Perfusion cultures of recombinant CHO cells: Effects on specific productivity, production stability, and protein glycosylation

We have utilized the inducible MMTV-GR promoter system in recombinant Chinese hamster ovary (CHO) cells to demonstrate the synergistic increase in productivity for culture in a high cell density perfusion bioreactor system as compared to culture in a batch bioreactor. Retention of suspension-adapted CHO cells was achieved by inclined sedimentation. Secreted alkaline phosphatase (SEAP) was expressed as the reporter glycoprotein. The large improvement in total SEAP production due to the combined effects of perfusion culture and induction at high cell density was most apparent in the specific productivity of the cell line; with more than a five fold increase in the specific productivity for perfusion culture with high cell density induction as compared to batch culture.; We have determined the independent impact of two protein over-production methods on the glycosylation profile of SEAP produced in CHO cells. The two over-production methods studied were gene amplification and culture operating strategy. Despite an increased specific productivity of almost 10-fold, the gene-amplified cell line demonstrated no significant differences in the glycosylation profile isolated from the reporter protein as compared to those samples isolated from the unamplified cell line. The impact of production method was studied using the unamplified cell line. The semi-continuous perfusion cultures demonstrated both increased specific productivity and increased degree of sialylation compared to samples isolated from batch or repeated fed-batch cultures. In addition, the relative glycan content of SEAP samples cultured in long-term semi-continuous perfusion cultures was more than two fold greater than the relative glycan content of SEAP samples produced in long-term repeated fed-batch cultures and more than five fold greater than that from cultures in one liter batch bioreactors.; Finally, we have studied the observed decay of specific productivity of the recombinant cell line in long term perfusion, semi-continuous perfusion, and repeated fed-batch cultures. The final specific productivity at the end of the long term culture was typically less than 75% of the maximum specific productivity. In this work, we have presented a correlation between the specific growth rate and the observed decay of specific protein production and provided a mathematical description of this correlation.