Monoclonal production

The procedures for performing monoclonal fusions have been described elsewhere (Harlow and Lane 1988). It is important that the individual performing the fusion be adept at mammalian cell culture and have a record of successM monoclonal production. A good mouse is too precious to waste due to the incompetence of a monoclonal facility. 

Immunoaffinity chromatography utilizes the high specificity of antigen—antibody interactions to achieve a separation. The procedure typically involves the binding, to a soHd phase, of a mouse monoclonal antibody which reacts either directly with the protein to be purified or with a closely associated protein which itself binds the product protein. The former approach has been appHed in the preparation of Factor VIII (43) and Factor IX (61) concentrates. The latter method has been used in the preparation of Factor VIII (42) by immobilization of a monoclonal antibody to von WiHebrand factor [109319-16-6] (62), a protein to which Factor VIII binds noncovalenfly. Further purification is necessary downstream of the immunoaffinity step to remove... 

Committee For Proprietary Medicinal Products, Xotes to Applicants for Marketing Authorisations on the Production and Quality Control of Monoclonal... 

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. 

Bioprocess plants are an essential part of food, fine chemical and pharmaceutical industries. Use of microorganisms to transform biological materials for production of fermented foods, cheese and chemicals has its antiquity. Bioprocesses have been developed for an enoimous range of commercial products, as listed in Table 1.1. Most of the products originate from relatively cheap raw materials. Production of industrial alcohols and organic solvents is mostly originated from cheap feed stocks. The more expensive and special bioprocesses are in the production of antibiotics, monoclonal antibodies and vaccines. Industrial enzymes and living cells such as baker s yeast and brewer s yeast are also commercial products obtained from bioprocess plants. 

Inhibition of inflammatory cytokines (Fig. 2) Humanized monoclonal anti-TNF antibodies (Infliximab (Remicade ), Adalimumab (Humira )) bind with high selectivity to human TNF-a and neutralize its activity. Thereby, infliximab decreases the effects of enhanced TNF levels during inflammatory disease such as production of proteases, chemokines, adhesion molecules, cyclooxygenase products (prostaglandins), and proinflammatory molecules such as interleukin-1 and -6. The antibodies may also recognize membrane-bound TNF-a on lymphocytes and other immune cells. These cells may subsequently become apoptotic or are eliminated via Fc-receptor-mediated phagocytosis. 

Two main strategies are presently used to suppress immune responses (summarized in Fig. 3). The first focuses on cytokines, the central mediators of the immune system. Efficient inhibition of cytokine production can be achieved by glucocorticoids. Specific anticytokine strategies include the use of monoclonal antibodies, soluble receptors, or receptor constructs. 

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

The main disadvantage of all these systems is the Hmitation of scale-up. Monoclonal antibodies are produced by multiplying the hollow fiber systems and stirred tank reactors with membrane aeration are known up to 100 liter. Small quantities of product can be produced by these systems but they are not suitable for real industrial scale-up. 

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

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