# US 4,391,749 A · Method for the purification of collagens

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- Number: US 4,391,749 A
- Document ID: US4391749A
- Status: Granted
- Kind: A
- Application number: 06/313,326
- Filed: 1981-10-19
- Granted: 1983-07-05
- Earliest claimed: 1981-10-19
- Assignees: La Jolla Cancer Research Foundation
- Inventors: Eva S. Engvall; Erkki I. Ruoslahti
- Web page: https://patentgrep.com/patent/US4391749A

## CPC

- B01D 15/3804: {Affinity chromatography}
- B01J 20/286: Phases chemically bonded to a substrate, e.g. to silica or to polymers
- B01J 20/3204: {Inorganic carriers, supports or substrates}
- B01J 20/321: {consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds}
- B01J 20/3212: {consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds}
- B01J 20/3274: {Proteins, nucleic acids, polysaccharides, antibodies or antigens}
- C07K 14/78: Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
- B01J 2220/54: Sorbents specially adapted for analytical or investigative chromatography
- B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28)
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- 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

Collagen affinity matrices are prepared, useful for separating collagenous substances from other high molecular weight proteinaceous substances. Fibronectin is fragmentated chemically or by enzymatic digestion, etc. Fragments of fibronectin, which retain the collagen-binding site but are severed from other binding sites of intact fibronectin, are selected and coupled to solid support material.

## Claims

### Claim 1

Affinity matrix material for separating collagen from other high molecular weight proteinaceous substances comprising:
solid chromatographic support material; and
at least one fragment of fibronectin which retains thereupon collagen binding sites, said fragment having been severed from other binding sites of intact fibronectin, said fragment being coupled with said solid chromatographic support material leaving collagen binding sites thereof exposed to selectively bind collagenous material when used chromatographically without binding interfering high molecular weight non-collagenous proteinaceous substances.

### Claim 2 (depends on 1)

The affinity matrix material of claim 1 wherein at least one of said fragments has a molecular weight of about 30 kd characterized as having the collagen specificity of such molecular weight fragment resulting from digestion of fibronectin with trypsin.

### Claim 3 (depends on 1)

The affinity matrix material of claim 1 wherein at least one of said fragments has a molecular weight of about 45 kd characterized as having collagen specificity of such molecular weight fragment resulting from digestion of fibronectin with chymotrypsin.

### Claim 4 (depends on 1)

The affinity matrix material of claim 1 wherein at least one of said fragments consists essentially of the sites of fibronectin which bind collagen, said fragment not including the sites of fibronectin which bind other high molecular weight proteinaceous substances.

### Claim 5 (depends on 1, 2, 3, 4)

The affinity matrix material of claim 1, 2, 3 or 4 wherein the solid chromatographic support material is Sepharose.

### Claim 6 (depends on 1, 2, 3, 4)

The affinity matrix material of claim 1, 2, 3 or 4 wherein the solid chromatographic support material is agarose, dextran, cellulose, polystyrene or glass.

### Claim 7

In the chromatographic method for separating proteinaceous substances from one another, in which a mixture of such substances is passed by way of a solution over a chromatographic material, the improvement comprising separating collagen from other proteinaceous sustances by passing the proteinaceous material containing solution over an affinity matrix material comprising:
solid chromatographic support material; and
at least one fragment of fibronectin which retains thereupon collagen binding sites, said fragment having been severed from other binding sites of intact fibronectin, said fragment being coupled with said solid chromatographic support material leaving callogen binding sites thereof exposed to selectively bind collagenous material when used chromatographically without binding interfering high molecular weight non-collagenous proteinaceous substances.

### Claim 8 (depends on 7)

The chromatographic method of claim 7 wherein at least one of said fragments has a molecular weight of about 30 kd characterized as having the collagen specificity of such molecular weight fragment resulting from digestion of fibronectin with trypsin.

### Claim 9 (depends on 7)

The chromatographic method of claim 7 wherein at least one of said fragments has a molecular weight of about 45 kd characterized as having collagen specificity of such molecular weight fragment resulting from digestion of fibronectin with chymotrypsin.

### Claim 10 (depends on 7)

The chromatographic method of claim 7 wherein at least one of said fragments consists essentially of the sites of fibronectin which bind collagen, said fragment not including the sites of fibronectin which bind other high molecular weight proteinaceous substances.