Hybridoma preparation

All of the processes and considerations described for polyclonal antibody production are also relevant to monoclonal antibody production. The only variation between the two, with respect to the the immunization process, is species selection. Monoclonal antibodies are most commonly produced in mice because of the commercial availability of mouse myeloma cells that are particularly suited as partner cells in hybridoma preparation (discussed below). 

Figure 13.5 Outline of the production strategy of CEA-SCAN. The antibody-producing hybridoma cell line was originally obtained by standard methods of hybridoma generation. Spleen-derived murine B-lymphocytes were fused with murine myeloma calls. The resulting stable hybridomas were screened for the production of anti-CEA monoclonals. The clone chosen produces an IgG anti-CEA antibody. Note that the finished product outlined above is not radiolabelled. The freeze-dried antibody preparation (which has a shelf life of 2 years at 2-8 °C) is reconstituted immediately prior to its medical use. The reconstituting solution contains 99mTc, and is formulated to facilitate direct conjugation of the radiolabel to the antibody fragment...

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. 

In addition to recombinant biopharmaceuticals, animal cell culture is used to produce various other biologically-based pharmaceuticals. Chief amongst these are a variety of vaccines and hybridoma cell-produced monoclonal antibodies (Chapter 10). Earlier interferon preparations were also produced in culture by a particular lymphoblastoid cell line (the Namalwa cell line), which was found to naturally synthesize high levels of several interferon-as (Chapter 4). 

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. 

Perfusion conhgurations can grow cells for up to two months, without having to reseed the cells. Consequently the process poses a risk of contamination. However, it has been shown that the preparation of some antibodies by means of hybridoma cell culture, which may last up to 30 days, can be realized with little or no contaminahon. 

Figure 10.3. Schematic representation of monoclonal antibody production using immortalized hybrid cells that secrete antibodies selective for the target antigen. The mortal, immune B cells Isolated from mice immunized with a target antigen are fused with myeloma, immortal B cells that express defective antibodies. The selecting of antigen-specific, immortal hybrid cells (hybridomas) results in identification of unique clones of cells that express antibodies with high specificity and affinity (monoclonal antibodies). These cells are cloned and expanded for large-scale monoclonal antibody preparations.

Media basal RPMI 1640 medium requires supplementation with sodium pyruvate, L-glutamine, and penicillin/streptomycin before use. The supplements are supplied as concentrates of 50X or 100X, and the appropriate amounts should be added. Standard tissue culture media for hybridoma production contain 5%, 10%, or 15% fetal bovine serum (FBS see Note 6). Sufficient quantities should be prepared in advance and a sterility check should be performed on them prior to use. 

The problem is that if an individual antibody-producing cell is isolated and grown in culture, its descendants have a limited lifespan that severely limits their use for the routine preparation of monoclonal antibodies. In 1975, Milstein and Kohler discovered how monoclonal antibodies of almost any desired antigen specificity can be produced indefinitely and in large quantities. Their method was to fuse a B lymphocyte producing antibody of the desired specificity with a cell derived from a cancerous lymphocyte tumor, called a myeloma cell, which is immortal. The cell fusion is called a hybridoma, which is both immortal and secretes the same specific antibody originally encoded by the B lymphocyte. 

Depending on the use of the mAbs, certain adaptations may be required for their preparation. When large quantities of mAbs are required, in vivo production of the antibody in ascitic fluid is not practical, because it will require the use of a large number of animals. Thus, it is often easier to cultivate the antibodies in an appropriate in vitro culture medium. However, given the strict nutritional requirements of the hybridomas and their fragility in the face of osmolality, pH variations, and the accumulation of metabolites, the production of large quantities of antibodies in vitro will necessitate special care. 

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