Fixed-bed bioreactors packed with macrocarriers show great potential to be used for vaccine process development and large-scale production due to distinguishing features of low shear force, high cell adhering surface area, and easy replacement of culture media in situ. As an initial step of utilizing this type of bioreactors for Pseudorabies virus production (PRV) by African green monkey kidney (Vero) cells, we developed a tube-fixed-bed bioreactor in the previous study, which represents a scale-down model for further process optimization. By using this scale-down model, here we evaluated impacts of two strategies (use of serum-free medium and low cell inoculum density) on PRV production, which have benefits of simplifying downstream process and reducing risk of contamination. We first compared Vero cell cultures with different media, bioreactors and inoculum densities, and conclude that cell growth with serum-free medium is comparable to that with serum-containing medium in tube-fixed-bed bioreactor, and low inoculum density supports cell growth only in this bioreactor. Next, we applied serum-free medium and low inoculum cell density for PRV production. By optimization of time of infection (TOI), multiplicity of infection (MOI) and the harvesting strategy, we obtained total amount of virus particles ~ 9 log10 TCID50 at 5 days post-infection (dpi) in the tube-fixed-bed bioreactor. This process was then scaled up by 25-fold to a Xcell 1-L fixed-bed bioreactor, which yields totally virus particles of 10.5 log10 TCID50, corresponding to ~ 3 × 105 doses of vaccine. The process studied in this work holds promise to be developed as a generic platform for the production of vaccines for animal and human health.

中文翻译：

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在低细胞接种密度的固定床生物反应器中使用无血清培养基生产伪狂犬病病毒

由于具有低剪切力、高细胞粘附表面积和易于原位更换培养基的显着特点，装有大载体的固定床生物反应器在疫苗工艺开发和大规模生产方面显示出巨大的潜力。作为利用这种类型的生物反应器通过非洲绿猴肾 (Vero) 细胞生产伪狂犬病病毒 (PRV) 的第一步，我们在之前的研究中开发了管固定床生物反应器，它代表了进一步缩小模型流程优化。通过使用这种缩小模型，我们在这里评估了两种策略（使用无血清培养基和低细胞接种密度）对 PRV 生产的影响，这些策略具有简化下游工艺和降低污染风险的好处。我们首先将 Vero 细胞培养物与不同的培养基进行比较，生物反应器和接种密度，并得出结论，无血清培养基的细胞生长与管固定床生物反应器中含血清培养基的细胞生长相当，低接种密度仅支持该生物反应器中的细胞生长。接下来，我们应用无血清培养基和低接种细胞密度来生产 PRV。通过优化感染时间 (TOI)、感染复数 (MOI) 和收获策略，我们在管固定床生物反应器中获得了感染后 5 天 (dpi) 的病毒颗粒总量 ~ 9 log10 TCID50。然后将该过程放大 25 倍，用于 Xcell 1-L 固定床生物反应器，其产生的病毒颗粒总量为 10.5 log10 TCID50，相当于约 3 × 105 剂量的疫苗。