Use of copper and glutamate in cell culture for production of polypeptides
View Patent ↗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.
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 0.7 and 3 μM copper and between 2.5 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 in said medium that lacks copper and glutamate.
2. 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 0.7 and 3 μM copper and between 2.5 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 in said medium that lacks copper and glutamate.
3. The method of claim 2 , wherein the first temperature range comprises a temperature range that is approximately 30 to 42 degrees Celsius.
4. The method of claim 2 , wherein the second temperature range comprises a temperature range that is approximately 25 to 41 degrees Celsius.
5. The method of claim 2 , including a second shifting step subsequent to said first shifting step comprising shifting said culture to a third temperature or temperature range, wherein at least one temperature of the third temperature range is lower than the lowest temperature of the second temperature range.
6. The method of claim 5 , wherein the third temperature range comprises a temperature range that is approximately 25 to 40 degrees Celsius.
7. The method of claim 1 , wherein the medium comprises an initial glutamine concentration, wherein the initial glutamine concentration of the cell culture medium is less than or equal to 4 mM.
8. The method of claim 1 , wherein the cell culture is further supplemented with approximately 2 grams per liter glucose.
9. The method of claim 1 , wherein the cell culture is further supplemented with supplementary components.
10. The method of claim 9 , wherein the supplementary components are provided in a feed medium.
11. The method of claim 10 , wherein the supplementary components are provided at multiple intervals.
12. The method of claim 1 , wherein the cell culture medium is defined.
13. The method of claim 12 , wherein the defined cell culture medium does not contain added serum or hydrolysates.
14. The method of claim 12 , wherein the defined cell culture medium is protein-free.
15. The method of claim 1 , wherein the cell culture medium comprises between 0.7 and 1.5 μM copper.
16. The method of claim 15 , wherein the cell culture medium comprises 1 μM copper.
17. The method of claim 1 , wherein the cell culture medium comprises between 3 and 7 mM glutamate.
18. The method of claim 17 , wherein the cell culture medium comprises approximately 5 mM glutamate.
19. 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 0.7 and 3 μM copper and between 2.5 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 in said medium that lacks copper and glutamate.
20. 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 0.7 and 3 μM copper and between 2.5 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 in said medium that lacks copper and glutamate.
21. The method of claim 19 , wherein the increased glycosylation pattern of the expressed polypeptide comprises an increase in total sialylation.
22. The method of claim 19 , wherein the cell culture medium is defined.
23. The method of claim 22 , wherein the defined cell culture medium does not contain added serum or hydrolysates.
24. The method of claim 22 , wherein the defined cell culture medium is protein-free.
25. The method of claim 19 , wherein the cell culture medium comprises between 0.7 and 1.5 μM copper.
26. The method of claim 25 , wherein the cell culture medium comprises 1 μM copper.
27. The method of claim 1 , wherein the polypeptide is TNFR-Ig.
28. The method of claim 1 , wherein the polypeptide is TNFR-Fc.
29. The method of claim 1 , wherein the polypeptide is a polypeptide selected from the group consisting of: an enzyme, a clotting factor, a receptor, an antibody, a hormone, a regulatory factor, an antigen, and a binding agent.
30. The method of claim 29 , wherein the polypeptide is an anti-Abeta antibody.
31. The method of claim 1 , wherein the step of culturing comprises culturing the mammalian cells in a batch culture.