Hybridoma cells

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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. 

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. 

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. 

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 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)... 

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. 

The cell fusion mixture is transferred to a culture medium containing hypoxanthine, aminopterin and thymidine (HAT medium). Unflised myeloma cells are unable to grow as they lack HGPRT. Unflised normal spleen cells can grow but their proliferahon is limited and they eventually die out. The hybridoma cell can proliferate in the HAT medium as the normal spleen cell supplies the enzyme which enables the hybridoma to utilize extracellular hypoxanthine. 

Shirai, Y., Yamaguchi, M., Kobayashi, A., Nishi, A., Nakamura, H., and Murakami, H., Change in Growth Kinetics of Hybridoma Cells Entrapped in Collagen Gel Affected by Alkaline Supply, Cytotechnol., 14 129 (1994)... 

Monoclonal antibody, an antibody produced by a single clone of cells (specifically, a single clone of hybridoma cells) and therefore a single pure homogeneous type of antibody that binds to the same target. 

With the exception of gel filtration, all modes of biochromatography are, in principle, adaptable to EBA. Separations based on ion exchange [27], Protein A affinity [28] and IMAC [29] have been published. The list of feed includes bacterial fermentation broth [30], cell homogenate [31] and renatured inclusion bodies [32], yeast fermentation broth [29] and cell homogenate [27] as well as mammalian or hybridoma cell culture broth [33]. EBA is also an accepted method in the production of medicinal products. Although, considering the problems in initial processing discussed earlier... 

Fig. 15.7 Glycosylation of an antibody produced in tobacco plants expressing a human 3(l,4)-galactosyltransferase. As illustrated for Guy sl3 in Fig. 15.4, when the monoclonal antibody Mgr48 is produced in wild type tobacco plants (left panel), its glycosylation is structurally different and more heterogeneous than that of its mammalian counterpart (lower panel). When this antibody is produced in tobacco plants expressing the human galactosyltransferase (right panel), 30% of its N-glycans show terminal N-acetyllactosamine sequences identical to those carried by this antibody when it is produced in hybridoma cells.

Over half of all biopharmaceuticals thus far approved are produced in recombinant E. coli or S. cerevisiae. Industrial-scale bacterial and yeast fermentation systems share many common features, an overview of which is provided below. Most remaining biopharmaceuticals are produced using animal cell culture, mainly by recombinant BHK or CHO cells (or hybridoma cells in... 

Monoclonal antibody technology entails isolation of such B-lymphocytes, with subsequent fusion of these cells with transformed (myeloma) cells. Many of the resultant hybrid cells retain immortal characteristics, while producing large quantities of the monospecific antibody. These hybridoma cells can be cultured long term to effectively produce an inexhaustible supply of the monoclonal antibody of choice. 

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