# US 10,864,227 B2 · Enzymatic preparation of increased anticoagulant bovine sourced heparin

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- Number: US 10,864,227 B2
- Document ID: US10864227B2
- Status: Granted
- Kind: B2
- Application number: 16/496,149
- Filed: 2018-03-20
- Published: 2020-04-30
- Granted: 2020-12-15
- Earliest claimed: 2017-03-20
- Assignees: Rensselaer Polytechnic Institute
- Applicants: Rensselaer Polytechnic Institute
- Inventors: Li Fu; Kevin Li; Daisuke Mori; Makoto Hirakane; Jonathan S. Dordick; Robert J. Linhardt
- Web page: https://patentgrep.com/patent/US10864227B2

## CPC

- A61K 31/727: Heparin; Heparan
- C12N 9/13: {transferring sulfur containing groups (2.8)}
- C12P 19/26: Preparation of nitrogen-containing carbohydrates
- C12Y 208/02: Sulfotransferases (2.8.2)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Y: ENZYMES

## Abstract

A heparin structure with increased anticoagulant activity and method of making the same are disclosed. A heparin sample is provided and treated with a heparan sulfate sulfotransferase in an enzymatic reaction to add sulfuryl groups from a sulfuryl group source to the heparin sample, resulting in a heparin structure having above about 8% more 3-O-sulfo groups relative to wild-type bovine intestinal heparin. The added sulfuryl groups modify the heparin structure and increase the sample's binding to antithrombin III and its anticoagulant activity to be more similar and a viable alternative to porcine intestinal heparin. The modified heparin exhibits an anti-FXa activity and an anti-FIIa activity greater than about 180 U/mg, and a ratio of the anti-FXa activity to the anti-FIIa activity of about 0.9 to about 1.1, consistent with U.S. Pharmacopeia (USP) heparin activity specifications.

## Claims

### Claim 1

A heparin structure exhibiting increased anticoagulant activity, the heparin comprising the structure:
wherein:
a+d is about 14 to about 18;
b is 2;
c is about 2 to about 8;
X and X′ are SO3−, H, or combinations thereof;
Y and Y′ are SO3−, COCH3, H, or combinations thereof; and
Z is SO3−, H, or combinations thereof.

### Claim 2 (depends on 1)

The heparin structure according to claim 1, wherein X and X′ in b and c is about 4H or about 4 SO3−.

### Claim 3 (depends on 1)

The heparin structure according to claim 1, wherein Y and Y′ in b and c is about 6 SO3−, about 1 COCH3, and about 1H.

### Claim 4 (depends on 1)

The heparin structure according to claim 1, wherein Z is about 0.8 SO3− and about 0.2H.

### Claim 5 (depends on 1)

The heparin structure according to claim 1, wherein the heparin structure includes above about 8% more 3-O-sulfo groups relative to wild-type bovine intestinal heparin.

### Claim 6 (depends on 5)

The heparin structure according to claim 5, wherein the heparin structure includes about 15% to about 25% more 6-O-sulfo groups and about 8% to about 20% more 3-O-sulfo groups relative to wild-type bovine intestinal heparin.

### Claim 7 (depends on 1)

The heparin structure according to claim 1, wherein the heparin structure includes an anti-FXa activity and an anti-FIIa activity greater than about 180 U/mg, and the ratio of the anti-FXa activity to the anti-FIIa activity is about 0.9 to about 1.1.