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NOTES Influence of Ammonium on Growth, Metabolism, and Productivity of a Continuous Suspension Chinese Hamster Ovary Cell Culture Henrik A. Hansen and Claus Emborg' Center for Process Biotechnology, Department of Biotechnology, Block 223, Technical University of Denmark, DK-2800 Lyngby, Denmark
A recombinant DNA Chinese hamster ovary (CHO) cell line which produces tissue-type plasminogen activator (t-PA) was cultivated continuously in suspension with a constant dilution rate of 0.5 day-'. The cultivation consisted of four phases with four different ammonium chloride concentrations (0,2.5,5, and 7.5 mM) in the feed medium, causing a reactor ammonium concentration of up to 8 mM. Cell growth was not inhibited by these high ammonium concentrations, as cell densities of around 2.3 X lo6 cells mL-l were established. In contrast, the production of t-PA was reduced under high ammonium concentration. The decrease in specific t-PA production could be due to either a negative ammonium influence on productivity or a limitation of medium components, e.g., amino acids. Cell metabolism was changed under high ammonium concentrations, seen most clearly by a decrease in specific ammonium production by a factor of 8 and an increase in specific alanine production of 30%.
Introduction Recombinant DNA Chinese hamster ovary (CHO) cell lines have increasing industrial importance in pharmaceutical productions of complex proteins. Currently there is increasing interest in investigationsof mammalian cell metabolism because such knowledge is necessary in the design of production processes (Miller and Blanch, 1991). In this context, ammonium secreted by mammalian cells in culture is often considered to be a serious problem because of its toxicity to cells or inhibition of cell growth and productivity (Newlandet al., 1990;Kurano et al., 1990; Hayter et al., 1991; Schlaeger and Schumpp, 1989). In this paper, we investigate the growth, metabolism, and production of a CHO cell line expressing the tissue-type plasminogen activator (t-PA) in continuous culture. The cells were cultivated in suspension with different ammonium concentrationsin the feed medium to the chemostat. Materials and Methods Cell Line. The investigationwas undertaken using the t-PA-expressing CHO cell line TF-70 kindly provided by Kabi Pharmacia (Stockholm,Sweden). The cell line was a constructionof a dihydrofolatereductase deficient (dhfr) mutant transfected with t-PA/dhfr and amplified in the presence of methotrexate. Medium. Standard Dulbecco's Modified Eagle's powder medium (DMEM) (Gibco, Paisley, UK) and Ham's F12 powder medium (Sigma, St. Louis, MO) 1:l were dissolved in Milli-Q water, and the following were added: D-glucose (Merck, Darmstadt, Germany), to a final concentration of 25 mM; 44 mM sodium bicarbonate (Merck); 10 mM HEPES (Gibco). The medium was then sterilized by filtration through a 0.22-pm filter and stored at 4 "C. Immediately prior to use, each liter of medium was
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to whom correspondence should be addressed.
8756-7938/94/3010-0121$04.50/0
supplemented with 50 mL of fetal calf serum (Batch 3506, Biochrom, Berlin, Germany), 5 mL of 10% (w/v) Pluronic F68 (Fluka, Buchs, Switzerland), 100 000 units L-l penicillin, and 100mg L-' streptomycin (Biological Industries, Kibbutz Beth Haemek, Israel). As will be described later, different concentrations of ammonium chloride (Merck) were added to the medium during the cultivation. Cultivation. The suspensionCHO cells were cultivated in a 2-L reactor (1.5-L working volume) in the chemostat mode, with dilution rate 0.5 day-', head space aeration DOT 40%, pH 7.2, 37 "C, and 80 rpm stirring, using techniques previously described (Hansen et al., 1993). To obtain steady-statestate conditions,the cultivation system was operated for about seven residence times before changes were made. The cultivationwas composed of four phases (I-IV), which differed in feed medium ammonium concentration (0, 2.5, 5, and 7.5 mM). Assays. Cell numbers, glucose, lactate, ammonium ions, and free amino acids were determined as previously described (Hansen et al., 1993;Hansen and Emborg, 1992). The t-PA concentration was determined by a four-layer sandwich enzyme-linked immunosorbent assay (ELISA) method. Microtiter plates were coated with goat antihuman melanoma t-PA antibodies (Biopool, Umei, Sweden). Wells were washed and blocked with Triton X-100. Then, appropriately diluted two-chain t-PA standards (Biopool) or culture supernatants were incubated for 2 h at 37 "C. After washing, mouse t-PA antibodies (Novo Nordisk, Bagsvaerd, Denmark) were incubated for 1h at ambient temperature. After another washing step, peroxidase-conjugated rabbit anti-mouse Ig antibodies (Dako, Glostrip, Denmark) were incubated for 1 h at ambient temperature before the last washing. Wells were developed with o-phenylenediamine and the OD492 was read with an Anthos Reader 2001 (Anthos Labtec Instruments, Salzburg, Austria).
0 1994 American Chemical Society and American Institute of Chemical Engineers
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=E
2 w
3.0
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2.5
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30
2.0
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25
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20
1.5
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1.0 0.5
-
0.0
-
0 c Y
u)
0
n
35
10 5 0
0
250
500
750
1000
1250
1500
Time ( h )
Figure 1. Development of continuous CHO cell culture. After batch cultivation for 67 h, the medium feed was started with a dilution rate of 0.5 day-1. Four phases (I-IV) with different feed ammonium concentrations(0,2.5,5.0,and 7.5 mM) are indicated. ( 0 )viable cells, ( 7 )glucose, (V)lactate, and (A)ammonium.
Calculations. Specific metabolite consumption (production) rate (qc) was determined at the steady state by qc =
(C, - C)D XV
where CF is the concentration of the metabolite in the feed medium, C is the concentration of the metabolite in the reactor, D is the dilution rate, and xv is the viable cell concentration. If qc is negative, the component is produced. The reported steady-state points are calculated as averages of samples taken during the last 3 days in each of the four phases (I-IV), just before a change in inlet medium was made. Because of the low residual glutamine concentration throughout the cultivation, no attempts to correct for spontaneous glutamine decomposition were made.
Results and Discussion Cell density during 2 months of CHO cell cultivation are illustrated in Figure 1. The cultivation was undertaken in batch mode for the first 67 h. In this period, the viable cell density increased from 0.27 X lo6 to 1.9 X lo6 cells mL-1. Then the continuous operation was started with a constant dilution rate of 0.5 day1. It is indicated in the graph that the cultivation is composed of four phases (IIV) with different concentrations of ammonium ions (0, 2.5,5.0,and 7.5 mM) in the feed medium, i.e., the medium ammonium concentration was increased by steps of 2.5 mM after 383, 693, and 1027.5 h of cultivation. Despite the increase in ammonium concentration during the cultivation, the cell density continued to increase until phase 111, where a viable cell density of around 2.3 X lo6 cells mL-' was achieved. This cell density was also kept in phase IV when the inlet ammonium concentration was 7.5 mM (reactor concentration 8 mM). The viability was near 100% throughout the cultivation. The cell densities achieved are higher than those reached with another suspension CHO cell line cultivated continuously in glucose-reduced (and limited) RPMI 1640 medium without serum, where a maximum viable cell density0.5 X lo6 cells mL-' was obtained (Hayter et al., 1992). The ammonium concentration during that cultivation, less than 2 mM, cannot be considered inhibitory. A few investigations report on ammonium growth inhibition in CHO batch cultures. Kurano et al. (1990) found an ammonium inhibition constant of 8 mM; growth was not inhibited by initial ammonium chloride concentrations
Table 1. Steady-State Parameters for CHO Cell Culture phase (CF-) I I1 I11 IV (0 mM) (2.5 mM) (5.0 mM) (7.5 mM)
t-PA (ng mL-l) glutamine (mM) alanine (mM) glycine (mM)
85 0.097 0.75 0.47 -1.15
76 0.140 1.03 0.46 -0.78
40 0.099 1.16 0.59 -0.36
45 0.126 1.40 0.56 -0.39
230
182
176
180
-328
-288
-234
-237
1.4 29.1
1.6 21.7
1.3 19.4
1.3 18.6
Qala
-8.7
-9.5
-9.5
-11.3
Qglr
-2.9
-2.1
-3.0
-2.7
Qt-PA
(ng (106cells)-'
h-l)
Qglc
(nmol (lo6cells)-' h-l) Qlac
(nmol (106 cells)-' h-1) Yhc/g~c (mol m o l 9 4&
(nmol (lo6cells)-' h-l)
(nmol (lo6cells)-' h-l) (nmol (106cells)-' h-1)
of up to 2 mM, but was reduced with an initial ammonium concentration of 4.5 mM (Hayter et al., 1991). Schlaeger and Shumpp (1989) found that CHO cells were relatively insensitive to inhibition by ammonium. In their study, 8-10 mM ammonium was required to give 50% inhibition of growth. For hybridomas in continuous culture, Miller et al. (1988) reported that the cells could adapt to ammonium concentration that were considered toxic when initially added in batch cultures. It is also possible that the cells in this investigation adapted to the high level of ammonium, which was slowly increased during the cultivation. Along with the increase in cell density from phases 1-111, the glucose concentration decreased, ending at level around 3 mM. The lactate concentration oscillated around 27 mM (Figure 1). Although qglcand qhcdecreased from phase I to IV phase (Table l),the YlaClglccalculation indicates that the metabolism of glucose apparently did not change to be more enconomic with respect to energy generation by moving from glycolytic to oxidative ATP production. Broth concentrations of consumed amino acids relative to medium concentrations are shown in the histogram in Figure 2. It is seen that glutamine, having a medium concentration of 2.5 mM, was extensively consumed in all four phases of the cultivation; however, with broth concentrations around 0.1 mM, it was not totally depleted. Since there were more cells in the later phases of the cultivation, this apparently constant situation actually represented a 35% decrease in qgh (Table 1). For most
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Figure 2. Reactor amino acid concentrations relative to feed medium concentrations for the four phases with different feed ammonium concentrations (columns from left to right: 0, 2.5, 5.0, and 7.5 mh4 ammonium). of the amino acids there was a tendency to lower broth concentrations in phases I11 and IV with the high ammonium concentrations and higher cell densities. Three amino acids, asparagine, glutamate, and aspartate, normally not considered essential, had low feed medium concentrations, 0.05 mM. Of these amino acids, asparagine was depleted throughout the culture, glutamate was depleted in phase IV, and aspartatewas depleted in phases I11 and IV. The trend for the amino acids that were consumed was that their specific consumption rates were highest in phase I with the lowest cell density and no ammonium in the feed (Figure 3). Glycine and alanine were the only two amino acids produced. The broth concentration for glycine was around 0.5 mM, and for alanine it was increasing from 0.7 to 1.4 mM through phases I-IV. The increase in alanine concentration was partly caused by a 30% increase in q h (Table 1). In another investigation with CHO cells, alanine, serine, and glutamate were produced during the entire cultivation, and aspartate was produced during one of the steady states (Hayter et al., 1992). A simple hypothesis might explain the differences seen in glutamate and aspartate production. Glutamine is well recognized as an energy-generating substrate for cultured cells (glutaminolysis) (McKeehan, 1986). If the cells are supplied with more glutamine than is necessary, excess intermediary catabolites are excreted, as seen with glutamate and aspartate. In the chemostat experiment with glutamate and aspartate production, glucose was a limiting substrate and the mole ratio of glucose to glutamine in the medium was 1.3-2.1. In the present experiment, the g1ucose:glutamineratio is 10,which might favor a better utilization of glutamine, i.e., it is unfavorable for the cells to excrete glutamate and aspartate. Serine is converted to glycine when the &carbon atom of serine is transferred to tetrahydrofolate. Tetrahydrofolate derivatives serve as donors of one-carbon units in a variety of biosyntheses. The difference seen in the secretion of either serine or glycine may have a background in the need for one-carbon units. A more likely hypothesis, however, is that in the cultivation with serine production, the medium was supplemented with 0.1 pM methotrexate. Methotrexate inhibits dihydrofolate reductase, causing a depression of the synthesis of tetrahydrofolate from dihydrofolate. The lack of tetrahydrofolate can block the conversion of serine to glycine, causing serine excretion. In the present study, the conversion of serine to glycine is not blocked and glycine is the metabolite which is secreted.
I
4 c
Figure 3. Specific amino acid consumption rates for the four phases with different feed ammonium concentrations(columns from left to right: 0, 2.5, 5.0, and 7.5 mM ammonium). 40
30
20
10
-
0 0
2
4
6
8
10
Ammonium conc. (mM)
Figure 4. Specificammonium production vs reactor ammonium concentration. The ammonium concentrations are seen in Figure 1. In phases I and 11, the ammonium concentration reaches levels around 2.5 and 5 mM, respectively. This is 2.5 mM more than the concentrations in the respective feed media. In phases I11and IV, the ammonium concentrations were around 6 and 8mM, only 1and 0.5 mM higher, respectively, than the levels in the feed media (5 and 7.5 mM). This observation apparently indicates that the metabolism of the cells was affected by the high ammonium concentrations in the bioreactor. This is confirmed by the calculations of q- depicted in Figure 4. Apparently, the cells have a mechanism that changes the metabolism, so that less ammonium is generated during growth under high ammonium concentrations. In phases I-IV, q& increased by 30 5%. The same kind of observation has been made earlier for a hybridoma cell line by Miller et al. (1988). They explained the q& increase and qa" decrease under high broth ammonium concentrations by a shift from use of the glutamate dehydrogenase pathway to use of the alanine aminotransferase pathway in the conversion of glutamate to 2-oxoglutarate. This explanation also applied to the present investigation, and the mechanism might be of a general nature. Proline and cysteinelcystine cannot be detected with the OPA HPLC method, and unfortunately methionine and tryptophan could not be quantified because these two components coeluted under the chromatographic conditions used. In phases I11 and IV of the cultivation, the peak representing methionine and tryptophan in the
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chromatogram disappeared, implying that limitation by these metabolites might have occurred. The t-PA concentrations measured with an ELISA technique and calculation of specific production rates are shown in Table 1. The product concentration was highest in phase I with no ammonium in the feed and was a factor 2 lower in phases I11 and IV when the ammonium concentration was high. Because of the higher cell densities in phases 111and IV, Qt-PA was a factor of 3 lower. One reason could be the loss of productivity as can be found for hybridomas in continuous cultivations (Frame and Hu, 1990). However, no indication of a loss of productivity was found in other low ammonium, continuous cultivations with this cell line (results not shown).A more likely hypothesis, therefore, is that high ammonium concentration has a negative influence on productivity. This is opposite the results for a hybridoma, where the increased ammonium concentration that caused growth inhibition in batch culture had no effect on specific antibody production (McQueen and Bailey, 1990). A third explanation is that the culture in the phases with the lower productivity was depleted of metabolites necessary for t-PA production. One could imagine that the cells have a preference for growth compared to produciton; limiting metabolites, e.g., the amino acids methionine,tryptophan, asparagine, glutamate, and/or aspartate, might be channelled to cell growth.
Hansen, H. A.; Emborg, C. Experimental Design in the Development and Characterizationof a High-PerformanceLiquid ChromatographicMethod for Amino Acids. J . Chromatogr.
Acknowledgment The excellent technical assistance of A. Jensen and M. Bjerre is gratefully acknowledged. The CHO cell line was kindly provided by Kabi Pharmacia (Stockholm, Sweden). The help with tests for mycoplasma and the gift of t-PA antibodies from Novo Nordisk (Bagsvaerd, Denmark) are also acknowledged. The study was supported by the Nordic Programme on Bioprocess Engineering under the auspices of the Nordic Fund for Technology and Industrial Development.
Miller, W. M.; Blanch, H. W. Regulation of Animal Cell Metabolismin Bioreactors. In Animal Cell Bioreactors; Ho, C. S., Wang, D. I. C., Ed.; Butterworth-Heinemann: Boston,
Literature Cited Frame, K. K.; Hu, W.-S. The Loss of Antibody Productivity in ContinuousCulture of Hybridoma Cells. Biotechnol.Bioeng.
Accepted October 12,1993."
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Abstract published in Advance ACS Abstracts, December 15,
1993.
