分析测试学报2017，Vol.36Issue(12)：1458-1463,6.DOI:10.3969/j.issn.1004-4957.2017.12.007
MALDI-TOF质谱准内标法及其用于rhTPO复杂糖一致性研究
MALDI-TOF MS with Quasi-internal Calibration Method and Its Study on Batch-to-Batch Consistency in Complex Glycan of rhTPO
摘要
Abstract
The relative contents of four kinds of glycan forms in the inovator and biosimilar products of recombinant human thromboplastin (rhTPO) were accurately determined using matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) with quasi-internal calibration,the consistencies between products and among batches were evaluated.The N-glycan,sialic acid and O-glycan of rhTPO were removed off with different endoglycosidase,respectively,bovine serum albumin (BSA)used as the standard was pointed target separate with the sample.The molecular weights of the intact glycoproteins and the different forms of deglycosylated proteins were determined by MALDI-TOF MS with quasi-internal calibration.The glycan contents in samples were calculated by the molecular weight differences among different deglycosylated samples.Finally,the consistency of the inovator and biosimilar products were evaluated.The multi-charge peaks of BSA were used for the quasi-internal calibration of MALDI-TOF MS.And their calibration relative standard deviations(RSDs) were all no more than 0.075％,and the RSDs among different calibrations were among 0.001％-0.004％.The method deviations away from the sequence molecular weight were less than 0.01％.For the biosimilar drug,the relative contents of N-glycan,sialic acid,O-glycan,and glycation were 24.6％,2.9％,9.0％ and 5.1％,respectively,and the batch-to-batch consistency was quite good.The total glycan contents were consistent with those reported in the literature.For the innovator drug,the relative contents of N-glycan,sialic acid,O-glycan and glycation were 25.6％,2.9％,7.9％ and 3.5％,respectively.The total glycan content was lower than that reported in the literature.The relative contents of glycosylation between the innoventor products and the biosimilar products were almost the same but the relative contents of glycation showed some differences.Glycation of rhTPO has not been reported up-to-now,the differences of the relative contents of glycation might be the important factors for the different rhTPO manufacturers,the glycation differences were mainly caused by the differences of fermentation process.关键词
重组人血小板生成素(rhTPO)/基质辅助激光解吸附电离飞行时间质谱(MALDI-TOF MS)/准内标法/糖含量/糖化Key words
recombinant human thrombopoietin (rhTPO)/matrix assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF MS)/quasi-internal calibration/glycan contents/glycation分类
化学化工引用本文复制引用
刘悦玫,侯利平,杨梅,孟晓光,魏开华..MALDI-TOF质谱准内标法及其用于rhTPO复杂糖一致性研究[J].分析测试学报,2017,36(12):1458-1463,6.基金项目
国家高技术研究发展计划863项目(2014AA020906) （2014AA020906）
国家科技部重大仪器专项(2012YQ18011710) （2012YQ18011710）
蛋白质组学国家重点实验室课题(SKLP-O201412) （SKLP-O201412）
