Process for the preparation of polymer conjugates

• US 7,365,127 B2
• Filed: 02/04/2005
• Issued: 04/29/2008
• Est. Priority Date: 02/04/2005
• Status: Expired due to Fees

• Current Assignee: Belrose Pharma, Inc.
• Original Assignee: Enzon Pharmaceuticals Inc. (Icahn Associates Holding LLC)
• Inventors: Zhao, Hong, Wu, Dechun
• Primary Examiner(s): Gulakowski; Randy
• Assistant Examiner(s): Asinovsky; Olga
• Application Number:
  US11/051,009
• Publication Number:
  US 20060178474A1
• Time in Patent Office: 1,180 Days
• Field of Search: 525/118,162,54.1 424/178.1
• US Class Current: 525/118
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol

Abstract

A process for efficiently preparing polymer conjugates such as branched PEG-polypeptide conjugates, in large scale without column chromatography clean up of the activated PEG linkers to remove impurities is disclosed.

Claims

1. A process for preparing an activated polymer having a multifunctional linking moiety thereon, comprising:

a) reacting an activatable polymer residue with an activating agent capable of providing a leaving group thereon to provide a first reaction mixture containing an activated polymer residue and said activatable polymer residue;

b) reacting said first reaction mixture with a multi-functional linking moiety having a protecting group thereon which is unreactive with said activated polymer residue under conditions wherein said activated polymer residue of the first reaction mixture is present in excess with respect to said multi-functional linking moiety to form a second reaction mixture containing said first reaction mixture and an intermediate polymer containing the protecting group resulting from said reacting of said activated polymer residue with said multi-functional linking moiety;

c) quenching said second reaction mixture with a sufficient amount of a first quenching agent to inactivate the activated polymer residue therein forming a third reaction mixture containing said intermediate polymer;

d) adding a sufficient amount of a second quenching agent to said third reaction mixture to inactivate the activatable polymer residue therein and form a fourth reaction mixture containing said intermediate polymer;

e) removing the protecting group from the intermediate polymer and neutralizing said fourth reaction mixture to form a fifth reaction mixture containing a deprotected intermediate polymer; and

f) reacting said fifth reaction mixture with a compound capable of activating the deprotected intermediate polymer therein for linking to a biologically active moiety and form a sixth reaction mixture containing an activated polymer having a multifunctional linking moiety thereon.

2. The process of claim 1, further comprising reacting said sixth reaction mixture with a biologically active compound, targeting moiety or diagnostic agent to form polymer conjugates.

3. The process of claim 2, further comprising isolating polymer conjugates.

4. The process of claim 3, wherein said isolating is carried out by diafiltration or size exclusion chromatography.

5. The process of claim 2, wherein the polymer conjugates are of the formula:

(R)_n-L-D

wherein;

R is a polymer residue;

L is a multi-functional aliphatic linking moiety;

D is a member of the group consisting of biologically active moieties, targeting moieties and diagnostic agents; and

n is a positive integer.

6. The process of claim 5, wherein R is a polyalkylene oxide.

7. The process of claim 6, wherein said polyalkylene oxide is polyethylene glycol.

8. The process of claim 7, wherein said polyethylene glycol has a weight average molecular weight of from about 200 to about 120,000 daltons.

9. The process of claim 8, wherein said polyethylene glycol has a weight average molecular weight of from about 2,000 to about 80,000 daltons.

10. The process of claim 9, wherein said polyethylene glycol has a weight average molecular weight of from about 10,000 to about 40,000 daltons.

11. The process of claim 1, wherein said activatable polymer residue is selected from the group consisting of mPEG-OH, mPEG-NH₂, mPEG-CO₂H, mPEG-SO₂, and mPEG-halogen.

12. The process of claim 11, wherein said activatable polymer residue is mPEG-OH.

13. The process of claim 1, wherein said activated polymer residue is selected from the group consisting of

14. The process of claim 13, wherein the activated polymer residue is SC-PEG

15. The process of claim 1, wherein said multi-functional linking moiety having at least one functional group being unreactive with said activated polymer residue is selected from the group consisting of substituted alkyl diamines, triamines, natural and unnatural amino acid derivatives and malonic ester derivatives.

16. The process of claim 15, wherein said multi-functional linking moiety having at least one functional group being unreactive with said activated polymer residue is selected from lysine, diamino alkyls, dihydroxyalkyls, and dithioakyls.

17. The process of claim 15, wherein the multi-functional aliphatic linking moiety having at least one functional group being unreactive with said activated polymer residue is lysine, a lysine ester or a lysine ethyl ester.

18. The process of claim 1, wherein said first quenching agent is a compound containing a free amine, free thiol or free hydroxyl group.

19. The process of claim 18, wherein said first quenching agent is selected from the group consisting of cysteine, benzyl amine, n-butyl amine, phenylethylamine, C-terminal protected amino acids and mixtures thereof.

20. The process of claim 1, wherein said second quenching agent is a compound containing a silyl group or an acid chloride.

21. The process of claim 20, wherein said second quenching agent is selected from the group consisting of TBDMSiCl, TMSiCl, MeI, MeSO₄, CF₃SO₃Me, and Me₃OBF₄.

22. The process of claim 21, wherein said second quenching agent is TBDMSiCl.

23. The process of claim 1, wherein the compound capable of activating the deprotected intermediate polymer conjugate is N-hydroxysuccinimide (NHS).

24. The process of claim 2, wherein the biologically active compound is selected from proteins, peptides polypeptides, enzymes, oligonucleotides, single chain binding antigens (SCA'"'"'s), antibodies, antibody fragments and natural or synthetic medicinal chemicals.

25. The process of claim 24, wherein the protein is an interferon.

26. The process of claim 25, wherein the interferon is α

, β

or γ

interferon.

27. The process of claim 2, wherein the polymer conjugates are selected from the group consisting of

28. The process of claim 1, wherein the activatable polymer residue is mPEG-OH;

the activating agent capable of providing a leaving group is N-hydroxysuccinimidyl;

the activated polymer residue is mPEG-succinimidyl carbonate, the first quenching agent is benzyl amine;

the second quenching agent is TBDMSiCl; and

the compound capable of activating the deprotected intermediate polymer for linking to a biological moiety is N-hydroxysuccinimidyl.

29. The process of claim 28 wherein the N-hydroxysuccinimidyl activated intermediate polymer conjugate is further reacted with a biologically active compound.

30. A process for preparing an activated polymer having a multifunctional linking moiety thereon, comprising:

a) reacting an activatable polymer residue with an activating agent capable of providing a leaving group thereon to provide a first reaction mixture containing an activated polymer residue and said activatable polymer residue;

b) reacting said first reaction mixture with a multi-functional linking moiety having a protecting group thereon which is unreactive with said activated polymer residue under conditions wherein said activated polymer residue of the first reaction mixture is present in excess with respect to said multi-functional linking moiety to form a second reaction mixture containing said first reaction mixture and an intermediate polymer containing the protecting group resulting from said reacting of said activated polymer residue with said multi-functional linking moiety;

c) quenching said second reaction mixture with a sufficient amount of a first quenching agent to inactivate the activatable polymer residue therein forming a third reaction mixture containing said intermediate polymer;

d) adding a sufficient amount of a second quenching agent to said third reaction mixture to inactivate the activated polymer residue therein and form a fourth reaction mixture containing said intermediate polymer;

e) removing the protecting group from the intermediate polymer and neutralizing said fourth reaction mixture to form a fifth reaction mixture containing a deprotected intermediate polymer; and

f) reacting said fifth reaction mixture with a compound capable of activating the deprotected intermediate polymer therein for linking to a biologically active moiety and form a sixth reaction mixture containing an activated polymer having a multifunctional linking moiety thereon.