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Production of Monoclonal Antibodies by Hybridoma Cells in a Flat Sheet Membrane Bioreactor J. Hagedorn and F. Kargi* Biotechnology Engineering Laboratory, Department of Chemical Engineering, Washington University, St. Louis, Missouri 63130
T h e purpose of this study was to investigate hybridoma growth and monoclonal antibody formation in a flat sheet membrane bioreactor. The effects of several different molecular weight cutoff membranes on growth and antibody formation were investigated. Nutrient and toxic product diffusion through the membranes were quantified, and the kinetic and physical constants of the system were determined.
Introduction Mammalian cell culture is used to produce a variety of products including vaccines, lymphokines, growth factors, hormones, and monoclonal antibodies (Birch et al., 1985). To produce large quantities of monoclonal antibodies, the relevant B-lymphocytes must be isolated and grown by using cell culture techniques. However, it is difficult to grow lymphocytes in cell culture for extended periods. To overcome this problem, new hybrid cells are produced by fusing B-lymphocyte and myeloma cells. The resulting hybridoma cells produce antibodies and can be grown in cell culture for much longer periods of time. There are two basic approaches to the cultivation of hybridoma cells, in vivo and in vitro. In vivo cell culture involves the injection of cells into the peritoneal cavity of mice. An advantage of this method is that the antibodies produced are very concentrated. However, the antibody mixture obtained is contaminated with foreign mouse antibody, and production of large quantities of antibody would be labor intensive and expensive. Each kilogram of antibody would require the use of 1000040 000 mice. The in vitro method of hybridoma cultivation involves propagation of cells in an artificial growth medium and harvest of the antibody from the culture supernatant. The advantage of this method is that the antibody obtained is relatively free of contaminating mouse antibody. Also, the culture conditions can be controlled to increase the rate of monoclonal antibody production, and the process can be more easily scaled up. The reactors used for in vitro cultivation of mammalian cells include roller bottles, mechanically stirred reactors, airlift reactors, immobilized-cell reactors, and membrane reactors (Lavery, et al., 1985; Sever, 1987). Membrane bioreactors have significant advantages over other reactor configurations. The major advantages are continuous removal of low molecular weight toxic metabolites, continuous addition of nutrients through the membranes, high cell and product concentrations, and gentle hydrodynamic conditions. In this study, a flat sheet membrane bioreactor was used to determine the effect of nutrient and toxic product transfer across the membrane on hybridoma growth and monoclonal antibody formation. Several different molecular weight cutoff membranes were tested, and the
physical and kinetic constants of the system were determined. Materials and Methods The cell line used in this study was a mouse-mouse hybridoma cell (HDP1) producing an IgG monoclonal antibody. The cells were cultured in Dulbecco's Modified Eagles' Medium with high bicarbonate and high glucose (DME HG/HB). The medium formulation was DME (HG/HB) (4.5 g/L glucose), 5 % (v/v) horse serum, 4 mM L-glutamine,1mM nonessential amino acids, 100 units/mL penicillin, and 100 pg/mL streptomycin. The cells were cultured in spinner flasks, which were placed in a COZ incubator with a 5 74 COz-enriched atmosphere. The temperature was maintained at 36.5 "C. Analytical Methods. Cell concentrations were determined by counting with use of a hemocytometer. Cell viability was determined by trypan blue exclusion. The concentrations of glucose, lactate, and ammonia were determined by enzymatic assays (Sigma Chemical Co., 1988ac). The monoclonal antibody concentrations were determined by an indirect enzyme linked immunosorbant assay (ELISA). Dissolved oxygen (DO) and pH levels were determined by sampling and immediately measuring with DO and pH probes. Reactor Configuration. Figure 1 shows the flat sheet membrane reactor used in this study. The reactor consisted of a flat sheet membrane (polysulfone) placed between two cylindrical glass chambers. Silicone rubber gaskets and stainless steel flanges were used to clamp the chambers together to achieve an aseptic seal. Each chamber had two ports, one for sampling and one for air circulation. The reactor was sterilized by completely filling it with distilled water and autoclaving for 30 min at 121 "C. After cooling, the water was removed, and nutrient media was added to both chambers. Oxygen was supplied to the liquid by surface aeration with 5% COz-enriched air. The air stream was passed through two bacterial air vents before entering the reactor. The reactor was mixed by placing it on an orbital shaker at 55-60 rpm. The reactor and shaker were placed in a COz incubator to maintain constant temperature and pH. The membranes used in this research had a molecular weight cutoff that retained the monoclonal antibodies in the culture chamber. Nutrients such as glucose and glutamine could diffuse into the culture chamber, and toxic products such as lactate and ammonia could diffuse out.
8756-7938/90/3006-0220$02.50/0 0 1990 American Chemical Society and American Institute of Chemical Engineers
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The performance of the membrane bioreactor with different MW cutoff (50 000 and 100 000) membranes was compared to a system without a membrane. In the system without a membrane, a piece of silicone gasket material was inserted between the two chambers in place of a membrane. One chamber was filled with 200 mL of DME (HG/HB) with 5% serum. The reactor was surface aerated at a rate of 400 mL/min, and the pH was maintained within the range of 7.0-7.4. Cells were added to the chamber after centrifuging and resuspending in fresh media to yield an initial cell concentration of 0.4 X 106 cells/mL. In the next experiment, a DDS (polysulfone) 50000 MW cutoff membrane was used. Both chambers of the reactor were filled with 200 mL of DME (HG/HB) with 5 % serum. Cells were inoculated into one chamber at an initial cell concentration of 0.4 X lo6 cells/mL. The culture conditions were identical with those of the batch reactor without membrane. The membrane area available for transport was 11 cm2, and the available membrane area to reactor volume 'ratio was 0.055 cm2/cm3. A Millipore 100 000 MW cutoff membrane (polysulfone) was used in the final experiment. Both chambers of the reactor were filled with 250 mL of DME (HG/ HB) with 5 % serum. Cells were added to one chamber at a concentration of 0.5 X 106 cells/mL. The culture conditions were identical with those of the two previous experiments. The membrane area available for transport was 14 cm2, and the membrane surface area to reactor volume ratio was 0.056 cm2/cm3.
Results and Discussion A comparison of the viable cell concentrations for the three experiments is depicted in Figure 2. The 100 000 MW cutoff membrane system achieved the highest viable cell concentration (2.9 X lo6 cells/mL), followed by the 50 OOO MW cutoff membrane system (1.8 X lo6 cells/ mL), and then the batch comparison reactor (1.2 X 106 cells/mL). As shown in Table I, the initial specific growth rate was also highest in the 100 OOO MW cutoff reactor. The glucose levels in the culture reservoir for the three experiments are shown in Figure 3. Glucose was used as
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Figure 2. Viable cell concentration profiles for different MW cutoff membrane reactors: (A)100 OOO; ( 0 )50 OOO; ( 0 )membrane free. ble I. Parameter Values for the Flat Sheet Membrane actor Experiments. 50 OOO 100 OOO batch reactor MWCO MWCO (no membrane) membrane membrane UM,h-' 0.015 0.024 0.030 YX~S, cells/mmol 0.77 X 108 1.0 X 108 1.2 X 108 YL,x,mmol/cell 2.7 X 10-8 1.2X 10-8 0.8 X 1O-e 1.0 X 10-8 0.2 X lo4 0.7 X 10" PAb,av,(pg O f Ab/ (cell h)) KMG m/s 1.2 x 10-7 1.6 x 10-7 CAb,fp Ng/mL 0.16 1.5 4.2 Parameter Estimation Yx/s= AX/AS where AX = total cells produced overall A S = total glucose consumed overall Y L / X= hL/AX where AL = total lactate produced overall AX = the overall total cells produced = AAb/(Xalbi.,&t) where AAb = antibody produced in At &.ble,av = average viable cell concentration for the interval t At = time interval of interest
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where S M=~nutrient reservoir glucose at time 1 S M=~nutrient reservoir glucose at time 2 S = culture glucose V M= volume of nutrient reservoir AM = membrane area available for transport The integral was numerically solved.
u=-1 dXT X, dt where XT = total cell concentration XV = viable cell concentration a MWCO = molecular weight cutoff.
a general indicator of the nutrient concentrations, although other nutrients such as glutamine or serum proteins could
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Figure 3. Culture reservoir glucose concentration profiles for different MW cutoff membrane reactors: (A)100 OOO; (0) 50 OOO; (0) membrane free.
be limiting. After 7 days, the glucose concentration in both membrane reactors was less than 1 mM, and the cultures were probably nutrient limited. At the end of the comparison reactor experiment, the glucose concentration was 7 mM. Therefore, either another nutrient was limiting or toxic product inhibition was occurring. The glucose permeabilities for the 100000 and 50000 and MW cutoff membranes were found to be 1.6 X 1.2 X lo-' m/s, respectively. Since the glucose permeability was higher for the 100 000 MW cutoff membrane, it is likely that the transport of other nutrients such as glutamine or serum proteins was higher also. This additional transport probably accounts for the higher cell densities and specific growth rates in the 100 000 MW cutoff reactor. The two largest proteins required for the growth and maintenance of mammalian cells are reported to be transferrin (MW = 80 000) and albumin (MW = 67 000) (Cima, 1987). Although both of these proteins were initially present in the culture reservoir, the 100 000 MW cutoff membrane would allow additional transport. The membranes used in this research had a MW cutoff distribution instead of a sharp cutoff. The 50 000 MW cutoff membrane would probably allow some transport of transferrin and albumin but much less than the 100 000 MW cutoff membrane. A membrane with a MW cutoff greater than 100000 was not used because of possible loss of antibody (MW = 150 000) into the nutrient chamber. Figure 4 shows the glucose concentrations in the culture and nutrient reservoirs for the 50000 MW cutoff membrane experiment. Glucose was transported across the membrane at a rate of 0.19 mmollday; however a large concentration driving force developed. This concentration difference could be reduced by using a higher membrane surface area to reactor volume ratio. The lactate levels in the culture reservoir for the experiments are depicted in Figure 5. The lactate levels in the membrane reactors were generally lower than the lactate concentrations reached at the end of the comparison reactor experiment. For a few days, the lactate levels in the 100 000 MW cutoff system were higher than the lactate levels in the comparison reactor. The increased lactate concentrations were due to the high cell densities achieved in the membrane reactor. The rate of lactate
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Figure 4. Culture and nutrient reservoir glucose concentration profiles for 50 000 MW cutoff membrane experiment.
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Figure 5. Lactate concentration profiles for different MW cutoff membrane reactors: (A)100 000; (0) 50 000; (0) membrane free.
production during this period exceeded the rate of transport across the membrane. The higher cell concentrations in the membrane reactors are partially due to the transport of lactate and other toxic products across the membrane. However, toxic product inhibition was still occurring in the membrane reactor systems. The lactate concentrations in the culture and nutrient reservoirs are shown in Figure 6. Lactate was transported across the membrane at a rate of 0.37 mmol/day. Again, a large concentration driving force developed across the membrane, and it could be reduced by increasing the membrane surface area to reactor volume ratio. Figure 7 depicts the monoclonal antibody concentrations that were obtained in the three experiments. In both membrane reactor experiments, essentially all the antibody was retained in the culture chamber. The highest total antibody concentration and specific productivity were obtained with the 100000 MW cutoff membrane system (Table I). By day 10, the antibody concentration in the 100 000 MW cutoff reactor was 2.3 times
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Figure 6. Culture and nutrient reservoir lactate concentration profiles for 50 000 MW cutoff membrane experiment. 5
Figure 8. Variation of monoclonal antibody productivities with specific growth rate of cells.
body is produced mainly as a secondary metabolite. Another possible explanation is that higher levels of a stimulating factor for antibody production could have built up in the culture media after maximum cell density was reached. A third explanation is that antibody is continuously produced by the cells but is only slowly excreted. After a maximum cell density is reached, the number of nonviable or "leakyncells starts to increase. The increased antibody levels in the supernatant during this period could be due to release from the leaky nonviable cells. Researchers at Celltech also found that the production of entibody increases during the death phase (Birch et al., 1987). However, they measured intracellular antibody levels during the course of an experiment and found low antibody concentrations. Therefore, one of the first two explanations seems to be more plausible.
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Figure 7. Monoclonal antibody concentration profiles for different MW cutoff membrane reactors: (A)100 000; ( 0 )50 000; (0) membrane free.
greater than that of the 50 000 MW cutoff reactor and 14 times greater than the maximum antibody concentration in the comparison reactor. Other researchers also obtained higher antibody titers when higher molecular weight cutoff membranes were used (Evans et al., 1986). Hollow-fiber membranes with molecular weight cutoffs of 10 OOO, 30 OOO, and 70 OOO were compared, and the 70 OOO MW cutoff resulted in the highest antibody concentration (Evans et al., 1986). The maximum rate of antibody production appears to occur after the maximum viable cell concentrations were reached or during the period of slow growth. Figure 8 depicts variation of specific antibody productivity with the specific growth rate for the 50 OOO MW cutoff membrane system. I t can be seen that the productivity is much higher at low growth rates. There are several possible explanations for the observed antibody kinetics. The cells could actually be producing more antibody during periods of stress, nutrient limitation, and slow growth. This would indicate that the anti-
Membrane reactors provide continuous addition of nutrients and continuous removal of toxic products from a cell culture. This results in higher cell concentrations and specific antibody productivities than obtained with batch culture without membranes. It was found that the HDPl cell line produced antibodies at higher rates during periods of slow growth and adverse culture conditions. A membrane bioreactor with a 100 OOO MW cutoff membrane was found to achieve higher cell concentrations and specific antibody productivities than those obtained with a 50 000 MW cutoff membrane reactor or a batch reactor without a membrane. With use of a 100 000 MW cutoff membrane reactor, final monoclonal antibody concentrations were improved by a factor of 14 as compared to a membrane-free culture.
C.4b.f
KMG PAb
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Notation final antibody concentration (pg/mL) glucose permeability (m/s) specific antibody productivity (pg of Ag/(cell h)) maximum specific growth rate (h-1) cells produced/mmol of glucose consumed mmol of lactate produced/cell produced
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Acknowledgment This study was supported by a grant from the state of Missouri and the Monsanto Co. We would like to thank Dr. Julian Fleischman of the Washington University Medical School for donating the hybridoma cell line and for his expert advice.
Literature Cited Birch, J. R.; Thompson, P.; Lambert, K.; Borastan, R. In Large Scale Mammalian Cell Culture; Feder, J., Tolbert, W., Eds.; Academic Press: New York, 1985; pp 1-15. Birch, J. R.; Thompson, P. W.; Borastan, R.; Oliver, S.; Lambert, K. Plant and Animal Cells Process Possibilities; 1987; pp 162-171. Cima, L. G. Anchorage Dependent Mammalian Cell Culture in Hollow Fiber Reactors. Ph.D. Thesis, University of California a t Berkeley, 1987.
Evans, T. L.; Hart, S. M.; Nguyen, H. T.; Coulter, C. Large Scale Production of Monoclonal Antibodies Using Hollow Fiber Reactors with Specific MW Cutoffs. Poster Presentation a t TCA Conference, 1986. Lavery, M.; Dearns, M. J.; Price, D. J.; Emery, A. N. Physical Conditions During Batch Culture of Hybridomas in Laboratory Scale Stirred Tank Reactors. Deo. Biol. Stand. 1985, 60, 199-206. Sever, S. In Commercial Production of Monoclonal Antibodies; Marcel Dekker: New York, 1987; pp 49-71. Sigma Chemical Co. Sigma Glucose Diagnostic Kit, Procedure 115; Sigma Chemical Co.: St. Louis, MO, 1988a. Sigma Chemical Co. Sigma Pyruvate/Lactate Diagnostic Kit, Procedure 726; Sigma Chemical Co.: St. Louis, MO, 1988b. Sigma Chemical Co. Sigma Ammonia Diagnostic Kit, Procedure 170-UV; Sigma Chemical Co.: St. Louis, MO, 1988. Accepted March 16, 1990.
