Increasing IFN-[gamma] productivity in CHO cells through CDK inhibition

Abstract

Approximately 60-70% of all recombinant human glycoproteins are produced in Chinese Hamster Ovary (CHO) cells. Production in CHO cells, however, is often plagued by low productivity when compared with other host cell lines, including bacteria and yeast. For this reason, investigating ways of improving the productivity of CHO cells producing recombinant proteins has been an active area of research for many decades. The induction of growth arrest is one such area that shows particular promise. Through the use of siRNA and chemical cyclin dependent kinase (CDK) inhibitors, we have developed new methods to improve and better understand recombinant protein production during growth arrest. In this study, we have shown that the specific inhibition of the CDK2-CcnE complex through chemical inhibition leads to growth arrest and a subsequent increase in specific productivity. In addition, we have shown that the knockdown of CcnEl alone leads to increases in specific productivity. With the advent of improved shRNA expression systems, we believe that the targeted knockdown of CcnE1 has the potential to induce growth arrest and improve total recombinant protein production The relationship between growth-arrested cell cycle phase and productivity is very poorly understood. In this work, we have used various CDK inhibitors to better understand the relationship between growth-arrested cell cycle phase, specific growth rate, and productivity. We have shown that increases in specific productivity are cell-cycle independent following growth arrest induced by CDK inhibition. Instead, specific productivity increases correlate strongly with a decreasing specific growth rate. Lastly, in this work, we have identified an interesting CDK2 inhibitor that inhibits mitosis and induces a subsequent growth arrest. Following its addition, we observe a decrease in specific growth rate, an increase in DNA content, and a drastic increase in the specific productivity of a recombinant protein (IFN-[gamma]). We used this inhibitor to increase total IFN-[gamma] productivity by 73% in a modified batch culture. With the development of an optimized feed medium, we believe that this CDK2 inhibitor could also be used to increase recombinant protein production in fed-batch cultures.