Fed-batch process for production of erythropoietin in human embryonic retina cells that express adenovirus E1A
AssigneeCrucell Holland B.V.
InventorChristopher A. Yallop
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Abstract
The invention provides processes for recombinant production of erythropoietin (EPO) in a human embryonic retina cell that expresses at least an adenoviral E1A protein, wherein said EPO is produced at high concentrations and wherein said EPO as produced has a high average sialic acid content per EPO molecule.
Claims19
A process for recombinant production of erythropoietin (EPO) in a human embryonic retina cell that expresses at least an adenoviral E1A protein, wherein said EPO is produced at a concentration of at least 3000 eU/ml, and wherein said EPO as produced has an average sialic acid content per EPO molecule of at least 7, the process comprising:
- a) providing an immortalized human embryonic retina cell that expresses at least an adenoviral E1A protein, said cell further comprising a nucleic acid encoding a sialyltransferase under control of a heterologous promoter, and said cell further comprising nucleic acid encoding EPO under control of a heterologous promoter;
- b) culturing the cell in a serum-free culture medium in a fed-batch process, wherein at least once during said culturing according to needs of nutrients glucose and at least one medium component selected from the group consisting of glutamine, leucine, serine, isoleucine, arginine, methionine, and cystine are added depending on the requirements of the cells;
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- c) harvesting EPO from the culture medium, wherein the EPO is present in the culture medium at a concentration of at least 3000 eU/ml and wherein said EPO as produced has an average sialic acid content per EPO molecule of at least 7.
A process for recombinant production of erythropoietin (EPO) at a concentration of at least 5000 eU/ml, the process comprising:
- a) providing a human embryonic retina cell that expresses at least an adenoviral E1A protein, and said cell further comprising nucleic acid encoding EPO under control of a heterologous promoter;
- b) culturing the cell in a culture medium in a fed-batch process, wherein at least once during said culturing according to needs of nutrients at least one essential amino acid and at least one carbohydrate are added depending on the requirements of the cells, adding, in an end concentration in mmoles/l of freshly added component per 10×106 cells/ml, between 4.0 and 8.0 for glucose, and between 1.17 and 3.47 for glutamine;
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- c) harvesting EPO from the culture medium, wherein the EPO is present at a concentration of at least 5000 eU/ml.
Dependencies are inferred from the claim text.