Relative quantitation strategy of glycans in tocilizumab labeled based on mass spectrometry probes d0/d15-CEBTPP: Monitoring glycan changes in rat serum

Glycosylation is crucial for the effector functions of therapeutic monoclonal antibodies (mAbs). However, the specific glycoprofile of tocilizumab, a humanized anti-interleukin-6 (IL-6) receptor mAb, remains inadequately characterized in the current literature. To address this, we designed and synthesized a novel pair of stable isotope-labeled mass spectrometry (MS) probes: d0/d15-[4-(1-carboxyethyl)phenyl]methyl-triphenylphosphonium (d0/d15-CEBTPP). Featuring a permanent positive charge, these probes not only significantly enhance the ionization efficiency and detection sensitivity of glycans in MS positive-ion mode, but also effectively neutralize the intrinsic negative charge of sialic acid residues, thereby substantially improving the structural stability of acidic glycans during analysis. Consequently, a novel relative quantification strategy for glycans was established by coupling d0/d15-CEBTPP isotopic labeling with ultra-high performance liquid chromatography-high-resolution MS (UHPLC-HRMS). Methodological validation using a sialoglycopeptide (SGP) model demonstrated excellent linearity ( R 2 = 0.9990), reproducibility (coefficient of variation (CV) = 3.50%), and accuracy (relative error (RE) = 0.76%) across a 10-fold molar mixing range. Application of this strategy to the comprehensive characterization of tocilizumab successfully identified 23 N-glycans, including 14 fucosylated and 7 sialylated glycoforms. Furthermore, the method was successfully applied in a rat model, precisely tracking the dynamic fluctuations of targeted serum glycan levels from 2 min to 36 h post-administration. Overall, this study establishes a robust strategy for the efficient and reliable relative quantification of trace glycans in complex biological matrices without relying on glycan standards. This work offers a powerful analytical tool for comprehensive glycosylation evaluation in biopharmaceuticals and disease-related comparative glycomics.