earticle

영어

Glycosylation of monoclonal antibody (mAb) therapeutics has critical roles such as biological activity, stability, plasma half-life, and immunogenicity. Therefore, detailed glycomic analyses are important to assess quality, safety, and potency of mAbs. Although mass spectrometry (MS) has emerged as a powerful tool for qualitative glycan analysis, MS-based quantitative analysis for glycome is still limited due to the absence of analytical platform and authentic standards. This study aims to develop the method for quantitative analysis of glycan and site-specific glycoforms directly from mAbs for QA/QC assessment using multiple reaction monitoring (MRM). Here, tryptic glycopeptide on trastuzumab, representative mAb, was selected as a standard for optimization of MRM transitions and instrument parameters of C18-UHPLC/triple quadrupole (QqQ). Three major tryptic glycopeptides having the same peptide moiety (EEQYNSTYR) with three different glycans (G0F, G1F, and G2F) on mAbs were targeted and quantified using MRM. In addition, we measured the absolute amounts of major glycans (G0F, G1F, and G2F) on each mAb (adalimumab, bevacizumab, infliximab, rituximab, and trastuzumab) using FLD response of 2-aminobenzamide (2-AB) labeled glycan standards. Consequently, we found that the abundance of glycopeptide calculated by MRM has highly correlated with absolute amounts of 2-AB labeled glycan by LC-FLD. The developed MRM method can be applied to evaluate mAbs variants and/or biosimilars for both developmental and regulatory purposes.