药学学报, 2021, 56(9): 2360-2366
引用本文:
李梦林#, 朱文文#, 张金兰*. 基于N-聚糖和完整糖肽的单克隆抗体药物糖基化修饰表征[J]. 药学学报, 2021, 56(9): 2360-2366.
LI Meng-lin#, ZHU Wen-wen#, ZHANG Jin-lan*. N-Glycans and intact glycopeptide-based characterization of N-glycosylation of monoclonal antibody drugs[J]. Acta Pharmaceutica Sinica, 2021, 56(9): 2360-2366.

基于N-聚糖和完整糖肽的单克隆抗体药物糖基化修饰表征
李梦林#, 朱文文#, 张金兰*
中国医学科学院、北京协和医学院药物研究所, 天然药物活性物质与功能国家重点实验室, 北京 100050
摘要:
近年来生物制药产业快速增长,其中单克隆抗体药物的市场规模增速显著,对其进行精确的结构表征和质量控制的需求日益突出。作为抗体药非常重要的翻译后修饰,糖基化对单抗药的疗效、稳定性、免疫原性都具有重要的影响。对糖基化修饰进行表征的主要方式之一是液相色谱串联质谱技术,但该方法目前主要集中在中高丰度的聚糖表征,对低丰度的糖基化修饰研究较少。本研究建立了基于RapiFluor-MS试剂标记的单克隆抗体药物的N-聚糖定性和定量分析技术,该方法样品处理时间短,灵敏度高,不仅能够表征3种单抗药阿达木单抗、贝伐珠单抗和曲妥珠单抗的主要糖型,而且对低丰度糖基化修饰能够准确的表征并定量分析。基于该方法,作者研究了上述3种单抗药主要糖型,并对不同批次的样品中N-聚糖的相对丰度进行了对比。同时在完整糖肽水平对N-聚糖的连接位点和糖型进行解析,进一步丰富了单抗药的N-聚糖结构信息。基于RapiFluor-MS试剂标记的单克隆抗体药物的N-聚糖定性和定量分析技术能够实现对单抗药糖基化修饰的深度表征和控制。
关键词:    液质联用      糖基化修饰      阿达木单抗      贝伐珠单抗      曲妥珠单抗      游离N-聚糖      完整糖肽     
N-Glycans and intact glycopeptide-based characterization of N-glycosylation of monoclonal antibody drugs
LI Meng-lin#, ZHU Wen-wen#, ZHANG Jin-lan*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences&Peking Union Medical College, Beijing 100050, China
Abstract:
In recent years, the biopharmaceutical industry has grown rapidly, and the market size of monoclonal antibody drugs has increased significantly. Accurate structural characterization and quality control are the supporting technologies for the development of monoclonal antibody drugs. As a significant post-translational modification of antibody drugs, glycosylation has an important influence on its efficacy, stability, and immunogenicity. The existing literature usually uses liquid chromatography-mass spectrometry to perform major glycosylation modifications of monoclonal antibody drugs. Characterization, there are few studies on low-abundance glycosylation, but the characterization and control of low-abundance glycosylation cannot be ignored. In this study, we have established a qualitative and quantitative analysis technology for N-glycans based on RapiFluor-MS reagent-labeled monoclonal antibody drugs. This method has a short sample processing time and high sensitivity. It can not only characterize the main glycoforms of three monoclonal antibody drugs (adalimumab, bevacizumab, and trastuzumab) but also can quantify low-abundance N-glycans. The results of the study showed that the main glycoforms specified in the Pharmacopoeia could be detected in different batches of monoclonal antibody drugs, but the content of N-glycans in different batches of samples is not identical. After that, we analyzed the N-glycans connection sites and glycoforms at the intact glycopeptide level, further enriching the N-glycans structure information of the monoclonal antibody. The qualitative and quantitative analysis technology of N-glycans based on RapiFluor-MS reagent-labeled monoclonal antibody drugs can realize the in-depth characterization and control of glycosylation modification of monoclonal antibody drugs.
Key words:    LC-MS/MS    glycosylation    adalimumab    bevacizumab    trastuzumab    free N-glycan    intact glycopeptide   
收稿日期: 2021-04-27
DOI: 10.16438/j.0513-4870.2021-0639
基金项目: 国家重点研发计划(高灵敏度糖蛋白鉴定方法研发及其在药物杂质分析中的应用,2018YFF0212504);中国医学科学院医学与健康科技创新工程(药物药效和安全性相关的关键分析新技术研究,2016-I2M-3-017).
通讯作者: 张金兰,Tel:86-10-83154880,Fax:86-10-63017757,E-mail:zhjl@imm.ac.cn
Email: zhjl@imm.ac.cn
相关功能
PDF(675KB) Free
打印本文
0
作者相关文章
李梦林#  在本刊中的所有文章
朱文文#  在本刊中的所有文章
张金兰*  在本刊中的所有文章

参考文献:
[1] Ecker DM, Jones SD, Levine HL. The therapeutic monoclonal antibody market[J]. MAbs, 2015, 7:9-14.
[2] Zhao CX, Hu ZW, Cui B. Recent advances in monoclonal antibody-based therapeutics[J]. Acta Pharm Sin (药学学报), 2016, 52:837-847.
[3] Liu LM. Antibody glycosylation and its impact on the pharmacokinetics and pharmacodynamics of monoclonal antibodies and Fc-fusion proteins[J]. J Pharm Sci, 2015, 104:1866-1884.
[4] Junttila TT, Parsons K, Olsson C, et al. Superior in vivo efficacy of afucosylated trastuzumab in the treatment of HER2-amplified breast cancer[J]. Cancer Res, 2010, 70:4481-4489.
[5] Yu M, Brown D, Reed C, et al. Production, characterization and pharmacokinetic properties of antibodies with N-linked mannose-5 glycans[J]. MAbs, 2012, 4:475-487.
[6] Kirchhoff CF, Wang XZM, Conlon HD, et al. Biosimilars:key regulatory considerations and similarity assessment tools[J]. Biotechnol Bioeng, 2017, 114:2696-2705.
[7] Zhang L, Luo S, Zhang BL. Glycan analysis of therapeutic glycoproteins[J]. mAbs, 2016, 8:205-215.
[8] Huhn C, Selman MH, Ruhaak LR, et al. IgG glycosylation analysis[J]. Proteomics, 2009, 9:882-913.
[9] Zhang H, Cui Wd, Gross ML. Mass spectrometry for the biophysical characterization of therapeutic monoclonal antibodies[J]. FEBS Lett, 2014, 588:308-317.
[10] Wohlschlager T, Scheffler K, Forstenlehner IC, et al. Native mass spectrometry combined with enzymatic dissection unravels glycoform heterogeneity of biopharmaceuticals[J]. Nat Commun, 2018, 9:1713.
[11] Fornelli L, Ayoub D, Aizikov K, et al. Middle-down analysis of monoclonal antibodies with electron transfer dissociation orbitrap fourier transform mass spectrometry[J]. Anal Chem, 2014, 86:3005-3012.
[12] Leymarie N, Griffin PJ, Jonscher K, et al. Interlaboratory study on differential analysis of protein glycosylation by mass spectrometry:the ABRF glycoprotein research multi-institutional study 2012[J]. Mol Cell Proteomics, 2013, 12:2935-2951.
[13] Taylor PC, Keystone EC, van der Heijde D, et al. Baricitinib versus placebo or adalimumab in rheumatoid arthritis[J]. New Engl J Med, 2017, 376:652-662.
[14] Loupakis F, Cremolini C, Masi G, et al. Initial therapy with FOLFOXIRI and bevacizumab for metastatic colorectal cancer[J]. N Engl J Med, 2014, 371:1609-1618.
[15] Modi S, Saura C, Yamashita T, et al. Trastuzumab deruxtecan in previously treated HER2-positive breast cancer[J]. New Engl J Med, 2019, 382:610-621.
[16] Yu C, Zhang F, Xu G, et al. Analytical similarity of a proposed biosimilar BVZ-BC to bevacizumab[J]. Anal Chem, 2020, 92:3161-3170.
[17] Schiel JE, Rogstad SM, Boyne MT. Comparison of Traditional 2-AB Fluorescence LC-MS/MS and automated LC-MS for the comparative glycan analysis of monoclonal antibodies[J]. J Pharm Sci, 2015, 104:2464-2472.
[18] Stadlmann J, Pabst M, Kolarich D, et al. Analysis of immunoglobulin glycosylation by LC-ESI-MS of glycopeptides and oligosaccharides[J]. Proteomics, 2008, 8:2858-2871.