治疗性单克隆抗体SIBP-A N-糖谱鉴定方法的建立及验证  

Development and verification of an N-glycans identification method for therapeutic monoclonal antibody SIBP-A

作　　者：张琳 沈培玉 袁宁 李清弘 陈慧聪 邱建华 李翱翔 马雷钧 梁红远 ZHANG Lin;SHEN Peiyu;YUAN Ning;LI Qinghong;CHEN Huicong;QIU Jianhua;LI Aoxiang;MA Leijun;LIANG Hongyuan(Shanghai Institute of Biological Products,Co.,Ltd.,Shanghai 200051,China)

机构地区：[1]上海生物制品研究所有限责任公司,上海200051

出　　处：《中国生物制品学杂志》2025年第1期27-33,共7页Chinese Journal of Biologicals

基　　金：上海市2021年度“科技创新行动计划”生物医药领域科技支持项目(21S11909900)。

摘　　要：目的建立超高液相-荧光(ultra-high performance liquid chromatography-fluorescence detection,UPLC-FLD)方法鉴定治疗性单克隆抗体SIBP-A的N-糖型修饰,并对方法进行验证,以确定该方法可用于该单克隆抗体的放行和稳定性检测。方法以治疗性单克隆抗体SIBP-A为研究对象,基于超高液相色谱-飞行时间质谱(ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry,UPLC-Q-Tof-MS)技术对其N-糖型进行鉴定,再用UPLC-FLD方法对其N-糖型进行定量分析;对建立的UPLC-FLD方法的专属性、重复性、中间精密度、线性和范围、耐用性进行验证,同时进行-30、10℃条件下样品稳定性试验,并与室温条件下处理后样品的检测结果进行比较。结果UPLC-FLD方法主要检测到10种糖型分子,质谱鉴定主要糖型为M3B、F(6)A1、A2、F(6)A2、Man5、F(6)A2[6]G(4)1、F(6)A2[3]G(4)1、A2[6]G(4)1、A2[3]G(4)1和F(6)A2G(4)2。SIBP-A正常出峰,80%乙腈溶液在其出峰处无明显干扰峰;平行制备的6份样品检测结果中不含半乳糖的寡糖峰面积百分比之和相对标准偏差(relative standard deviation,RSD)为0.8%;2人3次检测结果中不含半乳糖的寡糖峰面积百分比之和的RSD为1.4%;在320~480μg样品范围内,不含半乳糖的寡糖总峰面积之和与样品蛋白含量呈线性关系,线性决定系数(R^(2))为0.97;pH 4.4、4.5、4.6流动相A下不含半乳糖寡糖峰面积百分比之和的RSD分别为0.2%、0.1%、0.1%,不同pH流动相A下不含半乳糖的寡糖峰面积百分比之和的RSD为0.8%;处理后样品-30、10℃分别避光放置24 h后,不含半乳糖的寡糖峰面积百分比之和的RSD分别为0.5%、0.6%。结论基于UPLC-Q-Tof-MS技术可有效鉴定治疗性单克隆抗体SIBP-A的N-糖类型,建立的UPLC-FLD方法可有效对其N-糖型进行定量分析,且该方法专属性、重复性、中间精密度、线性及范围、耐用性良好,检测-30、10℃存放样品的稳定性也均良好,可�Objective To develop and verify an ultra-high performance liquid chromatography-fluorescence detection(UPLCFLD)system for the measurement of N-glycans of therapeutic monoclonal antibody SIBP-A,in order to validate the method for the release and stability detection of the monoclonal antibodies.Methods The therapeutic monoclonal antibody SIBP-A was selected as the research subject,the N-glycans of which were identified by using ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry(UPLC-Q-Tof-MS),and then were quantitatively analyzed by UPLCFLD.The specificity,repeatability,intermediate precision,linearity and range,as well as robustness of the developed UPLCFLD method were verified.The stability tests of samples at-30 and 10℃were performed,and the detection results were compared with those of samples treated at room temperature.Results Ten main types of glycan molecules were detected by UPLC-FLD,which were identified as M3B,F(6)A1,A2,F(6)A2,Man5,F(6)A2[6]G(4)1,F(6)A2[3]G(4)1,A2[6]G(4)1,A2[6]G(4)1,A2[3]G(4)1 and F(6)A2G(4)2 by mass spectrometry.The peak of SIBP-A was normal,and 80%acetonitrile solution registered no significant interference peak at the sample peak.The relative standard deviation(RSD)of the sum of galactosefree oligosaccharides'peak area percentage was 0.8%among six parallel samples.In the three tests by two operators,the RSD of the sum of galactose-free oligosaccharides'peak area percentage was 1.4%.There existed a linear relationship between the protein content within a sample range of 320 to 480μg and the sum of peak areas for galactose-free oligosaccharides with the R^(2)of 0.97.The RSDs of the sum of galactose-free oligosaccharide peak area percentage were determined to be0.2%,0.1%,and 0.1%at pH 4.4,4.5,and 4.6 mobile phase A,respectively,and the RSD of the percentage of overall galactose-free oligosaccharide peak area across different pH mobile phase A was found to be 0.8%.Additionally,the RSDs of the sum of galactose-free oligosaccharide peak area per

关 键 词：治疗性单克隆抗体 超高液相色谱-飞行时间质谱 超高液相-荧光技术 N-糖型 稳定性 

分 类 号：R917[医药卫生—药物分析学]