Vero细胞乙型脑炎灭活疫苗制备过程中制造工艺及检定技术的优化

发布时间：2018-04-08 18:25

  本文选题：乙型脑炎病毒P_3株　切入点：Vero细胞　出处：《大连医科大学》2011年硕士论文

【摘要】：目的： 为了提高产品的质量,制造出更经济、更安全、更有效乙型脑炎灭活疫苗,我们从生产和检定两个方面着手：一是简化生产程序,优化制备疫苗过程中的各个环节,选择最简单、最便宜又高效的方式方法进行生产,使乙脑疫苗更加安全、有效,且价格更便宜；二是完善检定技术,寻找最合适检测方法,减少干扰因素,使检测方法简单,条件易于控制。制定严格的操作规程,使检定结果最接近真实值,更能体现疫苗的真实情况,制造出更安全的人用乙型脑炎疫苗。 方法： 1.复苏Vero细胞,并在细胞瓶中传代、扩增,建立主细胞库和工作细胞库。 2.将乙脑病毒鼠脑P3株感染Vero细胞,制备乙型脑炎病毒Vero细胞P3株(P3MV株),建立主种子批和工作种子批。 3.用乙型脑炎病毒P3MV株接种Vero细胞,在适宜温度下培养,当细胞病变达到+++-++++时,收获病毒液。 4.将收获的病毒液合并、灭活、浓缩、纯化,经稀释制备成乙脑疫苗成品,再分装、冻干 5.按照《中国药典三部》2005版的要求,对制备疫苗的各个环节及半成品、成品进行检定。 结果： 1.扩增细胞,经过传代扩增,将Vero细胞131代定为主细胞库,135代为工作细胞库； 2.制备毒种库,乙型脑炎病毒Vero细胞P3株,经检测毒力稳定,免疫原性好,对现在流行的病毒株都具有良好的保护作用,建立病毒库,将5代设为主种子批,8代设为工作种子批； 3.收获病毒液,在接种病毒后每日观察细胞病变,当细胞病变达到+++-++++时收获病毒液,每日收获1次,连续收获3-5天,合并后加入甲醛灭活。 4.将灭活实验合格的病毒收获液超滤浓缩50倍。 5.采用凝胶层析的方法纯化疫苗,去除杂蛋白,降低不良反应的发生。 6.将纯化后原液稀释分装,制备成冻干或者水针。 7.按照药典的要求对成品进行全面检定。 结论： 1.使用Vero细胞作为乙脑疫苗的生产基质,细胞学稳定,没有外源因子污染和致瘤性的危险,且培养简单、使用代次高。 2.选用灭活工艺生产疫苗。采用经典的甲醛作为灭活剂灭活乙脑病毒,按照1:2000的浓度加入甲醛,在4℃作用28天,通过细胞培养-动物法进行灭活验证。 3.采用Sepharose6FF凝胶过滤层析纯化疫苗,其分离条件温和、重现性好,还具有较高的回收率。 4.采用细胞蚀斑法代替小鼠法进行病毒滴度检测,试验周期短、操作简单、成本低。
[Abstract]:Objective:In order to improve the quality of the products and to produce a more economical, safer and more effective type B encephalitis inactivated vaccine, we should start from two aspects of production and verification: first, simplify the production process and optimize the various links in the preparation of the vaccine.Choose the simplest, cheapest and most efficient way to make the je vaccine safer, more effective, and cheaper; second, perfect the verification technology, look for the most appropriate detection method, reduce interference factors, and make the detection method simple.Conditions are easy to control.Make strict operation regulations, make the verification results closest to the true value, more can reflect the true situation of the vaccine, and make a safer vaccine for people with Japanese encephalitis.Methods:1.Vero cells were resuscitated, subcultured in cell flask, amplified, and the main cell bank and working cell bank were established.2.Vero cells were infected with Japanese encephalitis virus (je) P3 strain, and the main seed batch and working seed batch were established by preparing P3 strain of Japanese encephalitis virus (Vero) strain P3MV.3.Vero cells were inoculated with Japanese encephalitis virus (P3MV) strain and cultured at suitable temperature.4.Combine, inactivate, concentrate and purify the harvested viral solution, then dilute and prepare the finished product of je vaccine.5.According to the requirements of 2005 edition of Chinese Pharmacopoeia, the preparation of vaccine, semi-finished products and finished products were verified.Results:1.After subculture, 131-generation Vero cells were selected as working cell banks.2.The virus seed bank and P3 strain of Japanese encephalitis virus (Vero) cell were prepared. The virulence and immunogenicity of P3 strain of Japanese encephalitis virus were stable and immunogenicity was good. The virus bank was established and 8 generations of primary seed batches were set up as working seed batches.3.After inoculating the virus, the cytopathic effect was observed daily. When the cytopathic effect reached -, the virus solution was harvested once a day for 3-5 days, then added formaldehyde inactivated.4.Concentration 50 times of the viral harvest solution qualified for the inactivation test.5.The vaccine was purified by gel chromatography to remove the impurity protein and reduce the occurrence of adverse reactions.6.The purified solution was diluted and divided into freeze-dried or water needle.7.According to the requirements of the Pharmacopoeia, the finished product is fully verified.Conclusion:1.Using Vero cells as the production matrix of Japanese encephalitis vaccine, the cytology is stable, there is no risk of exogenous factor contamination and tumorigenicity, and the culture is simple, and the use of the vaccine is high.2.Use inactivation process to produce vaccine.The classical formaldehyde was used as the inactivated agent to inactivate Japanese encephalitis virus. Formaldehyde was added in the concentration of 1: 2000 and exposed at 4 鈩，

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