Antibody production on the process scale demands tens of grams of highly pure product. This product must be free from contaminants such as viruses that may cause adverse events in patients. Chromatography is used to create highly pure products for therapeutics in the treatment of numerous diseases. This section describes the procedure for the production of a virus-free chimeric monoclonal antibody, Cetuximab.
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Cetuximab is a chimeric monoclonal antibody with a high specificity for human epidermal growth factor receptor. This antibody is currently used to treat patients with metastatic cancers. Several different methods have been tested to produce a virus-free antibody product; one such method using a five-step chromatographic procedure is described below. In this scaled-down example, the antibody is grown in serum-free production media and spiked with the Moloney ecotropic murine leukemia virus (MuLV). This model envelope retrovirus is approximately 80–110 nm in diameter.
The purification process involves a five-step chromatographic procedure utilizing strong anion and strong cation exchange chromatography (shown to the side). A known titer of the MuLV, here 1.9 × 106 plaque-forming units (PFU) per ml, is spiked into the antibody preparation and loaded onto an anionic column. This column strongly binds contaminants such as DNA and RNA, and the effluent contains purified protein and MuLV at a lower concentration than in the original solution (5.75 × 105 PFU/ml). The effluent is then loaded onto the cation exchange column, where the antibody binds to the column. The column is washed free of contaminants, and the antibody is then eluted by applying a pH gradient. This five-step method successfully yields purified antibody and reduces viral load eightfold, thus meeting the U.S. Food and Drug Administration safety requirements.
Chromatography process flow diagram for antibody purification and virus removal.
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