Batch cultures of a hybridoma cell line performed with in situ ammonia removal

ELSEVIER

Batch cultures of a hybridoma cell line performed with in situ ammonia removal Markus Schneider, Mounia El Alaoui, Urs von Stockar, and Ian W. Marison

Institute of Chemical Engineering, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland

Batch cultures of a mouselmouse hybridoma cell line secreting a dimeric &A were performed in combination with a membrane process for the continuous removal of ammonia from the culture medium. The latter involves hydrophobic porous membranes with an associated pH gradient. The process was shown to be efficient reducing the ammonia concentration by approximately 5070 by comparison to control experiments. Two series of experiments were pegormed with an initial glutamine concentration of 4 mM and 10 m.+t, respectively. The growth characteristics, antibody productivity, and quality of antibody assayed by Western blotting were not changed by ammonia removal; however, the cellular energy metabolism was afSected by reduction of ammonia levels. At the lower initial glutamine concentration, ammonia removal caused an increased glutamine consumption and reduced lactate production. No effect upon glucose uptake was observed, thus resulting in a considerably reduced yield of lactate from glucose. At the high initial glutamine concentration, the reduced ammonia concentration led to a decrease in glucose and glutamine consumption as well as lactate production. This resulted in only a slight increase in the yield of lactate from glucose. Despite the abundance of literature concerning ammonia inhibition of mammalian cell cultures, little has been reported concerning the effect of continuous ammonia removal from

such cultures. 0 1997 by Elsevier Science Inc.

Keywords: Hybridoma; ammonia; glutamine; ammonia removal; hydrophobic porous membrane

Introduction

A considerable amount of literature exists concerning the inhibitory or toxic effects resulting from ammonia or ammonium accumulation in mammalian cell cultures.1-18 Re- duced growth rates and lower maximal cell densities were reported for a range of cell lines by the addition of ammonium concentrations as low as l-5 mM; however, many cell lines can readily adapt to much higher ammonium concentrations particularly in continuous culture.7~‘3 In contrast, specific antibody production by hybridomas has been reported as relatively unaffected by increasing ammonium concentrations.6s10 A pH-dependence of the ammonia toxicity has also been observed by several authors.5*‘4,‘5 These authors found a pronounced increase in ammonia toxicity at

Address reprint requests to Dr. Ian W. Marison, Institute of Chemical Engineering Swiss Federal Institute of Technology Lausanne, CH-1015 Lausanne, Switzerland Received 7 March 1996; accepted 8 May 1996

higher pH values and concluded that unprotonated ammonia is responsible for the inhibitory effects. Elevated ammonia concentrations may further affect cellular metabolism and influence the intracellular processing of a protein and the quality of the product secreted.16 It has further been shown that the glycosylation pattern of a recombinant protein was affected by the ammonia concentration.‘7-‘9

The actual mechanism of ammonia and ammonium ion toxicity is still unclear. Unprotonated NH, readily diffuses across most cellular membranes, thus equilibrating rapidly any gradient in chemical potential of NH,. In contrast, NH; diffuses across membranes only very slowly but is easily transported by several transport proteins.12T20*21 Some further hypotheses are discussed in review articles.8*22*63 A number of explanations have been postulated to explain cell death resulting from ammonia toxicity.23*24

Ammonia accumulation in the culture medium is due to amino acid metabolism, in particular, that of glutamine, a major nutrient in addition to glucose for most animal cells in culture. Glutamine is usually supplied at concentrations between 2-5 mM.2*25-34 The cellular degradation of glutamine

Enzyme and Microbial Technology 20:266-276, 1997 Q 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

0141-0229/97/$17.00 PII SO141-0229(96)00122-6

has been intensively studied by many authors.35-38 This mainly occurs in the mitochondrial matrix involving the removal of the amid0 group of glutamine to yield glutamate which is catalyzed by glutaminase. The second step is the removal of the o-amino group which leads to a-ketoglu- tarate. This reaction can either be catalyzed by glutamate dehydrogenase which results in the liberation of a second ammonia molecule or a transaminase. The latter transfers the amino group to an o-keto acid (mainly pyruvate) which leads to alanine accumulation in the culture medium. Using nuclear magnetic resonance, it has been shown39 that most of the ammonia accumulating in the culture was derived from the amido group of glutamine. In addition to ammonia release due to cellular metabolism, ammonia production by the chemical decomposition of glutamine to pyrrolidonecar- boxylic acid also had to be taken into account.40s41

Two strategies have been applied to reduce ammonia accumulation in culture media: (1) prevention of ammonium production by the controlled addition of glutamine and glucose, 22,30*4244 the use of stable derivatives of glutamine,4s,46 transformation of cells with the glutamine synthetase gene to enable growth in the absence of glutamine,“’ or adaptation to growth in glutamine-free, nonammonia- genie media;48*49 and (2) ammonium removal using ion- exchange resins and membranes to selectively adsorb ammonium ions.50-57*6s

In the present study, an efficient and simple method using hydrophobic porous membranes58 has been applied to batch suspension cultures of a hybridoma cell line for ammonia removal. This enabled an investigation into the effects of ammonia on cell growth, antibody productivity, and metabolism.

Materials and methods

Principle of the ammonia removal process

Ammonia was removed from the culture by diffusion across a hydrophobic porous membrane by a newly developed process.58.59 Only the unprotonated volatile ammonia (NH,) is able to cross the pores of the membrane freely and be trapped in an aqueous acid by irreversible protonation. The driving force for ammonia transfer is the partial pressure difference of this species across the membrane. The principle of the process is given in Figure 1. The mass transfer characteristics of this system in different bioreactor configurations have been published earlier.58

Cell cultures

A mouse/mouse hybridoma (Zac3) secreting monoclonal dimeric IgA antibodies against Vibrio cholerue was used for all experiments.60

Media

Cells were routinely grown in a serum- and protein-free medium (FMX-TurbodomaTM, Cell Culture Technologies, Zurich, Switzer- land) supplemented with n-glucose (1 g l-l), to provide an initial glucose concentration of 16-18 mu. In specified cases, cells were grown in RPMI1640 (Gibco BRL, Basle, Switzerland) supplemented with 10% fetal calf serum (Inotech, Dottikon, Switzerland) for comparison.

cell culture strip solution

aqueous phosphoric acid

membrane

Figure 1 Schematic representation of the membrane process for ammonia removal. In the cell culture (left), ammonia is in equilibrium with ammonium. The equilibrium lies on the side of the protonated form at pH 7-7.5. Volatile, unprotontated ammonia is able to diffuse across the hydrophobicporous membrane and will be immediately and irreversibly prdonated in the acid on the nonculture side. Since the resulting ionic ammnoium cannot cross the membrane, it remains trapbed in the acid

Cultivation techniques

Flask cultures. Cells were cultivated in T25-flasks (Falcon) containing 6 ml medium in an incubator at 37°C under an atmosphere containing 5% CO, in the same serum- and protein-free medium as in the bioreactor. Different concentrations of ammonium chloride (0, 1 .O, 2.5, and 7.5 mM) were added before inoculation in order to evaluate the effect of ammonia on the behavior of the cells. Ten T-flasks of every series with the same initial’ ammonium concentration were inoculated with 0.5 x lo5 cells ml-’ and cultivated in parallel. Sampling was done by harvesting one T-flask of every series at intervals for analysis.

Membrane bioreactor cultures. Batch cultures were performed in a membrane bioreactor purposely built with a working volume of 2.5 1. The reactor consisted of a cylindrical vessel equipped with a marine impeller and draft tube. A flat PTFE-membrane sheet

Table 1 Batch culture experiments in membrane reacter with and without ammonia removal system

Trial Ammonia removal Initial glutamine

concentration

1 Without 2 Without 3 With 4 With 5 Without 6 Without 7 With 8 With

4rnM 4rnM 4rnM 4rnM

IOrnM 10 rnM 10 rnM 10 rnM

Batch cultures of a hybridoma cell line performed with in situ: M. Schneider et al.

Enzyme Microb. Technol., 1997, vol. 20, March 269

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with a nominal pore diameter of 0.2 pm (TF200, Gehnan Sciences, Ann Arbor, MI) was placed at the base of the reactor between the glass vessel and a support made from PVDF (Figure 2). Aqueous phosphoric acid (0.5 mol 1-l) was pumped below the membrane by means of a gear pump (Watson-Marlow Ltd., Falmouth, Cornwall, UK). Control experiments without ammonia removal were performed in the same bioreactor in the absence of the membrane at the base of the vessel. The temperature was maintained at 37°C by means of a heating mantle and an external heating bath. pH was controlled between 7.4 and 7.1 using a CO&carbonate buffer system. The CO, concentration of the surface aeration was varied between O-10%. Dissolved oxygen concentration (DO) was mea- sured by means of a polarographic oxygen electrode (Ingold, Ur- dorf, Switzerland). The DO was controlled at 60% air saturation by intermittent sparging of pure oxygen. Agitation (200 mint) was performed with a three-blade marine impeller with a diameter of 60 mm (Ekato, Mumpf, Switzerland). Four different experimental conditions were investigated in duplicate (Table 1). The first series of experiments was performed with an initial glutamine concentration of 4 IIIM with and without ammonia removal (Expts. l-4) and the second series with an initial glutamine concentration of 10 mM with and without ammonia removal (Expts. 5-8).

Inocula were prepared in T175-tissue culture flasks (Falcon) at 37°C under an atmosphere of 5% CO,. Cells from seven T175- flasks (each containing 130 ml cell suspension were harvested by centrifugation at 145 g for 5 min at 5’C and used to inoculate the bioreactor.

Analytical methods Viable cell concentration was determined microscopically using a hemocytometer and the trypan blue dye exclusion method. Glu-

-IL

- Acid flow

Acid comparkment ‘Support (PVDF)

Figure 2 Schematic representation of the membrane reactor consisting of a cylindrical stirred glass vessel equipped with draft tube and heating mantle for thermostatization. The membrane was placed at the base of the vessel between the glass and a support made from PVDF. Acid was pumped through the compartment below the membrane by means of a gear pump

case, glutamine, ammonia, and lactate were determined using stan- dard enzyme methods (Boehringer Mannheim, Mannheim, Ger- many). Glutamine was deaminated first by glutaminase (Sigma Chemical Co., St., Louis, MO) and the liberated ammonia determined enzymically. The concentrations of glucose, glutamine, lactate, alanine, glutamate, and pyrrolidone carboxylic acid were determined using a new HPLC technique.6’ The first-order reaction rate constant of glutamine decomposition kinetics was determined by integrating the glutamine concentration in T-flask samples in order to correct the amount of glutamine taken up and ammonia produced for this influence:

4g Wdec dt = -kgl,. [g InI

[g lnl,, = -Qn . &g 1nlW (2)

The glutamine concentration as a function of time was ap- proximated with a polynomial function of the 4th degree. The reaction rate constant was assumed to be constant during the culture.

Antibody (IgA) concentrations were determined by means of a sandwich-ELISA method. Plates (96-well) (Im- mulon M129B IV, Dynatec) were coated with the first antibody (Sigma) and directed against the heavy chains (o) of the antibodies secreted by the hybridomas. The second biotinylated antibody (Amersham Life Sciences, Amersham, Buckinghamshire, UK) was assayed by means of streptavi- dine and horseradish peroxidase (Amersham). Polyacryl- amide gel electrophoresis under denaturating conditions (SDS) was performed with subsequent Western blotting. Western blotting was performed using biotinylated antibodies directed against the heavy chains (Sigma). Biotinylated molecular weight standards (Biorad, Hemel Hempstead, England) were run in parallel for comparison.

To calculate the specific antibody production rate, the following differential equation was used and integrated:

Specific antibody production rate:

(4)

Results

Tissue flask cultures

For the control experiment, in the absence of added ammonium chloride, the cells reached a final density of 5.5 x lo5 cells ml-’ approximately 80 h after inoculation. This corre- sponded to a specific growth rate of 0.041 h-’ and a dou- bling time of 17 h (Figure 3). The stationary phase was very short with the viable cell density dropping very rapidly after reaching the maximum. Addition of 1.0 mM ammonium chloride prior to inoculation did not change the growth behavior significantly. Addition of 2.5 mM ammonium resulted in a 20% reduction in the maximum cell density and a reduction in the specific growth rate to 0.037 h-l. Addition

270 Enzyme Microb. Technol., 1997, vol. 20, March


” 0 20 40 60 80 100 120 140 160

time (h)

Figure 3 Viable cell densities of the hybridoma cell line Zac3 determined in T25-flasks with different concentrations of ammonium chloride added before inoculation. Symbols: 0 mM NH&I(O); 1.0 mM NH,CI (A); 2.5 mM NH&I (0); and 7.5 mM NH&I (A). The viability (ratio of viable cell number to total cell number) was 95% from 0 mM NH&I, 90% for 1 .O mM NH&I, and 85-90% for 2.5 mM NH&I during the growth phase

of 7.5 mM ammonium chloride completely prevented growth with a complete loss of viability within about 30 h.

Bioreactor cultures

Serum-free medium. The viable cell concentrations for the first series of experiments (Expts. l-4) involving an initial glutamine concentration of 4 mM with (Expt. 3) and without (Expt. 1) ammonia removal are shown in Figure 4A. Both curves show the same pattern. The cells reached a final density of about 7 x lo5 cells ml-’ 75 h after inoculation which corresponds to a specific growth rate of 0.035 h-l. The specific growth rates for experiments l-4 ranged between 0.033 and 0.037 h-l. After the maximal cell density was attained, a sharp decline in viable cell density was observed. The total ammonia concentration was efficiently reduced from a maximal value during the decline phase of 3.2 mM in the control experiment to 1.9 mu by means of the membrane process (Figure 4’). A higher rate of ammonia removal could be achieved by increasing the surface area of the hydrophobic membrane; however, the aim here was to reduce the ammonia concentration in the medium to below 2 mM. That concentration has been shown to be well below potentially toxic levels for the Zac3 cell line. The antibody concentration reached a final value of 17-18 Fg ml-’ which corresponds to a specific antibody production rate of 0.4 pg cell-’ h-’ in all four cultures performed with an initial glutamine concentration of 4 mM with production limited to the growth phase (Figure 43.). No differences were observed as a result of ammonia removal. Figure 5 shows a Western blot performed with samples taken from (Expts. 14. The band pattern is the same for all samples. The fastest migrating band represents monomeric IgA, a strong intermediate band that represents dimeric antibodies while the slowest migrating bands represent oligomers. The migration and intensity of the different species was the same with and without ammonia removal.

“.B

40 60 80 100 3

time (h) !O

Figure 4 Results of the batch cultures performed in combination with the membrane process (Expt. 3, A) and without the process (Expt. 1, 0) with the lower initial glutamine concentration of 4 mM. Shown are the viable cell concentrations (A), the antibody concentrations determined by ELISA (B), and the total ammonia concentration K). The viability (ratio of viable cell number to total cell number) was always higher than 90% during the growth phase

In the second series of experiments (Expts. 5-8) performed with an initial glutamine concentration of 10 mM, similar results were obtained to those with an initial glutamine concentration of 4 mM. In both cases with and without the membrane process, a slightly prolonged lag phase of about 10 h was observed. Otherwise, the growth behavior was the same. The specific growth rates again ranged around 0.035 h-l. The viable cell densities obtained from Expts. 5 and 7 are shown in Figure 6A. The total ammonia concentration was reduced from 5.0 to 2.8 mM in the decline phase (Figure 6C). The rate of ammonia removal was limited by the available hydrophobic membrane surface rather than the efficiency of the membrane process itself. Clearly


S 1234 56788

Figure 5 Western blot performed with samples from Expts. l-4 with and without ammonia removal. Two samples were used from Expt. 1 (lane 1 and 2) and two from Expt. 2 (lane 3 and 4). Both experiments were performed without ammonia removal. Two samples were taken from Expt. 3 (lane 5 and 6) and from Expt. 4 (lane 7 and 8). Both were done in combination with the membrane process. The first and the last lane of the gel were molecular weight standards (marked with “S”). The strong bands marked with “D” are due to dimeric IgA molecules; the bands marked with “M” are due to monomers. No differences in the band pattern can be attributed to the reduced ammonia concentration

an increased rate could have been achieved by increasing the surface area of the hydrophobic membrane. The final antibody concentrations ranged from 20-22 l.r,g ml-i (Fig- ure 6B) which corresponds to specific antibody production rates of 0.4-0.5 pg cell-’ h-‘; however, in contrast to the first series of experiments (Figure #b), the antibody concentration continued to increase during the decline phase. Western blots showed no effect of ammonia removal on the band pattern and were similar to those obtained from the first series of experiments (not shown).

Although neither growth nor antibody productivity was significantly affected by reducing the ammonia concentration, for low and high initial glutamine concentrations, some differences were found in the uptake of glucose and glutamine and in the production of lactate during the growth phase (Figure 7). Since no effect on growth behavior was observed, the quantities consumed or produced can be directly compared. In the case of lower initial glutamine concentrations (4 mM), the glucose consumption rate was unaffected by ammonia removal while lactate production was reduced by 20% which lead to a reduction of the mean lactate yield coefficient (Y,aC,slo ratio of mole lactate released mol-’ glucose consumed) from 2.1 to 1.5. Glutamine consumption increased by about 10% as a result of ammonia removal. About 25% of the glutamine decrease was due to chemical decomposition of glutamine with and without ammonia removal. The values of the mean ratio _of mol ammonia released mol-’ glutamine consumed (YNH;s~n) without ammonia removal were around 0.85 in Expts. 1 and 2, respectively. Since the quantity of ammonia removed across the membrane is not known, this ratio cannot be given for Expts. 3 and 4. For the higher initial glutamine concentrations (10 mM), the levels of glucose consumption and lactate production were only slightly increased (5-10%) in comparison to the first experimental series without ammonia removal (Expts. 5 and 6 compared with 1 and 2).

-I

0 20 40 60 80 100 120

time (h)

Figure 6 Results of the batch cultures performed in combination with the membrane process (Expt. 7 A) and without the process (Expt. 5, ??i) with the higher glutamine concentration of 10 mM. Shown are the viable cell concentrations (A), the antibody concentrations determined by ELISA (B), and the total ammonia concentration K). The viability (ratio of viable cell number to total cell number) was always higher than 75% during the growth phase

Glutamine consumption was not significantly reduced. With the combined membrane process, glucose consumption and lactafe production decreased both by about 20%. The value for Ykki, increased from 1.8 to 2.0 due to ammonia removal (Expts. 5 and 6 compared with 7 and 8). Glutamine consumption did not decrease significantly due to the reduced ammonia concentration. Approximately 50% of the total quantity of glutamine used during the growth phase was due to chemical decomposition. The values for YNHJa in without the membrane process at the higher initial glutamine concentration are approximately 1.0 for Expts. 5 and 6.

Growth in serum-supplemented medium. Batch cultures performed in serum-supplemented medium provided similar


Batch cultures of a hybridoma ce// line performed with in situ: M. Schneider et al.

I ??glucose 0 lactate ??glutamine E9 ammonia I

Figure 7 Consumption of glucose and glutamine and production of lactae and ammonia during the growth phase of the different batch cultures performed with and without ammonia removal. In the case of glutamine, the lower part of the bar represents the fraction actually metabolized by the cells (dark) and the upper part is the fraction decomposed chemically (light). Values for ammonia are not given for Expts. 3, 4,7, and 8 since the amount of ammonia removed into the acid solution of the membrane process cannot be determined

results to those in serum-free medium. The cells grew to a maximum density of 1 x lo6 cells ml-’ and an antibody concentration of about 45 p,g ml-‘. Ammonia accumulation could be efficiently reduced from a final concentration of 4 mM to 1.7 mM, in the death phase. Glucose and glutamine consumption were similar; however, lactate production_ in the presence of the membrane process was lower with Y,ac, glc decreasing from 1.5 to 1.1 (results not shown).

Glutamine decomposition kinetics

Glutamine decomposition in FM&TurbodomarM followed first-order kinetics. The first-order reaction rate constant was determined to be 0.0030 h-r which corresponds to a glutamine half-life of 9.6 days.

Discussion

T-flasks cultures

The effect of ammonia on the hybridoma cell line Zac3 grown in the serum- and protein-free medium was a reduction of the maximal cell density and the specific growth rate. Ammonia clearly has an inhibitory or toxic effect on this cell line with a reduction in growth at ammonium chloride concentrations as low as 2.5 mM and complete inhibition at 7.5mM. The aim of these preliminary experiments was to estimate the effect of ammonia on the growth behavior of this cell line at concentrations that can easily accumulate during batch culture conditions since large variations have been reported from one cell line to another,3 however, these cultures were undertaken by addition of a range of ammonia concentrations to the medium. Consequently, the effect on

cell growth and metabolism may not be similar to that observed due to intracellularly produced ammonia.

Batch cultures pedormed in the bioreactor with and without ammonia removal The main objective of these experiments was to study the effect of intracellular ammonia on cell growth and metabolism under controlled conditions. Unlike the T-flask experiments, no externally supplied ammonia was added. This could have important consequences since it has been reported that intracellularly produced ammonia leads to different physiological effects than when externally supplied to the medium.21,63 Such experiments were undertaken in re- actors by comparison of batch cultures grown under iden- tical conditions in the presence and absence of the membrane process.

Growth behavior. Experiments performed in the membrane reactor in serum- and protein-free medium showed that with an initial glutamine concentration of 10 mM, ammonia accumulated to 5 mM by the end of the growth phase (Figure 6C). Reduction of the ammonia concentration to 2.7 mM by means of the membrane process did not affect growth. This is in contrast to T-flask experiments (Figure 3) in which the addition of 2.5 mM ammonium reduced growth by about 20% while growth wa$ completely inhibited by the addition of 7.5 mM ammonium. These results are in agreement with those of Capiaumont et a1.s6 and indicate that the effect of ammonia accumulating slowly during growth is less harmful than the effect of ammonia added before inoculation possibly as a result of adaptation of the cells. Recent studies in our laboratory suggest that the low maximum cell densities achieved were due to a growth limitation by essential amino acids, particularly isoleucine, leu- tine, and valineW rather than accumulation of ammonia to toxic levels. In contrast, ammonia accumulation has been shown to be a problem in high-density qultures.52 The latter showed that removing ammonia from immobilized cell cultures resulted in an increase in viable cell density from 0.75 to 2.5 x lo7 cells ml-’ together with a ten-fold increase in antibody activity.

Antibody productivity and quality of the secreted antibodies. Specific antibody productivity did not change when ammonia was removed from the culture medium over the range of initial glutamine concentrations studied. This is in agreement with results published by several authors that the specific antibody productivity does not change with ammonia concentration. 6,10 In contrast, Rather et al. I * found a critical ammonia concentration for optimal specific antibody productivity of 2-2.5 mM for the hybridoma cell line ClE3; however, they could not explain why the specific antibody productivity should be reduced at ammonia concentrations below the optimal value. Furthermore in the present study, the addition of higher initial glutamine concentrations (10 mM) had no effect on specific antibody productivity although cells continued to secrete antibodies during the decline phase which leads to an increase in the final antibody concentration. This is in contrast to Omasa et a1.33 who presented data in which high initial glutamine concen-


Papers

trations increased the specific antibody production rate with an optimum concentration of 25 mM. In summary for Zac3, the specific antibody productivity is not sensitive to changes in total ammonia or glutamine concentrations over the range explored.

Western blots revealed no differences in migration pattern of the secreted antibodies during the different phases of batch growth, at different concentrations of accumulating ammonia, nor for different initial glutamine concentrations (Figure 5). A major effect of variations in culture conditions is the glycosylation of secreted protein,19 however, slight changes in the molecular composition of the carbohydrate side chains would not be detectable by Western blotting. It has been reported ” that protein glycosy lation may be altered or completely inhibited by ammonia particularly when the pH was increased to 8.4, a value at which most of the ammonia is present in the unprotonated form. In the present work, experiments were performed at a pH of 7.1-7.4, thus the concentration of NH, was low and probably did not reach levels which could significantly affect glycosylation.

Metabolic effects of ammonia removal. Although growth behavior and antibody production were shown to be unaffected by ammonia and glutamine concentrations over the range studied, significant differences in glutamine and glucose uptake and lactate production were found (Figure 7). Although the values show some degree of uncertainty, each experimental condition was performed twice; thus, a gen- eral tendency in the changes can be clearly deduced. At low initial glutamine concentrations, no effect on glucose uptake was observed while lactate production decreased by 20% and glutamine uptake increased slightly when ammonia was removed. This led to a decrease in Flaclglc from 2.1 to 1.5. The higher value represents the theoretical maximum in which glucose is almost completely converted to lactate by glycolysis with glutamine acting as the main energy source.3’y62 Since growth was not altered by the different experimental conditions, changes in uptake and production terms can directly be compared to one another. Although it is not possible to make detailed mass balances with the data obtained, it is clear that since the amount of lactate formed decreased and the amount of glutamine consumed increased, either some other carbon-containing product must have been formed, CO2 production increased, or the el- emental composition of the biomass changed as a result of ammonia removal. Detailed metabolic studies possibly with radioactively labelled intermediates would be required to clarify this situation.

Reduction in the ammonia concentration by application of the membrane process resulted in an increased uptake of glutamine which is in agreement with some reports,” while in contrast to others.’ An inhibition of ammonia release by elevated ammonia concentrations has also been reported.6,10 The latter cannot be deduced from the present work since the amount of ammonia removed across the membrane is not known.

With higher initial glutamine concentrations (10 mM>, in the absence of ammonia removal, a slightly decreased glutamine uptake and small increase in lactate production and glucose consumption were observed compared with the experiments carried out with 4 mM initial glutamine concen-

tration (Expts. 5 and 6 compared with Expts. 1 and 2). Higher glutamine concentrations have been reported to increase its own uptake,. 14 but chemical decomposition be- comes very important in this case; however, care must be taken since the relative contribution of chemical decomposition is more important for high concentrations of glutamine. Thus for high initial glutamine concentrations (10 mM), the total amount of glutamine consumed during the growth phase is higher than with cultures containing 4 mM glutamine although the amount actually metabolized by the cells decreased. The influence of glutamine decomposition on glutamine uptake and ammonia production has been discussed in detail by Ozturk and Palsson.4” These authors confirm the present results that the error made by neglecting glutamine decomposition may attain 200% or more; however, most calculations of the level of chemical decomposition of glutamine have used integration of the concentration assuming a constant reaction rate constant. This leads to approximate values only since the reaction rate constant has been shown to be strongly dependent on pH and medium composition, factors which may vary during a culture.40 Ammonia removal resulted in reduced lactate production for cultures with 10 mM glutamine; however, both glucose and glutanjne consumption decreased slightly with the result that Y,ac,g,c increased from 1.8 to 2.0. This is clearly different from the result with low initial glutamine concentrations and is not readily explained. These changes in yield values, although small, are reproducible since the errors have been estimated to be less than 3%; thus, metabolism of glucose and glutamine appears to be very sensitive to the concentration of these substrates as well as to changes in the ammonia concentration.

Previous studies on ammonia toxicity and the effect of glutamine concentration on cell growth and productivity have been undertaken by maintenance of a constant glutamine concentration followed by the addition of ammonia to the medium. This method has been used since raising the ammonia concentration by addition of higher levels of glutamine does not allow separation of the effects of ammonia and glutamine. The present paper describes how the two parameters may be studied independently by the application of a membrane process for ammonia removal, thus avoiding the external addition of ammonia, a situation which has no physiological significance.

Conclusions.

The removal of ammonia by means of a membrane process did not improve the growth and productivity in batch cultures of a mouse/mouse hybridoma cell line in comparison to control experiments without the process. The sharp decline of viable cell densities at the end of the growth phase cannot be attributed to ammonia inhibition, but must have been provoked by nutrient limitation (probably by essential amino acids) and/or inhibition by some component other than ammonia; however, cellular metabolism was affected by reducing the ammonia concentration in the medium. Re- moving ammonia reduced lactate production and influenced glutamine uptake depending on the initial glutamine concentration while glucose consumption was practically un- changed. The membrane process was shown to be efficient.



The presence of the PTFE membranes did not affect the process in any way and the ammonia concentration could be reduced without removing other medium components. Mammalian cell cultures performed in combination with the ammonia removal process could be very advantageous in the case of highly ammonia-sensitive cell lines for high cell density cultures and the maintenance of defined product quality such as glycosylation of secreted proteins. Further- more, application of ammonia removal to mammalian cell cultures allows the effects caused by glutamine and ammonia to be studied independently, a feature which should provide important insights to mammalian cell metabolism.

Acknowledgments

This work was partly founded from the Swiss Priority Pro- gram for Biotechnology. L. Vallotton and A. Neuen- schwander are thanked for manufacturing the reactor parts and Dr. W. van Gulik for carefully reading and correcting the manuscript.

List of symbols 18.

ATP ELISA Ig A PFK PTFE PVDF SDS DO

Kdl k 0

Adenosine triphosphate Enzyme-linked immunosorbent assay Immunoglobulin A Phosphofructokinase Polytetrafluorethylene Polyvinylidenedifuloride Sodium dodecylsulfate Dissolved oxygen concentration % air saturation Antibody concentration mg ml-’ First-order reaction rate constant of glutamine decomposition h-r Specific antibody productivity pg cell-’ h-r Viable cell concentration cells ml-’ Mean yield of lactate from glucose mol mole1 Mean yield of ammonia from glutamine mol mol-’

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Batch cultures of a hybridoma cell line performed with in situ ammonia removal