Sodium lactate 첨가에 의한 재조합 CHO 세포에서의 erythropoietin 생산성 증대

Theories and Applications of Chem. Eng., 2002, Vol. 8, No. 1

최연숙, 김익영, 김홍진*, 김익환

고려대학교 생명공학원, 중앙대학교 약학대학*

Enhancement of Erythropoietin Production in

Recombinant Chinese Hamster Ovary Cells by Sodium Lactate Addition

Yeon Sook Choi, Ick Young Kim, Hong Jin Kim*, Ik-Hwan Kim

Graduate School of Biotechnology, Korea University

*College of Pharmacy, Chung Ang University

Abstract

The stabilization of optimum pH for cells can cause a higher production rate of erythropoietin (EPO) and a good growth rate with the prolonged culture span in recombinant Chinese hamster ovary (r-CHO) cells. Our strategy for stabilizing the optimum pH in this study is to reduce the lactate production by adding sodium lactate to a culture medium. When 40 mM sodium lactate was added, a specific growth rate was decreased by approximately 22% as compared with the control culture. However, the culture longevity was extended to 187 h, and more than a 2.7-fold increase in a final accumulated EPO concentration was obtained at 40 mM of sodium lactate. On the condition that caused the high production of EPO, a specific glucose consumption rate and lactate production rate decreased by 23.3% and 52%, respectively. Activity of lactate dehydrogenase (LDH) in r-CHO cells increased and catalyzed the oxidation of lactate to pyruvate at the addition of 40 mM sodium lactate.

Introduction

Mammalian cell cultures are used extensively to produce proteins for a therapeutic use because many human proteins of the investigative and clinical value require folding events and post-translational modifications, such as glycosylation, which are unable to be carried out in bacteria. The production process of recombinant proteins depends on a high cell yield, a prolonged culture span, steady productivity, and consistent glycosylation. Waste products excreted by a mammalian cell itself have the significant effects on a cell growth and the production of metabolites. The major waste products are lactate, which is produced mainly from glycolysis for energy acquisition and in a small amount from glutamine metabolism, and ammonium, which is excreted when amino acids are transported into the cell. Lactate accumulation often exceeds the buffering capacity of a medium, thereby lowering a culture pH value and inhibiting a cell growth. Because mammalian cells grow rapidly in vitro only over a very narrow pH range within 0.2 to 0.4 pH units of the optimum.

In this study, our objective is to decrease the formation of lactate by the design of an initial medium that would provide a starting environment to achieve an optimal cell growth and product production. We expected that the lactate added to a medium could alter cell metabolism. Because it was reported that cell metabolism was affected by the environmental conditions such as pH and the concentration of metabolites (Ozturk and Palsson, 1991). We investigated whether the addition of sodium lactate could cause higher EPO productivity in r-CHO cell cultures, and also examined effects of the lactate addition on lactate production and glucose consumption. Finally, the relationship between LDH activity and lactate addition was checked.

화학공학의 이론과 응용 제8권 제1호 2002년

Materials and Methods

Minimum Essential Medium- (MEM-□) (Gibco, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco, USA) and 20 nM methotrexate (Sigma, USA) was used for the cultivation of r-CHO cells. Concentrations of lactate and glucose in culture media were measured with a YSI model 2700 Biochemistry Analyzer (Yellow Springs Instruments, USA). To monitor an external pH, a sample of 1.0 mL was taken and immediately measured by a pH meter 440 (Corning, USA). Cell viability was determined by counting in a hemacytometer using the trypan blue exclusion. Biological activity of EPO was examined by using an EPO-dependent cell line, human leukemia F-36E (Riken Cell Bank RCB0776, Japan), that could be maintained in the presence of EPO. Lactate dehydrogenase (LDH) activity was determined by a diagnostic kit (Sigma, USA).

Results and Discussion

We examined effects of the added lactate on growth, metabolism, and protein productivity of r-CHO cells producing recombinant human EPO in batch cultures with sodium lactate of 0, 5, 10, 20, 40, or 80 mM because the addition of sodium lactate was considered as a new strategy for the stabilization of culture pH in the present work. In our study of r-CHO cells, it seems that the specific growth rate gradually decreases with an increase of lactate addition concentration. From Figure 1A, however, no significant reduction in the specific growth rate was found up to 20 mM sodium lactate addition. The specific growth rate was not decreased by approximately 22% until the concentration of sodium lactate increased to 40 mM. We judged that the decreased specific growth rate in 40 mM would be profitable for EPO production because of the following reason. If a desirable product results from the expression of an amplified recombinant gene, the cellular energy required to synthesize this protein could be a substantial fraction of the total energy capacity. When cells grow rapidly, however, cellular energy sources may be diverted to the synthesis of materials necessary for cell growth rather than EPO production. Therefore, fast growth of r-CHO cells may lower EPO production. When the addition concentration increased further to 80 mM, much depressed growth rate was observed. In this result, the inhibitory concentration for a 50% decrease (IC-50) of r-CHO cells is estimated as 80 mM sodium lactate, which indicates that r-CHO cells are far less sensitive to lactate than other cells (Ozturk et al., 1992). On the contrary, a culture span was extended in lactate-addition cultures compared with a control culture. According to Figure 1A, the culture longevity was extended for between 50 and 100 h at high concentrations of lactate. Industrial trends toward high density cultures have required elongating a culture span. Therefore, it is valuable to find a way of elongating a culture span. From our study, we discovered that further prolonged culture span could be induced by the addition of sodium lactate.

All tested lactate concentrations except 80 mM produced EPO far more than that in the absence of sodium lactate, as illustrated in Figure 1B. For 80 mM lactate addition, the specific EPO production rate was 1.65 times higher than that in a control. However, the apparent volumetric production of EPO was similar to that in a control because the specific growth rate was below 0.02 h-1. The specific EPO production rate of 0.48 □/106 cells per hour in a control increased significantly to 1.21 □/106 cells per hour in the presence of 40 mM. Consequently, more than a 2.7-fold increase in final accumulated EPO concentration was obtained by adding 40 mM sodium lactate. The improvement of EPO production could be acquired by prolonged cell viability and increased specific EPO production rate. Therefore, 174% increase of final EPO concentration observed in 40 mM lactate addition was due to the elongated longevity of about 80 h and 152% increase of specific EPO production rate. In addition, the stimulation of EPO production observed here may be partially the result of reduced proliferation resulting in reduction of the cellular demand for ATP, because more than one post-transcriptional step in EPO synthesis may be affected by enhanced ATP availability. In conclusion, healthy cells, longer production period, and higher specific EPO production rate can be major factors to produce much EPO, and these factors can be accomplished by adding sodium lactate below 80 mM.

Figure 1. Effect of sodium lactate concentration on the cell growth (A) and the EPO production (B) of r-CHO cells in MEM-□. All cultures were initiated at an inoculum of 1□105cells/mL and performed in 6-well plates for 8 days. Sodium lactate was supplemented into cultures at the concentrations indicated.

The entire cell metabolism could be altered in the presence of lactate, due to followed causes. The first cause is the intracellular acidification induced by lactate. Therefore, it has been believed that lowering lactate accumulation would enhance cell growth and protein production, and the reduction of lactate production has been considered as one of the important goals in mammalian cell culture technology. The second cause is the effect of lactate in itself, especially important in the disturbance of the metabolic pathways in which it is produced, or involved (Cruz et al., 2000). We observed only a marginal increase of external pH at 5 and 10 mM of the added lactate concentrations (Figure 2A). However, the decrease rate of pH lowered on the condition that more than 20 mM sodium lactate was added. This result implies that the addition of sodium lactate supports the stabilization of culture pH. The small change in external pH is profitable for long-term cultivation as well as EPO production. Figure 2 also shows effects of the added lactate concentrations on specific rates in glucose consumption and lactate production. There was a significant decrease in specific glucose consumption rate at lactate concentrations varying from 0 to 80 mM, as shown in Figure 2B. The specific consumption rate of glucose decreased by 23.2% under the addition of 40 mM sodium lactate. While the specific production rate of lactate decreased by 52% (Figure 2C). These results from Figure 2B and C may be mainly due to lactate effect in itself. We expected that the added lactate could lead glucose to TCA cycle, because lactate could be converted into pyruvate which could enter the TCA cycle. The ratio of lactate produced to glucose consumed, L/G, is an important index of glycolysis. The effect of the added lactate concentrations on L/G is shown in Figure 2C. L/G decreased progressively as the added lactate concentration increased up to 40 mM. The decreased L/G indicates that a larger proportion of consumed glucose may enter the TCA cycle (Ljunggren and Haggstrom, 1994). Judging from the above results, the action of lactate is likely to have a profound effect on glycolysis, so a good amount of glucose may enter the TCA cycle at high lactate concentrations. In result, an increase in energy efficiency may occur. However, the elevated energy is not likely to be diverted for cell growth. It is likely to be used for the synthesis of EPO instead. Flickinger et al. (1992) also suggested that additional ATP could accelerate energy-consuming steps in biosynthetic pathway of protein.

It is shown in Figure 3 that LDH activity of r-CHO cells added 40 mM sodium lactate is higher than those in both the control and the 40 mM sodium chloride addition. The addition of 40 mM sodium chloride, which had same osmolarity as 40 mM sodium lactate, did not enhance LDH activity like the addition of lactate. The result in sodium chloride addition was preferably close to the control. The above results prove indirectly that the results obtained by addition of sodium lactate differ from the others in many respects because of lactate effect in itself rather than only osmolarity effect.

Figure 2. Effect of sodium lactate concentration on the decrease rate of pH (A), the specific glucose consumption rate (B), and the specific lactate production rate and the ratio lactate produced to glucose consumed (C). r-CHO cells were inoculated at 1□105 cells/mL and cultured in 6-well plates for 8 days. Sodium lactate was supplemented into cultures at the concentrations indicated.

Figure 3. Lactate dehydrogenase (LDH) activities based on the oxidation of lactate to pyruvate (A) and the reduction of pyruvate to lactate (B) in r-CHO cells. LDH activities were measured after 27 and 92 hours. Cells were inoculated at 1□105 cells/mL in 100 mm dishes and cultured in MEM-□	supplemented with 40 mM sodium chloride or 40 mM sodium lactate. No supplement including	sodium chloride and sodium lactate was added in the control experiment.

References

Cruz HJ, Freitas CM, Alves PM, Moreira JL, Carrondo MJT. 2000. Effects of ammonia and lactate on growth, metabolism, and productivity of BHK cells. Enzyme Microb. Technol. 27:43-52.

Flickinger MC, Goebel NK, Bibila T, Boyce-Jacino S. 1992. Evidence for posttranscriptional stimulation of monoclonal secretion by L-glutamine during slow hybridoma growth. J. Biotechnol. 22:201-226.

Ljunggren J, Haggstrom L. 1994. Catabolic control of hybridoma cells by glucose and glutamine limited fed batch cultures. Biotechnol. Bioeng. 44:808-818.

Ozturk SS, Palsson BO. 1991. Growth, metabolic, and antibody production kinetics of hybridoma cell culture: 1. Analysis of data from controlled batch reactors. Biotechnol. Prog. 7:471-480.

Ozturk SS, Riley MR, Palsson BO. 1992. Effects of ammonia and lactate on hybridoma growth, metabolism, and antibody production. Biotechnol. Bioeng. 39:418-431.