PEG-urate oxidase conjugates and use thereof

• US 8,618,267 B2
• Filed: 04/08/2011
• Issued: 12/31/2013
• Est. Priority Date: 08/06/1998
• Status: Expired due to Fees

• Current Assignee: Horizon Therapeutics USA, Inc. (Amgen Inc.), Duke University
• Original Assignee: Duke University, Mountain View Pharmaceuticals, Inc.
• Inventors: Williams, L. David, Hershfield, Michael S., Kelly, Susan J., Saifer, Mark G. P., Sherman, Merry R.
• Primary Examiner(s): Saidha, Tekchand
• Application Number:
  US13/083,152
• Publication Number:
  US 20130052677A1
• Time in Patent Office: 998 Days
• Field of Search: 530/412, 435/189
• US Class Current: 530/412
• 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 urate oxidase (uricase) covalently coupled to poly(ethylene glycol) or poly(ethylene oxide) (both referred to as PEG), wherein an average of 2 to 10 strands of PEG are conjugated to each uricase subunit and the PEG has an average molecular weight between about 5 kDa and 100 kDa. The resulting PEG-uricase conjugates are substantially non-immunogenic and retain at least 75% of the uricolytic activity of the unmodified enzyme.

Claims

1. A method for isolating a tetrameric form of urate oxidase (uricase) from a solution of purified uricase comprising tetrameric uricase and uricase aggregates, said method comprising:

(a) separating the solution into fractions on at least one separation column at a pH between about 9 and about 10.5, wherein said column is selected from the group consisting of an ion-exchange column and a size-exclusion column; and

(b) recovering from said column one or more fractions that contain isolated tetrameric uricase, wherein greater than 90% of said isolated uricase in said one or more fractions is in a tetrameric form.

2. The method of claim 1, wherein said separating is performed at a pH of 10.2.

3. The method of claim 1, wherein said separating is performed at a pH of 10 to 10.5.

4. The method of claim 1, further comprising analyzing said one or more fractions to determine at least one property thereof selected from the group consisting of the presence of said tetrameric unease and the absence of said unease aggregates.

5. The method of claim 4, wherein said analyzing comprises at least one analytical method selected from the group consisting of chromatography, centrifugation, light scattering and electrophoresis.

6. The method of claim 5, wherein said analytical method is high performance liquid chromatography.

7. The method of claim 1, wherein the uricase in said solution is selected from the group consisting of recombinant mammalian uricase, uricase from Candida utilis, recombinant soybean uricase, uricase from Arthrobacter globiformis, porcine uricase, and baboon uricase.

8. The method of claim 7, wherein said uricase is one or more of chimeric, amino-terminally truncated, carboxyl-terminally truncated, amino-terminally and carboxyl-terminally truncated, and mutated.