Systems for factor VIII processing and methods thereof

Patent metadata

Field	Value
Publication number	US-9611310-B2
Application number	US-201113809285-A
Country	US
Kind code	B2
Filing date	Jul 11, 2011
Priority date	Jul 9, 2010
Publication date	Apr 4, 2017
Grant date	Apr 4, 2017

Abstract

The present invention provides methods of reducing nonprocessed Factor VIII or a chimeric polypeptide comprising Factor VIII comprising co-transfecting in a host cell a polynucleotide encoding Factor VIII with a polynucleotide encoding a protein convertase, where the endogenous processing enzymes of the host cell are insufficient to convert all of the Factor VIII to its processed isoform; expressing a proprotein convertase from a second polynucleotide in the host cell; and reducing the nonprocessed Factor VIII by processing with said proprotein convertase.

First claim

What is claimed is:

1. A method for decreasing nonprocessed Factor VIII (FVIII) in a culturing medium, comprising contacting a nonprocessed FVIII polypeptide expressed from a polynucleotide in a host cell with proprotein convertase subtilisin/kexin type 5 (PC5),
wherein the PC5 is expressed from a second polynucleotide in the host cell expressing the FVIII,
wherein the host cell is a mammalian cell, and
wherein the PC5 processes the nonprocessed FVIII polypeptide to a processed FVIII polypeptide, which comprises a FVIII heavy chain and a FVIII light chain associated by a non-covalent bond.

2. The method of claim 1 , wherein the proprotein convertase cleaves Arginine in the nonprocessed FVIII polypeptide at an amino acid residue corresponding to amino acid 1648 of SEQ ID NO: 6 or amino acid 754 of SEQ ID NO: 2.

3. The method of claim 1 , wherein the FVIII heavy chain and the FVIII light chain are associated by a metal-ion mediated non-covalent bond.

4. The method of claim 1 , wherein the level of the nonprocessed FVIII polypeptide is decreased such that more than 75%, 80%, 85%, 90%, 95%, and 100% of the FVIII is the processed FVIII polypeptide.

5. The method of claim 1 , wherein the nonprocessed FVIII polypeptide is expressed as full-length FVIII or partial or full B-domain deleted FVIII.

6. The method of claim 1 , wherein the FVIII polypeptides are fused to an immunoglobulin constant region or a portion thereof.

7. The method of claim 6 , wherein the immunoglobulin constant region or a portion thereof comprises a neonatal Fc Receptor (FcRn) binding domain.

8. The method of claim 1 , wherein the polynucleotide encoding the nonprocessed FVIII polypeptide and the second polynucleotide encoding PC5 are located on the same vector or on two different vectors.

9. The method of claim 1 , wherein the host cell is a CHO cell, a HEK293 cell, a HKB11 cell, or a BHK cell.

10. The method of claim 1 , wherein the polynucleotide encoding the nonprocessed FVIII polypeptide and the second polynucleotide encoding the PC5 are expressed from two different vectors.

11. The method of claim 1 , wherein the processed FVIII polypeptide is produced in a large manufacturing process.

12. The method of claim 1 , wherein the PC5 comprises an amino acid sequence at least 80% identical to the sequence set forth as SEQ ID NO: 18 and wherein the PC5 is capable of cleaving full-length FVIII or B domain-deleted FVIII.

13. The method of claim 1 , wherein the PC5 comprises an amino acid sequence at least 95% identical to the sequence set forth as SEQ ID NO: 18 and wherein the PC5 is capable of cleaving full-length FVIII or B domain-deleted FVIII.

14. The method of claim 1 , wherein the PC5 comprises the amino acid sequence set forth as SEQ ID NO: 18.

15. The method of claim 1 , wherein the processed FVIII polypeptide is further cleaved by thrombin and is activated.

16. The method of claim 1 , wherein the FVIII polypeptide comprises human full-length FVIII.

17. The method of claim 1 , wherein the FVIII polypeptide comprises human B domain-deleted FVIII.

18. The method of claim 6 , wherein the immunoglobulin constant region or portion thereof comprises an Fc fragment.

19. The method of claim 6 , wherein the host cell further comprises a second polynucleotide encoding a second immunoglobulin constant region or portion thereof, which comprises a neonatal Fc Receptor (FcRn) binding domain.

20. The method of claim 19 , wherein the second immunoglobulin constant region or portion thereof comprises an Fc fragment.

21. The method of claim 8 , wherein each of the polynucleotide encoding the nonprocessed FVIII polypeptide and the second polynucleotide encoding the PC5 is operably linked to a promoter.

22. The method of claim 8 , wherein the polynucleotide encoding the nonprocessed FVIII polypeptide and the second polynucleotide encoding the PC5 are operably linked to a single promoter.

23. A method of increasing the yield of processed Factor VIII polypeptides in a host cell comprising contacting a nonprocessed FVIII polypeptide expressed from a polynucleotide in a host cell with proprotein convertase subtilisin/kexin type 5 (PC5), wherein the PC5 is expressed from a second polynucleotide in the host cell, wherein the host cell is a mammalian cell, and wherein the PC5 processes the nonprocessed FVIII polypeptide to a processed FVIII polypeptide, which comprises a FVIII heavy chain and a FVIII light chain associated by a non-covalent bond.

24. The method of claim 23 , wherein the polynucleotide encoding the nonprocessed FVIII polypeptide and the second polynucleotide encoding the PC5 are on the same vector or on the different vectors.

25. A method of reducing the level of nonprocessed Factor VIII polypeptides in a host cell comprising contacting a nonprocessed FVIII polypeptide expressed from a polynucleotide in the host cell with PC5 expressed from a second polynucleotide in the host cell, wherein the host cell is a mammalian cell, and wherein the PC5 processes the nonprocessed FVIII polypeptide to a processed FVIII polypeptide, which comprises a FVIII heavy chain and a FVIII light chain.

Assignees

• Low Susan C
• Peters Robert T
• Bioverativ Therapeutics Inc

Inventors

• Low Susan C
• Peters Robert T

Classifications

• C07K2319/30

  Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto · CPC title

• C07K14/755 Primary

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

• C12N9/6424

  Serine endopeptidases (3.4.21) · CPC title

• C07K2319/02

  containing a signal sequence · CPC title

• C12N9/60

  from yeast · CPC title