# US 9,611,312 B2 · Preparation and/or formulation of proteins cross-linked with polysaccharides

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

- Number: US 9,611,312 B2
- Document ID: US9611312B2
- Status: Granted
- Kind: B2
- Application number: 13/988,462
- Filed: 2011-11-22
- Published: 2013-11-07
- Granted: 2017-04-04
- Earliest claimed: 2010-11-23
- Assignees: Elastagen Pty. Limited
- Applicants: Jens Sommer-Knudsen
- Inventors: Jens Sommer-Knudsen
- Web page: https://patentgrep.com/patent/US9611312B2

## CPC

- A61L 27/227: {Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24}
- A61K 8/64: Proteins; Peptides; Derivatives or degradation products thereof
- A61K 8/735: {Mucopolysaccharides, e.g. hyaluronic acid; Derivatives thereof}
- A61L 27/20: Polysaccharides
- A61L 27/22: Polypeptides or derivatives thereof {, e.g. degradation products}
- A61L 27/26: Mixtures of macromolecular compounds
- A61L 27/50: Materials characterised by their function or physical properties {, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials}
- A61L 27/58: Materials at least partially resorbable by the body
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 17/00: Drugs for dermatological disorders
- A61P 17/02: for treating wounds, ulcers, burns, scars, keloids, or the like
- A61P 19/00: Drugs for skeletal disorders
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 27/02: Ophthalmic agents
- A61Q 19/08: Anti-ageing preparations
- C07K 14/765: Serum albumin, e.g. HSA
- C07K 14/78: Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
- C08B 11/08: with hydroxylated hydrocarbon radicals; Esters, ethers, or acetals thereof
- C08B 11/12: substituted with carboxylic radicals {, e.g. carboxymethylcellulose [CMC]}
- C08B 33/04: Ethers
- C08B 37/0009: {alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof}
- C08B 37/0021: {Dextran, i.e. (alpha-1,4)-D-glucan; Derivatives thereof, e.g. Sephadex, i.e. crosslinked dextran}
- C08B 37/0024: {beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof}
- C08B 37/0033: {Xanthan, i.e. D-glucose, D-mannose and D-glucuronic acid units, saubstituted with acetate and pyruvate, with a main chain of (beta-1,4)-D-glucose units; Derivatives thereof}
- C08B 37/0063: {Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan}
- C08B 37/0069: {Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof}
- C08B 37/0072: {Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates}
- C08B 37/0075: {Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof}
- C08B 37/0084: {Guluromannuronans, e.g. alginic acid, i.e. D-mannuronic acid and D-guluronic acid units linked with alternating alpha- and beta-1,4-glycosidic bonds; Derivatives thereof, e.g. alginates}
- C08B 37/0096: {Guar, guar gum, guar flour, guaran, i.e. (beta-1,4) linked D-mannose units in the main chain branched with D-galactose units in (alpha-1,6), e.g. from Cyamopsis Tetragonolobus; Derivatives thereof}
- C08H 1/00: Macromolecular products derived from proteins (food proteins A23; glue, gelatine C09H)
- C08H 1/06: derived from horn, hoofs, hair, skin or leather
- C08L 1/28: Alkyl ethers
- C08L 1/284: {with hydroxylated hydrocarbon radicals}
- C08L 1/286: {substituted with acid radicals, e.g. carboxymethyl cellulose [CMC] (C08L1/282 takes precedence)}
- C08L 3/14: Amylose derivatives; Amylopectin derivatives
- C08L 5/00: Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
- C08L 5/02: Dextran; Derivatives thereof
- C08L 5/04: Alginic acid; Derivatives thereof
- C08L 5/08: Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
- C08L 5/10: Heparin; Derivatives thereof
- C08L 89/00: Compositions of proteins; Compositions of derivatives thereof
- C08L 89/06: derived from leather or skin {, e.g. gelatin}
- A61K 2800/91: Injection
- A61L 2400/06: Flowable or injectable implant compositions
- A61L 2430/34: for soft tissue reconstruction
- 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)
- 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
- A61Q: SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY 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)
- C08B: POLYSACCHARIDES; DERIVATIVES THEREOF (polysaccharides containing less than six saccharide radicals attached to each other by glycosidic linkages C07H; fermentation or enzyme-using processes C12P19/00; sugar industry C13; production of cellulose D21)
- C08H: DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS (polysaccharides C08B; natural rubber C08C; natural resins or their derivatives C09F; bituminous materials C10)
- C08L: COMPOSITIONS OF MACROMOLECULAR COMPOUNDS (compositions based on polymerisable monomers C08F, C08G; artificial filaments or fibres D01F; textile treating compositions D06)

## Abstract

Therapeutic compositions and/or formulations are provided, comprising: at least one cross-linked protein matrix, wherein the at least one cross-linked protein matrix comprises at least one protein residue and at least one saccharide-containing residue, and methods of producing the same. The cross-linked protein matrix may be derived from cross-linking a full length or substantially full length protein, such as tropoelastin, elastin, albumin, collagen, collagen monomers, immunoglobulins, insulin, and/or derivatives or combinations thereof, with a saccharide containing cross-linking agent, such as a polysaccharide cross-linking agent derived from, for example, hyaluronic acid or a cellulose derivative. The therapeutic compositions may be administered topically or by injection. The present disclosure also provides methods, systems, and/or kits for the preparation and/or formulation of the compositions disclosed herein.

## Claims

### Claim 1

A composition, comprising at least one cross-linked protein matrix, wherein the at least one cross-linked protein matrix comprises:
i) between 1-250 mg/mL of at least one protein residue derived from at least one amine-bearing protein;
ii) at least one saccharide-containing cross-linking residue derived from at least one saccharide-containing cross-linking molecule comprising one or more carboxyl groups; and
iii) at least one intermolecular cross-linkage comprising an amide bond between an amine of the at least one protein residue and a carboxyl group of the at least one saccharide-containing cross-linking molecule.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the composition is an injectable composition.

### Claim 3 (depends on 1)

The composition of claim 1, wherein the composition is extrudable through needles of 18G to 31G to a length of between about 5 cm to about 30 cm and the extruded composition substantially holds together without surface support.

### Claim 4 (depends on 1)

The composition of claim 1, wherein the composition is extrudable to a length of between 5 cm to 30 cm, 10 cm to 20 cm; or 15 cm to 30 cm without further surface support when extruded through a fine gauge needle and wherein the extruded composition is substantially coherent and substantially holds together.

### Claim 5 (depends on 1)

The composition of claim 1, wherein the composition retains sufficient cohesiveness after needle extrusion such that strings of extruded composition do not break during extrusion.

### Claim 6 (depends on 1)

The composition of claim 1, wherein the at least one cross-linked protein matrix comprises a 50-99.99% full length protein residue or a full length protein residue.

### Claim 7 (depends on 1)

The composition of claim 1, wherein the composition enables the at least one protein residue to retain its full length, and wherein the composition is needle injectable, retains a coherent structure, and is sufficiently cross-linked to slow the composition's resorption in vivo.

### Claim 8 (depends on 1)

The composition of claim 1, wherein the at least one protein residue is 50-99.99% full length or full length.

### Claim 9 (depends on 1)

The composition of claim 1, wherein the composition is tissue compatible, enhances tissue in-growth, enhances tissue re-growth, or combinations thereof.

### Claim 10 (depends on 1)

The composition of claim 1, wherein the at least one saccharide-containing cross-linking residue has one or more of the following properties: substantially bioavailable, substantially biodegradeable, substantially bioabsorbable, or substantially bioresorbable.

### Claim 11 (depends on 1)

The composition of claim 1, wherein the at least one saccharide-containing cross-linking residue is derived from or comprises the residue of hyaluronic acid, a cellulose derivative, carboxy cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxy-propylcellulosecarboxymethyl amylose, xanthan gum, guar gum, α-glucan, β-glucan, β-1,4-glucan, β-1,3-glucan, alginates, carboxymethyl dextran, a glycosaminoglycan derivative, chondroitin-6-sulfate, dermatin sulfate, heparin, heparin sulfate, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic) acid, tricalcium phosphate, 1-hydroxyapatite, pharmaceutically acceptable salts of, derivatives of, or combinations thereof.

### Claim 12 (depends on 1)

The composition of claim 1, wherein the at least one protein residue is derived from or comprises the residue of tropoelastin, elastin, albumin, collagen, collagen monomers, immunoglobulins, insulin, derivatives of, or combinations thereof.

### Claim 13 (depends on 1)

The composition of claim 1, wherein the concentration of the at least one protein residue is in the range of between 1-200 mg/mL.

### Claim 14 (depends on 1)

The composition of claim 1, wherein the at least one cross-linked protein matrix is derived from or comprises the reside of a synthetic protein substantially identical to a naturally occurring human protein.

### Claim 15 (depends on 1)

The composition of claim 1, wherein the at least one cross-linked protein matrix is prepared from at least one saccharide-containing cross-linking molecule comprising at least 5% of activated carboxyl groups, modified carboxyl groups, or combinations thereof.

### Claim 16 (depends on 1)

The composition of claim 1, wherein the number of protein units not incorporated into the at least one cross-linked protein matrix and left unbound is at least 1%, 3%, 5%, or 7%.

### Claim 17 (depends on 1)

The composition of claim 1, wherein the at least one cross-linkage further comprises a spacer group.

### Claim 18 (depends on 17)

The composition of claim 17, wherein the spacer group comprises a glycol moiety, an ethylene oxide moiety, a polyamine, a polyol, or combinations thereof.

### Claim 19 (depends on 1)

The composition of claim 1, wherein the concentration of the at least one protein residue is in the range of between 5-30 mg/mL.

### Claim 20 (depends on 1)

The composition of claim 1, wherein the concentration of the at least one saccharide-containing cross-linking residue is in the range of between 1-30 mg/mL.

### Claim 21 (depends on 20)

The composition of claim 20, wherein the concentration of the at least one protein residue is in the range of between 25-50 mg/mL.

### Claim 22 (depends on 21)

The composition of claim 21, wherein:
i) the at least one protein residue is derived from or comprises the residue of tropoelastin; and
ii) the at least one saccharide-containing cross-linking residue is derived from or comprises the residue of hyaluronic acid.

### Claim 23 (depends on 8)

The composition of claim 8, wherein said at least one protein residue is devoid of intramolecular cross-links.

### Claim 24

A method of preparing a cross-linked protein composition, comprising:
cross-linking at least one amine-bearing protein with at least one saccharide-containing cross-linking molecule comprising one or more carboxyl groups to form at least one cross-linked protein matrix;
wherein the at least one cross-linked matrix comprises:
i) at least one protein residue; and
ii) at least one saccharide-containing cross-linking residue; and
iii) at least one intermolecular cross-linkage comprising an amide bond between the amine of the at least one amine-bearing protein and the carboxyl group of the at least one saccharide-containing cross-linking molecule.

### Claim 25 (depends on 24)

The method of claim 24, wherein the prepared cross-linked protein composition is an injectable composition.

### Claim 26 (depends on 24)

The method of claim 24, wherein the prepared cross-linked protein composition is extrudable through needles of 18G to 31G to a length of between 5-30 cm and the extruded composition substantially holds together without surface support.

### Claim 27 (depends on 24)

The method of claim 24, wherein the prepared cross-linked protein composition retains sufficient cohesiveness after needle extrusion such that strings of extruded composition do not break during extrusion.

### Claim 28 (depends on 24)

The method of claim 24, wherein the at least one protein residue is 50-99.99% full length, or full length.

### Claim 29 (depends on 28)

The method of claim 28, wherein said at least one protein residue is devoid of intramolecular cross-links.

### Claim 30 (depends on 24)

The method of claim 24, wherein the at least one saccharide-containing cross-linking residue has one or more of the following properties: substantially bioavailable, substantially biodegradeable, substantially bioabsorbable, or substantially bioresorbable.

### Claim 31 (depends on 24)

The method of claim 24, wherein the at least one saccharide-containing cross-linking residue is derived from or comprises the residue of hyaluronic acid, a cellulose derivative, carboxy cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxy-propylcellulosecarboxymethyl amylose, xanthan gum, guar gum, α-glucan, β-glucan, β-1,4-glucan, β-1,3-glucan, alginates, carboxymethyl dextran, a glycosaminoglycan derivative, chondroitin-6-sulfate, dermatin sulfate, heparin, heparin sulfate, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic) acid, tricalcium phosphate, 1-hydroxyapatite, pharmaceutically acceptable salts of, derivatives of, or combinations thereof.

### Claim 32 (depends on 24)

The method of claim 24, wherein the at least one protein residue is derived from or comprises the residue of tropoelastin, elastin, albumin, collagen, collagen monomers, immunoglobulins, insulin, derivatives of, or combinations thereof.

### Claim 33 (depends on 24)

The method of claim 24, wherein the prepared cross-linked protein comprises a reside of a synthetic protein substantially identical to a naturally occurring human protein.

### Claim 34 (depends on 24)

The method of claim 24, wherein the number of protein units not incorporated into the at least one cross-linked protein matrix and left unbound in the prepared cross-linked protein composition is at least 1%, 3%, 5%, or 7%.

### Claim 35 (depends on 24)

The method of claim 24, wherein the at least one saccharide-containing cross-linking molecule comprises at least 5% of activated carboxyl groups, modified carboxyl groups, or combinations thereof.

### Claim 36 (depends on 24)

The method of claim 24, wherein the prepared cross-linked protein composition comprises a concentration in the range of between 1-250 mg/mL of the at least one protein residue derived from the at least one amine-bearing protein.

### Claim 37 (depends on 24)

The method of claim 24, wherein the prepared cross-linked protein composition comprises a concentration in the range of between 5-30 mg/mL of the at least one protein residue.

### Claim 38 (depends on 24)

The method of claim 24, wherein the prepared cross-linked protein composition comprises a concentration in the range of between 1-30 mg/mL of the at least one saccharide-containing cross-linking residue.

### Claim 39 (depends on 38)

The method of claim 38, wherein the prepared cross-linked protein composition comprises a concentration in the range of between 25-50 mg/mL of the at least one protein residue.

### Claim 40 (depends on 39)

The method of claim 39, wherein:
i) the at least one protein residue is derived from or comprises the residue of tropoelastin; and
ii) the at least one saccharide-containing cross-linking residue is derived from or comprises the residue of hyaluronic acid.