Manufacture of pharmaceutical proteins from hybridomas and other cell substrates

Murine monoclonal antibodies are now routinely produced from highly expressing hybridoma cell lines for human clinical applications. Epstein Barr Virus transformed human lymphocytes are used to produce human monoclonal antibodies but these cells have generally exhibited low expression and poor stability in culture when compared with typical murine hybridomas. Alternative cells are therefore finding wider usage. Backfusions and tribrids are being used to increase and/or prolong the expression of monoclonal antibodies. In addition, genetically engineered cells producing antibodies with human constant and murine variable regions may reduce antigenicity of the chimeric protein used for injection. Pharmaceutical acceptability of the final product is also strongly dependent on culture and purification methods that are used in the manufacturing process. Large scale perfusion technology has allowed high density, efficient monoclonal production in the absence of antibiotics in low serum or serum free media. These methods reduce the cell protein/DNA challenge to the purification process by dead and dying cells and also prevent product degradation by reducing product residence time within the bioreactor. Gentle purification procedures that avoid extremes of pH and salt concentration can preserve structural integrity and biological activity of the protein and can provide high yields and purity in excess of 99%. Such purification procedures alone or in combination with specific viral inactivation steps can be validated to have the capability overall to reduce virus concentration by a factor of 10(8) to 10(12). The combination of carefully developed cell substrates, with advanced perfusion culture and purification technology can consistently produce acceptable protein-based pharmaceutical products in compliance with cGMP's.