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Temperature shift as a process optimization step for the production of pro-urokinase by a recombinant Chinese hamster ovary cell line in high-density perfusion culture
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 97, No. 4, 239–243. 2004
Temperature Shift as a Process Optimization Step for the Production of Pro-urokinase by a Recombinant Chinese Hamster Ovary Cell Line in High-Density Perfusion Culture ZHAO-LIE CHEN,1* BEN-CHUAN WU,1 HONG LIU,1 XING-MAO LIU,1 AND PEI-TANG HUANG1 Department of Cell Engineering, Institute of Biotechnology, 20 Dongdajie Street, Fengtai, Beijing 100071, P.R. China1 Received 25 November 2003/Accepted 19 January 2004
Based on the effects of temperature shift on the cell cycle, apoptosis and metabolism of a recombinant Chinese hamster ovary (rCHO) cell line (CL-11G) producing pro-urokinase (pro-UK) in batch cultures, the potential of temperature shift as a tool in the optimization of the perfusion culture of CL-11G cells for the production of pro-UK was examined. The proportion of CL-11G cells in the G0/G1 phase in static cultures increased from 56.4% to 82.8% following a temperature shift from 37°C to 31°C. Conversely, the proportion of CL-11G cells in the S phase decreased from 34.8% to 11.6%. The specific growth rate of CL-11G cells reflected the effect of temperature on the cell cycle and decreased from 0.024 h–1 at 37°C to 0.006 h–1 at 31°C. Continuous exposure to the non-permissive temperature of 31°C led to a marginal increase in apoptosis. The specific proUK productivity of CL-11G cells increased by 74% at 34°C compared with controls at 37°C in batch cultures. CL-11G cells immobilized with Cytopore 1 in a 5-l bioreactor initiated at 37°C and temperature shifted to 34°C exhibited an average 17% increase in viable cell density and an average 47% increase in pro-UK production. These results demonstrated that temperature shift offers the prospect of enhancing the productivity of pro-UK in high-density perfusion culture. [Key words: perfusion culture, process optimization, pro-urokinase, recombinant Chinese hamster ovary cell line]
densities, the process optimization requirements for highdensity perfusion cultures are somewhat different. However, the effects of temperature reduction on high-density perfusion processes have hardly been studied (1, 10). Chuppa et al., who investigated temperature set points of 34°C, 35.5°C and 37°C, found that reducing the fermentor temperature to 34°C resulted in several advantages in the control of highdensity perfusion culture of rCHO cells, including a higher cell density in oxygen limited reactor, a lower perfusion rate, improved product quality, simplified pH control, among others (1), but Takahashi et al. demonstrated that cultivation of hybridoma cells at 33°C resulted in the loss of specific monoclonal antibody productivity (10). In the present study, we determined the potential of fermentor temperature as a tool in the optimization of perfusion cultures, and demonstrated that reduced fermentor temperature results in increased cellular productivity of prourokinase (pro-UK) in a porous microcarrier perfusion culture of an rCHO cell line.
Mammalian cells are most commonly cultured at 37°C, which is the human body core temperature. This set point is so widely accepted that temperature has received little attention as a process optimization variable. However, since the culture temperature would affect such cellular events as growth, viability, protein synthesis and metabolism, it is an important factor that needs to be studied to realize an efficient process for protein production by animal cell culture (1). Most published studies have focused on the response of cells to a decrease in temperature in batch culture performance. It has been reported that a reduction in operating temperature results in growth arrest, metabolism decrease and improved viability with variable effects on productivity in batch cultures (2–9). For example, cultivation of recombinant Chinese hamster ovary (rCHO) cells producing erythropoietin at low temperature resulted in enhanced specific productivity (7), while rCHO cells secreting a humanized anti-4-1BB monoclonal antibody demonstrated no increase in specific productivity after a temperature downshift from 37°C to 30°C (9). In contrast to batch cultures characterized by low cell
MATERIALS AND METHODS Cell line and cell cultures The rCHO cell line, CL-11G, which produced pro-UK, is a recombinant cell line derived from a dihydrofolate reductase minus (dhfr–) CHO-K1 host. The cells were genetically engineered to secrete recombinant pro-UK using
* Corresponding author. e-mail: [email protected] phone: +86-10-63841526 fax: +86-10-63833521 239
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a dhfr/methotrexate selection method similar to that used by Kaufman and Sharpe (11). Routine T-flask cell passage was carried out at 80–90% confluence every 3–5 d at a 1 : 3–1 :4 split ratio in an amino acid-enriched D-MEM/F-12 (1:1, v/v) supplemented with 1.0 mM methotrexate (Sigma Chemical, St. Louis, MO, USA) and 1% (v/v) fetal calf serum (HyClone, Logan, UT, USA). Batch cultures were carried out in T-flasks containing 10 ml of culture medium. Cultures were inoculated with cells taken from the mid-exponential phase of a culture. Two set of experiments were performed. In set 1, cultures were grown at 37°C for the first 24 h, and then either maintained at this temperature, or the temperature was reduced to 35°C, 34°C, 33°C and 31°C for a further 3 d. In set 2, cultures were grown at 37°C for the first 2 d, and then either maintained at 37°C or shifted to 35°C, 34°C, 33°C and 31°C respectively, for a further 6 d with the serum-containing medium being refreshed daily. Spinner flask cultures were performed in two 250-ml Bellco spinner flasks (Bellco Glass, Vineland, NJ, USA) with a stirring rate of 25 rpm. CL-11G cells were seeded at 3.4´ 105 cells/ml and Cytopore 1 (Amersham Pharmacia Biotech, Uppsala, Sweden) was added to the spinner flasks at 2 g/l. The two spinner flask cultures were grown at 37°C for the first 8 d, and then either maintained at this temperature for the duration of the culture, or shifted to 34°C for a further 11 d. Cultures were fed daily with an 85% volume of fresh serum-free medium, 11G-SG-SFM, which was prepared by adding insulin, a trace element mixture, a lipid mixture, ascorbic acid and pluronic F68 to amino acid-enriched D-MEM/F-12 (1:1, v/v) (12). Two long-term perfusion processes were carried out in a 5-l stirred tank bioreactor equipped with a 75-mm spin filter (B. Braun Biotech International, Melsungen, Germany) using an initial cell density of 3–4 ´105 cells/ml and 4 g/l of Cytopore 1. Agitation was set at 50 rpm and perfusion was started on the second day of cultivation. Dissolved oxygen was controlled at 40% air saturation through bubble-free membrane aeration with air and/or oxygen, and the pH controlled between 7.1–7.2 through addition of NaHCO3. The temperature was set at 37°C for a period of 35–39 d, and then shifted to 34°C for a further 40–49 d. Cell counting methods A modified colorimetric assay based on thiazolyl blue (MTT) conversion (13) was employed for estimation of the numbers of cells in the spinner flask and perfusion cultures. Briefly, 0.1 ml of MTT stock solution (Sigma; 5 mg/ml in PBS) was added to 1-ml samples taken directly from the culture vessels or to 1-ml samples diluted with fresh medium, with 1 ml of fresh medium being used as a reference (blank control), in Nunc disposable tubes (Nunc, Roskilde, Denmark). The tubes were incubated at 37°C in a CO2 incubator for 10 h, then 1 ml of Triton X-100/dimethylformamide/distilled water (1: 2 : 3, v/v) was added
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to each tube which was then incubated for a further 12 h. The absorbance of the converted dye was measured at 570 nm. The correlation between MTT conversion by viable CL-11G cells and the viable cell number was determined by the trypan blue exclusion test. The trypan blue exclusion method was used for counting cells in T-flasks. Flow cytometry analysis Cell samples obtained from T-flask and perfusion cultures, containing approximately 5´105 cells, were washed twice with PBS and fixed with a mixture of 6 ml of 70% ice ethanol in 1 ml of D-MEM/F-12 and 1 ml of fetal calf serum for 1 h. Labeling was carried out according to the method described by Moore et al. (4), with minor modifications. Briefly, cell samples were pelleted and resuspended in 50 ml of a reaction solution containing 0.2 M potassium cacodylate, 25 mM Tris–HCl, 0.25 mg/ml BSA, 2.5 mM CoVl2, 25 units of terminal transferase (Boehringer Mannheim, Mannheim, Germany), and 500 pM FITCddUTP (Boehringer Mannheim). Samples were incubated at 37°C for 30 min, washed twice in PBS and then treated with 1 mg/ml RNAse (DNAse free; Invitrogen Life Science, Carlsbad, CA, USA) in PBS and stained with 100 mg/ml propidium iodide (Sigma) for 1 h at 37°C. Flow cytometry analysis was carried out using a Becton-Dickinson FACScan equipped with a 40-mW argon laser. Fluorescence data was collected for 20,000 cells for each sample. Cell-cycle distribution analysis was performed using the software package CellQuest (Becton-Dickinson, San Jose, CA, USA). Analysis of glucose, lactate and pro-UK The glucose and lactate concentrations in the culture supernatant were measured by the YSI 2700 Select Biochemistry Analyzer (Yellow Springs Instruments, Yellow Springs, OH, USA). The fibrinolytic activities of pro-UK were determined by an in vitro fibrin lysis assay (14) using the national UK standard as a reference.
RESULTS AND DISCUSSION Growth profile and pro-UK production of CL-11G cells in T-flask cultures at 37°C It has been well documented that a culture temperature below 37°C inhibits cell growth (5, 6), and protein production by some cell lines appears to be growth-associated (15, 16). Accordingly, the first step in determining the potential of temperature shift as a process optimization step for the production of pro-UK by a recombinant CHO cell line was to investigate the correlation between the cell growth kinetics and pro-UK expression of CL-11G cells. The time profiles of cell growth and pro-UK production for CL-11G cells showed that the production of pro-UK in batch culture at 37°C continued dur-
FIG. 1. Growth and specific rate of pro-UK production of CL-11G cells in batch culture at 37°C. Symbols: open circles, viable cell density; solid circles, total cell density; open squares, specific pro-UK production rate; solid squares, pro-UK concentration. Data are means± SD (n = 3).
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TABLE 1. Effects of temperature on cell cycle and growth of CL-11G in batch cultures
TABLE 2. Effects of temperature on specific pro-UK production rate, specific glucose consumption rate and specific lactate production rate in batch cultures
Specific growth rate G2/M S G0/G1 (h–1) 37 56.38 8.80 34.82 0.024 35 66.51 8.52 24.96 0.021 34 74.88 5.54 19.58 0.019 33 78.64 5.52 15.83 0.016 31 82.83 5.62 11.55 0.006 Results are expressed as the mean value of duplicate samples with a CV of no more than 10%.
Temperature qpro-UK qLac qGlc (°C) (IU/106 cells/d) (mmol/106 cells/d) (mmol/106 cells/d) 37 304 ± 54 3.61 ± 0.19 5.53 ± 0.91 35 407 ± 197 3.50 ± 0.46 5.62 ± 0.83 3.37 ± 0.41 5.29 ± 0.75 34 528 ± 76a 33 348 ± 101 3.48 ± 0.32 5.16 ± 0.47 31 387 ± 58 3.23 ± 0.23 5.25 ± 0.66 Data are means ± SD (n = 3). a Significantly different from cells cultured at 37°C (p<0.01).
ing the phase of non-growth and even partial death, and the specific pro-UK production rate (qpro-UK) was constant during the different growth phases (Fig. 1). The observation indicated that pro-UK production by CL-11G cells was not growth-associated, in contrast with, for example, tissue-type plasminogen activator and interferon-g production which appeared to be growth-associated under similar conditions (15, 16). Effects of temperature on cell cycle, growth and metabolism To investigate the effect of temperature on the specific metabolic rate of CL-11G cells, two sets of experiments were performed. Set 1 was performed comparing the effects of shifting the temperature from 37°C to 35°C, 34°C, 33°C and 31°C on the cell cycle and growth of CL-11G cells in batch cultures. As indicated by the data in Table 1, the proportion of CL-11G cells in the G0/G1 phase in batch culture increased from 56.4% to 82.8% following the temperature shift from 31°C to 37°C. Conversely, the proportion of CL-11G cells in the S phase decreased from 34.8% to 11.6%. The specific growth rate of CL-11G cells reflected the effect of temperature on the cell cycle and decreased from 0.024 h–1 at 37°C to 0.006 h–1 at 31°C (Table 1). Continuous exposure of CL-11G cells to a temperature of 31°C for 3 d resulted in approximately 4.2% of the cell population undergoing apoptosis, versus 1.8% at 37°C as estimated by flow cytometry analysis, without morphological changes (Fig. 2). This is in disagreement with previous ob-
servations by Moore et al., which demonstrated a significant suppression of apoptotic cell death in CHO cell cultures following a downshift of the cultivation temperature from 37°C to 30°C for 9–13 d (4). One explanation for this discrepancy is the difference in the period of exposure to the reduced temperature and the apoptosis checking set point. Set 2 was performed comparing the effects of culture temperature on the specific metabolic rates of CL-11G cells in T-flasks with daily refreshment of the medium to avoid nutrient limitation. Table 2 presents the specific pro-UK production rate, specific glucose consumption rate (qGlc) and specific lactate production rate (qLac) of CL-11G cells at different culture temperatures. Both glucose and lactate specific metabolic rates were marginally reduced corresponding with a culture temperature downshift. The specific glucose consumption rate decreased from 3.61 mmol/106 cells/d at 37°C to 3.23 mmol/106 cells/d at 31°C and the decrease in specific lactate production rate was presented by average values of 5.53 mmol/106 cells/d at 37°C to 5.25 mmol/106 cells/d at 31°C. A strong dependence of glucose metabolism on temperature was noted by Reuveny et al. (17) and by Sureshkumar and Mutharasan (6). The latter reported more than a 200% decrease in the specific glucose consumption rate at 33°C. However, due to the difference in the cell line used and the complexity of the medium formulation, this disagreement with our findings is difficult to explain. While the observed
Temperature (°C)
Cell cycle phase (%)
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FIG. 2. Morphology and fluorescence micrograph of CL-11G cells in T-flask cultures at 31°C or 37°C for a period of 3 d (magnification: 200´). (A) 37°C; (B) 31°C; (C) 31°C, stained with annexin V and propidium iodide.
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TABLE 3. Effects of temperature on specific pro-UK production rate, specific glucose consumption rate, specific lactate production rate and specific growth rate in immobilized cultures Specific Temperature qpro-UK qGlc qLac growth ratea (°C) (IU/106 cells/d) (mmol/106 cells/d) (mmol/106 cells/d) (h–1) 37 369 ± 84 3.21 ± 0.26 4.82 ± 0.61 0.017 34 558 ± 112 3.05 ± 0.41 4.29 ± 0.75 0.012 Data are means ±SD (n = 3). a Results are expressed as the mean value of duplicate samples with a CV of no more than 15%.
effect of temperature on the glucose and lactate metabolism was only marginal, the specific pro-UK production rate was significantly increased at 34°C compared to that at 37°C (Table 2). CL-11G cells immobilized on Cytopore 1 showed a parallel response in both growth and metabolism to that in batch cultures after shifting the temperature from 37°C to 34°C. The specific pro-UK productivity of CL-11G cells increased by 51% at 34°C compared with controls at 37°C, whereas only a marginal effect of temperature on the glucose and lactate metabolism was observed (Table 3). It is well documented that the intracellular transcription level of foreign genes is one of the crucial factors for the efficient expression of target proteins. Yoon et al. demonstrated that the effect of low culture temperature on specific productivity in rCHO cells has a direct bearing on the transcription level of foreign genes (7, 9). Accordingly, the beneficial effect of lowering the culture temperature on specific productivity appears to depend on cell type and target proteins and it may depend on the expression vectors used and the integration site of a foreign gene. The increase in specific pro-UK productivity seen in CL11G cells grown at 34°C prompted us to examine the potential of temperature as a tool in the optimization of perfusion culture to enhance pro-UK production by CL-11G cells. Therefore, two long-term perfusion processes, which continued for 75 and 88 d, were conducted in a 5-l stirred tank bioreactor equipped with a spin filter. The time profiles of cell density, pro-UK concentration and perfusion rate in a selected run are shown in Fig. 3. The temperature was shifted to 34°C after a period of 39 d of cultivation at 37°C. The average viable cell density in these two temperatures
FIG. 3. Time profiles of key process variables in a 5-l fermentation conducted at two different temperatures. Symbols: solid circles, viable cell density; open circles, pro-UK concentration; squares, perfusion rate.
was 1.64 ´106 cells/ml and 1.92 ´106 cells/ml, respectively. This 17.1% increase in viable cell density at 34°C resulted in an average 47% increase in pro-UK production and an average 40% increase in specific pro-UK productivity of CL-11G cells compared with the culture at 37°C. Although the growing CL-11G cells occupied the inner spaces of the carriers and formed clumps on the surface of the carriers, as shown in Fig. 4, the viability remained constant at around 90%. From a process optimization viewpoint, all process variables were positively affected or unaffected by the lower temperature except for the specific growth rate. The results of present study demonstrated that temperature shift offers the prospect of enhancing the productivity of pro-UK by a recombinant CHO cell line in high-density perfusion culture. Thus, attention should be focused on culture temperature as a process optimization variable for protein production in animal cell culture. ACKNOWLEDGMENTS The authors would like to thank the Ministry of Science and Technology of the People’s Republic of China for financially supporting this research, grant no. 2001AA215461.
FIG. 4. Growth of CL-11G cells immobilized with Cytopore 1 in continuously perfused culture. (A) Cytopore 1 (magnification: 200´); (B) CL11G cell clumps and Cytopore 1 (magnification: 100 ´).
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REFERENCES 1. Chuppa, S., Tsai, Y. S., Yoon, S., Shackieford, S., Rozales, C., Bhat, R., Tsay, G., Matanguihan, C., Konstantinov, K., and Naveh, D.: Fermentor temperature as a tool for control of high-density perfusion cultures of mammalian cells. Biotechnol. Bioeng., 55, 328–338 (1997). 2. Furukawa, K. and Ohsuye, K.: Effect of culture temperature on a recombinant CHO cell line producing a C-terminal a-amidating enzyme. Cytotechnology, 26, 153–164 (1998). 3. Kaufmann, H., Mazur, X., Fussenegger, M., and Bailey, J. E.: Influence of low temperature on productivity, proteome and protein phosphorylation of CHO cells. Biotechnol. Bioeng., 63, 573–582 (1999). 4. Moore, A., Mercer, J., Dutina, G., Donahue, C. J., Bauer, K. D., Mather, J. P., Etcheverry, T., and Ryll, T.: Effects of temperature of cell cycle, apoptosis and nucleotide polls in CHO cell batch cultures. Cytotechnology, 23, 47–54 (1997). 5. Weidemann, R., Ludwig, A., and Kretzmer, G.: Low temperature cultivation — a step towards process optimization. Cytotechnology, 15, 111–116 (1994). 6. Sureshkumar, G. K. and Mutharasan, R.: The influence of temperature on a mouse-mouse hybridoma growth and monoclonal antibody production. Biotechnol. Bioeng., 37, 292–295 (1991). 7. Yoon, S. K., Song, J. Y., and Lee, G. M.: Effect of low culture temperature on specific productivity, transcription level, and heterogeneity of erythropoietin in Chinese hamster ovary cells. Biotechnol. Bioeng., 82, 289–298 (2003). 8. Schatz, S. M., Kerschbaumer, R. J., Gerstenbauer, G., Kral, M., Dorner, F., and Scheiflinger, F.: Higher expression of Fab antibody fragments in a CHO cell line at reduced temperature. Biotechnol. Bioeng., 84, 433–438 (2003). 9. Yoon, S. K., Kim, S. H., and Lee, G. M.: Effect of low cul-
10.
11.
12.
13. 14. 15.
16. 17.
243
ture temperature on specific productivity and transcription level of anti-4-1BB antibody in recombinant Chinese hamster ovary cells. Biotechnol. Prog., 19, 1383–1386 (2003). Takahashi, K., Ueda, H., and Suzuki, E.: Temperature characteristics of mammalian cell perfusion culture, p. 217–222. In Funatsu, K., Shirai, Y., and Matsushita, T. (ed.), Animal cell technology: basic and applied aspects, vol. 8. Kluwer Academic Publishers, Dordrecht, The Netherlands (1997). Kaufman, R. J. and Sharpe, P. A.: Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase complementary DNA gene. J. Mol. Biol., 159, 601–621 (1982). Chen, Z. L., Wu, B. C., Jia, X. H., Li, W. Q., Liu, H., and Xiao, C. Z.: Development of serum-free media for genetically-engineered CHO cells producing prourokinase. Chin. J. Biotechnol., 12, 169–175 (1996). Jia, X. H. and Xiao, C. Z.: Thiazolyl blue method for the estimation of the number of cells cultivation in microcapsules. Bull. Mil. Med. Sci., 17, 207–211 (1993). Jespersen, J. and Astrup, T.: A study of the fibrin plate assay of fibrinolytic agents. Optimal conditions, reproducibility and precision. Haemostasis, 13, 301–315 (1983). Hayter, P. M., Curling, E. M. A., Baines, A. J., Jenkins, N., Salmon, I., Strange, P. G., and Bull, A. T.: Chinese hamster ovary cell growth and interferon production kinetics in stirred batch culture. Appl. Microbiol. Biotechnol., 34, 559–564 (1991). Rhodes, M. and Birch, J.: Large-scale production of proteins from mammalian cells. Bio/Technology, 6, 518–523 (1988). Reuveny, S., Velez, D., Macmillan, J., and Miller, L.: Factors affecting cell growth and monoclonal antibody production in stirred reactors. J. Immunol. Methods, 86, 53–59 (1986).
Temperature Shift as a Process Optimization Step for the Production of Pro-urokinase by a Recombinant Chinese Hamster Ovary Cell Line in High-Density Perfusion Culture ZHAO-LIE CHEN,1* BEN-CHUAN WU,1 HONG LIU,1 XING-MAO LIU,1 AND PEI-TANG HUANG1 Department of Cell Engineering, Institute of Biotechnology, 20 Dongdajie Street, Fengtai, Beijing 100071, P.R. China1 Received 25 November 2003/Accepted 19 January 2004
Based on the effects of temperature shift on the cell cycle, apoptosis and metabolism of a recombinant Chinese hamster ovary (rCHO) cell line (CL-11G) producing pro-urokinase (pro-UK) in batch cultures, the potential of temperature shift as a tool in the optimization of the perfusion culture of CL-11G cells for the production of pro-UK was examined. The proportion of CL-11G cells in the G0/G1 phase in static cultures increased from 56.4% to 82.8% following a temperature shift from 37°C to 31°C. Conversely, the proportion of CL-11G cells in the S phase decreased from 34.8% to 11.6%. The specific growth rate of CL-11G cells reflected the effect of temperature on the cell cycle and decreased from 0.024 h–1 at 37°C to 0.006 h–1 at 31°C. Continuous exposure to the non-permissive temperature of 31°C led to a marginal increase in apoptosis. The specific proUK productivity of CL-11G cells increased by 74% at 34°C compared with controls at 37°C in batch cultures. CL-11G cells immobilized with Cytopore 1 in a 5-l bioreactor initiated at 37°C and temperature shifted to 34°C exhibited an average 17% increase in viable cell density and an average 47% increase in pro-UK production. These results demonstrated that temperature shift offers the prospect of enhancing the productivity of pro-UK in high-density perfusion culture. [Key words: perfusion culture, process optimization, pro-urokinase, recombinant Chinese hamster ovary cell line]
densities, the process optimization requirements for highdensity perfusion cultures are somewhat different. However, the effects of temperature reduction on high-density perfusion processes have hardly been studied (1, 10). Chuppa et al., who investigated temperature set points of 34°C, 35.5°C and 37°C, found that reducing the fermentor temperature to 34°C resulted in several advantages in the control of highdensity perfusion culture of rCHO cells, including a higher cell density in oxygen limited reactor, a lower perfusion rate, improved product quality, simplified pH control, among others (1), but Takahashi et al. demonstrated that cultivation of hybridoma cells at 33°C resulted in the loss of specific monoclonal antibody productivity (10). In the present study, we determined the potential of fermentor temperature as a tool in the optimization of perfusion cultures, and demonstrated that reduced fermentor temperature results in increased cellular productivity of prourokinase (pro-UK) in a porous microcarrier perfusion culture of an rCHO cell line.
Mammalian cells are most commonly cultured at 37°C, which is the human body core temperature. This set point is so widely accepted that temperature has received little attention as a process optimization variable. However, since the culture temperature would affect such cellular events as growth, viability, protein synthesis and metabolism, it is an important factor that needs to be studied to realize an efficient process for protein production by animal cell culture (1). Most published studies have focused on the response of cells to a decrease in temperature in batch culture performance. It has been reported that a reduction in operating temperature results in growth arrest, metabolism decrease and improved viability with variable effects on productivity in batch cultures (2–9). For example, cultivation of recombinant Chinese hamster ovary (rCHO) cells producing erythropoietin at low temperature resulted in enhanced specific productivity (7), while rCHO cells secreting a humanized anti-4-1BB monoclonal antibody demonstrated no increase in specific productivity after a temperature downshift from 37°C to 30°C (9). In contrast to batch cultures characterized by low cell
MATERIALS AND METHODS Cell line and cell cultures The rCHO cell line, CL-11G, which produced pro-UK, is a recombinant cell line derived from a dihydrofolate reductase minus (dhfr–) CHO-K1 host. The cells were genetically engineered to secrete recombinant pro-UK using
* Corresponding author. e-mail: [email protected] phone: +86-10-63841526 fax: +86-10-63833521 239
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a dhfr/methotrexate selection method similar to that used by Kaufman and Sharpe (11). Routine T-flask cell passage was carried out at 80–90% confluence every 3–5 d at a 1 : 3–1 :4 split ratio in an amino acid-enriched D-MEM/F-12 (1:1, v/v) supplemented with 1.0 mM methotrexate (Sigma Chemical, St. Louis, MO, USA) and 1% (v/v) fetal calf serum (HyClone, Logan, UT, USA). Batch cultures were carried out in T-flasks containing 10 ml of culture medium. Cultures were inoculated with cells taken from the mid-exponential phase of a culture. Two set of experiments were performed. In set 1, cultures were grown at 37°C for the first 24 h, and then either maintained at this temperature, or the temperature was reduced to 35°C, 34°C, 33°C and 31°C for a further 3 d. In set 2, cultures were grown at 37°C for the first 2 d, and then either maintained at 37°C or shifted to 35°C, 34°C, 33°C and 31°C respectively, for a further 6 d with the serum-containing medium being refreshed daily. Spinner flask cultures were performed in two 250-ml Bellco spinner flasks (Bellco Glass, Vineland, NJ, USA) with a stirring rate of 25 rpm. CL-11G cells were seeded at 3.4´ 105 cells/ml and Cytopore 1 (Amersham Pharmacia Biotech, Uppsala, Sweden) was added to the spinner flasks at 2 g/l. The two spinner flask cultures were grown at 37°C for the first 8 d, and then either maintained at this temperature for the duration of the culture, or shifted to 34°C for a further 11 d. Cultures were fed daily with an 85% volume of fresh serum-free medium, 11G-SG-SFM, which was prepared by adding insulin, a trace element mixture, a lipid mixture, ascorbic acid and pluronic F68 to amino acid-enriched D-MEM/F-12 (1:1, v/v) (12). Two long-term perfusion processes were carried out in a 5-l stirred tank bioreactor equipped with a 75-mm spin filter (B. Braun Biotech International, Melsungen, Germany) using an initial cell density of 3–4 ´105 cells/ml and 4 g/l of Cytopore 1. Agitation was set at 50 rpm and perfusion was started on the second day of cultivation. Dissolved oxygen was controlled at 40% air saturation through bubble-free membrane aeration with air and/or oxygen, and the pH controlled between 7.1–7.2 through addition of NaHCO3. The temperature was set at 37°C for a period of 35–39 d, and then shifted to 34°C for a further 40–49 d. Cell counting methods A modified colorimetric assay based on thiazolyl blue (MTT) conversion (13) was employed for estimation of the numbers of cells in the spinner flask and perfusion cultures. Briefly, 0.1 ml of MTT stock solution (Sigma; 5 mg/ml in PBS) was added to 1-ml samples taken directly from the culture vessels or to 1-ml samples diluted with fresh medium, with 1 ml of fresh medium being used as a reference (blank control), in Nunc disposable tubes (Nunc, Roskilde, Denmark). The tubes were incubated at 37°C in a CO2 incubator for 10 h, then 1 ml of Triton X-100/dimethylformamide/distilled water (1: 2 : 3, v/v) was added
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to each tube which was then incubated for a further 12 h. The absorbance of the converted dye was measured at 570 nm. The correlation between MTT conversion by viable CL-11G cells and the viable cell number was determined by the trypan blue exclusion test. The trypan blue exclusion method was used for counting cells in T-flasks. Flow cytometry analysis Cell samples obtained from T-flask and perfusion cultures, containing approximately 5´105 cells, were washed twice with PBS and fixed with a mixture of 6 ml of 70% ice ethanol in 1 ml of D-MEM/F-12 and 1 ml of fetal calf serum for 1 h. Labeling was carried out according to the method described by Moore et al. (4), with minor modifications. Briefly, cell samples were pelleted and resuspended in 50 ml of a reaction solution containing 0.2 M potassium cacodylate, 25 mM Tris–HCl, 0.25 mg/ml BSA, 2.5 mM CoVl2, 25 units of terminal transferase (Boehringer Mannheim, Mannheim, Germany), and 500 pM FITCddUTP (Boehringer Mannheim). Samples were incubated at 37°C for 30 min, washed twice in PBS and then treated with 1 mg/ml RNAse (DNAse free; Invitrogen Life Science, Carlsbad, CA, USA) in PBS and stained with 100 mg/ml propidium iodide (Sigma) for 1 h at 37°C. Flow cytometry analysis was carried out using a Becton-Dickinson FACScan equipped with a 40-mW argon laser. Fluorescence data was collected for 20,000 cells for each sample. Cell-cycle distribution analysis was performed using the software package CellQuest (Becton-Dickinson, San Jose, CA, USA). Analysis of glucose, lactate and pro-UK The glucose and lactate concentrations in the culture supernatant were measured by the YSI 2700 Select Biochemistry Analyzer (Yellow Springs Instruments, Yellow Springs, OH, USA). The fibrinolytic activities of pro-UK were determined by an in vitro fibrin lysis assay (14) using the national UK standard as a reference.
RESULTS AND DISCUSSION Growth profile and pro-UK production of CL-11G cells in T-flask cultures at 37°C It has been well documented that a culture temperature below 37°C inhibits cell growth (5, 6), and protein production by some cell lines appears to be growth-associated (15, 16). Accordingly, the first step in determining the potential of temperature shift as a process optimization step for the production of pro-UK by a recombinant CHO cell line was to investigate the correlation between the cell growth kinetics and pro-UK expression of CL-11G cells. The time profiles of cell growth and pro-UK production for CL-11G cells showed that the production of pro-UK in batch culture at 37°C continued dur-
FIG. 1. Growth and specific rate of pro-UK production of CL-11G cells in batch culture at 37°C. Symbols: open circles, viable cell density; solid circles, total cell density; open squares, specific pro-UK production rate; solid squares, pro-UK concentration. Data are means± SD (n = 3).
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TABLE 1. Effects of temperature on cell cycle and growth of CL-11G in batch cultures
TABLE 2. Effects of temperature on specific pro-UK production rate, specific glucose consumption rate and specific lactate production rate in batch cultures
Specific growth rate G2/M S G0/G1 (h–1) 37 56.38 8.80 34.82 0.024 35 66.51 8.52 24.96 0.021 34 74.88 5.54 19.58 0.019 33 78.64 5.52 15.83 0.016 31 82.83 5.62 11.55 0.006 Results are expressed as the mean value of duplicate samples with a CV of no more than 10%.
Temperature qpro-UK qLac qGlc (°C) (IU/106 cells/d) (mmol/106 cells/d) (mmol/106 cells/d) 37 304 ± 54 3.61 ± 0.19 5.53 ± 0.91 35 407 ± 197 3.50 ± 0.46 5.62 ± 0.83 3.37 ± 0.41 5.29 ± 0.75 34 528 ± 76a 33 348 ± 101 3.48 ± 0.32 5.16 ± 0.47 31 387 ± 58 3.23 ± 0.23 5.25 ± 0.66 Data are means ± SD (n = 3). a Significantly different from cells cultured at 37°C (p<0.01).
ing the phase of non-growth and even partial death, and the specific pro-UK production rate (qpro-UK) was constant during the different growth phases (Fig. 1). The observation indicated that pro-UK production by CL-11G cells was not growth-associated, in contrast with, for example, tissue-type plasminogen activator and interferon-g production which appeared to be growth-associated under similar conditions (15, 16). Effects of temperature on cell cycle, growth and metabolism To investigate the effect of temperature on the specific metabolic rate of CL-11G cells, two sets of experiments were performed. Set 1 was performed comparing the effects of shifting the temperature from 37°C to 35°C, 34°C, 33°C and 31°C on the cell cycle and growth of CL-11G cells in batch cultures. As indicated by the data in Table 1, the proportion of CL-11G cells in the G0/G1 phase in batch culture increased from 56.4% to 82.8% following the temperature shift from 31°C to 37°C. Conversely, the proportion of CL-11G cells in the S phase decreased from 34.8% to 11.6%. The specific growth rate of CL-11G cells reflected the effect of temperature on the cell cycle and decreased from 0.024 h–1 at 37°C to 0.006 h–1 at 31°C (Table 1). Continuous exposure of CL-11G cells to a temperature of 31°C for 3 d resulted in approximately 4.2% of the cell population undergoing apoptosis, versus 1.8% at 37°C as estimated by flow cytometry analysis, without morphological changes (Fig. 2). This is in disagreement with previous ob-
servations by Moore et al., which demonstrated a significant suppression of apoptotic cell death in CHO cell cultures following a downshift of the cultivation temperature from 37°C to 30°C for 9–13 d (4). One explanation for this discrepancy is the difference in the period of exposure to the reduced temperature and the apoptosis checking set point. Set 2 was performed comparing the effects of culture temperature on the specific metabolic rates of CL-11G cells in T-flasks with daily refreshment of the medium to avoid nutrient limitation. Table 2 presents the specific pro-UK production rate, specific glucose consumption rate (qGlc) and specific lactate production rate (qLac) of CL-11G cells at different culture temperatures. Both glucose and lactate specific metabolic rates were marginally reduced corresponding with a culture temperature downshift. The specific glucose consumption rate decreased from 3.61 mmol/106 cells/d at 37°C to 3.23 mmol/106 cells/d at 31°C and the decrease in specific lactate production rate was presented by average values of 5.53 mmol/106 cells/d at 37°C to 5.25 mmol/106 cells/d at 31°C. A strong dependence of glucose metabolism on temperature was noted by Reuveny et al. (17) and by Sureshkumar and Mutharasan (6). The latter reported more than a 200% decrease in the specific glucose consumption rate at 33°C. However, due to the difference in the cell line used and the complexity of the medium formulation, this disagreement with our findings is difficult to explain. While the observed
Temperature (°C)
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FIG. 2. Morphology and fluorescence micrograph of CL-11G cells in T-flask cultures at 31°C or 37°C for a period of 3 d (magnification: 200´). (A) 37°C; (B) 31°C; (C) 31°C, stained with annexin V and propidium iodide.
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TABLE 3. Effects of temperature on specific pro-UK production rate, specific glucose consumption rate, specific lactate production rate and specific growth rate in immobilized cultures Specific Temperature qpro-UK qGlc qLac growth ratea (°C) (IU/106 cells/d) (mmol/106 cells/d) (mmol/106 cells/d) (h–1) 37 369 ± 84 3.21 ± 0.26 4.82 ± 0.61 0.017 34 558 ± 112 3.05 ± 0.41 4.29 ± 0.75 0.012 Data are means ±SD (n = 3). a Results are expressed as the mean value of duplicate samples with a CV of no more than 15%.
effect of temperature on the glucose and lactate metabolism was only marginal, the specific pro-UK production rate was significantly increased at 34°C compared to that at 37°C (Table 2). CL-11G cells immobilized on Cytopore 1 showed a parallel response in both growth and metabolism to that in batch cultures after shifting the temperature from 37°C to 34°C. The specific pro-UK productivity of CL-11G cells increased by 51% at 34°C compared with controls at 37°C, whereas only a marginal effect of temperature on the glucose and lactate metabolism was observed (Table 3). It is well documented that the intracellular transcription level of foreign genes is one of the crucial factors for the efficient expression of target proteins. Yoon et al. demonstrated that the effect of low culture temperature on specific productivity in rCHO cells has a direct bearing on the transcription level of foreign genes (7, 9). Accordingly, the beneficial effect of lowering the culture temperature on specific productivity appears to depend on cell type and target proteins and it may depend on the expression vectors used and the integration site of a foreign gene. The increase in specific pro-UK productivity seen in CL11G cells grown at 34°C prompted us to examine the potential of temperature as a tool in the optimization of perfusion culture to enhance pro-UK production by CL-11G cells. Therefore, two long-term perfusion processes, which continued for 75 and 88 d, were conducted in a 5-l stirred tank bioreactor equipped with a spin filter. The time profiles of cell density, pro-UK concentration and perfusion rate in a selected run are shown in Fig. 3. The temperature was shifted to 34°C after a period of 39 d of cultivation at 37°C. The average viable cell density in these two temperatures
FIG. 3. Time profiles of key process variables in a 5-l fermentation conducted at two different temperatures. Symbols: solid circles, viable cell density; open circles, pro-UK concentration; squares, perfusion rate.
was 1.64 ´106 cells/ml and 1.92 ´106 cells/ml, respectively. This 17.1% increase in viable cell density at 34°C resulted in an average 47% increase in pro-UK production and an average 40% increase in specific pro-UK productivity of CL-11G cells compared with the culture at 37°C. Although the growing CL-11G cells occupied the inner spaces of the carriers and formed clumps on the surface of the carriers, as shown in Fig. 4, the viability remained constant at around 90%. From a process optimization viewpoint, all process variables were positively affected or unaffected by the lower temperature except for the specific growth rate. The results of present study demonstrated that temperature shift offers the prospect of enhancing the productivity of pro-UK by a recombinant CHO cell line in high-density perfusion culture. Thus, attention should be focused on culture temperature as a process optimization variable for protein production in animal cell culture. ACKNOWLEDGMENTS The authors would like to thank the Ministry of Science and Technology of the People’s Republic of China for financially supporting this research, grant no. 2001AA215461.
FIG. 4. Growth of CL-11G cells immobilized with Cytopore 1 in continuously perfused culture. (A) Cytopore 1 (magnification: 200´); (B) CL11G cell clumps and Cytopore 1 (magnification: 100 ´).
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REFERENCES 1. Chuppa, S., Tsai, Y. S., Yoon, S., Shackieford, S., Rozales, C., Bhat, R., Tsay, G., Matanguihan, C., Konstantinov, K., and Naveh, D.: Fermentor temperature as a tool for control of high-density perfusion cultures of mammalian cells. Biotechnol. Bioeng., 55, 328–338 (1997). 2. Furukawa, K. and Ohsuye, K.: Effect of culture temperature on a recombinant CHO cell line producing a C-terminal a-amidating enzyme. Cytotechnology, 26, 153–164 (1998). 3. Kaufmann, H., Mazur, X., Fussenegger, M., and Bailey, J. E.: Influence of low temperature on productivity, proteome and protein phosphorylation of CHO cells. Biotechnol. Bioeng., 63, 573–582 (1999). 4. Moore, A., Mercer, J., Dutina, G., Donahue, C. J., Bauer, K. D., Mather, J. P., Etcheverry, T., and Ryll, T.: Effects of temperature of cell cycle, apoptosis and nucleotide polls in CHO cell batch cultures. Cytotechnology, 23, 47–54 (1997). 5. Weidemann, R., Ludwig, A., and Kretzmer, G.: Low temperature cultivation — a step towards process optimization. Cytotechnology, 15, 111–116 (1994). 6. Sureshkumar, G. K. and Mutharasan, R.: The influence of temperature on a mouse-mouse hybridoma growth and monoclonal antibody production. Biotechnol. Bioeng., 37, 292–295 (1991). 7. Yoon, S. K., Song, J. Y., and Lee, G. M.: Effect of low culture temperature on specific productivity, transcription level, and heterogeneity of erythropoietin in Chinese hamster ovary cells. Biotechnol. Bioeng., 82, 289–298 (2003). 8. Schatz, S. M., Kerschbaumer, R. J., Gerstenbauer, G., Kral, M., Dorner, F., and Scheiflinger, F.: Higher expression of Fab antibody fragments in a CHO cell line at reduced temperature. Biotechnol. Bioeng., 84, 433–438 (2003). 9. Yoon, S. K., Kim, S. H., and Lee, G. M.: Effect of low cul-
10.
11.
12.
13. 14. 15.
16. 17.
243
ture temperature on specific productivity and transcription level of anti-4-1BB antibody in recombinant Chinese hamster ovary cells. Biotechnol. Prog., 19, 1383–1386 (2003). Takahashi, K., Ueda, H., and Suzuki, E.: Temperature characteristics of mammalian cell perfusion culture, p. 217–222. In Funatsu, K., Shirai, Y., and Matsushita, T. (ed.), Animal cell technology: basic and applied aspects, vol. 8. Kluwer Academic Publishers, Dordrecht, The Netherlands (1997). Kaufman, R. J. and Sharpe, P. A.: Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase complementary DNA gene. J. Mol. Biol., 159, 601–621 (1982). Chen, Z. L., Wu, B. C., Jia, X. H., Li, W. Q., Liu, H., and Xiao, C. Z.: Development of serum-free media for genetically-engineered CHO cells producing prourokinase. Chin. J. Biotechnol., 12, 169–175 (1996). Jia, X. H. and Xiao, C. Z.: Thiazolyl blue method for the estimation of the number of cells cultivation in microcapsules. Bull. Mil. Med. Sci., 17, 207–211 (1993). Jespersen, J. and Astrup, T.: A study of the fibrin plate assay of fibrinolytic agents. Optimal conditions, reproducibility and precision. Haemostasis, 13, 301–315 (1983). Hayter, P. M., Curling, E. M. A., Baines, A. J., Jenkins, N., Salmon, I., Strange, P. G., and Bull, A. T.: Chinese hamster ovary cell growth and interferon production kinetics in stirred batch culture. Appl. Microbiol. Biotechnol., 34, 559–564 (1991). Rhodes, M. and Birch, J.: Large-scale production of proteins from mammalian cells. Bio/Technology, 6, 518–523 (1988). Reuveny, S., Velez, D., Macmillan, J., and Miller, L.: Factors affecting cell growth and monoclonal antibody production in stirred reactors. J. Immunol. Methods, 86, 53–59 (1986).