使用微载体生物反应器技术制备Sabin株脊髓灰质炎灭活疫苗的研究及其产品有效性及安全性的研究分析
发布时间：2018-07-25 07:14
【摘要】：生物反应器微载体大规模培养技术是疫苗大规模制备的核心技术,也是生物技术药物生产最关键和最具挑战性的技术。我国生物反应器微载体大规模培养技术研究起步较晚、培养规模较低、未建立完善的逐级放大细胞及病毒培养和后处理工艺技术。建立可应用于多种病毒疫苗生产的Vero细胞生物反应器微载体大规模培养产业化技术及其后处理工艺,对未来新型疫苗的研制具有重要价值。脊髓灰质炎(以下简称“脊灰”)是由脊灰病毒感染引起的传播广泛且危害极大的急性传染病。疫苗的使用是其有望成为继全球消灭天花后二十一世纪第一个即将被消灭的儿童传染病。随着全球消灭脊灰进程的推进,OPV相关病例(Vaccine-Associated Paralytic Poliomyelitis, VAPP)和疫苗衍生的脊灰病毒(Vaccine-Derived Poliovirus Virus, VDPV)引起的病例日益引起人们的重视。逐步使用序贯免疫的方式代替脊髓灰质炎减毒活疫苗(oral polio vaccine, OPV),并最终完全使用脊髓灰质炎灭活疫苗(Inactivated Poliomyelitis Vaccine, IPV)进行免疫,避免VAPP和VDPV的产生,是《全球消灭脊灰战略终结计划》的要求。全球根除脊灰的战略重点之一,是满足发展中国家逐步使用IPV的需求。而目前使用野毒株(Salk株)生产IPV需要达到生物安全水平3级(Biosafety laboratory level-3, BSL-3)的要求,全世界消灭脊灰后,需要达到更高的生物安全条件。对于疫苗生产厂家来说,技术难度高、造价昂贵。因而世界卫生组织(WHO)鼓励疫苗厂家研发减毒株IPV,特别是Sabin株脊髓灰质炎灭活疫苗(Inactivated Poliomyelitis Vaccine, Sabin strain, sIPV),作为消灭脊灰的最后武器。此外,随着纳入扩大免疫计划(Expanded Program on Immunization, EPI)中疫苗种类的不断增加,婴幼儿接种次数增多、接种负担加重。制备多价联合疫苗具有预防多种目标疾病,减少接种针次,简化免疫程序,提高接种率,降低交叉感染机会,为广大家长和儿童乐于接受,而且节约各种费用,有利于EPI的推广等优越性。sIPV疫苗的制备需要使用大量抗原,其用量是OPV的数十到数百倍。因此,大规模病毒培养制备病毒抗原是研制sIPV的关键。本研究旨在进行Vero细胞生物反应器微载体大规模培养相关技术的研究开发,建立细胞培养逐级放大工艺、病毒培养和后处理工艺,以大规模地制备疫苗抗原,便于sIPV疫苗在我国计划免疫中的尽快应用推广,同时应用于DTaP-sIPV联合疫苗的研发中。我们采用WHO提供的Vero细胞开展微载体生物反应器大规模培养技术的研究,并完成了：1)在生物安全2级(BSL-2)条件下,以Vero细胞为培养基质,脊灰病毒Sabin株(Ⅰ型Sabin株、Ⅱ型Sabin株和Ⅲ型Pfizer株)为培养病毒,通过优化生物反应器培养方法和发酵工艺参数,建立了Vero细胞在7升、75升和550升生物反应器中的微载体培养工艺和消化放大工艺,以及脊灰病毒550升反应器培养工艺。2)探索疫苗后处理技术,建立了稳定的疫苗后处理工艺,包括多级过滤澄清、超滤浓缩、凝胶过滤和离子交换层析纯化、除菌过滤和甲醛灭活等制备工艺,使疫苗蛋白去除率99%以上,疫苗蛋白含量(不高于10μg/剂量)、DNA残留量(《50pg/剂)等均达到或超过了现行中国药典及WHO标准,疫苗纯度达到95%以上。3)制备出了高、中、低不同配比的sIPV,通过效价测定、免疫原性、安全性、稳定性试验研究,证明其具有良好的安全性、免疫原性和稳定性,各项指标均达到规程要求。4)氢氧化铝分别吸附百白破5个组分抗原和sIPV3个型抗原,应用疫苗联合技术制备出实验性DTPa-sIPV四联疫苗。恒河猴免疫原性试验表明DTPa-sIPV各抗原组分间无明显的干扰作用,免疫效果良好；急性毒性试验、全身过敏试验和长期毒性试验表明此联合疫苗具有良好的安全性。综上,我们建立了Vero细胞生物反应器微载体大规模培养技术,使用该技术制备的sIPV疫苗和DTPa-sIPV四联疫苗均达到新型疫苗研制的相关要求,可用于未来大规模sIPV及其四联疫苗的生产。
[Abstract]:Large scale culture technology of bioreactor microcarrier is the core technology of large-scale vaccine preparation, and it is also the most critical and most challenging technology in the production of biotechnological drugs. The production of Vero cell bioreactor, which can be used in the production of a variety of virus vaccines, is of great value to the development of a new type of new vaccine. A large acute infectious disease. The vaccine is used to be the first child to be eradicated in twenty-first Century after the global elimination of smallpox. With the promotion of the global polio eradication process, the OPV related cases (Vaccine-Associated Paralytic Poliomyelitis, VAPP) and vaccine derived poliovirus (Vaccine-Derived Poliovirus) Virus, VDPV) causes more and more people to pay attention to. Sequential immunization is gradually used instead of oral polio vaccine (OPV), and eventually the poliomyelitis inactivated vaccine (Inactivated Poliomyelitis Vaccine, IPV) is completely immunized to avoid VAPP and VDPV. It is "the global eradication ridge". One of the strategic priorities for the global eradication of polio is to meet the needs of developing countries to gradually use IPV, and the current use of wild strain (Salk strain) to produce IPV needs to reach the 3 level of biosafety level (Biosafety laboratory level-3, BSL-3), and the worldwide elimination of polio needs to achieve higher biological safety. All conditions. For vaccine manufacturers, the technical difficulty is high and the cost is expensive. Therefore, the WHO (WHO) encourages the vaccine manufacturers to develop the attenuated strain IPV, especially the Sabin poliomyelitis inactivated vaccine (Inactivated Poliomyelitis Vaccine, Sabin strain, sIPV), as the final weapon to eliminate polio. The number of vaccines in Expanded Program on Immunization (EPI) increased, the number of vaccinated infants increased and the burden of inoculation increased. The preparation of multivalent combined vaccines could prevent multiple target diseases, reduce inoculation times, simplify immune procedures, increase inoculation rate, reduce the chance of cross infection, and be willing to accept for the majority of parents and children. It also saves a variety of costs, and is beneficial to the promotion of the promotion of EPI. The preparation of the.SIPV vaccine needs to use a large amount of antigen, the amount of which is tens to hundreds times of OPV. Therefore, the key to the development of the virus antigen in the large-scale virus culture is the key to the development of the sIPV. This study aims at the research of the large-scale culture related technology of the Vero cell biological counter reactor microcarrier. The development of the cell culture stage by step amplification process, virus culture and post-processing technology to produce vaccine antigen on a large scale facilitates the rapid application of sIPV vaccine in China's planned immunization as soon as possible. At the same time, it is applied to the research and development of DTaP-sIPV combined vaccine. We use Vero cells provided by WHO to develop microcarrier bioreactor for large-scale culture. The research of breeding technology was completed: 1) under the condition of biological safety 2 (BSL-2), Vero cells were used as culture matrix and poliovirus Sabin strain (type I type Sabin strain, type II Sabin strain and type III Pfizer strain) as culture virus. By optimizing the bioreactor culture method and fermentation process parameters, the Vero cells were established in 7 liters, 75 litres and 550 liters of biological reaction. The microcarrier culture technology, the digestion and amplification process and the poliovirus 550 liter reactor culture process.2) explored the post treatment technology of the vaccine. The stable post treatment process was established, including multistage filtration clarification, ultrafiltration concentration, gel filtration and ion exchange chromatography purification, bacteriophage filtration and formaldehyde inactivation. The removal rate of the vaccine protein was above 99%, the content of the vaccine protein (not higher than 10 g/), the DNA residue (<50pg/), etc. all reached or exceeded the current Chinese Pharmacopoeia and the WHO standard, and the purity of the vaccine reached more than 95%.3). The high, medium and low ratio sIPV were prepared, and the test of the titer, immunogenicity, safety and stability proved that it had been tested. With good safety, immunogenicity and stability, each index reached the requirements of the regulation.4) aluminum hydroxide was used to adsorb 5 components of the antigen and sIPV3 antigen of pertusis, and the experimental DTPa-sIPV quadruple vaccine was prepared by the combination of vaccine. The immunogenicity of Ganges RIver monkey showed that there was no obvious interference between the components of the antigen of DTPa-sIPV. The immunization effect is good; acute toxicity test, systemic allergy test and long-term toxicity test show that the combined vaccine has good safety. In summary, we have established the Vero cell bioreactor microcarrier large-scale culture technology, the sIPV vaccine and the DTPa-sIPV quadruple vaccine prepared by this technique all reach the new vaccine development phase. It can be used for future large-scale sIPV and its quadruple vaccine production.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R392
本文编号：2143044
[Abstract]:Large scale culture technology of bioreactor microcarrier is the core technology of large-scale vaccine preparation, and it is also the most critical and most challenging technology in the production of biotechnological drugs. The production of Vero cell bioreactor, which can be used in the production of a variety of virus vaccines, is of great value to the development of a new type of new vaccine. A large acute infectious disease. The vaccine is used to be the first child to be eradicated in twenty-first Century after the global elimination of smallpox. With the promotion of the global polio eradication process, the OPV related cases (Vaccine-Associated Paralytic Poliomyelitis, VAPP) and vaccine derived poliovirus (Vaccine-Derived Poliovirus) Virus, VDPV) causes more and more people to pay attention to. Sequential immunization is gradually used instead of oral polio vaccine (OPV), and eventually the poliomyelitis inactivated vaccine (Inactivated Poliomyelitis Vaccine, IPV) is completely immunized to avoid VAPP and VDPV. It is "the global eradication ridge". One of the strategic priorities for the global eradication of polio is to meet the needs of developing countries to gradually use IPV, and the current use of wild strain (Salk strain) to produce IPV needs to reach the 3 level of biosafety level (Biosafety laboratory level-3, BSL-3), and the worldwide elimination of polio needs to achieve higher biological safety. All conditions. For vaccine manufacturers, the technical difficulty is high and the cost is expensive. Therefore, the WHO (WHO) encourages the vaccine manufacturers to develop the attenuated strain IPV, especially the Sabin poliomyelitis inactivated vaccine (Inactivated Poliomyelitis Vaccine, Sabin strain, sIPV), as the final weapon to eliminate polio. The number of vaccines in Expanded Program on Immunization (EPI) increased, the number of vaccinated infants increased and the burden of inoculation increased. The preparation of multivalent combined vaccines could prevent multiple target diseases, reduce inoculation times, simplify immune procedures, increase inoculation rate, reduce the chance of cross infection, and be willing to accept for the majority of parents and children. It also saves a variety of costs, and is beneficial to the promotion of the promotion of EPI. The preparation of the.SIPV vaccine needs to use a large amount of antigen, the amount of which is tens to hundreds times of OPV. Therefore, the key to the development of the virus antigen in the large-scale virus culture is the key to the development of the sIPV. This study aims at the research of the large-scale culture related technology of the Vero cell biological counter reactor microcarrier. The development of the cell culture stage by step amplification process, virus culture and post-processing technology to produce vaccine antigen on a large scale facilitates the rapid application of sIPV vaccine in China's planned immunization as soon as possible. At the same time, it is applied to the research and development of DTaP-sIPV combined vaccine. We use Vero cells provided by WHO to develop microcarrier bioreactor for large-scale culture. The research of breeding technology was completed: 1) under the condition of biological safety 2 (BSL-2), Vero cells were used as culture matrix and poliovirus Sabin strain (type I type Sabin strain, type II Sabin strain and type III Pfizer strain) as culture virus. By optimizing the bioreactor culture method and fermentation process parameters, the Vero cells were established in 7 liters, 75 litres and 550 liters of biological reaction. The microcarrier culture technology, the digestion and amplification process and the poliovirus 550 liter reactor culture process.2) explored the post treatment technology of the vaccine. The stable post treatment process was established, including multistage filtration clarification, ultrafiltration concentration, gel filtration and ion exchange chromatography purification, bacteriophage filtration and formaldehyde inactivation. The removal rate of the vaccine protein was above 99%, the content of the vaccine protein (not higher than 10 g/), the DNA residue (<50pg/), etc. all reached or exceeded the current Chinese Pharmacopoeia and the WHO standard, and the purity of the vaccine reached more than 95%.3). The high, medium and low ratio sIPV were prepared, and the test of the titer, immunogenicity, safety and stability proved that it had been tested. With good safety, immunogenicity and stability, each index reached the requirements of the regulation.4) aluminum hydroxide was used to adsorb 5 components of the antigen and sIPV3 antigen of pertusis, and the experimental DTPa-sIPV quadruple vaccine was prepared by the combination of vaccine. The immunogenicity of Ganges RIver monkey showed that there was no obvious interference between the components of the antigen of DTPa-sIPV. The immunization effect is good; acute toxicity test, systemic allergy test and long-term toxicity test show that the combined vaccine has good safety. In summary, we have established the Vero cell bioreactor microcarrier large-scale culture technology, the sIPV vaccine and the DTPa-sIPV quadruple vaccine prepared by this technique all reach the new vaccine development phase. It can be used for future large-scale sIPV and its quadruple vaccine production.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R392
【参考文献】
相关期刊论文 前2条
1 吴燕;倪政;;脊髓灰质炎疫苗相关病例的流行现状、负担及应对措施[J];中国公共卫生管理;2009年06期
2 侬亮;姜述德;;应用减毒株和强毒株试制脊髓灰质炎灭活疫苗的比较[J];中国生物制品学杂志;1992年02期
，本文编号：2143044
本文链接：https://www.wllwen.com/yixuelunwen/jichuyixue/2143044.html
