Glycoform Separation and Characterization of Cetuximab Variants by Middle-up Off-Line Capillary Zone Electrophoresis-UV/Electrospray Ionization-MS - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Analytical Chemistry Année : 2015

Glycoform Separation and Characterization of Cetuximab Variants by Middle-up Off-Line Capillary Zone Electrophoresis-UV/Electrospray Ionization-MS

Résumé

Monoclonal antibodies (mAbs) are highly complex glycoproteins that present a wide range of microheteroge-neities that requires multiple analytical methods for full structure assessment and quality control. Capillary zone electrophoresis-Mass spectrometry couplings (CZE-MS), especially by Electrospray (ESI), appear 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 performed CZE separation, followed by ESI-MS infusion of the different fractions using CESI interface as nanoESI infusion platform. As the aim is to conserve electrophoretic resolu-tion and complete compatibility with ESI-MS without sample treatment, Hydroxypropylcellulose (HPC) coated capillary was used to prevent analyte adsorption and assymetric CZE conditions involving different BGE at both ends of capillary have been developed. The efficiency of our strategy was validated with the separation of cetuximab charge variant by middle-up approach. Molecular weights were measured for six charge variants detected in the CZE separation of cetuximab subunits. The three first 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 elec-trophoretic 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 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 structure. This work highlights the potential of CZE technology to perform separation of mAbs especially when they carry sialic acid carbohy-drates.
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Dates et versions

hal-01981309 , version 1 (14-01-2019)

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Michael Biacchi, Rabah Gahoual, Nassur Said, Alain Beck, Emmanuelle Leize-Wagner, et al.. Glycoform Separation and Characterization of Cetuximab Variants by Middle-up Off-Line Capillary Zone Electrophoresis-UV/Electrospray Ionization-MS. Analytical Chemistry, 2015, 87 (12), pp.6240-6250. ⟨10.1021/acs.analchem.5b00928⟩. ⟨hal-01981309⟩
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