Production of fully processed and functional factor X in a furin-secreting mammalian expression system

Patent metadata

Field	Value
Publication number	US-9873892-B2
Application number	US-201514824927-A
Country	US
Kind code	B2
Filing date	Aug 12, 2015
Priority date	Aug 12, 2014
Publication date	Jan 23, 2018
Grant date	Jan 23, 2018

Abstract

Disclosed herein are methods for production of fully-processed mature Factor X in an expression system producing a controlled amount of furin between 50 U/mL and 300 U/mL of culture supernatant. Also disclosed are transformed cells, expression systems, and expression vectors for the expression of furin and Factor X.

First claim

What is claimed is:

1. An isolated eukaryotic transformed cell comprising:
a first exogenous nucleic acid encoding a human furin and a second exogenous nucleic acid encoding a human Factor X, such that the transformed cell expresses and secretes the human furin and the human Factor X into a culture supernatant, wherein the human furin is secreted at a concentration of about 60 U/mL to about 300 U/mL in the culture supernatant after culture for about 36 to about 78 hours and at least 85% of the human Factor X is fully processed, and wherein the first exogenous nucleic acid and the second exogenous nucleic acid are present on one or two plasmids.

2. The isolated eukaryotic transformed cell of claim 1 , wherein the plasmid(s) is a non-viral expression vector.

3. The isolated eukaryotic transformed cell of claim 1 , wherein the plasmid(s) is a viral expression vector.

4. The isolated eukaryotic transformed cell of claim 1 , wherein the human furin is secreted at a concentration of about 100 to about 300 U/mL in the culture supernatant after culture for about 36 to about 78 hours.

5. The isolated eukaryotic transformed cell of claim 4 , wherein at least 90% of the human Factor X produced by the transformed cell is fully processed.

6. The isolated eukaryotic transformed cell of claim 1 , wherein the human furin is secreted at a concentration of at least about 90 to about 100 U/mL in the culture supernatant after culture for about 36 to about 78 hours.

7. The isolated eukaryotic transformed cell of claim 6 , wherein at least 95% of the human Factor X produced by the transformed cell is fully processed.

8. The isolated eukaryotic transformed cell of claim 1 , wherein the human furin concentration in the culture supernatant reaches about 60 to about 300 U/mL after culture for about 42 to about 72 hours.

9. The isolated eukaryotic transformed cell of claim 1 , wherein the cell is selected from the group consisting of a Chinese hamster ovary (CHO) cell, a human embryonic kidney cell, a primate kidney cell, a fibroblast, and a mouse myeloma cells.

10. The isolated eukaryotic transformed cell of claim 1 , further comprising an exogenous nucleic acid encoding a human vitamin K epoxide reductase (VKOR).

11. An isolated eukaryotic transformed cell comprising:
a first expression vector comprising an exogenous nucleic acid encoding a human furin; wherein the transformed cell secretes the human furin into the culture supernatant at a concentration of about 60 U/mL to about 300 U/mL after culture for about 36 to about 78 hours, and wherein the first expression vector is a plasmid.

12. The isolated eukaryotic transformed cell of claim 11 , wherein the cell is selected from the group consisting of a Chinese hamster ovary (CHO) cell, a human embryonic kidney cell, a primate kidney cell, a fibroblast, and a mouse myeloma cells.

13. The isolated eukaryotic transformed cell of claim 11 , further comprising an exogenous nucleic acid encoding a human vitamin K epoxide reductase (VKOR).

14. The isolated eukaryotic transformed cell of claim 11 , wherein the transformed cell further comprises an exogenous nucleic acid encoding a protein, and wherein the protein comprises an Arg-(Lys/Arg)-Arg motif and is cleavable by the human furin.

15. The isolated eukaryotic transformed cell of claim 14 , wherein the protein is von Willebrand Factor, Factor II, Factor IX, Factor X, Protein C, Protein S, or Protein Z.

16. The isolated eukaryotic transformed cell of claim 14 , wherein the exogenous nucleic acid encoding the protein is on a second expression vector.

Assignees

• Baxalta Inc
• Baxalta GmbH

Inventors

• Böhm Ernst
• Horling Franziska
• Koehn Jadranka
• Dockal Michael

Classifications

• A61P7/04

  Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents · CPC title

• C12Y304/21006 Primary

  Coagulation factor Xa (3.4.21.6) · CPC title

• C12N9/6432

  Coagulation factor Xa (3.4.21.6) · CPC title

• C12Y304/21075

  Furin (3.4.21.75) · CPC title

• A61K38/4846 Primary

  Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38) · CPC title