b型流感嗜血杆菌结合疫苗游离多糖含量检测高效阴离子交换色谱-脉冲安培法的建立及验证  

Development and verification of high performance anion exchange chromatography with pulse amperometry detector for determination of free polysaccharide content in Haemophilus influenzae type b conjugate vaccine

作　　者：赵丹[1] 李茂光[1] 毛琦琦[1] 李亚南[1] 陈苏京 叶强[1] ZHAO Dan;LI Mao-guang;MAO Qi-qi;LI Ya-nan;CHEN Su-jing;YE Qiang(National Institutes for Food and Drug Control,NHC Key Laboratory of Research on Quality and Standardization of Biotech Products,Beijing 102629,China)

机构地区：[1]中国食品药品检定研究院国家卫生健康委员会生物技术产品检定方法及其标准化重点实验室,北京102629

出　　处：《中国生物制品学杂志》2022年第1期79-84,共6页Chinese Journal of Biologicals

基　　金：国家科技重大专项(2018ZX09738-005)。

摘　　要：目的建立b型流感嗜血杆菌(Haemophilus influenzae type b,Hib)结合疫苗游离多糖含量检测的超速离心联合高效阴离子交换色谱-脉冲安培(high performance anion exchange chromatography with pulsed amperometric detector,HPAEC-PAD)法,并对该方法进行验证及初步应用。方法将供试品经超速离心后,收集上清获得游离多糖,加入水解液使供试品水解约16 h后,采用CarboPacPA-104 mm×250 mm分析柱与CarboPacPA-104 mm×50 mm保护柱,氢氧化钠-醋酸钠溶液梯度洗脱,检测供试品游离多糖含量。对该方法进行专属性、线性、精密度、稳定性、准确度验证,确定定量限。用建立的方法检测13个批次Hib结合疫苗游离多糖含量,同时与乙醇沉淀法进行比较。结果Hib多糖标准品在0.77~12.26μg/mL范围内,与峰面积呈良好的线性关系,测定5次,线性相关系数均大于0.995;方法定量限为1.875 ng/mL;精密度、稳定性的RSD均小于5%;高、中、低3个浓度的平均加标回收率为100.26%,RSD为3.96%。13个批次Hib结合疫苗中的游离多糖含量为6.76%~16.74%。结论该方法准确可靠,可用于检测Hib结合疫苗游离多糖含量,同时也为其他多糖蛋白结合疫苗游离多糖提取和检测提供了思路。Objective To develop,verify and preliminarily apply a method for determination of free polysaccharide content in Haemophilus infiuenzae type b conjugate vaccine by ultracentrifugation combined with high performance anion exchange chromatography with pulse amperometry detector(HPAEC-PAD).Methods The samples were subjected to ultracentrifugation,of which the supernatant was collected,hydrolyzed with hydrolytic solution for about 16 h,loaded onto CarboPacPA-104 mm×250 mm analytical column and CarboPacPA-104 mm×50 mm guard column,eluted by gradient elution with sodium hydroxide/sodium acetate,and determined for free polysaccharide content.The method was verified for specificity,linearity,precision,stability and accuracy,and determined for quantitative limit.The free polysaccharide contents in 13 batches of Hib conjugate vaccine were determined by the method,and compared with those by ethanol precipitation.Results Hib polysaccharide standards at concentrations of 0.77~12.26μg/mL showed a good linear relationship with peak area,with a linear correlation coefficient of more than 0.995 in five tests.The quantitative limit of the method was 1.875 ng/mL.Both the RSDs in precision and stability tests were less than 5%.The average spike recovery rate of samples at high,moderate and low concentrations was 100.26%,with a RSD of 3.96%.The free polysaccharide contents in 13 batches of Hib conjugate vaccine were 6.76%~16.74%.Conclusion The developed method is accurate and reliable,and may be used for determination of the free polysaccharide content of Hib conjugate vaccine,which provides an idea for the extraction and determination of free polysaccharide in other polysaccharide protein conjugate vaccines.

关 键 词：高效阴离子交换色谱-脉冲安培法 超速离心 B型流感嗜血杆菌 游离多糖 

分 类 号：O657.75[理学—分析化学]