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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2022, Vol. 42 Issue (6): 66-75    DOI: 10.13523/j.cb.2201016
综述     
单克隆抗体生产过程中二硫键还原成因及预防方法
张莘迪1,范长伟,宋晓清2,徐翠云,黄凤杰2,*()
1.中国药科大学生命科学与技术学院 南京 211198
2.上海复宏汉霖生物技术股份有限公司 上海 200233
Root Cause and Prevention of Monoclonal Antibody Disulfide Bonds Reduction During Biopharmaceutical Manufacturing Process
ZHANG Xin-di1,FAN Chang-wei2,SONG Xiao-qing2,*(),XU Cui-yun2,HUANG Feng-jie1
1. College of Life Science and Technology, China Pharmaceutical University, Nanjing 211198, China
2. Shanghai Henlius Biotech, Inc., Shanghai 200233, China
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摘要：

单克隆抗体生产过程中二硫键的还原是生物制药领域中的一个常见技术难题,可产生低分子量碎片,影响产品质量,导致蛋白纯度降低、稳定性下降,影响药物的安全性和有效性。抗体二硫键还原实质上是由细胞内的硫氧还蛋白系统和谷胱甘肽系统引起的可逆氧化还原反应,并与具体生产过程参数有关。近年来,随着抗体药物和哺乳动物细胞培养工艺规模的发展,二硫键还原问题频繁发生。为解决此问题,研究人员不断尝试并建立了多种预防方法以保证产品质量。概述了抗体二硫键结构、二硫键还原的主要成因及生产过程中的形成因素,重点阐述了消除或减缓抗体二硫键还原的方法、对策,并列举了几种可行的过程分析技术,以期为单克隆抗体药物生产制造工艺的进一步优化提供参考。
关键词： 抗体;  二硫键;  氧化还原;  工艺开发;  过程分析技术    
Abstract:

Monoclonal antibody disulfide bonds reduction has been a common issue in biopharmaceutical process, which could produce low-molecular weight fragments, affect product quality, and lead to decreased protein purity and stability. Moreover, it could also affect the safety and effectiveness of drugs. Antibody disulfide bonds reduction is a reversible redox reaction caused by intracellular thioredoxin system and glutathione system, and is related to the specific production process. In recent years, with the development of antibody drugs and mammalian cell culture scale, disulfide bonds reduction is observed more frequently. To solve this problem, scientists have been constantly developing mitigation strategies to ensure the product quality. In this paper, the antibody disulfide bonds structure, cause of disulfide bonds reduction and influencing factors in manufacturing process were summarized. It is focused on the prevention methods in the manufacturing process. Additionally, several feasible process analysis techniques are listed so as to provide reference for the further development of monoclonal antibody drugs in manufacturing.
Key words: Antibody    Disulfide bond    Oxidation and reduction    Process development    Process analytical technology
收稿日期: 2022-01-14 出版日期: 2022-07-07
ZTFLH:  Q819  
通讯作者: 黄凤杰     E-mail: hfj@cpu.edu.cn
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张莘迪
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黄凤杰

引用本文:

张莘迪,范长伟,宋晓清,徐翠云,黄凤杰. 单克隆抗体生产过程中二硫键还原成因及预防方法[J]. 中国生物工程杂志, 2022, 42(6): 66-75.

ZHANG Xin-di,FAN Chang-wei,SONG Xiao-qing,XU Cui-yun,HUANG Feng-jie. Root Cause and Prevention of Monoclonal Antibody Disulfide Bonds Reduction During Biopharmaceutical Manufacturing Process. China Biotechnology, 2022, 42(6): 66-75.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2201016    或    https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I6/66

图1  抗体二硫键结构及还原后的形式
图2  硫氧还蛋白系统和谷胱甘肽系统还原抗体二硫键机制
方法 原理 操作步骤 参考文献
敲除TXN1基因 抑制酶表达 细胞株开发阶段 [43]
添加酶抑制剂(ATG、ATM、Cu2+、EDTA、L型胱氨酸等) 抑制酶活性 培养&收获阶段 [18,33,37,44-45]
降低pH 降低酶反应速率 培养&收获阶段 [37,40]
低温储存 降低酶反应速率 收获阶段 [6]
提高DO设定点 使可逆的氧化还原
反应转为氧化方向		 培养阶段 [47]
向储存容器通入空气、添加H2O2 使可逆的氧化还原
反应转为氧化方向		 收获阶段 [28,38]
使用密封式离心机、控制深层过滤压差 减少酶的数量 收获阶段 [38,50]
缩短储存时间 缩短酶反应时间 收获阶段 [6]
Protein A亲和层析用氧化还原缓冲液洗涤 将已经还原的抗体重新氧化 纯化阶段 [52-53]
表1  生产过程中抗体二硫键还原的防治方法
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