Glycoform Separation and Characterization of Cetuximab
Variants by Middle-up Off-Line Capillary Zone Electrophoresis-UV/Electrospray
Ionization-MS
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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′)<sub>2</sub> 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′)<sub>2</sub> 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