The influence of cyclic nucleotides on hybridoma growth and monoclonal antibody production

Summary The influence of cyclic AMP and cyclic GMP, known regulatory mediators of cellular response, on hybridoma growth and monoclonal antibody production is studied. The cGMP-treated cells exhibited 41% higher specific antibody secretion rate, resulting in 52% higher antibody yields. Addition of 1 mM cAMP inhibited cellular growth but enhanced the specific production rate by 37%.     

Media for hybridoma growth and monoclonal antibody production

For the economical production of monoclonal antibodies (MAbs), the cell-culture medium must be optimized for three different phases: growth of the hybridomas, MAb productivity of the hybridomas, and MAb purification or downstream processing. Medium improvements are necessary to meet these requirements for large-scale MAb production. Information bearing on this issue is being addressed in two research areas, cell biology and biochemical engineering, and is reviewed in this article.     

Relationship between hybridoma growth and monoclonal antibody production.

Factors affecting cell growth and antibody production in a mouse hybridoma were investigated. Antibody was produced during the growth and decline phases of a batch culture with an increase in the specific rate of antibody production during the decline phase. The specific rate of antibody production was also increased in cells arrested by 2 mM thymidine, suggesting that cell proliferation and antibody production can be uncoupled. Reduced serum concentrations resulted in lower cell growth rates but increased antibody production rates. However, this trend was reversed in hybridomas which had been arrested by thymidine, since the highest antibody production rate was associated with high serum concentrations. Likewise, in proliferating cells, the optimum pH for antibody production (pH 6.8) was lower than the optimum pH for cell growth (pH 7.2), whereas in thymidine-blocked cells, the highest antibody production rate was at pH 7.2. High antibody production rates and product yields were also associated with low growth rates in continuous cultures. The possibility that antibody was under cell cycle control was investigated in synchronized hybridoma cultures. Antibody production occurred during G1 and G2 with a decline in the M phase and evidence of a further decline in the S phase. Thus antibody production was not restricted to the G1 and S phase in this hybridoma.     

A flow-cytometric analysis of hybridoma growth and monoclonal antibody production

A flow cytometric kinetic study of hybidoma growth and monoclonal antibody production is presented, along with the influence of glutamine on intracellular responses such as (relative) cell size, and cell RNA and total protein content. Specific findings are: (1) RNA content remained constant throughout the growth phase, then fell drastically as the cells entered the stationary phase. Also, in stationary phase, RNA content of antibody-producing cells was higher than for those not secreting antibody. (2) The cell size was constant and maximal throughout exponential phase, and diminished monotonically during later stages. (3) Average protein and antibody cellular content declined dramatically upon glutamine exhaustion. Thus, relative RNA levels and cell size provided quantitative determinants of both cell growth state and antibody secretion conditions. These results encourge consideration of structured kinetic studies which recognize the quality of the biophase.     

Effect of temperature on nucleotide pools and monoclonal antibody production in a mouse hybridoma

The specific monoclonal antibody productivity (q(Mab)) of a murine hybridoma (CC9C10) increased with incubation temperature in the range 33 degrees C to 39 degrees C. q(Mab) was constant at each temperature and was independent of the phase of culture. The q(Mab) increased 97% at 39 degrees C and decreased by 21% at 33 degrees C compared with controls at 37 degrees C. Specific rates of substrate (glucose and glutamine) utilization and byproduct (lactate and ammonia) formation also increased with temperature but the yield coefficient, Y(Lac/Llc') was constant for 33 degrees C to 39 degrees C and Y(Amm/Gin) was constant for 37 degrees C to 39 degrees C. Y(Amm/Gin) at 33 degrees C was lower than the control. Changes in specific nucleotide concentrations and ratios were monitored by analysis of intracellular nucleotide pools. The NTP ratio, (ATP + GTP)/(UTP + CTP), increased and the U-ratio (UTP/UDP-GNac) decreased during the course of each culture, whereas the adenylate energy charge, (ATP + 0.5ADP)/(ATP + ADP + AMP), remained relatively constant at a value 0.8. The relative content of UDP-/N acetyl galactosamine, UDP-N acetyl glucosamine, and NAD increased with incubation temperature, whereas the relative ATP content, SA(ATP + ADP + AMP)/SU (UTP + UDP-sugars) ratio, purine/pyrimidine, ATP/GTP, and U-ratio decreased at higher incubation temperatures. It is possible that these nucleotide parameters may have a regulatory role in the changes of q(Mab) observed at the higher temperatures. (c) 1994 John Wiley & Sons, Inc.     

The role of vitamins and amino acids on hybridoma growth and monoclonal antibody production

A balanced supplementation method was applied to develop a serum and protein- free medium supporting hybridoma cell batch culture. The aim was to improve systematically the initial formulation of the medium to prevent limitations due to unbalanced concentrations of vitamins and amino acids. In a first step, supplementation of the basal formulation with 13 amino acids, led to an increase of the specific IgA production rate from 0.60 to 1.07 pg cell⁻¹ h⁻¹. The specific growth rate remained unchanged, but the supplementation enabled maintenance of high cell viability during the stationary phase of batch cultures for some 70 h. Since IgA production was not growth- related, this resulted in an approximately4-fold increase in the final IgA concentration, from 26.6 to 100.2 mgl⁻¹. In a second step, the liposoluble vitamins E and K₃ were added to the medium formulation. Although this induced a slightly higher maximal cell concentration, it was followed by a sharp decline phase with the specific IgA production rate falling to 0.47 pg cell⁻¹ h⁻¹. However, by applying a second cycle of balanced supplementation with amino acids this decline phase could be reduced and a high cell viability maintained for over 300 h of culture. In this vitamin- and amino acid- supplemented medium, the specific IgA production rate reached a value of 1.10 pg cell⁻¹h⁻¹ with a final IgA concentration of 129.8 mgl⁻¹. The latter represents an increase of approximately5-fold compared to the non- supplemented basal medium. This revised version was published online in August 2006 with corrections to the Cover Date.     

Continuous hybridoma growth and monoclonal antibody production in hollow fiber reactors-separators

Growth of a hybridoma culture, along with production of monoclonal antibody, was demonstrated over extended periods in polysulfone hollow fiber membrane modules. The molecular weight cutoffs of the membranes were 70,000, 50,000, and 100,000 daltons. The hybridoma cell line, designated 65/26, produced IgG (2b/kappa) directed at mouse thymus cell surface antigen, TL.1. Cell growth occurred in the shell space of the reactor, using supplemented RPMI 1640 (20% fetal bovine serum) supplied from a separate reservoir vessel through the hollow fiber lumen. The reservoir contained 125 mL media, which was changed every 4 days. Concentrations of immunoglobulin were determined by an enzyme immunoassay (using protein A and alkaline phosphatase-labeled antibody conjugate). For the 10K, 50K, and 100K hollow fiber membrane modules, the maximum IgG concentrations detected in the 2.5-mL shell space were 47.5-80, 510, and 740 mug/mL, respectively. In the 125-mL reservoir for the 100K hollow fiber membrane module, the IgG concentration was measured at 260 mug/mL These values compare with an IgG concentration of 1 mug/mL when grown in a standard tissue culture flask and 3.2-7.6 mug/mL when grown in 100 ml media in a spinner flask. In addition, 10K and 50K hollow fiber membrane modules were run in a mode that decreased the fetal bovine serum supplement with time. Differences between these systems suggest that it is possible to obtain high IgG accumulation rates, both during and after the exponential growth phase of the hybridoma population.     

Transient kinetics of hybridoma growth and monoclonal antibody production in serum-limited cultures

A quantitative study of the influence of initial serum concentration on hybridoma growth rate, maximum viable and total cell yield, and specific antibody production rate is presented. The specific growth rate varied in a Monod fashion with initial serum levels (2-10% FCS), giving K(m) = 1.6 v/v% and mu(max) = 0.92 d(-1). The maximum cell yields (total and viable) were linear with initial serum level, indicating stoichiometric as well as kinetic limitation by serum component(s). The specific antibody production rate for each individual run fitted well to a non-growth-associated model. However, the non-growth-associated parameter varied monotonically with initial serum concentration, suggesting the catalytic role of serum component(s) in antibody production. Also, glutamine was utilized inefficiently, with at least a third of it secreted back into the culture supernate in the form of glutamate. While very simple model equations describe the specific growth and product formation rate for an individual batch run, the larger picture indicates need for a more detailed unstructured or structured model.     

Kinetic analysis of hybridoma growth and monoclonal antibody production in semicontinuous culture

Hybridoma I.13.17 was grown in semicontinuous culture in an attempt to investigate the steady-state concentrations of key components of monoclonal antibody (MAb) synthesis (e.g., intracellular MAb, IgG messenger RNAs) at different dilution rates between 0.008 and 0.055 h(-1). There was a general trend of increasing steady-state levels of total cytoplasmic RNA, total cell-associated MAb or cytoplasmic MAb, DNA synthesis rate, cellular metabolic activity, heavy (H-) and light (L-) chain IgG mRNAs with the increase in dilution rates. Increase in the half-lives of H- and L-chain mRNAs with increase in dilution rates may be sufficient to account for their increasing levels found under the same conditions. The specific growth rate was profoundly affected by the dilution rate, particularly near the lower end of the dilution rate range. Linear relationships were observed between the steady-state amounts of total cell-associated MAb and the relative levels of H- and L-chain mRNAs. Material balances on intracellular MAb demonstrated an increasing percentage of antibody not released into the growth medium (e.g., stored within the cell or anchored to the cell membrane) with increasing dilution rate. The MAb production rate per cell decreased significantly with the increase in dilution rates. No correlation was found between the relative levels of H- or L-chain mRNAs and the specific MAb production rate. Possible implications of rate-limiting steps in MAb synthesis and secretion are discussed.     

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.     

Effects of peptone on hybridoma growth and monoclonal antibody formation

Hybridoma WuT3 secreting a monoclonal antibody against T lymphocytes was grown in RPMI 1640 medium supplemented with 1% human serum. The effect of the concentration of peptone, as an additive, was investigated on cell growth, monoclonal antibody formation, and cell metabolism over 0–10 g l^–1 range. It was found that 1–5 g l^–1 peptone can significantly promote the growth of cells and increase the formation of monoclonal antibody, especially at 3–5 g l^–1, when both the accumulating level and secretion rate of monoclonal antibody are higher than that at other peptone concentrations. Based on glucose, lactate and ammonia analysis data, the efficiency of glycolysis was assessed and the utilization of amino acids was more efficient at 3–5 g l^–1 peptone. The cell growth and monoclonal antibody formation were inhibited at higher peptone concentrations, e.g. 10 g l^–1.     

Effects of growth factors on cell proliferation and monoclonal antibody production of batch hybridoma cultures

Summary Effects of growth factors such as EGF, FGF and IL-2 on cell proliferation and monoclonal antibody production in a hybridoma cell line adapted to a completely defined serum-free medium were determined in batch cultures. The results indicate that the presence of growth factors in the medium enhances the antibody secretion without significantly affecting the growth rate. The specific antibody secretion rate of cells grown in serum-free medium supplemented with growth factors was 35% higher than those grown in serum-free medium alone.     

Serum-free media in hybridoma culture and monoclonal antibody production

The replacement of serum in hybridoma cultures is considered. The focus is on the effects of serum-free media on hybridoma growth and monoclonal antibody secretion. Comparative literature data with serum supplemented cultures are discussed with an analysis of serum-free formulations and selection rules for the serum-free ingredients. In general, serum-free media which are "lipid rich" can sustain cell growth rates approaching that of serum supplemented cultures. Specific antibody secretion rate, however, is usually higher in serum-free media, irrespective of the lipid content.     

Effect of initial cell density on hybridoma growth, metabolism, and monoclonal antibody production.

A murine hybridoma cell line (167.4G5.3) was cultivated in batch mode with varying inoculum cell densities using IMDM media of varying fetal bovine serum concentrations. It was observed that maximum cell concentrations as well as the amount of monoclonal antibody attainable in batch mode were dependent on the inoculum size. Specifically, cultures with lower inoculum size resulted in lower cell yield and lower antibody concentrations. However, in the range of 10(2) to 10(5) cells per ml, the initial cell density affected the initial growth rate by a factor of only 20%. Furthermore, specific monoclonal antibody production rates were independent of initial cell density and the serum concentration. Glutamine was the limiting nutrient for all the cultures, determining the extent of growth and the amount of antibody produced. Serum was essential for cell growth and cultures with initial cell concentrations up to 10(6) cells per ml could not grow without serum. However, when adapted, the cells could grow in a custom-made serum-free medium containing insulin, transferrin, ethanolamine, and selenium (ITES) supplements. The cells adapted to the ITES medium could grow with an initial growth rate slightly higher than in 1.25% serum and the growth rate showed an initial density dependency-inocula at 10(3) cells per ml grew 30% slower than those at 10(4) or 10(5). This difference in growth rate was decreased to 10% with the addition of conditioned ITES medium. The addition of conditioned media, however, did not improve the cell growth for serum-containing batches.     

Rapamycin reduces hybridoma cell death and enhances monoclonal antibody production

Rapamycin was used as a medium additive to slow the progression of CRL 1606 hybridomas through the cell cycle, under the hypothesis that such a modulation might reduce cell death. Cell cycle distributions for CRL hybridomas in the G1 phase of the cell cycle ranged from 20% to 35% during batch, fed-batch, and continuous culture experiments, independent of culture time, dilution rate, growth rates, or death rates. Rapamycin, an mTOR signaling inhibitor, immunosuppressant, and G1-phase arresting agent, was identified and tested for efficacy in restraining cell cycle progression in CRL 1606 hybridoma cultures. However, in the presence of 100 nM rapamycin, the percentage of cells in the G1 phase of the cell cycle during fed-batch cultures was only increased from 28% to 31% in control cultures to 37% to 48% for those with rapamycin. Accordingly, rapamycin only slightly reduced culture growth rate. Instead, the use of rapamycin more notably kept viability higher than that of control cultures by delaying cell death for 48 h, thereby enabling viable proliferation to higher maximum viable cell densities. Furthermore, rapamycin enhanced specific monoclonal antibody production by up to 100% during high-viability growth. Thus, over the course of 6-day fed-batch cultivations, the beneficial effects of rapamycin on viable cell density and specific productivity resulted in an increase in final monoclonal antibody titer from 0.25 to 0.56 g/L (124%). As rapamycin is reported to influence a much broader range of cellular functions than cell cycle alone, these findings are more illustrative of the influence that signal transduction pathways related to mTOR can have on overall cell physiology and culture productivity.     

Effects of CO2 and osmolality on hybridoma cells: growth, metabolism and monoclonal antibody production

CO2 partial pressure (pCO2) in industrial cell culture reactors may reach 150–200 mm Hg, which can significantly inhibit cell growth and recombinant protein production. Due to equilibrium with bicarbonate, increased pCO2 at constant pH results in a proportional increase in osmolality. Hybridoma AB2-143.2 cell growth rate decreased with increasing pCO2 in well-plate culture, with a 45% decrease at 195 mm Hg with partial osmolality compensation (to 361 mOsm kg- 1). Inhibition was more extensive without osmolality compensation, with a 63% decrease in growth rate at 195 mm Hg and 415 mOsm kg-1. Also, the hybridoma death rate increased with increasing pCO2, with 31- and 64-fold increases at 250 mm Hg pCO2 for 401 and 469 mOsm kg- 1, respectively. The specific glucose consumption and lactate production rates were 40–50% lower at 140 mm Hg pCO2. However, there was little further inhibition of glycolysis at higher pCO2. The specific antibody production rate was not significantly affected by pCO2 or osmolality within the range tested. Hybridomas were also exposed to elevated pCO2 in continuous culture. The viable cell density decreased by 25–40% at 140 mm Hg. In contrast to the well-plate cultures, the death rate was lower at the new steady state at 140 mm Hg. This was probably due to higher residual nutrient and lower byproduct levels at the lower cell density (at the same dilution rate), and was associated with increased cell-specific glucose and oxygen uptake. Thus, the apparent effects of pCO2 may vary with the culture system. VMdZ and RK contributed equally to the results in this article. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mathematical modeling and analysis of monoclonal antibody production by hybridoma cells

An attempt has been made to mathematically describe and analyze monoclonal antibody (MAB) productivity of hybridoma cells, with particular emphasis on continuous cultures under unsteady-state conditions. A simple and unstructured general kinetic model that takes account of productivity loss during long-term cultivation, cell proliferation, and the effects of nutrients and toxic products is proposed. The model is verified with data of continuous culture from five different cell lines under a wide range of experimental conditions. Analysis of these results showed that for a reliable assessment of effects of different factors and for comparison of kinetic data on MAB production it is important to consider possible loss of MAB productivity, the time dependence of which can be modeled by an exponential function plus a constant term. Variations of nutrient concentration, particularly that of glucose, glutamine, and serum, can significantly alter MAB production under certain conditions. These effects can be described in terms of saturation kinetic and/or noncompetitive inhibition kinetics. (c) 1996 John Wiley & Sons, Inc.     

The influence of the chemical composition of cell culture material on the growth and antibody production of hybridoma cells

The multiplication and antibody production of murine hybridoma cells cultured on five different polymer membranes were tested and compared with conventional tissue culture polystyrene (TCPS). Membranes were prepared from polyacrylonitrile (PAN) and acrylonitrile copolymerized with N-vinylpyrrolidone (NVP20, NVP30), Na-methallylsulfonate (NaMAS) and N-(3-amino-propyl-methacrylamide-hydrochloride) (APMA). Cell number and antibody concentration were quantified as criteria for viability and productivity. Adhesion of hybridoma cells was characterized by vital and scanning electron microscopy. The results suggest that a strong adhesion of cells, observed on APMA and TCPS, increased cell growth but reduced monoclonal antibody production. In contrast membranes with lowered adhesivity such as NVP20 provided favourable conditions for monoclonal antibody production. In addition it was shown that this membrane also possessed a minor fouling as indicated by the low decrease of water flux across the membrane after protein adsorption. It was concluded that NVP20 could be a suitable material for the development of hollow fibre membranes for bioreactors.     

A three-phase pattern in growth, monoclonal antibody production, and metabolite exchange in a hybridoma bioreactor culture

The use of partial cubic spline data interpolation for the calculation of volumetric metabolite exchange rates suggested the existence of three distinct metabolic phases during bioreactor culture of a hybridoma cell line. During phase 1, a rapid amino acid uptake rate and ammonia release rate were observed. The growth rate was low and glutamine synthetase activity fell. In phase 2, maximum growth rate and minimum glutamine assimilation and ammonium production rates were observed. Attempts to corroborate the apparent ammonia assimilation in this phase using (15)NH(4)Cl resulted in low incorporation rates into alanine and glutamine. Maximum glutamine synthetase activity took place during this period. Maximum antibody production rate was observed during phase 3 during which peaks in glutamine assimilation, ammonia release, and glutamine synthetase activity were observed. The apparent existence of the three phases prompted us to carry out Northern blot analysis of glutamine synthetase RNA at appropriate times during the process. This revealed a pattern of appearance and dis-appearance of mRNA consistent with the three phases indicated by the fermentation parameters. (c) 1993 John Wiley & Sons, Inc.     

Enhanced monoclonal antibody production in hybridoma cells by LPS and Anti-mIgG

This paper reports on a methodology for increasing proliferation and monoclonal antibody (mAb) production in hybridoma cultures. The 55-6 murine B cell hybridoma line (CD40 and CD19-deficient expression) was treated with increasing concentrations of lipopolysaccharide (LPS). Expression of CD69, CD40, and CD19 surface antigens on 55-6 cells did not show significant changes from untreated cells. The specific growth rate decreased at higher concentrations of LPS, but the monoclonal antibody production rate was highest at the highest LPS concentration assayed. These data are in agreement with the lowest growth rate found at this concentration of LPS. Furthermore, cells were cultured with anti-mouse surface immunoglobulin G antibody (anti-mIgG) plus LPS to find out whether LPS-derived signals and anti-mIgG stimuli are synergistic. CD69, CD40, and CD19 expression was greater than for either untreated cells (control culture) or cells stimulated with LPS alone. Moreover, LPS stimulation in combination with anti-mIgG enhanced both the growth rate and IgG2a production over the control culture and cells stimulated with LPS alone. Maximum antibody concentration increased almost 500% compared to the control and about 100% with respect to culture stimulated with LPS alone. The maximum specific IgG2a production rate was about 300% higher than in the control culture and about 30% higher than in culture stimulated only with LPS.