反相高效液相色谱法检测重组类病毒颗粒疫苗原液中二硫苏糖醇的残留量  

Determination of residual DL-dithiothreitol content in recombinant virus-like particle vaccine by reverse phase-high performance liquid chromatography

作　　者：张学峰[1] 张靖[1] 靳玉琴[1] 陈实[1] 唐芳[1] 李启明[1] 

机构地区：[1]北京生物制品研究所有限责任公司,北京101111

出　　处：《中国生物制品学杂志》2015年第12期1316-1319,共4页Chinese Journal of Biologicals

基　　金：国家科技部重大新药创制(2013ZX09402302)

摘　　要：目的反相高效液相色谱法(reverse phase-high performance liquid chromatography,RP-HPLC)检测重组类病毒颗粒(virus-like particles,VLPs)疫苗原液中二硫苏糖醇(DL-dithiothreitol,DTT)的残留量,并对方法进行验证及初步应用。方法按设定的色谱条件对检测重组VLPs疫苗原液中DTT含量的RP-HPLC法进行专属性、精密性、准确性验证,确定方法的最佳线性范围及最低检测限,用该方法检测3批重组HPV单价疫苗原液中的DTT残留量。结果该方法检测DTT时,不受溶液中溶剂和蛋白的干扰;线性范围为1.5~15.0μg/ml(R^2=0.997),最低检测限为1.5μg/ml;该方法检测不同浓度DTT的平均相对标准偏差(relative standard deviation,RSD)均<5%;重组HPV疫苗原液分别加入3、5、10μg/ml的DTT对照品溶液后,经该方法检测的回收率在80%~120%之间。3批次重组HPV单价疫苗原液中的DTT含量均小于1.5μg/ml。结论本实验的RP-HPLC法专属性、精密性良好,且准确可靠,可用于重组HPV疫苗原液中DTT残留量的检测。Objective To determine the residual DL-dithiothreitol（DTT）content in the bulk of virus-like particle（VLP）vaccine by reverse phase-high performance liquid chromatography（RP-HPLC）, and verify and preliminarily apply the method. Methods Under designed condition, the HPLC was verified for specificity, precision and accuracy, and determined for linear range and minimum detection limit. The residual DTT contents in three batches of monovalent recombinant human papilloma virus（HPV）vaccine were determined by the method. Results The determination result of residual DTT were not interfered by the solvent and protein in solution. The linear range and minimum detection limit of RP-HPLC were 1. 5 ~ 15 μg / ml（R^2 = 0. 997） and 1. 5 μg / ml respectively. The mean relative standard deviations（RSDs）of determination results of DTT at various concentrations were less than 5%. The recovery rates of vaccine bulks added with 3, 5 and 10 μg / ml of DTT control were 80% ~ 120%. The DTT contents in three batches of recombinant HPV vaccine were less than 1. 5 μg / ml. Conclusion The developed HPLC method was accurate and reliable, which showed high specificity and precision, and might be used for the determination of residual DTT content in bulk of recombinant HPV vaccine.

关 键 词：反相高效液相色谱法 重组类病毒颗粒疫苗 二硫苏糖醇 

分 类 号：O657.72[理学—分析化学] R392-33[理学—化学]