Growth, metabolic, and antibody production kinetics of hybridoma cell culture: 2. Effects of serum concentration, dissolved oxygen concentration, and medium pH in a batch reactor.

The effects of serum, dissolved oxygen (DO) concentration, and medium pH on hybridoma cell physiology were examined in a controlled batch bioreactor using a murine hybridoma cell line (167.4G5.3). The effect of serum was also studied for a second murine hybridoma cell line (S3H5/gamma 2bA). Cell growth, viability, cell density, carbohydrate and amino acid metabolism, respiration and energy production rates, and antibody production rates were studied. Cell growth was enhanced and cell death was decreased by increasing the serum level. The growth rates followed a Monod-type model with serum being the limiting component. Specific glucose, glutamine, and oxygen uptake rates and specific lactate and ammonia production rates did not change with serum concentrations. Amino acid metabolism was slightly influenced by the serum level. Cell growth rates were not influenced by DO between 20% and 80% air saturation, while the specific death rates were lowest at 20-50% air saturation. Glucose and glutamine uptake rates increased at DO above 10% and below 5% air saturation. Cell growth rate was optimal at pH 7.2. Glucose and glutamine uptake rates, as well as lactate and ammonia production rates, increased above pH 7.2. Metabolic rates for glutamine and ammonia were also higher below pH 7.2. The consumption or production rates of amino acids followed the glutamine consumption very closely. Cell-specific oxygen uptake rate was insensitive to the levels of serum, DO, and pH. Theoretical calculations based on experimentally determined uptake rates indicated that the ATP production rates did not change significantly with serum and DO while it increased continually with increasing pH. The oxidative phosphorylation accounted for about 60% of total energy production. This contribution, however, increased at low pH values to 76%. The specific antibody production rate was not growth associated and was independent of serum and DO concentrations and medium pH above 7.20. A 2-fold increase in specific antibody production rates was observed at pH values below 7.2. Higher concentrations of antibody were obtained at high serum levels, between 20% and 40% DO, and at pH 7.20 due to higher viable cell numbers obtained.     

Effects of ammonia and lactate on hybridoma growth, metabolism, and antibody production

The influence of ammonia and lactate on cell growth, metabolic, and antibody production rates was investigated for murine hybridoma cell line 163.4G5.3 during batch culture. The specific growth rate was reduced by one-half in the presence of an initial ammonia concentration of 4 mM. Increasing ammonia levels accelerated glucose and glutamine consumption, decreased ammonia yield from glutamine, and increased alanine yield from glutamine. Although the amount of antibody produced decreased with increasing ammonia concentration, the specific antibody productivity remained relatively constant around a value of 0.22 pg/cell-h. The specific growth rate was reduced by one-half at an initial lactate concentration of 55 mM. Although specific glucose and glutamine uptake rates were increased at high lacatate concentration, they showed a decrease after making corrections for medium osmolarity. The yield coefficient of lactate from glucose decreased at high lactate concentrations. A similar decrease was observed for the ammonia yield coefficient from glutamine. At elevated lactate concentrations, specific antibody productivities increased, possibly due to the increase in medium osmolarity. The specific oxygen uptake rate was insensitive to ammonia and lactate concentrations. Addition of ammonia and lactate increased the calculated metabolic energy production of the cells. At high ammonia and lactate, the contribution of glycolysis to total energy production increased. Decreasing external pH and increasing ammonia concentrations caused cytoplasmic acidification. Effect of lactate on intracellular pH was insignificant, whereas increasing osmolarity caused cytoplasmic alkalinization.     

Effects of dissolved oxygen on hybridoma cell growth, metabolism, and antibody production kinetics in continuous culture.

The effects of dissolved oxygen concentration (DO) on hybridoma cell physiology were examined in a continuous stirred tank bioreactor with a murine hybridoma cell line (167.4G5.3). Dissolved oxygen concentration was varied between 0% and 100% air saturation. Cell growth and viability, carbohydrate, amino acid, and energy metabolism, oxygen uptake, and antibody production rates were investigated. Cell growth was inhibited at both high and low DO. Cells could grow at 0% DO and maintain viability under a nitrogen atmosphere. Cell viability was higher at low DO. Glucose, glutamine, and oxygen consumption rates changed little at DO above 1% air saturation. However, the metabolic uptake rates changed below 1% DO, where growth became oxygen limited, and a Km value of 0.6% DO was obtained for the specific oxygen uptake rate. The metabolic rates of glucose, glutamine, lactate, and ammonia increased 2-3-fold as the DO dropped from 1% to 0%. Amino acid metabolism followed the same general pattern as that of glutamine and glucose. Alanine was the only amino acid produced. The consumption rates of amino acids changed little above 1% DO, but under anaerobic conditions the consumption rates of all amino acids increased severalfold. Cells obtained most of their metabolic energy from glutamine oxidation except under oxygen limitation, when glucose provided most of the energy. The calculated ATP production rate was only slightly influenced by DO and rose at 0% DO. Antibody concentration was highest at 35% DO, while the specific antibody production rate was insensitive to DO.     

Effect of hypoosmotic stress on hybridoma cell growth and antibody production

To investigate the response of hybridoma cells to hypoosmotic stress, S3H5/gamma2bA2 and DB9G8 hybridomas were cultivated in the hypoosmolar medium [Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% serum] resulting from sodium chloride subtraction. Both hybridomas showed similar responses to hypoosmotic stress in regard to cell growth and antibody production. The cell growth and antibody production at 276 mOsm/kg were comparable to those at 329 mOsm/kg (standard DMEM). Both cells grew well at 219 mOsm/kg, though their growth and antibody production were slightly decreased. When the osmolality was further decreased to 168 mOsm/kg, the cell growth did not occur. When subjected to hyperosmotic stress, both cells displayed significantly enhanced specific antibody productivity (q(Ab)). However, the cells subjected to hypoosmotic stress did not display enhanced q(Ab). Taken together, both hyperosmotic and hypoosmotic stresses depressed the growth of S3H5/gamma2bA2 and DB9G8 hybridomas. However, their response to hypoosmotic stress in regard to q(Ab) was different from that to hyperosmotic stress. (c) 1997 John Wiley & Sons, Inc. Biotechnol Biong 55: 565-570, 1997.     

Enhanced specific antibody productivity of hybridomas resulting from hyperosmotic stress is cell line-specific

Hybridomas with non-growth-associated antibody production are thought to exhibit enhanced specific monoclonal antibody productivity (q _MAb) when subjected to hyperosmotic stress. Two hybridoma cell lines exhibiting non-growth-associated antibody production, S3H5/2bA2 and DB9G8 hybridomas, are cultivated in a batch mode using hyperosmolar media resulting from sodium chloride addition. Their response to hyperosmotic stress regarding q _MAb is quite different, though they show similar depression of cell growth in hyperosmolar media. The q _MAb of S3H5/2bA2 cells in a hyperosmolar medium (396 mOsm/kg, 10% fetal bovine serum (FBS)) is enhanced by approximately 180% when compared with that in a standard medium (283 mOsm/kg, 10% FBS), while q _MAb of DB9G8 cells in the same hyperosmolar medium is enhanced by only 10%. Thus, the enhanced q _MAb of hybridomas exhibiting non-growth-associated antibody production resulting from hyperosmotic stress is cell line-specific.     

Growth, metabolic, and antibody production kinetics of hybridoma cell culture: 1. Analysis of data from controlled batch reactors.

A mouse-mouse hybridoma cell line (167.4G5.3) was cultivated in a 1.5-L stirred-tank bioreactor under constant pH and dissolved oxygen concentration. The transient kinetics of cell growth, metabolism, and antibody production were followed by biochemical and flow cytometric methods. The cell-specific kinetic parameters (growth and metabolic rates) as well as cell size were constant throughout the exponential phase. Intracellular protein and RNA content followed a similar trend. Cell growth stopped when the glutamine in the medium was depleted. Glucose could not substitute for glutamine, as glucose consumption ceased after glutamine depletion. Ammonia and lactate production followed closely glutamine and glucose consumption, respectively. Alanine, glutamate, serine, and glycine were produced but other amino acids were consumed. The cells are estimated to obtain about 45% of the total energy from glycolysis, with the balance of the metabolic energy provided by oxidative phosphorylation. The antibody was produced at a constant rate in both the exponential and decline phases of growth. The intracellular antibody content of the cells remained relatively constant during the exponential phase of growth and decreased slightly afterwards.     

Loss of antibody productivity is highly reproducible in multiple hybridoma subclones

An immunoglobulin G (IgG(2b)) producing hybridoma cell line (S3H5/gamma2bA2) was cloned and subcloned. Twenty subclones were grown in parallel while being adapted in a stepwise fashion to serum-free medium. Following adaptation to serum-free medium, it was found that 16 of the 20 subclones remained at a relatively constant proportion of nonproducing cells. Three of the remaining subclones transiently deviated from this balance but eventually returned toward this population composition. One subclone continued to lose productivity. A population balance was reached at approximately 8% of the population being nonproducers. The loss of antibody productivity was thus highly reproducible. (c) 1993 John Wiley & Sons, Inc.     

Enhancement of monoclonal antibody production by immobilized hybridoma cell culture with hyperosmolar medium

To determine the effect of hyperosmotic stress on the monoclonal antibody (MAb) production by calcium-alginate-immobilized S3H5/gamma2bA2 hybridoma cells, the osmolalities of medium in the MAb production stage were varied through the addition of NaCI. The specific MAb productivity (q(MAb)) of immobilized cells exposed to abrupt hyperosmotic stress (398 mOsm/kg) was increased by 55% when compared with that of immobilized cells in the control culture (286 mOsm/kg). Furthermore, this enhancement of q(MAb) was not transient. Abrupt increase in osmolality, however, inhibited cell growth, resulting in no increase in volumetric MAb productivity (r(MAb)). On the other hand, gradual increase in osmolality allowed further cell growth while maintaining the enhanced q(MAb) immobilized cells. The q(MAb) immobilized cells at 395 mOsm/kg was 0.661 +/- 0.019 mug/10(6) cells/h, which is almost identical to that of immobilized cells exposed to abrupt osmotic stress. Accordingly, the r(MAb) was increased by ca. 40% when compared with that in the control immobilized cell culture. This enhancement in i(MAb) of immobilized S3H5/gamma2bA2 hybridoma cells by applying gradual osmotic stress suggests the potential of using hyperosmolar medium in other perfusion culture systems for improved MAb production. (c) 1995 John Wiley & Sons, Inc.     

An unstructured kinetic model of macromolecular metabolism in batch and fed-batch cultures of hybridoma cells producing monoclonal antibody

Growth profiles of the batch and fed-batch culture of hybridoma cells producing monoclonal antibody were simulated using an unstructured model. The model describes the production of cellular macromolecules and monoclonal antibody, the metabolism of glucose and glutamine with the production of lactate and ammonia, and the profiles of cell growth in batch and fed-batch culture. Equations describing the cells arrested in G1 phase [T.I. Linardos, N. Kalogerakis, L.A. Behie, Biotechnol. Bioeng. 40 (1992) 359–368; E. Suzuki, D.F. Ollis, Biotechnol. Bioeng. 34 (1989) 1398–1402] were included in this model to describe the increase of the specific antibody productivity in the near-zero specific growth rate, which was observed in the recent experiments in fed-batch cultures of this study and the semi-continuous culture of hybridoma cells [S. Reuveny, D. Velez, L. Miller, J.D. Macmillan, J. Immnol. Methods 86 (1986) 61–69]. This model predicted the increase of specific antibody production rate and the decline of the specific production rate of cellular macromolecules such as DNA, RNA, protein, and polysaccharide in the late exponential and decline phase of batch culture and at lower specific growth rates in the fed-batch culture.     

Immobilization can improve the stability of hybridoma antibody productivity in serum-free media

Hybridoma cells (S3H5/gamma2bA2) were cultivated in spinner flasks with 1% serum media and serum-free media. Monoclonal antibody productivity was maintained in 1% serum media. However, cells in serum-free media showed a decrease in antibody productivity, and it completely disappeared in IMDM-based low protein medium. This loss of antibody productivity was not observed when the cells were immobilized in alginate beads. In fact, immobilization enhanced the specific MAb productivity.     

Effects of ammonia and lactate on growth, metabolism, and productivity of BHK cells

The aim of the present work was to study the effect of ammonia and lactate on growth, metabolism, and productivity of BHK cells producing a recombinant fusion protein. Results show that cell growth was reduced with the increase in ammonia or lactate: k(1/2) of 1.1 mM and 3.5 mM for stirred and stationary cultures, respectively, for ammonia and of 28 mM for both stationary and stirred cultures for lactate, were obtained. The cell-specific consumption rates of both glucose (q(Glc)) and glutamine (q(Gln)) increased, whereas that of oxygen (q(O2)) decreased, with the increase in ammonia or lactate concentrations. The cell-specific production rates of lactate (q(Lac)) increased with an increase in ammonia concentration; similarly for the cell-specific production rates of ammonia (q(Amm)), which also increased with an increase in lactate concentration; on the other hand, both q(Lac) and q(Amm) markedly decreased when lactate or ammonia concentrations were increased, respectively; lactate was consumed at lactate concentrations above 30 mM and ammonia was consumed at ammonia concentrations above 5 mM. In vivo (31)P NMR experiments showed that ammonia and lactate affect the intracellular pH, leading to intracellular acidification, and decrease the content in phosphomonoesters, whereas the cell energy state was maintained. The effect of lactate on cell growth and q(Gln) is partially due to osmolarity, on q(Glc) and q(Amm) is entirely due to osmolarity, but on q(Lac) is mainly due to lactate effect per se. An increase in ammonia from 0 to 20 mM induced a 50% reduction in specific productivity, whereas an increase in lactate from 0 to 60 mM induced a 40% decrease.     

Reduction of ammonia and lactate through the coupling of glutamine synthetase selection and downregulation of lactate dehydrogenase-A in CHO cells

Chinese hamster ovary (CHO) cell cultivation for production of therapeutic proteins is accompanied by production of metabolic wastes, mostly ammonia and lactate. To reduce ammonia production, the glutamine synthetase (GS) system was used to develop therapeutic monoclonal antibody (mAb)-producing CHO cells (SM-0.025). Additionally, the lactate dehydrogenase-A (LDH-A) was downregulated with shRNA to reduce lactate production in SM-0.025. The resulting mAb-producing cell lines (#2, #46, and #52) produced less ammonia than the host cell line during the exponential phase due to GS protein overexpression. LDH-A downregulation in SM-0.025 not only reduced lactate production but also further reduced ammonia production. Among the three LDH-A-downregulated clones, clone #2 had the highest mAb production along with significantly reduced specific lactate and ammonia production rates compared to those in SM-0.025. Waste reduction increased the galactosylation level of N-glycosylation, which improved mAb quality. LDH-A downregulation was also successfully applied to the host cell lines (CHO K1 and GS knockout CHO-K1). However, LDH-A downregulated host cells could not survive the pool-selection process wherein glutamine was excluded and methionine sulfoximine was added to the media. Taken together, LDH-A downregulation in the mAb-producing cell line generated with the GS system successfully reduced both ammonia and lactate levels, improving mAb galactosylation. However, LDH-A downregulation could not be applied to host cell lines because it hampered the selection process of the GS system.

     

Examination of serum and bovine serum albumin as shear protective agents in agitated cultures of hybridoma cells.

A murine hybridoma cell line (167.4G5.3) was cultured in batch mode using IMDM containing different serum concentrations and bovine serum albumin (BSA). Cell growth and death, metabolism and antibody production were studied in these cultures. The cells were more susceptible to shear in the stationary and in the decline phase of growth as evidenced by higher death rates. Cell growth was best at high serum concentrations with high specific growth and low specific death rates. When BSA was used instead of serum in IMDM, no protective effect was observed. Cell metabolism and monoclonal antibody production rates were not influenced by the level of serum or by BSA. The use of serum in commercial serum-free media (OPTI-MEM) also resulted in no change in both growth and death rates.     

Production of monoclonal antibody using free-suspended and immobilized hybridoma cells: Effect of serum

The effect of serum on cell growth and monoclonal antibody (MAb) productivity was studied in a repeated fedbatch mode using both free-suspended and immobilized S3H5/gamma2bA2 hybridoma cells. In the suspension culture, serum influenced the cell growth rate but not the specific MAb productivity. The average specific growth rate of the suspension culture in medium containing 10% serum was approximately 0.99 +/- 0.12 day(-1) (+/-standard deviation), while that in medium containing 1% serum was approximately 0.73 +/- 0.12 day(-1). The specific MAb productivity was almost constant at 3.69 +/- 0.57 mug/10(6) cells/day irrespective of serum concentration reached a maximum at ca. 1.8 x 10(6) cells/mL of medium in 10% serum medium, and the cell concentration was gradually reduced to 1%. The specific MAb productivity of the immobilized cells was more than three times higher than that of the free-suspended cells. The amount of serum in the medium did not influence the specific MAb production rate of the immobilized cells. The maintenance of high cell concentration and the enhanced specific MAb productivity of the immobilized cell culture resulted in a higher volumetric MAb productivity. In addition, MAb yield in the immobilized cell culture with medium containing 1% serum was 2.2 mg/mL of serum, which was approximately three times higher than that in the suspension culture.     

A mathematical model of the cell cycle of a hybridoma cell line

A one-dimensional age-based population balance model of the cell cycle is proposed for a mouse-mouse hybridoma cell line (mm321) producing immunoglobulin G antibody to paraquat. It includes the four conventional cell cycle phases, however, G1 is divided into two parts (G1a and G1b). Two additional phases have been added, a non-cycling state G1', and a pre-death phase D. The duration of these additional phases is determined by cumulative glutamine content and ammonia concentration, respectively. It is assumed that glutamine is only consumed during G1 and antibody is only produced during G1b and S, the kinetics are assumed to be zero-order. Glucose is consumed throughout the cell cycle at a rate that is dependent upon its prevalent concentration. Ammonia and lactate are produced in direct proportion to glutamine and glucose consumption, respectively. Parameters in the model have been determined from experimental data or from fitting the model to post-synchronisation data. The model thus fitted has been used to successfully predict this cell lines behaviour in conventional batch culture at different initial glutamine concentrations, and in chemostat culture at steady-state and in response to a glutamine pulse. The model predicts viable cell, glutamine, glucose and lactate kinetics well, but there are some discrepancies in the prediction for ammonia and antibody. Overall, the results obtained support the assumptions made in the model relating to the regulation of cell cycle progression. It is concluded that this approach has the potential to be exploited with other cell lines and used in a model-based control scheme.     

Hybridoma growth and antibody production as a function of cell density and specific growth rate in perfusion culture

Steady state metabolic parameters for hybridoma cell line H22 were determined over a wide range of cell densities and specific growth rates in a filtration based homogeneous perfusion reactor. Operating the reactor at perfusion rates of 0.75, 2.0, and 2.9 day(-1)(each at four different specific growth rates), viable cell densities as high as 2 x 10(7) cells/mL were obtained. For the cell line under investigation, the specific monoclonal antibody production rate was found to be a strong function of the viable cell density, increasing with increasing cell density. In contrast, most of the substrate consumption and product formation rates were strong functions of the specific growth rate. Substrate metabolism became more efficient at high cell densities and low specific growth rates. The Specific rates of metabolite formation and the apparent yields of lactate from glucose and ammonia from glutamine decreased at low specific growth rates and high cell densities. While the specific oxygen consumption rate was independent of the specific growth rate and cell density, ATP production was more oxidative at lower specific growth rate and higher cell density. These observed shifts are strong indications of the production potential of high-density perfusion culture. (c) 1995 John Wiley & Sons, Inc.     

Application of hypoosmolar medium to fed-batch culture of hybridoma cells for improvement of culture longevity

Cell culture longevity in fed-batch culture of hybridomas is often limited by elevated medium osmolality caused by repeated nutrient feeding. Shotwise feeding of 10x Dulbecco's modified Eagle's medium (DMEM) concentrates elevated the osmolality of medium up to 540 mOsm/kg at the end of fed-batch culture of S3H5/gamma2bA2 hybridoma which is known to be lethal to most hybridomas. S3H5/gamma2bA2 hybridoma has been shown to grow without significant growth depression at 219 mOsm/kg in DMEM supplemented with 10% fetal bovine serum. To improve culture longevity in fed-batch cultures of S3H5/gamma2bA2 hybridoma, a hypoosmolar medium (223 mOsm/kg) was used as an initial basal medium. The use of hypoosmolar medium delayed the onset of severe cell death resulting from elevated osmolality and allowed one more addition of 10x DMEM concentrates to the culture. As a result, a final antibody concentration obtained was 121.5 microg/mL which is approximately 1.5-fold higher compared to fed-batch culture using a standard medium (335 mOsm/kg). When compared to batch culture, a more than 5-fold increase in the final antibody concentration was achieved. Taken together, the use of hypoosmolar medium as an initial medium in fed-batch culture improved culture longevity of S3H5/gamma2bA2 hybridoma, resulting in a substantial increase in the final antibody concentration.     

Effect of anchorage dependency on growth rate and monoclonal antibody production of hybridoma cells

Hybridoma cells (S3H5/2bA2) are found to grow either in suspension or as attached to the surface of cell culture T flask. Cell growth rates and monoclonal antibody (MAB) production rates of both suspended and attached cells were examined. Although the percentage of viable cells was higher for the attached cells, cells growing in suspension showed almost the same charateristics as cells attached to the flasks with respect to cell growth and MAB production rate. Cell attachment increased with increasing serum concentrations up to 5% and remained essentially constant at cell densities of about 2·10⁵/cm².No differences in cell growth rate and MAB production could be attributed to anchorage dependent growth.     

Transient responses of hybridoma cells to lactate and ammonia pulse and step changes in continuous culture

Ammonia and lactate are the major byproducts from mammalian cells grown in medium containing glutamine and glucose. Both can be toxic to cells, and may limit the productivity of commercial bioreactors. The transient and steady-state responses of hybridoma growth and metabolism to lactate and ammonia pulse and step changes in continuous suspension culture have been examined. No inhibition was observed at 40 mM lactate. Cell growth was inhibited by 5 mM ammonia, but the cells were able to adapt to ammonia concentrations as high as 8.2 mM. Ammonia production decreased and alanine production increased in response to higher ammonia concentrations. Increased ammonia concentrations also inhibited glutamine and oxygen consumption. The specific oxygen consumption rate decreased by an order of magnitude after an ammonia pulse to 18 mM. Under these conditions, over 90% of the estimated ATP production was due to glycolysis and a large fraction of glutamine was converted to lactate.     

The effects of microcarrier culture on recombinant CHO cells under biphasic hypothermic culture conditions

A Chinese hamster ovary (CHO) cell line, producing recombinant secreted human placental alkaline phosphatase (SEAP) was investigated under three different culture conditions (suspension cells, cells attached to Cytodex 3 and Cytopore 1 microcarriers) in a biphasic culture mode using a temperature shift to mild hypothermic conditions (33 °C) in a fed-batch bioreactor. The cell viability in both the suspension and the Cytodex 3 cultures was maintained for significantly longer periods under hypothermic conditions than in the single-temperature cultures, leading to higher integrated viable cell densities. For all culture conditions, the specific productivity of SEAP increased after the temperature reduction; the specific productivities of the microcarrier cultures increased approximately threefold while the specific productivity of the suspension culture increased nearly eightfold. The glucose and glutamine consumption rates and lactate and ammonia production rates were significantly lowered after the temperature reduction, as were the yields of lactate from glucose. However, the yield of ammonia from glutamine increased in response to the temperature shift.