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.     

Enhancement effects of BSA and linoleic acid on hybridoma cell growth and antibody production

The effects of linoleic acid and bovine serum albumin on hybridoma cell growth and antibody production were investigated. In dish cultivation, linoleic acid on its own promoted cell growth when used at concentrations below 50 mg L^–1, but strongly inhibited growth at a concentration of 100 mg L^–1 on more. However, linoleic acid bound to bovine serum albumin did not inhibit cell growth, even at a concentration as high as 100 mg L^–1. Also, linoleic acid did not affect the specific antibody production rate, with or without bovine serum albumin. In order to elucidate the enhancement of antibody production by bovine serum albumin, fractions were prepared by ultrafiltration (98% molecular weight cut-offs, 50,000 and 17,000) and the effects of the fractionation on antibody production were studied in batch cultivation. The high-molecular-weight fraction (50,000) promoted antibody production whereas the low-molecular-weight fraction (17,000) inhibited it. In continuous cultivation, the high-molecular-weight fraction was also found to enhance antibody production.     

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.     

A study of hybridoma cell growth and antibody production kinetics in continuous culture

Summary A detailed study of cell growth and antibody production kinetics in continuous culture found that the specific rate of antibody production reached a maximum saturated profile at a specific growth rate less than the maximum. This observation is novel and of importance in the understanding of the mechanism of antibody production and/or antibody transport.     

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.     

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.     

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.     

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.     

Influence of amino acids on hybridoma cell viability and antibody secretion

It is generally accepted that the phase of cell decline observed in batch culture of mammalian cells is related to exhaustion of medium nutrients (principally glucose and glutamine) and/or to waste products accumulation. In the present paper, we have studied the influence of glutamine on the proliferation of mouse hybridoma cells. We showed that repeated addition of glutamine prolonged the life span of the culture and significantly increased the secretion of monoclonal antibody. Flow cytometry analysis suggests that this effect of glutamine is related to a delay in cell death rather than to a stimulation of proliferation.Addition of glutamine and glucose failed however to prevent the death of the culture. Determinations of amino acid consumption in glutamine-supplemented samples and experiments carried out with complementary sources of amino acids (e.g. tryptose phosphate) strongly suggest that amino acid supply is a critical factor governing cell growth and productivity.     

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%.     

Observations consistent with autocrine stimulation of hybridoma cell growth and implications for large-scale antibody production

Existence of autocrine growth factors (aGFs) may influence the serum requirement for growth of hybridoma cells and thus significantly influence process economics. For the murine hybridoma cell line S3H5/2bA2, critical inoculum density (cID) and serum requirement for growth were inversely related for cultivation in both T flasks and spinner flasks. In spinner flasks, an inoculum density of 10⁶ cells/ml was necessary for the cells to grow in RPMI 1640 medium without serum supplement, and an inoculum density of 10³ cell/ml was necessary in RPMI 1640 medium with 10% serum. In T flasks, where the local cell density is higher than in spinner flasks, an inoculum density of 10⁶ cells/ml was necessary for the cells to grow in RPMI 1640 medium without serum supplement, and an inoculum density of 1 cell/ml was also necessary in RPMI 1640 medium with 10% serum. Further, immobilized cells at high local cell density could grow under conditions where cells in T flasks at corresponding overall cell density could not grow. The cells at high inoculum density were less sensitive to shear induced by mechanical agitation than the cells at low inoculum density. Taken together these observations support the existence of secreted aGF(s) by the hybridoma cell line used. Since the specific MAb production rate was independent of cultivation method and inoculum density, the existence of autocrine growth factors would suggest that the use of immobilized cells should improve the economics of MAb production.     

Glutamine-limited batch hybridoma growth and antibody production: experiment and model

The influence of glutamine (a major energy source) on both hybridoma growth and monoclonal antibody production was examined. A series of batch experiments were performed in T-flasks containing initial glutamine levels ranging from 0.5 to 4.0 mM in RPMI 1640 with 20% v/v fetal calf serum. The maximum final cell concentration increased with initial glutamine levels in the range of 0.5-2 mM; further glutamine increases had little or no effect. Earlier studies in our laboratories demonstrated that serum component(s) strongly influence the maximum specific growth rate. Here, the present studies reveal also the stoichiometric limitation by glutamine in the later stages of growth when its concentration is drastically reduced. For 0.5 to 1.5 mM initial glutamine, complete substrate utilization coincided with the cessation of cell growth and the onset of the death phase. For initial glutamine concentrations higher than 2.0 mM, growth halted prior to glutamine exhaustion, presumably because serum or RPMI component(s) were exhausted. The specific antibody secretion rate was essentially non-growth-associated above a critical low glutamine concentration in both the growth and death phases. At or below this critical value, an apparent emergence of stoichiometnc or energy limitation resulted in a dramatic drop in the secretion rate to zero. A simple unstructured model was developed that simulates these trends well. All parameters were determined using only subsets of the data. Nevertheless, these parameter values provided simulations in good agreement with all the glutamine-limited cultures.     

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.     

Antibody production and growth of mouse hybridoma cells in Nutridoma media supplements

Traditionally, cell culturists have relied upon the addition of serum to culture medium for the growth and maintenance of cell lines. However, many aspects of the use of serum in tissue culture are problematic. Cell culture supplements that circumvent the need for serum are readily available and provide a consistent protein composition. This defined environment allows the antibody to be more easily purified from culture supernatants. Nutridoma media supplements were formulated to support the growth of lymphoblastoid cells in a defined culture environment. In this study, Nutridoma media supplements were tested in parallel with serum-containing cultures to determine if Nutridoma supplemented medium is effective in supporting hybridoma cell growth and antibody production in three hybridoma cell lines. Data, based on cell growth and antibody production, show the importance of basal media selection when serum is replaced with Nutridoma media supplements. SDS-PAGE results show that cell supernatants from Nutridoma supplemented cultures contain very few contaminating proteins.     

Influence of dissolved oxygen concentration on growth,mitochondrial function and antibody production of hybridoma cells in batch culture

The effects of dissolved oxygen concentration on hybridoma cell growth, metabolism, and antibody production were studied. A mouse hybridoma cell line producing an IgG1 directed at a consensus -interferon was grown in batch cultures in a 5 dm³ stirred bioreactor at dissolved oxygen (DO) concentrations of 5, 30, 90 and 95% or air saturation. High oxygen tension (95% of air saturation) reduced specific growth rate without affecting cell viability. At lower dissolved oxygen levels, specific growth rates were approximately independent of DO, although changes in mitochondrial function and antibody production were observed. Flow cytometry assays of a fluorescent mitochondria-specific marker (Rhodamine 123) show significant single-cell heterogeneities during late exponential growth and greater average fluorescence in cultures grown at 95% DO. The quantity of cell-surface immunoglobulin, measured by an immunofluorescent flow cytometric technique, was the same at high (95%) and low (5%) dissolved oxygen concentrations. Myeloma cells of the type used in constructing the above hybridoma line were much less sensitive to dissolved oxygen level. Specific respiration rate, pyruvate utilization rate, cytochrome oxidase activity, and succinate-cytochrome c oxidoreductase activity were significantly greater (62–116%) for the hybridoma cells than for the myeloma cells in T-flask cultures.     

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.     

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.