Methods of cell culture
Polypeptide preparations having target levels of glycans, and methods of producing such polypeptide preparations using putrescine, are described.
1. A method of producing a recombinant protein preparation having a target value of fucosylated glycans, the method comprising:
(a) providing a cell genetically engineered to express a recombinant protein;
(b) culturing the cell in a culture medium comprising 0.1 mg/L to 10 mg/L putrescine under conditions in which the cell expresses the recombinant protein; and
(c) harvesting a preparation of the recombinant protein produced by the cell that meets the target value of the fucosylated glycans
wherein the target value of fucosylated glycans is a level at least 10% higher than a level of fucosylated glycans in a preparation produced by culturing the cell in the medium not comprising 0.1 mg/L to 10 mg/L putrescine.
2. The method of claim 1 , wherein the recombinant protein is a recombinant therapeutic protein.
3. The method of claim 1 , wherein the recombinant protein is a recombinant therapeutic antibody.
4. The method of claim 1 , wherein the target value is a level of fucosylated glycans in a reference therapeutic protein.
5. The method of claim 1 , wherein the target value is a level of fucosylated glycans in a reference therapeutic antibody.
6. The method of claim 1 , further comprising evaluating a level of fucosylated glycans in the recombinant protein preparation.
7. The method of claim 1 , wherein the target value is:
(a) 70% to 100% fucosylated glycans.
8. The method of claim 1 , wherein the culture medium further comprises one or more of lysine, ammonium, DMSO, copper, glucose, a growth factor, a vitamin, a lipid, and a peptone.
9. The method of claim 4 , wherein the reference therapeutic protein is selected from the group consisting of: abatacept, abciximab, adalimumab, aflibercept, alefacept, alemtuzumab, basiliximab, bevacizumab, belatacept, certolizumab, cetuximab, daclizumab, eculizumab, efalizumab, entanercept, gemtuzumab, ibritumomab, infliximab, muromonab-CD3, natalizumab, omalizumab, palivizumab, panitumumab, ranibizumab, rilonacept, rituximab, tositumomab, and trastuzumab.
10. A method of producing a recombinant protein preparation, the method comprising:
(a) providing a target value of fucosylated glycans;
(b) providing a cell genetically engineered to express a recombinant protein;
(c) culturing the cell in a culture medium comprising 0.1 mg/L to 10 mg/L putrescine under conditions in which the cell expresses the recombinant protein;
(d) harvesting a preparation of the recombinant protein produced by the cell; and
(e) formulating the preparation into a drug product if the preparation meets the target value of fucosylated glycans
wherein the target value of fucosylated glycans is a level at least 10% higher than a level of fucosylated glycans in a preparation produced by culturing the cell in the medium not comprising 0.1 mg/L to 10 mg/L putrescine.
11. The method of claim 10 , further comprising evaluating a level of fucosylated glycans in the recombinant protein preparation.
12. The method of claim 10 , wherein the culture medium further comprises one or more of lysine, ammonium, DMSO, copper, glucose, a growth factor, a vitamin, a lipid, and a peptone.
13. The method of claim 6 or 11 , further comprising recording the evaluated level of fucosylated glycans in a batch record for the preparation.
14. A method of increasing a level of fucosylated glycans in a recombinant protein preparation, the method comprising:
(a) providing a cell genetically engineered to express a recombinant protein;
(b) culturing the cell in a culture medium comprising 0.1 mg/L to 10 mg/L putrescine under conditions in which the cell expresses the recombinant protein; and
(c) harvesting a preparation of the recombinant protein produced by the cell, wherein the preparation has a level of fucosylated glycans, that is at least 10% higher than a level of fucosylated glycans in a preparation of the recombinant protein produced by culturing the cell in the medium not comprising 0.1 mg/L to 10 mg/L putrescine.
15. The method of claim 14 , further comprising measuring a level of fucosylated glycans.
16. The method of claim 14 , wherein the culture medium further comprises one or more of lysine, ammonium, DMSO, copper, glucose, a growth factor, a vitamin, a lipid, and a peptone.
17. The method of claim 15 , further comprising recording the evaluated level of fucosylated glycans in a batch record for the preparation.
18. The method of any one of claims 1 , 10 , or 14 , wherein the culturing step comprises a first stage and a second stage, wherein the first stage comprises culturing the cell in the culture medium comprising a first level of putrescine, and the second stage comprises culturing the cell in the culture medium comprising a second level of putrescine which is increased relative to the first level.
19. The method of claim 18 , wherein the second level is 0.1 mg/L to 10 mg/L putrescine.
20. The method of claim 18 , wherein the first stage comprises culturing the cell in the first level of putrescine for 1 to 8 days.
21. The method of claim 20 , wherein the second stage comprises culturing the cell in the second level of putrescine for 1 to 12 days.
22. The method of any one of claims 1 , 10 , or 14 , wherein the cell is a Chinese Hamster Ovary (CHO) cell.
23. The method of any one of claims 1 , 10 or 14 , wherein the recombinant protein is selected from the group consisting of: abatacept, abciximab, adalimumab, aflibercept, alefacept, alemtuzumab, basiliximab, bevacizumab, belatacept, certolizumab, cetuximab, daclizumab, eculizumab, efalizumab, entanercept, gemtuzumab, ibritumomab, infliximab, muromonab-CD3, natalizumab, omalizumab, palivizumab, panitumumab, ranibizumab, rilonacept, rituximab, tositumomab, and trastuzumab.
24. The method of any one of claims 1 , 10 , or 14 , wherein the culturing step comprises adding putrescine to the culture medium.