Method for Preparation of Site-Specific Protein Conjugates

• US 20150216995A1
• Filed: 04/21/2015
• Published: 08/06/2015
• Est. Priority Date: 04/11/2003
• Status: Active Grant

• Current Assignee: Rezolute, Inc.
• Original Assignee: AntriaBio, Inc.
• Inventors: Hinds, Kenneth, Lewis, Danny, Schmidt, Paul, Campbell, Kathleen M.
• Granted Patent: US 9,789,196 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 38/28 Insulins
• 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: A61K 9/5031 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C07K 14/62 Insulins

Abstract

The invention is directed to a single-step method for rapidly and efficiently preparing protein-polymer conjugates, including an insulin-polymer conjugate. According to the method of the present invention, a protein and hydrophilic polymer are contacted in the presence of at least one organic solvent and at least one metal chelator, under conditions that promote the formation of a conjugate of the protein and polymer. Thus, the invention is directed to the site-specific modification of selected proteins, such as insulin, with poly(ethylene glycol) at residue PheB1. The invention also provides a pharmaceutical formulation for encapsulating the conjugate in a biodegradable polymer.

Claims

1. An insulin-PEG conjugate comprising PEG linked to insulin at the PheB1 amino terminus via a secondary amine.

2. The insulin-PEG conjugate of claim 1, wherein the insulin comprises human insulin.

3. The insulin-PEG conjugate of claim 1, wherein the PEG comprises an amino-reactive PEG derivative selected from the group consisting of an aldehyde, a N-hydroxy succinimide, a PNP-carbonate, and a benzotrizole terminated hydrophilic polymer.

4. The insulin-PEG conjugate of claim 1, wherein the PEG is a linear polymer.

5. The insulin-PEG conjugate of claim 1, wherein the PEG is a branched polymer.

6. The insulin-PEG conjugate of claim 1, wherein the PEG has a molecular weight in the range of 500 to 50,000 Da.

7. The insulin-PEG conjugate of claim 1, wherein the PEG is a mPEG succinimidyl active ester of a propionic acid linker mPEG-SPA.

8. The insulin-PEG conjugate of claim 1, wherein the PEG is monomethoxy PEG-aldehyde.

9. The insulin-PEG conjugate of claim 1, wherein the insulin comprises insulin analogs, homologs, or derivatives.

10. The insulin-PEG conjugate of claim 1, wherein the conjugate comprises an unreacted GlyA1 amino terminus and an unreacted LysB29 amino terminus.

11. A poly(lactide-co-glycolide) (PLGA) microsphere encapsulating an insulin-PEG conjugate, wherein the insulin-PEG conjugate comprises PEG linked to insulin at the PheB1 amino terminus via a secondary amine.

12. An insulin-PEG conjugate produced according to a method comprising:

contacting insulin with PEG in the presence of at least one organic solvent and at least one metal chelator, under conditions that promote the formation of a conjugate of the insulin and PEG; and

isolating the conjugate.

13. The insulin-PEG conjugate of claim 12, wherein the at least one organic solvent is selected from the group consisting of ethanol, methanol, DMSO, dioxane, DMF, and NMP.

14. The insulin-PEG conjugate of claim 12, wherein the at least one metal chelator is selected from the group consisting of a metal ion chelator, EDTA, deferoxamine (DEF), diethylenetriamine pentaacetic acid (DTPA), and bis(aminoethyl)glycolether N,N,N′

N′

-tetraacetic acid (EGTA).

15. The insulin-PEG conjugate of claim 12, wherein the method comprises forming the insulin-PEG conjugate by a predominant reaction of insulin at residue PheB1 without protecting insulin at GlyA1 and LysB29 residues.

16. The insulin-PEG conjugate of claim 12, wherein the insulin and the PEG are contacted at a pH from about 5.0 to about 7.5.

17. A F5000 insulin-PEG conjugate comprising PEG linked to insulin at the PheB1 amino terminus via a secondary amine.

18. The F5000 insulin-PEG conjugate of claim 17, wherein the insulin-PEG conjugate is encapsulated by a biodegradable polymer microsphere.

19. The F5000 insulin-PEG conjugate of claim 18, wherein the biodegradable polymer comprises a poly(lactide), poly(glycolide), poly(d,l-lactide-co-glycolide), poly(caprolactone), poly(orthoester), copolymer of a poly(ester) and a poly(ether), or copolymer of a poly(lactide) and a poly(ethylene glycol).

20. The F5000 insulin-PEG conjugate of claim 18, wherein the biodegradable polymer is poly(lactide-co-glycolide).

21. A composition for preparing a pharmaceutical formulation, the composition comprising:

an organic solvent;

a metal chelator;

a first mono-PEGylated insulin conjugate comprising PEG linked to insulin at the PheB1 amino terminus via a secondary amine;

a second mono-PEGylated insulin conjugate comprising PEG linked to insulin at either the GlyA1 amino terminus or the LysB29 amino terminus;

an unreacted insulin protein; and

a di-PEGylated insulin conjugate, wherein;

the first mono-PEGylated insulin conjugate comprises 95% or greater of the total of the first mono-PEGylated insulin conjugate and the second mono-PEGylated insulin conjugate.