# US 9,040,664 B2 · Materials and methods for preparing protein-polymer conjugates

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 9,040,664 B2
- Document ID: US9040664B2
- Status: Granted
- Kind: B2
- Application number: 10/553,570
- Filed: 2004-04-08
- Published: 2007-04-12
- Granted: 2015-05-26
- Earliest claimed: 2003-04-11
- Assignees: AntriaBio, Inc.
- Applicants: Kenneth Hinds; Danny Lewis; Paul Schmidt; Kathleen M. Campbell
- Inventors: Kenneth Hinds; Danny Lewis; Paul Schmidt; Kathleen M. Campbell
- Web page: https://patentgrep.com/patent/US9040664B2

## CPC

- A61K 47/60: the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
- A61K 9/5031: {obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)}
- A61K 38/28: Insulins
- A61P 1/18: for pancreatic disorders, e.g. pancreatic enzymes
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- C07K 14/62: Insulins
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## 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

### Claim 1

A method for preparing a site-specific insulin-PEG conjugate comprising:
(a) contacting insulin protein with PEG in an aqueous solution in the presence of at least one organic solvent selected from the group consisting of ethanol, methanol, DMSO, dioxane, DMF, and NMP and at least one metal chelator selected from the group consisting of metal ion chelators, EDTA, deferoxamine (DEF), diethylenetriamine pentaacetic acid (DTPA), and bis(aminoethyl)glycolether N,N,N′,N′-tetraacetic acid (EGTA), to form the site-specific insulin-PEG conjugate by a predominant reaction of the insulin protein at residue PheB1 without protecting the insulin protein at GlyA1 and LysB29 residues, and
(b) isolating the conjugate.

### Claim 2 (depends on 1)

The method of claim 1, wherein the insulin protein comprises human insulin.

### Claim 3 (depends on 1)

The method 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.

### Claim 4 (depends on 1)

The method of claim 1, wherein the PEG and insulin protein are contacted at a molar ratio of about 10:1-1:1.

### Claim 5 (depends on 1)

The method of claim 1, wherein the organic solvent is present at a concentration of about 0.1 to 10%.

### Claim 6 (depends on 1)

The method of claim 1, wherein the insulin protein and the PEG are contacted at a pH of about 5.0-7.5.

### Claim 7 (depends on 1)

The method of claim 1, wherein the metal chelator is present at a concentration of about 0.1-10 mM.

### Claim 8 (depends on 1)

The method of claim 1, wherein the insulin protein and the PEG are contacted at a temperature of about 4-50° C.

### Claim 9 (depends on 1)

The method of claim 1, wherein the method further comprises the step of quenching formation of the conjugate prior to the isolating of the conjugate.

### Claim 10 (depends on 1)

The method of claim 1, further comprising the step of:
(c) encapsulating the isolated conjugate in a biodegradable polymer.