Production of mouse monoclonal antibodies using a continuous cell culture fermenter and protein G affinity chromatography.
The production of anti-alpha-fetoprotein monoclonal antibodies for diagnostic use was carried out in a stirred tank fermenter equipped with a double membrane stirrer for bubble free aeration and continuous medium perfusion. A serum-free medium supplemented with 4 mM L-glutamine and 2.0 milligrams glucose with a protein content of only 780 micrograms/ml was used for the production process. The harvested antibodies were concentrated 50-fold using a tangential ultrafiltration system and were then purified in a one step purification process by protein G affinity chromatography. The purity of the final product (90%) was controlled by SDS-polyacrylamide gel electrophoresis, gel exclusion chromatography and isoelectric focussing. For further quality controls of the product the immunoglobulin subclass and the isoelectric point were determined and the specificity of the purified mAb was tested by RIA using 125I labelled alpha-fetoprotein. 1.87 g of purified monoclonal antibodies were produced (90% purity) within 2 weeks. It was found that the use of this type of stirred tank fermenter combined with a one step purification process using protein G affinity chromatography represents a suitable method for the fast production of medium scale quantities (500 mg-5 g) of monoclonal antibodies for diagnostic use.
Zamboni
A
A
Research Department 1, Byk Gulden Italia, Cormano MI.
Giuntini
I
I
Gianesello
D
D
Maddalena
F
F
Rognoni
F
F
Herbst
D
D
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D002460:Cell Line; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D005285:Fermentation; D006825:Hybridomas / Q000378:metabolism*; D051379:Mice; D009419:Nerve Tissue Proteins
D000911:Antibodies, Monoclonal; C029152:G-substrate; D009419:Nerve Tissue Proteins