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目的对高压液相分子排阻层析-十八角激光散射仪(high performance size exclusion chromatography-Multiangle laser-light scattering,HPSEC-MALLS)联用技术分析b型流感嗜血杆菌荚膜多糖重均分子量(weight-average molecular weight,Mw)及其分布的方法进行验证。方法用已知Mw的普鲁兰多糖标准品对方法的准确性、精密度进行验证。采用该方法对15批b型流感嗜血杆菌荚膜多糖进行Mw及分子量分布的测定,并分析b型流感嗜血杆菌荚膜多糖分子大小指标分配系数(KD)与Mw的相关性。结果普鲁兰多糖标准品的Mw检测值与Mw标示值的相对偏差均小于5%(除P-20外);精密性相对标准偏差(relative standard clevia-tion,RSD)均小于0.5%,且色谱峰及分子量分布趋势完全重叠。15批b型流感嗜血杆菌荚膜多糖Mw(3次检测的平均值)为2.681×105~6.488×105 g/mol,与KD值无明显相关性。结论 HPSEC-MALLS法具有良好的准确性与精密度,可更直观、真实地反映样品的分子量分布,本实验为多糖及结合疫苗质量控制方法的优化提供了参考。
Objective To analyze the relationship between the weight average molecular weight of Haemophilus influenzae type b capsular polysaccharide (HPS) and high-pressure liquid chromatography-high angle laser mass spectrometry (HPSEC-MALLS) (weight-average molecular weight, Mw) and its distribution method to verify. Methods The accuracy and precision of the method were verified using pullulan standards of known Mw. The method was used to determine the Mw and molecular weight distribution of 15 batches of Haemophilus influenzae type b (Hib) Haemophilus influenzae capsular polysaccharides, and to analyze the correlation between molecular size distribution coefficient (KD) and Mw of Haemophilus influenzae type b capsular polysaccharide. Results The relative deviation (Mw) of the pullulan standard was less than 5% (except P-20). The relative standard deviations (RSDs) were all less than 0.5% Peaks and molecular weight distribution trends overlap completely. 15 batches of Haemophilus influenzae type b capsular polysaccharide Mw (3 times the average value) was 2.681 × 105 ~ 6.488 × 105 g / mol, and KD values were not significantly correlated. Conclusion The HPSEC-MALLS method has good accuracy and precision, which can more directly and truly reflect the molecular weight distribution of the sample. This experiment provides a reference for the optimization of polysaccharide and conjugate vaccine quality control methods.