生物反应器培养BHK-21悬浮细胞增殖伪狂犬病病毒工艺优化

Optimization of BHK-21 Suspension Cells to Propagate Pseudorabies Virus in Bioreactor

旨在筛选伪狂犬病病毒(PRV)敏感的BHK-21细胞并分析其生长和病毒增殖特性,优化反应器中BHK-21悬浮细胞的培养和病毒增殖条件,建立生物反应器培养BHK-21悬浮细胞增殖PRV工艺。本研究利用响应面和单因素优化法,以细胞生长动力学特性、TCID_(50)病毒滴度等参数为指标,优化1.2 L生物反应器中BHK-21悬浮细胞的最佳培养和增殖病毒条件,在5 L生物反应器中进一步批培养验证。结果显示,筛选获得PRV高敏感的BHK-21-02贴壁细胞和BHK-21-XF02悬浮细胞各1株,BHK-21-XF02悬浮细胞在含3%血清的SLM-BHK低血清培养基和SFM-BHK无血清培养基中均能实现良好的生长和病毒增殖。利用响应面法优化得到1.2 L反应器最佳培养条件为接种密度1.20×10~6cells·mL^(-1)、搅拌转速120 r·min^(-1)、DO值40%,5 L反应器批培养72 h细胞密度可达(7.61±0.18)×10~6 cells·mL^(-1)、细胞活率为(96.93±1.18)%。利用单因素法优化得到1.2 L反应器最佳病毒增殖条件为MOI 0.001、培养温度37℃、细胞密度2.0×10~6cells·mL^(-1)、搅拌转速80 r·min^(-1),5 L反应器批培养接毒后48 h病毒滴度达到最大值(7.13±0.11)lgTCID_(50)·mL^(-1)。本研究可为PRV疫苗相关研究和规模化生产提供参考。

In this study,we screened the BHK-21 cells which were sensitive to pseudorabies(PRV) and analyzed their growth and virus production characteristics.The conditions of cell culture and virus production in the reactor were optimized,so we finally established the PRV propagation process of BHK-21 suspension cells in bioreactor.The optimal culture and virus propagation conditions of BHK-21 suspension cells in 1.2 L bioreactor based on cell growth dynamics and TCID_(50) virus titer,were optimized by response surface methodology and single factor optimization method,and further batch culture was carried out in 5 L bioreactor.The results showed that one of adherent cells named BHK-21-02 and one of suspension cells named BHK-21-XF02 with high PRV sensitivity were screened.BHK-21-XF02 suspension cells could achieve good growth and virus propagation in serum-low medium containing 3% serum and serum-free medium.Using response surface methodology,the optimum cell culture conditions of 1.2 L reactor were as follows:inoculation density was 1.20×10~6 cells·mL^(-1),stirring speed was 120 r·min^(-1) and DO was 40%,the cell density could reach(7.61±0.18)×10~6 cells·mL^(-1) and the cell viability was(96.93±1.18)% after batch culture in 5 L reactor for 72 hours.Using single factor method,the optimal conditions of virus propagation in 1.2 L reactor were as follows:MOI was 0.001,temperature was 37 ℃,cell density for virus inoculation was 2.0×10~6cells·mL^(-1) and stirring speed was 80 r·min^(-1).Under conditions,the virus titer reached(7.13±0.11) lgTCID_(50)·mL^(-1) at 48 hours in 5 L reactor batch culture.Furthermore,all process steps can be fully scaled up to industrial quantities for commercial manufacturing of PRV vaccines.