HL60-pNL3.2报告基因热原检查法测定疫苗热原的适用性研究

Adaptability Research of HL60-pNL3.2 Reporter Gene Assay in Pyrogen Detection of Vaccines

关键词:  热原测定  报告基因法  水痘减毒活疫苗  流感病毒裂解疫苗  冻干甲型肝炎减毒活疫苗

Key words:  pyrogen detection  reporter gene assay  live attenuated varicella vaccine  inactivated influenza vaccine  lyophilized live attenuated hepatitis A vaccine

摘要:

目的 探讨HL60-pNL3.2报告基因热原检查法在水痘减毒活疫苗、流感病毒裂解疫苗、冻干甲型肝炎减毒活疫苗热原测定中的应用。方法 参照中国药典“体外热原检查法(报告基因法)”公示稿，计算3种疫苗最大有效稀释倍数(maximum valid dilution，MVD)，干扰实验探讨3种疫苗对该方法测定热原是否存在干扰。参照中国药典2020年版通则9101对该方法测定3种疫苗热原进行方法学验证，用经验证的方法测定3种疫苗热原含量。结果 水痘减毒活疫苗、流感病毒裂解疫苗、冻干甲型肝炎减毒活疫苗MVD分别为1 000，200和1 000倍。在MVD范围内，内毒素(endotoxin，LPS)在3种疫苗中的回收率在52.5%~110.1%。方法学验证结果显示该方法可用于3种疫苗热原测定，LPS浓度与其化学光强度(RLU值)相关系数(R²)均>0.98；LPS浓度在1~250 EU·mL^–1，回收率结果在76.4%~192.8%。LPS浓度>5 EU·mL^–1时，重复性变异度均<25%；该方法用于3种疫苗热原测定的检测限和定量限在0.05~0.13 EU·mL^–1。用建立的HL60-pNL3.2报告基因热原检查法测定3种疫苗热原含量，均低于污染物限值。结论 HL60-pNL3.2报告基因热原检查法具有不使用动物、操作简单、快速、检测热原谱广，可对热原进行定量的优点，可用于水痘减毒活疫苗、流感病毒裂解疫苗和冻干甲型肝炎减毒活疫苗热原测定。

Abstract:

OBJECTIVE To explore the application of HL60-pNL3.2 reporter gene assay in pyrogen detection of live attenuated varicella vaccine, inactivated influenza vaccine and lyophilized live attenuated hepatitis A vaccine. METHODS According to the publication draft named as "in vitro pyrogen test(reporter gene assay)" in Chinese Pharmacopoeia, maximum valid dilution(MVD) of three kinds of vaccines was calculated, and the interference test was conducted to explore whether these vaccines interfere with the pyrogen determination. According to the general rule 9101 from Chinese Pharmacopoeia 2020, the assay in the pyrogen detection of the three vaccines was validated, using the methods to determine the pyrogen content of three vaccines. RESULTS The MVD of live attenuated varicella vaccine, inactivated influenza vaccine and lyophilized live attenuated hepatitis A vaccine respectively were 1 000, 200 and 1 000 times. The recovery rates of endotoxin(LPS) in the three kinds of vaccine were between 52.5% and 110.1%. The methodological validation results showed that the method could be used for the determination of pyrogens in three vaccines, and the correlation coefficient(R²) between LPS concentration and its chemical light intensity(RLU value) was > 0.98, the recovery rates were between 76.4% and 192.8% when LPS concentration was between 1 and 250 EU·mL^–1. The coefficient of variation of measured results was less than 25% when LPS concentration was more than 5 EU·mL^–1. The limit of detection and the limit of quantitation were between 0.05 and 0.13 EU·mL^–1. Then, the pyrogen of these vaccines was determined by HL60-pNL3.2 reporter gene assay, and the pyrogen concentration all was less than contaminant limit concentration. CONCLUSION HL60-pNL3.2 reporter gene assay has the advantages of no animal, simple and rapid operation, with wide pyrogen spectrum and can quantitative pyrogen, which can be used to detect pyrogen of live attenuated varicella vaccine, inactivated influenza vaccine and lyophilized live attenuated hepatitis A vaccine.