Purification of factor VIII using a conductivity gradient

Patent metadata

Field	Value
Publication number	US-9488625-B2
Application number	US-201113325778-A
Country	US
Kind code	B2
Filing date	Dec 14, 2011
Priority date	Dec 15, 2010
Publication date	Nov 8, 2016
Grant date	Nov 8, 2016

Abstract

The present specification discloses methods of eluting a protein from a chromatography column.

First claim

The invention claimed is:

1. A method of collecting an elution from a column, said method comprising the steps of:
a) applying a sample comprising a Factor VIII protein to a cation exchange chromatography column, wherein said Factor VIII protein is retained by said cation exchange chromatography column;
b) applying a mobile phase to said cation exchange chromatography column, said mobile phase comprising a buffered solution, wherein said mobile phase establishes a conductivity gradient of from about 4 mS/cm to about 90 mS/cm in said cation exchange chromatography column;
c) starting a single fraction eluate collection, wherein said single fraction eluate collection includes a single start conductivity value and a single stop conductivity value, wherein said single start conductivity value starts when an inlet conductivity value measured for said mobile phase of step b) enters said cation exchange chromatography column at about 25.0 mS/cm to about 27.0 mS/cm and when an outlet conductivity value measured for said mobile phase of step b) enters said cation exchange chromatography column at about 19.0 mS/cm to about 21.0 mS/cm; and
d) stopping said single faction eluate collection when said single stop conductivity value measured for said mobile phase of step b) at the inlet is from about 41.0 mS/cm to about 43.0 mS/cm and said single stop conductivity value measured for said mobile phase of step b) at the outlet is from about 34.5 mS/cm to about 36.5 mS/cm; and wherein said single fraction eluate collection comprises 83% Factor VIII protein activity retained by said cation exchange chromatography column in step a).

2. The method of claim 1 , wherein in step (b) said mobile phase establishes a conductivity gradient of from about 8 mS/cm to about 90 mS/cm.

3. The method of claim 1 , wherein said Factor VIII protein is a recombinant protein or a protein from an organism or a transgenic organism.

4. A method of collecting an elution from a column, the method comprising the steps of:
a) applying a sample comprising a Factor VIII protein to an anion exchange chromatography column, wherein said Factor VIII protein is retained by said anion exchange chromatography column;
b) applying a mobile phase to said anion exchange chromatography column, said mobile phase comprising a buffered solution, wherein said mobile phase establishes a conductivity gradient of from about 4 mS/cm to about 80 mS/cm in said anion exchange chromatography column;
c) starting a single fraction eluate collection, wherein said single fraction eluate collection includes a single start conductivity value and a single stop conductivity value, wherein said single start conductivity value starts when an inlet conductivity value measured for said mobile phase of step b) enters said anion exchange chromatography column at about 27.8 mS/cm to about 29.8 mS/cm and when an outlet conductivity value measured for said mobile phase of step b) enters said anion exchange chromatography column at about 21.0 mS/cm to about 23.0 mS/cm; and
d) stopping said single fraction eluate collection when said single stop conductivity value measured for said mobile phase of step b) at the inlet is from about 51.0 mS/cm to about 53.0 mS/cm and said single stop conductivity value measured for mobile phase of step b) at the outlet is from about 45.5 mS/cm to about 47.5 mS/cm; and wherein said single fraction eluate collection comprises 83% Factor VIII protein activity retained by said anion exchange chromatography column in step (a).

5. The method of claim 4 , wherein said Factor VIII protein is a recombinant protein or a protein from an organism or a transgenic organism.

Assignees

• Felgenhauer Martin
• Maierhofer Helmut
• Mison Dominique
• Desponds Arnaud
• Baxalta GmbH
• Baxalta Inc

Inventors

• Felgenhauer Martin
• Maierhofer Helmut
• Mison Dominique
• Desponds Arnaud

Classifications

• G01N2030/8831

  involving peptides or proteins · CPC title

• B01D15/166

  Fluid composition conditioning, e.g. gradient · CPC title

• C07K1/18

  Ion-exchange chromatography · CPC title

• G01N30/96 Primary

  using ion-exchange (G01N30/02, G01N30/90 take precedence) · CPC title

• C07K14/755

  Factors VIII {, e.g. factor VIII C (AHF), factor VIII Ag (VWF)} · CPC title