Method for the preparation of growth hormone and antagonist thereof having lower levels of isoform impurities thereof
The present invention is directed generally to recombinant methods for making a desired polypeptide. These method(s) yield a polypeptide product containing reduced levels of isoform impurities thereof. In particular, the present invention is directed to (1) a recombinant method for preparing growth hormone with reduced isoform impurities thereof and (2) a recombinant method for preparing a growth hormone antagonist, such as pegvisomant, and its protein intermediate, also having reduced isoform impurities thereof. More specifically, the isoform impurities that are decreased by methods of the present invention are the trisulfide and des-phe isoform impurities of growth hormone and growth hormone antagonist (or its intermediate), respectively.
1. A process for decreasing the amount of an impurity produced in recombinant production of a growth hormone antagonist polypeptide B-2036 of SEQ ID NO:1 in genetically modified host cells containing cellular component(s), the process comprising the step of:
(a) contacting a chelating agent with a cell lysate or cell paste lysate comprising (1) said impurity, (2) said growth hormone antagonist polypeptide, (3) said cellular component(s) and (4) combinations thereof to decrease said amount of said impurity,
wherein said impurity is a trisulfide isoform of said polypeptide.
2. A process for decreasing the amount of an impurity produced in recombinant production of a growth hormone antagonist polypeptide B-2036 of SEQ ID NO:1 in genetically modified host cells containing cellular component(s), the process comprising the step of:
(a) contacting a chelating agent with a cell lysate or cell paste lysate comprising (1) said impurity, (2) said growth hormone antagonist polypeptide, (3) said cellular component(s) and (4) combinations thereof to decrease said amount of said impurity,
wherein said impurity is a des-phe isoform of said polypeptide.
3. The process of claim 1 , wherein said chelating agent is selected from the group consisting of EDTA, EGTA, and DTPA.
4. The process of claim 3 , wherein said chelating agent is EDTA.
5. The process of claim 1 , wherein said chelating agent is provided in a buffer.
6. The process of claim 4 , wherein said EDTA has an initial concentration from about 2 mM to about 10 mM.
7. The process of claim 5 , wherein said buffer is selected from the group consisting of Tris, phosphate, HEPES, citric acid, triethylamine, and histidine.
8. The process of claim 7 , wherein said buffer is Tris.
9. The process of claim 8 , wherein said Tris buffer has an initial Tris concentration from about 1 mM to about 200 mM.
10. The process of claim 5 , wherein after said contacting step (a) said buffer has a pH from about 6 to about 9.
11. The process of claim 1 , wherein the temperature is maintained from about 0° C. to about 35° C.
12. The process of claim 11 , wherein the temperature is maintained from about 2° C. to about 15° C.
13. The process of claim 2 , wherein said chelating agent is selected from the group consisting of EDTA, EGTA, and DTPA.
14. The process of claim 13 , wherein said chelating agent is EDTA.
15. The process of claim 2 , wherein said chelating agent is provided in a buffer.
16. The process of claim 14 , wherein said EDTA has an initial concentration from about 2 mM to about 10 mM.
17. The process of claim 15 wherein said buffer is selected from the group consisting of Tris, phosphate, HEPES, citric acid, triethylamine, and histidine.
18. The process of claim 17 , wherein said buffer is Tris.
19. The process of claim 18 , wherein said Tris buffer has an initial Tris concentration from about 1 mM to about 200 mM.
20. The process of claim 15 , wherein after said contacting step (a) said buffer has a pH from about 6 to about 9.
21. The process of claim 2 , wherein the temperature is maintained from about 0° C. to about 35° C.
22. The process of claim 21 , wherein the temperature is maintained from about 2° C. to about 15° C.