2P-32 PDF
酵素化学的合成法の為のモノクローナル抗体の糖ペプチドの定量的解析
We have developed an effective, sensitive method for quantitative glycopeptides profiling using stable isotope labeling and MALDI-TOF MS. In addition, we performed a rearrangement of Fc N-glycan on monoclonal antibodies (mAbs) from a heterogeneous N-glycosylation pattern to a homogeneous N-glycan structure using chemoenzymatic approaches with two types of endo-β-N-acetylglucosaminidases (ENG’ases), that one works as a hydrolase, another is used as a glycosynthase. We used an anti-Her2 antibody produced in transgenic silkworm cocoon, which has a mixture of the glycans, non-fucosylated pauci-mannose type (Man2-3GlcNAc2), high-mannose type (Man4-9GlcNAc2), and complex type (Man3GlcNAc3-4) as a starting material. For efficient chemoenzymatic synthesis of glycoengineered mAb, it is important to characterize the substrate specificities of selected endo-β-N-acetylglucosaminidases (ENG’ases). Thus, we analyzed ENG’ase activity for mAbs using the above glycopeptides labeling technique, in which the hydrolyzed glycoforms could be estimated based on the overlapping MS spectra of mAb glycopeptides with/without the reaction of ENG’ase (endoH, endoD, endoM, endoS, and endoLL). In this analysis, the decreased amount of glycopeptides hydrolyzed by ENG’ase could be observed indirectly, but the glycoform on the mAb as a product of the ENG’ase reaction could be detected directly. As the result, we found the ENG’ase activity to cleave heterogeneous N-glycans on mAb.