# US 4,391,746 A · Blood-coagulation-promoting products and methods of preparing them

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- Number: US 4,391,746 A
- Document ID: US4391746A
- Status: Granted
- Kind: A
- Application number: 06/374,835
- Filed: 1982-05-05
- Granted: 1983-07-05
- Earliest claimed: 1980-05-27
- Assignees: Cutter Laboratories, Inc.
- Inventors: Gautam Mitra; Michael H. Coan; Shohachi Wada
- Web page: https://patentgrep.com/patent/US4391746A

## CPC

- C12N 9/644: {Coagulation factor IXa (3.4.21.22)}
- C12N 9/647: {Blood coagulation factors not provided for in a preceding group or according to more than one of the proceeding groups}
- C12Y 304/21022: Coagulation factor IXa (3.4.21.22)
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- Y10S 530/83: Plasma; serum
- Y10S 530/831: Cohn fractions
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES
- 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)
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

Blood-coagulation-promoting products substantially free of thrombin are prepared from human blood plasma by contacting a human blood plasma fraction containing coagulation Factors II, VII, IX and X with an anion exchanger to adsorb the coagulation Factors, which are subsequently eluted from the anion exchanger. The eluate is treated to generate a substance having Factor VIII Inhibitor Bypassing Activity and being substantially free of thrombin, and activated Factor X.

## Claims

### Claim 1

A method of producing a blood-coagulation-promoting preparation substantially free of thrombin from human blood plasma, which comprises
(a) contacting a human blood plasma fraction containing coagulation Factors II, VII, IX, and X with an anion exchanger to adsorb the coagulation Factors on the anion exchanger,
(b) eluting the adsorbed coagulation Factors from the anion exchanger,
(c) treating the eluate containing the coagulation Factors to generate a Factor VIII Inhibitor Bypassing Activity (FEIBA) substance substantially free of thrombin, and
(d) treating the eluate to stop the generation of a FEIBA substance.

### Claim 2 (depends on 1)

The method of claim 1 wherein the fraction of Step (a) is Cohn Effluent I.

### Claim 3 (depends on 1)

The method of claim 1 wherein the anion exchanger of Step (a) is DEAE Sephadex.RTM..

### Claim 4 (depends on 1)

The method of claim 1 which further includes the step of selectively washing the anion exchanger of Step (a) to remove inhibitors to the generation of a FEIBA sustance without eluting the adsorbed coagulation Factors.

### Claim 5 (depends on 4)

The method of claim 4 wherein the anion exchanger is washed selectively with aqueous ammonium bicarbonate at a concentration sufficient to remove said inhibitors but insufficient to elute said coagulation Factors.

### Claim 6 (depends on 5)

The method of claim 5 wherein the anion exchanger further is washed selectively with an aqueous solution having an ionic strength sufficient to remove said inhibitors but insufficient to elute said coagulation Factors.

### Claim 7 (depends on 6)

The method of claim 6 wherein the ionic strength of the aqueous solution is less than about 0.3.

### Claim 8 (depends on 6)

The method of claim 6 wherein the aqueous solution is aqueous sodium chloride.

### Claim 9 (depends on 1)

The method of claim 1 wherein the eluate is treated in Step (c) with a source of calcium ions in an amount sufficient to generate substantial amounts of FEIBA substance but insufficient to generate substantial amounts of thrombin at a temperature and pH and for a period of time sufficient to generate a FEIBA substance.

### Claim 10 (depends on 9)

The method of claim 9 wherein the temperature in Step (c) is about 0.degree.-30.degree. C.

### Claim 11 (depends on 9)

The method of claim 9 wherein the temperature in Step (c) is about 5.degree.-20.degree. C.

### Claim 12 (depends on 9)

The method of claim 9 wherein the temperature in Step (c) is about 8.degree.-14.degree. C.

### Claim 13 (depends on 9)

The method of claim 9 wherein the temperature in Step (c) is about 10.degree. C.

### Claim 14 (depends on 9)

The method of claim 9 wherein the pH in Step (c) is about 6 to 9.

### Claim 15 (depends on 9)

The method of claim 9 wherein the pH in Step (c) is about 7 to 8.

### Claim 16 (depends on 9)

The method of claim 9 wherein the concentration of free calcium ions is about 0.000025-0.0008 mole per liter of solution.

### Claim 17 (depends on 9)

A pharmaceutical composition comprising the product of claim 9.

### Claim 18 (depends on 1)

The method of claim 1 wherein the adsorbed coagulation Factors are selectively eluted with aqueous ammonium bicarbonate in an amount sufficient to elute the coagulation Factors.

### Claim 19 (depends on 18)

The method of claim 18 wherein the eluate is
(a) treated to remove ammonium bicarbonate, and
(b) treated with free calcium ions in a concentration of about 0.0005-0.0008 mole per liter of solution to generate a FEIBA substance.

### Claim 20 (depends on 19)

The method of claim 19 wherein the eluate is lyophilized to remove ammonium bicarbonate.

### Claim 21 (depends on 19)

A pharmaceutical composition comprising the product of claim 19.

### Claim 22 (depends on 1)

The method of claim 1 wherein the adsorbed coagulation Factors are selectively eluted with an aqueous solution having an ionic strength sufficient to elute the coagulation Factors.

### Claim 23 (depends on 22)

The method of claim 22 wherein the ionic strength is about 0.35-2.0.

### Claim 24 (depends on 22)

The method of claim 22 wherein the aqueous solution is aqueous sodium chloride.

### Claim 25 (depends on 22)

The method of claim 22 wherein the eluate is held at a temperature and pH and for a time sufficient to generate a FEIBA substance.

### Claim 26 (depends on 25)

A pharmaceutical composition comprising the product of claim 25.

### Claim 27 (depends on 22)

The method of claim 22 wherein the eluate is treated with a source of calcium ions in an amount sufficient to generate a FEIBA substance but insufficient to generate substantial amounts of thrombin at a temperature and pH and for a time sufficient to generate a FEIBA substance.

### Claim 28 (depends on 27)

The method of claim 27 wherein the amount of free calcium ions is about 0.000025-0.0005 mole per liter.

### Claim 29 (depends on 27)

A pharmaceutical composition comprising the product of claim 27.

### Claim 30

A lyophilized blood-coagulation-promoting composition substantially free of thrombin comprising a product prepared by a process which includes the steps of
(a) contacting a human blood plasma fraction containing coagulation Factors II, VII, IX, and X with an anion exchanger to adsorb said coagulation Factors on the anion exchanger,
(b) eluting the adsorbed coagulation Factors from the anion exchanger,
(c) treating the eluate containing the coagulation Factors to generate a Factor VIII Inhibitor Bypassing Activity (FEIBA) substance substantially free of thrombin,
(d) treating the eluate to stop the generation of a FEIBA substance, and
(e) lyophilizing the eluate.

### Claim 31 (depends on 30)

A pharmaceutical preparation comprising the composition of claim 30.

### Claim 32 (depends on 30)

The composition of claim 30 wherein the process further includes the step of subjecting the eluate of Step (d) to sterile filtration.

### Claim 33 (depends on 32)

A pharmaceutical preparation comprising the composition of claim 32.

### Claim 34 (depends on 30)

The composition of claim 30 wherein the FEIBA concentration is at least about 60 Units per milliliter.

### Claim 35 (depends on 30)

The preparation of claim 30 wherein the FEIBA:thrombin ratio is at least about 1000:1.

### Claim 36 (depends on 30)

The preparation of claim 30 wherein the FEIBA:thrombin ratio is at least about 50:1.

### Claim 37 (depends on 30)

The preparation of claim 30 which is essentially free of thrombin.

### Claim 38 (depends on 30)

The preparation of claim 30 wherein the FEIBA:Factor Xa ratio is at least about 45:1.

### Claim 39 (depends on 30)

The preparation of claim 30 wherein the FEIBA:Factor II ratio, the FEIBA:Factor VII ratio, the FEIBA:Factor IX ratio, and the FEIBA:Factor X ratio are about 10:1 to 0.1:., respectively.