Improved sample preparation method for glycan analysis of glycoproteins by CE‐LIF and CE‐MS

Abstract

CE is a high-resolution separation technique broadly used in the biotechnology industry for carbohydrate analysis. The standard sample preparation protocol for CE analysis of glycans released from glycoproteins generally requires derivatization times of overnight at 37 degrees C, using > or =100 fold excess of fluorophore reagent, 8-aminopyrene-1,3,6-trisulfonic-acid, if the sample is unknown, or it is a regulated biotherapeutic product, possibly containing terminal sialic acid(s). In this paper, we report on significant improvements for the standard CE sample preparation method of glycan analysis. By replacing the conventionally used acetic acid catalyst with citric acid, as low as 1:10 glycan to fluorophore molar ratio (versus the typical 1:> or =100 ratio) maintained the >95% derivatization yield at 55 degrees C with only 50 min reaction time. Terminal sialic acid loss was negligible at 55 degrees C during the derivatization process, and indicating that the kinetics of labeling at 55 degrees C was faster than the loss of sialic acid from the glycan. The reduced relative level of 8-aminopyrene-1,3,6-trisulfonic-acid simplified the removal of excess reagent, important in both CE-LIF (electrokinetic injection bias) and CE-MS (ion suppression). Coupling CE- ESI-MS confirmed that the individual peaks separated by CE corresponded to single glycans and increased the confidence of structural assignment based on glucose unit values.