# US 10,246,485 B2 · Method to concentrate von willebrand factor or complexes thereof

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- Number: US 10,246,485 B2
- Document ID: US10246485B2
- Status: Granted
- Kind: B2
- Application number: 15/034,907
- Filed: 2014-11-07
- Published: 2016-10-06
- Granted: 2019-04-02
- Earliest claimed: 2013-11-08
- Assignees: CSL Limited
- Applicants: Csl Limited
- Inventors: Holger Lind; Sonja Beckmann-Scheld; Katharina Propp
- Web page: https://patentgrep.com/patent/US10246485B2

## CPC

- C07K 1/32: as complexes
- A61K 38/36: Blood coagulation or fibrinolysis factors
- A61K 38/37: Factors VIII
- C07K 14/755: Factors VIII {, e.g. factor VIII C (AHF), factor VIII Ag (VWF)}
- C07K 14/76: Albumins
- C07K 2319/31: fusions, other than Fc, for prolonged plasma life, e.g. albumin
- 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)
- 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

The present invention relates to a method for concentrating von Willebrand Factor (VWF) from an aqueous solution comprising precipitating the VWF by providing calcium ions and phosphate ions and the subsequent resolubilization of the VWF by means of an aqueous solution comprising a calcium complexing compound.

## Claims

### Claim 1

A method for concentrating von Willebrand Factor (VWF) from an aqueous solution comprising the steps of
(a) precipitating the VWF by adding calcium ions and phosphate ions to the aqueous solution,
(b) separating the precipitate formed in step (a), and
(c) resolubilizing the precipitate isolated in step (b) by contacting the precipitate with an aqueous solution comprising a calcium complexing agent, wherein
the VWF comprises a full-length VWF polypeptide.

### Claim 2 (depends on 1)

The method according to claim 1, wherein
(i) the phosphate ions are at a concentration between 1 mM and less than 10 mM, and the calcium ions are at a concentration of at least 16 mM, or
(ii) the phosphate ions are at a concentration of at least 10 mM, and the calcium ions are at a concentration of at least 10 mM.

### Claim 3 (depends on 2)

The method according to claim 2, wherein the concentration of the phosphate ions is at most 50 mM.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the concentration of the phosphate ions is 5 mM and the concentration of the calcium ions is between 50 mM and 60 mM including the endpoints of that range.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the concentration of the calcium complexing agent ranges from at least 120 mM up to the limit of solubility of the calcium complexing agent.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the calcium complexing agent is EDTA or EGTA.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the aqueous solution used in step (c) is a buffer.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the temperature of the precipitation step ranges from 21° C. to 37° C.

### Claim 9 (depends on 1)

The method according to claim 1, wherein the VWF:RCo/VWF:Ag ratio in the resolubilized precipitate is above 80% of the VWF:RCo/VWF:Ag ratio in the aqueous starting solution in step (a).

### Claim 10 (depends on 1)

The method according to claim 1, wherein the yield of VWF:RCo in the resolubilized precipitate is at least 75%.