Controlling and predicting monoclonal antibody production in hollow-fiber bioreactors A. Handa.Corrigan, S. Nikolay, D. Jeffery*, B. Heffernan* and A. Young* D e p a r t m e n t o f Microbiology, University o f Surrey, Guildford; * Wellcome Diagnostics, Langley Court, B e c k e n h a m , Kent, U K
A simple optimization strategy is described which enables monoclonal antibody (MCA) production in hollow-fiber bioreactors to be controlled and predicted. The MCA production rate is demonstrated to increase linearly with the uptake rates of glucose and glutamine and with the production rates of lactate and ammonia. The uptake and production rates of these metabolites can, in turn, be predicted from the pumping rates of basal medium to the bioreactor. We recommend a period of 2 weeks at the start of the cultivation when intensive assaying and monitoring should be carried out. After this period, the medium flow rate and MCA production rate may be predicted by linear extrapolation.
Keywords: Optimization; assaying; monitoring; hollow fiber bioreactor
Introduction As early as 1972, it was demonstrated that hollow-fiber bioreactors could be used for the cultivation of high densities of animal cells.~ In recent years, hollow-fiber bioreactors have increasingly been used for the production of milligram to gram quantities of monoclonal antibodies (MCA). 2-4 The advantages, production capabilities, and economics of various hollow-fiber bioreactor configurations have been discussed in previous publications.2-5 The main advantages of using these bioreactors are small space requirements, concentrated products, low serum requirements, and low labor and capital investment costs. Downstream purification is made cheaper and easier because the hollow-fiber bioreactor product is not only more concentrated but also contains lower concentrations of contaminants than the MCA produced from ascites or conventional stirredtank and airlift bioreactors. We have recently cultivated hybridoma cells in hollow-fiber bioreactors using serum-free medium. The concentrated MCA obtained was purified rapidly with ProsepA, resulting in excellent purity and activity. 6 The disadvantages of hollow-fiber bioreactors in-
Address reprint requests to Mr. Simon Nikolay at the Wolfson Cytotechnology Laboratory, The University of Surrey, Guildford, GU2 5XH, UK Received 20 November 1990; revised 12 June 1991
58
Enzyme Microb. Technol., 1992, vol. 14, January
clude poor understanding of mass transfer characteristics, optimization techniques, and scale-up procedures. The heterogeneous distribution of cells and the concentration polarization of proteins in the bioreactors have been demonstrated to result in reduced cell numbers and bioreactor productivities. 7 Improved productivities can be achieved by periodically reversing the direction of medium flow through the lumen of the fibers.7,8 The Celltronics (New Brunswick Ltd., UK) hollowfiber bioreactor, which employs a medium reversal strategy, has been shown to result in better productivities than other hollow-fiber bioreactor configurations available on the market. 8 A major problem with the large-scale production of mammalian proteins in high-density perfusion culture systems has been to determine the most favorable environment for both cell growth and productivity. In hollow-fiber bioreactors, Endotronics (USA) recommend a nutrient feed and waste removal strategy to control metabolic parameters such as glucose and lactic acid concentrations. This is combined with additional control of pH and dissolved oxygen. 9 Handa-Corrigan and Nikolay 8,~° have proposed a comprehensive optimization strategy adapted from Endotronics which can be used for any hybridoma cell line in different hollowfiber bioreactor configurations. In summary, they have demonstrated that by intensive asssaying (of glucose, glutamine, ammonia, lactate, and MCA) and control of physical parameters (pH, temperature, and dissolved oxygen), the MCA productivity from any hybridoma © 1992 Butterworth-Heinemann
Monoclonal antibody production in hollow-fiber bioreactors: A. Handa-Corrigan et al. cell line may be controlled to achieve linear productivity over a period of 1 to 2 months. 8'~° In this paper, we aim to show that a simple hollow-fiber bioreactor (lacking sophisticated facilities for monitoring and controlling pH and dissolved oxygen) can be used to control and predict the production of MCA from hybridoma cells.
AJR / CO2 SUPPLY TO JET PUMP
WASTE OUT
9 PRODUCT OUT FROM EC
BIOREACTOR: HOLLOW FIBRE CARTRIDGE
Materials and methods
Cell line
SERUMSUPPLEMENTED MEDIA JNTOEC
A mouse × mouse hybridoma cell line designated WDI was utilized in this study.
Figure 1 Schematic representation of the Celltronics hollowfiber bioreactor flowpath
PINCH VALVE FOR M E D I U M FLOW REVERSAL IN IC
CIRCULATION PUMP
Cell cultivation and hollow-fiber bioreactor perfusion media Cells were maintained and expanded in RPMI 1640 supplemented with 450 mg dl-l glucose, 5 mM glutamine, and 5% fetal calf serum. The intracapillary space (IC) of the hollow-fiber reactor was perfused with RPMI 1640 basal medium supplemented with 450 mg dl- ~glucose and 5 mM glutamine. The extracapillary space (EC) was perfused with the same serum-supplemented medium formulation described above. All media were devoid of antibiotics.
Hollow-fiber bioreactor inoculation and operating procedures MCA production was carried out in a Celltronics hollow-fiber reactor (New Brunswick Scientific). A diagrammatic representation of the perfusion circuit is shown in Figure 1. Detailed inoculation and operational protocols have been described previously. 8 Briefly, a total of 2 x 108 viable cells suspended in 50 ml serumsupplemented medium were pumped into the EC space at 200 ml h -l. Immediately after inoculation, fresh basal medium was continuously pumped into the medium reservoir at 20 ml h-1 and waste removed from the reservoir at the same rate. A continuous feed of serum-supplemented medium was pumped into the EC space at 1 ml h -1, and harvest containing MCA product was removed from the EC at the same rate. The basal medium was firstly oxygenated by a jet-pump and subsequently circulated through the IC at an initial rate of 100 ml h-1. To prevent build-up of metabolite gradients, the medium flow through the IC was periodically reversed every 60 min at the start of the cultivation. As the production run progressed, the pumping, circulation, and medium-reversal rates were changed according to the metabolic requirements of the cells. Temperature was controlled at 37°C in the Celltronics incubator. The pH was maintained at 7. I by adjusting the amount of CO2 delivered to the system or by increasing the basal medium flow rate. The pH changes in the Celltronics can only be monitored visually due to the color changes in the medium. We carried out off-line pH determinations using a flow-through pH cell (Pharmacia) to verify whether visual determinations were sufficient.
Optimization and control strategy Optimization and control of MCA production was effected by changing the rates at which media were supplied and/or product and waste removed during the course of the cultivation. Routine biochemical and product assays were carded out to verify the metabolic state of the culture. The assay results were then used to decide on changing one or more of the following pump rates: I. When glucose or glutamine concentrations fell below 150 mg dl-i and 2.5 mM, respectively, the basal medium rate was increased. 2. When lactate and ammonia concentrations were above 150 mg dl -t and 2.5 mM, respectively, basal medium addition, waste removal, and medium reversal rates were all increased. 3. When the concentration of MCA in the harvest stream was on the increase, the supply of serumsupplemented medium to the EC was also increased.
Metabolite assays Glucose concentrations were determined using Glucose HK Uni-Kit III (Roche) and a Cobas-Bio automated analyzer (Roche Diagnostics). Lactate concentrations were determined using a lactate reagent kit (Sigma 826-UV) and the Cobas-Bio automated analyzer. Ammonia concentrations were determined with an ammonia probe (Kent Industrial). Glutamine was enzymatically converted to free ammonia with glutaminase (Sigma) and glutamine concentrations were determined from the following equation: [Glutamine] = [total ammonia] -
[background ammonia]
Monoclonal antibodies were determined quantitatively using a ProAnaMabs column (Perstorp Biolytica) attached to an HPLC. Peak area was used to calculate MCA concentration using Nelson software programmed with data from an externally generated standard curve.
Enzyme Microb. Technol., 1992, vol. 14, January
59
Papers 1.2
Table 1 MCA yields in five bottles collected during the course of a 28-day cultivation in the Celltronics hollow-fiber bioreactor
a 1
Days post inoculation
Bottle number
Vol. (ml)
10 18 22 26 28
1 2 3 4 5
450 950 840 870 420
[MCA] (mg m1-1) 0.121 0.631 0.924 0.961 1.010 Total MCA (mg)
Total MCA (rag)
0.8
0.6
54.45 599.45 776.16 836.07 424.20 2,690.33
i
0.4
0.2 I
0 2
0
4
6
8
10
MCA PRODUCTION RATE mg/hr
Results
MCA productivity for a 28-day hollow-fiber cultivation
G
0.5
L U
The MCA secreted by the hybridoma cells in the EC space was harvested continuously into sterile 0.5- and 1.0-1 duran bottles• Five harvest bottles were collected during the course of the cultivation, with a total MCA yield of 2.69 g (Table 1). The MCA production rate increased from approximately 0.02 to 9.0 mg h- ~at the end of the cultivation (Figure 2). The averaged daily MCA production and media utilization was calculated from the following data: Total MCA produced = 2690.33 mg
T u P T A
K E R A T E m M / h r
b 0.4
0.3
0.2
o.1
O
i
I
I
I
2
4
6
8
10
MCA PRODUCTION RATE mg/hr
Total basal medium consumption = 106.65 1 Figure 3 Monoclonal antibody production rates vs glucose (a) and glutamine (b) uptake rates in the Celltronics hollow-fiber bioreactor
Total FCS consumption = 350 ml Total production run length = 28 days Averaged MCA production day= 96 mg 3.8 1-l basal medium 12.5 ml-' FCS
Effects of the optimization and control strategy As with other cell lines tested in our laboratory, the WD1 hybridoma showed three significant results with respect to MCA production:
M
c A P
R O D O C T I O N R A T E
10
8
S
Predicting MCA production in hollow-fiber reactors
4
j/
2
m
g / h r
0 100
200
300
400
500
600
TIME (HOURS)
Figure 2 Monoclonal anUbody production rate for a 28-day Celltronics hollow-fiber cultivation
60
1. The MCA production rate was directly proportional to the uptake rates of glucose and glutamine (Figure 3a and b). For each milligram of MCA produced per hour, approximately 0.5 mM glutamine and I mM glucose were utilized• 2. The MCA production rate was directly proportional to the production rates of lactate and ammonia (Figure 4a and b). For each milligram of MCA produced per hour, approximately 0.5 mM ammonia and 1.5 mM lactate were produced. 3. A molar conversion of glutamine to ammonia occurred throughout the cultivation period (Figure 5a). Each mole of glucose was converted to approximately 1.5 mol of lactate (Figure 5b).
Enzyme Microb. Technol., 1992, vol. 14, January
The simple, linear relationship between MCA production rates and metabolite uptake and production rates allows extrapolation to be carded out easily• It must be emphasized, however, that for any particular cell line, intensive assaying and monitoring must be carded out for at least the first couple of weeks of a run in order to establish the initial phase of the linear profile. It must also be pointed out that we have not, as yet,
Monoclonal antibody production in hollow-fiber bioreactors: A. Handa-Corrigan et al. (e.g. other amino acids) may also be important and are recommended. The secreted MCA is maintained at a high concentration because the feed of serum-supplemented medium is not increased unnecessarily. The linear relationship observed for this cell line (and for others that we have studied s'~°) allows for simple extrapolation and prediction of MCA productivity in hollow-fiber bioreactors. In summary, the following relationships exist for MCA production in hollow-fiber bioreactors using the optimization strategy recommended by Handa-Corrigan and Nikolay; at a specific time (t) in the bioreactor:
1.6 a 1.4 1.2
1 O.8
0.6 0.4 0.2
i
O 0
i
2 4 MCA PRODUCTION
i
i
6 8 R A T E mg/hr
lO
Glucose uptake rate oc MCA production rate Glutamine uptake rate
0.8 M M O N
I
oc basal medium pump rate
b or,
0.4
A P R O D R A T E
Lactate production rate Ammonia production rate
0.3
oc basal medium pump rate
0.2
The MCA production rate at any other time (x) during the cultivation may therefore be predicted from the above relationships. The following strategy is recom-
0.1 / h r
MCA production rate
2
4
MCA PRODUCTION
6
8
R A T E mg/hr
Figure 4 Monoclonal antibody production rates vs lactate (a) and a m m o n i a (b) production rates in the Celltronics hollow-fiber bioreactor
G L U T U P T A K
0.6
a 0.4
0.3
E
demonstrated whether these linear relationships are maintained in longer-term cultivations (greater than 3 months). O An alternative method of control and prediction is based solely on the IC pump rate, which we have shown to follow closely with the metabolite uptake/production rates (Figure 6a-d). IC pump rates may therefore be extrapolated to predict and achieve the relevant productivities. Alternatively, an algorithm developed by EndotronicsZ° may be utilized for computer control of the culture. Discussion and conclusion In the work presented here, and in previous studies, we have demonstrated that MCA production from hybridoma cells cultivated in hollow-fiber bioreactors increases linearly with the uptake rates of glucose and glutamine, with the production rate of ammonia, and sometimes also with that of lactate. The process optimization and control strategy recommended for hollowfiber bioreactors ensures that the cells are provided with appropriate nutrients (such as glucose and glutamine) and that accumulating waste products (such as ammonia and lactate) are removed from the vicinity of the cells. Monitoring and control of other metabolites
R
A T E
0.2
0.1 / h r
0
0.1 0.2 0.3 0.4 A M M O N I A P R O D R A T E rnM/hr
(3 L U C U P
T
0.5
1.2 1
0.8
A K
E R A T
E In M / h r
0.6 0.4
0.2 0 O
o.6 1 LACTATE PROD RATE
1.6
mM/hr
Figure 5 Glutamine uptake rates vs a m m o n i a production rates (a) and glucose uptake rates vs lactate production rates (b) for the Celltronics hollow-fiber cultivation
Enzyme Microb. Technol., 1992, vol. 14, January
61
Papers 300
0.8
A M 1,4 O N I A
a 260
0.4
2o0
180 lOO 8,1
0
100
IO0
300 400 TIME (HOURS)
800
800
2
260
I C
200
F E E O
16Q 100
O4,
O~
i
100
200
l
R A T E
T / 60
!
300 400 TIME (HOURS)
l
i
SOQ
60Q
SO A~'
8 0
30Q
1.=
100
I h r
O 700
c
II10 0.|
0.1
60
0
0.3
R A T E
0.1
Ill@
0.4
|00
P R O D
O.3
3O4
0.8
h ¢
0 tO0
• L U C
1.2
T A K E
o.a
l
i
100
:tOO
i
i
300 400 TIME (HOURS)
I
i
600
804
1
R A T E ~-1 I h r
.y x
d
x
0 TOO
3OO
260 SO0
o.8
160
0.4
IOQ
0.,I
8O
N'-X~X= 0 0 100
NX= x 200
xI
i
300 400 TIME (HOURS)
600
I Io0
! C F E E D
T E
T I b f
700
Figure 6 IC pump rates plotted against the uptake rates of glutamine (a) and glucose (b) and the production rates of lactate (c) and ammonia (d) in the Celltronics hollow-fiber bioreactor
mended for the control and prediction of MCA in hollow-fiber bioreactors: 1. Carry out intensive assaying, monitoring, and control of metabolic and physical parameters for a 2-week period (as described in Materials and methods). 2. Calculate and plot graphs of metabolic rates and IC feed rates versus time for the 2-week period (see F i g u r e 6). Predict the medium flow rate and metabolic rate for a later time (x) during the cultivation. 3. Plot graphs of metabolic rates versus MCA production rates for the 2-week period (see F i g u r e s 3 and 4). Extrapolate and predict MCA production rate for any of the metabolic rates predicted at time x. 4. Use predicted medium flow rates in the hollowfiber bioreactor for the rest of the cultivation period. Further assaying of metabolic parameters is not necessary. MCA assays should be carried out throughout the cultivation. The simple, linear relationships observed allow for rapid extrapolation and prediction of MCA productivity. This technique is particularly useful for perfusion culture systems where sampling and determination of cell yields is not possible. In the future we aim to implement on-line analysis and computer control for such process-intensified mammalian cell culture systems. Uptake rate = G x - F t G - E ( G x - F t G o )
1
62
-
E
Production rate =
FtL - LoE
1-E
where Gx = (FmGrn) + (FsGs)
Enzyme Microb. Technol., 1992, vol. 14, January
(3)
Ft = Fm + Fs E = exp(-FtT/V) Fm Fs
Gm
= medium flow rate (ml h-l) = serum-supplemented medium flow rate (ml h- 1) = concentration of glucose or glutamine in the medium (mM)
Go
= previous glucose or glutamine value (mM)
Gs
= concentration of glucose or glutamine in the serum-supplemented medium (mM)
G = current glucose or glutamine value (mM) L = current lactate, ammonia (mM), or MCA (mg ml-l) value Lo
= previous lactate, ammonia (mM), or MCA (rag ml-]) value
T = time since last sample (h)
(I)
(2)
V = volume of the system
Monoclonal antibody production in hollow-fiber bioreactors: A. Handa-Corrigan et al. Endotronics have successfully implemented this algorithm in their production-scale hollow-fiber reactors (e.g. the Acusyst P-3X). The simple, linear relationships observed allow for rapid extrapolation and prediction of MCA productivity. This technique is particularly useful for peffusion culture systems where sampling and determination of cell yields is not possible. In the future we aim to implement on-line analysis and computer control for such process-intensified mammalian cell culture systems.
3
4 5 6 7 8
References 1 2
Knazek, R. A., Gullino, P. M., Kohler, P. O. and Dedrick, R. L. In Vitro Sci. 1972, 178, 65-66 Handa-Corrigan, A., Nikolay, S. and Spier, R. E. in Advances in Animal Cell Biology and Technology for Bioprocesses (Spier,
9 10
R. E., Griffiths, J. B., Stephenne, J. and Crooy, P. S., eds) Butterworth Ltd., UK, 1987, p. 378 Tiebout, R. F., Van der Meet, W. G. J. and Zeijlemaker, W. P. in Proceedings of the 4th European Congress on Biotechnology, The Netherlands, 1987. Publishers BV, Amsterdam, 1987 Hirschel, M. D. and Gruenberg, M. L. in Large Scale Cell Culture Technology (Lydersen, B. J., ed) Macmillan, New York, 1987, pp. 113-144 Handa-Corrigan, A. Bio/Technol. 1988, 6, 784-786 Handa Corrigan, A., Chadd, M., Garcia de Castro, A. and Zhang, S. A defined serum-free medium for diverse cell culture applications. Presented at the 1lth ESACT Meeting, UK, 1991 Piret, J. M. and Cooney, C. L. Biotech. Bioeng. 1990, 36, 902-910 Nikolay, S., Garcia de Castro, A., Chadd, M. and HandaCorrigan, A. Presented at ESACT, France, May 1990 Anderson, B. G. and Gruenberg, M. L. in Commercial Production of Monoclonal Antibodies: A Guide for Scale-Up (Seaver, S. S., ed) Marcel Dekker, New York, 1987, pp. 175-195 Handa-Corrigan, A., Nikolay, S. and Spier, R. E. Presented at ESACT, France, May 1990
Enzyme Microb. Technol., 1992, vol. 14, January
63
A simple optimization strategy is described which enables monoclonal antibody (MCA) production in hollow-fiber bioreactors to be controlled and predicted. The MCA production rate is demonstrated to increase linearly with the uptake rates of glucose and glutamine and with the production rates of lactate and ammonia. The uptake and production rates of these metabolites can, in turn, be predicted from the pumping rates of basal medium to the bioreactor. We recommend a period of 2 weeks at the start of the cultivation when intensive assaying and monitoring should be carried out. After this period, the medium flow rate and MCA production rate may be predicted by linear extrapolation.
Keywords: Optimization; assaying; monitoring; hollow fiber bioreactor
Introduction As early as 1972, it was demonstrated that hollow-fiber bioreactors could be used for the cultivation of high densities of animal cells.~ In recent years, hollow-fiber bioreactors have increasingly been used for the production of milligram to gram quantities of monoclonal antibodies (MCA). 2-4 The advantages, production capabilities, and economics of various hollow-fiber bioreactor configurations have been discussed in previous publications.2-5 The main advantages of using these bioreactors are small space requirements, concentrated products, low serum requirements, and low labor and capital investment costs. Downstream purification is made cheaper and easier because the hollow-fiber bioreactor product is not only more concentrated but also contains lower concentrations of contaminants than the MCA produced from ascites or conventional stirredtank and airlift bioreactors. We have recently cultivated hybridoma cells in hollow-fiber bioreactors using serum-free medium. The concentrated MCA obtained was purified rapidly with ProsepA, resulting in excellent purity and activity. 6 The disadvantages of hollow-fiber bioreactors in-
Address reprint requests to Mr. Simon Nikolay at the Wolfson Cytotechnology Laboratory, The University of Surrey, Guildford, GU2 5XH, UK Received 20 November 1990; revised 12 June 1991
58
Enzyme Microb. Technol., 1992, vol. 14, January
clude poor understanding of mass transfer characteristics, optimization techniques, and scale-up procedures. The heterogeneous distribution of cells and the concentration polarization of proteins in the bioreactors have been demonstrated to result in reduced cell numbers and bioreactor productivities. 7 Improved productivities can be achieved by periodically reversing the direction of medium flow through the lumen of the fibers.7,8 The Celltronics (New Brunswick Ltd., UK) hollowfiber bioreactor, which employs a medium reversal strategy, has been shown to result in better productivities than other hollow-fiber bioreactor configurations available on the market. 8 A major problem with the large-scale production of mammalian proteins in high-density perfusion culture systems has been to determine the most favorable environment for both cell growth and productivity. In hollow-fiber bioreactors, Endotronics (USA) recommend a nutrient feed and waste removal strategy to control metabolic parameters such as glucose and lactic acid concentrations. This is combined with additional control of pH and dissolved oxygen. 9 Handa-Corrigan and Nikolay 8,~° have proposed a comprehensive optimization strategy adapted from Endotronics which can be used for any hybridoma cell line in different hollowfiber bioreactor configurations. In summary, they have demonstrated that by intensive asssaying (of glucose, glutamine, ammonia, lactate, and MCA) and control of physical parameters (pH, temperature, and dissolved oxygen), the MCA productivity from any hybridoma © 1992 Butterworth-Heinemann
Monoclonal antibody production in hollow-fiber bioreactors: A. Handa-Corrigan et al. cell line may be controlled to achieve linear productivity over a period of 1 to 2 months. 8'~° In this paper, we aim to show that a simple hollow-fiber bioreactor (lacking sophisticated facilities for monitoring and controlling pH and dissolved oxygen) can be used to control and predict the production of MCA from hybridoma cells.
AJR / CO2 SUPPLY TO JET PUMP
WASTE OUT
9 PRODUCT OUT FROM EC
BIOREACTOR: HOLLOW FIBRE CARTRIDGE
Materials and methods
Cell line
SERUMSUPPLEMENTED MEDIA JNTOEC
A mouse × mouse hybridoma cell line designated WDI was utilized in this study.
Figure 1 Schematic representation of the Celltronics hollowfiber bioreactor flowpath
PINCH VALVE FOR M E D I U M FLOW REVERSAL IN IC
CIRCULATION PUMP
Cell cultivation and hollow-fiber bioreactor perfusion media Cells were maintained and expanded in RPMI 1640 supplemented with 450 mg dl-l glucose, 5 mM glutamine, and 5% fetal calf serum. The intracapillary space (IC) of the hollow-fiber reactor was perfused with RPMI 1640 basal medium supplemented with 450 mg dl- ~glucose and 5 mM glutamine. The extracapillary space (EC) was perfused with the same serum-supplemented medium formulation described above. All media were devoid of antibiotics.
Hollow-fiber bioreactor inoculation and operating procedures MCA production was carried out in a Celltronics hollow-fiber reactor (New Brunswick Scientific). A diagrammatic representation of the perfusion circuit is shown in Figure 1. Detailed inoculation and operational protocols have been described previously. 8 Briefly, a total of 2 x 108 viable cells suspended in 50 ml serumsupplemented medium were pumped into the EC space at 200 ml h -l. Immediately after inoculation, fresh basal medium was continuously pumped into the medium reservoir at 20 ml h-1 and waste removed from the reservoir at the same rate. A continuous feed of serum-supplemented medium was pumped into the EC space at 1 ml h -1, and harvest containing MCA product was removed from the EC at the same rate. The basal medium was firstly oxygenated by a jet-pump and subsequently circulated through the IC at an initial rate of 100 ml h-1. To prevent build-up of metabolite gradients, the medium flow through the IC was periodically reversed every 60 min at the start of the cultivation. As the production run progressed, the pumping, circulation, and medium-reversal rates were changed according to the metabolic requirements of the cells. Temperature was controlled at 37°C in the Celltronics incubator. The pH was maintained at 7. I by adjusting the amount of CO2 delivered to the system or by increasing the basal medium flow rate. The pH changes in the Celltronics can only be monitored visually due to the color changes in the medium. We carried out off-line pH determinations using a flow-through pH cell (Pharmacia) to verify whether visual determinations were sufficient.
Optimization and control strategy Optimization and control of MCA production was effected by changing the rates at which media were supplied and/or product and waste removed during the course of the cultivation. Routine biochemical and product assays were carded out to verify the metabolic state of the culture. The assay results were then used to decide on changing one or more of the following pump rates: I. When glucose or glutamine concentrations fell below 150 mg dl-i and 2.5 mM, respectively, the basal medium rate was increased. 2. When lactate and ammonia concentrations were above 150 mg dl -t and 2.5 mM, respectively, basal medium addition, waste removal, and medium reversal rates were all increased. 3. When the concentration of MCA in the harvest stream was on the increase, the supply of serumsupplemented medium to the EC was also increased.
Metabolite assays Glucose concentrations were determined using Glucose HK Uni-Kit III (Roche) and a Cobas-Bio automated analyzer (Roche Diagnostics). Lactate concentrations were determined using a lactate reagent kit (Sigma 826-UV) and the Cobas-Bio automated analyzer. Ammonia concentrations were determined with an ammonia probe (Kent Industrial). Glutamine was enzymatically converted to free ammonia with glutaminase (Sigma) and glutamine concentrations were determined from the following equation: [Glutamine] = [total ammonia] -
[background ammonia]
Monoclonal antibodies were determined quantitatively using a ProAnaMabs column (Perstorp Biolytica) attached to an HPLC. Peak area was used to calculate MCA concentration using Nelson software programmed with data from an externally generated standard curve.
Enzyme Microb. Technol., 1992, vol. 14, January
59
Papers 1.2
Table 1 MCA yields in five bottles collected during the course of a 28-day cultivation in the Celltronics hollow-fiber bioreactor
a 1
Days post inoculation
Bottle number
Vol. (ml)
10 18 22 26 28
1 2 3 4 5
450 950 840 870 420
[MCA] (mg m1-1) 0.121 0.631 0.924 0.961 1.010 Total MCA (mg)
Total MCA (rag)
0.8
0.6
54.45 599.45 776.16 836.07 424.20 2,690.33
i
0.4
0.2 I
0 2
0
4
6
8
10
MCA PRODUCTION RATE mg/hr
Results
MCA productivity for a 28-day hollow-fiber cultivation
G
0.5
L U
The MCA secreted by the hybridoma cells in the EC space was harvested continuously into sterile 0.5- and 1.0-1 duran bottles• Five harvest bottles were collected during the course of the cultivation, with a total MCA yield of 2.69 g (Table 1). The MCA production rate increased from approximately 0.02 to 9.0 mg h- ~at the end of the cultivation (Figure 2). The averaged daily MCA production and media utilization was calculated from the following data: Total MCA produced = 2690.33 mg
T u P T A
K E R A T E m M / h r
b 0.4
0.3
0.2
o.1
O
i
I
I
I
2
4
6
8
10
MCA PRODUCTION RATE mg/hr
Total basal medium consumption = 106.65 1 Figure 3 Monoclonal antibody production rates vs glucose (a) and glutamine (b) uptake rates in the Celltronics hollow-fiber bioreactor
Total FCS consumption = 350 ml Total production run length = 28 days Averaged MCA production day= 96 mg 3.8 1-l basal medium 12.5 ml-' FCS
Effects of the optimization and control strategy As with other cell lines tested in our laboratory, the WD1 hybridoma showed three significant results with respect to MCA production:
M
c A P
R O D O C T I O N R A T E
10
8
S
Predicting MCA production in hollow-fiber reactors
4
j/
2
m
g / h r
0 100
200
300
400
500
600
TIME (HOURS)
Figure 2 Monoclonal anUbody production rate for a 28-day Celltronics hollow-fiber cultivation
60
1. The MCA production rate was directly proportional to the uptake rates of glucose and glutamine (Figure 3a and b). For each milligram of MCA produced per hour, approximately 0.5 mM glutamine and I mM glucose were utilized• 2. The MCA production rate was directly proportional to the production rates of lactate and ammonia (Figure 4a and b). For each milligram of MCA produced per hour, approximately 0.5 mM ammonia and 1.5 mM lactate were produced. 3. A molar conversion of glutamine to ammonia occurred throughout the cultivation period (Figure 5a). Each mole of glucose was converted to approximately 1.5 mol of lactate (Figure 5b).
Enzyme Microb. Technol., 1992, vol. 14, January
The simple, linear relationship between MCA production rates and metabolite uptake and production rates allows extrapolation to be carded out easily• It must be emphasized, however, that for any particular cell line, intensive assaying and monitoring must be carded out for at least the first couple of weeks of a run in order to establish the initial phase of the linear profile. It must also be pointed out that we have not, as yet,
Monoclonal antibody production in hollow-fiber bioreactors: A. Handa-Corrigan et al. (e.g. other amino acids) may also be important and are recommended. The secreted MCA is maintained at a high concentration because the feed of serum-supplemented medium is not increased unnecessarily. The linear relationship observed for this cell line (and for others that we have studied s'~°) allows for simple extrapolation and prediction of MCA productivity in hollow-fiber bioreactors. In summary, the following relationships exist for MCA production in hollow-fiber bioreactors using the optimization strategy recommended by Handa-Corrigan and Nikolay; at a specific time (t) in the bioreactor:
1.6 a 1.4 1.2
1 O.8
0.6 0.4 0.2
i
O 0
i
2 4 MCA PRODUCTION
i
i
6 8 R A T E mg/hr
lO
Glucose uptake rate oc MCA production rate Glutamine uptake rate
0.8 M M O N
I
oc basal medium pump rate
b or,
0.4
A P R O D R A T E
Lactate production rate Ammonia production rate
0.3
oc basal medium pump rate
0.2
The MCA production rate at any other time (x) during the cultivation may therefore be predicted from the above relationships. The following strategy is recom-
0.1 / h r
MCA production rate
2
4
MCA PRODUCTION
6
8
R A T E mg/hr
Figure 4 Monoclonal antibody production rates vs lactate (a) and a m m o n i a (b) production rates in the Celltronics hollow-fiber bioreactor
G L U T U P T A K
0.6
a 0.4
0.3
E
demonstrated whether these linear relationships are maintained in longer-term cultivations (greater than 3 months). O An alternative method of control and prediction is based solely on the IC pump rate, which we have shown to follow closely with the metabolite uptake/production rates (Figure 6a-d). IC pump rates may therefore be extrapolated to predict and achieve the relevant productivities. Alternatively, an algorithm developed by EndotronicsZ° may be utilized for computer control of the culture. Discussion and conclusion In the work presented here, and in previous studies, we have demonstrated that MCA production from hybridoma cells cultivated in hollow-fiber bioreactors increases linearly with the uptake rates of glucose and glutamine, with the production rate of ammonia, and sometimes also with that of lactate. The process optimization and control strategy recommended for hollowfiber bioreactors ensures that the cells are provided with appropriate nutrients (such as glucose and glutamine) and that accumulating waste products (such as ammonia and lactate) are removed from the vicinity of the cells. Monitoring and control of other metabolites
R
A T E
0.2
0.1 / h r
0
0.1 0.2 0.3 0.4 A M M O N I A P R O D R A T E rnM/hr
(3 L U C U P
T
0.5
1.2 1
0.8
A K
E R A T
E In M / h r
0.6 0.4
0.2 0 O
o.6 1 LACTATE PROD RATE
1.6
mM/hr
Figure 5 Glutamine uptake rates vs a m m o n i a production rates (a) and glucose uptake rates vs lactate production rates (b) for the Celltronics hollow-fiber cultivation
Enzyme Microb. Technol., 1992, vol. 14, January
61
Papers 300
0.8
A M 1,4 O N I A
a 260
0.4
2o0
180 lOO 8,1
0
100
IO0
300 400 TIME (HOURS)
800
800
2
260
I C
200
F E E O
16Q 100
O4,
O~
i
100
200
l
R A T E
T / 60
!
300 400 TIME (HOURS)
l
i
SOQ
60Q
SO A~'
8 0
30Q
1.=
100
I h r
O 700
c
II10 0.|
0.1
60
0
0.3
R A T E
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Ill@
0.4
|00
P R O D
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3O4
0.8
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0 tO0
• L U C
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T A K E
o.a
l
i
100
:tOO
i
i
300 400 TIME (HOURS)
I
i
600
804
1
R A T E ~-1 I h r
.y x
d
x
0 TOO
3OO
260 SO0
o.8
160
0.4
IOQ
0.,I
8O
N'-X~X= 0 0 100
NX= x 200
xI
i
300 400 TIME (HOURS)
600
I Io0
! C F E E D
T E
T I b f
700
Figure 6 IC pump rates plotted against the uptake rates of glutamine (a) and glucose (b) and the production rates of lactate (c) and ammonia (d) in the Celltronics hollow-fiber bioreactor
mended for the control and prediction of MCA in hollow-fiber bioreactors: 1. Carry out intensive assaying, monitoring, and control of metabolic and physical parameters for a 2-week period (as described in Materials and methods). 2. Calculate and plot graphs of metabolic rates and IC feed rates versus time for the 2-week period (see F i g u r e 6). Predict the medium flow rate and metabolic rate for a later time (x) during the cultivation. 3. Plot graphs of metabolic rates versus MCA production rates for the 2-week period (see F i g u r e s 3 and 4). Extrapolate and predict MCA production rate for any of the metabolic rates predicted at time x. 4. Use predicted medium flow rates in the hollowfiber bioreactor for the rest of the cultivation period. Further assaying of metabolic parameters is not necessary. MCA assays should be carried out throughout the cultivation. The simple, linear relationships observed allow for rapid extrapolation and prediction of MCA productivity. This technique is particularly useful for perfusion culture systems where sampling and determination of cell yields is not possible. In the future we aim to implement on-line analysis and computer control for such process-intensified mammalian cell culture systems. Uptake rate = G x - F t G - E ( G x - F t G o )
1
62
-
E
Production rate =
FtL - LoE
1-E
where Gx = (FmGrn) + (FsGs)
Enzyme Microb. Technol., 1992, vol. 14, January
(3)
Ft = Fm + Fs E = exp(-FtT/V) Fm Fs
Gm
= medium flow rate (ml h-l) = serum-supplemented medium flow rate (ml h- 1) = concentration of glucose or glutamine in the medium (mM)
Go
= previous glucose or glutamine value (mM)
Gs
= concentration of glucose or glutamine in the serum-supplemented medium (mM)
G = current glucose or glutamine value (mM) L = current lactate, ammonia (mM), or MCA (mg ml-l) value Lo
= previous lactate, ammonia (mM), or MCA (rag ml-]) value
T = time since last sample (h)
(I)
(2)
V = volume of the system
Monoclonal antibody production in hollow-fiber bioreactors: A. Handa-Corrigan et al. Endotronics have successfully implemented this algorithm in their production-scale hollow-fiber reactors (e.g. the Acusyst P-3X). The simple, linear relationships observed allow for rapid extrapolation and prediction of MCA productivity. This technique is particularly useful for peffusion culture systems where sampling and determination of cell yields is not possible. In the future we aim to implement on-line analysis and computer control for such process-intensified mammalian cell culture systems.
3
4 5 6 7 8
References 1 2
Knazek, R. A., Gullino, P. M., Kohler, P. O. and Dedrick, R. L. In Vitro Sci. 1972, 178, 65-66 Handa-Corrigan, A., Nikolay, S. and Spier, R. E. in Advances in Animal Cell Biology and Technology for Bioprocesses (Spier,
9 10
R. E., Griffiths, J. B., Stephenne, J. and Crooy, P. S., eds) Butterworth Ltd., UK, 1987, p. 378 Tiebout, R. F., Van der Meet, W. G. J. and Zeijlemaker, W. P. in Proceedings of the 4th European Congress on Biotechnology, The Netherlands, 1987. Publishers BV, Amsterdam, 1987 Hirschel, M. D. and Gruenberg, M. L. in Large Scale Cell Culture Technology (Lydersen, B. J., ed) Macmillan, New York, 1987, pp. 113-144 Handa-Corrigan, A. Bio/Technol. 1988, 6, 784-786 Handa Corrigan, A., Chadd, M., Garcia de Castro, A. and Zhang, S. A defined serum-free medium for diverse cell culture applications. Presented at the 1lth ESACT Meeting, UK, 1991 Piret, J. M. and Cooney, C. L. Biotech. Bioeng. 1990, 36, 902-910 Nikolay, S., Garcia de Castro, A., Chadd, M. and HandaCorrigan, A. Presented at ESACT, France, May 1990 Anderson, B. G. and Gruenberg, M. L. in Commercial Production of Monoclonal Antibodies: A Guide for Scale-Up (Seaver, S. S., ed) Marcel Dekker, New York, 1987, pp. 175-195 Handa-Corrigan, A., Nikolay, S. and Spier, R. E. Presented at ESACT, France, May 1990
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