GlycoformGlycoformSeparation and Characterization of Separation and Characterization of CetuximabCetuximabVariants by MiddleVariants by Middle--up up OffOff--line CEline CE--UV/ESIUV/ESI--MSMS

摘要

mAbs are highly complex glycoproteins that present a wide range of microheterogeneities that requires multiple analytical methods for full structure assessment and quality control. Here we report an original strategy to perform off-line CE-ESI-MS using CE-UV/fraction collection technology to performed CE separation, followed by ESI-MS infusion of the different fractions using CESI interface as nanoESI infusion platform. The efficiency of our strategy was validated with the separation of cetuximab charge variant by middle-up approach. MWs were measured for six charge variants detected in the CE separation of cetuximab subunits. Two F_(c/2) C-terminal lysine variants were identified and separated. Regarding F(ab')2, 8 glycoforms were detected and separated in three different peaks following the presence of NGNA residues in some glycan structure.