TY - JOUR
T1 - Efficient laboratory-scale production of monoclonal antibodies using membrane-based high-density cell culture technology
AU - Trebak, Mohamed
AU - Chong, Jae Min
AU - Herlyn, Dorothee
AU - Speicher, David W.
PY - 1999/11/19
Y1 - 1999/11/19
N2 - 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 (107-108 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. Copyright (C) 1999 Elsevier Science B.V.
AB - 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 (107-108 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. Copyright (C) 1999 Elsevier Science B.V.
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U2 - 10.1016/S0022-1759(99)00122-2
DO - 10.1016/S0022-1759(99)00122-2
M3 - Article
C2 - 10594354
AN - SCOPUS:0032758496
VL - 230
SP - 59
EP - 70
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
SN - 0022-1759
IS - 1-2
ER -