Multi-armed, monofunctional, and hydrolytically stable derivatives of poly(ethylene glycol) and related polymers for modification of surfaces and molecules

• US 8,354,477 B2
• Filed: 08/03/2010
• Issued: 01/15/2013
• Est. Priority Date: 01/10/1995
• Status: Expired due to Fees

• Current Assignee: Nektar Therapeutics Incorporated
• Original Assignee: Nektar Therapeutics Incorporated
• Inventors: Harris, J. Milton, Veronese, Francesco Maria, Caliceti, Paolo, Schiavon, Oddone
• Primary Examiner(s): Harlan, Robert D.
• Application Number:
  US12/849,683
• Publication Number:
  US 20100298496A1
• Time in Patent Office: 896 Days
• Field of Search: 525/419, 525/425, 514/424, 435/188, 435/94.3, 435/177, 435/180, 435/181
• US Class Current: 525/419
• 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: C08G 65/329 with organic compounds
• CPC Class Codes: C08G 65/48 Polymers modified by chemical after-treatment
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: Y10S 530/815 Carrier is a synthetic polymer
• CPC Class Codes: Y10S 530/816 Attached to the carrier via a bridging agent

Abstract

Multi-armed, monofunctional, and hydrolytically stable polymers are described having the structure

wherein Z is a moiety that can be activated for attachment to biologically active molecules such as proteins and wherein P and Q represent linkage fragments that join polymer arms poly_a and poly_b, respectively, to central carbon atom, C, by hydrolytically stable linkages in the absence of aromatic rings in the linkage fragments. R typically is hydrogen or methyl, but can be a linkage fragment that includes another polymer arm. A specific example is an mPEG disubstituted lysine having the structure

where mPEG_a and mPEG_b have the structure CH₃O—(CH₂CH₂O)_nCH₂CH₂— wherein n may be the same or different for poly_a- and poly_b- and can be from 1 to about 1,150 to provide molecular weights of from about 100 to 100,000.

Claims

1. A method for preparing a conjugate of a purified branched water-soluble polymer, comprising:

a. providing an impure polymer composition comprising

(i) a branched water-soluble polymer having the structure;

2. The method of claim 1 wherein Z is carboxyl.

3. The method of claim 1, wherein said polymeric impurities further comprise a mono-substituted mPEG intermediate.

4. The method of claim 3, wherein the mono-substituted mPEG intermediate is mPEG-mono-substituted lysine.

5. The method of claim 1, wherein said purifying further comprises:

loading the impure polymer composition onto an ion exchange chromatography medium to provide a loaded medium,

washing the polymeric impurities from said loaded medium using an aqueous eluent under conditions effective to elute said impurities from said medium,

adjusting the conditions of the aqueous eluent to effect elution of said branched water-soluble polymer from the medium,

eluting said branched water-soluble polymer from said medium to provide an aqueous solution comprising the purified branched water-soluble polymer in essentially pure form, and recovering the purified branched water-soluble polymer from said aqueous solution.

6. The method of claim 1, wherein said branched water soluble polymer has a molecular weight ranging from about 10,000 daltons to about 50,000 daltons.

7. The method of claim 6, wherein the branched water soluble polymer has a molecular weight of about 40,000 daltons.

8. The method of claim 1, wherein mPEG_a and mPEG_b are the same.

9. The method of claim 1 including step c., wherein the Z-activated moiety is an active ester.

10. The method of claim 9, wherein the active ester is a succinimidyl ester.

11. The method of claim 1, including step c., wherein the Z-activated moiety is selected from the group consisting of trifluoroethylsulfonate, isocyanate, isothiocyanate, active carbonate, aldehyde, sulfone, vinyl sulfone, malemide, iodoacetamide, and iminoester.

12. method of claim 11, wherein the Z-activated moiety is an active carbonate selected from succinimidyl carbonate, p-nitrophenylcarbonate, and trichlorophenylcarbonate.

13. The method of claim 1, wherein the biologically active molecule is selected from the group consisting of a peptide, a protein, a nucleotide, a polynucleotide, a lipid, and a small molecule drug.

14. The method of claim 1, wherein the nucleophilic group on the biologically active molecule is selected from amino, thiol, and hydroxyl.

15. The method of claim 14, wherein the nucleophilic group on the biologically active molecule is amino.

16. The method of claim 1, wherein the biologically active molecule is an interferon.

17. The method of claim 16, wherein the interferon is an interferon-α

.

18. The method of claim 17, wherein the nucleophilic group is an amino group.

19. A method according to claim 1 including step c., wherein:

(i) the branched water soluble polymer in a.(i) is a mPEG-disubstituted lysine branched polymer having the structure;