GLYCOLYSIS-INHIBITING SUBSTANCES IN CELL CULTURE

• US 20080108106A1
• Filed: 11/02/2007
• Published: 05/08/2008
• Est. Priority Date: 11/03/2006
• Status: Active Grant

• Current Assignee: Wyeth LLC (Pfizer Inc.)
• Original Assignee: Wyeth (Pfizer Inc.)
• Inventors: Luan, Yen, Wang, Wenge
• Granted Patent: US 8,192,951 B2
• Time in Patent Office: Days
• US Class Current: 435/69.1
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2510/02 Cells for production
• CPC Class Codes: C12N 5/0018 Culture media for cell or tissue culture media for specific animal cell type C12N5/06

Abstract

An improved system for large scale production of proteins and/or polypeptides in cell culture is provided. In accordance with the present invention, cells expressing the protein or polypeptide of interest are grown in media that comprise a glycolysis-inhibiting substance. Additionally and/or alternatively, cells expressing the protein or polypeptide of interest are grown in media in which glutamine is limited. The use of such a system allows high levels of protein or polypeptide production and lessens accumulation of undesirable metabolic waste products such as lactate. Proteins and polypeptides expressed in accordance with the present invention may be advantageously used in the preparation of pharmaceutical, immunogenic, agricultural or other commercial compositions.

Claims

1. A cell culture medium comprising a glycolysis-inhibiting substance.

2. The medium of claim 1 wherein the glycolysis-inhibiting substance is 2-deoxyglucose.

3. The medium of claim 2, wherein 2-deoxyglucose is present at between approximately 0.25 grams per liter and 1 gram per liter.

4. The medium of claim 2, further comprising glucose, wherein the ratio of 2-deoxyglucose to glucose is equal to or less than about 1 to 9.

5. The medium of claim 1, further comprising glutamine at less than approximately 13 mM.

6. The medium of claim 1, wherein the medium further comprises glucose present at approximately 8 g/L or above.

7. The medium of claim 1, wherein the cell culture medium has a medium characteristic selected from the group consisting of:

(i) a cumulative amino acid concentration greater than about 70 mM, (ii) a molar cumulative glutamine to cumulative asparagine ratio of less than about 2, (iii) a molar cumulative glutamine to cumulative total amino acid ratio of less than about 0.2, (iv) a molar cumulative inorganic ion to cumulative total amino acid ratio between about 0.4 to 1, (v) a combined cumulative glutamine and cumulative asparagine concentration between about 16 and 36 mM, and combinations thereof.

8. The medium of claim 1, further comprising manganese at a concentration between approximately 10 and 600 nM.

9. The medium of claim 1, wherein the medium is defined.

10. The medium of claim 1, wherein the glycolysis-inhibiting substance is selected from the group consisting of 2-deoxyglucose, di(2-ethyl hexyl)phosphate, tributyl phosphate, dodecyl phosphate, 2-dimethylamino ethyl ester of (diphenyl methyl)-phosphoric acid, [2-(diphenyl phosphinyloxy)ethyl]trimethyl ammonium iodide, iodoacetate, fluoroacetate, and combinations thereof.

11. A method of producing a polypeptide in cell culture comprising steps of:

culturing mammalian cells that contain a gene encoding a polypeptide of interest, which gene is expressed under conditions of cell culture, in a cell culture medium comprising a glycolysis-inhibiting substance; and

maintaining the culture under conditions and for a time sufficient to permit expression of the polypeptide such that lactate levels of the culture are lower than lactate levels produced under otherwise identical conditions in an otherwise identical medium that lacks the glycolysis-inhibiting substance.

13. The method of claim 11 or 12, wherein the glycolysis-inhibiting substance is 2-deoxyglucose.

14. The method of claim 13, wherein 2-deoxyglucose is present in the cell culture medium at between approximately 0.25 grams per liter and 1 gram per liter.

15. The method of claim 13, wherein the cell culture medium further contains glucose, and wherein the ratio of 2-deoxyglucose to glucose is equal to or less than about 1 to 9.

16. The method of claim 11 or 12, wherein the cell culture medium further comprises glutamine at less than approximately 13 mM.

17. The method of claim 11 or 12, wherein the cell culture medium further comprises glucose, and wherein the level of glucose is maintained at approximately 8 g/L or above.

18. The method of claim 11 or 12, wherein the cell culture medium has a medium characteristic selected from the group consisting of:

(i) a cumulative amino acid concentration greater than about 70 mM, (ii) a molar cumulative glutamine to cumulative asparagine ratio of less than about 2, (iii) a molar cumulative glutamine to cumulative total amino acid ratio of less than about 0.2, (iv) a molar cumulative inorganic ion to cumulative total amino acid ratio between about 0.4 to 1, (v) a combined cumulative glutamine and cumulative asparagine concentration between about 16 and 36 mM, and combinations thereof.

19. The method of claim 11 or 12, wherein the cell culture medium further comprises manganese at a concentration between approximately 10 and 600 nM.

20. The method of claim 11 or 12, wherein the step of providing comprises providing at least 500 liters of culture.

21. The method of claim 11 or 12, wherein the cell culture is further provided with supplementary components.

22. The method of claim 21, wherein the supplementary components are provided in a feed medium.

23. The method of claim 21, wherein the supplementary components comprise 2-deoxyglucose.

24. The method of claim 21 wherein the supplementary components are selected from the group consisting of hormones and/or other growth factors, particular ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds usually present at very low final concentrations), amino acids, lipids, glucose or other energy source, and combinations thereof.

31. The method of claim 11 or 12, wherein the glycolysis-inhibiting substance is selected from the group consisting of 2-deoxyglucose, di(2-ethyl hexyl)phosphate, tributyl phosphate, dodecyl phosphate, 2-dimethylamino ethyl ester of (diphenyl methyl)-phosphoric acid, [2-(diphenyl phosphinyloxy)ethyl]trimethyl ammonium iodide, iodoacetate, fluoroacetate, and combinations thereof.

32. The method of claim 11 or 12 wherein the polypeptide is selected from the group consisting of:

an enzyme, a receptor, an antibody, a hormone, a regulatory factor, an antigen, a binding agent, a clotting factor, a growth factor, and combinations thereof.

33. The method of claim 32, wherein the polypeptide is an antibody.

34. The method of claim 33, wherein the antibody is an anti-ABeta antibody.

35. The method of claim 33, wherein the antibody binds to an epitope more strongly, with more specificity, or both, than an antibody produced under otherwise identical conditions in an otherwise identical medium that lacks the glycolysis-inhibiting substance.

12. 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, which gene is expressed under conditions of cell culture, in a cell culture medium comprising a glycolysis-inhibiting substance;

maintaining the culture at a first temperature range for a first period of time;

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; and

maintaining the culture for a second period of time under conditions and for a time sufficient to permit expression of the polypeptide, wherein the lactate levels of the culture are lower than lactate levels observed under otherwise identical conditions in an otherwise identical medium that lacks the glycolysis-inhibiting substance.

25. The method of claim 12, wherein the first temperature range is 30 to 42 degrees Celsius.

26. The method of claim 12, wherein the second temperature range is 25 to 41 degrees Celsius.

27. The method of claim 12, wherein the step of shifting comprises shifting when the viable cell density of the culture is within a range of about 6×

10⁶ cell/mL to 12×

10⁶ cells/mL, such that the cells will begin to take up lactate during the second period of time.

28. The method of claim 12, wherein the step of shifting comprises shifting when the lactate concentration of the culture is less than about 8 grams per liter, such that the cells will begin to take up lactate during the second period of time.

29. The method of claim 12, including a second shifting step subsequent to the first shifting step comprising shifting the 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.

30. The method of claim 29, wherein the third temperature range is 25 to 40 degrees Celsius.