摘要
【背景】口蹄疫疫苗质量评价方法对疫苗生产企业和监管部门进行质量控制尤为重要,146S抗原含量是评价口蹄疫疫苗质量的关键指标。蔗糖密度梯度离心法(sucrose density gradient centrifugation,SDGC)是公认经典的测定口蹄疫146S抗原含量的方法,但存在检测耗时长、过程复杂、重复性差等缺点,影响了疫苗的质量监测。口蹄疫灭活疫苗146S抗原含量高效液相体积排阻色谱法(size-exclusion high-performance liquid chromatography,SE-HPLC)是一种简便、快速、自动化程度高、高效的测定方法。【目的】在初步建立的采用SE-HPLC测定口蹄疫灭活疫苗146S抗原含量方法的基础上,针对不同类型、不同浓缩纯化生产工艺制备的口蹄疫灭活疫苗,进一步确认SE-HPLC法在检测过程中的普适性势在必行。【方法】利用口蹄疫146S抗原标准品建立SE-HPLC法标准曲线,求得回归方程,用于检测样品的146S含量。采用SE-HPLC法和SDGC法分别检测146S抗原标准品1倍、2倍、4倍、8倍、16倍稀释的5个样品和市场上随机选取的22批疫苗,计算146S抗原含量,对比分析两种方法的相关性。用SE-HPLC法,3次重复检测不同企业生产的134批口蹄疫灭活疫苗146S抗原含量,通过色谱图特异性、检测值相对标准偏差(relative standard deviation,RSD)分析SE-HPLC法的重复性,评价该方法的适用性,分析市场流通疫苗的总体质量情况。【结果】SE-HPLC法建立的标准曲线,峰面积与146S抗原含量的线性关系良好(R^2=0.9981,n=8)。口蹄疫146S抗原标准品5个稀释样品和22批口蹄疫疫苗的146S抗原含量检测结果表明,口蹄疫146S抗原含量检测SDGC法和SE-HPLC法高度正相关(RS^2=0.9994,nS=5;Rv 2=0.9602,nv=22)。134批口蹄疫灭活疫苗中,146S抗原含量相对标准偏差RSD<5%的疫苗批次分别占疫苗总批次(134批)、单价苗总批次(18批)、双组分及双价苗总批次(76批)、三价苗总批次(40批)的81.34%、72.22%�
【Background】As vaccine manufacturers and regulatory departments,the quality evaluation method of foot-and-mouth disease(FMD)vaccine is particularly important in quality control,where the 146S antigen is the key index.For the quantification of 146S antigen in FMD vaccine,sucrose gradient density centrifugation(SGDC)is recognized as a classical method but the process is complex,time-consuming and poorly repeatable,which affect the quality monitor of vaccines.Size-exclusion high-performance liquid chromatography(SE-HPLC)is previously reported as a simple,rapid,highly automated and efficient technology for analysis of 146S content.【Objective】A method of 146S antigen determination in inactivated FMD vaccine by SE-HPLC has been preliminarily established.However,the applicability,repeatability and effectiveness of this method are unknown in the FMD vaccines produced by different manufacturers with different concentration and purification processes.Hence,it is imperative to confirm the universality of the SE-HPLC method.【Method】Here,the standard curve and regression equation of SE-HPLC method were established with FMD 146S antigen standard,which were used for 146S antigen detection of samples.Moreover,using five samples with 1,2,4,8 and 16 times dilution of 146S antigen standard and 22 batches of vaccine randomly selected in the market,the 146S antigen was detected by SE-HPLC and SDGC respectively,and the correlation between the two methods was analyzed.Further,134 batches of inactivated FMD vaccines products from different manufacturers in the market were detected three times each for 146S content by SE-HPLC.By analysis of chromatogram specificity of SE-HPLC method and relative standard deviation of 146S content,we confirmed its repeatability and reliability in evaluating all types of vaccine.Finally,146s content was operated for the quality of FMD vaccines in the market.【Result】The standard curve of SE-HPLC method showed good linearity between peak area and concentration of FMD 146S antigen standard(R^2
作者
朱元源
徐嫄
邹兴启
杨延丽
刘丽丽
万建青
李翠
徐璐
张乾义
夏应菊
王兆
郎洪武
王琴
张松平
赵启祖
ZHU YuanYuan;XU Yuan;ZOU XingQi;YANG YanLi;LIU LiLi;WAN JianQing;LI Cui;XU Lu;ZHANG QianYi;XIA YingJu;WANG Zhao;LANG HongWu;WANG Qin;ZHANG SongPing;ZHAO QiZu(China Institute of Veterinary Drug Control,Beijing 100081;National Key Laboratory of Biochemical Engineering/Institute of Process Engineering,Chinese Academy of Sciences,Beijing 100190)
出处
《中国农业科学》
CAS
CSCD
北大核心
2019年第20期3695-3704,共10页
Scientia Agricultura Sinica
基金
国家重点研发计划资助(2018YFC1200501),国家重点研发计划资助(2016YFD0501500)
关键词
高效液相体积排阻色谱法
口蹄疫灭活疫苗
146S
疫苗质量
size-exclusion high-performance liquid chromatography
inactivated foot-and-mouth disease vaccine
146S
quality of the vaccine
