title: Cetuximab Fab and Fc N-glycan fast characterization using IdeS digestion and liquid chromatography coupled to electrospray ionization mass spectrometry.
abstract: Antibodies and related products represent one of the fastest growing areas of new drug development within the pharmaceutical industry. Monoclonal antibodies (mAbs) undergo many posttranslational modifications (PTMs) that must be extensively characterized. Here we described a rapid mass spectrometry (MS) method for the characterization of cetuximab glycosylation. The reported analytical technique is based on the use of a cystein protease, immunoglobulin-degrading enzyme of Streptococcus pyogenes that allows a fast limited proteolysis of the mAb with low material consumption. The resulting large fragments are analyzed by ultrahigh-performance liquid chromatography combined to an electrospray ionization mass spectrometer and a time-of-flight analyzer (ESI-TOF). Cetuximab is a potent chimeric mouse/human antibody worldwide approved for the treatment of colon and head and neck cancers. This antibody, produced by SP2/0 murine myeloma cells, is N-glycosylated both in the Fc and Fab moieties, which have been shown to impact on safety and PK/PD and considered as a critical quality attribute. The method can also be applied for biosimilars, biobetters, and next-generation antibodies and Fc-fusion proteins.
authors.affiliation: Physico-Chemistry Department, Centre d'Immunologie Pierre-Fabre, Saint Julien-en-Genevois, France.
authors.forename: Marie-Claire
authors.initials: MC
authors.lastname: Janin-Bussat
authors.forename: Laure
authors.initials: L
authors.lastname: Tonini
authors.forename: Céline
authors.initials: C
authors.lastname: Huillet
authors.forename: Olivier
authors.initials: O
authors.lastname: Colas
authors.forename: Christine
authors.initials: C
authors.lastname: Klinguer-Hamour
authors.forename: Nathalie
authors.initials: N
authors.lastname: Corvaïa
authors.forename: Alain
authors.initials: A
authors.lastname: Beck
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry*; D002021:Buffers; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D000068818:Cetuximab; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D004229:Dithiothreitol / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D007141:Immunoglobulin Fc Fragments; D051379:Mice; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000737:chemistry; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry* / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D019163:Reducing Agents / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D001426:Bacterial Proteins; D002021:Buffers; D007141:Immunoglobulin Fc Fragments; C442815:Mac-1-like protein, Streptococcus; D011134:Polysaccharides; D019163:Reducing Agents; D009439:Neuraminidase; D003546:Cysteine Endopeptidases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D000068818:Cetuximab; D004229:Dithiothreitol