# US 2014/0193386 A1 · Method for enzymatic cross-linking of a protein > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0193386 A1 - Document ID: US20140193386A1 - Status: Published application - Kind: A1 - Application number: 14/181,841 - Filed: 2014-02-17 - Published: 2014-07-10 - Earliest claimed: 2008-06-18 - Assignees: Lifebond Ltd. - Applicants: LifeBond Ltd. - Inventors: Orahn PREISS-BLOOM; Guy TOMER; Nadav BRAMSON - Web page: https://patentgrep.com/patent/US20140193386A1 ## CPC - C12N 9/1044: {Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract A method for cross-linking albumin for use as a sealant or glue for a biological system, for example to induce hemostasis and/or prevent leakage of any other fluid from a biological tube or tissue, such as lymph for example. The cross-linked albumin may optionally and preferably be applied as part of a bandage for example. In other embodiments, the present invention provides a method of enzymatically cross-linking globular proteins, by altering the structure of the protein to improve the accessibility of the protein to the cross-linking enzyme. ## Claims ### Claim 1 A gel composition comprising denatured albumin, transglutaminase, a chaotropic agent and a disulfide bond reducing agent in a pH range of from 6 to 9. ### Claim 2 (depends on 1) The composition of claim 1, wherein said transglutaminase is calcium independent. ### Claim 3 (depends on 2) The composition of claim 2, wherein said transglutaminase is microbial transglutaminase. ### Claim 4 (depends on 3) The composition of claim 3, wherein a protein content of said transglutaminase is present in an amount from about 0.0001% to about 2% w/w of the composition. ### Claim 5 (depends on 4) The composition of claim 4, wherein said transglutaminase is present in an amount of from about 0.01% to about 1% w/w of the composition. ### Claim 6 (depends on 5) The composition of claim 5, wherein said transglutaminase is present in an amount of from about 0.1% to about 1% w/w of the composition. ### Claim 7 (depends on 6) The composition of claim 6, wherein said transglutaminase is present in an amount of from about 0.5% to about 1.5% w/w of the composition. ### Claim 8 (depends on 1) The composition of claim 1, wherein said disulfide bond reducing agent comprises one or more of a thiol containing reducing agent, dithiothreitol (DTT), cystein, glutathione, a phosphine-containing agent or a combination thereof. ### Claim 9 (depends on 8) The composition of claim 8 wherein said disulfide bond reducing agent comprises 2-mercaptoethanol. ### Claim 10 (depends on 8) The composition of claim 8, wherein said phosphine containing agent comprises tris(2-carboxyethyl) phosphine (TCEP). ### Claim 11 (depends on 1) The composition of claim 1, wherein said chaotropic agent is selected from the group consisting of urea, guanidinium chloride, and lithium perchlorate. ### Claim 12 (depends on 11) The composition of claim 11, wherein said urea is present in a range of from 3M to 6M. ### Claim 13 (depends on 1) The composition of claim 1, further comprising a salt. ### Claim 14 (depends on 13) The composition of claim 13 wherein said salt comprises sodium chloride. ### Claim 15 (depends on 14) The composition of claim 14, wherein said sodium chloride is present in an amount of from 0.25M to 1M. ### Claim 16 (depends on 1) The composition of claim 1, wherein said chaotropic agent comprises urea and wherein said disulfide bond reducing agent comprises at least one of TCEP, DTT or 2-mercaptoethanol. ### Claim 17 (depends on 1) The composition of claim 1, wherein said albumin is present in a concentration from 1% to 25%. ### Claim 18 (depends on 17) The composition of claim 17, wherein said albumin is present in a concentration from 1% to 10%. ### Claim 19 (depends on 1) The composition of claim 1, wherein said denatured albumin comprises heat denatured albumin. ### Claim 20 (depends on 1) The composition of claim 1, wherein a concentration of transglutaminase is in the range of from about 5 to about 200 enzyme units (U/g) of total composition. ### Claim 21 (depends on 20) The composition of claim 20, wherein said concentration of transglutaminase is in the range of from about 15 to about 55 enzyme units (U/g) of total composition. ### Claim 22 (depends on 21) The composition of claim 21, wherein said concentration of transglutaminase is in the range of from about 25 to about 45 enzyme units (U/g) of total composition. ### Claim 23 (depends on 1) A medical sealant, comprising the composition of claim 1. ### Claim 24 A method of preparation of a gel composition, comprising: providing albumin with a pH value of from 4 to 6; heat denaturing said albumin to form denatured precipitated albumin; dissolving said denatured precipitated albumin with a disulfide bond reducing agent at a pH value of no more than 3 or of at least 9 to form reduced soluble albumin; adjusting a pH value of said reduced albumin to be between 6 and 9; and combining transglutaminase with said reduced albumin to form a combination. ### Claim 25 (depends on 24) The method of claim 24, further comprising applying a salt to said albumin to prevent precipitation. ### Claim 26 (depends on 25) A product produced according to the method of claim 25. ### Claim 27 (depends on 26) A method of treatment of a subject in need of treatment with a medical sealant or glue, comprising applying the product of claim 26 to a tissue of the subject as a medical sealant or glue. ### Claim 28 (depends on 27) The method of claim 27, wherein said applying comprises applying in a medical application selected from the group consisting of reinforcement of surgical repair lines; provision of fluid-stasis; prevention of lymphorrhea; prevention of cerebro-spinal fluid (CSF) leakage; prevention of anastomotic dehiscence; and sealing of an attachment between a tissue and a material. ### Claim 29 (depends on 27) The method of claim 27, wherein said medical sealant is provided in a form selected from the group consisting of a gel, a spray, a strip, a patch, and a bandage. ### Claim 30 (depends on 29) The method of claim 29, wherein said applying the product comprises applying the product in gel form as a medical glue. ### Claim 31 (depends on 1) Use of the composition of claim 1 for preparation of a tissue engineering scaffold. ### Claim 32 A gel composition comprising heat denatured albumin, transglutaminase, a chaotropic agent and a disulfide bond reducing agent in a pH range of from 6 to 9. ### Claim 33 A method of preparation of a gel composition, comprising: providing albumin with a pH value of from 6 to 9; denaturing said albumin with a chaotropic agent to form denatured albumin; treating said denatured albumin with a disulfide bond reducing agent at a pH value of 6 to 9 to form reduced albumin; and combining transglutaminase with said reduced albumin to form a combination. ### Claim 34 (depends on 33) The method of claim 33, further comprising adding a salt to said albumin to prevent precipitation before or during said denaturing. ### Claim 35 (depends on 33) The method of claim 33, wherein said denaturing said albumin comprises heat denaturing. ### Claim 36 (depends on 33) The method of claim 33, wherein said chaotropic agent comprises urea.