Correct prediction of protein elution behavior on an ion-exchange resin depends largely on an accurate description of the binding models, in terms of both the model form and the model parameters. At low coverages, several widely-used isotherms approach linear behavior, but at higher surface coverages, encountered during saturation loading, the deviation from linearity and approach to a plateau level deviate among different models. This behavior leads to different peak shapes for different models when saturation levels of loading are reached. In the current work, several models, including steric mass action (SMA), modified Langmuir and a colloidal model, were examined for prediction of elution peak shape from low loading to saturation of protein load onto the column. The elution peak shape is found to be dependent not only upon the protein size and its physiological properties but also on details of the resin morphology. This is demonstrated using lysozyme, cytochrome c and a monoclonal antibody on different resins (SP Sepharose FF, Fractogel EMD SO3-, Capto S, Capto SP ImpRes, and Poros HS ). To address another important aspect of mechanistic modelling, namely accurate and easy estimation of isotherm parameters, the binding models have been also evaluated based on the capacity and ease of estimation of parameters by independent measurements. Among the models evaluated, the colloidal model parameters were easily obtained from pulse and breakthrough curves. The colloidal isotherm descriptions depend mainly on two independently-measured parameters: (a) the adsorption equilibrium, Keq, which captures protein–surface interactions and (b) the Yukawa parameter, Bpp, which captures lateral protein-protein interactions, particularly electrostatic ones, with the range determined by the Debye parameter κ. Keq is independent of Bpp and was easily obtained from retention factors for pulse injections at different ionic strengths. Bpp plays a more dominant role at higher surface coverage and was determined from breakthrough curves at different ionic strengths. Some of the systems such as lysozyme on Fractogel were not adequately described by the existing binding models. For such systems isotherms were approximated by frontal analysis using the characteristic point method and insights were examined for possible mechanisms.

Dependence of elution peak shape on binding models and resin structure in ion-exchange chromatography of proteins

255th National Meeting (2018)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.