title: Improved isoelectric focusing chromatography on strong anion exchange media via a new model that custom designs mobile phases using simple buffers.
abstract: Isoelectric chromatofocusing (ICF), a mode of chromatography by which proteins are separated based on changes in their charge state with pH, is widely used at analytical scales and finding increasing interest in biologics manufacturing due to its exceptional resolving power. Here, a method is described for using simple monoprotic and diprotic buffers to create stable mobile phases for sample loading on a strong anion exchange column and for achieving an elution pH gradient of desired shape covering any pH range from pH 10.0 to 3. The buffers used are selected to satisfy cost constraints, and to permit facile detection of eluted biologics by UV spectroscopy and mass spectrometry. The method exploits a new model described here that combines multiple-chemical and adsorption-equilibria theory to enable in silico tailoring of elution pH profiles using mixtures of these simple buffers. It is shown to provide a versatile platform for optimizing and conducting ICF of protein mixtures on strong anion exchange media.
authors.affiliation: Department of Chemical and Biological Engineering, Michael Smith Laboratories, University of British Columbia, 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: Charles
authors.initials: C
authors.lastname: Haynes
mesh_terms: D000665:Ampholyte Mixtures; D002021:Buffers; D002852:Chromatography, Ion Exchange / Q000379:methods*; D007475:Ion Exchange Resins; D007525:Isoelectric Focusing / Q000379:methods*
chemical_list: D000665:Ampholyte Mixtures; D002021:Buffers; D007475:Ion Exchange Resins
keywords: chromatography; downstream processing; ion-exchange chromatography; multiple chemical equilibria; purification