title: Top-down and middle-down approach by fraction collection enrichment using off-line capillary electrophoresis - mass spectrometry coupling: Application to monoclonal antibody Fc/2charge variants.
abstract: The characterization of complex protein mixtures represents one of the biggest challenge in many research fields such as biological or biopharmaceutical sciences. Out of all categories, monoclonal antibodies (mAbs) and related products drawn the most interest due to their strong therapeutic potency and specificity. Because of their intrinsic complexity due to a large number of micro-heterogeneities, there is a crucial need for analytical methods to provide comprehensive in-depth characterization of these proteins. In this work, we developed a methodology using CE-UV/MALDI-MS to perform top-down or middle-down characterization after fraction collection enrichment applied to intact protein and mAbs samples. The performance of the method was evaluated with the rapid separation of three intact protein mixture. Good robustness of CZE separation and quality of MALDI-MS spectra and MALDI-ISD spectra of each protein confirms the usefulness of sample enrichment to obtain adequate quantity of deposed protein for top-down analysis and the proof of principle of the method. In a second step, the method was applied to the middle-down characterization of Fc/2 cetuximab variants. Identification of around 9% sequence coverage of Fc/2 cetuximab fragments allows to conclude on the feasibility of the strategy for middle-down characterization of Fc/2 cetuximab variants using CE-UV/MALDI-MS. Moreover, MALDI-ISD fragmentation of Fc/2 cetuximab variants confirm separation phenomenon based on the formation of Fc/2 dimers with and without C-terminal truncation.
authors.affiliation: Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS), UMR 7140 (Unistra-CNRS), Université de Strasbourg, France; Laboratoire de Biochimie Biologie Moléculaire Nutrition Métabolisme, UF 3544 Dépistage Néonatal, CHRU Nancy, France.
authors.forename: Michael
authors.initials: M
authors.lastname: Biacchi
authors.affiliation: Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS), UMR 7140 (Unistra-CNRS), Université de Strasbourg, France.
authors.forename: Nassur
authors.initials: N
authors.lastname: Said
authors.affiliation: Centre d'immunologie Pierre Fabre, Saint-Julien-en-Genevois, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Beck
authors.affiliation: Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS), UMR 7140 (Unistra-CNRS), Université de Strasbourg, France.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Leize-Wagner
authors.affiliation: Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS), UMR 7140 (Unistra-CNRS), Université de Strasbourg, France. Electronic address: yfrancois@unistra.fr.
authors.forename: Yannis-Nicolas
authors.initials: YN
authors.lastname: François
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000302:isolation & purification; D000068818:Cetuximab / Q000032:analysis* / Q000302:isolation & purification; D019281:Dimerization; D019075:Electrophoresis, Capillary / Q000379:methods*; D007141:Immunoglobulin Fc Fragments / Q000032:analysis / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000911:Antibodies, Monoclonal; D007141:Immunoglobulin Fc Fragments; D000068818:Cetuximab
keywords: Capillary electrophoresis-mass spectrometry; Enrichment; MALDI-ISD; Middle-down; Monoclonal antibody; Top-down