Aggregate-free protein compositions and methods of preparing same

• US 20050014240A1
• Filed: 08/30/2004
• Published: 01/20/2005
• Est. Priority Date: 08/06/1998
• Status: Abandoned Application

• Current Assignee: Horizon Therapeutics USA, Inc. (Amgen Inc.), Duke University, Mountain View Pharmaceuticals, Inc.
• Original Assignee: Mountain View Pharmaceuticals, Inc.
• Inventors: Sherman, Merry R., Williams, L. David, Saifer, Mark G.P.
• Application Number:
  US10/928,370
• Publication Number:
  US 20050014240A1
• Time in Patent Office: Days
• US Class Current: 435/191
• 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 13/12 of the kidneys
• CPC Class Codes: A61P 19/06 Antigout agents, e.g. antihyperuricemic or uricosuric agents
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: C12N 9/0046 with oxygen as acceptor (1.7.3)
• CPC Class Codes: C12N 9/0048 Uricase (1.7.3.3)
• CPC Class Codes: C12N 9/0093 acting on CH or CH2 groups (1.17)
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Y 107/03003 Factor-independent urate hydroxylase (1.7.3.3), i.e. uricase

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

1-33. -33. (cancelled).

34. A conjugate comprising a therapeutic protein covalently linked to poly(ethylene glycol) or poly(ethylene oxide), wherein the protein in said conjugate is substantially free of aggregates.

35. The conjugate of claim 34, wherein said protein is selected from the group consisting of a naturally occurring protein, a recombinant protein and a mutant protein that has substantially the structure of a naturally occurring protein.

36. The conjugate of claim 34, wherein said protein is a growth factor or a growth hormone.

37. The conjugate of claim 34, wherein said protein is an interferon.

38. The conjugate of claim 37, wherein said interferon is interferon-alpha.

39. The conjugate of claim 34, wherein said protein is an Immunoglobulin G (IgG).

40. The conjugate of claim 34, wherein said poly(ethylene glycol) is monomethoxy poly(ethylene glycol).

41. The conjugate of claim 34, wherein said protein is conjugated to said poly(ethylene glycol) or poly(ethylene oxide) via a linkage selected from the group consisting of a urethane (carbamate) linkage, a secondary amine linkage and an amide linkage.

42. The conjugate of claim 34, wherein said poly(ethylene glycol) or poly(ethylene oxide) has a molecular weight of between about 10 kDa and about 60 kDa.

43. The conjugate of claim 34, wherein the average number of strands of said poly(ethylene glycol) or poly(ethylene oxide) that are covalently linked to said protein is between 2 and 12.

44. The conjugate of claim 34, wherein said poly(ethylene glycol) or poly(ethylene oxide) is linear.

45. The conjugate of claim 34, wherein said poly(ethylene glycol) or poly(ethylene oxide) is branched.

61. A pharmaceutical composition comprising the conjugate of claim 34 and one or more pharmaceutically acceptable excipients.

62. The pharmaceutical composition of claim 61, wherein said composition is stabilized by lyophilization and dissolves upon reconstitution to provide a solution suitable for parenteral administration.

46. A method for preparing a protein composition that is substantially free of aggregates of said protein, comprising:

(a) preparing a solution of a protein in a suitable solvent;

(b) separating said protein solution by a separation process, whereby a plurality of fractions of said protein is obtained;

(c) assessing the level of aggregation of said protein during or following said separation; and

(d) separating the fractions of said protein that contain substantially aggregated protein from those fractions that are substantially free of aggregates of said protein.

47. The method of claim 46, wherein the aggregation of said protein in said individual fractions is assessed by measuring the light scattering of each of said fractions.

48. The method of claim 46, wherein said separation process is selected from the group consisting of ion-exchange chromatography, size-exclusion chromatography and ultrafiltration.

49. The method of claim 46, wherein said protein is selected from the group consisting of a naturally occurring protein, a recombinant protein and a mutant protein that has substantially the structure of a naturally occurring protein.

50. The method of claim 46, wherein said protein is a growth factor or a growth hormone.

51. The method of claim 46, wherein said protein is an interferon.

52. The method of claim 51, wherein said interferon is interferon-alpha.

53. The method of claim 46, wherein said protein is an Immunoglobulin G (IgG).

54. A method for preparing a conjugate of a therapeutic protein having reduced immunogenicity, comprising:

(a) preparing a protein that is substantially free of aggregates of said protein according to the method of claim 46;

and

(b) covalently linking said protein to one or more poly(ethylene glycol) or poly(ethylene oxide) molecules.

55. The method of claim 54, wherein said poly(ethylene glycol) is monomethoxy poly(ethylene glycol).

56. The method of claim 54, wherein said protein is conjugated to said poly(ethylene glycol) or poly(ethylene oxide) via a linkage selected from the group consisting of a urethane (carbamate) linkage, a secondary amine linkage and an amide linkage.

57. The method of claim 54, wherein said poly(ethylene glycol) or poly(ethylene oxide) has a molecular weight of between about 10 kDa and about 60 kDa.

58. The method of claim 54, wherein the average number of strands of said poly(ethylene glycol) or poly(ethylene oxide) that are covalently linked to said protein is between 2 and 12.

59. The method of claim 54, wherein said poly(ethylene glycol) or poly(ethylene oxide) is linear.

60. The method of claim 54, wherein said poly(ethylene glycol) or poly(ethylene oxide) is branched.