An N-terminal BAR-like domain in Num1 mediates its assembly into patches and its interaction with dynein to promote spindle translocation into the mitotic yeast bud.

This case study addresses the difficulty in achieving high level expression and production of a small, very positively charged recombinant protein. The novel challenges with this protein include the protein's adherence to the cell surface and its inhibitory effects on Chinese hamster ovary (CHO) cell growth. To overcome these challenges, we utilized a multi-prong approach. We identified dextran sulfate as a way to simultaneously extract the protein from the cell surface and boost cellular productivity. In addition, host cells were adapted to grow in the presence of this protein to improve growth and production characteristics. To achieve an increase in productivity, new cell lines from three different CHO host lines were created and evaluated in parallel with new process development workflows. Instead of a traditional screen of only four to six cell lines in bioreactors, over 130 cell lines were screened by utilization of 15 mL automated bioreactors (AMBR) in an optimal production process specifically developed for this protein. Using the automation, far less manual intervention is required than in traditional bench-top bioreactors, and much more control is achieved than typical plate or shake flask based screens. By utilizing an integrated cell line and process development incorporating medium optimized for this protein, we were able to increase titer more than 10-fold while obtaining desirable product quality. Finally, Monte Carlo simulations were performed to predict the optimal number of cell lines to screen in future cell line development work with the goal of systematically increasing titer through enhanced cell line screening.

No abstract