsorbed γ-globulin was displaced by lysozyme and eluted earlier as in single-component breakthrough curves whereas the elution time of lysozyme was almost identical. However, the breakthrough curve of lysozyme was flatter in the presence of γ-globulin due to the diffusional hindrance in the mixture.