Purification of Proteins With Cationic Surfactant

• US 20090317889A1
• Filed: 04/12/2006
• Published: 12/24/2009
• 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
• Granted Patent: US 9,534,013 B2
• Time in Patent Office: Days
• US Class Current: 435/191
• 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 target protein comprising identifying a target protein and contacting a solution comprising the solubilized target protein and one or more solubilized contaminating proteins with one or more cationic surfactants in an amount effective to selectively precipitate the one or more contaminating proteins.

2. The method of claim 1, further comprising the step of recovering the solubilized target protein.

3. The method of claim 1 wherein at least one of the one or more cationic surfactants is an amphipathic ammonium compound.

4. The method of claim 3 wherein the amphipathic ammonium compound is selected from the group consisting of quaternary ammonium compounds of the general formula QN+;

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

salts thereof.

5. The method of claim 4 wherein the amphipathic ammonium compound is selected from the group consisting of cetyl pyridinium salts, stearamide-methylpyridinium salts, lauryl pyridinium salts, cetyl quinolynium salts, lauryl aminopropionic acid methyl ester salts, lauryl amino propionic acid metal salts, lauryl dimethyl betaine, stearyl dimethyl betaine, lauryl dihydroxyethyl betaine and benzethonium salts.

6. The method of claim 5 wherein the amphipathic ammonium compound is selected from hexadecylpyridinium chloride, dequalinium acetate, hexadecylpyridinium chloride, 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, and dichloro-benzyldimethyl-alkylammonium chloride, tetradecyl trimethylammonium bromide, dodecyl trimethylammonium bromide, cetyl trimethylammonium bromide, lauryl dimethyl betaine stearyl dimethyl betaine, and lauryl dihydroxyethyl betaine.

7. The method of claim 5 wherein the amphipathic ammonium compound is a cetylpyridinium salt.

17. The method of claim 1 wherein the target protein is a recombinant protein.

18. The method of claim 17 wherein the recombinant protein is an enzyme.

19. The method of claim 17 wherein the target protein is selected from the group consisting of an antibody, a uricase, an interferon-beta, 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.

20. The method of claim 19 wherein the target protein is a uricase.

23. The method of claim 17 wherein the target protein is an antibody.

24. The method of claim 23 wherein the antibody is a single chain antibody.

25. The method of claim 17 wherein the target protein is an interferon.

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

28. The method of claim 27 wherein the one or more cationic surfactants are added to a concentration of from 0.01% to 0.5%.

29. The method of claim 27 wherein the one or more cationic surfactants are added to a concentration of from 0.03% to 0.2%.

37. The method of claim 1, wherein the solution is substantially free of polyanions;

solid supports and aggregates of the contaminating proteins with other molecules.

38. The method of claim 37, wherein the cationic surfactant is a cetylpyridinium salt.

39. The method of claim 37, wherein the cetylpyridinium salt is cetylpyridinium chloride.

40. The method of claim 1, wherein the target protein has an isoelectric point greater than or equal to 7.

41. A purified protein prepared according to the method of claim 1.

42. A purified uricase prepared according to the method of claim 1.

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47. A method for purifying a target protein comprising the steps of:

a. identifying a target protein;

b. contacting a solution comprising solubilized target protein and one or more solubilized contaminating proteins with one or more cationic surfactants in an amount effective to selectively precipitate the one or more contaminating proteins; and

c. recovering the soluble target protein.

48. A method of increasing the percentage of a target protein in a solution of proteins comprising the steps of

a. obtaining a solution of a plurality of proteins, wherein the proteins in solution comprise the target protein and contaminating proteins, and the target protein comprises a first percentage by weight of the total protein in the solution;

b. contacting the solution with one or more cationic surfactants in an amount effective to selectively precipitate the contaminating proteins;

wherein the target protein in the solution of step b comprises a second percentage by weight of the total protein, and the second percentage is greater than the first percentage.