重组戊型肝炎疫苗类病毒颗粒纯度高效分子排阻色谱检测方法的建立、验证及应用

Validation and application of purity test method for recombinant hepatitis E vaccine virus particles by SEC-HPLC

目的建立检测重组戊型肝炎疫苗类病毒颗粒(hepatitis E virus-like particle,HE-VLP)纯度的高效分子排阻色谱法(size-exclusion high performance liquid chromatography,SEC-HPLC),并对该方法进行验证及应用。方法利用TSK G5000 PW色谱柱(7. 5 mm×30 cm)和LC-20AT高效液相色谱系统检测HE-VLP纯度,并对该方法的专属性、线性、重复性、中间精密度、耐用性进行验证,确定检测限及定量限。应用建立的方法检测戊型肝炎病毒(hepatitis E virus,HEV)疫苗原液以及纯化工艺过程中粗纯液、超滤液、超滤透过液、层析纯化液纯度;将蛋白原液与铝佐剂进行吸附制备疫苗半成品,应用建立的方法检测游离蛋白含量。结果空白溶剂在设定的积分范围内无特异峰出现;样品浓度在50~500μg/mL时,溶液浓度与吸收峰峰面积呈线性相关,相关系数R2=0. 999;6次重复进样峰面积相对标准偏差(RSD)为0. 47%;不同实验人员间隔7 d对相同样品进行5次检测,RSD均小于2. 0%;将样品浓度2μg/mL,进样体积20μL,即0. 04μg定为检测限,将样品浓度4μg/mL,进样体积20μL,即0. 08μg定为定量限;耐用性试验中,PBS浓度在0. 05~0. 001 mol/L范围内,保留时间RSD为0. 89%,峰面积RSD为0. 94%。该方法可在20 min内完成检测,原液纯度均不低于95%;随着蛋白纯化步骤的进行,纯度逐步显著提高;吸附完全性检测显示灵敏度较为理想。结论建立的SEC-HPLC法各项指标均符合要求,可用于HE-VLP蛋白的纯度检测。

Objective To develop a size-exclusion high performance liquid chromatography(SEC-HPLC) assay for determination of purity of hepatitis E virus-like particles(HE-VLPs). Methods The purity of HE-VLPs was determined by TSK G5000 PW column(7. 5 mm × 30 cm) chromatography and LC-20 AT high performance liquid chromatography(HPLC) system. The method was validated for specificity,linearity,reproducibility,intermediate precision and durability,and determined for limit of detection(LOD)and limit of quantitation(LOQ). The purities of bulk of HEV vaccine as well as samples collected during crude purification,ultrafiltration and further purification by chromatography were determined by the developed method. Final bulk was prepared by adsorption of the bulk with aluminum adjuvant and determined for protein content by the developed method. Results There was no specific peak in the set range of blank solution,while the sample concentration at a range of 50 ~ 500 μg/mL showed linear relationship to the absorption peak area(R2 = 0. 999). The relative standard deviation(RSD) of peak area in six tests was 0. 47%. However,the RSD of results of the same samples in five tests by two laboratory personnel on two working days at an interval of 7 d was less than 2. 0%. The LOD and LOQ were served as 0. 04 μg(sample concentration 2 μg/mL,sample load 20 μL) and0. 08 μg(sample concentration 4 μg/mL,sample load 20 μL) respectively. In durability test,the RSDs of retention time and peak area were 0. 89% and 0. 94% respectively at a PBS concentration of 0. 05 ~ 0. 001 mol/L. Conclusion All the indicators of developed SEC-HPLC method met the relevant requirements,indicating that the method might be used for determination of purity of HE-VLPs.