Claims for Patent: 6,114,146

Summary for Patent: 6,114,146

Title:	Expression plasmid, a fusion protein, a transfected eukaryotic cell line, a method of producing foreign proteins, a foreign protein preparation as well as a pharmaceutical composition
Abstract:	The invention describes an expression plasmid containing a dicistronic transcription/translation unit, which unit comprises a sequence for a foreign protein and a sequence for a fusion protein, the fusion protein containing at least one selection marker and at least one amplification marker. Further described is a method of producing foreign proteins by using the plasmids according to the invention, as well as cell lines transformed with the plasmid according to the invention.
Inventor(s):	Herlitschka; Sabine E. (Salzburg, AT), Schlokat; Uwe (Orth/Donau, AT), Falkner; Falko-Guenter (Orth/Donau, AT), Dorner; Friedrich (Vienna, AT)
Assignee:	Baxter Aktiengesellschaft (Vienna, AT)
Application Number:	08/557,210
Patent Claims:	1. A method for producing a protein comprising: (a) transfecting an eukaryotic cell line expressing an endogenous dihydrofolate reductase (dhfr) gene with an expression plasmid to obtain clones, wherein said expression plasmid comprises a dicistronic transcription/translation unit comprising: (i) a sequence encoding said protein; and (ii) a sequence encoding a fusion protein comprising the coding sequence of a dominant selection marker gene and a dihydrofolate reductase (dhfr) gene that expresses a wild-type dhfr phenotype as an amplification marker, wherein said sequence encoding said fusion protein is located 3' to said sequence encoding said protein, and wherein said dicistronic transcription/translation unit further comprises an internal ribosome binding site between the sequence encoding the protein and the sequence encoding the fusion protein, (b) isolating said clones obtained by said transfection step controlled by said selection marker, (c) amplifying said selected clones in a first amplification, (d) subsequently further amplifying said clones in a further amplification controlled by said amplification marker, thereby expressing said protein, and (e) obtaining said expressed protein. 2. A method as set forth in claim 1, wherein said first amplification occurs while selecting said clones obtained. 3. A method as set forth in claim 1, wherein said selection process is effected by using hygromycin B and said further amplification is effected by using methotrexate. 4. A method as set forth in claim 1, wherein said eukaryotic cell line transfected with said expression plasmid is selected from the group consisting of a Chinese Hampster Ovary cell line, a human kidney cell line, and a human liver cell line. 5. A method as set fourth in claim 1, wherein said eukaryotic cell lines transfected with said expression plasmid are selected from the group consisting of CHO, 293 and human liver cell lines, such as SK-HEP-1 and Chang liver. 6. A method as set forth in claim 1, wherein said protein is a recombinant blood coagulation factor or a viral protein. 7. A method as set forth in claim 1, wherein said protein is recombinant human prothrombin, recombinant human factor VIII, recombinant human FVIIIdB928, a recombinant human factor IX, recombinant human protein C, recombinant human von Willebrand factor or recombinant human serum albumin. 8. A method according to claim 1, wherein said internal ribosome binding site is the 5'-untranslated region of the encephalomyocarditis virus (EMCV 5' UTR). 9. A method according to claim 1, wherein said sequence encoding the protein is 5' and said sequence encoding the fusion protein is 3' from said internal ribosome binding site. 10. A method according to claim 1, further comprising a spacer sequence, said spacer sequence separating said dominant selection marker gene and said dihydrofolate reductase (dhfr) gene of said sequence encoding a fusion protein. 11. A method according to claim 1, wherein said dominant selection marker is selected from the group consisting of adenosine deaminase, hygromycin phosphotransferase and neomycin transferase. 12. A method according to claim 1, wherein said sequence encoding a protein comprises a sequence encoding a human plasma protein, a viral protein, a derivative thereof or a fragment thereof. 13. A method according to claim 12, wherein said sequence encoding a protein comprises a sequence encoding human prothrombin cDNA. 14. A method according to claim 12, wherein said sequences encoding a protein comprises a sequence encoding human factor VIII cDNA. 15. A method according to claim 12, wherein said sequences encoding a protein comprise a sequence encoding human factor VIII dB928 cDNA. 16. A method according to claim 12, wherein said sequences encoding a protein comprise a sequence encoding human factor IX cDNA. 17. A method according to claim 12, wherein said sequences encoding a protein comprise a sequence encoding human protein C cDNA. 18. A method according to claim 12, where said sequences encoding a protein comprise a sequence encoding human von Willebrand factor cDNA. 19. A method according to claim 1, wherein the expression plasmid is selected from the group consisting of pCMVFII/EDH-Sp, pCMVFII/EDHGly and pCMVFII/EDHPro. 20. A method according to claim 1, wherein the expression plasmid is selected from the group consisting of pCMVFVIIIc/EDH-Sp, pCMVFVIIIc/EDHGly and pCMVFVIIIc/EDHPro. 21. A method according to claim 1, wherein the expression plasmid is selected from the group consisting of pCMVFVIIIdB928/EDH-Sp, pCMVFVIIIdB928/EDHGly and pCMVFVIIIdB928/EDHPro. 22. A method according to claim 1, wherein the expression plasmid is selected from the group consisting of pCMVFIX/EDH-Sp, pCMVFIX/EDHGly and pCMVFIX/EDHPro. 23. A method according to claim 1, wherein the expression plasmid is selected from the group consisting of pCMV-PCwt-EDH-Sp, pCMV-PCwt-EDHGly, pCMV-PCwt-EDHPro, pMCV-PCpt. mut.-EDH-Sp, pCMV-PCpt.mut.-EDHPro and pCMV-PCpt. mut.-EDHGly. 24. A method according to claim 1, wherein the expression plasmid is selected from the group consisting of pAct-vWF-EDH-Sp, pACT-vWF-EDHPro and pACT-vWF-EDHGly. 25. A method according to claim 1, wherein said expression plasmid comprises at least one expression cassette containing DNA sequences selected from the group consisting of SEQ.ID.No. 6, SEQ.ID.No. 7 and SEQ.ID.No. 8. 26. A method as set forth in claim 5, wherein said eukaryotic cell line is 293 or SK-HEP-1. 27. A method for producing a protein comprising: (a) transfecting SK-HEP-1 cells with an expression plasmid to obtain clones, wherein said expression plasmid comprises a dicistronic transcription/translation unit comprising: (i) a sequence encoding said protein, and (ii) a sequence encoding a fusion protein comprising the coding sequence of a dominant selection marker gene and a dihydrofolate reductase (dhfr) gene that expresses a wild-type dhfr phenotype as an amplification marker, wherein said sequence encoding said fusion protein is located 3' to said sequence encoding said protein, and wherein said dicistronic transcription/ translation unit further comprises an internal ribosome binding site between the sequence encoding the protein and the sequence encoding the fusion protein, (b) isolating said clones expressing said protein, and (c) obtaining said expressed protein. 28. A method according to claim 27, wherein said protein is selected from the group consisting of prothrombin, factor VIII, factor VIII deletion mutant, factor IX, protein C, von Willebrand factor and serum albumin. 29. A method for producing a protein comprising: (a) transfecting SK-HEP-1 cells with an expression plasmid comprising the coding sequence of a blood factor protein selected from the group consisting of prothrombin, factor VIII, factor VIII dB928, a factor VIII deletion mutant, Factor IX, protein C, von Willebrand factor, serum albumin, a derivative thereof and fragment thereof to obtain clones, wherein said expression plasmid comprises a dicistronic transcription/translation unit comprising: (i) a sequence encoding said protein, and (ii) a sequence encoding a fusion protein comprising the coding sequence of a dominant selection marker gene and a dihydrofolate reductase (dhfr) gene that expresses a wild-type dhfr phenotype as an amplification marker, wherein said sequence encoding said fusion protein is located 3' to said sequence encoding said protein, and wherein said dicistronic transcription/translation unit further comprises an internal ribosome binding site between the sequence encoding the protein and the sequence encoding the fusion protein, (b) isolating said clones expressing said protein, and (c) obtaining said expressed protein. 30. A method for producing a polypeptide having factor VIII activity comprising: (a) transfecting SK-HEP-1 cells with an expression plasmid comprising the coding sequence of a polypeptide having factor VIII activity selected from the group consisting of full-length factor VIII, a factor VIII deletion mutant, factor VIII dB928, and a factor VIII derivative to obtain clones, wherein said expression plasmid comprises a dicistronic transcription/translation unit comprising: (i) a sequence encoding said polypeptide having factor VIII activity, and (ii) a sequence encoding a fusion protein comprising the coding sequence of a dominant selection marker gene and a dihydrofolate reductase (dhfr) gene that expresses a wild-type dhfr phenotype as an amplification marker, wherein said sequence encoding said fusion protein is located 3' to said sequence encoding said polypeptide having factor VIII activity and wherein said dicistronic transcription/translation unit further comprises an internal ribosome binding site between the sequence encoding said polypeptide having factor VIII activity and the sequence encoding the fusion protein, (b) isolating said clones expressing said polypeptide having factor VIII activity, and (c) obtaining said expressed polypeptide having factor VIII activity. 31. A method for producing a protein comprising: (a) transfecting an eukaryotic cell line expressing an endogenous dihydrofolate reductase (dhfr) gene with an expression plasmid to obtain clones, wherein said expression plasmid comprises a dicistronic transcription/translation unit comprising: (i) a sequence encoding said protein, wherein said protein is recombinant human prothrombin, recombinant human factor VIII, recombinant human FVIIIdB928, a recombinant human factor IX, recombinant human protein C, recombinant human von Willebrand factor or recombinant human serum albumin, and (ii) a sequence encoding a fusion protein comprising the coding sequence of a dominant selection marker gene and a dihydrofolate reductase (dhfr) gene that expresses a wild-type dhfr phenotype as an amplification marker, wherein said sequence encoding said fusion protein is located 3' to said sequence encoding said protein, and wherein said dicistronic transcription/translation unit further comprises an internal ribosome binding site between the sequence encoding the protein and the sequence encoding the fusion protein, (b) isolating said clones obtained by said transfection step controlled by said selection marker, (c) amplifying said selected clones in a first amplification, (d) subsequently further amplifying said clones in a further amplification controlled by said amplification marker, thereby expressing said protein, and (e) obtaining said expressed protein.

Details for Patent 6,114,146

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Takeda Pharmaceuticals U.s.a., Inc.	VONVENDI	von willebrand factor (recombinant)	For Injection	125577	December 08, 2015		2015-11-14