Purification of proteins
This application relates to methods for purification of proteins such as antibodies using a hydrophilic polymer (e.g., PEG), a fatty acid (e.g., caprylic acid), and/or another agent (e.g., calcium chloride).
1. A method for isolating a protein of interest from nucleic acids and other host cell proteins present in cell culture supernatant comprising said protein, the method comprising:
(a) combining said cell culture supernatant with a first precipitating agent to remove precipitated proteins and/or other components to produce a secondary cell culture supernatant, the first precipitating agent being calcium chloride and/or a fatty acid;
(b) introducing a precipitating polymer into said secondary cell culture supernatant to precipitate the protein of interest from the secondary cell culture supernatant.
2. The method of claim 1 , wherein the precipitating polymer is polyethylene glycol (PEG).
3. The method of claim 2 , wherein the PEG is selected from PEG2000, PEG4000, PEG6000 and PEG20000.
4. The method of claim 2 , wherein the PEG is introduced into the secondary cell culture supernatant in an amount of from about 5 to about 30%.
5. The method of claim 1 , wherein the pH in step (b) is about from 4 to 9.
6. The method of claim 5 , wherein the pH is from 5.0 to 8.5.
7. The method of claim 1 , wherein the first precipitating agent is a fatty acid used with a salt.
8. The method of claim 7 , wherein the fatty acid is caprylic acid.
9. The method of claim 8 wherein the fatty acid is present in step (a) in a concentration of from about 0.1% to about 10% (v/v).
10. The method of claim 9 wherein the fatty acid is present in step (a) in a concentration of about 1%.
11. The method of claim 1 , wherein the pH in step (a) is from about 4 to about 6.
12. The method of claim 11 , wherein the pH in step (a) is from about 4.5 to about 5.5.
13. The method of claim 7 , wherein the salt is sodium acetate or sodium citrate.
14. The method of claim 1 , wherein the first precipitating agent is calcium chloride.
15. The method of claim 1 further comprising subsequent to steps (a) and (b), subsequently precipitating impurities with calcium chloride as step (c).
16. The method of claim 15 , wherein the concentration of calcium chloride is from about 10 mM to about 400 mM.
17. The method of claim 15 , wherein the pH in step (c) is from about 6 to about 9.
18. The method of claim 1 , further comprising a subsequent precipitation using cold ethanol.
19. The method of claim 15 , further comprising a subsequent precipitation using cold ethanol.
20. The method of claim 1 , wherein the protein of interest is an antibody.
21. The method of claim 1 , wherein the antibody is immunoglobulin G.
22. The method of claim 1 , wherein said method is carried out using a two-step precipitation reactor, optionally wherein the two steps are continuous.
23. The method of claim 1 , performed without using chromatography.
24. The method of claim 15 , performed without using chromatography.
25. The method of claim 19 , performed without using chromatography.
26. The method of claim 14 , wherein the PEG is introduced into the secondary cell culture supernatant in an amount of from about 10 to about 21%.