WAVETM生物反应器灌注培养生产种子细胞的开发和应用

Development and application of perfusion culture producing seed cells in WAVETM bioreactor

摘要:

近年来，国内中国仓鼠卵巢细胞 (Chinese hamster ovary，CHO) 生产罐的培养规模已达上千升，国外已达上万升，最终的生产罐前需要多级摇瓶、种子罐进行种子细胞扩增，扩增效率较低，严重影响了抗体、融合蛋白等生物制品的生产效率。文中利用WAVETM波浪式生物反应器，通过灌注培养的方法，成功地实现了种子细胞的高效扩增。WAVETM波浪式生物反应器灌注培养方法制备种子细胞，CHO细胞密度高达2.28×107 cells/mL时仍处于指数生长期且细胞活力大于95％，以此细胞作为种子细胞，4×105 cells/mL接种于另一个WAVETM生物反应器进行流加培养，最大活细胞密度仍可达1.73×107 cells/mL。通过此种扩增方式，1台WAVETM 20/50即可为1 000 L或2 000 L的生产罐提供种子细胞，种子细胞的扩增倍数 (Split ratio) 可以达到1∶50~1∶100倍，与传统不锈钢罐种子细胞扩增倍数1∶2~1∶10相比，可以显著减少2~3级种子罐，种子细胞的扩增时间减少7~9 d，极大地提高生产效率。

Abstract:

In recent years, Chinese hamster ovary (CHO) production vessel volume has reached more than 1 000 L in Chinese biopharms, and 10 000 L in foreign big biopharms, such as Lonza and Genetech. In general, there are some steps seed bioreactor for seed expansion, which decreases the efficiency of production process. In this work, a perfusion-based process was developed to drastically increase the split ratio during the scale-up of CHO cell cultures. Fed-batch cultures were inoculated with cells propagated in either batch or perfusion cultures that grown in disposable Cellbags using the WAVETM Bioreactor system. The higher cell concentration of 2×107 cells/mL with 95% viability allowed to increase the split ratio to about 1:50~1:100 for inoculum propagated in perfusion culture. The method described here could reduce the number of required expansion steps and eliminate two or three bioreactors. Disposable perfusion bioreactor with only a few liters working volume have the potential to directly inoculate volumes of up to 1 000 liters. This would allow to shorten process time in these bioreactors, which often are the bottleneck in plant throughput.