微载体无血清培养水痘-带状疱疹病毒的效果

Effect of serum-free culture of varicella zoster virus on microcarriers

目的探讨利用生物反应器微载体无血清培养水痘-带状疱疹病毒(varicella-zoster virus,VZV)的效果。方法利用生物反应器,将人二倍体细胞2BS培养至在微载体上形成单层后,分别采用BD008无血清培养基及含有2%新生牛血清的MEM维持液按MOI为0.004~0.008感染VZV毒种;当细胞呈现70%细胞病变效应(cytopatho-genic effect,CPE)时,无血清培养的细胞沉淀经PBS洗涤1次,含血清培养基培养的细胞洗涤1~3次后收获,分别制备3批VZV原液;噬斑法检测病毒滴度,ELSIA法检测牛血清白蛋白残留量,并对制备的水痘减毒活疫苗(live attenuated varicella vaccine,VarV)进行热稳定性检测。结果洗涤1~3次获得的3批含血清培养VZV原液滴度从5.00 lgPFU/mL降至4. 12 lgPFU/mL;牛血清白蛋白残留量分别为203. 2、120. 3和81.2 ng/mL,前2批超出合格范围(≤100 ng/mL)。洗涤1次获得的3批无血清培养VZV原液滴度分别为4.92、5.00和5.30 lgPFU/mL;牛血清白蛋白残留量分别为75.2、82.3和71.9 ng/mL,均值低于洗涤1及2次含血清培养VZV原液(P <0.05)。3批无血清培养的VarV热稳定性均符合要求。结论获得的无血清培养的VZV原液滴度及牛血清白蛋白残留量均符合《中国药典》三部(2015版)标准,本研究为提高VarV质量及生物反应器微载体工艺改进提供了参考。

Objective To investigate the effect of serum-free culture of varicella zoster virus(VZV) on microcarriers in bioreactor. Methods The 2 BS cells were cultured on microcarriers in a bioreactor at a certain density to form a single layer,infected with VZV at a MOI of 0. 004 ~ 0. 008 and cultured in serum-free medium BD008,using the MEM containing 2% newborn calf serum as control. When 70% of the cytopathogenic effect(CPE) appeared,the sediment of cells cultured in serum-free medium BD008 was washed once with PBS,while that in serum-containing medium MEM for1 ~ 3 times,and harvested,each of which was prepared into three batches of bulk. The virus titer was determined by plaque assay,while the residual bovine serum albumin by ELISA. The prepared live attenuated varicella vaccine(VarV)was tested for heat stability. Results The titers of three batches of bulk prepared with the VZV cultured in serumcontaining medium decreased from 5. 00 to 4. 12 lgPFU/mL after being washed for 1 ~ 3 times,while the residual bovine serum albumin contents were 203. 2,120. 3 and 81. 2 ng/mL respectively,of which the former two were more than the standard for qualification (≤ 100 ng/mL). However,the titers of three batches of bulk prepared with the VZV culture in serum-free medium were 4. 92,5. 00 and 5. 30 lgPFU/mL respectively after being washed for one time.However,the residual bovine serum albumin content were 75. 2,82. 3 and 71. 9 ng/mL respectively,of which the mean was lower than those of bulk prepared with VZV cultured in serum-containing medium after being washed for 1 and2 times(P < 0. 05). All the heat stabilities of the three batches of VarV prepared with VZV cultured in serum-free medium met the relevant requirements. Conclusion The titer and residual bovine serum albumin content of vaccine prepared with VZV cultured in serum-free medium met the requirements in Chinese Pharmacopoeia(Vol Ⅲ,2015 edition). The study provided a reference for improvement of quality of VarV and microcarrier procedure in bioreactor.