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=Biotechnology and Bioprocess Engineering 2007, 12: 60-72 Enhancement of Erythropoietin Production in Recombinant Chinese Hamster Ovary Cells by Sodium Lactate Addition q É=_çç= ÜçÉQI= åÇ=fâJeï å=háãNG= 1 College of life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea 2 Microbiology Section, College of Pharmacy, Chung-Ang University, Seoul 156-756, Korea 3 Division of Applied Chemical Engineering and Bio Engineering, Hanyang University, Seoul 133-791, Korea 4 Department of Microbiological Engineering, Konkuk University, Seoul 143-701, Korea Abstract The stabilization of optimum pH for cells can cause a higher erythropoietin (EPO) production rate and a good growth rate with the prolonged culture span in recombinant Chinese hamster ovary (r-CHO) cells. Our strategy for stabilizing the opti- mum pH in this study is to reduce the lactate production by adding sodium lactate to a culture medium. When 40 mM so- dium 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 con- centration 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 dehydro- genase (LDH) in r-CHO cells increased and catalyzed the oxidation of lactate to pyruvate, together with the reverse reaction, at the addition of 40 mM sodium lactate. The addition of 40 mM sodium lactate caused the positive effects on a cell growth and an EPO production in the absence of carbon dioxide gas as well as in the presence of carbon dioxide gas by reducing the accumulation of lactate. KSBB Keywords: erythropoietin, high productivity, lactate, CHO, lactate dehydrogenase, pH ====INTRODUCTION Periodic replenishment of glucose and glutamine through a fed-batch operation is commonly practiced to overcome their Mammalian cell cultures including the culture of Chinese exhaustion. Waste products excreted by a mammalian cell hamster ovary cells are used extensively to produce proteins itself have the significant effects on a cell growth and the for a therapeutic use [1-6]. The production process of re- production of metabolites. The major waste product is lactate, combinant proteins depends on a high cell yield, a prolonged which is produced mainly from glycolysis for energy acqui- culture span, steady productivity, and consistent glycosyla- sition and in a small amount from glutamine metabolism, tion. The depletion of nutrients and the accumulation of cel- and ammonium, which is excreted when amino acids are lular waste products are two major factors limiting cell metabolized in cells [7,8]. The concentration of lactate in growth in mammalian cell cultures. As a result, the cell den- mammalian cell cultures depends on the concentration of sity in a conventional batch culture is usually low and the glucose, a type of cell, and a mode of bioreactor operation. culture span is short. Consequently, the amount of a product Lactate inhibitions to mammalian cells are mainly attributed is small, which is undesirable for the production process of to the acidification of a medium. Namely, lactate accumula- recombinant proteins. Both glucose and glutamine are the tion often exceeds the buffering capacity of a medium, major carbon and energy sources used by mammalian cells, thereby lowering a culture pH value and inhibiting a cell and these nutrients are often depleted during the cultivation. growth. Because mammalian cells grow rapid in vitro only over a very narrow pH range within 0.2 to 0.4 pH units of *Corresponding author the optimum, cell growth rates decrease at high lactate con- Tel: +82-2-3290-3447 Fax: +82-2-927-9028 centrations [9,10]. However, as compared to ammonium, the e-mail: [email protected] inhibition of cell growth by lactate occurs at relatively higher Biotechnol. Bioprocess Eng. 61= concentrations [11]. The toxic effects of lactate on mammal- lactate was significantly decreased through a rational me- ian cell cultures are well known and widely reported on a dium design and a stoichiometric feeding of nutrients [25]. number of different cell lines [9,11-13]. For CRL-1606 hy- In this study, our objective is to decrease the formation of bridoma cells, lactate was found to be the only environ- lactate by the design of an initial medium that would provide mental parameter that significantly inhibited antifibronectin a starting environment to achieve an optimal cell growth and monoclonal antibody production, but the cell growth was product production. We expected that the lactate added to a slightly inhibited even at 40 mM lactate [14]. Stimulation of medium could alter cell metabolism. Because it was reported the cell growth by lactate concentrations up to 22 mM and that cell metabolism was affected by the environmental con- inhibition above 28 mM lactate were reported in hybridoma ditions such as pH and the concentration of metabolites cells (VII H-8) [15]. Miller et al. did not see any inhibition [26,27]. We investigated whether the addition of sodium of lactate on the cell growth over the range of lactate concen- lactate could cause higher EPO productivity in r-CHO cell tration considered [16]. In addition, they observed almost no cultures, and also examined effects of the lactate addition on change in metabolic rates when lactate concentration was lactate production and glucose consumption. Finally, the changed from 25 to 44 mM. Ozturk et al. reported that a relationship between LDH activity and lactate addition was specific growth rate of mouse hybridoma 167.4G5.3 cells was reduced by one-half at an initial lactate concentration of 55 mM [11]. A tolerance toward lactate is specific in cell lines [16]. A difference in the effects of lactate on different MATERIALS AND METHODS cell types may lie on the sensitivity of key enzymes to the glucose metabolic pathway and the metabolic shift in re- Cells and Their Maintenance sponse to the adverse environment. As previously mentioned, some cell lines such as hybri- r-CHO cell line producing EPO used in this study was doma are characterized with respect to the effects of lactate. provided by Dr. H. J. Hong from the Korea Research Insti- However, less information is available on the influence of tute of Bioscience and Biotechnology, Daejeon, Korea. lactate on the growth and productivity of Chinese hamster Minimum Essential Medium-α (M EM -α) (Gibco, USA) ovary (CHO) cells. CHO cells are the most frequently used supplemented with 10% (v/v) fetal bovine serum (FBS) mammalian host for biomedical products and they can ex- (Gibco) and 20 nM methotrexate (Sigma, USA) was used for press a wide variety of recombinant proteins. CHO cells the cultivation of r-CHO cells. No antibiotic was added to have two significant advantages: (1) high productivity and the medium. Cells were routinely cultured on 25-cm2 T- (2) ability to synthesize oligosaccharide chain structures flasks (Nunc, Denmark) and grown in a model MCO-175 resembling those of the natural product. In addition, CHO incubator (Sanyo, Japan) at 37oC under atmosphere contain- cells are not apoptotic and eventually die by necrotic death ing 5% CO2 and 95% humid air. Cells in an exponential [17]. The choice of nonapoptotic cells may, therefore, be phase were used to seed at 105 cells/mL in 6-well plates (2.5 beneficial to the production of proteins. EPO is a growth mL per well) in duplicate in media supplemented with vari- factor that stimulates the proliferation and differentiation of ous concentrations of indicated chemicals. erythroid precursor cells to more mature erythrocytes [18]. This is one of the first recombinant proteins to make a suc- cessful trip from the laboratory to the clinical use. Recombi- nant human EPO has been approved for the clinical treat- For experiments in the addition of lactate, sodium lactate ment of anemia associated with chronic renal failure, as well (Sigma) was dissolved in MEM-α at 400 mM, and then the as nonrenal anemia [18,19]. Change in a pH value by the pre-determined volume of 400 mM sodium lactate was added accumulation of lacate alters the protein processing, include- to a culture medium to obtain the required final concentration ing glycosylation, and the secretion [20,21]. Many studies at the start of cel cultures. In this study, culture media with 0, have described strategies to reduce the accumulation of lac- 5, 10, 20, 40, and 80 mM sodium lactate were used. Using the tate in mammalian cell cultures. The usual strategy is to re- same batch of cel s, the culture with the same concentration of place the "spent" medium with a fresh medium. However, sodium chloride, which would give roughly the same osmolar- this strategy increases the use of serum, reduces the yield of ity as that with sodium lactate, was also set up for the purpose a product on serum, and ultimately raises the cost of produc- of distinguishing between lactate effect and osmolarity effect. tion. Glacken et al. introduced a strategy, which was to feed To test the effect of osmolarity, sodium chloride (Sigma) was glucose into the cultures at a controlled low rate [22]. Be- added to a medium at the concentrations of 27.5, 47.5, 62.5, cause an increase in the concentration of glucose from 0.2 to 72.5, and 87.5 mM. These concentrations of sodium chloride 10 mM reduces the flux of glucose through the pentose- would give those osmolarities as 380, 420, 450, 470, and 500 phosphate pathway by 50%, it preferably increases the flux mOsm/kg because the measured osmolarity of culture me- of glucose through glycolysis to lactic acid [23]. In addition, dium was found to be approximately 325 mOsm/kg. Cells Eagle et al. demonstrated that the substitution of more cultured in the medium with normal osmolarity (325 mOsm/ slowly utilized sugars, such as fructose, galactose, and mal- kg) were inoculated into media with varying osmolarities and tose, results in a small amount of the accumulation of lactate cultured in 6-wel plates (Nunc). Several aliquots of fresh me- [24]. Xie and Wang also reported that a production rate of dium were transferred to a series of conical tubes and their pH were adjusted using sterile NaOH and HCl. An aliquot of a containing LDH isoenzymes 1 to 5 was used as an aid in the prepared suspension of cel s was added to these tubes to identification of isoenzymes. After the electrophoresis, sepa- achieve a final cell density of 105 cells/mL. Each cell suspen- rated LDH isoenzymes were visualized by using a colorimet- sion was pipetted in 2.5 mL into each wel of 6-well plate. ric detection (Procedure No. 705-EP, Sigma). The stained During the cultivation, the supernatant of cell cultures was gel was fixed in a methanol-acetic acid solution (75 metha- taken at regular intervals and frozen for later analysis. Cells nol : 5 glacial acetic acid : 20 water). were counted for obtaining a growth profile. Al experiments were duplicated. Lactate Dehydrogenase Activity Lactate dehydrogenase (LDH) activity was determined by a diagnostic kit (Sigma). There were two procedures for deter- Concentrations of lactate and glucose in culture media mination of LDH activity. The first procedure was based on were measured with a YSI model 2700 Biochemistry Ana- the oxidation of lactate to pyruvate [lactate + NAD → pyru- lyzer (Yellow Springs Instruments, USA). Osmolarity was vate + NADH]. LDH catalyzes the oxidation of lactate to py- measured by a freezing point depression on an Osmomat 030 ruvate with the simultaneous reduction of NAD. Formation of (Gonotec, Germany). To monitor an external pH, a sample NADH results in an increase in absorbance at 340 nm. The of 1.0 mL was taken and immediately measured by a pH increasing rate of absorbance at 340 nm is directly propor- meter 440 (Corning, USA). Cell viability was determined by tional to LDH activity in a sample. To determine LDH activity, counting in a hemacytometer under a phase contrast micro- the supernatant of cell lysates was prepared by following the scope using the trypan blue exclusion. procedure used in the detection of LDH isoenzyme. Assay reagent contained 50 mmol/L lactate, 7 mmol/L NAD, 0.05% Bioassay for EPO-Quantitative Analysis sodium azide, and nonreactive stabilizer and fillers (pH 8.9). The prepared cel extract (50 μL) was added to a assay solu- Biological activity of EPO was examined by using an tion (1 mL, 30oC) and mixed by gentle inversion. After the EPO-dependent cell line, human leukemia F-36E (Riken incubation for 30 sec at 30oC, the absorbance was read at 340 Cell Bank RCB0776, Japan), that could be maintained in the nm by a spectrophotometer (Cary 300 Bio, Varian, Australia). presence of EPO [28]. F-36E cell density in each well of 96- This was initial absor-bance. Continuous incubation was per- well plate was initially fixed to 1 × 105 cells/mL. The stan- formed and the absorbances of 30 and 60 sec following initial dard of EPO (Boehringer Mannheim, Germany) and the su- absorbance reading were recorded. The absorbance reading pernatant of each sample were diluted serially with F-36E after 60 sec was final absorbance. Subtraction of initial ab- cell broth and 100 μL of the dilution was assayed for the sorbance from final absorbance was calculated to obtain ‘ab- biological activity of EPO by measuring the viable cell den- sorbance change per minute'. LDH activity was determined as sity of EPO-dependent cells. After incubation for 4 days, we follows: LDH activity [U/L] = (‘absorbance change per min- performed MTT assay to measure viability, proliferation, ute' × total reaction mixture volume × 1,000) / (millimolar and activation of cells [29]. One hundred microliters of the absorptivity of NADH at 340 nm × sample volume × light- cultured sample was incubated once more with 10 μL of 5 path). One unit of LDH activity was defined as the amount of mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium enzyme that would catalyze the formation of one micromole bromide (Sigma) in 96-well plates at 37oC for 3 h. Then 100 of NADH per minute. Specific activity of LDH was expressed μL of 0.04 M HCl in iso-propyl alcohol (Duksan, Korea) as units per 106 cel s. LDH activity was measured by monitor- was added to each well, and the mixture was pipetted to dis- ing the rate at which the substrate, pyruvate, is reduced to lac- solve the insoluble blue formazan crystals. The plate contain- tate [pyruvate + NADH → lactate + NAD]. Since the reduc- ing the dissolved blue formazan was read at an optical density tion is coupled with the oxidation of reduced form of NADH, of 570 nm. Sample was run in duplicate. this reaction is measured in terms of the decreasing rate of absorbance at 340 nm. Potassium phosphate buffer (2.85 mL, Electrophoretic Fraction and Colorimetric Detection 0.1 mol/L, pH 7.5) and 50 μL cell extract were pipetted di- of Lactate Dehydrogenase Isoenzymes rectly into a glass vial containing 0.2 mg reduced form of NAD. The mixture was mixed wel , then left at 25oC for 20 Cells for this assay were harvested, centrifuged (1,000 min. This was transferred from a glass vial to a square cuvet rpm, 2 min), and washed twice in ice-cold phosphate buff- (1-cm lightpath) capable of holding 3 mL, and 0.1 mL sodium ered saline (PBS). After the second washing, the viable cell pyruvate solution (22.7 mmol/L in 0.1 mol/L potassium phos- density was determined. Intracellular components were ex- phate buffer) was also added to that cuvet. The absorbance tracted by ultra-sonicating the cell pellet in 0.5 mL of PBS. was read and recorded at 340 nm at 30 sec intervals for 3 min The obtained cell lysates were centrifuged at 13,000 × g for using water as reference. LDH activity in this second proce- 5 min, and LDH isoenzyme present in the supernatants was dure was determined as follows: LDH activity [U/mL] = (‘ab- separated by adding to 1% agarose (Bethesda Research sorbance change per minute') / (0.001 × sample volume × Laboratories, USA) gel and electrophoresing in 50 V for 2 h lightpath), where: 0.001 = ‘absorbance change per minute' using a mini gel migration system (Mupid-21, Cosmo Bio, equivalent to 1 unit of LDH activity in 3 mL volume with 1- Japan). An electrophoretic marker (LDH Isotrol, Sigma) cm lightpath at 25oC.

Biotechnol. Bioprocess Eng. 63= ior was similar to that at pH 7.08. In addition, a variation of final cell density appeared. In result, an optimal plateau for good cell yield was obtained between pH 7.08 and 7.36, and a maximum specific growth rate was observed at pH 7.36, whereas the specific EPO production rate increased sharply above pH 6.80, reaching a maximum at pH 7.15 (Fig. 1B). Both higher EPO production rate and good cell growth were the cause of maximum EPO production. When the initial external pH decreased to 6.80, the specific EPO production rate was 43.8% of that at pH 7.15. The results from Fig. 1 verify the reports that pH has a direct effect on the cells pro- ducing EPO [20,31]. In general, external pH influences in- tracellular pH, and the change in intracellular pH can affect the activity of cytosolic enzymes or alter the metabolism of cells [32]. Moreover, the pH change in an endoplasmic re- ticulum and/or a Golgi apparatus could alter protein glycosy- lation and secretion [21]. Gramer and Goochee also reported that a sialidase activity in CHO cell lysates was active and stable at pH 7 [33]. Our results, together with these reports mentioned above, could account for the effect of initial ex- ternal pH on the specific EPO production rate. In result, we concluded that the pH stabilization was necessary for good EPO production. The usual strategies for stabilizing the op- timal pH in mammalian cell cultures are to replace the "spent" medium with fresh medium and to titrate the pH of Fig. 1. Effect of initial external pH on cell growth (A) and EPO culture media with alkaline. However, the one increases se- production (B) of r-CHO cells in MEM-α. Cells were in- rum usage and reduces the yield of product on serum, thus oculated at 1 × 105 cells/mL and grown in 6-well plates increasing production costs, while the other increases media osmolarity, which ultimately causes cell lysis. Like this, the existing strategies have many problems. Therefore, in this study, we introduced a new strategy, which was to add so- RESULTS AND DISCUSSION dium lactate as a culture supplement. Inhibitory Effect of External pH on Cell Growth and Increased EPO Production with Addition of Sodium Protein Productivity The major by-product of energy metabolism is lactate, We examined effects of the added lactate on growth, me- which arises from glycolysis. The accumulation of lactate tabolism, and protein productivity of r-CHO cells producing often exceeds the buffering capacity of a culture medium, recombinant human EPO in batch cultures with sodium lac- thereby lowering the culture pH. Subsequently, this may tate of 0, 5, 10, 20, 40, or 80 mM because the addition of cause undesirable effects on cell growth, since mammalian sodium lactate was considered as a new strategy for the sta- cells grow rapidly in vitro only over a very narrow pH range bilization of culture pH in the present work. In our study of within 0.2 to 0.4 pH units of the optimum. Unlike insect and r-CHO cells, it seems that the specific growth rate gradually primary mammalian cells, transformed mammalian cells decreases with an increase of lactate addition concentration. convert a large amount of glucose to lactate [30]. A previ- From Fig. 2A, however, no significant reduction in the spe- ously investigated CHO cell line was converted more than cific growth rate was found up to 20 mM sodium lactate 97% of the consumed glucose into lactate. Effects of initial addition. The specific growth rate was not decreased by ap- external pH on cell growth and protein productivity of r- proximately 22% until the concentration of sodium lactate CHO cells transfected for the human EPO synthesis were increased to 40 mM. We judged that the decreased specific examined in batch cultures. Fig. 1A shows the growth results growth rate in 40 mM would be profitable for EPO produc- of r-CHO cells seeded at 105 cells per mL and grown over a tion because of the following reason. If a desirable product 6 day-period at various pH values. Poor cell growth occurred results from the expression of an amplified recombinant at the extremes of tested pH. At pH 6.80, the specific growth gene, the cellular energy required to synthesize this protein rate decreased dramatically by the acidification of a culture could be a substantial fraction of the total energy capacity. medium, but the culture span was prolonged because of a When cells grow rapidly, however, cellular energy sources retarded nutrient depletion. When initial culture pH was 7.51, may be diverted to the synthesis of materials necessary for cells had a long lag time initially, but they were able to adapt cell growth rather than EPO production. Therefore, fast to pH 7.51 within a few days and subsequently their behav- growth of r-CHO cells may lower EPO production. When

significantly to 1.21 μg/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 par- tially 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 produc- tion rate can be major factors to produce much EPO, and these factors can be accomplished by adding sodium lactate The entire cell metabolism could be altered in the pres- ence 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 reduc- tion 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 im- portant in the disturbance of the metabolic pathways in which it is produced, or involved [34]. We observed only a Fig. 2. Effect of sodium lactate concentration on cell growth (A) marginal increase of external pH at 5 and 10 mM of the and EPO production (B) of r-CHO cells in MEM-α. All cul- added lactate concentrations (Fig. 3A). However, the de- tures were initiated at an inoculum of 1 × 105 cells/mL crease rate of pH was lowered on the condition that more and performed in 6-well plates for 8 days. Sodium lactate than 20 mM sodium lactate was added. This result implies was supplemented into cultures at the concentrations in- 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. Fig. 3 also shows effects of the added lactate concentrations on the addition concentration increased further to 80 mM, much specific rates in glucose consumption and lactate production. depressed growth rate was observed. In this result, the in- There was a significant decrease in specific glucose con- hibitory concentration for a 50% decrease (IC-50) of r-CHO sumption rate at lactate concentrations varying from 0 to 80 cells is estimated as 80 mM sodium lactate, which indicates mM, as shown in Fig. 3B. The specific consumption rate of that r-CHO cells are far less sensitive to lactate than other glucose decreased by 23.2% as 40 mM sodium lactate was cells [11]. On the contrary, a culture span was extended in added, while the specific production rate of lactate decreased lactate-addition cultures compared with a control culture. by 52% (Fig. 3C). These results from Figs. 3B and 3C may According to Fig. 2A, the culture longevity was extended for be mainly due to lactate effect in itself. In general, continu- between 50 and 100 h at high concentrations of lactate. In- ous mammalian cell lines have a metabolic disadvantage of dustrial trends toward high density cultures have required being unable to completely oxidize glucose to CO2 and H2O, elongating a culture span. Therefore, it is valuable to find a although they are well accepted as reliable and safe produc- way of elongating a culture span. From our study, we dis- tion hosts for manufacturing pharmaglycoproteins. In other covered that further prolonged culture span could be induced words, most of the glucose (95 to 99.8%) is oxidized to py- by the addition of sodium lactate. ruvate and finally to lactate [35-37]. The reason why con- All tested lactate concentrations except 80 mM produced tinuous cells oxidize glucose incompletely that the enzy- EPO far more than that in the absence of sodium lactate, as matic connection between glycolysis and tricarboxylic acid illustrated in Fig. 2B. For 80 mM lactate addition, the spe- (TCA) cycle has yet to be known [38]. However, we ex- cific EPO production rate was 1.65 times higher than that in pected that the added lactate could lead glucose to TCA cy- a control. However, the apparent volumetric production of cle, because lactate could be converted into pyruvate which EPO was similar to that in a control because the specific could enter the TCA cycle. The yield of lactate produced growth rate was below 0.02 h−1. The specific EPO produc- from glucose consumed, YL/G, is an important index of gly- tion rate of 0.48 μg/106 cells per hour in a control increased colysis. The effect of the added lactate concentrations on

Biotechnol. Bioprocess Eng. 65= Fig. 3. Effect of sodium lactate concentration on decrease rate Fig. 4. Ratio of viable cells in the absence of atmospheric CO2 of pH (A), specific glucose consumption rate (B), and to in the presence of atmospheric CO2 (A), and change of specific lactate production rate and ratio lactate produced pH (ΔpH) and accumulated lactate concentration of r- to glucose consumed (C). r-CHO cells were inoculated at CHO cells in the absence of atmospheric CO2 at the cul- 1 × 105 cells/mL and cultured in 6-well plates for 8 days. ture time of 39.5 h (B). r-CHO cells were inoculated at 2 × Sodium lactate was supplemented into cultures at the 105 cells/mL in 6-well plates and cultured in MEM-α concentrations indicated. supplemented with 0 or 40 mM sodium lactate for 4 days. YL/G is shown in Fig. 3C. YL/G decreased progressively as the tralized by increasing the bicarbonate concentration (Na- added lactate concentration increased up to 40 mM. The 3 ↔ Na+ + HCO3 ). The increased HCO3 concentration decreased YL/G indicates that a larger proportion of con- forces the equilibrium of CO2/bicarbonate buffer system sumed glucose may enter the TCA cycle [39]. Judging from toward the left until equilibrium is reached at pH 7.4. Due to results above, the action of lactate is likely to have a pro- the equilibrium attained between dissolved CO2 and bicar- found effect on glycolysis, so a good amount of glucose may bonate in this way, the pH of culture medium is maintained enter the TCA cycle at high lactate concentrations. In result, to the proper physiological level in buffer capacity. The ab- an increase in energy efficiency may occur. However, the sence of atmospheric CO2 allows the equilibrium of CO2/ elevated energy is not likely to be diverted for cell growth. It bicarbonate buffer system to the left, eventually eliminating is likely to be used for the synthesis of EPO instead. Flick- 3 from the medium. However, HCO3 is necessary for inger et al. [40] and Leno et al. [41] also suggested that addi- the good growth in CHO cells, because H+ transport system tional ATP could accelerate energy-consuming steps in bio- of CHO cells requires Na+, Cl−, and HCO − 3 [42]. Therefore, synthetic pathway of protein. the absence of atmospheric CO2 inhibits cell growth. Never- theless, it must be worth considering a free-CO2 condition Effect of 40 mM Sodium Lactate Addition on r-CHO within the limits of the possibility, since the use of atmos- Cells in the Absence of Carbon Dioxide pheric CO2 increases costs. In this study, we examined whether the addition of 40 mM sodium lactate could im- The pH of cell culture medium is controlled by CO2/ bi- prove the growth of r-CHO cells in the absence of atmos- carbonate buffer system (H2O + CO2 ↔ H2CO3 ↔ H+ + pheric CO2. The ratio of viable cells in the absence of at- 3 ). The net result of increasing atmospheric CO2 is to mospheric CO2 to in the presence of atmospheric CO2 is depress the pH, so the effect of elevated CO2 tension is neu- shown in Fig. 4A. In the control experiment, where no so-

concentration in the presence of 40 mM sodium lactate was 232% of that in the absence of sodium lactate (Fig. 5B). These results imply the positive effect of added 40 mM so- dium lactate on the growth and EPO production of r-CHO cells on the condition without atmospheric CO2. Comparison between Sodium Chloride and Sodium Lactate Addition in r-CHO Cell Cultures The addition of sodium lactate increases the osmolarity, for example, at the addition of 40 mM sodium lactate, the osmolarity is approximately 400 mOsm/kg. However, the osmolarity in the range of 270 330 mOsm/kg is known to be quite acceptable for most cells [43]. In addition, growth de- pression in hypertonic medium has been reported for MCL1 cells and other hybridoma cells [44,45]. Therefore, we had to investigate whether the results in Figs. 2 and 3 were due to the sodium lactate addition itself or the osmolarity induced by the addition of sodium lactate. In advance of this investi- gation, we examined the effect of medium osmolarity on cell growth and EPO production of r-CHO cells over the range of 325 to 500 mOsm/kg. With an increase of the medium osmolarity, the specific growth rate and maximum cell density decreased (Fig. 6A). The specific growth rates at 380, 420, and 450 mOsm/kg decreased by 16, 31, and 46% as compared with the control (325 mOsm/kg), respectively. Fig. 5. Viable cell density (A, closed symbols), glucose concen- The inhibition of cell growth was extreme at 500 mOsm/kg. tration (A, open symbols), and EPO production (B) of r- The result at 500 mOsm/kg could partially explain the CHO cells in MEM-α supplemented with 0 (circle symbol) exceptional phenomena observed in Figs. 2B and 3B when or 40 (reversed triangle symbol) mM sodium lactate for 4 80 mM sodium lactate (approximately 480 mOsm/kg) was days. Cells were inoculated at 2 × 105 cells/mL in 6-well added. The result of cell growth in Fig. 6A was somewhat plates and cultured in the absence of atmospheric CO2. different from the observation by Kurano et al. [46] that CHO cell growth was not affected by osmolarity below 390 mOsm/kg. In general, hybridomas are known to exhibit an dium lactate was added, the viable cell density in the absence increased specific antibody production rate when subjected of atmospheric CO2 decreased by 51.9% compared with that to environmental stress, as hyperosmotic pressure [26,44, in the presence of atmospheric CO2 at the culture time of 45,47-56]. However, the increase of osmolarity decreases the 39.5 h and decreased significantly by 74.9% at 62.5 h. How- specific growth rate [44,57,58]. Therefore, some "trade-off" ever, the viable cell density in the absence of atmospheric relationship between cell growth and protein production is CO2 was 88.7% of that in the presence of atmospheric CO2 likely to exist [59]. We observed a similar response for the at 39.5 h on the condition that 40 mM of sodium lactate was specific EPO production of r-CHO cells (Fig. 6B). However, present. The addition of 40 mM sodium lactate also caused Glacken et al. observed a decrease of specific antibody pro- the small change of external pH by reducing lactate produc- duction rate in CRL-1606 cells under osmolarity conditions tion, as shown in Fig. 4B. In other words, the added sodium increased by the addition of sodium lactate [35]. The in- lactate is likely to replace the role of atmospheric CO2 by fluence of osmolarity on protein productivity seems to be decreasing lactate accumulation. When atmospheric CO2 cell line dependent [60]. Increasing osmolarities from 325 to was absence, the poor viability in the medium without so- 420 mOsm/kg increased the specific EPO production rate, as dium lactate decreased dramatically to zero, as shown in Fig. shown in Fig. 6B. The specific EPO production rate in cells 5A. This result indicates that the absence of CO2 inhibits the exposed to 420 mOsm/kg hyperosmotic stress was increased growth of r-CHO cells. However, r-CHO cells in the culture by 74% when compared with that of cells in the culture with medium containing 40 mM sodium lactate remained at the 325 mOsm/kg, and the specific EPO production rates be- concentration of 3.52 × 105 cells/mL even at the culture time tween 420 and 470 mOsm/kg formed a plateau. However, at of 89 h. In addition, the glucose consumption in the presence 500 mOsm/kg, the specific EPO production rate dropped to of 40 mM sodium lactate was slower than that in the absence 85.8% of 420 mOsm/kg. Oh et al. demonstrated that the of sodium lactate. This result indirectly shows that the effi- osmolarity effect acted quite specifically on Na+-dependent ciency of energy production is increased by the addition of transport systems, while there was no significant increase in sodium lactate even on the condition without atmospheric the uptake by Na+-independent transport systems [50]. Ac- CO2. Moreover, because of the elongated culture span, EPO cordingly, the increase in osmolarity might enhance the

Biotechnol. Bioprocess Eng. 67= Fig. 6. Effect of medium osmolarity on cell growth (A) and EPO Fig. 7. Comparison of cell growth (A) and specific glucose con- production (B) of r-CHO cells in MEM-α. Cells were in- sumption rate (B) of r-CHO cells in MEM-α supplemented oculated at 1 × 105 cells/mL and grown in 6-well plates with 40 mM sodium chloride (40 mM-C) or 40 mM so- for 7 days. The growth medium became hyperosmotic dium lactate (40 mM-L) for 7 days. No supplement includ- through the addition of sodium chloride. ing sodium chloride and sodium lactate was added in the control experiment. Cells were inoculated at 1 × 105 cells/mL in 6-well plates. transport of nutrients, in particular, amino acids through Na+- dependent transport systems [49]. In addition, stressed cul- tures contain the enhanced levels of total RNA, of which ity by comparing the results under the same osmolarity. In 80% is ribosomal RNA (rRNA). Higher rRNA content result, we obtained no significant difference of cell growth could increase the translation rate of protein. Oh et al. also between lactate-containing culture and lactate-excluding reported that RNA/DNA ratio was always higher for cultures culture of the same osmolarity, as illustrated in Fig. 7A. with higher osmolarity. High RNA/DNA ratio gives us a Thus, it is obvious that lactate itself has very little influence crude indication that transcription levels might be elevated in on growth and the inhibition to cell growth is mainly due to osmotically stressed cells [50]. Furthermore, under the osmolarity. However, Fig. 7B showed that the specific glu- higher osmolarity, histones are more readily dissociated cose consumption rate decreased by 39.4% in addition of 40 from DNA, thus allowing easier accessibility of the chroma- mM sodium lactate and 12.1% in the same concentration of tin structure to RNA polymerase for transcription [61]. It sodium chloride. In addition, specific lactate production rate was also reported that the rate of transcription increased was also found to be inhibited by 57.7% in 40 mM sodium through the nucleosome at high osmolarity [62]. These re- lactate and 24.7% in 40 mM sodium chloride (Fig. 8B). ports can partially explain the reason why the increase in Namely, when 40 mM sodium lactate was added, the spe- osmolarity enhances the specific EPO production rate in our cific glucose consumption rate and lactate production rate were considerably lower than that observed in the equivalent To verify that the increase of specific EPO productivity concentration of sodium chloride. This indicates that the observed in Fig. 2B was the effect of sodium lactate rather effects of lactate on specific glucose consumption rate and than the osmolarity effect, 40 mM sodium lactate and so- lactate production rate are not due to the osmolarity induced dium chloride were used for adjusting medium osmolarity to by lactate addition alone. On any osmolarity adjusted by approximately 400 mOsm/kg. We estimated that the effects sodium chloride, YL/G remained constant in the range of of lactate observed in this study were mainly related to the 1.8 2.0 (data not shown). This result agrees with that from alteration in cell metabolism by sodium lactate itself rather the report of Ryu and Lee [63]. However, on the condition than the change in medium osmolarity induced by the added that sodium lactate was added, YL/G decreased as concentra- sodium lactate. Therefore, we attempted to separate the tion of sodium lactated increased, as shown in Fig. 3C. It is chemical effect of sodium lactate and the effect of osmolar- assumed that osmolarity drives the effect of lactate on cell effects of sodium lactate itself and osmolarity induced by it. Therefore, the effects of sodium lactate may be partly ex- plained by the increased osmolarity in the case of r-CHO cells. After "correcting" for osmolarity effects, sodium lac- tate itself was found to increase the specific EPO productiv- ity by 98%, and to decrease the specific glucose consump- tion rate and specific lactate production rate by 27 and 33%, respectively. If osmolarity was the only effect, the results of sodium lactate and sodium chloride observed in Figs. 7 and 8 would always be equal, but those of sodium lactate and so- dium chloride did not coincide. Relationship between Lactate Dehydrogenase Activity and Lactate Addition in r-CHO Cell Culture Lactate dehydrogenase catalyzes the interconversion of lactate and pyruvate. The enzyme is a tetrameric protein [64] and gives rise to five isoenzymes. In massive documentation that has followed the reports of Dewey and Conklin [65] and Van der Helm [66], it is generally agreed that human tissue can be grouped into three categories based on LDH isoen- zymes contents. LDH-1, -2, and -3 are prominent in tissues such as heart, kidney, brain, pancreas, erythrocytes, lympho- Fig. 8. Comparison of EPO (A) and lactate production (B) of r- cytes, platelets, and diaphragm. LDH-4 and LDH-5 are the CHO cells in MEM-α supplemented with 40 mM sodium major fractions of liver, skeletal muscle, and granulocytes. chloride (40 mM-C) or 40 mM sodium lactate (40 mM-L) Prostate, thyroid, lung, adrenal, gastric mucosa, and ovary for 7 days. No supplement including sodium chloride and are characterized by preponderance of fractions intermediate sodium lactate was added in the control experiment. to the other groups [67]. H represents the peptide associated Cells were inoculated at 1 × 105 cells/mL in 6-well plates. with the main cardiac enzyme (LDH-1) and M the peptide from skeletal muscle (LDH-5). The intermediate isoenzymes LDH-2, -3, and -4 are hybrids formed by random association growth, but lactate itself is the main reason for the specific of H and M subunits into tetramers. Thus, LDH-2, -3, and -4 glucose consumption rate and lactate production rate. In ad- would contain H3M, H2M2, and HM3 subunits [68]. LDH-1 dition, the change of pH in culture medium during the expo- moves farthest toward the anode during electrophoresis at nential phase was 0.16 from initial pH in 40 mM sodium pH 8.6 and LDH-5 is the extreme cathodic enzymes. LDH-1 lactate and 0.25 in the same concentration of sodium chlo- and LDH-2 are sometimes termed the fast isoenzymes and ride (Fig. 8B). This indicates that sodium lactate addition can LDH-4 and LDH-5 the slow enzymes. LDH-3 is an enzyme prevent the big drop of medium pH. The pH stabilization with intermediate electrophoretic mobility. LDH-X migrates induced by sodium lactate may be profitable for the long- between LDH-3 and LDH-4 on agarose gel [69]. Agarose term cultures of r-CHO cells. We also observed that the gel electrophoresis of LDH isoenzyme pattern in r-CHO addition of sodium lactate had the highest level of specific cells is shown in Fig. 9. We observed no change of LDH EPO production rate among all the experiments tested, as isoenzyme pattern between lactate-containing culture and shown in Fig. 8A. This increase in EPO productivity, on the lactate-excluding culture of r-CHO cells. Thus, it is obvious condition that 40 mM sodium lactate was added, was not that lactate itself has no influence on the isoenzyme pattern identical to the corresponding increase caused by the of LDH. Electrophoretic band for LDH isoenzyme type of r- osmolarity. Specific EPO production rate increased by 144% CHO cells was present in the LDH-3 and LDH-4 regions, in 40 mM sodium lactate and 46% in the same concentration whereas Blanco and Zinkham [69] observed that human ma- of sodium chloride as compared with the control. In addition, ture ovary tissue had five isoenzymes with the exception of the final accumulated EPO concentration from 40 mM so- extra isoenzyme, LDH-X. Taken together, our result ob- dium chloride supplemented culture showed the slightly tained in r-CHO cells originated from Chinese hamster ovary higher level than that of the control. However, the accumu- tissue is quite different from that of human mature ovary lated EPO in 40 mM sodium lactate was 1.9 times higher than tissue. We conclude from Fig. 9 that LDH isoenzyme type of that in the control. This proves indirectly that the increased r-CHO cells is LDH-X. It is generally known that H-type osmolarity of culture medium is not the only effector on LDH is better adapted to function in the oxidation of lactate EPO production. Although the exact mechanisms of actions to pyruvate, whereas M-type LDH is more suited to catalyze by the two-stress factors, sodium lactate itself and osmolarity, the reverse reaction [70]. However, Brooks et al. [71] have are unknown, it is evident that the influences of lactate addi- reported that lactate oxidation exceeded pyruvate reduction tion on EPO production and metabolisms are the combined by 10 40% in mitochondria isolated from liver, skeletal Biotechnol. Bioprocess Eng. 69= Fig. 9. Agarose gel electrophoretic pattern of LDH of r-CHO cells in MEM-α supplemented with 40 mM sodium chlo- ride (lanes 3 and 6) or 40 mM sodium lactate (lanes 4 and 7). No supplement including sodium chloride and sodium lactate was added in the isoenzyme-determina- tion of control (lanes 2 and 5). Cells were cultured for 27 (lanes 2, 3, and 4) and 92 (lanes 5, 6, and 7) h, respec- tively. Cells were inoculated at 1 × 105 cells/mL in 100 mm dishes. Lactate dehydrogenase isoenzymes were separated by electrophoresis on 1% agarose gel. A marker (lanes 1 and 8) containing 5 human lactate dehy- drogenase isoenzymes was used to aid in the identifica- tion of sample LDH isoenzymes separated by electropho- muscle, and heart. Heart and muscle mitochondria were noted by the prevalence of both LDH-1 (H4) and LDH-5 (M4), while liver mitochondria were distinguished by the Fig. 10. Lactate dehydrogenase (LDH) activity based on the presence of LDH-5 (M4). Taking these reports together, it is oxidation of lactate to pyruvate (A) and the reduction of likely that the ability to convert lactate to pyruvate is not pyruvate to lactate (B) in r-CHO cells. LDH activity was necessarily governed by only LDH isoenzyme pattern. measured after 27 and 92 h. Cells were inoculated at 1 Therefore, we expected that r-CHO cells would not only × 105 cel s/mL in 100 mm dishes and cultured in MEM-α reduce pyruvate but also oxidize lactate well despite LDH supplemented with 40 mM sodium chloride or 40 mM isoenzyme type of r-CHO cells presented between the LDH- sodium lactate. No supplement including sodium chlo- 3 and LDH-4 types. ride and sodium lactate was added in the control ex- Figs. 10A or 10B shows LDH activities based on the oxi- dation of lactate to pyruvate or the reduction of pyruvate to lactate, respectively. LDH activities in Fig. 10 were obtained to a similar pattern regardless of contrary reactions. It is result in sodium chloride addition was preferably close to the shown in Figs. 10A and 10B that LDH activity of r-CHO control. The results above prove indirectly that the results cells added 40 mM sodium lactate is higher than that in both obtained by addition of sodium lactate differ from others in the control and the 40 mM sodium chloride addition. How- many respects because of lactate effect in itself rather than ever, the difference of LDH activities at 27 h-culture time is only osmolarity effect. The measured activity in vitro of much smaller than that at 92 h-culture time. One of differ- LDH enzyme, which was obtained by the addition of 40 mM ences between 27 and 92 h-culture time is the remained con- sodium lactate, was high in the assay based on the reduction centration of glucose that is a major carbon source in culture of pyruvate (Fig. 10B) as well as in the assay based on the medium. About 4 mM of glucose remained at 27 h-culture oxidation of lactate (Fig. 10A). Then, why did lactate pro- time, while glucose was completely exhausted at 92 h- duction decrease in the culture with sodium lactate? The culture time (data not shown). This indicates that the notice- reason might be attributed to the difference between in vitro able enhancement of LDH activity by the added lactate does LDH assay condition and in vivo cell culture condition. The not occur in the presence of glucose, but the added lactate concentration of pyruvate used in LDH assay, based on the can influence LDH activity greatly when glucose is almost reduction of pyruvate, was 22.7 mM and there was no addi- consumed. The addition of 40 mM sodium chloride, which tion of lactate. However, the used culture medium contained had same osmolarity as 40 mM sodium lactate, did not en- 1 mM pyruvate and 40 mM sodium lactate. In other words, hance LDH activity like the addition of lactate. The when lactate concentration was low (nearly zero), LDH in r- CHO cells would mainly catalyze the reduction of pyruvate. ovary cells cultured in serum-free media. Biotechnol. However, on the condition that high concentration (40 mM Bioprocess Eng. 11: 319-324. in this study) of lactate was added, LDH was likely to cata- 2. Kim, J. S., M. K. Min, and E. C. Jo (2001) High-level lyze the oxidation of lactate at a considerable rate, since en- expression and characterization of single chain urokinase- zyme activity could be varied despite the same enzyme when type plasminogen activator (scu-PA) produced in recom- the environment of enzyme reaction changed. Therefore, a binant Chinese hamster ovary (CHO) cells. Biotechnol. considerable amount of lactate accumulation (about 10 mM) Bioprocess Eng. 6: 117-127. was in the control experiment contained only 1 mM pyruvate 3. Kato, H., T. Inoue, N. Ishii, Y. Murakami, M. Matsu- without the addition of lactate, and only 5 mM of lactate mura, T. Seya, and P. C. Wang (2002) A novel simple accumulation was observed in the case of the addition of 40 method to purify recombinant soluble human comple- mM sodium lactate (data not shown). From the results above, ment receptor type 1 (sCR1) from CHO cell culture. we also explained why specific glucose uptake rate de- Biotechnol. Bioprocess Eng. 7: 67-75. creased by the addition of 40 mM sodium lactate. LDH of r- 4. Chang, K. H., K. S. Kim, and J. H. Kim (1998) Analy- CHO cells is activated by the addition of 40 mM sodium sis of erythropoietin glycoform produced by recombi- lactate in our study. This activated LDH catalyzes the oxida- nant CHO cells using the lectin-blotting technique. tion of lactate to pyruvate with simultaneous reduction of Biotechnol. Bioprocess Eng. 3: 40-43. NAD to NADH, which will lead to a higher accumulation of 5. Bae, G. W., D. W. Jeong, H. J. Kim, G. M. Lee, H. W. NADH than in the control. The somewhat increased concen- Park, T. B. Choe, S. M. Kang, I. Y. Kim, and I. H. Kim tration of NADH will lower the specific consumption rate of (2006) High productivity of t-PA in CHO cells using glucose in our study, since glycolysis is inhibited at the pres- hypoxia response element. J. Microbiol. Biotechnol. ence of excess NADH. 6. Li, J., C. Menzel, D. Meier, C. Zhang, S. Dubel, and T. Jostock (2007) A comparative study of different vector designs for the mammalian expression of recombinant IgG antibodies. J. Immunol. Methods 318: 113-124. We suggest that the main reason for results obtained in 7. Choi, Y. S., D. Y. Lee, I. Y. Kim, S. Kang, K. Ahn, H. J. our study is a series of alterations of metabolism by the lac- Kim, Y. H. Jeong, G. T. Chun, J. K. Park, and I. H. Kim tate addition. Sodium lactate added with 40 mM lets a LDH (2000) Ammonia removal using hepatoma cells in enzyme catalyze the oxidation of lactate to pyruvate at a mammalian cell cultures. Biotechnol. Prog. 16: 760-768. relatively high rate, so the accumulation of NADH is in- 8. Kim, N. Y., Y. J. Lee, H. J. Kim, J. H. Choi, J. K. Kim, creased. The uptake rate of glucose becomes slow because of K. H. Chang, J. H. Kim, and H. J. Kim (2004) En- the NADH accumulation. The decreased consumption rate hancement of erythropoietin production from Chinese of glucose causes a high-energy yield and a small amount of hamster ovary (CHO) cells by introduction of the urea lactate accumulation, and then the acidification of a culture cycle enzymes, carbamoyl phosphate synthetase 1 and medium is suppressed. In addition, the pyruvate produced by ornithine transcarbamylase. J. Microbiol. Biotechnol. the oxidation of lactate is used for production of energy and formation of various amino acids. Consequently, EPO pro- 9. Lao, M. S. and D. Toth (1997) Effects of ammonium duction is enhanced at 40 mM lactate addition, since r-CHO and lactate on growth and metabolism of a recombinant cells can synthesize more proteins through enough amino Chinese hamster ovary cell culture. Biotechnol. 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