脑膜炎球菌疫苗多糖及多糖蛋白结合物分子量尺寸排阻色谱-多角度激光光散射联用检测方法的建立及验证  

Establishment and verification of size exclusion chromatography⁃multiangle laser⁃light scattering for detection of molecular weight of meningococcal polysaccharide and polysaccharide⁃protein conjugates

作　　者：邹剑 杨蕾 李茂光[2] 叶强[2] 焦旭雯[3] 梁蔚阳[3] 郭中平[4] 马晶 姚宇峰[5] 李炎 ZOU Jian;YANG Lei;LI Maoguang;YE Qiang;JIAO Xuwen;LIANG Weiyang;GUO Zhongping;MA Jing;YAO Yufeng;LI Yan(Sichuan Institute for Drug Control,NMPA Key Laboratory for Quality Control and Evaluation of Vaccine and Biological Products,Chengdu 610097,Sichuan Province,China;不详)

机构地区：[1]四川省药品检验研究院国家药品监督管理局疫苗及生物制品质量控制与评价重点实验室,四川成都6100972 [2]中国食品药品检定研究院,北京102629 [3]广东省药品检验所,广东广州510663 [4]国家药典委员会,北京100061 [5]中国医学科学院生物学研究所,云南昆明650118

出　　处：《中国生物制品学杂志》2024年第10期1230-1238,共9页Chinese Journal of Biologicals

基　　金：云南省科技攻关计划(202002AA100009);国家药典委员会课题(2020S05)。

摘　　要：目的建立尺寸排阻色谱-多角度激光光散射(size exclusion chromatography-multiangle laser-light scattering,SEC-MALLS)联用测定脑膜炎球菌多糖及多糖蛋白结合物分子量的方法,并进行验证及初步应用,以期进一步提高该品种质量控制水平。方法建立SEC-MALLS联用方法检测脑膜炎球菌多糖及多糖蛋白结合物分子量,色谱条件:流动相为0.9%氯化钠溶液,色谱分离柱为Shodex OHpak LB-806 HQ(300 mm×8.0 mm,13μm),流速为0.5 mL/min,柱温为35℃。对方法的系统适用性、准确性、精密性、耐用性进行验证,并用该方法检测不同厂家来源的样品,同时与现行药典质量标准方法进行相关性分析。结果普鲁兰糖标准品除STD P-100以外,STD P-50 STD P-800重均分子量(Mw)实测值与标示值相对误差和精密度小于5.0%,可作为系统适应性样品;重复性、中间精密度RSD均不大于5.0%。多糖及多糖蛋白结合物在配制完成48 h内稳定性良好。A、C、Y、W135群多糖平均Mw分别为257.5、377.3、399.7、305.5 kDa;A、C群多糖蛋白结合物分别为6438、23360 kDa。71批多糖样品中有5批次A、C群结果超趋势,其余批次批内和批间差异均在均值±3 SD的范围以内。SEC-MALLS法与现行药典质量标准方法测定结果除Y群多糖分配系数(KD)与Mw呈一定的负相关性外,两种方法在其余多糖及多糖蛋白结合物中相关性并不显著。结论SEC-MALLS方法能直接测定脑膜炎球菌疫苗多糖及多糖蛋白结合物分子量,具有操作简单、快速、准确、能一定程度反映分子结构信息等优点,有利于提升该关键质量参数的质量控制水平。Objective To establish a method for the determination of molecular weight of meningococcal polysaccharide and polysaccharide-protein conjugates by size exclusion chromatography-multiangle laser-light scattering(SEC-MALLS),and to verify and apply the method,so as to improve the quality control level of meningococcal vaccines.Methods A SEC-MALLS method was established to detect the molecular weight of polysaccharide and polysaccharide-protein conjugates of meningococcus.The chromatographic conditions were as follows:0.9%sodium chloride solution as the mobile phase;Shodex OHpak LB-806 HQ(300 mm×8.0 mm,13μm)as the column;the flow rate at 0.5 mL/min;the column temperature at 35℃.The systematic applicability,accuracy,precision and durability of the method were verified,and the samples from different manufacturers were detected by this method.In addition,the correlation between SEC-MALLS and the existing pharmacopoeia quality standard methods was analyzed.Results Except for STD P-100,the relative error and precision between the measured and marked values of weight-average molecular weight(Mw)of STD P-50 to STD P-800 were less than 5.0%,indicating that the pullulan standard could be used as the systematic adaptive sample.The RSDs of reproducibility and intermediate precision were not more than 5.0%.The polysaccharide and polysaccharide-protein conjugates had good stability within 48 h after preparation.The average Mw of polysaccharides from group A,C,Y and W135 were 257.5,377.3,399.7 and 305.5 kDa,while the polysaccharide-protein conjugates of group A and group C were 6438 and 23360 kDa,respectively.Among the 71 batches of polysaccharide samples,the results of group A and group C in five batches exceeded the trend,and the inner-and inter-batch differences of other batches were within the range of mean±3 SD.Except the KD value of polysaccharide in group Y had a certain negative correlation with Mw,there was no significant correlation between the two methods in the remaining polysaccharide and polysaccharide-protein c

关 键 词：多角度激光光散射 尺寸排阻色谱 凝胶渗透色谱 脑膜炎球菌多糖 多糖蛋白结合物 重均分子量 分子构象和粒径 

分 类 号：R917[医药卫生—药物分析学]