微载体灌注培养制备Vero细胞狂犬病疫苗

Preparation of Rabies Vaccine by Perfusion Culture of Vero Cells on Microcarriers

本文研究在生物反应器中用微载体连续灌注培养Vero细胞生产狂犬病毒制备技术.在5L体积的生物反应器中,加入含10g/L微载体的199培养基,接种Vero细胞至细胞浓度达到1×105/mL,培养7d后细胞可生长至6～7×106/mL,然后以感染复数(MOI)为0.01接种狂犬病毒VaG株,接毒后24h开始收获,连续收获12d左右,收获的病毒滴度范围在6.0～8.5log LD50/mL,收获的病毒原液经浓缩、灭活和纯化等步骤制备成疫苗,各项质量指标均达到<中国生物制品规程>2000年版要求.实验表明,用生物反应器微载体灌注培养制备人用Vero细胞狂犬病疫苗小试工艺可行.

To establish a technique for preparing rabies vaccine by perfusion culture on microcarriers in bioreactor, a 5 litre bioreactor was filled with 199 cell culture medium which contains 10g/L of microcarriers. Then Vero cells were inoculated with a final cell density of 1×105/mL . After 7 days of culture, cell density can reach 6~7×106/mL, then rabies virus VaG strain was inoculated with a MOI of 0.01. After 24h inoculation, the cultured viruses were harvested continuously for 12 days. The titer of collected virus in the culture medium was about 6.0 to 8.5 logLD50/mL. From these collected viruses, vaccines were prepared through the steps of concentration, inactivation and purification, The quality of these vaccines meets “The Chinese Requirements for Biologics Year 2000”. This lab-scale method for preparing rabies vaccine by perfusion culture on microcarries is viable.