甲胺化衍生结合基于硅氢化物固定相的正相色谱用于单克隆抗体药物的<i>N</i>-糖基化表征

引用本文: 汪耀, 梁高道, 韩清, 胡迅, 张启伟, 何振宇. 甲胺化衍生结合基于硅氢化物固定相的正相色谱用于单克隆抗体药物的N-糖基化表征[J]. 色谱, 2018, 36(7): 615-620. doi: 10.3724/SP.J.1123.2018.01026 shu

Citation: WANG Yao, LIANG Gaodao, HAN Qing, HU Xun, ZHANG Qiwei, HE Zhenyu. Methylamidation and normal phase chromatography with silica-hydride-based stationary phase for N-glycosylation characterization of monoclonal antibody drugs[J]. Chinese Journal of Chromatography, 2018, 36(7): 615-620. doi: 10.3724/SP.J.1123.2018.01026 shu

甲胺化衍生结合基于硅氢化物固定相的正相色谱用于单克隆抗体药物的N-糖基化表征

汪耀 ^1, ,
梁高道 ^1, ,
韩清 ^1, ,
胡迅 ^1, ,
张启伟 ^2, ,
何振宇 ^1,

1.
武汉市疾病预防控制中心, 湖北武汉 430015;
2.
江汉大学, 湖北武汉 430056
基金项目:
湖北省卫生计生委面上项目（WJ2017M198）.

摘要: 采用甲胺化衍生结合基于硅氢化物固定相的正相色谱（SiH-NPC）分析单抗的N-糖基化。样品经酶切、甲胺化衍生、纯化后由液相色谱-质谱进行分析。结果表明，相较于亲水相互作用色谱（HILIC），SiH-NPC分离机制不同，使用常规的无盐流动相即可实现高分离度，避免污染质谱，色谱柱结构稳定，使用寿命长，更适合快速分析。结合唾液酸衍生方法，SiH-NPC在液相色谱-质谱联用鉴定酸性糖和糖异构体方面呈现显著优势，在生物制药行业中具有重要的应用潜力。

关键词:

English

Methylamidation and normal phase chromatography with silica-hydride-based stationary phase for N-glycosylation characterization of monoclonal antibody drugs

1.
Wuhan Centers for Disease Prevention & Control, Wuhan 430015, China;
2.
Jianghan University, Wuhan 430056, China
Received Date: 16 January 2018
Fund Project:
General Program of Health and Family Planning Commission of Hubei Province(No. WJ2017M198).

Abstract: Monoclonal antibodies (mAbs) are efficacious therapeutic agents against various complex diseases. N-glycosylation characterization of mAbs is based primarily on fluorescence derivatization coupled with hydrophilic interaction liquid chromatography (HILIC). However, this method has some drawbacks. Presently, the N-glycosylation of mAbs was analyzed by methylamidation and silica-hydride-based normal phase chromatography (SiH-NPC). Samples were analyzed by liquid chromatography-mass spectrometry after enzymatic cleavage, methylamidation, and purification. SiH-NPC had some advantages compared to HILIC. It offered a novel separation mechanism that allowed for high resolution using salt-free mobile phases, avoiding contamination of the mass spectrometer. SiH-NPC may be applicable for rapid analysis. Its structure was more stable than that of HILIC, and it provided a long service life. In combination with the sialic acid derivatization, SiH-NPC presented a significant advantage in the analysis of sialylated glycans as well as isomeric oligosaccharides by liquid chromatography-mass spectrometry. The approach has potential applications in the biopharmaceutical industry.

Key words:

methylamidation
/ silica-hydride
/ liquid chromatography-mass spectrometry(LC-MS)
/ N-glycans
/ monoclonal antibodies(mAbs)

1. [1] Walsh G. Nat Biotechnol, 2014, 32(10):992
2. [2] Batra J, Rathore A S. Biotechnol Prog, 2016, 32(5):1091
3. [3] Jefferis R. Trends Pharmacol Sci, 2009, 30(7):356
4. [4] Bhatia H, Read E, Agarabi C, et al. Int J Pharm, 2016, 512(1):242
5. [5] Rathore A S, Reason A J, Weiskopf A. Biopharm Int, 2014, 27(7):34
6. [6] Cong Y T, Hu L H. Chinese Journal of Chromatography, 2016, 34(12):1186 丛宇婷, 胡良海, 色谱, 2016, 34(12):1186
7. [7] Melmer M, Stangler T, Schiefermeier M, et al. Anal Bioanal Chem, 2010, 398(2):905
8. [8] Pabst M, Altmann F. Proteomics, 2011, 11(4):631
9. [9] Szekrenyes A, Partyka J, Varadi C, et al. Methods Mol Biol, 2015, 1274:183
10. [10] Sanchez-De Melo I, Grassi P, Ochoa F, et al. J Proteomics, 2015, 127:225
11. [11] Zhao J, Li S, Li C, et al. Anal Chem, 2016, 88(14):7049
12. [12] Song T, Ozcan S, Becker A, et al. Anal Chem, 2014, 86(12):5661
13. [13] Liu X, Afonso L, Altman E, et al. Proteomics, 2008, 8(14):2849
14. [14] Desantos-Garcia J L, Khalil S I, Hussein A, et al. Electrophoresis, 2011, 32:3516
15. [15] Higel F, Seidl A, Demelbauer U, et al. MAbs, 2014, 6(4):894
16. [16] Liu X, Qiu H, Lee R K, et al. Anal Chem, 2010, 82(19):8300
17. [17] Zhang Q, Feng X, Li H, et al. Anal Chem, 2014, 86(15):7913
18. [18] Zhang Q, Li H, Feng X, et al. PLoS One, 2014, 9(4):e94232
19. [19] Alley W R Jr, Vasseur J A, Goetz J A, et al. J Proteome Res, 2012, 11(4):2282
20. [20] Wuhrer M, Koeleman C A, Hokke C H, et al. Rapid Commun Mass Spectrom, 2006, 20(11):1747
21. [21] Alley W R Jr, Madera M, Mechref Y, et al. Anal Chem, 2010, 82(12):5095

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甲胺化衍生结合基于硅氢化物固定相的正相色谱用于单克隆抗体药物的N-糖基化表征

English

Methylamidation and normal phase chromatography with silica-hydride-based stationary phase for N-glycosylation characterization of monoclonal antibody drugs

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通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

甲胺化衍生结合基于硅氢化物固定相的正相色谱用于单克隆抗体药物的N-糖基化表征

English

Methylamidation and normal phase chromatography with silica-hydride-based stationary phase for N-glycosylation characterization of monoclonal antibody drugs

CCS Chemistry：嵌段共聚物自组装成 “三明治”二维有机材料，实现纳米级压力传感功能

CCS Chemistry：颜徐州、鲍哲南： 你的皮肤可能是一片SPMs

CCS Chemistry：工作高效不疲劳，只须让催化剂动起来

CCS Chemistry：静电组装导向聚合- 聚电解质纳米凝胶量化制备新策略

CCS Chemistry：金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的？

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