Controlled cross-linking processing of proteins

Patent metadata

Field	Value
Publication number	US-9683011-B2
Application number	US-201514720331-A
Country	US
Kind code	B2
Filing date	May 22, 2015
Priority date	Sep 30, 2008
Publication date	Jun 20, 2017
Grant date	Jun 20, 2017

Abstract

A method of forming a cross-linked protein structures includes preparing a solution of protein dissolved in a benign solvent and forming an intermediate protein structure from the solution. The intermediate protein structure can be cross-linked by providing for a specific ratio of chemical cross-linking agents to form the cross-linked protein structure. The solution can be prepared by adding a cross-linker of N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) at a ratio of two-to-one of NHS to EDC to alcohol. PBS buffer (20×) can be added to the solution until the volume ratio of PBS buffer (20×) to alcohol is about one-to-one. About 16 percent by weight of protein can be dissolved in the solution. The solution can be electrospun to form an intermediate protein structure. After a period of time, the protein structure can be cross-linked to form the cross-linked protein structure.

First claim

What is claimed is:

1. A method of forming a protein structure comprising:
forming a solution comprising water, alcohol, salt, collagen, and a cross-linker;
forming a plurality of collagen fibers from the solution; and
after the passage of a period of time, crosslinking at least a portion of the plurality of collagen fibers to form a protein structure.

2. The method of claim 1 , wherein the cross-linker is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

3. The method of claim 2 , wherein the molar ratio of NHS to EDC is greater than about 1.5:1.

4. The method of claim 3 , wherein a concentration of EDC is about 200 nM and a concentration of NHS is about 400 nM.

5. The method of claim 3 , wherein the salt is a PBS buffer (20×) and a volume ratio of PBS buffer (20×) to alcohol is about 1:1.

6. The method of claim 5 , wherein the collagen is dissolved in the solution.

7. The method of claim 1 , wherein the plurality of collagen fibers are formed by an electrospinning method.

8. The method of claim 7 , wherein the electrospinning method results in the plurality of collagen fibers generally aligned after electrospinning.

9. The method of claim 1 , wherein the protein structure is placed in an environment where the relative humidity is between about 40% and about 60%.

10. The method of claim 1 , wherein the protein structure is immersed in a solution of salt, alcohol and water.

11. The method of claim 2 , wherein the molar ratio of NHS to EDC is about 1:1.

12. The method of claim 6 , wherein the collagen dissolved in the solution is less than about 25 percent by weight.

13. The method of claim 12 , wherein the collagen dissolved in the solution is less than about 16 percent by weight.

Assignees

• Univ Case Western Reserve

Inventors

• Wnek Gary E
• Meng Linghui

Classifications

• C07K1/1075 Primary

  by covalent attachment of amino acids or peptide residues · CPC title

• D01F1/10

  Other agents for modifying properties · CPC title

• C07K1/36

  by a combination of two or more processes of different types · CPC title

• C07K1/107

  by chemical modification of precursor peptides · CPC title

• D01F4/00

  Monocomponent artificial filaments or the like of proteins; Manufacture thereof · CPC title