BHK-21细胞悬浮驯化及对新城疫病毒悬浮培养工艺研究

BHK-21 cell Suspension Adaptation and Culture Process Establishment for Newcastle Disease Virus (NDV) Production with the Suspension Cell Line

关键词:  BHK-21  鸡新城疫病毒  反应器  悬浮培养

Key words:  BHK-21  Newcastle disease virus  Bioreactor  Suspension culture

摘要:

为建立新城疫病毒在BHK-21细胞的无血清全悬浮培养工艺以获得高滴度和高纯度的新城疫悬浮培养抗原，本研究通过悬浮培养驯化和筛选获得了形态良好、稳定传代的BHK-21-sc悬浮细胞株；该细胞以初始密度0.5×106 cells/mL接种，培养72 h可增殖到6×106cells/mL，细胞活率达95%。本研究以5 L生物反应器悬浮培养BHK-21-sc细胞，对鸡新城疫病毒La Sota株的接毒剂量、TPCK胰酶添加浓度、病毒培养温度、收获时间等工艺参数进行了摸索和优化；并在5L-16L-50L生物反应器中进行逐级放大，以优化后的鸡新城疫悬浮培养工艺进行3个批次病毒悬浮培养。最终确定鸡新城疫病毒La Sota株接种BHK-21-sc悬浮细胞株的悬浮培养工艺：BHK-21-sc细胞悬浮培养的第3天按照感染复数（multiplicity of infection，MOI）为0.005接种病毒，并添加终浓度为5 μg/mL的TPCK胰酶，于33℃培养72 h后收获病毒液。应用该悬浮培养工艺在5L、16L和50L反应器上悬浮培养BHK-21-sc悬浮细胞株生产鸡新城疫病毒HA滴度不低于9log2，病毒含量不低于106.0TCID50/0.1mL。表明BHK-21-sc细胞无血清全悬浮生产鸡新城疫病毒工艺稳定，可以实现逐级放大和规模化生产。

Abstract:

To increase the highly titer and purity of Newcastle disease virus (NDV) vaccine antigen by the establishment of BHK-21 cell suspension culture process, an adherent BHK-21 cell line was domesticated to be a suspension cell line BHK-21-sc with good morphology and stable proliferation. The density of the suspension cell line could increase from the initial density of 0.5×106 cells/mL to 6×106 cells/mL in 72 h and the viability of cell was above 95%. Incubation parameters, such as inoculation dose, TPCK trypsin concentration, virus proliferation temperature and harvest time were optimized on suspension cultured BHK-21-sc in 5L bioreactor. The optimized parameters were also verified 3 times in 16L and 50L bioreactor following BHK-21-sc cell culture amplifying process successively. The results showed that the best viral inoculation quantity was 0.005 of MOI. And when the suspending cells were cultured with 5μg/mL of TPCK trypsin at 33℃ for 72 h, the hemagglutination inhibition (HI) titers of NDV was above 1:512 and viral titer was more than 106.0 TCID50/0.1mL, respectively. In conclusion, the established NDV suspension culture process is stable and could be used for the production of NDV antigen by large-scale suspension culture in bioreactor.