Monoclonal antibodies, hybridomas production

Biotechnology products are pharmaceutical products prepared by industrial processes that involve the use of biological systems such as fermentation or tissue culture. For some industries, these processes involve the use of genetically engineered organisms. The preparation of products generally involves recombinant DNA, monoclonal antibody/hybridoma, continuous cell lines, and cellular therapy technology. 

Fig. 6.29. Outline of hybridoma creation and monoclonal antibody (MAb) production.

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. 

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. 

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

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

Knowing the importance of PGA for the ion exchange in the cell wall, we considered the production of monoclonal antibodies (Ab) to homopolygalacturonic acid. We produced one IgGl hybridoma, named 2F4, that appeared to be specific of PGA [11]. 

Generation of antibodies that can recognize and bind to specific viruses is straightforward. A sample of live or attenuated virus, or a purified component of the viral caspid, can be injected into animals to stimulate polyclonal antibody production (or to facilitate monoclonal antibody production by hybridoma technology). Harvested antibodies are then employed to develop specific immunoassays that can be used to screen test samples routinely for the presence of that specific virus. Immunoassays capable of detecting a wide range of viruses are available commercially. The sensitivity, ease, speed and relative inexpensiveness of these assays render them particularly attractive. 

The basis of monoclonal antibody production by hybridoma technology... 

Monoclonal antibodies can be produced not only in a cell culture but also in live animals. When injected into mice (in the peritoneal cavity, the gut), the hybridoma cells produce tumors containing an antibody-rich fluid called ascites fluid. Production in cell culture is usually preferred, as the ascites technique may be very painful to the animal and if replacement techniques exist, may be considered unethical. The process of producing monoclonal antibodies described above was invented by Georges Kohler. Cesar Milstein, and Niels Kaj Jeme in 1975 they shared the Nobel Prize in Physiology or Medicine in 1984 for the discovery (http //en.wikipedia.org/ wiki/Antibody). 

The next development was the production of monoclonal antibodies (MAbs) in the mid-1970s. This uses hybridoma technology, which involves the fusion of antibody-producing B cells to immortal myeloma cells. Figure 4.4 shows the preparation of MAbs using hybridoma techniques. A more detailed discussion of biopharmaceuticals production is presented in Section 10.5. 

A further advance in antibody technology is the development of transgenic mouse human strains. XenoMouse animals have been engineered in such a way that they now produce exclusively human antibodies rather than murine antibodies when immunized. The use of XenoMouse animals to produce MAbs avoids the need for any engineering of the antibody genes, since the products are already 100% human protein. XenoMouse animals are fully compatible with standard hybridoma technology and can be readily adopted by laboratories experienced in monoclonal antibody production [56]. 

Only a few fused cells actually produce antibodies. To identify these cells, the hybridomas have to be isolated and replicated by cloning (4). After the clones have been tested for antibody formation, positive cultures are picked out and selected by further cloning (5). This results in hybridomas that synthesize monoclonal antibodies. Finally, MAB production is carried out in vitro using a bioreactor. 

The culture of hybridomas, thereby producing monoclonal antibodies, may be undertaken by ascites production or by direct animal cell culture. Ascites production entails injection of the hybridoma cells into the peritoneal cavity of mice (the mice essentially serve as a live fermentation chamber). The transplanted hybridoma cells produce antibody as they grow. Ascitic fluid collects in the cavity, which contains high concentrations (up to 15 mg/ml) of the desired antibody. On average, 5 ml of this fluid can be extracted per mouse. Most of the earlier monoclonal antibody preparations were produced in this manner, e.g. OKT-3, the first monoclonal antibody to be approved for therapeutic use by the FDA (see later), is produced using this strategy. 

Figure 10.2. Overview of the production by hybridoma cell culture of monoclonal antibodies destined for pharmaceutical use. Refer to text for specific details...

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