title: Solvent modulated linear pH gradient elution for the purification of conventional and bispecific antibodies: Modeling and application.
abstract: Classical ion-exchange chromatography using a linear salt gradient to elute the adsorbed protein at fixed pH is the most common method to separate product-related impurities during downstream processing of biopharmaceuticals. Linear pH gradient elution provides a useful alternative by separating proteins in a linear pH gradient at fixed salt concentration. Although linear pH gradient elution provides excellent selectivity, it is rarely encountered in industrial purification processes. Here, a stoichiometric displacement model is used to characterize pH gradient elution based on simple linear gradient elution experiments. Protein retention behavior is described with respect to the pH dependencies of the characteristic binding charge and the equilibrium constant of the ion exchange reaction. Furthermore, the influence of solvent composition using PEG as a mobile phase modifier is investigated. Validity and applicability of the model are demonstrated for the purification of a conventional monoclonal antibody from soluble aggregates and for a novel bispecific antibody format containing a unique product-related impurity profile. pH step elution protocols are derived from model calculations without further optimization experiments necessary.
authors.affiliation: Institute of Biochemistry, University of Applied Sciences Mannheim, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Kluters
authors.affiliation: Institute of Molecular Biology and Cell Culture Technology, University of Applied Sciences Mannheim, Germany.
authors.forename: Mathias
authors.initials: M
authors.lastname: Hafner
authors.affiliation: Roche Pharmaceutical Research and Early Development, Large Molecule Research, Roche Innovation Center, Penzberg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: von Hirschheydt
authors.affiliation: Institute of Biochemistry, University of Applied Sciences Mannheim, Germany. Electronic address: c.frech@hs-mannheim.de.
authors.forename: Christian
authors.initials: C
authors.lastname: Frech
mesh_terms: D018033:Antibodies, Bispecific / Q000302:isolation & purification*; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange / Q000379:methods; D006863:Hydrogen-Ion Concentration; D008962:Models, Theoretical; D018892:Proton-Motive Force; D012965:Sodium Chloride; D012997:Solvents
chemical_list: D018033:Antibodies, Bispecific; D000911:Antibodies, Monoclonal; D012997:Solvents; D012965:Sodium Chloride
keywords: Bispecific antibodies; Linear pH gradient elution; Monoclonal antibodies; Product-related impurities; Solvent modulation