Efficient laboratory-scale production of monoclonal antibodies using membrane-based high-density cell culture technology

Monoclonal antibodies (MAbs) are important tools used in basic research as well as in the imaging and therapy of cancer. Many countries have limited t he use of animals for large-scale production of MAbs, obliging laboratories to find efficient in vitro alternatives to ascites production. In this rep ort we describe a protocol for laboratory-scale production of MAbs by cultu ring hybridoma cells in the two-chamber cell culture device CELLine 1000, T his culture flask supports high cell densities (10(7)-10(8) cells/ml) and g enerates high concentrations of MAbs (0.7-2.5 mg/ml). Two hybridomas produc ing MAbs directed against the gastrointestinal antigen GA733-2, GA733 MAb a nd CO17-1A MAb, were evaluated over culture periods of up to two months usi ng several alternative conditions. Two different sets of conditions are rep orted; the first using serum-supplemented medium (20% v/v) and the second u sing serum-free medium (SFM). Average weekly yields of the purified MAbs in serum-supplemented medium were 24 mg and 33 mg, and in SFM were 21 mg and 17 mg for GA733 MAb and CO17-1A MAb, respectively. Experimental variables t hat can affect antibody production and economy include: nutrient medium and cell compartment medium compositions (cell line dependent), the proportion of the cell compartment medium harvested every 3 days (50% to 80% with 80% optimal) and the frequency of nutrient medium changes (3 to 9 days with 6 days as most cost effective). Protein-A Sepharose purification followed by antigen-specific affinity purification showed that MAbs obtained from serum -supplemented cultures contain less than 0.6% of bovine Ige contamination, while MAbs obtained from serum-free cultures contained no extraneous IgG. I n addition, MAbs from both culture media were fully active (essentially 100 %) as measured by their ability to bind to an antigen column. In contrast, the same MAbs purified from ascites using Protein-A-Sepharose typically con tained a major portion of inactive IgG, This in vitro method for laboratory -scale production of MAbs (10 to 500 mg) proved to be simple, reproducible and cost effective. It represents a useful alternative to the in vivo produ ction of MAbs in mice. (C) 1999 Elsevier Science B.V. All rights reserved.