质谱方法实现抗体类药物糖链修饰的鉴定与定量研究

doi:10.13523/j.cb.20180104

中国生物工程杂志

2018, Vol. 38

Issue (1): 32-41 DOI: 10.13523/j.cb.20180104

技术与方法

质谱方法实现抗体类药物糖链修饰的鉴定与定量研究

黄怡^1,²,李晓宇²,田芳²,钱小红²,应万涛^1,²(

)

1 安徽医科大学合肥 230032
2 蛋白质组学国家重点实验室北京蛋白质组学研究中心国家蛋白质科学中心军事医学科学院放射与辐射医学研究所北京 102206

Identification and Quantitative Study on Glycosylation Chain Modification of Antibody Drugs by Mass Spectrometry

Yi HUANG^1,²,Xiao-yu LI²,Fang TIAN²,Xiao-hong QIAN²,Wan-tao YING^1,²(

)

1 Anhui Medical University,Hefei 230032,China
2 State Key Laboratory of Proteomics, Beijing Proteome Research Center,National Center for Protein Science,Beijing Institute of Radiation Medicine, Beijing 102206,China

全文: PDF(1843 KB) HTML

摘要： 目的

基于液质联用的串联质谱技术,建立基于完整糖肽水平的抗体药糖型定性定量分析方法,并用于生物仿制药糖型研究。

方法

基于液相色谱质谱技术,从完整糖肽水平对糖型进行定性定量分析,同时与传统超高效液相荧光检测(ultra performance liquid chromatography_fluorescence detection,UPLC-FLR)结果比较,获得最优分析策略,并用于曲妥珠单抗仿制药批次间糖型定性定量分析。

结果

UPLC-FLR方法定量分析了贝伐单抗7种糖型;液质联用方法定量19种糖型;经亲水相互作用色谱(hydrophilic interaction liquid chromatography,HILIC)富集后,定量22种糖型。曲妥珠单抗仿制药批次间糖型定量结果显示良好的一致性。

结论

基于液质联用的方法,具有速度快、灵敏度高、检测糖型种类多等优势,有望作为一种稳定的抗体药物糖蛋白位点特异性定量分析策略。

关键词： 超高效液相荧光检测; 液质联用; 抗体药物; 完整糖肽定量

Abstract: Objective:

To establish a qualitative and quantitative method for the determination of antibody glycosylation from the level of intact glycopeptides based on LC-MS, and to apply it to qualitative and quantitative analysis of biomimetic drugs.

Methods:

The qualitative and quantitative analysis of glycoforms was carried out from the level of intact glycopeptides, and compared with the results from UPLC-FLR, the traditional glycan analysis approach. The optimal analysis strategy was established and used for the analysis of N-glycans on antibody biosimilars.

Result:

The UPLC-FLR methods obtained the quantitative result of seven glycoforms from bevacizumab; While the method of mass spectrometry quantified 19 glycoforms. After enrichment by hydrophilic interaction liquid chromatography (HILIC), 22 glycan forms were quantified. The quantitative results of the glycosylation mass of the trastuzumab showed good agreement.

Conclusion:

Based on the method of LC-MS, it has the advantages of fast speed, high sensitivity and high detection of glycoforms, and it is expected to be a stable quantitative strategy for site - specific glycosylation analysis of biopharmaceuticals.

Key words: UPLC-FLR LC-MS Antibody drugs Intact glycopeptides quantification

收稿日期: 2017-07-18 出版日期: 2018-01-31

ZTFLH:

Q816

基金资助: 国家重点研发计划(2017YFF0205400);国家自然科学基金重点项目资助项目(81530021)

作者简介: 通讯作者应万涛。E-mail: proteomics@126.com

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	黄怡
	李晓宇
	田芳
	钱小红
	应万涛

引用本文:

黄怡,李晓宇,田芳,钱小红,应万涛. 质谱方法实现抗体类药物糖链修饰的鉴定与定量研究[J]. 中国生物工程杂志, 2018, 38(1): 32-41.

Yi HUANG,Xiao-yu LI,Fang TIAN,Xiao-hong QIAN,Wan-tao YING. Identification and Quantitative Study on Glycosylation Chain Modification of Antibody Drugs by Mass Spectrometry. China Biotechnology, 2018, 38(1): 32-41.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180104 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I1/32

图1 实验线路图

图2 贝伐单抗N-聚糖荧光标记色谱图

图3 贝伐单抗N-糖肽HN4H3F1_EEQYNSTYR二级谱图解析

图4 UPLC-FLR糖型定量结果

图5 Trypsin酶切后糖型定量结果

图6 HILIC富集后糖型定量结果

图7 三种方法糖型鉴定结果相关性分析

表1 三种方法糖型定量结果比较

表2 三种方法平行实验RSD值比较

表3 三个批次间曲妥珠单抗仿制药糖型定量结果比较

[1]	翁锦玉. 国际生物医药产业发展趋势分析.科技与创新,2017, 1(1): 1-2.
	Weng J Y.Analysis of international biomedical industry development trend . Science and Technology & Innovation,2017, 1(1): 1-2.
[2]	刘艳玲,刘晓志,赵伟,等.糖蛋白药物的多糖结构解析进展.生物技术进展,2016, 6(4): 244-248.
	Liu Y L,Liu X Z, Zhao W, et al.Progress on glycan analysis of therapeutic glycoprotein . Current Biotechnology,2016, 6(4): 244-248.
[3]	Yang X,Kim S M,Ruzanski R,et al.Μltrafast and high-throughput N-glycan analysis for monoclonal antibodies.MAbs Taylor & Francis,2016, 8(4): 706-717.
[4]	Reusch D, Haberger M, Maier B, et al.Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles-part 1:Separation-based methods.MAbs Taylor & Francis,2015, 7(1): 167-179. doi: 10.4161/19420862.2014.986000 pmid: 25524468
[5]	Yang N,Goonatilleke E,Park D,et al.Quantitation of site-specific glycosylation in manufactured recombinant monoclonal antibody drugs. Analytical Chemistry,2016, 88(14): 7091-7100. doi: 10.1021/acs.analchem.6b00963 pmid: 4955800
[6]	Planinc A,Dejaegher B,Vander Heyden Y,et al. Batch-to-batch N-glycosylation study of infliximab, trastuzumab and bevacizumab, and stability study of bevacizumab. Eur J Hosp Pharm,2017, 24(5): 286-292.
[7]	Sanchez-De Melo I,Grassi P,Ochoa F,et al. N-glycosylation profile analysis of trastuzumab biosimilar candidates by normal phase liquid chromatography and MALDI-TOF MS approaches . Journal of Proteomics,2015, 127: 225-233. doi: 10.1016/j.jprot.2015.04.012 pmid: 25907685
[8]	任跃明. 国内外生物技术类制品质量标准差异浅析. 中国执业药师, 2012, 9(9): 60-64. doi: 10.3969/j.issn.1672-5433.2012.09.012
	Ren Y M.Discussion on the official standards of biotechnological products in Chinese and European Pharmacopeias.China Licensed Pharmacist, 2012, 9(9): 60-64. doi: 10.3969/j.issn.1672-5433.2012.09.012
[9]	高凯, 徐志凯, 任跃明, 等. 关于我国药典单克隆抗体类生物治疗药物总论的思考. 中国生物工程杂志, 2014, 34(1): 127-134. doi: 10.13523/j.cb.20140117
	Gao K, Xu Z K, Ren Y M, et al.Points to consider for the general monograph of monoclonal antibody based biotherapeutics in chinese pharamacopeia .China Biotechnology, 2014, 34(1): 127-134. doi: 10.13523/j.cb.20140117
[10]	Liu M Q, Zeng W F, Fang P, et al.pGlyco 2.0 enables precision N-glycoproteomics with comprehensive quality control and one-step mass spectrometry for intact glycopeptide identification. Nature Communications, 2017, 8(1): 438. doi: 10.1038/s41467-017-00535-2 pmid: 5585273

[1]	史瑞,严景华. 抗新型冠状病毒单克隆中和抗体药物研发进展^*[J]. 中国生物工程杂志, 2021, 41(6): 129-135.
[2]	武瑞君,李治非,张鑫,濮润,敖翼,孙燕荣. 新冠病毒抗体药物研发进展及展望分析[J]. 中国生物工程杂志, 2020, 40(5): 1-6.
[3]	吝建华,韩君,徐寒梅. PD-1/PD-L1免疫检查点抗体药物制剂稳定性开发[J]. 中国生物工程杂志, 2020, 40(10): 35-42.
[4]	刘国芳,刘晓志,高健,王志明. 宿主细胞残留蛋白质对单克隆抗体药物质量影响及其质量控制 ^*[J]. 中国生物工程杂志, 2019, 39(10): 105-110.
[5]	李庆猛,李盛陶,王宁,高晓冬. 酵母来源α-1,2甘露糖转移酶Alg11的异源表达、纯化和活性分析 ^*[J]. 中国生物工程杂志, 2018, 38(6): 26-33.
[6]	杜力,刘晓志,高健,王志明. 抗体药物免疫原性评估的研究进展[J]. 中国生物工程杂志, 2018, 38(2): 89-94.
[7]	任华景, 刘晓志, 王志明, 高健. 中枢神经系统疾病治疗性抗体药物应用进展[J]. 中国生物工程杂志, 2016, 36(9): 54-58.
[8]	陈龙冠, 覃锦红, 黄云娜, 麦俊新, 谢秋玲. 信号肽优化对重组抗体分泌表达的影响及研究进展[J]. 中国生物工程杂志, 2016, 36(3): 77-81.
[9]	马杉姗, 马素永, 赵广荣. 中国抗体药物产业现状与发展前景[J]. 中国生物工程杂志, 2015, 35(12): 103-108.
[10]	王兰, 夏懋, 高凯. 抗体偶联药物的研究进展与质量控制[J]. 中国生物工程杂志, 2014, 34(4): 85-94.
[11]	刘伯宁. 治疗性单抗与抗体产业关键技术[J]. 中国生物工程杂志, 2013, 33(5): 132-138.
[12]	贾伟, 陈熙, 周春喜, 宋兰坤. 单克隆抗体仿制药物的结构分析策略[J]. 中国生物工程杂志, 2012, 32(10): 93-98.
[13]	顾江, 邹全明. 治疗耐甲氧西林金黄色葡萄球菌感染的抗体药物[J]. 中国生物工程杂志, 2012, 32(02): 96-99.
[14]	张莹,何金生,洪涛. 重组抗体药物研究进展及应用[J]. 中国生物工程杂志, 2009, 29(08): 102-106.

Viewed

Full text

Abstract

Cited

Shared

Discussed