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刊物信息

期刊名称:药物分析杂志
主管单位:中国科学技术协会
主办单位:中国药学会
承办:中国食品药品检定研究院
主编:金少鸿
地址:北京天坛西里2号
邮政编码:100050
电话:010-67012819,67058427
电子邮箱:ywfx@nicpbp.org.cn
国际标准刊号:ISSN 0254-1793
国内统一刊号:CN 11-2224/R
邮发代号:2-237
 

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抗EpCAM+CD3双特异性抗体关键表征解析

Analysis for key characterizations of anti-EpCAM+CD3 bispecific antibody

分类号:R917
出版年·卷·期(页码):2020,40 (4):620-632
DOI: 10.16155/j.0254-1793.2017.01.01
-----摘要:-------------------------------------------------------------------------------------------

目的: 解析抗EpCAM+CD3双特异性抗体(BsAb)的关键表征。方法: 以抗EpCAM+CD3双特异性抗体为研究对象,超高效液相色谱串联质谱联用技术(LC-MS/MS)检测相对分子质量、肽图(氨基酸序列)、N-糖基化位点、糖型及各糖型含量、二硫键位点;表面等离子体共振(SPR)技术测定其与上皮细胞粘附因子(EpCAM)、白细胞分化簇3(CD3)和Fc受体(FcRs)的亲和力。结果: 含糖基化的完整抗体、轻链、重链和单链的相对分子质量分别为128 521.78、24 117.83、50 808.85和53 585.87;不含糖完整抗体、轻链、重链和单链的相对分子质量分别为125 633.30、24 117.85、49 364.53和52 141.30;对象BsAb氨基酸序列与理论序列一致;N-糖基化位点位于重链Asn300和单链Asn326N-连接寡糖的糖型包括以岩藻糖化的双天线复杂N-糖(G0F占全部糖型含量77.10%)为主的11种糖型;对象BsAb内含13对二硫键;单链第Cys105位和第Cys249为游离巯基;SPR测定结果显示:对象BsAb与EpCAM、CD3的亲和力分别为2.71×10-8和5.94×10-7 mol·L-1,与FcγRⅠ、FcγRⅡa、FcγRⅡb、FcγRⅢa、FcγRⅢb和FcRn的亲和力分别为2.89×10-8、8.85×10-7、7.94×10-6、3.69×10-7、2.73×10-6和8.77×10-7 mol·L-1结论: 采用LC-MS/MS和SPR技术可有效解析抗EpCAM+CD3 BsAb的关键表征,为其质量控制和产品放行提供依据。

-----英文摘要:---------------------------------------------------------------------------------------

Objective: To analyze the key characterizations of anti-EpCAM+CD3 bispecific antibody (BsAb). Methods: The relative molecular mass,peptide mapping(amino acid sequence),site of N-glycosylation,N-glycan glycoform and content,and disulfide sites was analyzed with ultra-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The binding affinities with epithelial cell adhesion molecule(EpCAM),cluster of differentiation 3(CD3) and Fc receptors (FcRs) were determined with surface plasmon resonance (SPR). Results: Relative molecular masses of intact antibody,light chain(LC),heavy chain(HC) and single chain(SC) were 128 521.78,24 117.83,50 808.85,and 53 585.87,respectively. Deglycosylation relative molecular masses of intact antibody,light chain,heavy chain and single chain were 125 633.30,24 117.85,49 364.53,and 52 141.30,respectively. The amino acid sequence consistentence with theoretical sequence was confirmed. N-glycosylation sites located at the Asn300 of heavy chain and Asn326 of single chain. There were mainly 11 forms of N-glycans in sample,fucosylated double-antenna complex N-glycan took the vast majority,of which,the G0F glycan occupied 77.10% of the total glycans. Disulfide analysis results showed there was 13 disulfides in BsAb molecular,the Cys105 and Cys249 of single chain were free sulfhydryls. The binding affinities of sample with EpCAM and CD3 were 2.71×10-8 and 5.94×10-7 mol·L-1,respectively. The binding affinities of sample with FcγRⅠ,FcγRⅡa,FcγRⅡb,FcγRⅢa,FcγRⅢb and FcRn were 2.89×10-8,8.85×10-7,7.94×10-6,3.69×10-7,2.73×10-6 and 8.77×10-7 mol·L-1,respectively. Conclusion: The LC-MS/MS method and SPR technology can use to effectively and comprehensively analyze the key characterizations of anti-EpCAM+CD3 BsAb,and provide useful reference data for quality control and product release.

-----参考文献:---------------------------------------------------------------------------------------
[1] 张峰,王开芹,王兰.双特异性抗体研发进展[J].药物分析杂志,2019,39(1):78 ZHANG F,WANG KQ,WAGN L.Development in bispecific antibody[J].Chin J Pharm Anal,2019,39(1):78
[2] FAN GW,WANG ZJ,HAO MG,et al.Bispecific antibodies and their applications[J].J Hematol Oncol,2015,8(1):130
[3] BRINKMANN U,KONTERMANN RE.The making of bispecific antibodies[J].MAbs,2017,9(2):182
[4] SUURS FV,LUB-de HOOGE MN,de VRIES EGE,et al.A review of bispecific antibodies and antibody constructs in oncology and clinical challenges[J].Pharmacol Ther,2019,201:103
[5] LABRIJN AF,JANMAAT ML,REICHERT JM,et al.Bispecific antibodies:a mechanistic review of the pipeline[J].Nat Rev Drug Discov,2019,18(8):585
[6] YU SN,ZHANG J,YAN YX,et al.A novel asymmetrical anti-HER2/CD3 bispecific antibody exhibits potent cytotoxicity for HER2-positive tumor cells[J].J Exp Clin Cancer Res,2019,38(1):355
[7] XIANG T,CHUMSAE C,LIU H.Localization and quantitation of free sulfhydryl in recombinant monoclonal antibodies by differential labeling with 12C and 13C iodoacetic acid and LC-MS analysis[J].Anal Chem,2009,81(19):8101
[8] SPIESS C,ZHAI Q,CARTER PJ.Alternative molecular formats and therapeutic applications for bispecific antibodies[J].Mol Immunol,2015,67(2):956
[9] BECK A,WAGNER-ROUSSET E,AYOUB D,et al.Characterization of therapeutic antibodies and related products[J].Anal Chem,2013,85(2):715
[10] HUANG L,BIOLSI S,BALES KR,et al.Impact of variable domain glycosylation on antibody clearance:an LC/MS characterization[J].Anal Biochem,2006,349(2):197
[11] ALLEN D,BAFFI R,BAUSCH J,et al.Validation of peptide mapping for protein identity and genetic stability.Biologics and biotechnology section,pharmaceutical research and manufacturers of America[J].Biologicals,1996,24(3):255
[12] GATLIN CL,ENG JK,CROSS ST,et al.Automated identification of amino acid sequence variations in proteins by HPLC/microspray tandem mass spectrometry[J].Anal Chem,2000,72(4):757
[13] HIGEL F,SEIDL A,S?RGEL F,et al.N-glycosylation heterogeneity and the influence on structure,function and pharmacokinetics of monoclonal antibodies and Fc fusion proteins[J].Eur J Pharm Biopharm,2016,100:94
[14] LIU L.Antibody glycosylation and its impact on the pharmacokinetics and pharmacodynamics of monoclonal antibodies and Fc-fusion proteins[J].J Pharm Sci,2015,104(6):1866
[15] REUSCH D,TEJADA ML.Fc glycans of therapeutic antibodies as critical quality attributes[J].Glycobiology,2015,25(12):1325
[16] NICOLA B,EMMANUEL S,YOICHI M,et al.Optimizing anti-CD3 affinity for effective T cell targeting againt tumor cells[J].Eur J Immunol,2002,23:3102
[17] 张峰,武刚,于传飞.应用生物膜干涉技术测定治疗性单抗与新生儿Fc受体的亲和力[J].中国新药杂志,2016,25(16):1861 ZHANG F,WU G,YU CF.Detection of affinity between therapeutic monoclonal antibodies and neonatal Fc receptor using biolayer interferometry[J].Chin J New Drugs,2016,25(16):1861
[18] HEISS MM,MURAWA P,KORALEWSKI P,et al.The trifunctional antibody catumaxomab for the treatment of malignant ascites due to epithelialcancer:results of a prospective randomized phase Ⅱ/Ⅲ trial J].Int J Cancer,2010,127(9):2209

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