使用微載體生物反應(yīng)器技術(shù)制備Sabin株脊髓灰質(zhì)炎滅活疫苗的研究及其產(chǎn)品有效性及安全性的研究分析
發(fā)布時(shí)間：2018-07-25 07:14
【摘要】：生物反應(yīng)器微載體大規(guī)模培養(yǎng)技術(shù)是疫苗大規(guī)模制備的核心技術(shù),也是生物技術(shù)藥物生產(chǎn)最關(guān)鍵和最具挑戰(zhàn)性的技術(shù)。我國(guó)生物反應(yīng)器微載體大規(guī)模培養(yǎng)技術(shù)研究起步較晚、培養(yǎng)規(guī)模較低、未建立完善的逐級(jí)放大細(xì)胞及病毒培養(yǎng)和后處理工藝技術(shù)。建立可應(yīng)用于多種病毒疫苗生產(chǎn)的Vero細(xì)胞生物反應(yīng)器微載體大規(guī)模培養(yǎng)產(chǎn)業(yè)化技術(shù)及其后處理工藝,對(duì)未來(lái)新型疫苗的研制具有重要價(jià)值。脊髓灰質(zhì)炎(以下簡(jiǎn)稱“脊灰”)是由脊灰病毒感染引起的傳播廣泛且危害極大的急性傳染病。疫苗的使用是其有望成為繼全球消滅天花后二十一世紀(jì)第一個(gè)即將被消滅的兒童傳染病。隨著全球消滅脊灰進(jìn)程的推進(jìn),OPV相關(guān)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們的重視。逐步使用序貫免疫的方式代替脊髓灰質(zhì)炎減毒活疫苗(oral polio vaccine, OPV),并最終完全使用脊髓灰質(zhì)炎滅活疫苗(Inactivated Poliomyelitis Vaccine, IPV)進(jìn)行免疫,避免VAPP和VDPV的產(chǎn)生,是《全球消滅脊灰戰(zhàn)略終結(jié)計(jì)劃》的要求。全球根除脊灰的戰(zhàn)略重點(diǎn)之一,是滿足發(fā)展中國(guó)家逐步使用IPV的需求。而目前使用野毒株(Salk株)生產(chǎn)IPV需要達(dá)到生物安全水平3級(jí)(Biosafety laboratory level-3, BSL-3)的要求,全世界消滅脊灰后,需要達(dá)到更高的生物安全條件。對(duì)于疫苗生產(chǎn)廠家來(lái)說(shuō),技術(shù)難度高、造價(jià)昂貴。因而世界衛(wèi)生組織(WHO)鼓勵(lì)疫苗廠家研發(fā)減毒株IPV,特別是Sabin株脊髓灰質(zhì)炎滅活疫苗(Inactivated Poliomyelitis Vaccine, Sabin strain, sIPV),作為消滅脊灰的最后武器。此外,隨著納入擴(kuò)大免疫計(jì)劃(Expanded Program on Immunization, EPI)中疫苗種類的不斷增加,嬰幼兒接種次數(shù)增多、接種負(fù)擔(dān)加重。制備多價(jià)聯(lián)合疫苗具有預(yù)防多種目標(biāo)疾病,減少接種針次,簡(jiǎn)化免疫程序,提高接種率,降低交叉感染機(jī)會(huì),為廣大家長(zhǎng)和兒童樂(lè)于接受,而且節(jié)約各種費(fèi)用,有利于EPI的推廣等優(yōu)越性。sIPV疫苗的制備需要使用大量抗原,其用量是OPV的數(shù)十到數(shù)百倍。因此,大規(guī)模病毒培養(yǎng)制備病毒抗原是研制sIPV的關(guān)鍵。本研究旨在進(jìn)行Vero細(xì)胞生物反應(yīng)器微載體大規(guī)模培養(yǎng)相關(guān)技術(shù)的研究開(kāi)發(fā),建立細(xì)胞培養(yǎng)逐級(jí)放大工藝、病毒培養(yǎng)和后處理工藝,以大規(guī)模地制備疫苗抗原,便于sIPV疫苗在我國(guó)計(jì)劃免疫中的盡快應(yīng)用推廣,同時(shí)應(yīng)用于DTaP-sIPV聯(lián)合疫苗的研發(fā)中。我們采用WHO提供的Vero細(xì)胞開(kāi)展微載體生物反應(yīng)器大規(guī)模培養(yǎng)技術(shù)的研究,并完成了：1)在生物安全2級(jí)(BSL-2)條件下,以Vero細(xì)胞為培養(yǎng)基質(zhì),脊灰病毒Sabin株(Ⅰ型Sabin株、Ⅱ型Sabin株和Ⅲ型Pfizer株)為培養(yǎng)病毒,通過(guò)優(yōu)化生物反應(yīng)器培養(yǎng)方法和發(fā)酵工藝參數(shù),建立了Vero細(xì)胞在7升、75升和550升生物反應(yīng)器中的微載體培養(yǎng)工藝和消化放大工藝,以及脊灰病毒550升反應(yīng)器培養(yǎng)工藝。2)探索疫苗后處理技術(shù),建立了穩(wěn)定的疫苗后處理工藝,包括多級(jí)過(guò)濾澄清、超濾濃縮、凝膠過(guò)濾和離子交換層析純化、除菌過(guò)濾和甲醛滅活等制備工藝,使疫苗蛋白去除率99%以上,疫苗蛋白含量(不高于10μg/劑量)、DNA殘留量(《50pg/劑)等均達(dá)到或超過(guò)了現(xiàn)行中國(guó)藥典及WHO標(biāo)準(zhǔn),疫苗純度達(dá)到95%以上。3)制備出了高、中、低不同配比的sIPV,通過(guò)效價(jià)測(cè)定、免疫原性、安全性、穩(wěn)定性試驗(yàn)研究,證明其具有良好的安全性、免疫原性和穩(wěn)定性,各項(xiàng)指標(biāo)均達(dá)到規(guī)程要求。4)氫氧化鋁分別吸附百白破5個(gè)組分抗原和sIPV3個(gè)型抗原,應(yīng)用疫苗聯(lián)合技術(shù)制備出實(shí)驗(yàn)性DTPa-sIPV四聯(lián)疫苗。恒河猴免疫原性試驗(yàn)表明DTPa-sIPV各抗原組分間無(wú)明顯的干擾作用,免疫效果良好；急性毒性試驗(yàn)、全身過(guò)敏試驗(yàn)和長(zhǎng)期毒性試驗(yàn)表明此聯(lián)合疫苗具有良好的安全性。綜上,我們建立了Vero細(xì)胞生物反應(yīng)器微載體大規(guī)模培養(yǎng)技術(shù),使用該技術(shù)制備的sIPV疫苗和DTPa-sIPV四聯(lián)疫苗均達(dá)到新型疫苗研制的相關(guān)要求,可用于未來(lái)大規(guī)模sIPV及其四聯(lián)疫苗的生產(chǎn)。
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R392
本文編號(hào)：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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R392
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