Use of Copper Glutamate in Cell Culture for Production of Polypeptides
First Claim
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1. A method of producing a polypeptide in a cell culture comprising steps of:
- culturing mammalian cells that contain a gene encoding a polypeptide of interest in a cell culture medium comprising between approximately 0.5 and 5 μ
M copper and between approximately 1.7 and 33 mM glutamate under conditions and for a time sufficient to permit expression of the polypeptide, wherein the fraction of misfolded and/or aggregated polypeptide, relative to the total polypeptide produced, is decreased compared to the fraction of misfolded and/or aggregated polypeptide that would be observed under otherwise identical conditions in an otherwise identical medium that lacks copper and glutamate.
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Abstract
An improved system for large scale production of polypeptides in cell culture is provided. In accordance with the present invention, cells expressing a polypeptide of interest are grown in media that contain copper, glutamate or both. The use of such a system allows production of polypeptides in which misfolding and/or aggregation are reduced, and in which total glycosylation is increased. Polypeptides expressed in accordance with the present invention may be advantageously used in the preparation of pharmaceutical, agricultural or other commercial compositions.
102 Citations
43 Claims
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1. A method of producing a polypeptide in a cell culture comprising steps of:
culturing mammalian cells that contain a gene encoding a polypeptide of interest in a cell culture medium comprising between approximately 0.5 and 5 μ
M copper and between approximately 1.7 and 33 mM glutamate under conditions and for a time sufficient to permit expression of the polypeptide, wherein the fraction of misfolded and/or aggregated polypeptide, relative to the total polypeptide produced, is decreased compared to the fraction of misfolded and/or aggregated polypeptide that would be observed under otherwise identical conditions in an otherwise identical medium that lacks copper and glutamate.- View Dependent Claims (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 37, 38, 39, 40, 41, 42, 43)
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2. A method of producing a polypeptide in a cell culture comprising steps of:
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culturing mammalian cells that contain a gene encoding a polypeptide of interest in a cell culture medium comprising between approximately 0.5 and 5 μ
M copper and between approximately 1.7 and 33 mM glutamate;maintaining the culture at a first temperature range for a first period of time sufficient to allow the cells to reproduce to a viable cell density within a range of about 20%-80% of the maximal possible viable cell density if the culture were maintained at the first temperature range; shifting the culture to a second temperature range, wherein at least one temperature of the second temperature range is lower than the lowest temperature of the first temperature range; maintaining the culture for a second period of time under conditions and for a time sufficient to permit expression of the polypeptide, wherein the fraction of misfolded and/or aggregated polypeptide, relative to the total polypeptide produced, is decreased compared to the fraction of misfolded and/or aggregated polypeptide that would be observed under otherwise identical conditions in an otherwise identical medium that lacks copper and glutamate. - View Dependent Claims (3, 4, 5, 6)
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- 19. A cell culture medium comprising between approximately 0.5 and 5 mM copper and between approximately 1.7 and 33 mM glutamate.
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29. A method of producing a polypeptide in a cell culture comprising steps of:
culturing mammalian cells that contain a gene encoding a polypeptide of interest in a cell culture medium comprising between approximately 0.5 and 5 μ
M copper and between approximately 1.7 and 33 mM glutamate under conditions and for a time sufficient to permit expression of the polypeptide, wherein the glycosylation pattern of the expressed polypeptide is increased relative to the glycosylation pattern that would be observed on the expressed polypeptide under otherwise identical conditions in an otherwise identical medium that lacks copper and glutamate.- View Dependent Claims (31, 32, 33, 34, 35, 36)
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30. A method of producing a polypeptide in a cell culture comprising steps of:
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culturing mammalian cells that contain a gene encoding a polypeptide of interest in a cell culture medium comprising between approximately 0.5 and 5 μ
M copper and between approximately 1.7 and 33 mM glutamate;maintaining the culture at a first temperature range for a first period of time sufficient to allow the cells to reproduce to a viable cell density within a range of about 20%-80% of the maximal possible viable cell density if the culture were maintained at the first temperature range; shifting the culture to a second temperature range, wherein at least one temperature of the second temperature range is lower than the lowest temperature of the first temperature range; maintaining the culture for a second period of time under conditions and for a time sufficient to permit expression of the polypeptide, wherein the glycosylation pattern of the expressed polypeptide is increased relative to the glycosylation pattern that would be observed on the expressed polypeptide under otherwise identical conditions in an otherwise identical medium that lacks copper and glutamate.
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Specification