When two recombinant Chinese hamster ovary (rCHO) cell lines producing human thrombopoietin (hTPO), CHO/dhfr-B22-4 and W44, were cultivated in hyperosmolar media resulting from NaCl addition, their specific foreign protein productivity increased with medium osmolality. However, due to a simultaneous suppression of cell growth at elevated osmolality, no enhancement in the maximum foreign protein titer was made in batch cultures. To test the feasibility of using glycine betaine, known as a strong osmoprotective compound, for improved foreign protein production in hyperosmotic rCHO cell cultures, hyperosmotic batch cultures were carried out in the presence of 15 mM glycine betaine. Glycine betaine was found to have a strong osmoprotective effect on both rCHO cell lines. Inclusion of 15 mM glycine betaine in hyperosmolar medium enabled rCHO cell lines to grow at 571-573 mOsm/kg where they could not grow in the absence of glycine betaine. However, effect of glycine betaine inclusion in hyperosmolar medium on foreign protein production differed between rCHO cell lines. CHO/dhfr-B22-4 cells retained enhanced specific hTPO productivity ($q_{hTPO}$) in the presence of glycine betaine, and thereby, the maximum hTPO titer obtained at 573 mOsm/kg was increased by 72% over that obtained in the control culture with physiological osmolality (292 mOsm/kg). On the other hand, enhanced $q_{hTPO}$ of W44 at elevated osmolality decreased significantly in the presence of glycine betaine at a cost of the recovery of cell growth. As a result, the maximum antibody titer at 571 mOsm/kg was similar to that obtained in the control culture with physiological osmolality. Taken together, efficacy of the simultaneous use of hyperosmotic pressure and glycine betaine as a means to improve foreign protein production was variable among different rCHO clones.
When parental rCHO clones producing hTPO were subjected to higher methotrexate (MTX) level, clonal variations in regard to cell growth were apparent. For clones without any significant enhancement in $q_{hTPO}$ at higher MTX level, their growth was not depressed significantly and their cell size did not change significantly, either. On the other hand, for clones with significant enhancement in qhTPO at higher MTX level, their cell growth was depressed initially and recovered in successive passages. Furthermore, their cell size was increased, suggesting that changes in cell size may be indicative of gene amplification. When enhancement of $q_{hTPO}$ of 9 clones at higher MTX level was plotted with the extent of increase of their size, there was a linear correlation, suggesting that enhancement of $q_{hTPO}$ at higher MTX can be judged from the increase in cell size. Taken together, the data obtained here demonstrate that the selection of amplified CHO clones with enhanced qhTPO can be made based on their increased size and growth pattern, facilitating the development of high producing recombinant CHO cells.
During the culture of rCHO cells producing hTPO, necrosis soon after a period of exponential growth was induced, resulting in low hTPO titer. Arginine depletion in the culture medium was found to be mainly responsible for cell death. When the culture medium was fortified with arginine, both maximum cell concentration and hTPO titer were enhanced by approximately 5-fold, compared to those in the control culture. The favorable effect of arginine was also evident in the production of human erythropoietin and antibody. The arginine effect was shown in rCHO cells derived from another kind of CHO cell line (DG44), however, not in hybridoma cells, indicating higher level of arginine is required by rCHO cells than hybridoma cells. In addition, the arginine fortification was also effective in serum-free cultivation. Thus, the arginine-fortified medium can be generally used in rCHO cell cultures for the various heterologous protein production. The increased requirement of rCHO cells for arginine was primarily due to the enhancement of specific arginine consumption rate in CHO cells by heterologous protein production and gene over-expression. Since basal level of arginine in the conventional medium is relatively low with respect to the increased arginine consumption rate, the conventional medium should be elevated in arginine level for effective rCHO cell cultivation. Taken together, the use of the arginine-fortified medium was a effective means to improve foreign protein production by extending cell viability in rCHO cell cultures.'
