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128521.78、24117.83、50808.85和53585.87;不含糖完整抗体、轻链、重链和单链的相对分子质量分别为125633.30、24117.85、49364.53和52141.30;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Asn326;N-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。
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 chromatographytandem 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 128521.78,24117.83,50808.85,and 53585.87,respectively.Deglycosylation relative molecular masses of intact antibody,light chain,heavy chain and single chain were 125633.30,24117.85,49364.53,and 52141.30,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.
作者
张红梅
张峰
武刚
王文波
于传飞
段茂芹
黄璟
王兰
刘万卉
ZHANG Hong-mei;ZHANG Feng;WU Gang;WANG Wen-bo;YU Chuan-fei;DUAN Mao-qin;HUANG Jing;WANG Lan;LIU Wan-hui(School of Pharmcay,Yantai University,Yantai 264005,China;Division of Monoclonal Antibody,National Institutes for Food and Drug Control,Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products,Beijing 102629,China;School of Life Science and Biopharmaceutics,Shenyang Pharmaceutical University,Shenyang 110016,China)
出处
《药物分析杂志》
CAS
CSCD
北大核心
2020年第4期620-632,共13页
Chinese Journal of Pharmaceutical Analysis
基金
重大新药创制科技重大专项——“创新生物技术药评价及标准化研究”子课题3:免疫检查点单抗和其他创新生物技术药的质量评价方法和标准研究(2018ZX09101-001-003)。
