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
Mj. Keen et C. Hale, 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, Cytotechnology, 18(3), 1995, pp. 207-217
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.