Abstract
A protein-free growth medium (W38 medium) had previously been developed 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 (glutamine synthetase) as a selectable marker. Several GS-engineered NS0 cell lines expressing humanised monoclonal antibody grew in a modification of W38 medium which maintained GS-selection, supplemented with cholesterol, phosphatidylcholine and β-cyclodextrin. Further studies showed that additional glutamic acid, asparagine, ribonucleosides and choline 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 enable them to grow serum-free in the absence of cholesterol and β-cyclodextrin. Cholesterol-independent 9D4 (9D4.CF) cells grown in shake flask culture using an enriched protein-free medium (WNSD medium), supplemented with human recombinant insulin (Nucellin), reached a maximum cell density to 1.86×106 cells ml−1 producing 76.6 mg l−1 of antibody. CAMPATH-1H antibody produced using serum-free medium was found to be functionally activein vitro in the Antibody Dependant Cellular Cytotoxicity (ADCC) assay.
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Abbreviations
- C:
-
cholesterol
- CD:
-
cyclodextrin
- dhfr:
-
dihydrofolate reductase
- F68:
-
Pluronic F68
- GS:
-
glutamine synthetase
- MSX:
-
methionine sulphoximine
- P:
-
phosphatidylcholine
- PC-FBS:
-
phosphatidylcholine, cholesterol and foetal bovine serum
- RPMI:
-
RPMI 1640 medium
- ADCC:
-
Antibody-dependant cellular cytotoxicity
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Keen, M.J., Hale, C. The use of serum-free medium for the production of functionally active humanised monoclonal antibody from NS0 mouse myeloma cells engineered using glutamine synthetase as a selectable marker. Cytotechnology 18, 207–217 (1995). https://doi.org/10.1007/BF00767768
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DOI: https://doi.org/10.1007/BF00767768