title: Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography, Part II: pH gradient approach.
abstract: The cation exhange pH gradient approach was evaluated for the characterization of 10 model monoclonal antibodies including panitumumab, natalizumab, cetuximab, bevacizumab, trastuzumab, rituximab, palivizumab, adalimumab, denosumab and ofatumumab. This work shows that retention and resolution can be modelled in cation exchange pH gradient mode, based on only four initial runs (i.e. two gradient times and two mobile phase temperature). Only 6h were required for a complete method optimization when using a 100 mm × 4.6 mm strong cation exchange column. The accuracy of the predictions was excellent, with an average difference between predicted and experimental retention times of about 1%. The 10 model antibodies were successfully eluted in both pH and salt gradient modes, proving that both modes of elution can be considered as multi-product charge sensitive separation methods. For most of the compounds, the variants were better resolved in the salt gradient mode and the peak capacities were also higher in the salt gradient approach. These observations confirm that pH gradient approach may be of lower interest than salt gradient cation exchange chromatography for antibody characterization.
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d'Yvoy 20, 1211 Geneva 4, Switzerland. Electronic address: szabolcs.fekete@unige.ch.
authors.forename: Szabolcs
authors.initials: S
authors.lastname: Fekete
authors.affiliation: Center of Immunology Pierre Fabre, 5 Avenue Napoléon III, BP 60497, 74160 Saint-Julien-en-Genevois, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Beck
authors.affiliation: Budapest University of Technology and Economics, Department of Inorganic and Analytical Chemistry, Szt. Gellért tér 4., 1111 Budapest, Hungary.
authors.forename: Jenő
authors.initials: J
authors.lastname: Fekete
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d'Yvoy 20, 1211 Geneva 4, Switzerland.
authors.forename: Davy
authors.initials: D
authors.lastname: Guillarme
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006863:Hydrogen-Ion Concentration
chemical_list: D000911:Antibodies, Monoclonal
keywords: Cetuximab; Ion exchange; Method development; Monoclonal antibody; pH gradient