Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
Biotechnol Bioeng. 2021 Oct;118(10):3996-4013. doi: 10.1002/bit.27876. Epub 2021 Jul 20.
Seasonal influenza epidemics occur both in northern and southern hemispheres every year. Despite the differences in influenza virus surface antigens and virulence of seasonal subtypes, manufacturers are well-adapted to respond to this periodical vaccine demand. Due to decades of influenza virus research, the development of new influenza vaccines is relatively straight forward. In similarity with the ongoing coronavirus disease 2019 pandemic, vaccine manufacturing is a major bottleneck for a rapid supply of the billions of doses required worldwide. In particular, egg-based vaccine production would be difficult to schedule and shortages of other egg-based vaccines with high demands also have to be anticipated. Cell culture-based production systems enable the manufacturing of large amounts of vaccines within a short time frame and expand significantly our options to respond to pandemics and emerging viral diseases. In this study, we present an integrated process for the production of inactivated influenza A virus vaccines based on a Madin-Darby Canine Kidney (MDCK) suspension cell line cultivated in a chemically defined medium. Very high titers of 3.6 log (HAU/100 µl) were achieved using fast-growing MDCK cells at concentrations up to 9.5 × 10 cells/ml infected with influenza A/PR/8/34 H1N1 virus in 1 L stirred tank bioreactors. A combination of membrane-based steric-exclusion chromatography followed by pseudo-affinity chromatography with a sulfated cellulose membrane adsorber enabled full recovery for the virus capture step and up to 80% recovery for the virus polishing step. Purified virus particles showed a homogenous size distribution with a mean diameter of 80 nm. Based on a monovalent dose of 15 µg hemagglutinin (single-radial immunodiffusion assay), the level of total protein and host cell DNA was 58 µg and 10 ng, respectively. Furthermore, all process steps can be fully scaled up to industrial quantities for commercial manufacturing of either seasonal or pandemic influenza virus vaccines. Fast production of up to 300 vaccine doses per liter within 4-5 days makes this process competitive not only to other cell-based processes but to egg-based processes as well.
季节性流感在南北半球每年都会流行。尽管季节性亚型的流感病毒表面抗原和毒力存在差异，但制造商能够很好地应对这种周期性的疫苗需求。由于几十年来对流感病毒的研究，新流感疫苗的开发相对较为直接。与当前的 2019 年冠状病毒病大流行相似，疫苗生产是全球数十亿剂疫苗快速供应的主要瓶颈。特别是，基于鸡蛋的疫苗生产将难以安排，而且高需求的其他基于鸡蛋的疫苗也将出现短缺。基于细胞培养的生产系统能够在短时间内生产大量疫苗，并大大扩展了我们应对大流行和新出现的病毒性疾病的选择。在这项研究中，我们提出了一种基于在化学成分确定的培养基中培养的 Madin-Darby 犬肾（MDCK）悬浮细胞系生产灭活流感 A 病毒疫苗的综合工艺。在 1L 搅拌罐生物反应器中，使用快速生长的 MDCK 细胞，在高达 9.5×10 细胞/ml 的浓度下，用流感 A/PR/8/34 H1N1 病毒感染，可实现高达 3.6 个对数（HAU/100 μl）的高滴度。膜基空间排阻色谱与带硫酸化纤维素膜吸附剂的伪亲和色谱的组合，使病毒捕获步骤得到完全回收，病毒抛光步骤的回收率高达 80%。纯化的病毒粒子显示出均匀的粒径分布，平均直径为 80nm。基于 15μg 血凝素（单放射免疫扩散测定）的单价剂量，总蛋白和宿主细胞 DNA 的水平分别为 58μg 和 10ng。此外，所有工艺步骤都可以完全放大到商业规模，用于生产季节性或大流行流感病毒疫苗。在 4-5 天内，每升高达 300 剂的快速生产速度使该工艺不仅与其他基于细胞的工艺具有竞争力，而且与基于鸡蛋的工艺也具有竞争力。
