# US 6,465,624 B1 · Purification of von-Willebrand factor by cation exchanger chromatography

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

- Number: US 6,465,624 B1
- Document ID: US6465624B1
- Status: Granted
- Kind: B1
- Application number: 09/367,460
- Filed: 1999-10-21
- Granted: 2002-10-15
- Earliest claimed: 1997-02-27
- Assignees: Baxter Aktiengesellschaft
- Inventors: Bernhard Fischer; Öyvind L. Schönberger; Artur Mitterer; Christian Fiedler; Friedrich Dorner; Johann Eibl
- Web page: https://patentgrep.com/patent/US6465624B1

## CPC

- C07K 14/755: Factors VIII {, e.g. factor VIII C (AHF), factor VIII Ag (VWF)}
- A61P 7/04: Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
- 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)
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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)

## Abstract

Disclosed are a method of recovering vWF in which vWF at a low salt concentration is bound to a cation exchanger and vWF having a high specific activity is recovered by fractionated elution, as well as a preparation having purified vWF obtainable by this method.

## Claims

### Claim 1

Delete “vWF” and Insert “von Willebrand factor (vWF)” in line 1 of the claim.

### Claim 2 (depends on 1)

A method as set forth in claim 1, wherein said vWF is bound to said cation exchanger at a salt concentration of ≦250 mM, and wherein at least one vWF fraction containing high-molecular weight vWF multimers is eluted at a salt concentration of ≧300 mM.

### Claim 3 (depends on 2)

A method as set forth in claim 2, wherein at least one vWF fraction containing said high-molecular weight vWF multimers is recovered by elution in a buffer having a pH ranging from 5.0 to 8.5.

### Claim 4 (depends on 3)

A method as set forth in claim 3, wherein said pH of said buffer ranges between 6.0 and 8.0.

### Claim 5 (depends on 1)

A method as set forth in claim 1, further comprising removing low-molecular weight vWF multimers, proteolytic vWF degradation products and other proteins at a salt concentration of between ≧250 mM and ≦300 mM.

### Claim 6 (depends on 1)

A method as set forth in claim 1, wherein said cation exchanger is selected from the group consisting of a sulfopropyl group conjugated carrier and a carboxymethyl group conjugated carrier.

### Claim 7 (depends on 2)

A method as set forth in claim 2, wherein at least one vWF-containing fraction containing high-molecular weight vWF multimers is recovered having a specific platelet agglutinating activity of at least 65 U/mg of protein and a specific collagen binding activity of at least 65 U/mg of protein.

### Claim 8 (depends on 2)

A method as set forth in claim 2, wherein at least one vWF containing fraction recovered is substantially free from low-molecular weight vWF multimers, from vWF fragments with low specific vWF activity and from contaminating nucleic acids.

### Claim 9 (depends on 1)

A method as set forth in claim 1, wherein said vWF-containing solution is selected from the group consisting of plasma, a plasma fraction, cryoprecipitate, a supernatant and an extract of a recombinant cell culture and an enriched protein solution.

### Claim 10 (depends on 9)

A method as set forth in claim 9, wherein said enriched protein solution is obtained by a preceding purification step.

### Claim 11 (depends on 10)

A method as set forth in claim 10, wherein said preceding purification step is a chromatographic method.

### Claim 12 (depends on 11)

A method as set forth in claim 11, wherein said chromatographic method is selected from the group consisting of an anion exchange chromatography, an affinity chromatography and a combination thereof.

### Claim 13 (depends on 2)

A method as set forth in claim 2, further comprising subjecting said at least one vWF-containing fraction recovered to at least one virus inactivation or a virus depletion step.

### Claim 14

A preparation comprising purified von Willebrand factor (vWF) containing high-molecular weight vWF multimers, obtainable from a vWF-containing protein solution by cation exchange chromatography.

### Claim 15 (depends on 14)

A preparation as set forth in claim 14, said preparation being substantially free from low-molecular weight vWF multimers, from inactive vWF degradation products and from contaminating nucleic acids.

### Claim 16 (depends on 15)

A preparation as set forth in claim 15, wherein said vWF of said high-molecular weight vWF multimers has a specific platelet agglutinating activity of at least 65 U/mg of protein and a specific collagen binding activity of at least 65 U/mg of protein.

### Claim 17 (depends on 14)

A preparation as set forth in claim 14, wherein said preparation is substantially free from factor VIII and has a factor VIII content of <0.1%, based on the ratio of activity of vWF to factor VIII:C.

### Claim 18 (depends on 14)

A preparation as set forth in claim 14, wherein said preparation is virus-safe.

### Claim 19 (depends on 14)

A preparation as set forth in claim 14, wherein said preparation is present in a storage-stable form.

### Claim 20 (depends on 14)

A preparation as set forth in claim 14, wherein said preparation is formulated as a pharmaceutical preparation.

### Claim 21

A method of treating a patient suffering from a disorder, selected from the group consisting of phenotypic hemophilia and von Willebrand Syndrome, by administering an effective dose of a von Willebrand factor (vWF)-containing preparation to said patient, said vWF-containing preparation comprising purified vWF containing high-molecular weight vWF multimers with a factor VHII content of 1% (total protein) or less, obtained from a vWF-containing protein solution by cation exchange chromatography.

### Claim 22 (depends on 1)

The method of claim 1, wherein said vWF is bound to said cation exchanger at a salt concentration of ≦250 mm, and wherein the fractionated elution step comprises
(i) eluting said cation exchanger with a salt concentration of between ≧250 mm and ≦300 mm; and
(ii) eluting said cation exchanger with a salt concentration of ≧300 mm.