title: A new mixed-mode model for interpreting and predicting protein elution during isoelectric chromatofocusing.
abstract: Experimental data are combined with classic theories describing electrolytes in solution and at surfaces to define the primary mechanisms influencing protein retention and elution during isoelectric chromatofocusing (ICF) of proteins and protein mixtures. Those fundamental findings are used to derive a new model to understand and predict elution times of proteins during ICF. The model uses a modified form of the steric mass action (SMA) isotherm to account for both ion exchange and isoelectric focusing contributions to protein partitioning. The dependence of partitioning on pH is accounted for through the characteristic charge parameter m of the SMA isotherm and the application of Gouy-Chapman theory to define the dependence of the equilibrium binding constant Kbi on both m and ionic strength. Finally, the effects of changes in matrix surface pH on protein retention are quantified through a Donnan equilibrium type model. By accounting for isoelectric focusing, ion binding and exchange, and surface pH contributions to protein retention and elution, the model is shown to accurately capture the dependence of protein elution times on column operating conditions.
authors.affiliation: Michael Smith Laboratories, and the Department of Chemical and Biological Engineering, University of British Columbia, 231 Michael Smith Building, Vancouver, BC, Canada, V6T 1Z4.
authors.forename: Derek Y C
authors.initials: DY
authors.lastname: Choy
authors.forename: A Louise
authors.initials: AL
authors.lastname: Creagh
authors.forename: Eric
authors.initials: E
authors.lastname: von Lieres
authors.forename: Charles
authors.initials: C
authors.lastname: Haynes
mesh_terms: D002852:Chromatography, Ion Exchange / Q000379:methods*; D007525:Isoelectric Focusing / Q000379:methods*; D008956:Models, Chemical; D011506:Proteins / Q000302:isolation & purification*
chemical_list: D011506:Proteins
keywords: chromatography; downstream processing; ion-exchange chromatography; multiple chemical equilibria; purification