Investigations on hydrodynamic sensitivity of mammalian cells have focused mainly on the readout of lethal effect as determined by cell death or lysis. In this talk, we will present the first systematic investigation of sub-lethal responses of biopharmaceutical producing mammalian cells to hydrodynamic stress. In large-scale agitated bioreactors, cells constantly circulate between high hydrodynamic stress zone around the impeller and low hydrodynamic stress zone away from impellers. A novel scale-down model, which reproduces this repetitive stress was used to test GS-CHO cells in a fed-batch process. Lethal effects were monitored through measurement of viability and lactate dehydrogenase (LDH) release. As a focus of this study, a broad array of assays and measurements were used to detect sub-lethal effects, which could affect important process and product quality attributes. These sub-lethal effects include cell growth rate, peak cell density, monoclonal antibody (mAb) production rate, glucose metabolism, post-translational modifications, and product related impurities.

In general, GS-CHO cells showed strong resistance to hydrodynamic stress, even under ten days' repetitive exposure. Cell growth rate and peak cell density were not affected at the highest stress level tested. Reduction in productivity and glucose utilization was observed, but the impact was minimal. However, significant non-lethal and physiological responses were indeed observed at hydrodynamic stress level much lower than that needed to elicit cell death or productivity drop. Cell size became smaller under hydrodynamic stress at which growth rate was not affected and production rate was only minimally reduced. Product quality was found to be the most sensitive parameter to hydrodynamic force. mAb charge profile shifted where the percentage of acidic species increased and that of basic species decreased. A significant shift of glycosylation pattern was also observed. Detailed results, including process performance, product quality, and the threshold hydrodynamic stress that triggers each sublethal effects, will be presented. The implication of this study on large-scale bioreactor design and operation will also be discussed.