Methods for increasing recovery of an ADAMTS activity from a cell culture supernatant
View Patent ↗The present invention provides culture mediums that are useful for the expression of ADAMTS proteins, such as ADAMTS13. Methods for the expression and purification of ADAMTS proteins are also provided. In some embodiments, the mediums and methods of the invention are useful for the expression of ADAMTS proteins having high specific activities. Also provided are ADAMTS, e.g., ADAMTS13, protein compositions with high specific activities, which are expressed and purified according to the methods provided herein.
1. A method for increasing recovery of an ADAMTS activity from a cell culture supernatant, the method comprising the steps of:
(a) culturing a cell harboring a nucleic acid encoding an ADAMTS protein in a culture medium;
(b) removing a fraction of the supernatant from the culture;
(c) performing a filtration or centrifugation step to remove residual cells from the fraction of the supernatant removed from the culture, thereby forming a clarified supernatant;
(d) concentrating ADAMTS protein in the clarified supernatant by ultrafiltration, thereby forming a concentrated supernatant; and
(e) diafiltering the concentrated supernatant against a buffer comprising at least about 0.5 μM zinc and at least about 0.1 mM calcium; thereby preparing an ADAMTS composition with increased recovery of ADAMTS activity.
2. The method of claim 1 , wherein the step of culturing a cell comprises batch cell cultivation.
3. The method of claim 1 , wherein the step of culturing a cell comprises continuous cell cultivation.
4. The method of claim 1 , wherein the culture medium comprises at least 3 μM zinc and at least 0.5 mM calcium.
5. The method of claim 4 , wherein the culture medium comprises at least 5 μM zinc.
6. The method of claim 4 , wherein the culture medium contains at least 2 mg/L nicotinamide (vitamin B3).
7. The method of claim 4 , wherein the culture medium contains at least 7 mg/L nicotinamide (vitamin B3).
8. The method of claim 5 , wherein the culture medium contains at least 2 mg/L nicotinamide (vitamin B3).
9. The method of claim 5 , wherein the culture medium contains at least 7 mg/L nicotinamide (vitamin B3).
10. The method of claim 1 , wherein the diafiltration buffer contains at least 5 μM zinc and at least 2 mM calcium.
11. The method of claim 4 , wherein the diafiltration buffer contains at least 5 μM zinc and at least 2 mM calcium.
12. The method of claim 7 , wherein the diafiltration buffer contains at least 5 μM zinc and at least 2 mM calcium.
13. The method of claim 1 , wherein the ADAMTS protein in is ADAMTS13.
14. The method of claim 4 , wherein the ADAMTS protein is ADAMTS13.
15. The method of claim 7 , wherein the ADAMTS protein is ADAMTS13.
16. The method of claim 10 , wherein the ADAMTS protein is ADAMTS13.
17. The method of claim 11 , wherein the ADAMTS protein is ADAMTS13.
18. The method of claim 12 , wherein the ADAMTS protein is ADAMTS13.
19. The method of claim 13 , wherein the ADAMTS13 specific activity of the composition formed in step (e) is at least 80% of the ADAMTS13 specific activity of the clarified supernatant formed in step (c).
20. The method of claim 5 , wherein the culture medium contains from 5 μM to 12 μM zinc.
21. The method of claim 6 , wherein the culture medium contains from 5 μM to 12 μM zinc.
22. The method of claim 7 , wherein the culture medium contains from 5 μM to 12 μM zinc.
23. The method of claim 8 , wherein the culture medium contains from 5 μM to 12 μM zinc.
24. The method of claim 9 , wherein the culture medium contains from 5 μM to 12 μM zinc.
25. The method of claim 10 , wherein the culture medium contains from 5 μM to 12 μM zinc.
26. The method of claim 12 , wherein the culture medium contains from 5 μM to 12 μM zinc.
27. The method of claim 13 , wherein the culture medium contains from 5 μM to 12 μM zinc.
28. The method of claim 15 , wherein the culture medium contains from 5 μM to 12 μM zinc.
29. The method of claim 16 , wherein the culture medium contains from 5 μM to 12 μM zinc.
30. The method of claim 18 , wherein the culture medium contains from 5 μM to 12 μM zinc.
31. The method of claim 6 , wherein the culture medium contains from 2 mg/L to 10 mg/L nicotinamide (vitamin B3).
32. The method of claim 8 , wherein the culture medium contains from 2 mg/L to 10 mg/L nicotinamide (vitamin B3).