title: Capillary ion-exchange chromatography with nanogram sensitivity for the analysis of monoclonal antibodies.
abstract: Ion-exchange chromatography (IEC) is widely used for profiling the charge heterogeneity of proteins, including monoclonal antibodies (mAbs). Despite good resolving power and robustness, ionic strength-based ion-exchange separations are generally product specific and can be time consuming to develop. In addition, conventional analytical scale ion-exchange separations require tens of micrograms of mAbs for each injection, amounts that are often unavailable in sample-limited applications. We report the development of a capillary IEC (c-IEC) methodology for the analysis of nanogram amounts of mAb charge variants. Several key modifications were made to a commercially available liquid chromatography system to perform c-IEC for charge variant analysis of mAbs with nanogram sensitivity. We demonstrate the method for multiple monoclonal antibodies, including antibody fragments, on different columns from different manufacturers. Relative standard deviations of <10% were achieved for relative peak areas of main peak, acidic and basic regions, which are common regions of interest for quantifying monoclonal antibody charge variants using IEC. The results herein demonstrate the excellent sensitivity of this c-IEC characterization method, which can be used for analyzing charge variants in sample-limited applications, such as early-stage candidate screening and in vivo studies.
authors.affiliation: Protein Analytical Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA. Electronic address: rea.jennifer@gene.com.
authors.forename: Jennifer C
authors.initials: JC
authors.lastname: Rea
authors.affiliation: Early Stage Pharmaceutical Development, Genentech, Inc., South San Francisco, CA 94080, USA.
authors.forename: Benny S
authors.initials: BS
authors.lastname: Freistadt
authors.affiliation: Protein Analytical Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: McDonald
authors.affiliation: Protein Analytical Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Dell
authors.initials: D
authors.lastname: Farnan
authors.affiliation: Protein Analytical Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Yajun Jennifer
authors.initials: YJ
authors.lastname: Wang
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis*; D002852:Chromatography, Ion Exchange / Q000379:methods; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal
keywords: Antibodies; Capillary; Charge heterogeneity; Ion-exchange chromatography; Monoclonal