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1 生物工程学报 Chinese Journal of Biotechnology DOI: /j.cjb 徐珺等 /LC-MS 对奥马珠单抗与其生物类似物 CMAB007 的分析与结构特征 April 25, 2016, 32(4): Chin J Biotech, All rights reserved 医学与免疫生物技术 497 LC-MS CMAB007 徐珺 1,3, 张大鹏 1,2, 王皓 1,2, 郭怀祖 1, 俆俆,,,. LC-MS CMAB007., 2016, 32(4): Xu J, Zhang DP, Wang H, et al. Structural characterization and analysis of Omalizumab and its biosimilar CMAB007 by LC-MS. Chin J Biotech, 2016, 32(4): 摘要 : 哮喘是当今世界威胁公共健康的最主要的慢性肺部疾病, 作为治疗中重度和难治性哮喘的特效药奥马珠单抗治疗费用相对昂贵, 生物类似药 CMAB007 的研制可以降低治疗费用本研究用质谱分析一种奥马珠单抗生物类似药 CMAB007 与原研药的一致性, 分别从氨基酸分析肽图 N/C 端序列, 还原质谱, 寡糖含量, N 糖分析等层面对 CMAB007 和奥马珠单抗进行了系统的比对研究, 为进一步临床研究打下基础结果表明 CMAB007 和奥马珠单抗氨基酸序列一致, 赖氨酸剪切也基本一致, 带唾液酸修饰糖形和带核心岩藻糖形比例相近, 高甘露糖形比例差异不大,CMAB007 略低于奥马珠单抗因此 CMAB007 满足生物类似药在结构比对上一致性的要求, 有望成为国内首先上市的重组抗人 IgE 单克隆抗体药物 : 单克隆抗体, 奥马珠单抗, 哮喘, 液质联用, 生物类似药 Received: July 24, 2015; Accepted: September 28, 2015 Supported by: National Science and Technology Major Projects for "Major New Drugs Innovation and Development"(No X ), Shanghai Key Technologies R&D Program of Biological Medicine (No ). Corresponding author: Huaizu Guo. Tel: ; Fax: ; guohuaizu@163.com (No X ) (No )

2 498 ISSN CN /Q Chin J Biotech April 25, 2016 Vol.32 No.4 Structural characterization and analysis of Omalizumab and its biosimilar CMAB007 by LC-MS Jun Xu 1,3, Dapeng Zhang 1,2, Hao Wang 1,2, and Huaizu Guo 1,2 1 Shanghai Zhangjiang Biotechnology Co., Ltd., Shanghai , China 2 State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai , China 3 School of Life Sciences, Fudan University, Shanghai , China Abstract: We compared the similarity of Omalizumab (Xolair; a humanized anti-immunoglobulin E monoclonal antibody) and it s biosimilar CMAB007. An in depth characterization of a candidate biosimilar was carried out using a systematic approach, the approach provides a set of routine tools that combine accurate intact mass measurement, peptide mapping, and released glycan profiling. CMAB007 and Omalizumab had the same primary structure and exhibited almost the same content of C-terminal lysine variants. The types of detected free oligosaccharides were very similar, such as sialylation, fucosylation and high mannose types. CMAB007 could be considered as a highly similar molecular to Omalizumab and expected to be the first humanized anti-immunoglobulin E monoclonal antibody drug in China. Keywords: monoclonal antibody, Omalizumab, asthma, liquid chromatography mass spectrometry, biosimilar Tanox IgE IgE FcεRI [1-2] [3] IgE [4-5] 2003 FDA 2014 H1 (12 ) [6] [7] 1 CMAB007 [8] [9] [10] kda [11] [12] [13-14] FDA EMA CFDA

3 徐珺等 /LC-MS 对奥马珠单抗与其生物类似物 CMAB007 的分析与结构特征 499 [10] CFDA ( ) ( ) (Biosimilar) Remisima (CT-P13) [15] (EPAR The Erasmus PhD Association Rotterdam) ( / / ) N C ( ) (LC-ESI-Q-Tof) CMAB007 Omalizumab 1 材料与方法 1.1 材料 (HAc) (MeOH) (ACN) Fisher Scientific MS (NaI) (GFP) Sigma 90% (FA) Fluka MS (NH 4 HCO 3 ) (DTT) Sigma (18.2 MΩ) Millipore Milli-Q PVDF 0.45 um Agela N- (PNGaseF) CMAB007 IgE Omalizumab (Xolair ) 1.2 仪器设备 AKTA Purifier GE Xevo G2-S QTof Waters Biopharmalynx Waters Acquity UPLC H-Class Bio Waters 4 20 SANYO 80 SANYO milli-q Millipore Christ Thermo 1.3 方法 mg/ml 4 1 mol/l DTT MassPREP Desalting Column 2.1 mm 5 mm A 10% +1% B 50% +50% C D 0.4 ml/min V 40 V Biopharmalynx ( ) ESI cjb@im.ac.cn

4 500 ISSN CN /Q Chin J Biotech April 25, 2016 Vol.32 No mol/l +5 mmol/l EDTA+0.5 mol/l Tris (ph 8.3) 1 mol/l DTT 60 mmol/l (37 60 min) 2.9 mol/l 45 min 1 mol/l DTT 50 mmol/l NH 4 HCO 3 ( G25 ) ( / 1 50 (W/W)) (37 ) 4 h ph min A 10% +1% B 50% +50% C D 0.2 ml/min 45 (m/z) mg 30 kda 50 mmol/l (ph 8.0) 50 1 PNGaseF 100 μl h HILIC 30% +70% DMSO 2-AB 50 mg/ml 60 mg/ml 65 5 h HILIC ACQUITY UPLC BEH300 GLycan 1.7 μm 2.1 mm 150 mm 0 40 min B 30 mmol/l ph C 0.4 ml/min 60 (m/z) 结果与分析 2.1 单抗完整蛋白质谱分析 [16] IgE (CMAB007) ( 1) CMAB Da 图 1 Fig. 1 奥马珠单抗 ( 上 ) 和 CMAB007 ( 下 ) 完整蛋白去卷积处理图谱 Deconvoluted mass spectrum of intact Omalizumab (up) and CMAB007 (down).

5 徐珺等 /LC-MS 对奥马珠单抗与其生物类似物 CMAB007 的分析与结构特征单抗经 DTT 还原后的轻链重链分析 DTT 2 CMAB Da Da 162 Da G0F G1F Da G2F Biopharmalynx C FC [17] 图 2 Fig. 2 奥马珠单抗 ( 上 ) 和 CMAB007 ( 下 ) 轻链去卷积处理图谱 Deconvoluted mass spectrum of the light chain of Omalizumab (up) and CMAB007 (down). 图 3 Fig. 3 奥马珠单抗 ( 上 ) 和 CMAB007 ( 下 ) 重链去卷积处理图谱 Deconvoluted mass spectrum of the heavy chain of Omalizumab (up) and CMAB007 (down) cjb@im.ac.cn

502 ISSN 1000-3061 CN 11-1998/Q Chin J Biotech April 25, 2016 Vol.32 No.4 2.3 肽图分析 C 18 Biopharmalynx 1.3.3 CMAB 4 5 1) CMAB007 100% 100% 100% 99% 2) N- N- DIQLTQ SPSSLSASVGDR 18 CMAB007 N- 1 860.

6 502 ISSN CN /Q Chin J Biotech April 25, 2016 Vol.32 No 肽图分析 C 18 Biopharmalynx CMAB 4 5 1) CMAB % 100% 100% 99% 2) N- N- DIQLTQ SPSSLSASVGDR 18 CMAB007 N Da Da Da 图 4 CMAB007 质量肽图谱 TIC 图 Fig. 4 Total ion chromatogram of CMAB007 peptide mapping.

7 徐珺等 /LC-MS 对奥马珠单抗与其生物类似物 CMAB007 的分析与结构特征 503 图 5 Fig. 5 奥马珠单抗质量肽图谱 TIC 图 Total ion chromatogram of Omalizumab peptide mapping. N- EVQLVE SGGGLVQPGGSLR 19 CMAB007 N Da Da Da 3) C- C- CMAB % 93.5% 2.4 游离寡糖分析 -Fab Fc [18] (CDC) (ADCC) ADCC CDC cjb@im.ac.cn

8 504 ISSN CN /Q Chin J Biotech April 25, 2016 Vol.32 No.4 [19] CDC (Galactosylation) IgG1 CDC (Fucosylation) IgG1 ADCC (Man5 Man6 Man7) IgG1 ( G0F ) ADCC CDC [20] 6 7 CMAB007 2-AB CHO G0F G1F CMAB % 89.99% 0.38% 0.60% CMAB % 4.23% ( 0.1% Man5 Man6) CMAB007 图 6 CMAB007 游离寡糖荧光检测 FLR 积分图 ( 积分含量在 0.1% 以上 ) Fig. 6 Fluorescence chromatogram of released free glycans from CMAB007 (content above 0.1%).

9 徐珺等 /LC-MS 对奥马珠单抗与其生物类似物 CMAB007 的分析与结构特征 505 图 7 奥马珠单抗游离寡糖荧光检测 FLR 积分图 ( 积分含量在 0.1% 以上 ) Fig. 7 Fluorescence chromatogram of released free glycans from Omalizumab (content above 0.1%). 3 结论 (LC-ESI-Q-Tof) IgE CMAB007 N- C- CMAB007 CMAB007 Omalizumab ( ) IgE REFERENCES [1] Buhl R. Omalizumab (Xolair ) improves quality of life in adult patients with allergic asthma: a review. Resp Med, 2003, 97(2): [2] Gould HJ, Sutton BJ. IgE in allergy and asthma today. Nat Rev Immunol, 2008, 8(3): [3] Chan AC, Carter PJ. Therapeutic antibodies for autoimmunity and inflammation. Nat Rev Immunol, 2010, 10(5): [4] Simons F. Anaphylaxis. J Allergy Clin Immunol, 2008, 121(2): S402 S407. [5] Catley MC, Coote J, Bari M, et al. Monoclonal antibodies for the treatment of asthma. Pharmacol Therapeut, 2011, 132(3): [6] Maurer M, Rosén K, Hsieh H J, et al. Omalizumab cjb@im.ac.cn

10 506 ISSN CN /Q Chin J Biotech April 25, 2016 Vol.32 No.4 for the treatment of chronic idiopathic or spontaneous urticaria. N Engl J Med, 2013, 368(10): [7] Lai TW, Wang SB, Xu ZW, et al. Long-term efficacy and safety of omalizumab in patients with persistent uncontrolled allergic asthma: a systematic review and meta-analysis. Sci Rep, 2015, 5: [8] Qian WZ, Zhang XM, Li BH, et al. Development and characterization of a novel anti-ige monoclonal antibody. Biochem Biophys Res Commun, 2010, 395(4): [9] Reichert JM, Beck A, Iyer H. European Medicines Agency workshop on biosimilar monoclonal antibodies: July 2, 2009, London, UK. mabs, 2009, 1(5): [10] Beck A, Diemer H, Ayoub D, et al. Analytical characterization of biosimilar antibodies and Fc-fusion proteins. TrAC-Trends Anal Chem, 2013, 48: [11] Fekete S, Gassner AL, Rudaz S, et al. Analytical strategies for the characterization of therapeutic monoclonal antibodies. TrAC-Trends Anal Chem, 2013, 42: [12] Xie XG, Wu J. Study on status and countermeasures about biosimilars in China. China Biotechnol, 2012, 32(3): (in Chinese).,.., 2012, 32(3): [13] McCamish M, Woollett G. Worldwide experience with biosimilar development. mabs, 2011, 3(2): [14] Hirsch BR, Lyman GH. Biosimilars: a cure to the U.S. health care cost conundrum? Blood Rev, 2014, 28(6): [15] Jung SK, Lee KH, Jeon JW, et al. Physicochemical characterization of Remsima. mabs, 2014, 6(5): [16] Wei W, Luo JH, Bai Y. General considerations of quality study for biosimilar products containing recombinant monoclonal antibodies. Chin Pharm J, 2014, 49(18): (in Chinese).,,.., 2014, 49(18): [17] Antes B, Amon S, Rizzi A, et al. Analysis of lysine clipping of a humanized Lewis-Y specific IgG antibody and its relation to Fc-mediated effector function. J Chromatogr B Analyt Technol Biomed Life Sci, 2007, 852(1/2): [18] Wagner-Rousset E, Bednarczyk A, Bussat MC, et al. The way forward, enhanced characterization of therapeutic antibody glycosylation: comparison of three level mass spectrometry-based strategies. J Chromatogr B Analyt Technol Biomed Life Sci, 2008, 872(1/2): [19] Kuribayashi R, Hashii N, Harazono A, et al. Rapid evaluation for heterogeneities in monoclonal antibodies by liquid chromatography/mass spectrometry with a column-switching system. J Pharm Biomed Anal, 2012, 67 68: 1 9. [20] Goetze AM, Liu YD, Zhang ZQ, et al. High-mannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans. Glycobiology, 2011, 21(7): ( 本文责编陈宏宇 )

材料与方法仪器 ACQUITY UPLC Xevo TQ-S 三重四级杆液质联用仪 ( 美国 Waters 公司 ); 电喷雾离子源 (ESI), Masslynx 4.1 数据处理系统 (Waters);Thermo 低温离心机 ;KQ

材料与方法仪器 ACQUITY UPLC Xevo TQ-S 三重四级杆液质联用仪 ( 美国 Waters 公司 ); 电喷雾离子源 (ESI), Masslynx 4.1 数据处理系统 (Waters);Thermo 低温离心机 ;KQ [ 应用纪要 ] Rapid identification of 5 illegal additives in slimming health food by Xevo TQ-S 郝刚苏州市食品药品检验所, 苏州 15104 关键词超高效液相色谱 - 质谱, 多反应监测, 减肥类保健食品非法添加, 快速定性定量