THE USE OF SERUM-FREE MEDIUM FOR THE PRODUCTION OF FUNCTIONALLY ACTIVE HUMANIZED MONOCLONAL-ANTIBODY FROM NSO MOUSE MYELOMA CELLS ENGINEERED USING GLUTAMINE-SYNTHETASE AS A SELECTABLE MARKER

A protein-free growth medium (W38 medium) had previously been develope d for the NS0 mouse myeloma cell line which is cholesterol-auxotrophic . This paper describes the development of a protein-free growth medium for NS0 cells expressing humanised monoclonal antibody using GS (glut amine synthetase) as a selectable marker. Several GS-engineered NS0 ce ll lines expressing humanised monoclonal antibody grew in a modificati on of W38 medium which maintained GS-selection, supplemented with chol esterol, phosphatidylcholine and beta-cyclodextrin. Further studies sh owed that additional glutamic acid, asparagine, ribonucleosides and ch oline chloride improved cell growth. Amino acid analysis identified a number of amino acids that were being depleted from the culture medium . NS0 cell lines 9D4 and 2H5 expressing CAMPATH-1H were adapted to en able them to grow serum-free in the absence of cholesterol and beta-cy clodextrin. Cholesterol-independent 9D4 (9D4.CF) cells grown in shake flask culture using an enriched protein-free medium (WNSD medium), sup plemented with human recombinant insulin (Nucellin), reached a maximum cell density to 1.86 x 10(6) cells ml(-1) producing 76.6 mg l(-1) of antibody. CAMPATH-1H antibody produced using serum-free medium was fou nd to be functionally active in vitro in the Antibody Dependant Cellul ar Cytotoxicity (ADCC) assay.