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

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 conjugates.Conclusion SEC-MALLS can directly determine the molecular weight of polysaccharide and polysaccharide-protein conjugates of meningococcal vaccine with the advantages of fast,accurate and simple operation,and can reflect the molecular structure information to a certain extent,which is conducive to improve the quality control of this key quality parameter.