Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.
PLoS One. 2013 Sep 5;8(9):e72288. doi: 10.1371/journal.pone.0072288. eCollection 2013.
Influenza viruses are a major public health burden during seasonal epidemics and a continuous threat due to their potential to cause pandemics. Annual vaccination provides the best protection against the contagious respiratory illness caused by influenza viruses. However, the current production capacities for influenza vaccines are insufficient to meet the increasing demands. We explored the possibility to establish a continuous production process for influenza viruses using the duck-derived suspension cell line AGE1.CR. A two-stage bioreactor setup was designed in which cells were cultivated in a first stirred tank reactor where an almost constant cell concentration was maintained. Cells were then constantly fed to a second bioreactor where virus infection and replication took place. Using this two-stage reactor system, it was possible to continuously produce influenza viruses. Surprisingly, virus titers showed a periodic increase and decrease during the run-time of 17 days. These titer fluctuations were caused by the presence of defective interfering particles (DIPs), which we detected by PCR. Mathematical modeling confirmed this observation showing that constant virus titers can only emerge in the absence of DIPs. Even with very low amounts of DIPs in the seed virus and very low rates for de novo DIP generation, defective viruses rapidly accumulate and, therefore, represent a serious challenge for continuous vaccine production. Yet, the continuous replication of influenza virus using a two-stage bioreactor setup is a novel tool to study aspects of viral evolution and the impact of DIPs.
流感病毒是季节性流行期间的主要公共卫生负担，也是持续存在的威胁，因为它们有可能引发大流行。每年接种疫苗是预防流感病毒引起的传染性呼吸道疾病的最佳方法。然而，目前流感疫苗的生产能力不足以满足不断增长的需求。我们探索了使用源自鸭的悬浮细胞系 AGE1.CR 建立流感病毒连续生产工艺的可能性。设计了一个两阶段生物反应器装置，其中细胞在第一搅拌罐式反应器中培养，在该反应器中保持几乎恒定的细胞浓度。然后，细胞不断被输送到第二个生物反应器中，在那里进行病毒感染和复制。使用这种两阶段反应器系统，可以连续生产流感病毒。令人惊讶的是，在 17 天的运行时间内，病毒滴度呈现周期性的增加和减少。这些滴度波动是由缺陷干扰颗粒（DIP）的存在引起的，我们通过 PCR 检测到了这些颗粒。数学模型证实了这一观察结果，表明只有在不存在 DIP 的情况下才能出现恒定的病毒滴度。即使在种子病毒中存在非常低水平的 DIP 和非常低的从头生成 DIP 速率的情况下，缺陷病毒也会迅速积累，因此对连续疫苗生产构成严重挑战。然而，使用两阶段生物反应器装置连续复制流感病毒是研究病毒进化和 DIP 影响的新工具。
