Variable functions of bcl-2 in mediating bioreactor stress-induced apoptosis in hybridoma cells

Abstract

It has been demonstrated that the cell lines used for production of biopharmaceuticals are highly susceptible to apoptosis, and that over-expression of the bcl-2 oncogene can protect cells from death. Stress associated with the deprivation of nutrients has been shown to be the main cause of apoptosis in culture. We have extended these studies by investigating the mechanism of cell death under conditions of sub-optimal pH, shear stress and hyperosmolarity, and the protective action of bcl-2 over-expression. At pH 6, there was no clear evidence of protection from cell death. However, at pH 8, the viability of the bcl-2 transfected cells was about 20% higher relative to the control cells. Cultivation of control cells in a flat bottomed bioreactor with a magnetic stirrer bar without a pivot ring resulted in exposure of the cells to a high attrition effect. As a result, cell growth was retarded and a high level of cell death by apoptosis was observed. Under the same conditions, the bcl-2 transfected cell line exhibited a nearly five fold increase in viable cell number. This finding indicates that under apoptosis-suppressed conditions, shear stress can stimulate cell growth. Batch cultivation of both control and bcl-2 transfected cells in 350 and 400 mOsm media resulted in suppression of cell growth, athough the effect was most marked in the control cell line. Adaptation of control cells to 400 mOsm proved to be impossible to achieve. However, the bcl-2 transfected cells exhibited resistance to the osmotic stress resulting in long term adaptation to a high salt environment. Specific productivity of bcl-2 transfected cells grown in high osmolarity medium was 100% higher than that produced by non-adapted bcl-2 transfected cells grown in normal osmolarity medium. These results demonstrate that bcl-2 has a beneficial effect on hybridoma cultivation under a wide range of culture stresses.

Hyperosmotic stress induces autophagy and apoptosis in recombinant Chinese hamster ovary cell culture

“…Apoptotic cell death can also occur under various stressful conditions such as hyperosmolality, sodium butyrate addition, and shear stress (Kim and Lee, 2002;Perani et al, 1998).…”

Inhibitory effects of persistent apoptotic cells on monoclonal antibody production in vitro

“…33,34 However, to our knowledge, the phenomenon described here of substantially improved antibody production by hybridoma cells following removal of non-viable cells prior to culture seeding has not been reported previously. The effect is particularly intriguing because it was not limited to the initial population of cells from which the non-viable cells were removed.…”

Mcl‐1 overexpression leads to higher viabilities and increased production of humanized monoclonal antibody in Chinese hamster ovary cells

“…In commercial bioreactors, mammalian cells may experience conditions of nutrient or growth factor depletion, hydrodynamic stress, byproduct accumulation, elevated osmolality, and fluctuations in culture pH, all of which can signal the cells to die by apoptosis. 2,3 Premature apoptosis leads to decreased product yields, compromised product quality, and increased costs for downstream processing. 1,4,5 Apoptosis is a highly-regulated network of signaling pathways initiating from both inside and outside of the cell.…”