Methods for enhanced production of bone morphogenetic proteins
View Patent ↗Methods and processes for improved recombinant protein production are provided. The methods are useful for production of growth factors, particularly those of the TGF-β superfamily, including bone morphogenetic proteins (BMPs), such as BMP-2. Suitable host cells are cultured in media where iron is present at a concentration of at least 2.25 μM and if pyridoxal is present, it makes up less than 55% of the molar concentration of vitamin B6 in the media.
1. A method of BMP-2 production comprising the steps of:
i) culturing a suitable host cell comprising a DNA molecule encoding a BMP-2 in a culture medium comprising iron at a concentration of at least about 2.25 μM, a polyanionic compound, a total amino acid concentration of 15 mM to 40 mM, and if pyridoxal is present, it makes up less than about 50% of the molar concentration of vitamin B6 in the culture medium; and
ii) recovering the protein of interest wherein the host cell is cultured in a bioreactor having a capacity greater than about 3 L.
2. The method of claim 1 , wherein the iron is at a concentration of at least about 5 μM.
3. The method of claim 1 , wherein the culture medium can support a harvest density of at least about 2×10 6 cells/mL.
4. The method of claim 1 , where in the culture medium further comprises at least one vitamin B6 selected from the group consisting of pyridoxine, pyridoxamine, pyridoxine 5′-phosphate, pyridoxamine 5′-phosphate.
5. The method of claim 4 , wherein the culture medium has a total vitamin B6 concentration of at least about 15 μM.
6. The method of claim 4 , wherein the culture medium has a ratio of pyridoxal to pyridoxine of less than 1.2.
7. The method of claim 6 , wherein the ratio of pyridoxal to pyridoxine is less than 0.4.
8. The method of claim 1 , wherein the culture medium further comprises copper at a concentration of at least about 10 nM.
9. The method of claim 1 , wherein the culture medium further comprises zinc at a concentration of at least about 0.2 μM.
10. The method of claim 1 , wherein the polyanionic compound is dextran sulfate.
11. The method of claim 10 , wherein the dextran sulfate is present at a concentration of at least about 10mg/L.
12. The method of claim 10 , wherein the dextran sulfate is present at a concentration of at least about 100 mg/L.
13. The method of claim 11 , wherein the dextran sulfate has a molecular weight of between about 5,000 and 500,000 g/mole.
14. The method of claim 13 , wherein the dextran sulfate has a molecular weight of about 7,000 g/mole.
15. The method of claim 1 , wherein the culture medium comprises L-cystine at a concentration of at least about 0.5 mM.
16. The method of claim 15 , wherein the culture medium comprises L-glutamic acid at a concentration of at most about 0.3 mM.
17. The method of claim 1 , wherein the culture medium has an initial osmolarity of between about 260 and 360 mOsm.
18. The method of claim 1 , wherein the BMP-2 is a recombinant human BMP-2 (rhBMP-2).
19. The method of claim 1 , wherein the suitable host cell is selected from the group consisting of a COS cell, a CHO cell, a BHK cell, a Balb/c 3T3 cell, or a 293 cell.
20. The method of claim 19 , wherein the suitable host cell is a CHO cell.
21. The method of claim 20 , wherein the CHO cell has a reduced expression of the DHFR gene.
22. The method of claim 1 , wherein the suitable host cell is cultured in a bioreactor that has a capacity of at least about 160 L.
23. The method of claim 22 , wherein the bioreactor has a capacity of at least about 500 L.
24. The method of claim 23 , wherein the bioreactor has a capacity of at least about 2,500 L.
25. The method of claim 24 , wherein the bioreactor has a capacity of at least about 12,000 L.
26. The method of claim 23 , wherein the host cell is cultured by batch refeed, fed batch, or perfusion.
27. The method of claim 26 , where the host cell is cultured by batch refeed.
28. The method of claim 27 , where the host cell is cultured at an essentially constant temperature.
29. The method of claim 28 , where the temperature is about 36-38° C.
30. The method of claim 1 , further comprising the step of purifying the BMP-2 on a butyl sepharose resin.
31. The method of claim 30 , wherein the purification further comprises the steps of applying a conditioned culture medium containing the BMP-2 to a heparin-like resin, obtaining an eluate containing the BMP-2, and applying the eluate to the butyl sepharose resin.
32. A method of BMP-2 production comprising the steps of:
i) culturing a suitable host cell comprising a DNA molecule encoding a BMP-2 protein in a culture medium comprising iron at a concentration of at least about 2.25 μM, a polyanionic compound, a total amino acid concentration of 15 mM to 40 mM, and vitamin B6 at a concentration of at least about 15 μM, and if pyridoxal is present, it makes up less than about 50% of the molar concentration of vitamin B6 in the culture medium; and
ii) recovering a BMP-2 protein wherein the host cell is cultured in a bioreactor having a capacity greater than about 3 L.
33. The method of claim 32 , wherein the culture medium further comprises copper at a concentration of at least about 10 nM.
34. The method of claim 32 , wherein the culture medium further comprises zinc at a concentration of at least about 0.2 μM.
35. The method of claim 32 wherein the host cell is a CHO cell.
36. The method of claim 32 , wherein the BMP-2 is rhBMP-2.
37. The method of claim 32 , wherein the polyanionic compound is dextran sulfate at a concentration of at least about 10 mg/L.
38. The method of claim 32 , wherein the culture medium comprises L-cystine at a concentration of at least about 0.5 mM.
39. The method of claim 38 , wherein the culture medium further comprises L-glutamic acid at a concentration of at most about 0.3 mM.
40. The method of claim 32 , wherein the culture medium has an initial osmolarity of between about 260 and 360 mOsm.
41. A method of BMP-2 production comprising the steps of:
i) culturing a CHO cell comprising a DNA molecule encoding a BMP-2 protein in a batch refeed process in culture medium comprising iron at a concentration of at least about 2.25 μM, copper at a concentration of at least about 10 nM, L-cystine at a concentration of at least about 0.5 mM, dextran sulfate at a concentration of at least about 10 mg/L, a total amino acid concentration of 15 mM to 40 mM, and vitamin B6 at a concentration of at least about 15 μM, and if pyridoxal is present, it makes up less than about 50% of the molar concentration of vitamin B6 in the culture medium; and
ii) recovering a BMP-2 protein, wherein the host cell is cultured in a bioreactor having a capacity of at least about 160 L.
42. The method of claim 41 , wherein the culture medium further comprises zinc at a concentration of at least about 0.2 μM.
43. A method of BMP-2 production comprising the steps of:
i) culturing a suitable host cell comprising a DNA molecule encoding a BMP-2 in a culture medium comprising iron at a concentration of at least about 2.25 μM, a polyanionic compound, a total amino acid concentration of 15 mM to 40 mM, and if pyridoxal is present, it is present at a concentration of less than about 15 μM; and
ii) recovering the protein of interest wherein the host cell is cultured in a bioreactor having a capacity greater than about 3 L.
44. The method of claim 1 , wherein the culture medium comprises amino acids at a total concentration of about 15 mM to 35 mM.
45. The method of claim 1 , wherein the culture medium comprises amino acids at a total concentration of about 20 mM to 30 mM.
46. The method of claim 1 , wherein the culture medium comprises amino acids at a total concentration of about 25 mM.
47. The method of claim 1 , wherein the culture medium comprises amino acids at a total concentration of about 30 mM.