Establishment and verification of size exclusion chromatography-multiangle laser-light scattering for detection of molecular weight of meningococcal polysaccharide and polysaccharide-protein conjugates
10.13200/j.cnki.cjb.004311
- VernacularTitle: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
- Author:
ZOU Jian
- Publication Type:Journal Article
- Keywords:
Multiangle laser-light scattering;
Size exclusion chromatography;
Gel permeation chromatography;
Meningococcal polysaccharide;
Polysaccharide-protein conjugates;
Weight-average molecular weight;
Molecular conformation and particle size
- From:
Chinese Journal of Biologicals
2024;37(10):1230-1238
- CountryChina
- Language:Chinese
-
Abstract:
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, 13 μm) 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. 7and 305. 5 kDa, while the polysaccharide-protein conjugates of group A and group C were 6 438 and 23 360 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 KDvalue 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.
