Relationship between hybridoma growth and monoclonal antibody production P. M. Hayter Biological Laboratory, University o f K e n t at Canterbury, Kent, U K
N. F. Kirkby D e p a r t m e n t o f Chemical and Process Engineering, University o f Surrey, Guildford, Surrey, U K
R. E. Spier School o f Biological Sciences, University o f Surrey, Guildford, Surrey, U K
Factors affecting cell growth and antibody production in a mouse hybridoma were investigated. Antibody was produced during the growth and decline phases of a batch culture with an increase in the specific rate of antibody production during the decline phase. The specific rate of antibody production was also increased in cells arrested by 2 mM thymidine, suggesting that cell proliferation and antibody production can be uncoupled. Reduced serum concentrations resulted in lower cell growth rates but increased antibody production rates. However, this trend was reversed in hybridomas which had been arrested by thymidine, since the highest antibody production rate was associated with high serum concentrations. Likewise, in proliferating cells, the optimum pH for antibody production (pH 6.8) was lower than the optimum pH for cell growth (pH 7.2), whereas in thymidine-blocked cells, the highest antibody production rate was at pH 7.2. High antibody production rates and product yields were also associated with low growth rates in continuous cultures. The possibility that antibody was under cell cycle control was investigated in synchronized hybridoma cultures. Antibody production occurred during GI and G2 with a decline in the M phase and evidence of a further decline in the S phase. Thus antibody production was not restricted to the G1 and S phase in this hybridoma.
Keywords:Hybridoma;continuous culture; cell cycle; serum pH Introduction The potential for the large-scale use of monoclonal antibodies in clinical medicine, j downstream processing, 2'3 and more recently as catalysts 4 has resulted in considerable attention being directed towards the intensification of the monoclonal antibody production process. This requires knowledge of the influence of
Address reprint requests to Dr. Spier at the School of Biological Sciences, Universityof Surrey, Guildford, Surrey GU2 5XH, UK Received 2 September 1991; revised 2 December 1991
454
Enzyme Microb. Technol., 1992, vol. 14, June
the environment on hybridoma growth and antibody production kinetics. In many cases it has been found that antibody production is not restricted to the growth phase in batch cultures but continues during the stationary and decline phases of the culture. 5-9 Whereas in some cases the antibody production rate appears to be constant, 7'9 in others the specific rate of antibody production is higher during the stationary and decline phases than during the growth phase.l°'ll Many factors which perturb hybridoma growth also increase the specific rate of antibody production. 8,1z-16 It is not clear from these observations whether the increased rate of antibody production is a stress-related phenomenon, as has been suggested, s,16 or is due to
© 1992 Butterworth-Heinemann
Hybridoma growth and monoclonal antibody production: P. M. Hayter et al. enhanced antibody production at low growth rates. Some continuous culture studies indicate that the specific antibody production rate is higher at low specific growth rates, 6'8 but the precise relationship between cell growth and antibody production has not been elucidated. However, a possible explanation for this behavior is that antibody production is under cell cycle control. In many cell lines of lymphoid origin the production of antibody appears to be restricted to the G1 and S phases of the cell growth cycle. 17-21The duration of the cell cycle is determined by the length of the G1 phase, since the S, G2, and M phases are relatively c o n s t a n t . 22 Hence the proportion of cells residing in the G1 phase is dependent on the cell growth rate. If antibody production is either restricted to or occurs at a higher rate during the G1 phase in hybridomas, this could explain the enhanced antibody production rate at low cell growth rates or during stationary phase when the majority of cells are in the G1 phase. 23 In this paper we report an investigation of the relationship between hybridoma growth and monoclonal antibody production kinetics using batch, continuous, and synchronous culture techniques.
Materials and methods
Cell line The cell line used in this investigation was a mouse hybridoma producing IgG specific for paraquat. 24 This hybridoma was kindly provided by Dr. A. Wright (Central Toxicology Laboratory, ICI, Macclesfield).
suming that the deamination of 1 mol of glutamine liberates 1 mol of ammonia. The monoclonal antibody concentration was determined using a sandwich ELISA. Assay plates were coated overnight with sheep anti-mouse IgG (Scottish Antibody Production Unit) in 0.1 M sodium carbonate buffer, pH 9.5. The plates were washed 3 times with wash buffer (PBS, 0.1% Tween 20) and the samples and standards were applied diluted in sample buffer (PBS, 10% newborn calf serum). The standard was IgG from the 321 hybridoma purified using paraquatSepharose affinity chromatography. 25 The plates were incubated for 2 h and washed for a second time with wash buffer. Alkaline phosphatase conjugated sheep anti-mouse IgG was then added and the plates were incubated for 2 h. After a final wash, the substrate was applied (p-nitrophenyl phosphate; Sigma 104) and the color allowed to develop for 1 h. The absorbance was read at 410 nm using an Dynatech MR600 plate reader linked to a BBC microcomputer.
Batch culture Batch cultures were performed in 150-cm 2 tissue culture flasks (Falcon) containing 100-ml cultures. Cells were inoculated at 2 x 105 ml-~ and the cultures were incubated at 37°C in an atmosphere comprising 95% air and 5% CO2. Samples were removed daily for analysis. The specific growth rate (/z) and the specific rate of antibody production (qab) in batch cultures were calculated from the cell growth and antibody production curves.
Continuous culture Medium The medium used in this investigation was RPMI 1640. Unless stated otherwise, the medium was supplemented with 10% newborn calf serum derived from a single production batch (Seralab). In experiments where the pH was varied, the sodium bicarbonate concentration was reduced from 20 to 5 mM and 15 mM HEPES buffer was added. The medium was then adjusted to the required pH by the addition of HCI or NaOH.
Assays Cell numbers were determined using a modified Fuchs-Rosenthal counting chamber and cell viability by trypan blue dye exclusion. Glucose was determined by the o-toluidine method (Sigma assay no. 635), and lactate was determined using lactate dehydrogenase (Sigma assay no. 826-UV). Ammonia was determined using an ion selective probe (model 8002-8, Kent Instruments) calibrated with ammonium chloride standards. Glutamine was determined by the addition of an equal volume of 5 U ml-1 glutaminase (Grade 5 from E. coli, Sigma) in 0.1 M sodium acetate buffer, pH 4.9. After incubation for 30 min at 37°C, the ammonia concentration was determined using the ammonia probe. The glutamine concentration was calculated as-
Continuous culture was performed in a 1-1 bioreactor (Gallenkamp). The culture was agitated by a magnetically driven paddle (100 rev min -~, 60 mm × 10 mm). The pH was maintained between 7.0 and 7.1 by supplying 95% air and 5% CO2 through the reactor headspace at 3 1 h-1. Chemostat culture was performed under glutamine limitation with a feed glutamine concentration of 1 mM. Samples were removed daily for analysis.
Synchronous culture Hybridomas were synchronized by the double thymidine block method, z6 Cells in early to mid-exponential phase were arrested by the addition of 2 mM thymidine. After 30 h the cells were washed and resuspended in thymidine-free medium at 2.5 × 105 cells m1-1. After 6 h incubation 2 mM thymidine was added and the cells were incubated for a further 18 h. The cells were then washed and resuspended at 2.5 x 105 cells m1-1 in thymidine-free medium. The cell growth and antibody productivity were measured for 34 h.
Results and discussion
Batch cultures Cells seeded at 2 x 105 ml-1 reached a maximum cell density of 7-8 x 105 ml-t after 40-50 h after which
Enzyme Microb. Technol., 1992, vol. 14, June
455
Papers there was a rapid decline in cell viability (Figure 1). Glutamine was utilized rapidly by these cells and was completely consumed by the end of the growth phase. Ammonia accumulated to 2 mM by the end of the growth phase. Glucose was utilized only during the growth phase and was never completely consumed by the cells. The accumulation of lactate was also associated with the growth phase, and there were typically 1.5 mol of lactate formed for each mole of glucose consumed. As in previous reports on antibody production kinetics by hybridomas, 5-9 the production of antibody by the 321 hybridoma was not restricted to the growth phase of batch cultures but continued into the stationary and decline phases. Indeed, there was an increased specific rate of antibody production (qab) as cells entered the latter phase of the culture, and approximately 60% of the total antibody was produced at this time. It has been suggested that this apparent increase in the specific production rate is due to the release of accumulated antibody by dead cells, '~ but much of the evidence indicates that antibody production is associated with
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98%) at all dilution rates. Furthermore there was no evidence of cell adherence to the walls of the bioreactor, and hence the specific growth rate was considered to be equal to the dilution rate. The highest specific rate of antibody production occurred at a growth rate of 0.024 h I and declined with increasing dilution rate (Table I ). Other chemostat studies have shown a similar relationship between cell growth rate and antibody production/,a Similarly, in perfused cultures where cell growth was restricted, specific antibody production rates were found to be higher) 3'34 One explanation for these observations is that more of the cells' biosynthetic capacity is diverted towards the synthesis of biomass at high growth rates. Lower antibody yield coefficients for glutamine and glucose, Yahgin and Yahglc, were observed at high growth rates, which would ~e consistent with such a hypothesis (Table 2). Furthermore, there were significant changes in glucose and glutamine metabolism at different specific growth rates. The increased Ylac,glc at high specific growth rates is in accordance with other metabolic studies on hybridomas 8'ss's6 and suggests that cells de-
qantibody
(/~g 10 -s cells h -1) qglucose 5.4 4.6 3.8
0.21 0.28 0.34
qlactate qglutaEnine (/zmol 10-*
cells h -1)
0.12 0.24 0.40
qammonia
0.042 0.058 0.069
0.022 0.024 0.024
rive more ATP from glycolysis at high growth rates. This may be particularly important in the chemostat where the alternative energy source, glutamine, 37 was limiting. Glacken et al. 38have shown that the ammonia yield is lower under glutamine limitation, and indeed the yield coefficient Yammgin was considerably lower in the chemostat than in batch cultures. Furthermore, there was a decrease in Yamm,glnat higher growth rates, which may be a result of the increased utilization of glutamine for biomass production, glutamine being the nitrogen donor in several biosynthetic pathways, including nucleotide synthesis. 39'4° Whether the changes in antibody production can be attributed to these changes in cell metabolism at different growth rates has not been ascertained, but this is an area which merits further investigation.
Synchronized cell culture Alternatively, antibody production may be under cell cycle control, since antibody production has been reported to be restricted to the late G1 and S phases in several lymphoblastoid cell lines. 17-21 Based on this evidence, it might be postulated that the rate of antibody production is related to the proportion of cells in those particular phases of the cell cycle, which in turn will be affected by the cell growth rate. Indeed this assumption formed the basis of the recent cell cycle model of Suzuki and Ollis :1 which showed good correlation with experimental data. This theory was tested in the 321 hybridoma by synchronizing the cells with the double thymidine block technique. Cells that had been synchronized by the double thymidine block method had a cell cycle
Table 2 Antibody and metabolite yield coefficients at different growth rates
(h//"-1)
Yarnn('~nOItool -1)~lac,glc
Yab,gln (rag mg _ l~ab,glc
0.024 0.033 0.042 Batch
0.52 0.41 0.35 1.0
0.87 0.52 0.36 0.53
0.57 0.86 1.18 1.5
E n z y m e M i c r o b . T e c h n o l . , 1992, vol. 14, J u n e
0.142 0.090 0.062 0.140
459
Papers time of 13.5 h (Figure 9), which compared favorably with the previously determined minimum doubling time of 13.6 h (a growth rate of 0.051 h-l). After release from the thymidine block, the cells progressed through the S, G2, and M phases in 10.5 h, which suggests that the G 1 phase was 3 h at the maximum growth rate. The duration of the S phase was determined to be between 5 and 6 h by measuring the incorporation of 3H-thymidine in a synchronized culture (data not shown), leaving a period of 4.5-5.5 h for the G2 and M phases. The accumulation of antibody was monitored during synchronous cell growth. The rate of antibody production was not constant throughout the cell cycle; in particular, there was a marked and reproducible decline in the rate of antibody production during the period corresponding to the late G2 and M phases (Figure 10). This was not unexpected, since protein synthesis is suppressed during mitosis.42 Contrary to other reports, ~7-2~ antibody production was not restricted to the G 1 and S phases but continued through most of the G2 phase. In some experiments there was also a decrease in the rate of antibody production during the S phase (Figure lOb), although this was not always apparent, possibly due to a loss of synchrony during the G1 phase. High rates of antibody production during G1 and G2 with low rates in S and M have been described in some lymphoblastoid lines,43'44 and a model based on this assumption has been constructed by Faraday et al. 45and fitted to this data. Thus models which are based on the assumption that
0.6
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..-.e.
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6oi
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j " .
0
10
20
,
30
40
Time(hours} Figure 9 Cell growth and antibody accumlation in synchronized culture. Data are the mean of 3 experiments. Error bars are the standard deviation
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N. F. Kirkby D e p a r t m e n t o f Chemical and Process Engineering, University o f Surrey, Guildford, Surrey, U K
R. E. Spier School o f Biological Sciences, University o f Surrey, Guildford, Surrey, U K
Factors affecting cell growth and antibody production in a mouse hybridoma were investigated. Antibody was produced during the growth and decline phases of a batch culture with an increase in the specific rate of antibody production during the decline phase. The specific rate of antibody production was also increased in cells arrested by 2 mM thymidine, suggesting that cell proliferation and antibody production can be uncoupled. Reduced serum concentrations resulted in lower cell growth rates but increased antibody production rates. However, this trend was reversed in hybridomas which had been arrested by thymidine, since the highest antibody production rate was associated with high serum concentrations. Likewise, in proliferating cells, the optimum pH for antibody production (pH 6.8) was lower than the optimum pH for cell growth (pH 7.2), whereas in thymidine-blocked cells, the highest antibody production rate was at pH 7.2. High antibody production rates and product yields were also associated with low growth rates in continuous cultures. The possibility that antibody was under cell cycle control was investigated in synchronized hybridoma cultures. Antibody production occurred during GI and G2 with a decline in the M phase and evidence of a further decline in the S phase. Thus antibody production was not restricted to the G1 and S phase in this hybridoma.
Keywords:Hybridoma;continuous culture; cell cycle; serum pH Introduction The potential for the large-scale use of monoclonal antibodies in clinical medicine, j downstream processing, 2'3 and more recently as catalysts 4 has resulted in considerable attention being directed towards the intensification of the monoclonal antibody production process. This requires knowledge of the influence of
Address reprint requests to Dr. Spier at the School of Biological Sciences, Universityof Surrey, Guildford, Surrey GU2 5XH, UK Received 2 September 1991; revised 2 December 1991
454
Enzyme Microb. Technol., 1992, vol. 14, June
the environment on hybridoma growth and antibody production kinetics. In many cases it has been found that antibody production is not restricted to the growth phase in batch cultures but continues during the stationary and decline phases of the culture. 5-9 Whereas in some cases the antibody production rate appears to be constant, 7'9 in others the specific rate of antibody production is higher during the stationary and decline phases than during the growth phase.l°'ll Many factors which perturb hybridoma growth also increase the specific rate of antibody production. 8,1z-16 It is not clear from these observations whether the increased rate of antibody production is a stress-related phenomenon, as has been suggested, s,16 or is due to
© 1992 Butterworth-Heinemann
Hybridoma growth and monoclonal antibody production: P. M. Hayter et al. enhanced antibody production at low growth rates. Some continuous culture studies indicate that the specific antibody production rate is higher at low specific growth rates, 6'8 but the precise relationship between cell growth and antibody production has not been elucidated. However, a possible explanation for this behavior is that antibody production is under cell cycle control. In many cell lines of lymphoid origin the production of antibody appears to be restricted to the G1 and S phases of the cell growth cycle. 17-21The duration of the cell cycle is determined by the length of the G1 phase, since the S, G2, and M phases are relatively c o n s t a n t . 22 Hence the proportion of cells residing in the G1 phase is dependent on the cell growth rate. If antibody production is either restricted to or occurs at a higher rate during the G1 phase in hybridomas, this could explain the enhanced antibody production rate at low cell growth rates or during stationary phase when the majority of cells are in the G1 phase. 23 In this paper we report an investigation of the relationship between hybridoma growth and monoclonal antibody production kinetics using batch, continuous, and synchronous culture techniques.
Materials and methods
Cell line The cell line used in this investigation was a mouse hybridoma producing IgG specific for paraquat. 24 This hybridoma was kindly provided by Dr. A. Wright (Central Toxicology Laboratory, ICI, Macclesfield).
suming that the deamination of 1 mol of glutamine liberates 1 mol of ammonia. The monoclonal antibody concentration was determined using a sandwich ELISA. Assay plates were coated overnight with sheep anti-mouse IgG (Scottish Antibody Production Unit) in 0.1 M sodium carbonate buffer, pH 9.5. The plates were washed 3 times with wash buffer (PBS, 0.1% Tween 20) and the samples and standards were applied diluted in sample buffer (PBS, 10% newborn calf serum). The standard was IgG from the 321 hybridoma purified using paraquatSepharose affinity chromatography. 25 The plates were incubated for 2 h and washed for a second time with wash buffer. Alkaline phosphatase conjugated sheep anti-mouse IgG was then added and the plates were incubated for 2 h. After a final wash, the substrate was applied (p-nitrophenyl phosphate; Sigma 104) and the color allowed to develop for 1 h. The absorbance was read at 410 nm using an Dynatech MR600 plate reader linked to a BBC microcomputer.
Batch culture Batch cultures were performed in 150-cm 2 tissue culture flasks (Falcon) containing 100-ml cultures. Cells were inoculated at 2 x 105 ml-~ and the cultures were incubated at 37°C in an atmosphere comprising 95% air and 5% CO2. Samples were removed daily for analysis. The specific growth rate (/z) and the specific rate of antibody production (qab) in batch cultures were calculated from the cell growth and antibody production curves.
Continuous culture Medium The medium used in this investigation was RPMI 1640. Unless stated otherwise, the medium was supplemented with 10% newborn calf serum derived from a single production batch (Seralab). In experiments where the pH was varied, the sodium bicarbonate concentration was reduced from 20 to 5 mM and 15 mM HEPES buffer was added. The medium was then adjusted to the required pH by the addition of HCI or NaOH.
Assays Cell numbers were determined using a modified Fuchs-Rosenthal counting chamber and cell viability by trypan blue dye exclusion. Glucose was determined by the o-toluidine method (Sigma assay no. 635), and lactate was determined using lactate dehydrogenase (Sigma assay no. 826-UV). Ammonia was determined using an ion selective probe (model 8002-8, Kent Instruments) calibrated with ammonium chloride standards. Glutamine was determined by the addition of an equal volume of 5 U ml-1 glutaminase (Grade 5 from E. coli, Sigma) in 0.1 M sodium acetate buffer, pH 4.9. After incubation for 30 min at 37°C, the ammonia concentration was determined using the ammonia probe. The glutamine concentration was calculated as-
Continuous culture was performed in a 1-1 bioreactor (Gallenkamp). The culture was agitated by a magnetically driven paddle (100 rev min -~, 60 mm × 10 mm). The pH was maintained between 7.0 and 7.1 by supplying 95% air and 5% CO2 through the reactor headspace at 3 1 h-1. Chemostat culture was performed under glutamine limitation with a feed glutamine concentration of 1 mM. Samples were removed daily for analysis.
Synchronous culture Hybridomas were synchronized by the double thymidine block method, z6 Cells in early to mid-exponential phase were arrested by the addition of 2 mM thymidine. After 30 h the cells were washed and resuspended in thymidine-free medium at 2.5 × 105 cells m1-1. After 6 h incubation 2 mM thymidine was added and the cells were incubated for a further 18 h. The cells were then washed and resuspended at 2.5 x 105 cells m1-1 in thymidine-free medium. The cell growth and antibody productivity were measured for 34 h.
Results and discussion
Batch cultures Cells seeded at 2 x 105 ml-1 reached a maximum cell density of 7-8 x 105 ml-t after 40-50 h after which
Enzyme Microb. Technol., 1992, vol. 14, June
455
Papers there was a rapid decline in cell viability (Figure 1). Glutamine was utilized rapidly by these cells and was completely consumed by the end of the growth phase. Ammonia accumulated to 2 mM by the end of the growth phase. Glucose was utilized only during the growth phase and was never completely consumed by the cells. The accumulation of lactate was also associated with the growth phase, and there were typically 1.5 mol of lactate formed for each mole of glucose consumed. As in previous reports on antibody production kinetics by hybridomas, 5-9 the production of antibody by the 321 hybridoma was not restricted to the growth phase of batch cultures but continued into the stationary and decline phases. Indeed, there was an increased specific rate of antibody production (qab) as cells entered the latter phase of the culture, and approximately 60% of the total antibody was produced at this time. It has been suggested that this apparent increase in the specific production rate is due to the release of accumulated antibody by dead cells, '~ but much of the evidence indicates that antibody production is associated with
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10
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~
0.5. ¢-t
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-I00
-80
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-/+0
98%) at all dilution rates. Furthermore there was no evidence of cell adherence to the walls of the bioreactor, and hence the specific growth rate was considered to be equal to the dilution rate. The highest specific rate of antibody production occurred at a growth rate of 0.024 h I and declined with increasing dilution rate (Table I ). Other chemostat studies have shown a similar relationship between cell growth rate and antibody production/,a Similarly, in perfused cultures where cell growth was restricted, specific antibody production rates were found to be higher) 3'34 One explanation for these observations is that more of the cells' biosynthetic capacity is diverted towards the synthesis of biomass at high growth rates. Lower antibody yield coefficients for glutamine and glucose, Yahgin and Yahglc, were observed at high growth rates, which would ~e consistent with such a hypothesis (Table 2). Furthermore, there were significant changes in glucose and glutamine metabolism at different specific growth rates. The increased Ylac,glc at high specific growth rates is in accordance with other metabolic studies on hybridomas 8'ss's6 and suggests that cells de-
qantibody
(/~g 10 -s cells h -1) qglucose 5.4 4.6 3.8
0.21 0.28 0.34
qlactate qglutaEnine (/zmol 10-*
cells h -1)
0.12 0.24 0.40
qammonia
0.042 0.058 0.069
0.022 0.024 0.024
rive more ATP from glycolysis at high growth rates. This may be particularly important in the chemostat where the alternative energy source, glutamine, 37 was limiting. Glacken et al. 38have shown that the ammonia yield is lower under glutamine limitation, and indeed the yield coefficient Yammgin was considerably lower in the chemostat than in batch cultures. Furthermore, there was a decrease in Yamm,glnat higher growth rates, which may be a result of the increased utilization of glutamine for biomass production, glutamine being the nitrogen donor in several biosynthetic pathways, including nucleotide synthesis. 39'4° Whether the changes in antibody production can be attributed to these changes in cell metabolism at different growth rates has not been ascertained, but this is an area which merits further investigation.
Synchronized cell culture Alternatively, antibody production may be under cell cycle control, since antibody production has been reported to be restricted to the late G1 and S phases in several lymphoblastoid cell lines. 17-21 Based on this evidence, it might be postulated that the rate of antibody production is related to the proportion of cells in those particular phases of the cell cycle, which in turn will be affected by the cell growth rate. Indeed this assumption formed the basis of the recent cell cycle model of Suzuki and Ollis :1 which showed good correlation with experimental data. This theory was tested in the 321 hybridoma by synchronizing the cells with the double thymidine block technique. Cells that had been synchronized by the double thymidine block method had a cell cycle
Table 2 Antibody and metabolite yield coefficients at different growth rates
(h//"-1)
Yarnn('~nOItool -1)~lac,glc
Yab,gln (rag mg _ l~ab,glc
0.024 0.033 0.042 Batch
0.52 0.41 0.35 1.0
0.87 0.52 0.36 0.53
0.57 0.86 1.18 1.5
E n z y m e M i c r o b . T e c h n o l . , 1992, vol. 14, J u n e
0.142 0.090 0.062 0.140
459
Papers time of 13.5 h (Figure 9), which compared favorably with the previously determined minimum doubling time of 13.6 h (a growth rate of 0.051 h-l). After release from the thymidine block, the cells progressed through the S, G2, and M phases in 10.5 h, which suggests that the G 1 phase was 3 h at the maximum growth rate. The duration of the S phase was determined to be between 5 and 6 h by measuring the incorporation of 3H-thymidine in a synchronized culture (data not shown), leaving a period of 4.5-5.5 h for the G2 and M phases. The accumulation of antibody was monitored during synchronous cell growth. The rate of antibody production was not constant throughout the cell cycle; in particular, there was a marked and reproducible decline in the rate of antibody production during the period corresponding to the late G2 and M phases (Figure 10). This was not unexpected, since protein synthesis is suppressed during mitosis.42 Contrary to other reports, ~7-2~ antibody production was not restricted to the G 1 and S phases but continued through most of the G2 phase. In some experiments there was also a decrease in the rate of antibody production during the S phase (Figure lOb), although this was not always apparent, possibly due to a loss of synchrony during the G1 phase. High rates of antibody production during G1 and G2 with low rates in S and M have been described in some lymphoblastoid lines,43'44 and a model based on this assumption has been constructed by Faraday et al. 45and fitted to this data. Thus models which are based on the assumption that
0.6
~0.5 ~O.t~ "~ 0-3 = 0"2-
..-.e.
0.1 i
!
6oi
30 i
j " .
0
10
20
,
30
40
Time(hours} Figure 9 Cell growth and antibody accumlation in synchronized culture. Data are the mean of 3 experiments. Error bars are the standard deviation
460
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|
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