title: Glycoform Separation and Characterization of Cetuximab Variants by Middle-up Off-Line Capillary Zone Electrophoresis-UV/Electrospray Ionization-MS.
abstract: Monoclonal antibodies (mAbs) are highly complex glycoproteins that present a wide range of microheterogeneities that requires multiple analytical methods for full structure assessment and quality control. Capillary zone electrophoresis-mass spectrometry (CZE-MS) couplings, especially by electrospray ionization (ESI), appear to be really attractive methods for the characterization of biological samples. However, due to the presence of non- or medium volatile salts in the background electrolyte (BGE), online CZE-ESI-MS coupling is difficult to implement for mAbs isoforms separation. Here, we report an original strategy to perform off-line CZE-ESI-MS using CZE-UV/fraction collection technology to perform CZE separation, followed by ESI-MS infusion of the different fractions using the capillary electrophoresis-electrospray ionization (CESI) interface as the nanoESI infusion platform. As the aim is to conserve electrophoretic resolution and complete compatibility with ESI-MS without sample treatment, hydroxypropylcellulose (HPC) coated capillary was used to prevent analyte adsorption and asymmetric CZE conditions involving different BGE at both ends of the capillary have been developed. The efficiency of our strategy was validated with the separation of Cetuximab charge variant by the middle-up approach. Molecular weights were measured for six charge variants detected in the CZE separation of Cetuximab subunits. The first three peaks correspond to Fc/2 variants with electrophoretic resolution up to 2.10, and the last three peaks correspond to F(ab')2 variants with average electrophoretic resolution of 1.05. Two Fc/2 C-terminal lysine variants were identified and separated. Moreover, separation of Fc/2 fragments allowed the glycoprofiling of the variants with the characterization of 7 different glycoforms. Regarding the F(ab')2 domain, 8 glycoforms were detected and separated in three different peaks following the presence of N-glycolyl neuraminic acid residues in some glycan structures. This work highlights the potential of CZE technology to perform separation of mAbs especially when they carry sialic acid carbohydrates.
authors.affiliation: †Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS), UDS-CNRS UMR 7140, Université de Strasbourg, Strasbourg 67000, France.
authors.forename: Michael
authors.initials: M
authors.lastname: Biacchi
authors.affiliation: †Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS), UDS-CNRS UMR 7140, Université de Strasbourg, Strasbourg 67000, France.
authors.forename: Rabah
authors.initials: R
authors.lastname: Gahoual
authors.affiliation: †Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS), UDS-CNRS UMR 7140, Université de Strasbourg, Strasbourg 67000, France.
authors.forename: Nassur
authors.initials: N
authors.lastname: Said
authors.affiliation: ‡Centre d'Immunologie Pierre Fabre, Saint-Julien-en-Genevois 74164, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Beck
authors.affiliation: †Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS), UDS-CNRS UMR 7140, Université de Strasbourg, Strasbourg 67000, France.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Leize-Wagner
authors.affiliation: †Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS), UDS-CNRS UMR 7140, Université de Strasbourg, Strasbourg 67000, France.
authors.forename: Yannis-Nicolas
authors.initials: YN
authors.lastname: François
mesh_terms: D000068818:Cetuximab / Q000235:genetics / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary; D014644:Genetic Variation / Q000235:genetics; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000068818:Cetuximab