兽用灭活纯化狂犬病疫苗生产工艺的优化

目的优化兽用灭活纯化狂犬病疫苗的生产工艺。方法建立BHK21C13细胞主细胞库、工作细胞库,狂犬病病毒疫苗株LEP-Flury主种子批、工作种子批,并进行鉴定。优化悬浮培养工艺参数,并分析病毒原液浓缩、纯化和灭活过程中抗原及成品的稳定性。结果主细胞库、工作细胞库无外源因子污染,细胞形态、生长良好;狂犬病病毒主种子批和工作种子批无外源因子污染,病毒滴度最高可达107.8logLD50/ml。采用15L生物反应器,在优化的培养工艺条件下,细胞密度可达4.5×106个/ml,病毒按0.3MOI接种,灌流培养5次,病毒滴度平均可达106.8logLD50/ml。病毒原液合并后,经750KD中空纤维柱30倍浓缩,Sepharose Fast Flow6层析柱纯化,1/4000β-丙内酯灭活,期间加入稳定剂,纯化疫苗效价平均达(5.03±1.84)IU/头份,纯化的抗原量达(11.75±0.70)μg/头份,在4℃可保存24个月,在37℃可保存30d,在45℃可保存12h。结论优化的兽用灭活狂犬病疫苗的生产工艺可提高疫苗的安全性、有效性及稳定性。

Optimization of Production Procedure for Inactivated Purified Rabies Vaccine for Animal Use

Objective To optimize the production procedure for inactivated purified rabies vaccine for animal use. Methods The master and working BHK-21 CB cell banks as well as mater and working seed lots of rabies vaccine virus strain LEP-Flury were established and identified, the parameters of suspension culture procedure were optimized, and the stabilities of antigen during concentration, purification and inactivation and final product were analyzed. Results Both master and working cell banks were free from contamination with adventitious factors, and the morphology and growth of cells were normal. Both master and working virus seed lots were free from contamination with adventitious factors, and the virus titer reached 107. 8 logLD50 / ml at most. The cell density cultured in 15 L bioreactor under optimized condition reached 4. 5 × 106 cells / ml. The mean titer of virus inoculated at a MOI of 0. 3 and incubated by 5 rounds of perfused culture reached 106. 8 logLD50 / ml. After the virus bulk was pooled and 30-fold concentrated with a 750 KD hollow fiber column, then purified by Sepharose Fast Flow 6 chromatography and inactivated with 1 / 4 000 β-propiolactone, during which stabilizer was added, the obtained purified vaccine reached a mean titer of(5. 03 ± 1. 84)IU / dose and an antigen content of(11. 75 ± 0. 70)μg / dose, and showed good stability after storage at 4℃ for 24 months, at 37℃ for 30 d and after 45℃ for 12 h. Conclusion By the optimized production procedure, the safety, effectiveness and stability of inactivated rabies vaccine for animal use were enhanced.