title: Highly linear pH gradients for analyzing monoclonal antibody charge heterogeneity in the alkaline range: Validation of the method parameters.
abstract: Cation exchange chromatography has been routinely used for the quantification of monoclonal antibody (mAb) charge heterogeneity. A previously developed method utilizing pH gradients for the elution instead of salt gradients was validated according to current guidelines proposed by the ICH. The linearity, stability, accuracy, precision and the lower limit of quantification have been determined, using pure charge variant standards. The method is valid for the quantification of mAb samples with a charge heterogeneity between 1% and 50%. Three different approaches to obtaining pure standard material for the validation of bio-analytical methods for the quantification of charge heterogeneity of IgG are presented. These methods are based on salt gradient elution, pH gradient elution and displacement in cation exchange chromatography.
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, A-1190 Vienna, Austria; Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, No. 06-01 Centros, 138668 Singapore, Singapore.
authors.forename: Nico
authors.initials: N
authors.lastname: Lingg
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, A-1190 Vienna, Austria; Department of Pure and Applied Biochemistry, Lund University, Faculty of Engineering, Lund, Sweden.
authors.forename: Martina
authors.initials: M
authors.lastname: Berndtsson
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, A-1190 Vienna, Austria.
authors.forename: Beate
authors.initials: B
authors.lastname: Hintersteiner
authors.affiliation: Apeiron Biologics AG, Campus-Vienna-Biocenter 5, A-1030 Vienna, Austria.
authors.forename: Manfred
authors.initials: M
authors.lastname: Schuster
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, No. 06-01 Centros, 138668 Singapore, Singapore; Normandie Université, Laboratoire Glyco-MEV, EA 4358, Institut de Recherche et d'Innovation Biomédicale (IRIB), 76821 Mont-Saint-Aignan Cedex, France.
authors.forename: Muriel
authors.initials: M
authors.lastname: Bardor
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, A-1190 Vienna, Austria. Electronic address: alois.jungbauer@boku.ac.at.
authors.forename: Alois
authors.initials: A
authors.lastname: Jungbauer
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis*; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D006863:Hydrogen-Ion Concentration
chemical_list: D000911:Antibodies, Monoclonal
keywords: Ion-exchange chromatography; Monoclonal antibody variants; Validation; pH gradient