Development and optimisation of a procedure for the production of Parapoxvirus ovis by large-scale microcarrier cell culture in a non-animal, non-human and non-plant-derived medium.
Development and optimisation of a procedure for the production of Parapoxvirus ovis by large-scale microcarrier cell culture in a non-animal, non-human and non-plant-derived medium.
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DOI：
10.1016/j.vaccine.2008.01.032
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发表时间：
2008-03
期刊：
影响因子：
5.5
通讯作者：
M. Pohlscheidt;U. Langer;T. Minuth;B. Bödeker;H. Apeler;H. Hörlein;D. Paulsen;H. Rübsamen-Waigmann-H.-Rübsamen-Waigm
中科院分区：
文献类型：
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作者：
M. Pohlscheidt;U. Langer;T. Minuth;B. Bödeker;H. Apeler;H. Hörlein;D. Paulsen;H. Rübsamen-Waigmann-H.-Rübsamen-Waigm
为了生产化学灭活的绵羊副痘病毒（PPVO），在Cytodex®-3微载体上悬浮培养贴壁牛肾细胞系。灭活和纯化的病毒颗粒在几种动物模型中显示出免疫调节活性。PPVO通过双相分批工艺以3.5和10 L规模生产。通过无泡膜氧合经由具有中心双叶锚叶轮的管定子实现曝气。为了提高效率、工艺耐用性和安全性，对既定工艺进行了优化。细胞系适应于无蛋白质培养基（重组胰岛素除外），以提高生物安全性。成功地进行了直接细胞扩增的50 L中试工厂的规模。同时，特别强调不同的操作条件（细胞数量、感染复数（MOI）等）优化了双相分批工艺。和工艺管理（补料分批、透析等）。纯化的病毒颗粒的质量和浓度进行了评估，通过定量电子显微镜，残留的宿主细胞蛋白质和DNA含量，最后，在转基因小鼠模型中的生物活性。这种综合方法产生了一种新的、安全的、稳健的和高产的大规模生产工艺，称为“体积-扩增-补料”批次，细胞密度高达6- 7 e06个细胞/mL。通过随后将感染的细胞稀释到下一个工艺规模中，实现了总生产率增加40倍（与建立的双相分批工艺相关）。
For the production of a chemically inactivated Parapoxvirus ovis (PPVO), an adherent bovine kidney cell line was cultivated on Cytodex®-3 microcarriers in suspension culture. The inactivated and purified virus particles have shown immune modulatory activity in several animal models. PPVO was produced by a biphasic batch process at the 3.5 and 10L scale. Aeration was realised by bubble-free membrane oxygenation via a tube stator with a central two-blade anchor impeller. In order to increase efficiency, process robustness, and safety, the established process was optimised. The cell line was adapted to a protein-free medium (except recombinant insulin) in order to increase biosafety. A scale up to a 50L pilot plant with direct cell expansion was performed successfully. In parallel, the biphasic batch process was optimised with special emphasis on different operating conditions (cell number, Multiplicity of Infection (MOI), etc.) and process management (fed-batch, dialysis, etc.). The quality and concentration of the purified virus particles was assessed by quantitative electron microscopy, residual host cell protein and DNA-content and, finally, biologic activity in a transgenic mouse model. This integrated approach led to a new, safe, robust and highly productive large-scale production process, called “Volume-Expanded-Fed” Batch with cell densities up to 6–7e06cells/mL. By subsequent dilution of infected cells into the next process scale, an increase in total productivity by a factor of 40 (related to an established biphasic batch process) was achieved.