Aggregate-free urate oxidase for preparation of non-immunogenic polymer conjugates

• US 7,927,852 B2
• Filed: 08/03/2007
• Issued: 04/19/2011
• Est. Priority Date: 08/06/1998
• Status: Expired due to Fees

• Current Assignee: Horizon Therapeutics USA, Inc. (Amgen Inc.), Duke University
• Original Assignee: Mountain View Pharmaceuticals, Inc.
• Inventors: Sherman, Merry R., Saifer, Mark G. P., Kelly, Susan J., Williams, L. David, Hershfield, Michael S.
• Primary Examiner(s): Landsman; Robert
• Assistant Examiner(s): Dang; Ian
• Application Number:
  US11/882,750
• Publication Number:
  US 20080057048A1
• Time in Patent Office: 1,355 Days
• Field of Search: None
• US Class Current: 435/190
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61P 13/00 Drugs for disorders of the urinary system
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 19/06 Antigout agents, e.g. antihyperuricemic or uricosuric agents
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 9/0046 with oxygen as acceptor (1.7.3)
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates

Abstract

A naturally occurring or recombinant protein, especially a mutein of porcine urate oxidase (uricase), that is essentially free of large aggregates can be rendered substantially non-immunogenic by conjugation with a sufficiently small number of strands of polymer such that the bioactivity of the protein is essentially retained in the conjugate. Such conjugates are unusually well suited for treatment of chronic conditions because they are less likely to induce the formation of antibodies and/or accelerated clearance than are similar conjugates prepared from protein preparations containing traces of large aggregates.

Claims

1. A purified urate oxidase (uricase) wherein greater than 95% of said uricase is in the tetrameric and octameric forms.

2. The uricase of claim 1, wherein said uricase is a mammalian uricase.

3. The uricase of claim 2, wherein said uricase is porcine liver uricase, bovine liver uricase or ovine liver uricase.

4. The uricase of claim 1, wherein said uricase is recombinant.

5. The uricase of claim 4, wherein said uricase has the amino acid sequence of porcine, bovine, ovine or baboon liver uricase.

6. The uricase of claim 4, wherein said uricase is chimeric.

7. The uricase of claim 6, wherein said chimeric uricase contains portions of porcine liver uricase and portions of baboon liver uricase.

8. The uricase of claim 7, wherein said chimeric uricase is porcine uricase in which arginine residue 291 of SEQ ID NO:

1 has been replaced by lysine (R291K) and threonine residue 301 of SEQ ID NO;

1 has been replaced by serine (T301S) (PKS uricase).

9. The uricase of claim 4, wherein said uricase has the amino acid sequence as set forth in SEQ ID NO:

2, wherein tyrosine residue 97 has been replaced by histidine (Y97H).

10. The uricase of claim 1, wherein said uricase is a fungal or microbial uricase.

11. The uricase of claim 10, wherein said fungal or microbial uricase is isolated from Aspergillus flavus, Arthrobacter globiformis, Bacillus sp, or Candida utilis, or is a recombinant enzyme having the amino acid sequence of any one thereof.

12. The uricase of claim 1, wherein said uricase is an invertebrate uricase.

13. The uricase of claim 12, wherein said invertebrate uricase is isolated from Drosophila melanogaster or Drosophila pseudoobscura, or is a recombinant enzyme having the amino acid sequence of any one thereof.

14. The uricase of claim 1, wherein said uricase is a plant uricase.

15. The uricase of claim 14, wherein said plant uricase is isolated from root nodules of Glycine max or is a recombinant enzyme having the amino acid sequence thereof.

16. The uricase of claim 1, wherein greater than 97% of said uricase is in the tetrameric and octameric forms.

17. A purified chimeric urate oxidase (uricase), wherein greater than 95% of said uricase is in the tetrameric and octameric forms and wherein said chimeric uricase is porcine uricase in which arginine residue 291 of SEQ ID NO:

1 has been replaced by lysine (R291K) and threonine residue 301 of SEQ ID NO;

1 has been replaced by serine (T301S) (PKS uricase).

18. A conjugate comprising a purified urate oxidase (uricase) conjugated to poly(ethylene glycol) or poly(ethylene oxide), wherein greater than 95% of said uricase is in the tetrameric and octameric forms.

19. The conjugate of claim 18, wherein greater than 97% of said uricase is in the tetrameric and octameric forms.

20. The conjugate of claim 18, wherein said poly(ethylene glycol) is monomethoxy poly(ethylene glycol).

21. The conjugate of claim 18, wherein said uricase is conjugated to said poly(ethylene glycol) or poly(ethylene oxide) via a linkage selected from the group consisting of urethane (carbamate), secondary amine and amide.

22. The conjugate of claim 18, wherein said poly(ethylene glycol) or poly(ethylene oxide) has an average molecular weight of about 10 kDa to 60 kDa.

23. The conjugate of claim 22, wherein said poly(ethylene glycol) or poly(ethylene oxide) has an average molecular weight of about 10 kDa to 30 kDa.

24. The conjugate of claim 18, wherein the average number of strands of said poly(ethylene glycol) or poly(ethylene oxide) conjugated to said uricase is between about 2 and 12 per uricase subunit.

25. The conjugate of claim 24, wherein said average number of strands of said poly(ethylene glycol) or poly(ethylene oxide) is between about 6 and 10 per uricase subunit.

26. The conjugate of claim 25, wherein said average number of strands of said poly(ethylene glycol) or poly(ethylene oxide) is between about 7 and 9 per uricase subunit.

27. The conjugate of claim 18, wherein said poly(ethylene glycol) or poly(ethylene oxide) is linear.

28. The conjugate of claim 18, wherein said poly(ethylene glycol) or poly(ethylene oxide) is branched.

29. A purified fragment of urate oxidase (uricase), wherein said fragment is a recombinant uricase that has been truncated at the amino terminus, at the carboxyl terminus, or at both the amino and carboxyl termini, and wherein greater than 95% of said truncated uricase is in the tetrameric and octameric forms.

30. The purified uricase fragment of claim 29, wherein greater than 97% of said truncated uricase is in the tetrameric and octameric forms.

31. A conjugate comprising a purified urate oxidase (uricase) conjugated to poly(ethylene glycol) or poly(ethylene oxide), wherein greater than 95% of said uricase is in the tetrameric and octameric forms and wherein the average number of strands of said poly(ethylene glycol) or poly(ethylene oxide) conjugated to said uricase is between about 2 and about 10 per uricase subunit.

32. The conjugate of claim 31, wherein greater than 97% of said uricase is in the tetrameric and octameric forms.

33. The conjugate of claim 31, wherein said poly(ethylene glycol) or poly(ethylene oxide) has an average molecular weight of about 10 kDa to 60 kDa.

34. The uricase conjugate of claim 33, wherein said poly(ethylene glycol) or poly(ethylene oxide) has an average molecular weight of about 10 kDa to 30 kDa.

35. A conjugate comprising a purified urate oxidase (uricase) conjugated to poly(ethylene glycol) or poly(ethylene oxide), wherein greater than 95% of said uricase is in the tetrameric and octameric forms, wherein the average number of strands of said poly(ethylene glycol) or poly(ethylene oxide) conjugated to said uricase is between about 2 and 10 per uricase subunit and wherein said poly(ethylene glycol) or poly(ethylene oxide) has an average molecular weight of about 10 kDa to 60 kDa.

36. The uricase conjugate of claim 35, wherein greater than 97% of said uricase is in the tetrameric and octameric forms.

37. A pharmaceutical composition for lowering uric acid levels in a body fluid or tissue, comprising a purified urate oxidase (uricase) conjugated to poly(ethylene glycol) or poly(ethylene oxide) and a pharmaceutically acceptable carrier, wherein greater than 95% of said uricase is in the tetrameric and octameric forms.

38. The pharmaceutical composition of claim 37, wherein said composition is stabilized by lyophilization and dissolves upon reconstitution to provide a solution suitable for parenteral administration.

39. An isolated urate oxidase (uricase) prepared by a method comprising separating uricase aggregates larger than octamers from uricase tetramers and octamers and excluding such aggregates from the isolated uricase, wherein greater than 95% of said isolated uricase is in the tetrameric and octameric forms.

40. The uricase of claim 39, wherein greater than 97% of said isolated uricase is in the tetrameric and octameric forms.