Purification of proteins with cationic surfactant

• US 9,534,013 B2
• Filed: 04/12/2006
• Issued: 01/03/2017
• Est. Priority Date: 04/12/2006
• Status: Active Grant

• Current Assignee: Horizon Therapeutics USA, Inc. (Amgen Inc.)
• Original Assignee: Horizon Pharma Rheumatology LLC (Amgen Inc.)
• Inventors: Fischer, Meir, Harosh, Eliyahu
• Primary Examiner(s): STANFIELD, CHERIE MICHELLE
• Application Number:
  US11/918,292
• Publication Number:
  US 20090317889A1
• Time in Patent Office: 3,919 Days
• US Class Current: 1/1
• CPC Class Codes: C07K 1/30 by precipitation
• CPC Class Codes: C07K 14/565 IFN-beta
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/36 against blood coagulation factors
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C12N 9/0048 Uricase (1.7.3.3)
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione
• CPC Class Codes: C12Y 107/03003 Factor-independent urate hydroxylase (1.7.3.3), i.e. uricase

Abstract

The subject invention provides a method for purifying a target protein from a mixture comprising the target protein and contaminating protein, comprising the steps of exposing the mixture to an effective amount of a cationic surfactant such that the contaminating protein is preferentially precipitated and recovering the target protein. Proteins purified according to the method of the invention are also provided.

Claims

1. A method for purifying a uricase comprising:

a. obtaining a solution comprising a mixture of a solubilized uricase, one or more solubilized contaminating proteins and an alkaline buffer, wherein the uricase is positively charged under alkaline pH and has an isoelectric point greater than 7 and the one or more contaminating proteins has a polyanion charge;

b. contacting the solution comprising the mixture of the solubilized uricase and the one or more solubilized contaminating proteins with one or more cationic surfactants in an amount effective to preferentially precipitate the one or more contaminating proteins, thereby increasing the proportion of proteins remaining in solution represented by the uricase; and

c. recovering the uricase in solution after the preferential precipitation of step b;

wherein the method is performed in the absence of a solid support and wherein the one or more cationic surfactants is an amphipathic ammonium compound selected from the group consisting of a quaternary ammonium compound of the general formula QN+;

a paraffin chain primary ammonium compound of the general formula RNH3+; and

a salt thereof.

2. The method of claim 1, wherein the amphipathic ammonium compound is selected from the group consisting of a cetylpyridinium salt, a stearamide-methylpyridinium salt, a lauryl pyridinium salt, a cetylquinolynium salt, a lauryl aminopropionic acid methyl ester salt, a lauryl amino propionic acid metal salt, a lauryl dimethyl betaine, a stearyl dimethyl betaine, a lauryl dihydroxyethyl betaine and a benzethonium salt.

3. The method of claim 2, wherein the amphipathic ammonium compound is selected from the group consisting of hexadecylpyridinium chloride, dequalinium acetate, cetyltrimethylammonium chloride, mixed n-alkyl dimethyl benzylammonium chloride, cetylpyridinium chloride, N,N-dimethyl-N-[2-[2-[4-(1,1,3,3,-tetramethylbutyl)-phenoxy]ethoxy]ethyl]benzenemethanammonium chloride, alkyl-dimethylbenzyl-ammonium chloride, dichloro-benzyldimethyl-alkylammonium chloride, tetradecyl trimethylammonium bromide, dodecyl trimethylammonium bromide, cetyltrimethylammonium bromide, lauryl dimethyl betaine stearyl dimethyl betaine, and lauryl dihydroxyethyl betaine.

4. The method of claim 2, wherein the amphipathic ammonium compound is a cetylpyridinium salt.

5. The method of claim 1, wherein the uricase is a recombinant protein.

6. The method of claim 1, wherein the one or more cationic surfactants are added to a concentration of from 0.001% to 5.0%.

7. The method of claim 6, wherein the one or more cationic surfactants are added to a concentration of from 0.01% to 0.5%.

8. The method of claim 6, wherein the one or more cationic surfactants are added to a concentration of from 0.03% to 0.2%.

9. The method of claim 1, wherein the method does not depend upon the presence of polyanions, solid supports and aggregates of the contaminating proteins.

10. The method of claim 9, wherein the one or more cationic surfactants are a cetylpyridinium salt.

11. The method of claim 10, wherein the cetylpyridinium salt is cetylpyridinium chloride.

12. The method of claim 1, wherein the solution has a pH that is about the same as the isoelectric point of the uricase.

13. The method of claim 1, wherein the solution has a pH that is below the isoelectric point of the uricase.

14. The method of claim 1, wherein the solution has a pH that is above the isoelectric point of the target protein and within 2 pH units of the isoelectric point of the target protein.

15. The method of claim 14, wherein the pH of the solution is within 1 pH unit of the isoelectric point of the target protein.

16. The method of claim 1, wherein the solution has a pH that is between 7 and 11.

17. The method of claim 1, wherein the solution has a pH that is between 8 and 11.

18. The method of claim 1, further comprising:

dissolving, in the presence of an alkaline buffer, one or more inclusion bodies from a bacterial cell that expresses the uricase, thereby providing the solubilized uricase and the one or more solubilized contaminating proteins of step a.

19. A method for purifying a uricase, comprising:

a. dissolving, in the presence of an alkaline buffer, one or more inclusion bodies from a bacterial cell that expresses the uricase, thereby providing a solubilized uricase and one or more solubilized contaminating proteins;

b. obtaining a solution comprising the solubilized uricase, the one or more solubilized contaminating proteins and the alkaline buffer;

c. contacting the solution comprising the solubilized uricase, the one or more solubilized contaminating proteins and the alkaline buffer with one or more cationic surfactants in an amount effective to preferentially precipitate the one or more contaminating proteins, thereby increasing the proportion of proteins remaining in solution represented by the uricase; and

d. recovering the uricase in solution after the preferential precipitation of step c;

e. wherein;

(i) the method is performed in the absence of a solid support;

or

(ii) the one or more cationic surfactants are added to a concentration of from 0.03% to 0.2%;

or

(iii) both (i) and (ii); and

e. wherein the one or more cationic surfactants is an amphipathic ammonium compound selected from the group consisting of a cetylpyridinium salt, a stearamide-methylpyridinium salt, a lauryl pyridinium salt, a cetylquinolynium salt, a lauryl aminopropionic acid methyl ester salt, a lauryl amino propionic acid metal salt, a lauryl dimethyl betaine, a stearyl dimethyl betaine, a lauryl dihydroxyethyl betaine and a benzethonium salt.

20. The method of claim 19, wherein the one or more cationic surfactants are added to a concentration of from 0.03% to 0.1%.

21. The method of claim 19, wherein the amphipathic ammonium compound is a cetylpyridinium salt.

22. The method of claim 21, wherein the cetylpyridinium salt is cetylpyridinium chloride.

23. A method for purifying a target protein comprising:

a. obtaining a solution of a plurality of proteins, wherein the proteins in solution comprise the target protein, one or more contaminating proteins and an alkaline buffer,

wherein the target protein is positively charged under alkaline pH and has an isoelectric point greater than 7 and the one or more contaminating proteins has a polyanion charge;

b. contacting the solution with one or more cationic surfactants in an amount effective to preferentially precipitate the one or more contaminating proteins, thereby increasing the proportion of proteins remaining in solution represented by the target protein; and

c. recovering the target protein in solution after the preferential precipitation of step b;

wherein the method is performed in the absence of a solid support;

wherein the one or more cationic surfactants are added to a concentration of from 0.03% to 0.2%;

wherein the target protein is selected from the group consisting of an antibody, a uricase, a factor X inhibitor, an acid deoxyribonuclease II, an elastase, a lysozyme, a papain, a peroxidase, a pancreatic ribonuclease, a trypsinogen, a trypsin, a cytochrome c, an erabutoxin, staphylococcus aureus enterotoxin C1, an interferon and a monoamine oxidase A; and

wherein the one or more cationic surfactants is an amphipathic ammonium compound selected from the group consisting of a quaternary ammonium compound of the general formula QN+;

a paraffin chain primary ammonium compound of the general formula RNH3+; and

a salt thereof.

24. The method of claim 23, wherein the one or more cationic surfactants are added to a concentration of from 0.03% to 0.1%.

25. The method of claim 23, wherein the amphipathic ammonium compound is a cetylpyridinium salt.

26. The method of claim 25, wherein the cetylpyridinium salt is cetylpyridinium chloride.

27. The method of claim 23, wherein the target protein is a uricase.

28. The method of claim 23, wherein the amphipathic ammonium compound is selected from the group consisting of a cetylpyridinium salt, a stearamide-methylpyridinium salt, a lauryl pyridinium salt, a cetylquinolynium salt, a lauryl aminopropionic acid methyl ester salt, a lauryl amino propionic acid metal salt, a lauryl dimethyl betaine, a stearyl dimethyl betaine, a lauryl dihydroxyethyl betaine and a benzethonium salt.

29. The method of claim 28, wherein the amphipathic ammonium compound is selected from hexadecylpyridinium chloride, dequalinium acetate, cetyltrimethylammonium chloride, mixed n-alkyl dimethyl benzylammonium chloride, cetylpyridinium chloride, N,N-dimethyl-N-[2-[2-[4-(1,1,3,3,-tetramethylbutyl)-phenoxy]ethoxy]ethyl]benzenemethanammonium chloride, alkyl-dimethylbenzyl-ammonium chloride, dichloro-benzyldimethyl-alkylammonium chloride, tetradecyl trimethylammonium bromide, dodecyl trimethylammonium bromide, cetyltrimethylammonium bromide, lauryl dimethyl betaine stearyl dimethyl betaine, and lauryl dihydroxyethyl betaine.

30. The method of claim 23, further comprising:

dissolving one or more inclusion bodies from a bacterial cell that expresses the target protein, thereby providing the target protein, and the one or more contaminating proteins, in solution, of step a.