Fragmentation stability and retention time-shift obtained by LC-MS/MS to distinguish sialylated N -glycan linkage isomers in therapeutic glycoproteins.
Journal of pharmaceutical analysis(2023)
摘要
Sialylated -glycan isomers with α2-3 or α2-6 linkage(s) have distinctive roles in glycoproteins, but are difficult to distinguish. Wild-type (WT) and glycoengineered (mutant) therapeutic glycoproteins, cytotoxic T lymphocyte-associated antigen-4-immunoglobulin (CTLA4-Ig), were produced in Chinese hamster ovary cell lines; however, their linkage isomers have not been reported. In this study, -glycans of CTLA4-Igs were released, labeled with procainamide, and analyzed by liquid chromatography-tandem mass spectrometry (MS/MS) to identify and quantify sialylated -glycan linkage isomers. The linkage isomers were distinguished by comparison of 1) intensity of the -acetylglucosamine ion to the sialic acid ion (Ln/Nn) using different fragmentation stability in MS/MS spectra and 2) retention time-shift for a selective value in the extracted ion chromatogram. Each isomer was distinctively identified, and each quantity (>0.1%) was obtained relative to the total -glycans (100%) for all observed ionization states. Twenty sialylated -glycan isomers with only α2-3 linkage(s) in WT were identified, and each isomer's sum of quantities was 50.4%. Furthermore, 39 sialylated -glycan isomers (58.8%) in mono- (3 -glycans; 0.9%), bi- (18; 48.3%), tri- (14; 8.9%), and tetra- (4; 0.7%) antennary structures of mutant were obtained, which comprised mono- (15 -glycans; 25.4%), di- (15; 28.4%), tri- (8; 4.8%), and tetra- (1; 0.2%) sialylation, respectively, with only α2-3 (10 -glycans; 4.8%), both α2-3 and α2-6 (14; 18.4%), and only α2-6 (15; 35.6%) linkage(s). These results are consistent with those for α2-3 neuraminidase-treated -glycans. This study generated a novel plot of Ln/Nn versus retention time to distinguish sialylated -glycan linkage isomers in glycoprotein.
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关键词
LC-MS/MS,Linkage isomer,Sialylation,Therapeutic glycoprotein
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