摘要
目的：基于液质联用的串联质谱技术,建立基于完整糖肽水平的抗体药糖型定性定量分析方法,并用于生物仿制药糖型研究。方法：基于液相色谱质谱技术,从完整糖肽水平对糖型进行定性定量分析,同时与传统超高效液相荧光检测（ultra performance liquid chromatography_fluorescence detection,UPLC-FLR）结果比较,获得最优分析策略,并用于曲妥珠单抗仿制药批次间糖型定性定量分析。结果：UPLC-FLR方法定量分析了贝伐单抗7种糖型;液质联用方法定量19种糖型;经亲水相互作用色谱（hydrophilic interaction liquid chromatography,HILIC）富集后,定量22种糖型。曲妥珠单抗仿制药批次间糖型定量结果显示良好的一致性。结论：基于液质联用的方法,具有速度快、灵敏度高、检测糖型种类多等优势,有望作为一种稳定的抗体药物糖蛋白位点特异性定量分析策略。
Objective： To establish a qualitative and quantitative method for the determination of antibody glycosylation from the level of intact glycopeptides based on LC-MS, and to apply it to qualitative and quantitative analysis of biomimetic drugs. Methods： The qualitative and quantitative analysis of glycoforms was carried out from the level of intact glycopeptides, and compared with the results from UPLC-FLR, the traditional glycan analysis approach. The optimal analysis strategy was established and used for the analysis of N-glycans on antibody biosimilars. Result： The UPLC-FLR methods obtained the quantitative result of seven glycoforms from bevacizumab; While the method of mass spectrometry quantified 19 glycoforms. After enrichment by hydrophilic interaction liquid chromatography （ HILIC ）, 22 glycan forms were quantified. The quantitative results of the glycosylation mass of the trastuzumab showed good agreement. Conclusion： Based on the method of LC-MS, it has the advantages of fast speed, high sensitivity and high detection of glycoforms, and it is expected to be a stable quantitative strategy for site - specific glycosylation analysis of biopharmaceuticals.
作者
黄怡
李晓宇
田芳
钱小红
应万涛
HUANG Yi;LI Xiao-yu;TIAN Fang;QIAN Xiao-hong;YING Wan-tao(Anhui Medical University, Hefei 230032, China;State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Science, Beijing Institute of Radiation Medicine, Beijing 102206,China)
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2018年第1期32-41,共10页
China Biotechnology
基金
国家重点研发计划(2017YFF0205400)
国家自然科学基金重点项目(81530021)资助项目
