Cell cycle kinetics and monoclonal antibody productivity of hybridoma cells during perfusion culture

The flow-cytometric (FCM) analysis of bivariate DNA/lgG distributions has been conducted to study the cell cycle kinetics and monoclonal antibody (MAb) production during perfusion culture of hybridoma cells. Three different perfusion rates were employed to demonstrate the dependency of MAb synthesis and secretion on cell cycle and growth rate. The results showed that, during the rapid growth period of perfusion culture, the level of intracellular igG contents of hybridoma cells changed significantly at each perfusion rate, while the DNA histograms showing cell cycle phases were almost constant. Meanwhile, during the reduced growth period of perfusion culture, the fraction of cells in the S phase decreased, and the fraction cells in the G1/G0 phase increased with decreasing growth rate. The fraction of cells in the G2/M phase was relatively constant during the whole period of perfusion culture. Positive correlation was found between mean intracellular IgG contents and the specific MAb production rate, suggesting that the deletion of intracellular IgG contents by a flow cytometer could be used as a good indicator for the prediction of changes in specific MAb productivity following manipulation of the culture condition. (c) 1994 John Wiley & Sons, Inc.     

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.     

The action of caffeine on X-irradiated HeLa cells. IX. Hypothermic effects

Hypothermic enhancement of the lethal effect of 3.5 Gy of 220-kV X rays in the absence of caffeine as well as in its presence (4 mM) was examined at temperatures between 10 and 34 degrees C in monolayer cultures in the G1 phase of the cell cycle. Correction has been made for the toxicity of low temperatures, and of caffeine at low temperatures, by concomitantly measuring cell killing in unirradiated cells. In the absence of caffeine, incubation of irradiated cells for up to 34 h at temperatures in the range 15 to 30 degrees C (or possibly 34 degrees C) enhances killing compared to that observed at 38 degrees C; the amount of enhancement is about the same throughout this range, but is nil at 10 degrees C. The enhanced killing induced by caffeine at 38 degrees C decreases as the temperature is lowered to 15 degrees C; there is no enhancement at 10 degrees C. Less killing is manifested in the range 15 to 25 degrees C in the presence of caffeine than in its absence. Recovery (loss of sensitivity to caffeine) and fixation of potentially lethal damage were studied in late-S/G2-phase cells at reduced temperatures by delaying treatment with caffeine for increasing times after irradiation. As the temperature is progressively lowered to 20 degrees C, less recovery is manifested after 5 h of incubation; no recovery is detected in the range 10 to 20 degrees C. Despite extensive recovery at 34 degrees C, no fixation is observed at that (or any lower) temperature in G2-phase cells: the cells are able to recover essentially fully when returned to 38 degrees C. In addition, responses of unirradiated control series to incubation at low temperatures appear to differ from those reported by others for longer treatment times of different cell systems.     

Growth and production kinetics of human x mouse and mouse hybridoma cells at reduced temperature and serum content.

The growth and production kinetics of a mouse hybridoma cell line and a human-mouse heterohybridoma were analyzed under conditions of reduced temperature and serum content. The mouse hybridoma P24 had a constant cell specific production rate and RNA content, while the heterohybridoma 3D6-LC4 showed growth associated production kinetics and an increased RNA content at higher growth rates. This behaviour of 3D6-LC4 cells can be explained by the unusual cell cycle kinetics of this line, which can be arrested in any phase under growth limiting conditions, so that a low growth rate does not result in a greater portion of high producing G1-phase cells. Substrate limitation changes the cell cycle distribution of this cell line to a greater extent than low temperature or serum content, which indicates that this stress factor exerts a greater physiological control than assumed.     

Cell-cycle-dependent protein accumulation by producer and nonproducer murine hybridoma cell lines: a population analysis

Single-cell rates of accumulation of cellular protein have been determined as a function of total protein content using flow cytometry and population balance equations for exponentially growing murine hybridoma cells in the individual G(1), S(1) and G(2) + M cell cycle phases. A novel flow cytometric technique for the identification of hybridoma cells in mitosis was developed and implemented. The data were obtained from a producer cell line which synthesizes and secretes high levels of monoclonal antibodies, and from a nonproducer clone which does not synthesize and secrete substantial amounts of antibody. The results indicate that the kinetics of single-cell protein accumulation in these two cell lines are considerably different. In particular, low protein content G(1) phase producer cells were characterized by a rate of protein accumulation which was approximately five times higher than the mean rate observed for higher protein content producer cells cycle phase. In contrast, the rate of accumulation of protein increased continuously with totalprotein content for the G(1) phase nonproducer cells. S phase hybridoma cells were characterized by a considerably lower rate of protein accumulation which did not vary much with protein content for either cell line. Finally, G(2) + M phase producer cells demonstrated a negative rate of protein accumulation which indicates that the rates of protein synthesis. It was hypothesized that these differences in total protein accumulation are caused by differences in monoclonal antibody accumulation. The distribution of rates suggests the need for a segregated approach to the modeling of the kinetics of antibody production in hybridomas.     

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.     

Spermatogenesis in Hydra oligactis. II. How temperature controls the reciprocity of sexual and asexual reproduction

Hydra oligactis undergo two mutually exclusive modes of reproduction: at warm temperatures (18-22 degrees C) animals reproduce asexually by budding, while at cold temperatures (10-12 degrees C) gamete differentiation occurs. Using a monoclonal antibody which is specific for cells of the sperm lineage, it was discovered that under conditions where sperm differentiation does not occur (18-22 degrees C), cells continually enter the sperm pathway but progression down the pathway is prematurely halted, effectively blocking the production of sperm. To elucidate the mechanism by which completion of sperm differentiation is controlled, the cell cycle times of interstitial cells entering the sperm pathway at both the restrictive (18 degrees C) and permissive (10 degrees C) temperatures were examined. It was envisaged that at the restrictive temperature the cell cycle times of committed cells would lengthen as they proceeded down the pathway, leading to dilution and eventual loss of cells at later stages of sperm differentiation. This did not occur. Although cells of the sperm lineage were found overall to divide more slowly at 18 degrees C than at 10 degrees C, at both temperatures the cell cycle times shortened as cells proceeded further down the pathway, making a dilution mechanism untenable. The effect of high temperature on the survival of cells was then tested by subjecting animals to a heat shock. Within 12 hr of the increase in temperature, the total number of sperm lineage interstitial cells dropped 10-fold while the total numbers of epithelial and somatic interstitial cells remained virtually unchanged. A distinct consequence of this cell loss was the disappearance of cells furthest down the sperm pathway. It is proposed that as cells move down the sperm pathway, they become increasingly sensitive to high temperature which adversely affects their survival; the higher the temperature, the earlier in the pathway cells die. The lethal effect is abolished by lowering the temperature, allowing sperm differentiation to continue to completion. The possible adaptive advantages of temperature controlling gametogenesis are discussed.     

Effects of synchronization on CD40 expression and antibody production in hybridoma cells stimulated with anti-mIgG

In previous experiments with the 55-6 hybridoma cell line, we showed that cell stimulation with anti-mouse surface immunoglobulin G antibody (anti-mIgG) increased both CD40 expression and specific monoclonal antibody (mAb) production rate. Cell preincubation with lipopolysaccharide (LPS) prior to anti-mIgG stimulation enhanced these results. Moreover, the expression of both CD40 and surface immunoglobulin G (sIgG) were higher for cells in the G1 phase of the cell cycle. Therefore, to determine the relationship between cell cycle position, CD40 expression, and mAb productivity, in this work cells were synchronized in the G1 phase by thymidine block. In addition, synchronized cells were subjected to different treatments with anti-mIgG. Although synchronized cells showed a slight increase in both CD40 expression and maximum specific growth rate (mu max) compared with unsynchronized cells, specific productivity did not show significant changes. However, the stimulation of synchronized cells with anti-mIgG increased over 65% the expression of CD40 and over 50% the specific productivity in comparison with that obtained on unsynchronized cells after anti-mIgG stimulation. These data improved additionally over 15 and 60%, respectively, by adding 2 mM thymidine to the culture medium. These results suggest that the effect of the positive association between G1 phase, CD40 expression, and specific productivity is subordinated to the effect of anti-mIgG stimulation, which is enhanced by increasing the percentage of cells on the G1 phase of the cell cycle. Contrary to expectations, LPS preincubation of synchronized cells prior to anti-mIgG stimulation did not increase the specific productivity in comparison with non-preincubated cells, and the expression of CD40 was minor compared to that on non-preincubated cells.     

Growth and production modeling in hybridoma continuous cultures

Several experimental data on continuous cultures of hybridoma cells show that monoclonal antibody productivity is a decreasing function of dilution rate. It has been suggested that this unusual behavior may be due to the arrest of a fraction of cycling cells at a critical point of Phase G(1). Although this hypothesis has been recently investigated by using population balance models, mathematical analysis has been performed without accounting for the dynamics of the arrested cells properly. In this article, a more general and accurate approach is presented and new specific assumptions are introduced to characterize the arrest and the later progress through the cycle. Two different models (stochastic and deterministic) and two different critical points for the arrest (at the beginning and at the end of G(1)) are considered. The cell cycle parameters are estimated so that data predicted by the model fit those reported in the literature. In particular, the fraction of arrested cells, the cell arrest probability, and the mean cell generation time are computed as functions of the dilution rate. Results so far obtained predict that there is an optimal value of dilution rate for maximizing specific production rate of monoclonal antibody. (c) 1993 John Wiley & Sons, Inc.     

Induction of CD40 expression and enhancement of monoclonal antibody production on murine B cell hybridomas by cross-linking of IgG receptors

The 55-6 murine B cell hybridoma line not constitutively expressing CD40 was treated with increasing amounts of intact anti-mouse surface immunoglobulin G antibody (anti-mIgG) either not preincubated or preincubated for 48 h with lipopolysaccharide (LPS). In vitro, cross-linking of surface immunoglobulin G (sIgG) with the whole molecule of anti-IgG antibodies induced the expression of CD69, CD40, and CD19 surface antigens on 55-6 cells. The effect of sIgG ligation was dose-dependent, and preincubation with LPS enhanced their responsiveness to anti-mIgG stimulation. The expression of these surface molecules reached the maximum value during the first part of the cell cycle, corresponding to the position of the G1 peak of the DNA distribution. Stimulation of cells with anti-mIgG did not induce changes either in the number of viable cells or in the fraction of cells undergoing proliferation (mitosis). However, preincubation of 55-6 cells with LPS for 48 h before stimulation with anti-mIgG increased both the maximum specific growth rate (micromax) and the percentage of cells in the G2/M phase, in comparison with non-preincubated cells. Moreover, on cells preincubated with LPS prior to anti-mIgG treatment, specific IgG2a production rate was enhanced significantly compared to that obtained in control cultures. The correlation between the antibody production rate and the amount of IgG that is detectable on the cell surface was analyzed by flow cytometry. A good correlation between secreted and surface IgG was observed, and the results of cell cycle analyses demonstrated that the 55-6 hybridoma cell line has a substantially higher sIgG content in G1 phase.     

Cell cycle model for growth rate and death rate in continuous suspension hybridoma cultures

As a result of recent advances in flow cytometry, renewed interest is shown in modeling the kinetic behavior of cells in culture on the basis of cell cycle parameters. An important but often overlooked kinetic variable in hybridoma cultures is the cell death rate. Not only the overall cell growth but also the kinetics of nutrient metabolism and monoclonal antibody production have been shown to depend on the cell death rate in continuous suspension hybridoma cultures. The present study shows that the death rate in hybridoma cultures is proportional to the fraction of cells arrested in the G(1) phase of the cell cycle. The steady-state cell age distributions in the various phases of the division cycle have been calculated analytically. A simple mathematical model has been used to produce the profiles of the cycling and arrested cell fractions with respect to the dilution rate. The calculated steady-state growth rate, death rate, and viability profiles are shown to be in agreement with recently published experimental data from continuous suspension hybridoma cultures. (c) 1992 John Wiley & Sons, Inc.     

Modulation of cell cycle progression and of antibody production in mouse hybridomas by a nucleotide analogue

The nucleotide analogue 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG) has been identified as a powerful antiproliferative substance when acting on hybridoma cells. In the range of 10 nM to 100 nM concentrations this agent reduces cell growth rate, while its apoptosis-inducing activity is marginal. Marked induction of apoptosis can be observed at micromolar and higher order concentrations. In PMEG-supplemented media the cell cycle progression is perturbed, the flow-cytometric DNA profile shows a higher proportion of cells in the S and G2/M phases of the cell cycle. Concomitantly with the reduction of the growth rate, the specific monoclonal antibody production rate may rise by 20–27%. Addition of PMEG at the end of the exponential phase of a batch culture results in an enhancement of the final monoclonal antibody concentration. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of shear rate on hybridoma cell metabolism

The effect of shear rate on cell growth and monoclonal antibody production of hybridoma cells was studied. The dependence of agitation rate on antibody production is discussed by measuring the amount of monoclonal antibody in cells cultured by a spinner vessel. The effect of shear rate is also studied by exposing a homogeneous shear flow to hybridoma cells in a cone-and-plate viscometer. The dependence of shear rate on hybridoma cells was observed and the increase of antibody production was arised from the increase of secretion from cells.     

Blockade of Fc receptor-mediated binding to U-937 cells by murine monoclonal antibodies directed against a variety of surface antigens

The effect of murine IgG hybridoma antibodies directed against leukocyte antigens on the Fc receptor function of human cells was studied. For this purpose, the specific binding of 125I-labeled monomeric human IgG1 to a macrophage-like cell-line (U-937) was quantitated before and after incubation in the presence of murine monoclonal hybridoma antibodies. Four monoclonal hybridoma antibodies (A1G3, 23D6, 4F2, and 3A 10), each of which binds to different antigens on the surface of U-937 cells, rapidly and potently inhibited the specific binding of labeled IgG1 to these cells. Inasmuch as inhibition was mediated only by IgG antibodies with an intact Fc fragment and antibody activity against surface antigens found on U-937, inhibition appears to have resulted from the formation of a three-component complex composed of antibody bound by its Fab portion to antigen and by its Fc fragment to a Fc receptor. Equilibrium binding studies performed on treated cells confirmed that reduced Fc receptor-mediated binding was due to a reduction in the number of available receptors. Binding studies employing double isotope labeling methods demonstrated that about 0.5 to 1.0 Fc receptor was blocked for each molecule of intact antibody bound to a U-937 cell. Using several techniques, it was shown that most of the monoclonal antibody bound to cells and the Fc receptors blocked by antibody remained on the cell surface despite incubation at 37 degrees C for 3 hr. Thus, the loss of receptor function observed in these experiments was almost exclusively due to reversible receptor blockade rather than receptor internalization or degradation. The antibodies identified in these studies also markedly inhibited Fc receptors on one other human cell line (HL-60) as well as those on normal human peripheral blood monocytes.     

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.     

Preparation of human salivary alpha-amylase specific monoclonal antibody

A mouse hybridoma cell line which produced an anti-human salivary alpha-amylase monoclonal antibody was obtained by fusion between mouse spleen cells immunized with human salivary alpha-amylase and mouse myeloma cells, followed by screening the hybridoma cells by enzyme-linked immunosorbent assay. The hybridoma cell line (27-4-1) secreted IgG. The monoclonal antibody produced by the hybridoma showed no inhibitory effect on the activity of human salivary alpha-amylase. The specificity and reactivity of this monoclonal antibody were examined by determining the activities of human salivary and pancreatic alpha-amylases bound to the monoclonal antibody immobilized on polystyrene balls or by enzyme immunoassay with the monoclonal antibody conjugated with beta-D-galactosidase. The results revealed that the monoclonal antibody produced by the hybridoma cell line was specific for salivary alpha-amylase and absolutely unreactive to pancreatic alpha-amylase.     

Evidence for a role of heat-shock proteins in proliferation after heat treatment of synchronized mouse neuroblastoma cells

A role for heat-shock proteins (HSPs) in proliferation after heat treatment was considered in synchronized mouse neuroblastoma cells. For this purpose enhancement of HSP synthesis after heat treatment was inhibited by actinomycin D and the effect of this on cell cycle progression into mitosis and on cell survival was studied both in thermoresistant G1- and in thermosensitive late S/G2-phase cells. In G1-phase cells expression of basal and heat-induced HSP synthesis was the same as that in late S/G2-phase cells, which suggests that regulation of thermoresistance throughout the cell cycle is not directly linked with HSP synthesis. The synthesis of HSP36, HSP68, and HSP70 was enhanced after a 30-min treatment at 41-43 degrees C. Increase of HSP synthesis after heat shock was partly suppressed by the presence of 0.1 microgram/ml actinomycin D during heat treatment, while 0.2 micrograms/ml prevented enhancement of HSP synthesis completely. Suppression of heat-induced HSP synthesis by actinomycin D had the same concentration dependency in G1- and late S/G2-phase cells. Actinomycin D potentiated induction of mitotic delay by heat treatment (30 min, 42.5 degrees C) but only under conditions where it actually inhibited heat-induced enhancement of HSP synthesis. Heat-induced cell killing was also potentiated by actinomycin D. The potentiating effect of actinomycin D on heat-induced mitotic delay and on heat-induced cell killing was more pronounced in G1-phase cells than in late S/G2-phase cells. These results give evidence for a role of HSPs in the resumption of proliferation after heat treatment and suggest that heated G1-phase cells are more dependent on HSP synthesis for recovery of proliferation after heat treatment than heated late S/G2-phase cells.     

Engineering of a mammalian cell line for reduction of lactate formation and high monoclonal antibody production

Lactate and ammonia are the two major waste products formed during mammalian cell growth. Accumulation of these side products can have a negative effect on cell growth, and has drawn recent attention because of their inhibitory effects on the specific product synthesis rate. Our aim is to reduce lactate formation in the cell culture by genetically manipulating of the pathway of lactate synthesis with an aim to achieve high monoclonal antibody production. We have partially disrupted the LDH-A gene by homologous recombination in hybridoma cells (ATCC-CRL-1606). The cells that received the newly introduced DNA were selected by G418, and an LDH-deficient cell was identified by a screening method based on medium color changing in 96-well plates. A variant cell, LDH-neo21, was identified through this screening method and was characterized. The specific productivity of lactate by LDH-neo21 cells was 50% lower than that of parental cells. Intracellular LDH enzyme activity was significantly reduced. The cell growth was improved both in terms of cell density and cell viability. Total cell density potentially reached 5 x 10(6) cells/mL while the parental hybridoma cells had a cell density of 3.5 x 10(6) cells/mL, which represented a 30% increase. The antibody production of LDH-neo21 cells was threefold greater than that of parental cells during 5-day batch culture. Polymerase chain reaction (PCR) results showed that at least one copy of the LDH-A gene was disrupted in the LDH-neo21 cells. The variant of the hybridoma cell exhibited a significant advantage of reduced lactate formation in the cell culture with a high concentration of glucose, which led to a higher production of monoclonal antibody. 2001 John Wiley & Sons, Inc.