Identification of human cell lines for the production of human proteins by endogenous gene activation
AssigneeRoche Diagnostics GmbH
InventorsMichael Brandt, Reinhard Franze, Ulrich Pessara
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Abstract
A process for selecting human cells for the production of human proteins by endogenous gene activation allows human proteins to be produced in economically feasible quantities and in a form suitable for producing a pharmaceutical composition. Also disclosed is a process for producing human proteins in a cell line identified in this matter.
Claims15
Method for the selection of human cell lines for the preparation of human proteins by activation of a target gene endogenously present in the cell,
- characterized in that
- (a) a human cell line is tested for the presence of the following features:
- (i) a target gene with the desired nucleic acid sequence,
- (ii) at least 5 population doublings within 14 days in a suspension culture, and
- (iii) at least 5 population doublings within 14 days in a serum-free culture medium, and
- (b) a cell line satisfying features (i), (ii) and (iii) is used as a starting cell line for the endogenous activation of the target gene.
Method according to any one of the foregoing claims,
- characterized in that
- a human cell line is used which (v) shows no substantial endogenous expression of the target gene.
Method according to any one of the foregoing claims,
- characterized in that
- a human cell line is used which (vi) contains more than 2 chromosomal copies of the target gene.
Method according to any one of the foregoing claims,
- characterized in that
- a human cell line is used which (vii) synthesizes the cell protein with a glycosylation pattern comparable to the naturally occurring target protein.
Method according to any one of the foregoing claims,
- characterized in that
- a human cell line is used which (viii) is free of detectable infectious contamination.
Method according to any one of the foregoing claims for the preparation of a human factor selected from EPO, TPO, colony stimulating factors, proteins which affect blood coagulation, interferons, interleukins, chemokines, neurotrophic factors, proteins which affect bone growth, hedgehog proteins, tumor growth factor, growth hormones, ACTH, encephalins, endorphins, receptors and other protein-binding proteins.
Human cell line,
- characterized in that
- it contains a copy of an endogenous gene in operable linkage with a heterologous promoter active in the human cell and is capable of the production of at least 200 ng of the polypeptide encoded by the endogenous gene/106 cells/24 h.
Human cell line,
- characterized in that
- it contains several copies of an endogenous gene, each in operative connection with a heterologous promoter active in the human cell, and is capable of the production of at least 1000 ng of the polypeptide encoded by the endogenous gene/106 cells/24 h.
Dependencies are inferred from the claim text.