(74a) Cell Cycle Arrest Engineering to Enhance Production Phase in CHO Cell Culture

Chinese hamster ovary (CHO) cells are used to produce protein-based therapeutics including monoclonal antibodies, cytokines, vaccines and fusion proteins in bioreactors. Over the past decade, volumetric productivity of these therapeutics has greatly improved. Although this improvement has been accomplished primarily through media development and optimized process/feed conditions, a new era of improvement is possible based on genetic alterations to the cells themselves. One method developed to increase productivity and extend culture time is to allow the cells to grow rapidly initially and then reduce the cell's growth rate by lowering the culture temperature once a high cell number is reached. While this strategy is effective at reducing the growth of the cell it also generally reduces the metabolic activity. To avoid the latter, we can genetically engineer the cells to enter cell cycle arrest upon induction of the expression a cell cycle arrest protein. Here we compared the effectiveness of the induced expression of cell cycle arrest proteins on cell growth and productivity. The cyclin dependent kinase inhibitor, p21, is a protein known to inhibit the interaction of cdk-2 with cyclin E which is needed for the cell to progress past the G1 phase checkpoint. When expression of this protein is induced before a peak cell density is reached the cells become arrested in the G0/G1 phase of the cell cycle, where the energy consumed is primarily for protein production instead of DNA replication. By this method the culture can be maintained in a protein producing phase for an extended period of time before adverse conditions are present. To potentially lower the burden of p21 expression, we also examine the potential of improving the p21 protein activity. By expressing a more active p21 even more of the cell's energy can be devoted to producing product. Taken as a whole, we showed that with this cell cycle engineering method we can more precisely and efficiently control the proliferation of CHO cells for biotherapeutic protein production.