Propagation of a mouse myeloma cell line J558L producing human CD4 immunoglobulin G1.
Transfected mouse myeloma cells are of increasing interest for the production of a wide variety of solubilised recombinant fusion proteins. A stably transfected J558L mouse myeloma subclone (J558L-CD4) secreting human CD4-immunoglobulin type G1 receptor (CD4-H gamma 1) was employed as a model system for cell suspension culture and expression of chimaeric molecules. Cells were grown up to 3-5 x 10(6) cells/ml in serum-free and protein-reduced DHI medium consisting of a mixture of DMEM, HamF12 and IMDM media supplemented with transferrin, insulin, Primatone RL and Pluronic F68. Primatone RL was the essential growth-promoting factor in protein-free medium. The soluble CD4-H gamma 1 receptor, the production of which was not growth-associated, accumulated in the medium to concentrations of 40 micrograms/ml with a specific formation rate of 0.18 micrograms/10(6) cells/h in conventional cultures. The cell density was further increased by growing the cells in dialysis tubing or by using a perfusion system with cell retention. Because of the continuous exchange of nutrients and metabolic end-products average concentrations of 35 x 10(6) cells/ml were achieved. CD4-H gamma 1 accumulated in the dialysis tubing up to 1.3 mg/ml. After an initial rapid growth period, a ten-fold reduction in specific nutrient consumption rates and metabolic end-product formation was observed. Chimaeric proteins purified by protein G chromatography from conventional and perfusion cultures were indistinguishable when compared by SDS-PAGE, limited proteolysis and isoelectric focusing analysis (isoelectric point: 8.5-8.6).
Schlaeger
E J
EJ
Pharma Research, New Technologies, F. Hoffmann-LaRoche Ltd., Basle, Switzerland.
Schumpp
B
B
D000596:Amino Acids / Q000032:analysis; D000641:Ammonia; D000818:Animals; D000998:Antiviral Agents; D015704:CD4 Antigens / Q000096:biosynthesis* / Q000302:isolation & purification; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D005947:Glucose / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis* / Q000302:isolation & purification; D007142:Immunoglobulin gamma-Chains; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology; D007525:Isoelectric Focusing; D007773:Lactates / Q000096:biosynthesis; D019344:Lactic Acid; D051379:Mice; D009101:Multiple Myeloma / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D014162:Transfection; D014168:Transferrin / Q000494:pharmacology
D000596:Amino Acids; D000998:Antiviral Agents; D015704:CD4 Antigens; C063735:CD4-IgG1 hinge fusion protein, recombinant; D007143:Immunoglobulin Heavy Chains; D007142:Immunoglobulin gamma-Chains; D007328:Insulin; D007773:Lactates; D011993:Recombinant Fusion Proteins; D014168:Transferrin; D005973:Glutamine; D019344:Lactic Acid; D000641:Ammonia; D005947:Glucose