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Biotechnology Paper

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Biotechnology Paper

Biotechnology products are the yield of engineering labors that process biological material and agents to produce a modified biological substance. Examples of biotechnology drugs are monoclonal antibodies and recombinant DNA. Monoclonal antibodies are important reagents in the treatment and diagnosis of disease. They have been used for diagnosis of pregnancy, detection of presence and concentration of drugs in the blood, histocompatibility assay, and detecting shed tumor antigens. They are antibodies that are mass produced in a laboratory from a single clone that recognizes only one epitope of a single antigen. Recombinant DNA is DNA that has been created artificially and codes for a specific desired protein for harvest. New biotechnology drug discovery begins with identification of cellular and genetic factors that play a role in specific diseases. Chemical and biological substances that target these factors are likely to have drug-like effects and are searched for. If an antigen is identified as a disease causing agent, a monoclonal antibody could be an appropriate therapeutic agent to develop. Or if a single protein is likely to exert a pharmacological effect recombinant DNA may be appropriate.

After the desired chemical substances have been identified, research for production can begin. The processes of recombinant DNA production and Monoclonal antibody production are very different. Most antigens have many antibody binding sites, or epitopes. Therefore, many different antibodies may bind to any one antigen. Monoclonal antibodies are specific for only one antigen epitope. Epitope specificity brings several advantages including dose response predictability and uniformity of batches. In 1975 Kohler and Milstein developed a process that produced monoclonal antibody.

The generation of monoclonal antibodies requires a living organism, normally, a mouse. The first step in monoclonal antibody production is immunization of mice. The mice are injected with antigen. Then, the mice are allowed to produce antibody to the injected antigen. Serum antibody titer is analyzed by ELISA. After the mice have developed a maximal amount of antibody the mice are euthanized. The killed mice spleens are then harvested. Antibody producing spleen cells created by the mice are isolated and hybridized with mylomia cells. This is accomplished by co-centrifuging with polyethylene glycol, which causes cell membranes to fuse. Antibody producing spleen cells have a limited life span. Fusing these cells with cells derived from an immortal mylomia cell results in a hybrid cell that is capable of dividing unlimitedly.

Hybridoma cells are selectively isolated by utilizing the HAT (hypoxanthine-aminopterin - thymidine) selection growth media. In this process myloma-myloma and spleen cell-spleen cell fusions are eliminated leaving only spleen cell-myloma hybrids. The HAT growth medium contains aminopterin which is an inhibitor of the pathways that the homogenously fused cells use to make nucleotides and therefore DNA. So, the cells must use a bypass pathway that is defective in the myeloma cell line to which the normal antibody producing cells are fused. Because neither the myeloma nor the antibody producing cell will grow on its own only the hybrids grow. The hybridoma fusions are exclusively left on the growth media.

The fused cells then need to be separated for antigen epitope specificity. This can be accomplished by limiting dilution. This process dilutes a solution of hybridomas to a single hybridoma. The single hybridomas are cultured in wells until a large amount of antibody is produced. Each well now contains a monoclonal antibody producing colony of hybridoma cells. The antibodies of each well are tested to determine which antibody best binds the original antigen. The respective monoclonal antibody producing colonies are then cloned by limiting dilution to ensure monoclonality. Finally, the monoclonal antibodies are purified by antigen affinity chromatography and a monoclonal antibody has been effectively isolated and reproduced.

Recombinant DNA production also requires a living organism. In 1973 Boyer and Cohen developed a method to insert DNA from one organism into the DNA of bacteria. The first step in producing a recombinant DNA protein is to identify what portion of host DNA codes for a desired protein. After the DNA has been identified, the specified section of host DNA is removed and isolated using unambiguous DNA cutting proteins. The DNA fragment is inserted into a previously opened bacteria plasmid. The altered plasmid is inserted back into the bacteria. The plasmid replicates and produces many copies of the exogenous gene. Finally, the bacteria are stimulated to transcript the gene for the protein that it codes for and the protein can be harvested.

Infliximab is a monoclonal antibody produced by Centocor Inc. Infliximab is composed of human constant and murine variable antibody regions. Infliximab is produced by a recombinant cell line cloned from a single cell isolated by limiting diffusion and is purified by steps that inactivate and remove viruses. Infliximab binds specifically to Tumor Necrosis Factor Ñ"С (Ñ"nÑ"vTNF ÐŽV Ñ"С) Ñ"w TNF ÐŽV Ñ"С Ñ"nrelated biological activities include induction of inflammation factors such as interleukins 1 and 6 (IL-1 and IL-6), enhancement of leukocyte migration by increasing endothelial layer permeability and expression of adhesion molecules by endothelial cells and leukocytes, activation of neutrophil and eosinophil functional activity, induction of acute phase reactants and other liver proteins, as well as tissue destroying enzymes produced by synoviocytes and chondrocytes. TNF ÐŽV Ñ"С Ñ"nis found in elevated concentrations in tissues and fluids of patients with Rheumatoid arthritis, CrohnÐŽ¦s Disease, and ankylosing spondylitis. Infliximab binds exclusively to TNF - Ñ"СÑ"n and encourages antigen - antibody complex removal. Cells expressing membrane TNF - Ñ"СÑ"n are bound by Infliximab and are lysed.

Rheumatoid arthritis is an autoimmune disease that causes chronic inflammation of the joints. The cause of rheumatoid arthritis is unknown. Tissue inflammation and destruction of the joints affected by rheumatoid arthritis is mediated by TNF ÐŽV Ñ"С Ñ"|Ñ"n. Ñ"n The safety and efficacy of Infliximab treatment of rheumatoid arthritis has been assessed in two clinical trials. In the first trial, n=428 and placebo controlled, patients with active rheumatoid arthritis were treated with Methotrexate, an immunosuppressive agent and Infliximab. PatientÐŽ¦s received either Infliximab and Methotrexate or Placebo and Methotrexate. In this study, all patients



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