核酸酶在人用狂犬病疫苗(Vero细胞)生产中的筛选及安全性评价  

Screening and safety assessment of nucleases in the manufacture of human rabies vaccine(Vero cells)

作　　者：张海平 曾智 周慧明 刘宝峰 张怡轩[1] 石亮 Zhang Haiping;Zeng Zhi;Zhou Huiming;Liu Baofeng;Zhang Yixuan;Shi Liang(School of Life Sciences and Biopharmaceutical,Shenyang Pharmaceutical University,Shenyang 110016,China;Liquid Workshop,Jilin Huikang Biological Pharmaceutical Co.,Ltd.,Changchun 130012,China;Shandong Xinbo Drug Safety Evaluation Research Center,Dezhou 251500,China;Changchun Baike Biotechnology Co.,Ltd.,Changchun 130015,China)

机构地区：[1]沈阳药科大学生命科学与生物制药学院,沈阳110016 [2]吉林惠康生物药业有限公司原液车间,长春130012 [3]山东欣博药物安全评价研究中心,德州251500 [4]长春百克生物科技股份公司,长春130015

出　　处：《国际生物制品学杂志》2024年第6期371-377,共7页International Journal of Biologicals

基　　金：吉林省科技发展计划(20200404119YY)。

摘　　要：目的在生产以Vero细胞为基质的液体人用狂犬病疫苗过程中,添加不同厂家的核酸酶,筛选合适的核酸酶处理条件,评价该条件下液体人用狂犬病疫苗的安全性。方法温度25℃、37℃下,向病毒液中加入酶解浓度为0.15、0.30 U/ml的核酸酶,经层析系统纯化后采用β-丙内酯灭活,制备成原液及成品。采用PCR法和ELISA分别检测原液DNA残留量和核酸酶残留量,筛选出酶解温度和酶解浓度;采用PCR法分析原液中Vero细胞残留DNA片段和检测残留量;通过NIH法检测成品的效价来评价免疫原性,开展SD大鼠单次给药毒性试验和大耳白兔肌肉刺激性试验评价产品安全性。结果筛选酶解浓度为0.15 U/ml,酶解温度为25℃;残留DNA符合中国药典2020年版要求,核酸酶残留量在试剂盒检测下限0.2 ng/ml以下;残留DNA大于221 bp片段占DNA总残留量的比例少于25%;大鼠毒性试验和大耳白兔给药后未见异常,组织病理学检查均未见病理学改变。结论筛选到合适的酶解温度和酶解浓度,可将Vero细胞的DNA酶解成较小的片段,降低Vero细胞DNA残留量;动物安全性试验评价安全可靠,为Vero细胞基质疫苗的研发与应用奠定理论依据。Objective To evaluate the safety of liquid human rabies vaccine by adding nucleases from different manufacturers and screening suitable nuclease treatment conditions in the process of producing liquid human rabies vaccine based on Vero cells.Methods At 25℃and 37℃,nuclease at enzymolysis concentrations of 0.15 and 0.30 U/ml was added to the virus stock.After purification through the chromatographic system,β-propiolactone was utilized for inactivation to prepare the bulk and the final product.The PCR method and ELISA were employed to detect the DNA residue and nuclease residue in the bulk,and the enzymolysis temperature and concentration were screened.PCR was utilized to analyze the residual DNA fragments of Vero cells in the bulk and the residual amount was determined.The immunogenicity was evaluated by the NIH assay.The safety of the product was assessed by the single-dose toxicity test in SD rats and the muscle irritation test in large ear white rabbits.Results The selected concentration of enzymolysis was 0.15 U/ml,and the enzymolysis temperature was 25℃.The residual DNA fulfilled the requirements of the Chinese pharmacopoeia 2020 edition,and the residual nuclease was beneath the detection limit of 0.2 ng/ml.Fragments of residual DNA larger than 221 bp constituted less than 25%of the total residual DNA.The product supplemented with nuclease demonstrated better immunogenicity.No abnormality was identified in the toxicity tests on rats and large ear white rabbits,and no pathological alterations were observed in the histopathological examination.Conclusions The DNA of Vero cells is enzymatically hydrolyzed into smaller fragments,and the residual amount of DNA in Vero cells is decreased when the appropriate enzymolysis temperature and concentration are selected.The animal safety test proves the safety and reliability of the vaccine,laying a theoretical foundation for the development and application of Vero cells as vaccine matrix.

关 键 词：狂犬病疫苗 人用疫苗 核酸酶 筛选 Vero细胞DNA 安全性评价 

分 类 号：R37[医药卫生—病原生物学]