Tapia Felipe, Jordan Ingo, Genzel Yvonne, Reichl Udo
International Max Planck Research School for Advanced Methods in Process and Systems Engineering, Magdeburg, Germany.
Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.
PLoS One. 2017 Aug 24;12(8):e0182553. doi: 10.1371/journal.pone.0182553. eCollection 2017.
One important aim in cell culture-based viral vaccine and vector production is the implementation of continuous processes. Such a development has the potential to reduce costs of vaccine manufacturing as volumetric productivity is increased and the manufacturing footprint is reduced. In this work, continuous production of Modified Vaccinia Ankara (MVA) virus was investigated. First, a semi-continuous two-stage cultivation system consisting of two shaker flasks in series was established as a small-scale approach. Cultures of the avian AGE1.CR.pIX cell line were expanded in the first shaker, and MVA virus was propagated and harvested in the second shaker over a period of 8-15 days. A total of nine small-scale cultivations were performed to investigate the impact of process parameters on virus yields. Harvest volumes of 0.7-1 L with maximum TCID50 titers of up to 1.0×109 virions/mL were obtained. Genetic analysis of control experiments using a recombinant MVA virus containing green-fluorescent-protein suggested that the virus was stable over at least 16 d of cultivation. In addition, a decrease or fluctuation of infectious units that may indicate an excessive accumulation of defective interfering particles was not observed. The process was automated in a two-stage continuous system comprising two connected 1 L stirred tank bioreactors. Stable MVA virus titers, and a total production volume of 7.1 L with an average TCID50 titer of 9×107 virions/mL was achieved. Because titers were at the lower range of the shake flask cultivations potential for further process optimization at large scale will be discussed. Overall, MVA virus was efficiently produced in continuous and semi-continuous cultivations making two-stage stirred tank bioreactor systems a promising platform for industrial production of MVA-derived recombinant vaccines and viral vectors.
基于细胞培养的病毒疫苗和载体生产的一个重要目标是实现连续生产过程。这样的发展有可能降低疫苗生产成本，因为提高了体积生产率并减少了生产占地面积。在这项工作中，对改良痘苗病毒安卡拉株（MVA）的连续生产进行了研究。首先，建立了一个由两个串联的摇瓶组成的半连续两阶段培养系统作为小规模方法。禽AGE1.CR.pIX细胞系的培养物在第一个摇瓶中扩增，MVA病毒在第二个摇瓶中繁殖并收获，持续8至15天。总共进行了九次小规模培养，以研究工艺参数对病毒产量的影响。收获体积为0.7至1升，最大半数组织培养感染剂量（TCID50）滴度高达1.0×109个病毒粒子/毫升。使用含有绿色荧光蛋白的重组MVA病毒进行的对照实验的基因分析表明，该病毒在至少16天的培养过程中是稳定的。此外，未观察到可能表明缺陷干扰颗粒过度积累的感染单位的减少或波动。该工艺在一个由两个相连的1升搅拌罐生物反应器组成的两阶段连续系统中实现了自动化。实现了稳定的MVA病毒滴度，总生产量为7.1升，平均TCID50滴度为9×107个病毒粒子/毫升。由于滴度处于摇瓶培养的较低范围，将讨论大规模进一步工艺优化的潜力。总体而言，MVA病毒在连续和半连续培养中高效生产，使两阶段搅拌罐生物反应器系统成为生产MVA衍生重组疫苗和病毒载体的有前景的工业平台。
