Perfusion Cultures of Hybridoma Cells for Monoclonal Antibody Production FRANCISCO J. CASTILLO, LARRY J. MULLEN, JOHN C. THRIFT, AND BARRY C. GRANT XOMA Corporation Berkelq, California 94710
INTRODUCTION
Perfusion cultivations of mammalian cells allow for the maintenance of longterm, high-density, and high-productivity cultures.1,2Among the different perfusion systems, hollow fibers have been used for the production of biologicals?“ including monoclonal a n t i b o d i e ~ ,with ~ - ~ good results. However, few studies have gone beyond simply describing the performance of a given cell line in a particular perfusion system. These studies are of limited use for predicting the performance of other cell lines, even in the few cases where the mass transfer characteristics of the bioreactor are well understood. Hybridoma cells have nutritional requirements, metabolic rates, and product formation/secretion rates that are unique to each line even when derived from the same parental myeloma. Often, these characteristics are functions of the culture conditions, including the composition of the medium, the bioreactor type, and the way it is operated. These characteristics need to be studied and quantitated under reproducible conditions that will reflect actual production processes. Simple chemostats may be valuable tools when utilized for this purpose by studying hybridomas under continuous cultivation. The results obtained may then be used to predict the performance of the corresponding cell line in a perfusion reactor operated continuously for extended periods. The objective of this work is to describe the cultivation of hybridoma lines in industrial-scale perfusion culture, and to determine the value of chemostat-derived data in the interpretation of the results obtained, as a method for predicting the performance of hybridoma/bioreactor systems.
MATERIALS AND METHODS
The hybridoma cells used were all of murine-murine origin and are listed in TABLE1. The media used consisted of Dulbecco’s Modified Eagle’s Medium/Ham’s F-12 (1:l) containing 25 mM glucose and 4 or 6 mM glutamine and supplemented either with 5% fetal bovine serum (FBS) or with 20 nM sodium selenite, 20 JLM ethanolamine, 30 mg/L bovine transferrin, and 5 mg/L bovine insulin.1° For chemostat experiments, jacketed spinner flasks (Wheaton, Celstir 3000 mL), 1500 to 2000 mL working volume, were used. The covers were perforated and fitted with connectors and silicone tubing to allow continuous supply of medium and 72
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withdrawal of spent medium and cells. Ports were also made for removal of samples, addition of gas (5-10% COz in air), gas exhaust, inoculation of cells, and other additions. Gas inlets and outlets had 0.2-pm hydrophobic filters. The gases were applied to the liquid overhead to poise the medium before inoculation. Agitation was provided by magnetic floating stir bars rotating between 60 and 150 rpm. The temperature was maintained at 37 "C by pumping water through the jackets with a heating-circulator bath. Automatic addition of 7% sodium bicarbonate was used to control the pH within the desired values. Continuous perfusion runs were done in Acusyst-P hollow fiber systems (Endotronics, Incorporated). Samples, 5 to 10 mL, were withdrawn with syringes. Total and viable cells were counted in hemacytometers after diluting 1:l with 0.2% trypan blue. The values of pH, pCO2, PO*, and HC03 were measured immediately after sampling in a blood pH/gas analyzer (Corning). Glucose, lactate, and ammonia were determined by Sigma enzymatic procedure nos. 510, 876 UV, and 170 UV, respectively. Glutamine was estimated enzymatically by a modification of a previously described method." Monoclonal antibody (MAb) concentrations were determined by HPLC methods.IZGlucose and glutamine consumption rates were calculated based on the media TABLE1. Cell Lines Studiedu Cell Line
MAb Type
Parental Myeloma P3-NS1-1-Ag4-1 P3-NS1-1-Ag4-1 P3-NS1-1-Ag4-1 P3-X63-Ag8.653 SP2/0-Ag14
XM-A XM-D XM-G XM-I XM-SM "All splenocytes of BALB/c origin.
flow rates and residual values of the nutrients in the spent media. Oxygen consumption rates were continuously calculated by the computer controlling the Acusyst systems. The calculations were based on gas compositions and flow rates and on the differences in oxygen partial pressures between the inlet and outlet of the bioreactors measured in line by oxygen electrodes and in samples analyzed as described previously. Consumption and production rates were normalized to express the values per liter of bioreactor volume.
RESULTS AND DISCUSSION The MAb productivity and the oxygen consumption rate (OCR) for cell line XM-A in perfusion culture are shown in FIGURE 1. An apparent correlation between MAb productivity and OCR seems to exist, with an average yield between 7 and 7.5 mg of MAb per mmol of oxygen. Similar correlations were observed with all the cell lines studied and the corresponding OCRs and MAb yields are listed in TABLE2. As can be seen, the OCR for line XM-I was lower in serum-free medium, but the MAb yield remained very similar. Clearly, if the value of MAb/02 for a cell line is known,
ANNALS NEW YORK ACADEMY OF SCIENCES
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INTRODUCTION
Perfusion cultivations of mammalian cells allow for the maintenance of longterm, high-density, and high-productivity cultures.1,2Among the different perfusion systems, hollow fibers have been used for the production of biologicals?“ including monoclonal a n t i b o d i e ~ ,with ~ - ~ good results. However, few studies have gone beyond simply describing the performance of a given cell line in a particular perfusion system. These studies are of limited use for predicting the performance of other cell lines, even in the few cases where the mass transfer characteristics of the bioreactor are well understood. Hybridoma cells have nutritional requirements, metabolic rates, and product formation/secretion rates that are unique to each line even when derived from the same parental myeloma. Often, these characteristics are functions of the culture conditions, including the composition of the medium, the bioreactor type, and the way it is operated. These characteristics need to be studied and quantitated under reproducible conditions that will reflect actual production processes. Simple chemostats may be valuable tools when utilized for this purpose by studying hybridomas under continuous cultivation. The results obtained may then be used to predict the performance of the corresponding cell line in a perfusion reactor operated continuously for extended periods. The objective of this work is to describe the cultivation of hybridoma lines in industrial-scale perfusion culture, and to determine the value of chemostat-derived data in the interpretation of the results obtained, as a method for predicting the performance of hybridoma/bioreactor systems.
MATERIALS AND METHODS
The hybridoma cells used were all of murine-murine origin and are listed in TABLE1. The media used consisted of Dulbecco’s Modified Eagle’s Medium/Ham’s F-12 (1:l) containing 25 mM glucose and 4 or 6 mM glutamine and supplemented either with 5% fetal bovine serum (FBS) or with 20 nM sodium selenite, 20 JLM ethanolamine, 30 mg/L bovine transferrin, and 5 mg/L bovine insulin.1° For chemostat experiments, jacketed spinner flasks (Wheaton, Celstir 3000 mL), 1500 to 2000 mL working volume, were used. The covers were perforated and fitted with connectors and silicone tubing to allow continuous supply of medium and 72
CASTILLO et a[.: PERFUSION CULTURES
73
withdrawal of spent medium and cells. Ports were also made for removal of samples, addition of gas (5-10% COz in air), gas exhaust, inoculation of cells, and other additions. Gas inlets and outlets had 0.2-pm hydrophobic filters. The gases were applied to the liquid overhead to poise the medium before inoculation. Agitation was provided by magnetic floating stir bars rotating between 60 and 150 rpm. The temperature was maintained at 37 "C by pumping water through the jackets with a heating-circulator bath. Automatic addition of 7% sodium bicarbonate was used to control the pH within the desired values. Continuous perfusion runs were done in Acusyst-P hollow fiber systems (Endotronics, Incorporated). Samples, 5 to 10 mL, were withdrawn with syringes. Total and viable cells were counted in hemacytometers after diluting 1:l with 0.2% trypan blue. The values of pH, pCO2, PO*, and HC03 were measured immediately after sampling in a blood pH/gas analyzer (Corning). Glucose, lactate, and ammonia were determined by Sigma enzymatic procedure nos. 510, 876 UV, and 170 UV, respectively. Glutamine was estimated enzymatically by a modification of a previously described method." Monoclonal antibody (MAb) concentrations were determined by HPLC methods.IZGlucose and glutamine consumption rates were calculated based on the media TABLE1. Cell Lines Studiedu Cell Line
MAb Type
Parental Myeloma P3-NS1-1-Ag4-1 P3-NS1-1-Ag4-1 P3-NS1-1-Ag4-1 P3-X63-Ag8.653 SP2/0-Ag14
XM-A XM-D XM-G XM-I XM-SM "All splenocytes of BALB/c origin.
flow rates and residual values of the nutrients in the spent media. Oxygen consumption rates were continuously calculated by the computer controlling the Acusyst systems. The calculations were based on gas compositions and flow rates and on the differences in oxygen partial pressures between the inlet and outlet of the bioreactors measured in line by oxygen electrodes and in samples analyzed as described previously. Consumption and production rates were normalized to express the values per liter of bioreactor volume.
RESULTS AND DISCUSSION The MAb productivity and the oxygen consumption rate (OCR) for cell line XM-A in perfusion culture are shown in FIGURE 1. An apparent correlation between MAb productivity and OCR seems to exist, with an average yield between 7 and 7.5 mg of MAb per mmol of oxygen. Similar correlations were observed with all the cell lines studied and the corresponding OCRs and MAb yields are listed in TABLE2. As can be seen, the OCR for line XM-I was lower in serum-free medium, but the MAb yield remained very similar. Clearly, if the value of MAb/02 for a cell line is known,
ANNALS NEW YORK ACADEMY OF SCIENCES
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