N端焦谷氨酸环化封闭类单克隆抗体药物的N端测序方法

中国生物工程杂志

2013, Vol. 33

Issue (8): 75-83

技术与方法

N端焦谷氨酸环化封闭类单克隆抗体药物的N端测序方法

李明珠, 韩伟东, 邢光慧, 滕珍林, 薛国梅, 侯辰瑞, 阮宏强, 陈薇

中国科学院上海生命科学研究院蛋白质组学分析研究中心/上海中科新生命生物科技有限公司上海 200233

Research of N Terminal Sequencing Methods for Monoclonal Antibody Pharmaceuticals Blocked by Pyroglutamic Acid

LI Ming-zhu, HAN Wei-dong, Xing Guang-hui, TENG Zhen-lin, XUE Guo-mei, HOU Chen-rui, RUAN Hong-qiang, CHEN Wei

Research Center for Proteome Analysis Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences/Shanghai Applied Protein Technology Co.Ltd, Shanghai 200233, China

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摘要： 目的:对于直接用N端测序仪无法进行测序的N端焦谷氨酸环化封闭的抗体类药物,去除焦谷氨酸后实现对其N端序列的从头测序。方法:对抗体的重轻链分别进行N端测序无果,再通过LC-MSMS对其重轻链N端的焦谷氨酸进行确认,证明其为焦谷氨酸封闭。对抗体蛋白进行焦谷氨酸氨肽酶酶解后,再通过N端测序仪进行Edman降解法N端测序,获得抗体药物重轻链的N端氨基酸序列。结论:抗体药物的重轻链N端经过N端测序仪测序和LC-MSMS分析,确定均为焦谷氨酸封闭。再经过焦谷氨酸氨肽酶处理后进行N端测序仪从头测序证实抗体药物的N端序列与理论序列一致。

关键词： 单克隆抗体; 焦谷氨酸环化; Edman降解; N端测序; LC-MSMS

Abstract: Objective: Some monoclonal antibody pharmaceuticals with N terminal sequence blocked by pyroglutamic acid can't be sequenced directly by N terminal sequencer which is designed based on Edman degradation principle. Removal of the pyroglutamic acid to realize de novo sequencing of the antibody. Methods: First, using N terminal sequencer to test the light and heavy chains of antibody without treated. Second, confirm the existing of pyroglumatic acid by LC-MSMS. Last, Cutting pyroglumatic acid with pyroglumate aminopeptidase, and then, sequencing the light and heavy chains of antibody with N terminal sequencer. Conclusion: Antibody's light and heavy chains blocked with pyroglumatic acid can be proved by N terminal sequencer and LC-MSMS. And, N terminal sequence of the samples can be proved the same with theoretical one after removal of pyroglumatic acid.

Key words: Monoclonal antibody Pyroglumatic acid blocking Edman degradation N terminal sequencing LC-MSMS

收稿日期: 2013-02-21 出版日期: 2013-08-25

ZTFLH:

Q78

基金资助: 国家科技部科技型中小企业技术创新基金(12C26243102092)资助项目

通讯作者: 陈薇wchen@sibs.ac.cn E-mail: wchen@sibs.ac.cn

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引用本文:

李明珠, 韩伟东, 邢光慧, 滕珍林, 薛国梅, 侯辰瑞, 阮宏强, 陈薇. N端焦谷氨酸环化封闭类单克隆抗体药物的N端测序方法[J]. 中国生物工程杂志, 2013, 33(8): 75-83.

LI Ming-zhu, HAN Wei-dong, Xing Guang-hui, TENG Zhen-lin, XUE Guo-mei, HOU Chen-rui, RUAN Hong-qiang, CHEN Wei. Research of N Terminal Sequencing Methods for Monoclonal Antibody Pharmaceuticals Blocked by Pyroglutamic Acid. China Biotechnology, 2013, 33(8): 75-83.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I8/75

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