title: Unbiased in-depth characterization of CEX fractions from a stressed monoclonal antibody by mass spectrometry.
abstract: Characterization of charge-based variants by mass spectrometry (MS) is required for the analytical development of a new biologic entity and its marketing approval by health authorities. However, standard peak-based data analysis approaches are time-consuming and biased toward the detection, identification, and quantification of main variants only. The aim of this study was to characterize in-depth acidic and basic species of a stressed IgG1 monoclonal antibody using comprehensive and unbiased MS data evaluation tools. Fractions collected from cation ion exchange (CEX) chromatography were analyzed as intact, after reduction of disulfide bridges, and after proteolytic cleavage using Lys-C. Data of both intact and reduced samples were evaluated consistently using a time-resolved deconvolution algorithm. Peptide mapping data were processed simultaneously, quantified and compared in a systematic manner for all MS signals and fractions. Differences observed between the fractions were then further characterized and assigned. Time-resolved deconvolution enhanced pattern visualization and data interpretation of main and minor modifications in 3-dimensional maps across CEX fractions. Relative quantification of all MS signals across CEX fractions before peptide assignment enabled the detection of fraction-specific chemical modifications at abundances below 1%. Acidic fractions were shown to be heterogeneous, containing antibody fragments, glycated as well as deamidated forms of the heavy and light chains. In contrast, the basic fractions contained mainly modifications of the C-terminus and pyroglutamate formation at the N-terminus of the heavy chain. Systematic data evaluation was performed to investigate multiple data sets and comprehensively extract main and minor differences between each CEX fraction in an unbiased manner.
authors.affiliation: a Phys.-Chem. Analytics, Analytical Development and Characterization NBEs, Biopharmaceutical Product and Process Development, Biologics Technical Development and Manufacturing , Novartis Pharma AG, Postfach , Basel , Switzerland.
authors.forename: François
authors.initials: F
authors.lastname: Griaud
authors.affiliation: a Phys.-Chem. Analytics, Analytical Development and Characterization NBEs, Biopharmaceutical Product and Process Development, Biologics Technical Development and Manufacturing , Novartis Pharma AG, Postfach , Basel , Switzerland.
authors.forename: Blandine
authors.initials: B
authors.lastname: Denefeld
authors.affiliation: a Phys.-Chem. Analytics, Analytical Development and Characterization NBEs, Biopharmaceutical Product and Process Development, Biologics Technical Development and Manufacturing , Novartis Pharma AG, Postfach , Basel , Switzerland.
authors.forename: Manuel
authors.initials: M
authors.lastname: Lang
authors.affiliation: a Phys.-Chem. Analytics, Analytical Development and Characterization NBEs, Biopharmaceutical Product and Process Development, Biologics Technical Development and Manufacturing , Novartis Pharma AG, Postfach , Basel , Switzerland.
authors.forename: Héloïse
authors.initials: H
authors.lastname: Hensinger
authors.affiliation: b Genedata GmbH , Munich , Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Haberl
authors.affiliation: a Phys.-Chem. Analytics, Analytical Development and Characterization NBEs, Biopharmaceutical Product and Process Development, Biologics Technical Development and Manufacturing , Novartis Pharma AG, Postfach , Basel , Switzerland.
authors.forename: Matthias
authors.initials: M
authors.lastname: Berg
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D011994:Recombinant Proteins / Q000032:analysis
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins
keywords: 3-dimensional peptide map; CEX; IgG1; characterization; charge heterogeneity; glycation; mass spectrometry; monoclonal antibody; pyroglutamate; time-resolved deconvolution