Hybridoma

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In 1975, the first successful production of MAbs was reported (44). By fusing normal antibody-producing cells with a B-ceU tumor (myeloma), hybridoma cell lines resulted which produced antibodies having a specificity to only one deterrninant on an antigen ie, all the antibodies produced from the cell line are identical. These studies resulted in a standard approach to MAb production. In this approach, the hybridoma cells are produced in large quantities in culture and screened to select specific clones producing the desired MAb using an appropriate assay. The selected clones are then expanded in culture (or in animals), the cells are collected, and the MAbs are extracted and purified. 

OKT-3 Ortho hybridoma MA prevention of trans-plant rejection... 

Product formation kinetics in mammalian cells has been studied extensively for hybridomas. Most monoclonal antibodies are produced at an enhanced rate during the Gq phase of the cell cycle (8—10). A model for antibody production based on this cell cycle dependence and traditional Monod kinetics for cell growth has been proposed (11). However, it is not clear if this cell cycle dependence carries over to recombinant CHO cells. In fact it has been reported that dihydrofolate reductase, the gene for which is co-amplified with the gene for the recombinant protein in CHO cells, synthesis is associated with the S phase of the cell cycle (12). Hence it is possible that the product formation kinetics in recombinant CHO cells is different from that of hybridomas. 

Most hybridomas can be grown in batch suspension culture. Recombinant CHO cells can also be adapted for growth in suspension. However,... 

Mammalian Cells Unlike microbial cells, mammalian cells do not continue to reproduce forever. Cancerous cells have lost this natural timing that leads to death after a few dozen generations and continue to multiply indefinitely. Hybridoma cells from the fusion of two mammalian lymphoid cells, one cancerous and the other normal, are important for mammalian cell culture. They produce monoclonal antibodies for research, for affinity methods for biological separations, and for analyses used in the diagnosis and treatment of some diseases. However, the frequency of fusion is low. If the unfused cells are not killed, the myelomas 1 overgrow the hybrid cells. The myelomas can be isolated when there is a defect in their production of enzymes involved in nucleotide synthesis. Mammahan cells can produce the necessary enzymes and thus so can the fused cells. When the cells are placed in a medium in which the enzymes are necessaiy for survival, the myelomas will not survive. The unfused normal cells will die because of their limited life span. Thus, after a period of time, the hybridomas will be the only cells left ahve. 

A hybridoma can live indefinitely in a growth medium that includes salts, glucose, glutamine, certain amino acids, and bovine serum that provides essential components that have not been identified. Serum is expensive, and its cost largely determines the economic feasibihty of a particular ciilture system. Only recently have substitutes or partial replacements for serum been found. Antibiotics are often included to prevent infection of the culture. The pH, temperature and dissolved oxygen, and carbon dioxide concentration must be closely controlled. The salt determines the osmotic pressure to preserve the integrity of the fragile cell. 

Monoclonal antibodies are derived from a single, monospecific B cell clone. Monoclonal antibodies can be obtained from hybridoma cells that result from the fusion of antibody-producing B cells with immortal cells of a myeloma cell line. 

Biotech products based on recombinant DNA, gene expression or hybridoma/monoclonal antibody technologies. 

Vitamins and lipids are often required for animal cells to grow in serum-free medium. Phosphoethanolamine and ethanolamine are key additives that facilitate the growth of the mammary tumor cell line 64024 (Kano-Sueoka and Errick, 1981). In addition, ethanolamine promotes the growth of human lymphocytes and mouse hybridoma cells. Short-term cultures of human diploid lung and foreskin fibroblasts grow in medium that includes among its supplements soybean lecithin, cholesterol, sphingomyelin, and vitamin E. 

The experimental results for hybridoma and protozoa cells given as examples in Fig. 25 indicate that much higher stress (4 to 30 times) is required under laminar flow conditions of viscosimeters than in stirred vessels to achieve the same death rate k. Here the death rate k is defined as first order deactivation constant k = 1/t In (Nq/N), where N, is the initial and N the time-dependent number of living cells in special deactivation experiments under otherwise optimal living conditions. The stress in Fig. 25 was calculated with Eq. (28) for stirred vessels and with Eq. (1) for the viscosimeter. Our own results for hybri-... 

The results in Fig. 25 for hybridoma cells show that due to the low growth rate, which lies in the range of p < 0.02/h, animal cells could only be cultivated under very moderate stress conditions of it< 0.005 -0.05 N/m. That means that, also for very low-shear impellers such as large-blade impellers, the average power input has to be limited to P/V <30-50 W/m ... 

Protein precipitate, hybridoma cells Insect cells Mammalian Cells... 

Fig. 17. Response of CRL-8018 hybridoma cells to increasing levels of well-defined laminar shear in the concentric cylinder viscometer for 10 min. Spinner flask cultures were seeded with cells from routine T-flask cultures that were 3 days old. Cell samples were taken from the spinner flask cultures during late-exponential growth and sheared in the viscometer [17]...

Models based on Eqs. (47)-(50) have been used in the past to describe the disruption of unicellular micro-organisms and mammalian (hybridoma) cells [62]. The extent of cell disruption was measured in terms of loss of cell viability and was found to be dependent on both the level of stress (deformation) and the time of exposure (Fig. 25). All of the experiments were carried out in a cone and plate viscometer under laminar flow conditions by adding dextran to the solution. A critical condition for the rupture of the walls was defined in terms of shear deformation given by Eq. (44). Using micromanipulation techniques data were provided for the critical forces necessary to burst the cells (see Fig. 4)... 

Fig. 25. Total and viable cell concentrations of TB/C3 hybridomas versus duration of shear in a cone and plate viscometer (shear stress 208 Nm ). The error bars indicate the 95% confidence intervals [62]...

By far the greatest amount of work in the literature has been done on suspended hybridoma cells but the general tendency can be transferred to anchorage-dependent cell hnes. 

Most work on chemical stress was done with hybridoma cells since they are widely used for industrial relevant monoclonal antibody production but... 

The main reasons for the damage to cells in a reactor are the apparent shear forces and the collision of microcarriers with themselves and with turbulent eddies. In the literature studies are mainly focused on suspension cells and there again on hybridoma cells. The work reported in the hterature can be divided into two fields studies dealing with the influence of various stirrer speeds on cell viability and those investigating the influence of defined shear forces on cells with a viscosimeter. 

The shear forces are mainly in the range of 1 to lONm. This exposure causes cell death between 20 and 80% depending on the exposure duration which is between a few seconds and several hours. Studies performed in a bioreactor have an exposure duration of several days. The results are partly contradictory. Tramper et al. [30] found a critical stress level of 1.5 Nm" for insect cells, whereas Oh et al. [31] could not show an influence on hybridoma cells even at high stirrer speed. This shows that each cell line reacts different and that there is a necessity for defined stress systems if the results is to be comparable. 

Hybridomas Provide Long-Term Sources of Highly Useful Monoclonal Antibodies... 

The method involves cell fusion, and the resulting permanent cell line is called a hybridoma. Typically, B cells are obtained from the spleen of a mouse (or other suitable animal) previously injected with an antigen or mixture of antigens (eg, foreign cells). The B cells are... 

Grown In presence of HAT medium Hybridoma multiplies myeloma and B cells die... 

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