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无血清 Vero 细胞悬浮培养中新城疫病毒载体疫苗的工艺开发
发表日期:2021-12-02


Process Development for Newcastle Disease Virus-Vectored Vaccines in Serum-Free Vero Cell Suspension Cultures
无血清 Vero 细胞悬浮培养中新城疫病毒载体疫苗的工艺开发


The ongoing COVID-19 pandemic drew global attention to infectious diseases, attracting numerous resources for development of pandemic preparedness plans and vaccine platforms—technologies with robust manufacturing processes that can quickly be pivoted to target emerging diseases. Newcastle Disease Virus (NDV) has been studied as a viral vector for human and veterinary vaccines, but its production relies heavily on embryonated chicken eggs, with very few studies producing NDV in cell culture. Here, NDV is produced in suspension Vero cells, and analytical assays (TCID50 and ddPCR) are developed to quantify infectious and total viral titer. NDV-GFP and NDV-FLS (SARS-CoV-2 full-length spike protein) constructs were adapted to replicate in Vero and HEK293 suspension cultures using serum-free media, while fine-tuning parameters such as MOI, temperature, and trypsin concentration. Shake flask productions with Vero cells resulted in infectious titers of 1.07 × 108 TCID50/mL for NDV-GFP and 1.33 × 108 TCID50/mL for NDV-FLS. Production in 1 L batch bioreactors also resulted in high titers in culture supernatants, reaching 2.37 × 108 TCID50/mL for NDV-GFP and 3.16 × 107 TCID50/mL for NDV-FLS. This shows effective NDV production in cell culture, building the basis for a scalable vectored-vaccine manufacturing process that can be applied to different targets.




持续的 COVID-19 大流行引起了全球对传染病的关注,吸引了大量资源用于开发大流行防范计划和疫苗平台——这些技术具有强大的制造工艺,可以快速转向针对新出现的疾病。新城疫病毒 (NDV) 已被研究作为人类和兽用疫苗的病毒载体,但其生产严重依赖鸡胚,很少有研究在细胞培养中产生 NDV。在这里,NDV 是在悬浮的 Vero 细胞中产生的,分析测定 (TCID 50ddPCR) 用于量化感染性和总病毒滴度。NDV-GFP NDV-FLSSARS-CoV-2 全长刺突蛋白)构建体适用于使用无血清培养基在 Vero HEK293 悬浮培养物中复制,同时微调参数,如 MOI、温度和胰蛋白酶浓度. 摇瓶用Vero细胞生产导致1.07×10的感染滴度8 TCID 50 / mLNDV-GFP1.33×10 8 TCID 50NDV-FLS /毫升。在 1 L 批次生物反应器中的生产也导致培养物上清液的高滴度,NDV-GFP达到 2.37 × 10 8 TCID 50 /mL 3.16 × 10 7 TCID 50/mL 用于 NDV-FLS。这显示了细胞培养中有效的 NDV 生产,为可应用于不同目标的可扩展载体疫苗制造过程奠定了基础。

Furthermore, process intensification could increase the quantity of infective particles harvested, using technologies such as fed-batch or perfusion [48]. The lower infectious titers observed at later time points in bioreactors and shake flasks suggest that NDV could be a good candidate for production in perfusion mode, as the viruses could be continuously harvested before suffering a loss in infectivity due to temperature and shear stress in the bioreactor.

Therefore, we have successfully developed the upstream process and analytical methods for suspension Vero cell-based production of NDV, using the constructs NDV-GFP and NDV-FLS as models. Future steps include establishing a scalable purification protocol and testing different bioreactor production modes, such as fed-batch and perfusion, to move towards a complete process based on continuous manufacturing.


此外,使用分批补料或灌注等技术,过程强化可以增加收获的感染性颗粒的数量 [ 48 ]。在生物反应器和摇瓶中的稍后时间点观察到的较低感染滴度表明 NDV 可能是在灌注模式下生产的良好候选者,因为可以在由于生物反应器中的温度和剪切应力而丧失传染性之前连续收获病毒.

因此,我们使用构建体 NDV-GFP NDV-FLS 作为模型,成功开发了基于悬浮 Vero 细胞生产 NDV 的上游工艺和分析方法。未来的步骤包括建立可扩展的纯化方案和测试不同的生物反应器生产模式,如补料分批和灌注,以实现基于连续制造的完整工艺。

无血清 Vero 细胞悬浮培养中新城疫病毒载体疫苗的工艺开发无血清 Vero 细胞悬浮培养中新城疫病毒载体疫苗的工艺开发

关键词:新冠病毒,Vero悬浮培养,病毒疫苗生物工艺,生物反应器生产,疫苗生产平台,COVID-19,SARS-CoV-2,Newcastle Disease Virus,Vero suspension culture,viral vaccine bioprocess, bioreactor production, vaccine production platform,COVID-19, SARS-CoV-2

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