Trabelsi Khaled, Rourou Samia, Loukil Houssem, Majoul Samy, Kallel Héla
Viral Vaccines Research and Development Unit, Institut Pasteur de Tunis 13, Place Pasteur, BP 74, 1002 Belvédère, Tunis, Tunisia.
J Biotechnol. 2006 Jan 24;121(2):261-71. doi: 10.1016/j.jbiotec.2005.07.018. Epub 2005 Sep 8.
To improve rabies vaccine production by Vero cells, we have developed a strategy based on high cell density culture and optimization of virus yield. We have first optimized cell growth in spinner flask using a Taguchi's L8 experimental design. We analyzed the effects of the following factors: initial glucose and glutamine concentrations, Cytodex 1 concentration and the regulation of glucose level at 1 g l(-1). We have also investigated the effect of the following factor interactions: Cytodex 1 concentration/glutamine concentration, Cytodex 1 concentration/glucose concentration and glucose concentration/glutamine concentration. Statistical analysis of the collected data pointed to the initial glucose concentration, the regulation of glucose level at 1 g l(-1) and the interactions between Cytodex 1 concentration/initial glucose concentration and Cytodex 1 concentration/initial glutamine concentration as the parameters that affected cell growth. Using the optimal conditions determined earlier, we have studied Vero cell growth in a 7-l bioreactor and in batch culture, and obtained a cell density level equal to 3.6 +/- 0.2 x 10(6) cells ml-1. Cell infection with rabies virus (LP 2061/Vero strain) at a multiplicity of infection (MOI) of 0.3 using M199 medium supplemented with 0.2% bovine serum albumin (BSA), yielded a maximal virus titer equal to 8 +/- 1.6 x 10(7) Fluorescent Focus Units (FFU) ml-1. We have also studied Vero cell growth in a 7-l bioreactor using recirculation as a perfusion culture mode during cell proliferation step and perfusion for virus multiplication phase. In comparison to batch culture, we reached a higher cell density level that was equal to 10.1 +/- 0.5 x 10(6) cells ml-1. Cell infection under the conditions previously indicated, yielded 14l of virus harvest that had a virus titer equal to 2.6 +/- 0.5 x 10(7) FFU ml-1. The activity of the inactivated virus harvest showed a protective activity that meets WHO requirements.
为提高Vero细胞狂犬病疫苗的产量，我们制定了一种基于高细胞密度培养和优化病毒产量的策略。我们首先使用田口L8实验设计在转瓶中优化细胞生长。我们分析了以下因素的影响：初始葡萄糖和谷氨酰胺浓度、Cytodex 1浓度以及葡萄糖水平在1 g l(-1)时的调节。我们还研究了以下因素相互作用的影响：Cytodex 1浓度/谷氨酰胺浓度、Cytodex 1浓度/葡萄糖浓度以及葡萄糖浓度/谷氨酰胺浓度。对收集数据的统计分析表明，初始葡萄糖浓度、葡萄糖水平在1 g l(-1)时的调节以及Cytodex 1浓度/初始葡萄糖浓度和Cytodex 1浓度/初始谷氨酰胺浓度之间的相互作用是影响细胞生长的参数。利用先前确定的最佳条件，我们研究了Vero细胞在7升生物反应器中的生长以及分批培养情况，获得了细胞密度水平为3.6 +/- 0.2 x 10(6)个细胞/ml。使用补充有0.2%牛血清白蛋白（BSA）的M199培养基，以感染复数（MOI）为0.3用狂犬病病毒（LP 2061/Vero株）感染细胞，产生的最大病毒滴度为8 +/- 1.6 x 10(7)荧光聚焦单位（FFU）/ml。我们还研究了Vero细胞在7升生物反应器中的生长情况，在细胞增殖步骤中使用再循环作为灌注培养模式，在病毒增殖阶段进行灌注。与分批培养相比，我们达到了更高的细胞密度水平，等于10.1 +/- 0.5 x 10(6)个细胞/ml。在先前所述条件下感染细胞，收获了14升病毒，其病毒滴度为2.6 +/- 0.5 x 10(7) FFU/ml。灭活病毒收获物的活性显示出符合世界卫生组织要求的保护活性。
