Large-scale cultivation of hybridoma cells

The demand for large quantities of monoclonal antibodies has increased dramatically over the last few years to supply diagnostic kits for application areas with large markets, and for use in developing applications such as anti-tumour therapies and affinity chromatography. Scale-up from tissue and murine culture has not been a straightforward matter because of the special requirements of mammalian cells in culture. The sensitivity of hybridomas to their environment means that efficient mixing must be obtained at low shear rates, and that oxygen transfer must be adequate for high density cultures without aggressive sparging. The high fraction of serum required to satisfy undefined nutrient requirements needs to be reduced considerably in order to achieve economy of scale. This review discusses the technical barriers to scale-up and reports on successful attempts to produce large volumes of monoclonal antibodies.     

Karyological analysis of hybridoma lines producing monoclonal antibodies to viral antigens

The number of Hybridomes obtained from various sources rapidly increases at present. Clones producing monoclonal antibodies to influenza virus A/USSR/090/77 and to VEE-230 are generated in the laboratory of the Institute of Virology (Academy of Sciences of the USSR). The present work is devoted to the study of Hybridome karyotype by means of C-method for chromosome staining with the aim to reveal specific characteristics of these lines. Results of the investigation have shown that the count of chromosomes together with an examination of their C-staining picture permit proving a hybrid nature of the clones and identifying various Hybridomes by chromosome markers.     

Continuous cultivation of hybridoma cells in cell reactor

Summary Mouse hybridoma cells were grown on surface of glass disks in a cell reactor. It is shown that during continuous cultivation the cells retain their antibody-producing ability. The yield of the culture supernatant obtained in the reactor exceeds about 5 fold those obtained in flasks with equivalent area.     

Proteomics analysis of chinese hamster ovary cells undergoing apoptosis during prolonged cultivation

The degradation of environmental conditions, such as nutrient depletion and accumulation of toxic waste products over time, often lead to premature apoptotic cell death in mammalian cell cultures and suboptimal protein yield. Although apoptosis has been extensively researched, the changes in the whole cell proteome during prolonged cultivation, where apoptosis is a major mode of cell death, have not been examined. To our knowledge, the work presented here is the first whole cell proteome analysis of non-induced apoptosis in mammalian cells. Flow cytometry analyses of various activated caspases demonstrated the onset of apoptosis in Chinese hamster ovary cells during prolonged cultivation was primarily through the intrinsic pathway. Differential in gel electrophoresis proteomic study comparing protein samples collected during cultivation resulted in the identification of 40 differentially expressed proteins, including four cytoskeletal proteins, ten chaperone and folding proteins, seven metabolic enzymes and seven other proteins of varied functions. The induction of seven ER chaperones and foldases is a solid indication of the onset of the unfolded protein response, which is triggered by cellular and ER stresses, many of which occur during prolonged batch cultures. In addition, the upregulation of six glycolytic enzymes and another metabolic protein emphasizes that a change in the energy metabolism likely occurred as culture conditions degraded and apoptosis advanced. By identifying the intracellular changes during cultivation, this study provides a foundation for optimizing cell line-specific cultivation processes, prolonging longevity and maximizing protein production.     

Some aspects of hybridoma cell cultivation

Summary Two hybridoma cell lines were cultivated in an indirectly aerated 10-1 reactor in batch, fed-batch and continuous (perfusion) operations and in spinner flasks. The medium in the reactor was sampled either by an aseptic cross-flow filtration module integrated into a loop or by an in-situ tubular filter. The glucose concentration was monitored by an on-line flow injection analyzer and the ammonia concentration by an ion-selective electrode. Since the membrane transmission of the high-molecular components decreased during cultivation, the product, a monoclonal antibody, was enriched in the reactor. During cultivation, the concentrations of cells, viable cells, glucose, lactase, acetate, citrate, ammonia, urea, amino acids, proteins, and monoclonal antibodies were determined off-line. The specific growth rate, specific production, and consumption rates of the medium components were influenced considerably by the medium composition, especially by the type and amount of serum used.Offprint requests to: K. Schügerl     

Quantitative cytochemical analysis of immobilised hybridoma cells

Summary The activity of hybridoma cells immobilised in agarose gels is observed to decline with time in a manner consistent with oxygen diffusion limitation. The final thickness of the active cell layer is dependent on the initial cell density. The effect of diffusion resistance is more pronounced in medium without serum.     

Factors governing lactic acid formation in long term cultivation of hybridoma cells

Summary Effect of glucose concentration and pH on lactic acid formation was investigated in batch cultures of a hybridoma cell line. Lactate formation increases with growth rate. High glucose concentration leads to extensive lactate formation only during growth phase and not during stationary phase. Lactate formation also may serve to regulate extracellular pH to pH 6.8, provided conditions are favorable to maintain viability and if sufficient nutrients are present in the medium.     

Poly(ethylenimine)-reinforced liquid-core capsules for the cultivation of hybridoma cells

The mechanical stability of liquid-core alginate-poly-L-lysine (PLL) capsules used for encapsulation of hybridoma cells can be greatly enhanced by the inclusion of poly(ethylenimine) (PEI) in the hardening solution containing calcium chloride. The PEI can also reinforce the PLL-coated carboxymethyl-celluose liquid-core capsules. The cultivation of murein hybridoma CT04 in these two capsules was carried out. Cell concentrations higher than 10(8) cells/mL per capsule were obtained with ca. 80% of the specific antibody productivity as the freely suspended cells. These capsules could withstand severe agitation and aeration in an air-lift reactor over a period of 3 weeks with minimal damage. (c) 1992 John Wiley & Sons, Inc.     

Coiled tube membrane bioreactor for cultivation of hybridoma cells producing monoclonal antibodies.

A coiled tube membrane reactor was developed for the cultivation of mouse-mouse hybridoma cells producing monoclonal antibodies. The cell and antibody concentrations obtained in the membrane reactor were higher than that obtained in a batch spinner flask without a membrane. A mathematical model has been developed to describe the membrane transport coupled growth and product formation, and the physical and kinetic constants of the system were determined.     

Cell cycle,cell size and mitochondrial activity of hybridoma cells during batch cultivation

Cell cycle, cell size and rhodamine 123 fluorescence in cell populations of two batch cultures were analysed and quantified with a fluorescence-activated cell sorter (FACS). Two cultures derived from either exponential or stationary phase innocula were investigated in order to demonstrate the dependency of the subsequent cell growth on innoculum condition. The results demonstrated that the level of activity of cells in the innoculum culture could have a significant effect on cellular activity during the initial phase of the inoculated culture, as it advances through its growth cycle. Positive correlation was found between the cell size and mitochondrial activity (as measured by rhodamine 123 uptake) with S and G₂ fractions as the cell progressed through the cell cycle. The enumeration of the fractions of cell cycle phases has helped in prediction of the changes in cell numbers following perturbation of the culture condition.     

Comparative cytogenetic analysis of cells of mouse-mink inter-species hybridoma lines]

The comparative cytogenetic analysis of the interspecific mouse-mink hybridoma cells revealed a segregation of the great number of the mink chromosomes, inter- and intraline variability according to the number of cells with the mink DNA and its quantity in the cells. No characteristics of the mink chromosomal material distribution in the hybridoma cells which secreted the immunoglobulins of the American mink or lost its secretion were found. The great changes in the karyotype of the hybrid cells were revealed by in situ hybridization with 3H-labelled total mink DNA. Numerous insertions of the regions from the mink chromosomes to the mouse chromosomes and the appearance of the chromosomes not typical of the mink and mouse parent cells were observed. The number of cells with translocations of fragments from the chromosomes to the mouse one was observed to grow in the hybridoma cell lines cultivated for a long time. Synthesis of the lambda-L-chains of the mink immunoglobulin in the cells of line 7 was absent because they lost lambda-gene.     

A comparison of simple growth vessels and a specially designed bioreactor for the cultivation of hybridoma cells

Three tank type bioreactors of very simple design were compared to a commercially available laboratory-scale bioreactor, designed especially for mammalian cell culture, for their ability to support hybridoma growth and antibody production under batch culture conditions. The comparison reveals quite similar numbers for maximum viable cell densities and IgG production, despite large differences in vessel and agitator geometry and aeration mode. Furthermore, some data indicate that the hydrodynamic stress level in the growth vessels may influence the specific production rate of the cells and thus the overall productivity of the reactors.     

Microcalorimetric studies of hybridoma cells

In the present study, overall metabolism has been estimated in hybridoma cells by microcalorimetric measurement. Heat production rate was found to be 30-50 pW/cell at cell concentrations 0.65-4.5 x 10(5)/ml. High cell concentrations (1 x 10(6) cells/ml) caused unstable power-curves with an initial high peak and a rapid declining phase, whereas low cell concentrations (0.6-4.5 x 10(5) cells/ml) produced steady-state power-curves. Oxygen consumption was found to range between 1.5-6.1 x 10(-5) mol 02/cell/min, corresponding to about 80% of the total metabolic activity. The metabolic inhibitors sodium fluoride (50 nM), sodium azide (160 mM) and rotenone (0.1 mM) caused a reduction in overall cell metabolism of 60, 55 and 40% respectively.     

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.