title: Efficient laboratory-scale production of monoclonal antibodies using membrane-based high-density cell culture technology.
abstract: Monoclonal antibodies (MAbs) are important tools used in basic research as well as in the imaging and therapy of cancer. Many countries have limited the use of animals for large-scale production of MAbs, obliging laboratories to find efficient in vitro alternatives to ascites production. In this report we describe a protocol for laboratory-scale production of MAbs by culturing hybridoma cells in the two-chamber cell culture device CELLine 1000. This culture flask supports high cell densities (10(7)-10(8) cells/ml) and generates high concentrations of MAbs (0.7-2.5 mg/ml). Two hybridomas producing MAbs directed against the gastrointestinal antigen GA733-2, GA733 MAb and CO17-1A MAb, were evaluated over culture periods of up to two months using several alternative conditions. Two different sets of conditions are reported; the first using serum-supplemented medium (20% v/v) and the second using 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 that 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 IgG contamination, while MAbs obtained from serum-free cultures contained no extraneous IgG. In 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 contained 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 production of MAbs in mice.
authors.affiliation: The Wistar Institute, Room 151, 3601 Spruce Street, Philadelphia, PA 19104, USA.
authors.forename: M
authors.initials: M
authors.lastname: Trebak
authors.forename: J M
authors.initials: JM
authors.lastname: Chong
authors.forename: D
authors.initials: D
authors.lastname: Herlyn
authors.forename: D W
authors.initials: DW
authors.lastname: Speicher
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000302:isolation & purification; D000951:Antigens, Neoplasm; D001709:Biotechnology; D002417:Cattle; D015815:Cell Adhesion Molecules; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D003470:Culture Media; D016895:Culture Media, Serum-Free; D004340:Drug Contamination; D000071858:Epithelial Cell Adhesion Molecule; D005069:Evaluation Studies as Topic; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007158:Immunologic Techniques / Q000295:instrumentation; D008567:Membranes, Artificial; D051379:Mice
chemical_list: D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D003470:Culture Media; D016895:Culture Media, Serum-Free; D000071858:Epithelial Cell Adhesion Molecule; D008567:Membranes, Artificial