Production of monoclonals via hybridoma technology

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. 

Ascites production, however, suffers from a number of drawbacks. It is costly, and the product is contaminated by significant levels of various mouse proteins, rendering subsequent downstream processing more complex. As a result, monoclonal antibody production by standard animal cell culture techniques has become the method of choice for the production of pharmaceutical-grade monoclonal antibody preparations. 

Optimal fermentation parameters have been well established and air-lift, stirred tank, and hollow fibre systems have all been used. At commercial scale, fermentation volumes in excess of 1000 litres can be used, which can yield 100 g or more of final product. While hybridoma growth is straightforward, production levels of antibody can be quite low compared with ascites-based production systems. Typically, fermentation yields antibody concentrations of 0.1-0.5 mg/ml. Removal of cells from the antibody-containing media is achieved by centrifugation or filtration. An ultrafiltration step is then normally undertaken in order to concentrate the filtrate by up to 20-fold. 

Depending upon the intended application, the antibody may next be conjugated to specific molecular tags (e.g. a radionuclide or toxin). Finally, stabilizing agents (e.g. buffer components, glycine or sometimes human serum albumin) are added to the product. This is then aseptically filled into the final containers after sterile filtration. The product is then usually freeze-dried and sealed under an atmosphere of an inert gas. 

As with all biopharmaceuticals, strict and numerous QC checks are made upon the product during all stages of manufacture. Because some issues of GMP are particularly relevant or 

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