Systems for factor VIII processing and methods thereof
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.
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.