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.

Interference‐free mass spectrometric detection of capillary isoelectric focused proteins, including charge variants of a model monoclonal antibody

“…Nevertheless, 1500 mg/L for an MS analysis represents an excellent value compared to previous works regarding CZE‐MS analysis of antibodies. Exemplarily, Biacchi et al., 2015 demonstrated a glycoform separation and characterization by an offline coupling of CZE‐UV with ESI‐MS with a sample concentration of 5000 mg/L . Another common top‐down analysis technique for mAbs is provided by ultra‐high‐performance liquid chromatography (UHPLC)‐MS.…”

Fluorescent Labelling of Glycans with FRET‐Based Probes in a Gold(III)‐Mediated Three‐Component Coupling Reaction

“…[2][3][4][5][6][7][8] Specific glycosylation patterns are associated with numerous human diseases such as cancers, diabetes, Alzheimer's disease, hepatocellular carcinoma and kidney diseases. [9][10][11][12] To study the role of glycans in various biological events and disease developments, it is of importance to develop effective and selective methods for glycan modification.…”

Recent advances in CE‐MS coupling: Instrumentation, methodology, and applications

“…Even though the separation was finished within only 10 min and with excellent relative standard deviations of migration times (∼1%), a higher separation resolution is needed for a reliable data analysis . Similarly, offline CE‐UV/fraction collection method followed by ESI‐MS infusion was optimized for middle‐up characterization of charged variants of cetuximab subunits distinguishing three forms of Fc/2 fragments and three Fab2 fragments .…”

“…A total of 138 proteoforms were found, and 55 were quantitated. An original strategy to perform offline CE fraction collection technology followed by nanoliter flow ESI‐MS infusion was developed for the separation and characterization of glycoform variants of highly complex glycoprotein, therapeutic mAbs, cetuximab . An integrated microfluidic platform of CE‐MS was developed to analyze an intact lysine‐linked mAb drug conjugate in order to assess PTM and drug load variants.…”