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Effect of hydrostatic pressure on hybridoma cell metabolism
JOURNALOF FERMENTATION ANDBIOENGINEERING Vol. 80, No. 6, 619-621. 1995
Effect of Hydrostatic
Pressure on Hybridoma
Cell Metabolism
MUTSUMI TAKAGI,* KEN-ICHI OHARA, AND TOSHIOMI YOSHIDA International
Center for Cooperative Research in Biotechnology, Suita, Osaka 565, Japan
Osaka University, 2-1 Yamada-oka,
Received 21 June 199WAccepted 5 September 1995 The effects of hydrostatic pressure on hybridoma cell metabolism were investigated. In cell cultures grown at various constant hydrostatic pressures between 0.1 and 0.9MPa, little variation was observed in either cell growth or the specitic glucose consumption rates at different pressures. However, the rate of conversion from glucose to lactate decreased from 89 to 83%, while the specific glutamine consumption rate increased from 1.07 to 1.29 X lo-l3 mol/cell/h between the lowest and highest pressures. The specific production rate of monoclonal antibody (MAb) rose from 4.5 to 5.6 x lo-l3 g/cell/h in proportion to the pressure increase. The main factor affecting the conversion rate was not the hydrostatic pressure during cell propagation, but rather the hydrostatic pressure at which metabolism occurred. The specific glutamine consumption and MAb production rates were influenced by the hydrostatic pressure in both the growth and production phases of the cultivation.
[Key words:
hydrostatic
pressure,
hybridoma
cells, monoclonal
Hydrostatic pressure is one of the operational parameters which are effective at increasing the supply rate of dissolved oxygen in mammalian cell cultures. However, while the effects of pH, temperature, dissolved oxygen concentration and agitation rate on mammalian cultures have been well documented, there have been few reports describing the effect of hydrostatic pressure. We have recently demonstrated (1) that the conversion rate from glucose to lactate in human embryo lung cells at 0.25 MPa was less than that at 0.12MPa. In general, hydrostatic pressure affects the kinetics of enzymatic reactions (2) and it has been suggested that hydrostatic pressure has a repressive effect on membrane protein synthesis in Escherichia coli (3). In the present study, the effects of hydrostatic pressure on hybridoma cell culture using monoclonal antibody (MAb) productivity were investigated. Cells and medium Mouse-mouse hybridoma cells AFP-27, which produce MAb against human a-fetoprotein, were used (4). DMEM/F-12 medium (Gibco Co. Ltd., NY, USA) supplemented with 1.35 g/l glucose, 219 mg/l glutamine, 0.4 g/l Pluronic F-68, 10 ,ug/l insulin, 2 g/l BSA, 20 mg/l transfer-in, 10 ,&I ethanolamine, 2.44 g/l NaHC03, 5,000 U/l penicillin and 5 mg/l streptomycin was used. Cell growth culture A T-flask for suspension cell culture (75 cm2, Sumilon Co. Ltd., Tokyo) containing 28.5 ml of the medium was inoculated with 2.85 x 106 cells and incubated at 37°C for 65 h in a conventional CO2 incubator with 5% C02, or in a pressurized incubator as described below. Pressurized cultivation A pressurized incubator system (Fig. 1) which consisted of a stainless steel vessel (length, 120mm; inner diameter, 95 mm), closed using flanges fitted with a valve and a pressure gauge, and a valve and a PVA filter at the outlet and inlet ends, respectively, was constructed. The vessel was placed in a 37°C water bath to maintain the temperature. After placing a T-flask culture in the vessel, the inner gas phase was replaced 3 times with a gas mixture consisting of N2, 02 and CO2 for 5 min and then pressurized up to a
antibody]
desired value between 0.1 and 0.9 MPa. The composition of the gas mixture was adjusted so that the partial pressures of O2 and CO2 in the T-flask during the cultivation were 0.021 MPa and 0.005 MPa, respectively. The vessel was not opened until the end of the cultivation. After cell growth culture Cell maintenance culture at 0.1 or 0.9 MPa, cells were centrifuged (2,500 rpm, 5 min), resuspended to adjust the cell concentration to 1.OX lo6 cells/ml with fresh medium, and then incubated in a T-flask for 24 h at 0.1 and 0.9 MPa, respectively. In T-flask cultures containing 28.5 ml of medium, the cell concentration increased to a maximum of 1.1 x 10” cells/ml because of a limited supply of O2 supply and the cell concentration was maintained at a virtually constant level. The values in all figures are the means of duplicates. The results were reproducible. Analysis Viable cells were counted by the trypanblue dye exclusion method. Glucose and lactate concentrations were measured by the glucose-oxidase peroxidase and lactate-oxidase peroxidase methods, respectively, using an auto-analyzer (Biochemistry Analyzer 2700; YSI Inc., Ohio, USA). The glutamine and ammonium concentrations were determined by the L-asparginase glutamine-dehydrogenase method (F-kit L-glutamic acid; Boehringer Mannheim Yamanouchi Co. Ltd., Tokyo) and the indophenol method (Ammonia-Test Wako; Wako Pure Chemicals Co. Ltd., Osaka), respectively. MAb concentration was measured using an enzymelinked immunosorption assay (ELISA) method (4). Effect of hydrostatic
pressure on cell growth culture
Cell growth cultures were simultaneously carried out in a conventional CO2 incubator at 0.1 MPa and in the pressurized incubator at constant hydrostatic pressures of 0.1, 0.3, 0.5 and 0.9 MPa. Monitoring of the cell concentration in the conventional CO2 incubator cultivation revealed that at 0.1 MPa cells were growing logarithmically, even at 65 h (data not shown). As shown in Fig. 2, the pH could be maintained at a constant level in the pressurized vessel by adjusting the CO2 partial pressure, while the cell concentrations at 65 h did not differ with pressure. Although the specific glucose consumption rate was not affected by pressure, the specific lactate production
* Corresponding author. 619
620
J. FERMENT.BIOENG..
TAKAGI ET AL.
3
r+ r
8
1
1
1 7
1
I
r
2 FIG. 1. Schematic diagram of a pressurized incubator system for hybridoma cultures. 1, Pressurized vessel; 2, water bath; 3, gas inlet; 4, gas filter; 5, gas outlet; 6, pressure gauge; 7, T-flask; 8, mixed gas.
rate was slightly reduced as the pressure increased (Fig. 3). Consequently, the rate of conversion from glucose to lactate decreased from 89% to 83% as the pressure was raised from 0.1 MPa to 0.9 MPa. Both the specific glutamine consumption and ammonia production rates increased as pressure increased up to 0.5 MPa (Fig. 3). This may be due to a change in the enzymatic reaction rate since the thermal decomposition rate of glutamine was not affected by the pressure (data not shown). The specific MAb production rate increased markedly from 4.5 to 5.6 x lo-l3 g/cell/h as the hydrostatic pressure increased (Fig. 3). Effects of hydrostatic pressure during cell growth and To cell maintenance on cell maintenance cultures ascertain which intracellular processes were directly affected by pressure or cell metabolism kinetics, including enzyme reactions, maintenance cultivations without cell growth were conducted at 0.1 or 0.9 MPa using cells grown at 0.1 and 0.9 MPa, respectively. As shown in Fig. 4, the conversion rate in the maintenance culture was affected by hydrostatic pressure only during cell maintenance and not during cell growth cultures. On the other hand, the specific glutamine consumption rate and the specific production rates of ammonia and MAb were influenced by pressure in both the cell maintenance and cell growth cultures. The results indicate that higher hydrostatic pressure ,
.”
I
7.3 1
6.5L
E
0.0
’
’
’
’
’
0.2 0.4 0.6 0.8 Hydrostatic pressure (MI%)
’
1 .o
FIG. 2. Cell growth in pressurized cultures. Symbols: 0, initial cell concentration; 0, final cell concentration; A, final culture pH.
=r
m-
Hydrostatic
pressure (MPal
FIG. 3. Effect of hydrostatic pressure on cell growth cultures. Symbols: A, conversion from glucose to lactate; 0, specific glutamine consumption rate; 0, specific ammonia production rate; q , specific MAb production rate.
stimulated the specific MAb production rate in static cultures. Since almost all cells were suspended in this system and tissue plasminogen activator productivity by human embryo lung cells was slightly increased at 0.25 MPa compared with that at 0.12 MPa in a microcarrier submerged culture (l), this hydrostatic pressure effect on MAb productivity is expected to be present even in submerged cultures of hybrydoma cells. Consequently, this pressurized cultivation method may potentially be applicable to commercial MAb production processes for the purpose of not only increasing the oxygen supply rate, but also directly increasing MAb productivity. Generally, hydrostatic pressure influences the rate of an enzymatic reaction (2) according to the equation: d(lnk) _ _- Av# ~_ (1) RxT dP where k : reaction rate constant P : hydrostatic pressure (MPa) Av# : activation volume in the transition state (cm3/mol) T : temperature (“K) R : gas constant [ = 8.21 (MPa x cm3/mol/“K)] Using this equation, the rate of reaction with negative activation volume should increase at higher hydrostatic pressure. Solving Eq. 1 gives ‘=exp k,
(-Al”lx(P2-P,)/R/T)
(2)
NOTES
;
8.1ee.i
P s
0
t
20
Conversion
m
t
0
0
2
40
from
60
Q
Specific glutamine
80
glucose
6
to
100
lactate
8
10
(%)
12
consumption rate (10%mol/cell/h)
AZ
5 e.9ee.9 2 m 8.9-8.1 E
2 e.l+e.9 E
8.i-re.t 0
u)
g
Specific
2
ammonia
b
6
8
production
rate
10
12
(10”‘mmol/cell/h)
8.1-8.9 e.iee.1 0
Specific
10
20
MAb production
30
rate
40
50
(10~“mg/cell/h)
FIG. 4. Effect of hydrostatic pressure on cell metabolic rates in cell maintenance cultures during cell growth or cell maintenance. Cells grown at 0.1 or 0.9 MPa were resuspended to adjust the cell concentration to 1.Ox 106cells/ml and incubated for 24 h at 0.1 or 0.9 MPa, respectively. Thus, for example, “O.lWO.9” indicates the results of maintenance cultures at 0.9 MPa using cells grown at 0.1 MPa. (A) Conversion from glucose to lactate; (B) specific glutamine consumption rate; (C) specific ammonia production rate; (D) specific MAb production rate.
where k, and k2 are rate constants at pressures PI and Pz, respectively. The results (Fig. 4) indicate that the hydrostatic pressure influenced the rate of glucose to lactate conversion by changing the rates of certain reactions involved in glycolysis and the TCA cycle, while the overall glycolysis rate was not changed. The decrease in the conversion rate may be the result of a decrease in the
621
lactate dehydrogenase reaction rate. The enzyme reaction rate should decrease by 6%, as calculated by Eq. 2 using the results shown in Fig. 3, while the conversion rate was observed to decrease from 89% at 0.1 MPa to 84% at 0.9MPa. It was also observed that the rates of glycolytic reactions other than that of the conversion to lactate increased by at least 45%, from 11% at 0.1 MPa to 16% at 0.9MPa (Fig. 3). However, the transition-state activation volume of the lactate dehydrogenase reaction is reported to be from -4 to 35 (2), while the rate of decrease of this reaction with the pressure change from 0.1 MPa to 0.9 MPa is calculated to be less than 1% according to Eq. 2, a value which is much less than the observed change of 6%. Moreover, the transition-state activation volumes for reactions in glycolysis metabolism beyond pyruvate and the TCA cycle are reported to be larger than - 14, which may cause only a 0.5% increase in the glycolysis metabolism rate, except for that of conversion to lactate. Consequently, the change observed in the glycolysis rate can not be explained by enzyme reaction kinetics only. Under higher hydrostatic pressure, the specific glutamine consumption rate increased and the specific lactate production rate decreased, while the specific glucose consumption rate was not changed. These phenomena may suggest that the TCA cycle reaction rates increased through activation of the enzymes caused by a conformational change rather than an increase in the reaction rate constants. The specific glutamine consumption and MAb production rates in the cell maintenance cultures were influenced not only by the pressure during cell maintenance, but also by that during cell growth. The mRNA synthesis rate for a cell membrane protein in E. coli was reported to decrease markedly as the hydrostatic pressure increased from 0.1 MPa to 30 MPa (2). It is thus suggested that hydrostatic pressure may influence these rates by bringing about a change in the quantity of mRNA or of the enzyme itself. REFERENCES Takagi, M., Okumura, H., Okada, T., Kobayasbi, N., Kiyotn, T., and Ueda, K.: An oxygen supply strategy for the large-
scale production of tissue plasminogen activator by microcarrier cell culture. J. Ferment. Bioena.. 77. 301-306 (1994). Molid, M.: The theory of pressure effects on enzymes. Adv. Protein Chem., 34, 93-166 (1981). Nakashima, K., Horikosbi, K., and Mizuno, T.: Effects of hydrostatic pressure on the synthesis of outer membrane proteins in E. coli. Biosci. Biotech. Biochem., 59, 130-132 (1995). Frame, K. K. and Hu, W.: The loss of antibody productivity in continuous culture of hybridoma cells. Biotechnol. Bioeng., 35, 469-476 (1990).
Effect of Hydrostatic
Pressure on Hybridoma
Cell Metabolism
MUTSUMI TAKAGI,* KEN-ICHI OHARA, AND TOSHIOMI YOSHIDA International
Center for Cooperative Research in Biotechnology, Suita, Osaka 565, Japan
Osaka University, 2-1 Yamada-oka,
Received 21 June 199WAccepted 5 September 1995 The effects of hydrostatic pressure on hybridoma cell metabolism were investigated. In cell cultures grown at various constant hydrostatic pressures between 0.1 and 0.9MPa, little variation was observed in either cell growth or the specitic glucose consumption rates at different pressures. However, the rate of conversion from glucose to lactate decreased from 89 to 83%, while the specific glutamine consumption rate increased from 1.07 to 1.29 X lo-l3 mol/cell/h between the lowest and highest pressures. The specific production rate of monoclonal antibody (MAb) rose from 4.5 to 5.6 x lo-l3 g/cell/h in proportion to the pressure increase. The main factor affecting the conversion rate was not the hydrostatic pressure during cell propagation, but rather the hydrostatic pressure at which metabolism occurred. The specific glutamine consumption and MAb production rates were influenced by the hydrostatic pressure in both the growth and production phases of the cultivation.
[Key words:
hydrostatic
pressure,
hybridoma
cells, monoclonal
Hydrostatic pressure is one of the operational parameters which are effective at increasing the supply rate of dissolved oxygen in mammalian cell cultures. However, while the effects of pH, temperature, dissolved oxygen concentration and agitation rate on mammalian cultures have been well documented, there have been few reports describing the effect of hydrostatic pressure. We have recently demonstrated (1) that the conversion rate from glucose to lactate in human embryo lung cells at 0.25 MPa was less than that at 0.12MPa. In general, hydrostatic pressure affects the kinetics of enzymatic reactions (2) and it has been suggested that hydrostatic pressure has a repressive effect on membrane protein synthesis in Escherichia coli (3). In the present study, the effects of hydrostatic pressure on hybridoma cell culture using monoclonal antibody (MAb) productivity were investigated. Cells and medium Mouse-mouse hybridoma cells AFP-27, which produce MAb against human a-fetoprotein, were used (4). DMEM/F-12 medium (Gibco Co. Ltd., NY, USA) supplemented with 1.35 g/l glucose, 219 mg/l glutamine, 0.4 g/l Pluronic F-68, 10 ,ug/l insulin, 2 g/l BSA, 20 mg/l transfer-in, 10 ,&I ethanolamine, 2.44 g/l NaHC03, 5,000 U/l penicillin and 5 mg/l streptomycin was used. Cell growth culture A T-flask for suspension cell culture (75 cm2, Sumilon Co. Ltd., Tokyo) containing 28.5 ml of the medium was inoculated with 2.85 x 106 cells and incubated at 37°C for 65 h in a conventional CO2 incubator with 5% C02, or in a pressurized incubator as described below. Pressurized cultivation A pressurized incubator system (Fig. 1) which consisted of a stainless steel vessel (length, 120mm; inner diameter, 95 mm), closed using flanges fitted with a valve and a pressure gauge, and a valve and a PVA filter at the outlet and inlet ends, respectively, was constructed. The vessel was placed in a 37°C water bath to maintain the temperature. After placing a T-flask culture in the vessel, the inner gas phase was replaced 3 times with a gas mixture consisting of N2, 02 and CO2 for 5 min and then pressurized up to a
antibody]
desired value between 0.1 and 0.9 MPa. The composition of the gas mixture was adjusted so that the partial pressures of O2 and CO2 in the T-flask during the cultivation were 0.021 MPa and 0.005 MPa, respectively. The vessel was not opened until the end of the cultivation. After cell growth culture Cell maintenance culture at 0.1 or 0.9 MPa, cells were centrifuged (2,500 rpm, 5 min), resuspended to adjust the cell concentration to 1.OX lo6 cells/ml with fresh medium, and then incubated in a T-flask for 24 h at 0.1 and 0.9 MPa, respectively. In T-flask cultures containing 28.5 ml of medium, the cell concentration increased to a maximum of 1.1 x 10” cells/ml because of a limited supply of O2 supply and the cell concentration was maintained at a virtually constant level. The values in all figures are the means of duplicates. The results were reproducible. Analysis Viable cells were counted by the trypanblue dye exclusion method. Glucose and lactate concentrations were measured by the glucose-oxidase peroxidase and lactate-oxidase peroxidase methods, respectively, using an auto-analyzer (Biochemistry Analyzer 2700; YSI Inc., Ohio, USA). The glutamine and ammonium concentrations were determined by the L-asparginase glutamine-dehydrogenase method (F-kit L-glutamic acid; Boehringer Mannheim Yamanouchi Co. Ltd., Tokyo) and the indophenol method (Ammonia-Test Wako; Wako Pure Chemicals Co. Ltd., Osaka), respectively. MAb concentration was measured using an enzymelinked immunosorption assay (ELISA) method (4). Effect of hydrostatic
pressure on cell growth culture
Cell growth cultures were simultaneously carried out in a conventional CO2 incubator at 0.1 MPa and in the pressurized incubator at constant hydrostatic pressures of 0.1, 0.3, 0.5 and 0.9 MPa. Monitoring of the cell concentration in the conventional CO2 incubator cultivation revealed that at 0.1 MPa cells were growing logarithmically, even at 65 h (data not shown). As shown in Fig. 2, the pH could be maintained at a constant level in the pressurized vessel by adjusting the CO2 partial pressure, while the cell concentrations at 65 h did not differ with pressure. Although the specific glucose consumption rate was not affected by pressure, the specific lactate production
* Corresponding author. 619
620
J. FERMENT.BIOENG..
TAKAGI ET AL.
3
r+ r
8
1
1
1 7
1
I
r
2 FIG. 1. Schematic diagram of a pressurized incubator system for hybridoma cultures. 1, Pressurized vessel; 2, water bath; 3, gas inlet; 4, gas filter; 5, gas outlet; 6, pressure gauge; 7, T-flask; 8, mixed gas.
rate was slightly reduced as the pressure increased (Fig. 3). Consequently, the rate of conversion from glucose to lactate decreased from 89% to 83% as the pressure was raised from 0.1 MPa to 0.9 MPa. Both the specific glutamine consumption and ammonia production rates increased as pressure increased up to 0.5 MPa (Fig. 3). This may be due to a change in the enzymatic reaction rate since the thermal decomposition rate of glutamine was not affected by the pressure (data not shown). The specific MAb production rate increased markedly from 4.5 to 5.6 x lo-l3 g/cell/h as the hydrostatic pressure increased (Fig. 3). Effects of hydrostatic pressure during cell growth and To cell maintenance on cell maintenance cultures ascertain which intracellular processes were directly affected by pressure or cell metabolism kinetics, including enzyme reactions, maintenance cultivations without cell growth were conducted at 0.1 or 0.9 MPa using cells grown at 0.1 and 0.9 MPa, respectively. As shown in Fig. 4, the conversion rate in the maintenance culture was affected by hydrostatic pressure only during cell maintenance and not during cell growth cultures. On the other hand, the specific glutamine consumption rate and the specific production rates of ammonia and MAb were influenced by pressure in both the cell maintenance and cell growth cultures. The results indicate that higher hydrostatic pressure ,
.”
I
7.3 1
6.5L
E
0.0
’
’
’
’
’
0.2 0.4 0.6 0.8 Hydrostatic pressure (MI%)
’
1 .o
FIG. 2. Cell growth in pressurized cultures. Symbols: 0, initial cell concentration; 0, final cell concentration; A, final culture pH.
=r
m-
Hydrostatic
pressure (MPal
FIG. 3. Effect of hydrostatic pressure on cell growth cultures. Symbols: A, conversion from glucose to lactate; 0, specific glutamine consumption rate; 0, specific ammonia production rate; q , specific MAb production rate.
stimulated the specific MAb production rate in static cultures. Since almost all cells were suspended in this system and tissue plasminogen activator productivity by human embryo lung cells was slightly increased at 0.25 MPa compared with that at 0.12 MPa in a microcarrier submerged culture (l), this hydrostatic pressure effect on MAb productivity is expected to be present even in submerged cultures of hybrydoma cells. Consequently, this pressurized cultivation method may potentially be applicable to commercial MAb production processes for the purpose of not only increasing the oxygen supply rate, but also directly increasing MAb productivity. Generally, hydrostatic pressure influences the rate of an enzymatic reaction (2) according to the equation: d(lnk) _ _- Av# ~_ (1) RxT dP where k : reaction rate constant P : hydrostatic pressure (MPa) Av# : activation volume in the transition state (cm3/mol) T : temperature (“K) R : gas constant [ = 8.21 (MPa x cm3/mol/“K)] Using this equation, the rate of reaction with negative activation volume should increase at higher hydrostatic pressure. Solving Eq. 1 gives ‘=exp k,
(-Al”lx(P2-P,)/R/T)
(2)
NOTES
;
8.1ee.i
P s
0
t
20
Conversion
m
t
0
0
2
40
from
60
Q
Specific glutamine
80
glucose
6
to
100
lactate
8
10
(%)
12
consumption rate (10%mol/cell/h)
AZ
5 e.9ee.9 2 m 8.9-8.1 E
2 e.l+e.9 E
8.i-re.t 0
u)
g
Specific
2
ammonia
b
6
8
production
rate
10
12
(10”‘mmol/cell/h)
8.1-8.9 e.iee.1 0
Specific
10
20
MAb production
30
rate
40
50
(10~“mg/cell/h)
FIG. 4. Effect of hydrostatic pressure on cell metabolic rates in cell maintenance cultures during cell growth or cell maintenance. Cells grown at 0.1 or 0.9 MPa were resuspended to adjust the cell concentration to 1.Ox 106cells/ml and incubated for 24 h at 0.1 or 0.9 MPa, respectively. Thus, for example, “O.lWO.9” indicates the results of maintenance cultures at 0.9 MPa using cells grown at 0.1 MPa. (A) Conversion from glucose to lactate; (B) specific glutamine consumption rate; (C) specific ammonia production rate; (D) specific MAb production rate.
where k, and k2 are rate constants at pressures PI and Pz, respectively. The results (Fig. 4) indicate that the hydrostatic pressure influenced the rate of glucose to lactate conversion by changing the rates of certain reactions involved in glycolysis and the TCA cycle, while the overall glycolysis rate was not changed. The decrease in the conversion rate may be the result of a decrease in the
621
lactate dehydrogenase reaction rate. The enzyme reaction rate should decrease by 6%, as calculated by Eq. 2 using the results shown in Fig. 3, while the conversion rate was observed to decrease from 89% at 0.1 MPa to 84% at 0.9MPa. It was also observed that the rates of glycolytic reactions other than that of the conversion to lactate increased by at least 45%, from 11% at 0.1 MPa to 16% at 0.9MPa (Fig. 3). However, the transition-state activation volume of the lactate dehydrogenase reaction is reported to be from -4 to 35 (2), while the rate of decrease of this reaction with the pressure change from 0.1 MPa to 0.9 MPa is calculated to be less than 1% according to Eq. 2, a value which is much less than the observed change of 6%. Moreover, the transition-state activation volumes for reactions in glycolysis metabolism beyond pyruvate and the TCA cycle are reported to be larger than - 14, which may cause only a 0.5% increase in the glycolysis metabolism rate, except for that of conversion to lactate. Consequently, the change observed in the glycolysis rate can not be explained by enzyme reaction kinetics only. Under higher hydrostatic pressure, the specific glutamine consumption rate increased and the specific lactate production rate decreased, while the specific glucose consumption rate was not changed. These phenomena may suggest that the TCA cycle reaction rates increased through activation of the enzymes caused by a conformational change rather than an increase in the reaction rate constants. The specific glutamine consumption and MAb production rates in the cell maintenance cultures were influenced not only by the pressure during cell maintenance, but also by that during cell growth. The mRNA synthesis rate for a cell membrane protein in E. coli was reported to decrease markedly as the hydrostatic pressure increased from 0.1 MPa to 30 MPa (2). It is thus suggested that hydrostatic pressure may influence these rates by bringing about a change in the quantity of mRNA or of the enzyme itself. REFERENCES Takagi, M., Okumura, H., Okada, T., Kobayasbi, N., Kiyotn, T., and Ueda, K.: An oxygen supply strategy for the large-
scale production of tissue plasminogen activator by microcarrier cell culture. J. Ferment. Bioena.. 77. 301-306 (1994). Molid, M.: The theory of pressure effects on enzymes. Adv. Protein Chem., 34, 93-166 (1981). Nakashima, K., Horikosbi, K., and Mizuno, T.: Effects of hydrostatic pressure on the synthesis of outer membrane proteins in E. coli. Biosci. Biotech. Biochem., 59, 130-132 (1995). Frame, K. K. and Hu, W.: The loss of antibody productivity in continuous culture of hybridoma cells. Biotechnol. Bioeng., 35, 469-476 (1990).