LC-MS/MS在线部分还原法快速表征雷珠单抗一级结构
Rapid Characterization of Primary Structure of Ranibizumab by LC-MS/MS On-line Partial Reduction Method
李佳婧 1温学美 1刘菊 1潘红娟1
作者信息
- 1. 中国医药工业研究总院上海医药工业研究院,创新药物与制药工艺国家重点实验室,上海 201203
- 折叠
摘要
首次建立了在线部分三(2-羧乙基)膦还原法,通过LC-MS/MS 一次进样采集分析,同时对雷珠单抗(1批参比制剂和2批样品)的肽图和二硫键进行表征.结果表明,肽图序列覆盖度均可达100％,鉴定到的翻译后修饰主要为氧化、脱酰胺化和焦谷氨酸环化;除确认与理论连接一致的5对二硫键外,该方法能够鉴定到游离半胱氨酸和3对错配二硫键;烷基化试剂的加入能有效降低样品前处理过程中产生的错配概率.该研究采用在线处理方法,缩短了处理和分析时间,减少了人为引入的不可控因素,为高通量抗体结构的表征提供了参考.
Abstract
The peptide mapping and disulfide linkages of ranibizumab(including one batch of reference preparation and two batches of samples)were simultaneously characterized by the online partial reduction of tris-(2-carboxyethyl)phosphine(TCEP)after a single injection by LC-MS/MS for the first time.The results showed that the sequence coverage of peptide mapping could reach 100％,and the identified post translational modifications were mainly oxidation,deamidation and pyroglutamic acid cyclization.The five pairs of disulfide bonds were confirmed to be consistent with the theoretical connection.In addition,free cysteine and three pairs of scrambled disulfide bonds were identified by this method,and the probability of artifacts could be effectively reduced by adding a certain amount of alkylation reagents.This online method shortened the processing and analysis time,reduced the uncontrollable factors introduced by humans,and provided references for high-throughput antibody structure characterization.
关键词
雷珠单抗/质谱/二硫键/肽图Key words
ranibizumab/mass spectrometry/disulfide bond/peptide mapping引用本文复制引用
基金项目
中国医药工业研究总院创新基金(YJS2021008)
出版年
2023
NETL
NSTL
维普
万方数据