文献类型：期刊文献

中文题名：亲水作用色谱用于西妥昔单抗的N端糖链分析

英文题名：Hydrophilic Interaction Chromatography for the Analysis of N-sugar Chains in Cetuximab

作者：高宗林[1];曹旭妮[1]

机构：[1]华东理工大学食品科学与工程系生物反应器工程国家重点实验室,上海200237

年份：2020

卷号：42

期号：10

起止页码：1169

中文期刊名：化学试剂

外文期刊名：Chemical Reagents

收录：CSTPCD;;北大核心:【北大核心2017】；

基金：上海市科委科研计划项目(18142202600)。

语种：中文

中文关键词：亲水作用色谱(HILIC);2-氨基苯甲酰胺;N端糖链;西妥昔单抗;液质联用技术

外文关键词：HILIC;2-aminobenzamide;N-sugar chains;cetuximab;UPLC-MS

摘要：单克隆抗体的糖链对其功能活性具有重要的影响,因此开展糖链分析技术的研究具有十分重要的意义。采用亲水作用色谱荧光检测方法建立了N端糖链的定量分析技术。对于常见的3种N端糖链G0F、G1F、G2F,其线性范围分别为7.5×10^-11~1.5×10^-8、4.5×10^-11~9.0×10^-9以及7.5×10^-11~4.5×10^-9 mol/L;检测下限分别为2.8×10^-11、2.5×10^-11、2.6×10^-11 mol/L;保留时间和峰面积的RSD值均小于3%,方法的回收率均大于95%。采用酶切N端糖链和2-氨基苯甲酰胺标记法可以实现西妥昔单抗的这3种N端糖链的含量分析,分别为86.0、85.1、16.6μg/g。除了上述3种糖链,还利用超高效液相色谱-电喷雾离子化串联质谱联用技术进一步对西妥昔单抗其他6种相对丰度较高的N端糖链的种类进行解析。这些研究不仅可以应用于单抗药物的N端糖链的质控分析,而且对其糖链种类进行解析以实现更多的生物学研究。
The sugar chains of the monoclonal antibody have an important influence on its functional activity,so it is of great significance to carry out a research on sugar chains analysis.To this end,a quantitative analysis technique of N-sugar chains was established by hydrophilic interaction chromatography coupled with fluorescence detection.For the three common N-sugar chains G0F,G1F,G2F,their linear ranges were 7.5×10^-11~1.5×10^-8,4.5×10^-11~9.0×10^-9 and 7.5×10^-11~4.5×10^-9 mol/L while the detection limits were 2.8×10^-11,2.5×10^-11,2.6×10^-11 mol/L,respectively.The RSD values of reserved time and peak area were all found less than 3%,and their recoveries were greater than 95%.It meant that the established method was sufficient for the content analysis of these three N-sugar chains in a real sample,which were released by digesting cetuximab and further labeled with 2-aminobenzamide.Further results indicated they were 86.0,85.1,16.6μg/g protein,respectively.Besides,we also provided an UPLC-ESI-MS-MS method to determine other six relatively abundant N-sugar chains of cetuximab.This work can be not only applied for quality control analysis of the N-sugar chains of monoclonal antibodies,but also determination of the types of sugar chains for further biological research.