title: Monoclonal antibodies biosimilarity assessment using transient isotachophoresis capillary zone electrophoresis-tandem mass spectrometry.
abstract: Out of all categories, monoclonal antibody (mAb) therapeutics attract the most interest due to their strong therapeutic potency and specificity. Six of the 10 top-selling drugs are antibody-based therapeutics that will lose patent protection soon. The European Medicines Agency has pioneered the regulatory framework for approval of biosimilar products and approved the first biosimilar antibodies by the end of 2013. As highly complex glycoproteins with a wide range of micro-variants, mAbs require extensive characterization through multiple analytical methods for structure assessment rendering manufacturing control and biosimilarity studies particularly product and time-consuming. Here, capillary zone electrophoresis coupled to mass spectrometry by a sheathless interface (CESI-MS) was used to characterize marketed reference mAbs and their respective biosimilar candidate simultaneously over different facets of their primary structure. CESI-MS/MS data were compared between approved mAbs and their biosimilar candidates to prove/disconfirm biosimilarity regarding recent regulation directives. Using only a single sample injection of 200 fmol, CESI-MS/MS data enabled 100% amino acids (AA) sequence characterization, which allows a difference of even one AA between 2 samples to be distinguished precisely. Simultaneously glycoforms were characterized regarding their structures and position through fragmentation spectra and glycoforms semiquantitative analysis was established, showing the capacity of the developed methodology to detect up to 16 different glycans. Other posttranslational modifications hotspots were characterized while their relative occurrence levels were estimated and compared to biosimilars. These results proved the value of using CESI-MS because the separation selectivity and ionization efficiency provided by the system allowed substantial improvement in the characterization workflow robustness and accuracy. Biosimilarity assessment could be performed routinely with a single injection of each candidate enabling improvements in the biosimilar development pipeline.
authors.affiliation: a Laboratoire de spectrométrie de masse des interactions et des systèmes (LSMIS); CNRS - UMR 7140 , Université de Strasbourg , Strasbourg Cedex , France.
authors.forename: Rabah
authors.initials: R
authors.lastname: Gahoual
authors.forename: Michaël
authors.initials: M
authors.lastname: Biacchi
authors.forename: Johana
authors.initials: J
authors.lastname: Chicher
authors.forename: Lauriane
authors.initials: L
authors.lastname: Kuhn
authors.forename: Philippe
authors.initials: P
authors.lastname: Hammann
authors.forename: Alain
authors.initials: A
authors.lastname: Beck
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Leize-Wagner
authors.forename: Yannis N
authors.initials: YN
authors.lastname: François
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000032:analysis*; D061067:Antibodies, Monoclonal, Humanized / Q000032:analysis; D059451:Biosimilar Pharmaceuticals / Q000032:analysis*; D000068818:Cetuximab; D019075:Electrophoresis, Capillary / Q000379:methods*; D006031:Glycosylation; D057625:Isotachophoresis / Q000379:methods*; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000032:analysis; D011499:Protein Processing, Post-Translational; D015203:Reproducibility of Results; D020539:Sequence Analysis, Protein; D053719:Tandem Mass Spectrometry / Q000379:methods*; D000068878:Trastuzumab
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D059451:Biosimilar Pharmaceuticals; D011134:Polysaccharides; D000068878:Trastuzumab; D000068818:Cetuximab
keywords: AA, amino acid; CESI-MS, sheathless capillary electrophoresis-mass spectrometry; CHO, Chinese hamster ovary; CZE, capillary zone electrophresis; HC, heavy chain; LC, light chain; PTM, posttranslational modification; biobetter; biosimilar; cetuximab; complete sequence coverage; glycoforms semiquantitative analysis; glycosylations; mAb, monoclonal antibody; mass spectrometry; monoclonal antibodies; peptide mapping; posttranslational modifications; sheathless capillary electrophoresis; t-ITP, transient isotachophoresis; transient isotachophoresis; trastuzumab