Hybridoma Cell Culture Research Articles

Use of the Centritech Lab centrifuge for perfusion culture of hybridoma cells in protein-free medium.

As part of an effort to develop a suspension-culture perfusion-based process with high flow rate without the fouling and antibody retention inherent to filter-based cell-separation devices, we have evaluated and contributed to the development of the Centritech Lab centrifuge for the perfusion culture of hybridoma cells in protein-free medium. Culture start-ups showed that cell growth and monoclonal-antibody (MAb) production rates were similar in both a spinner flask and continuous centrifugation coupled to a bioreactor. The centrifuge efficiently separated viable cells from dead ones. Viable-cell recoveries were never below 98%, whereas dead-cell recoveries were usually around 80%. The cell content of the centrifuge supernatant and concentrate was strongly determined by the total amount of cells, viable and dead, in the culture broth, but an influence of the centrifugation parameters (feed rate, times of separation and discharge, and rotor speed) was observed. This understanding of the separation process inside the centrifuge is important and may apply to other similar devices. Monoclonal antibodies were not retained in the bioreactor during centrifugation perfusion. However, whereas similar growth rates were obtained in perfusion cultures using either continuous centrifugation or filtration, MAb concentrations were 35% lower in the former case. Utilization of the centrifuge in an intermittent fashion decreased the daily cell residence time outside the bioreactor, the daily pelleted-cell residence time in the centrifuge, and the frequency of cell passage to the centrifuge. This led to higher viable-cell numbers in the bioreactor and an accompanying increase in MAb concentrations, 225-250 mg of IgM L-1, equal to the performance of filter-based perfusion systems with the same cell line. It was hypothesized that having cells periodically packed at the bottom of the centrifuge insert (up to 800 x 10(6) cells mL-1) is deleterious to the culture by exposing the pelleted cells to prolonged nutrient limitations.

Interactive dual control of glucose and glutamine feeding in hybridoma cultivation

With the aim of developing an advanced computer control system for hybridoma cell cultures, on-line monitoring and control strategies for nutrient feeding in a fed-batch culture of a hybridoma cell line were investigated. Good correlation was obtained between the on-line monitoring and the off-line analysis of growth, and between the uptake rates of glucose, oxygen and glutamine, respectively. Using the variables monitored on-line, control of the glucose and glutamine concentrations in fed-batch culture was achieved. The characteristics of the glucose-limited fed-batch culture of hybridoma cells were also investigated. In combination with further research on cell physiology, the results of this study should accelerate the development of an advanced and reliable control system for hybridoma cell cultures.

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.

Intracellular flux analysis applied to the effect of dissolved oxygen on hybridomas

Quantitative estimates of intracellular fluxes and measurements of intracellular concentrations were used to evaluate the effect of dissolved oxygen (DO) concentration on CRL 1606 hybridoma cells in batch culture. The estimates of intracellular fluxes were generated by combining material balances with measurements of extracellular metabolite rates of change. Experiments were performed at DO levels of 60% and 1% air saturation, as well as under oxygen-limited conditions. Cell extracts were analyzed to evaluate the effect of DO on the intracellular concentrations of the glutamate dehydrogenase reactants, as well as the redox state of the pyridine nucleotides in the cytosol and mitochondria. The relationship between cell density and pyridine nucleotide redox state was also investigated. Dissolved oxygen concentration had a significant effect on nitrogen metabolism and the flux through glutamate dehydrogenase was found to reverse at low DO, favoring glutamate formation. The NAD in the cytosol and mitochondria was more reduced under low DO conditions while the cytosolic NAD was more oxidized at low DO. Cytosolic NAD was reduced at higher cell densities while the redox states of cytosolic NADP and mitochondrial NAD did not exhibit significant variation with cell density. These results point to the fundamental role of the intracellular oxidation/reduction state in cell physiology and the possibility of controlling physiological processes through modulation of the dissolved oxygen level or the oxidation/reduction potential of the culture.

Real‐time monitoring of protein secretion in mammalian cell fermentation: Measurement of monoclonal antibodies using a computer‐controlled HPLC system (BioCad/RPM)

On-line, "real-time" monitoring of product concentration is important for mammalian cell culture fermentation. The continuous measurement of monoclonal antibodies allows for instantaneous determination of cell productivity and effective manipulation of the fermentor operating conditions for optimal production. This article will present the evaluation and application of a BioCad/RPM system (Per Septive Biosystems) for rapid analysis of lgG concentration for hybridoma cell cultivation. Several commercial crossflow filtration devices are tested for low protein retention and fouling properties. A protein G column is used successfully for analyzing about 400 samples of lgG(1), without significant loss in separation efficiency. The Immuno Detection system is integrated into a computer-controlled 15-L fermentor. This fermentor could be operated in batch and perfusion modes with cell densities up to 20 million cells/mL. A continuous cell-free sample stream obtained by a hollow fiber filter system is introduced to the BioCad/RPM for analysis. The speed of this system allows for real-time monitoring even at high densities with fast dynamics. A murine hybridoma cell (A10G10) is cultivated in batch and continuous reactors and antibody concentration is measured continuously with complete sterility. The results are compared to offline measurements with good agreement.

Chromophore-assisted laser inactivation of subunits of the T-cell receptor in living cells is spatially restricted.

Chromophore-assisted laser inactivation (CALI) is a molecular photoablation technique that has been used to elucidate the in vivo roles of specific proteins in neural development. The interpretation of its effects on proteins in living cells relies on knowing how spatially restricted the CALI-induced damage is in vivo. To determine the spatial specificity of CALI in living cells, we have applied CALI to individual subunits of the T-cell receptor (TCR) complex on the surface of 2B4 hybridoma cells in culture and have examined the consequent structural and functional integrity of the TCR-alpha, TCR-beta and CD3-epsilon. The CALI of TCR-beta resulted in the disruption of the beta subunit and also resulted in a small effect on antibody binding alone to the neighboring TCR-alpha but caused no effect on another subunit, CD3-epsilon. Reciprocal experiments directing CALI to TCR-alpha and CD3-epsilon gave consistent results. No effects other than a simple loss of function were observed for any of these CALI experiments. These data demonstrate the extent of CALI-induced damage within a multisubunit complex in living cells and provide greater confidence for the future application of this technique to understanding in vivo function of proteins during complex cellular processes.

Cell growth and monoclonal antibody production in the presence of antigen and serum.

The impact that the continuous presence in the fermentation broth of the cognate antigen has on the serum-supplemented hybridoma cell cultures was investigated. Both soluble and immobilized antigen at various concentrations was applied. The cell line (ATCC TIB191) was cultured in a serum-supplemented PFHM-II medium in T-flasks. Sepharose gel beads provided the immobilization matrix, and bovine gamma-globulin was the carrier protein upon which the antigen, picric acid, was conjugated. Produced antibody, after elution from the beads by displacement with free picric acid, was measured with an ELISA. Soluble antigen--carrier protein conjugates showed no effect on the cultures, but the immobilized antigen had a strong influence on them. Cell growth rate and total antibody production decreased as the amount of immobilized antigen increased from 16:10 (units are in (mol of Ag/mol of carrier):(mg of carrier/mL of beads) to 36:40. Most importantly, the specific antibody production rate switched from growth phase-independent behavior in the antigen-free cultures to growth phase-dependent behavior in the immobilized antigen cultures. Furthermore, during the early stationary phase of the cultures with immobilized antigen, the specific antibody production was 20-100% higher relative to the production rate in antigen-free cultures. This increased specific antibody production rate would be particularly useful in increasing the volumetric productivity of perfusion-type bioreactors for hybridoma cell cultures.

Specific growth rate as a parameter for tracing growth-limiting substances in animal cell cultures

The specific growth rate (μ) reaches its maximum very early during culture (at 20 h), but declines again thereafter so that no exponential growth phase occurs in batch cultures of hybridoma cells. This growth rate limitation depends neither on exhaustion of any of the macro-nutrients, nor on inhibition by metabolic byproducts (Ljunggren and Häggström, 1994). Intermittent additions of serum, after 20 and 40 h of culture, resulted in exponential growth throughout the growth phase. Insulin was found to be the main component responsible for the growth rate increasing effect. Intermittent additions of serum or insulin to a dual substrate (glucose and glutamine) limited fed batch culture increased the growth rate also here, and the results indicate the existence of a minimum growth rate (about 0.02 h −1) at a threshold glutamine level (0.005 mM). Serum and insulin additions markedly enhanced the glucose consumption and lactate formation rates, a metabolic effect that was not coupled to the increase in μ. The reduced concentrations of glucose and glutamine in substrate limited fed batch cultures suppressed substrate consumption rates and byproduct formation (lactate, ammonium, alanine, other amino acids) even in the serum and insulin stimulated cultures and rendered the energy metabolism much more efficient than in batch culture. Further, the serum and insulin stimulated cells growing in substrate limited fed batch culture produced almost 4-times more antibodies, from the same amount of nutrients as supplied to the batch grown cells.

High viable cell concentration fed‐batch cultures of hybridoma cells through on‐line nutrient feeding

A hybridoma cell line was cultivated in fed-batch cultures using a low-protein, serum-free medium. On-line oxygen uptake rate (OUR) measurement was used to adjust the nutrient feeding rate based on glucose consumption, which was estimated on-line using the stoichiometric relations between glucose and oxygen consumption. Through on-line control of the nutrient feeding rate, not only sufficient were supplied for cell growth and antibody production, but also the concentrations of glucose and other important nutrients such as amino acids were maintained at low levels during the cell growth phase. During the cultivation, cell metabolism changed from high lactate production and low oxygen consumption to low lactate production and high oxygen consumption. As a result the accumulation of lactate was reduced and the growth phase was extended. In comparison with the batch cultures, in which cells reached a concentration of approximately 2 x 10(6) cells/mL, a very high concentration of 1.36 x 10(7) cells/mL with a high cell viability (>90%) was achieved in the fed-batch culture. By considering the consumption of glucose and amino acids, as well as the production of cell mass, metabolites, and antibodies, a well-closed material balance was established. Our results demonstrate the value of coupling on-line OUR measurement and the stoichiometric relations for dynamic nutrient feeding in high cell concentration fed batch cultures.

A kinetic model for product formation of microbial and mammalian cells

Growth of microbial and mammalian cells can be classified into substrate-limited and substrate-sufficient growth according to the relative availability of the substrate (carbon and energy source) and other nutrients. It has been observed for a number of microbial and mammalian cells that the consumption rate of substrate and energy (ATP) is generally higher under substratesufficient conditions than under substrate limitation. Accordingly, the product formation under substrate excess often exhibits different patterns from those under substrate limitation. The extent of increase or decrease in product formation may depend not only on the nature of limitation and cell growth rate but also on the residual substrate concentration in a relatively wide range. The product formation kinetic models existing in literature cannot describe these effects. In this study, the Luedeking-Piret kinetic is extended to include a term describing the effect of residual substrate concentration. The extended model has a similar structure to the kinetic model for substrate and energy consumption rate recently proposed by Zeng and Deckwer. The applicability of the extended model is demonstrated with three microbial cultures for the production of primary metabolites and three hybridoma cell cultures for the production of ammonia and lactic acid over a wide range of substrate concentration. The model describes the product formation in all these cultures satisfactorily. Using this model, the range of residual substrate concentration, in which the product formation is affected, can be quantitatively assessed. (c) 1995 John Wiley & Sons, Inc.

Proteolytic Activity of Hybridoma Cultures - a Simulation Study

Using a structured model written in the PC program “MATLAB”, we have studied the influence of proteolytic activity on antibody concentration in hybridoma cell cultures and the effect of addition of a sacrificial substrate, which the proteinase could degrade instead of the antibody. These simulations were carried out in a factorial experimental design including the variation of the individual rate constants for breakdown kinetics between different cell lines. The results show that the differences in these rate constants are very important for the increase in antibody yield when a sacrificial substrate is added and that proteolytic activity can be present, even though it is not revealed by the antibody curve, indicating that product degradation in cell cultures may be more wide spread than normally assumed. Apparent variations in productivity of antibody may be related to the productivity of proteolytic enzymes and the modification of the activity of these enzymes by medium components, rather than intrinsic changes in cell behavior.

Optimizing antibody production in batch hybridoma cell culture

Optimizing productivity by hybridoma cells relies partly on developing suitable methods for screening and selection of high producing cultures and on understanding regulation of antibody production. In this study, the behavior of hybridoma cells in batch culture was investigated using flow cytometry, and a simple model for antibody production was used to explain production data obtained from these cultures. Surface antibody fluorescence values were found to closely follow the decreasing trend of specific antibody secretion rate over the course of several batch cultures. Therefore, for the hybridoma cell lines studied here (ATCC HB124 and TIB138), surface immunofluorescence levels can be used to select high producing cells as well as to monitor culture productivity. Surface and intracellular antibody fluorescence values were also found to be correlated for cells exhibiting a bimodal distribution with respect to intracellular antibody content. The population of cells containing lower levels of intracellular antibody was determined to secrete significantly less antibody than the population possessing high intracellular antibody concentrations. Factors which influence antibody production rates and possible strategies for optimizing monoclonal antibody yield are discussed.

Death mechanisms of animal cells in conditions of intensive agitation

The question is addressed as to whether cells which are subject to high-energy dissipation rates in agitated bioreactors show an apoptotic response. Murine hybridoma cells in batch culture were agitated in bench-scale (1-L) bioreactors without gas sparging. At an energy dissipation rate of 1.5 W m(-3) there was no apparent damage. At 320 W m(-3) cell viability declined, and increasing proportions of the dead cells displayed the morphological features of apoptosis, but necrosis also remained as a significant mechanism of death. When cells were subjected to the intensive energy dissipation rate of 1870 W m(-3) in a bioreactor without gas headspace, the cell number dropped by 50% within 2 h and a subpopulation of smaller-sized cells emerged. This excluded trypan blue but showed some apoptotic characteristics such as reduced and condensed DNA content and low F-actin content. The incidence of apoptotic activity was further demonstrated by the appearance of numerous apoptotic bodies. Analysis of the cell cycles of both small and normal size populations indicated that greater proportions of S and G2 cells had become apoptotic and there was evidence of preferential survival of G1 cells. It is suggested that two mechanisms of cell death are apparent in hydrodynamically stressful situations, but their relative expression depends on the energy dissipation rate.

Methods for reducing the ammonia in hybridoma cell cultures

The factors which limit the proliferation of eukaryotic cells in vitro are still not well known. Ammonia is believed to be toxic for mammalian cell proliferation and secretion. We have tried two approaches to reducing the ammonia in the medium. We first limited the ammonia produced by the cells by replacing glutamine by glutamate. Then, we used two chemical engineering methods to eliminate accumulated ammonia. In one the used medium was passed through a natural cation exchanger: the clinoptilolite. In the other, the culture medium was passed through a hydrophobic microporous hollow fiber module. Replacing the glutamine by glutamate reduced the medium ammonia concentration. The physicochemical removal of ammonia induced a better cell growth, but not a better specific antibody secretion.

Cultivation of hybridoma cells in an inclined bioreactor

Murine hybridoma cells were grown in a bubble column that was inclined up to 45 degrees from vertical. Inclining the column by a few degrees separated the rising bubbles against the upper surface, leaving the bulk of the liquid bubble free. The liquid was circulated well by the rising bubbles, but collection of cells by rising bubbles and exposure of cells to bursting bubbles were minimized. Maximum viable cell count and exponential growth of the cells were not affected by inclination, but an inclination of 30 degrees gave an antibody titer of 42 mg/L, which more than doubled the yield of 17 mg/L in the vertical position. By comparison, the culture gave yields of 30 mg/L when grown in spinner flasks. The enhanced antibody production in the inclined bioreactor corresponded to a prolonged stationary phase of 45 h. (c) 1995 John Wiley & Sons, Inc.

Role of glutamine in hybridoma cell culture: Effects on cell growth, antibody production, and cell metabolism

The effects of glutamine on cell growth kinetics, monoclonal antibody productivity, and cell metabolism of hybridoma cells were investigated. The mouse-mouse hybridoma cell line V111 H-8, which produces mouse immunoglobulin (IgG 2a) was used as a model system. Glutamine showed a Monod-type effect on specific cell growth rate with a very small Monod constant of 0.089 (m m). A reciprocal relationship between initial glutamine concentration and specific cell death rate was observed. Monoclonal antibody (MAb) production was a strong function of glutamine concentration. Glutamine enhanced MAb production not only by stimulation of cell growth, but also by increasing the specific MAb productivity. Increasing glutamine concentration stimulated specific glutamine consumption rate and specific ammonium ion production rate. The specific glucose consumption rate decreased with increasing glutamine concentration up from 0 to 0.1 n m, and then increased. The specific lactate production rate decreased as glutamine concentration decreased.

High cell density perfusion cultures of anchorage-dependent Vero cells in a depth filter perfusion system

A depth filter perfusion system (DFPS) with polypropylene fibers had been demonstrated to support high density cultures of anchorage-independent hybridoma cells. The DFPS provides advantages of high surface-to-volume ratio of 450-600 cm(2)/cm(3), low cost set-up, easy operation and scale-up. To test the feasibility of using DFPS for high density cultures of anchorage-dependent cells, Vero cells were cultivated in the DFPS. Gelatin coating on polypropylene fibers in the DFPS was necessary to promote cell attachment and growth. Dissolved oxygen (DO) concentrations could be controlled by sparging air into the reservoir vessel through a filter sparger. When DO concentration was controlled above 40% of air saturation in the DFPS with 40 μm pore size, the maximum cell concentration as estimated on specific lactate production rate, was 3.81×10(7) cells/ml of the total reactor volume. This viable cell concentration is approximately 18 times higher than that obtained in a T-flask batch culture. Taken together, the results obtained here showed the potential of DFPS for high-density cultures of anchorage-dependent cells.

In vitro production of monoclonal antibodies in high concentration in a new and easy to handle modular minifermenter

This paper describes a new and easy to handle reusable minifermenter for high-density culture of hybridoma and other cells. The culture apparatus is composed of two modules: a 40 ml disposable cell culture and antibody production chamber (the ‘production module’) and a 550 ml medium reservoir (the ‘supply module’). The two modules are separated from each other by a dialysis membrane allowing passage of low molecular mass nutrients and metabolites. The monoclonal antibodies are produced and enriched in the production module. The outer part of this module is made from a thin gas-permeable silicone rubber membrane allowing exchange of gases (oxygen and carbon dioxide). To start the culture, the cells are injected into the production module through ports in the silicone rubber which are equipped with Luer Lock connectors. Samples can be removed in the same way. For culturing, the minifermenter is rolled on a roller apparatus in a carbon dioxide-supplied incubator. Depending on the individual properties of the hybridoma cells cultured, cell densities of more than 10 × 10 6 (in some cases up to 35 × 10 6) cells per ml and monoclonal antibody concentrations of several mg per ml can be obtained in the new minifermenter. On average, 61 mg (range: 9–159 mg) could be produced within 1–4 weeks. In terms of their properties the monoclonal antibodies produced in the new modular minifermenter were indistinguishable from antibodies prepared from ascitic fluid or from the supernatant of conventional stationary culture. The culture method is a useful alternative to the in vivo production method in mice. In addition, it represents a completely new, inexpensive and easy to handle general solution to the problem of culturing cells in high density and obtaining cellular products in high concentrations.

The culture of rat myeloma and rat hybridoma cells in a protein-free medium

Y0 is a rat x rat hybridoma cell line, which does not secrete immunoglobulin, produced using a fusion partner derived from the Y3 (Y3,Ag.1.2.3) rat myoloma cell line. Y0 and Y3 have both been widely used as fusion partners in the production of rat x rat hybridomas. Y0 has also been used in recombinant gene technology. Y0 cells grown in shake flask culture, using RPMI 1640 medium with 4mM l-glutamine and 5% foetal bovine serum, reached a maximal cell density of 1.5×10(6) cells ml(-1) with 86% viability. Y0 cells which has been adapted to grow in ABC protein-free medium reached a maximal density, in shake flask culture, of 8.75×10(5) cells ml(-1) with 79% viability. An improved protein-free medium, designated W38 medium, was developed. In shake flask culture, W38 medium supported Y0 cell growth to a density of 2.02×10(6) cells ml(-1) with 96% viability. Two Y3 hybridomas, YID 13.9.4 cells and SAM 618 cells were adapted to growth in W38 medium. For both hybridomas, cell growth and product yield in shake flask culture using W38 medium was superior to that obtained with serum-containing RPMI 1640 medium.

Production of Polyclonal and Monoclonal Antibodies Against Hyphae from Arbuscular Mycorrhizal Fungi

Polyclonal and monoclonal antibodies were produced against hyphae of the arbuscular mycorrhizal fungus Glomus monosporum. The polyclonal antibodies (pAbs) were raised in a rabbit by immunizing with hyphae. They were tested for their specificity by a dot-immunoblot assay (DIBA). After the third immunization, a distinct difference in the signal strength was observed between the antisera and the preimmune serum. The pAbs showed cross-reactions to a number of fungal species, both mycorrhizal and other. For the production of monoclonal antibodies (mAbs), mice were immunized intraperitoneally with hyphae. The resulting hybridoma cell culture supernatants were tested by an indirect immunolabeling procedure. For this purpose the hyphae were immobilized on silane-coated microscopic slides. The mAb 8A7 reacted with hyphae from all Glomus isolates tested so far. Cross-reactivities were not observed with hyphae from fungi of the family Acaulosporaceae, phytopathogenic fungi tested so far, or from spores from Glomus species.