# US 9,040,665 B2 · Controlled cross-linking processing of proteins > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,040,665 B2 - Document ID: US9040665B2 - Status: Granted - Kind: B2 - Application number: 13/430,562 - Filed: 2012-03-26 - Published: 2013-01-24 - Granted: 2015-05-26 - Earliest claimed: 2008-09-30 - Assignees: Case Western Reserve University - Applicants: Gary Wnek; Linghui Meng - Inventors: Gary Wnek; Linghui Meng - Web page: https://patentgrep.com/patent/US9040665B2 ## CPC - C07K 1/1075: {by covalent attachment of amino acids or peptide residues} - A61L 27/22: Polypeptides or derivatives thereof {, e.g. degradation products} - C07K 1/107: by chemical modification of precursor peptides - C07K 1/36: by a combination of two or more processes of different types - C07K 14/78: Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG] - D01F 1/10: Other agents for modifying properties - D01F 4/00: Monocomponent artificial filaments or the like of proteins; Manufacture thereof - 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) - A61L: METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES (preservation of bodies or disinfecting characterised by the agents employed A01N; preserving, e.g. sterilising, food or foodstuffs A23; preparations for medical, dental or toiletry purposes A61K) - D01F: CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS ## 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. ## Claims ### Claim 1 A method of forming a cross-linked protein structure, comprising: forming a protein solution by dissolving a protein in a solution of water, salt, alcohol, and a cross-linker, wherein the cross-linker is N-hydroxysuccinimide (“EDC”) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (“NHS”), and the protein is collagen; forming an intermediate protein structure from the protein solution; and after a period of time has elapsed, cross-linking at least a portion the intermediate protein structure to form the cross-linked protein structure. ### Claim 2 (depends on 1) The method of claim 1, wherein the molar ratio of NHS to EDC is about 1:1. ### Claim 3 (depends on 1) The method of claim 1, wherein the intermediate protein structure is formed by an electrospinning process. ### Claim 4 (depends on 1) The method of claim 1, wherein the intermediate protein structure is formed by an electrospraying process. ### Claim 5 (depends on 1) The method of claim 1, wherein the intermediate protein structure is formed by a gravitational feed process. ### Claim 6 (depends on 1) The method of claim 1, wherein the collagen dissolved in the solution is less than about 25 percent by weight. ### Claim 7 (depends on 6) The method of claim 6, wherein the collagen dissolved in the solution is about 16 percent by weight. ### Claim 8 (depends on 1) The method of claim 1, wherein the cross-linker is EDC and NHS, and the protein is gelatin. ### Claim 9 (depends on 8) The method of claim 8, wherein the molar ratio of NHS to EDC is 1:1. ### Claim 10 (depends on 8) The method of claim 8, wherein the intermediate protein structure is formed by an electrospinning process. ### Claim 11 (depends on 8) The method of claim 8, wherein the intermediate protein structure is formed by an electrospraying process. ### Claim 12 (depends on 8) The method of claim 8, wherein the intermediate protein structure is formed by a gravitational feed process. ### Claim 13 (depends on 8) The method of claim 8, wherein the gelatin dissolved in the solution is less than about 25 percent by weight. ### Claim 14 (depends on 13) The method of claim 13, wherein the gelatin dissolved in the solution is about 16 percent by weight.