Claims for Patent: 5,958,727

Summary for Patent: 5,958,727

Title:	Methods for modifying the production of a polypeptide
Abstract:	The present invention relates to methods for modifying the production of a polypeptide, comprising: (a) introducing a nucleic acid construct into a cell, wherein the cell comprises a DNA sequence encoding a polypeptide, under conditions in which the nucleic acid construct integrates into the genome of the cell at a locus not within the DNA sequence encoding the polypeptide to produce a mutant cell, wherein the integration of the nucleic acid construct modifies the production of the polypeptide by the mutant cell relative to the cell when the mutant cell and the cell are cultured under the same conditions; and (b) identifying the mutant cell with the modified production of the polypeptide.
Inventor(s):	Brody; Howard (Davis, CA), Yaver; Deborah S. (Davis, CA), Lamsa; Michael (Davis, CA), Hansen; Kim (Vaerlose, DK)
Assignee:	Novo Nordisk Biotech, Inc (Davis, CA)
Application Number:	08/928,692
Patent Claims:	1. A method of producing a polypeptide, comprising: (a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein (i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence, not within a second DNA sequence encoding a protein that negatively regulates transcription, translation or secretion of the polypeptide, and not within a third DNA sequence encoding a protease which hydrolyzes the polypeptide under the conditions; and (ii) the mutant cell produces more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and (b) recovering the polypeptide. 2. The method of claim 1, wherein the nucleic acid construct cannot homologous recombine with the first DNA sequence. 3. The method of claim 1, wherein the nucleic acid construct cannot homologous recombine with the locus. 4. The method of claim 1, wherein the locus is on a different chromosome than the first DNA sequence or on the same chromosome but at least 3,000 bps from the 5' or 3' terminus of the first DNA sequence. 5. The method of claim 1, wherein the nucleic acid construct is introduced by restriction enzyme-mediated integration. 6. The method of claim 1, wherein the nucleic acid construct comprises a selectable marker. 7. The method of claim 6, wherein the selectable marker is amdS, argB, bar, hygB, niaD, pyrG, sC, or trpC. 8. The method of claim 1, wherein the parent cell is a mammalian cell. 9. The method of claim 1, wherein the parent cell is a bacterial cell. 10. The method of claim 1, wherein the parent cell is a fungal cell. 11. The method of claim 10, wherein the fungal cell is a filamentous fungal cell. 12. The method of claim 11, wherein the filamentous fungal cell is selected from the group consisting of Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia, Tolypocladium, and Trichoderma. 13. The method of claim 10, wherein the fungal cell is a yeast cell. 14. The method of claim 1, wherein the polypeptide is a recombinant polypeptide. 15. The method of claim 1, wherein the polypeptide is a heterologous polypeptide. 16. The method of claim 1, wherein the polypeptide is a hormone, enzyme, receptor or portions thereof, antibody or portions thereof, or reporter. 17. The method of claim 16, wherein the enzyme is an oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase. 18. The method of claim 17, wherein the polypeptide is an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, or xylanase. 19. The method of claim 1, wherein the mutant cell has an increased uptake of an inorganic cofactor compared to the parent cell. 20. The method of claim 1, wherein the mutant cell has a more desirable morphology than the parent cell. 21. The method of claim 1, wherein the mutant cell produces higher yields of one or more secreted proteins than the parent cell. 22. The method of claim 1, wherein the mutant cell which has lost its ability to synthesize one or more essential metabolites. 23. The method of claim 1, wherein a phenotype of the mutant cell is observed only under certain conditions. 24. The method of claim 1, wherein the mutant cell exhibits an altered growth rate relative to the parent cell. 25. The method of claim 1, wherein the growth of the mutant cell is not inhibited by the overproduction of a desired polypeptide or metabolite when grown under conditions that induce high level production of the polypeptide or metabolite. 26. The method of claim 1, wherein the mutant cell is able to tolerate lower oxygen conditions than the parent cell. 27. The method of claim 1, wherein the mutant cell exhibits altered production of a transcriptional activator of a promoter than the parent cell. 28. The method of claim 1, wherein the mutant cell has a mutation in one or more of the genes of a signal transduction pathway of the parent cell. 29. The method of claim 1, wherein the mutant cell does not erroneously splice a cryptic intron. 30. The method of claim 1, wherein the nucleic acid construct is pDSY109, pDSY112, pMT1936, pDSY138, pDSY162, pDSY163, pDSY141, pSMO1204, pSMOH603, p4-8.1, p7-14.1, pHB220, pSMO717, pSMO321, pHowB571 or pSMO810. 31. The method of claim 1, wherein the locus is SEQ ID NO:9, SEQ ID NO:16, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:50, SEQ ID NO:56, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:71, SEQ ID NO:76, or a fragment thereof. 32. The method of claim 1, wherein the locus encodes a glucose transporter, mannitol-1-phosphate dehydrogenase, chitin synthase, heat shock protein, manganese superoxide dismutase, or a gene required for activation of pacC. 33. The method of claim 32, where the gene required for activation of pacC is a palB gene. 34. A method of producing a polypeptide, comprising (a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein (i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence, wherein the introduction of the nucleic acid construct disrupts a gene encoding an oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, or regulatory or control sequences thereof, other than a gene encoding a protease which hydrolyzes the polypeptide under the conditions; and (ii) the mutant cell produces more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and (b) recovering the polypeptide. 35. A method of producing a polypeptide, comprising (a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein (i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence, not within a second DNA sequence encoding a protein that negatively regulates transcription of the polypeptide, and not within a third DNA sequence encoding a protease which hydrolyzes the polypeptide under the conditions; and (ii) the mutant cell expresses more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and (b) recovering the polypeptide. 36. The method of claim 35 wherein the nucleic acid construct cannot homologous recombine with the first DNA sequence. 37. The method of claim 35, wherein the nucleic acid construct cannot homologous recombine with the locus. 38. The method of claim 35, wherein the locus is on a different chromosome than the first DNA sequence or on the same chromosome but at least 3,000 bps from the 5' or 3' terminus of the first DNA sequence. 39. A method of producing a polypeptide, comprising (a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein (i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence, wherein the introduction of the nucleic acid construct disrupts a gene encoding an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, a ligase, or regulatory or control sequences thereof, other than a gene encoding a protease which hydrolyzes the polypeptide under the conditions; and (ii) the mutant cell expresses more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and (b) recovering the polypeptide. 40. A method of producing a polypeptide, comprising (a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein (i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence, not within a second DNA sequence encoding a protein that negatively regulates translation of the polypeptide, and not within a third DNA sequence encoding a protease which hydrolyzes the polypeptide under the conditions; and (ii) the mutant cell synthesizes more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and (b) recovering the polypeptide. 41. The method of claim 40, wherein the nucleic acid construct cannot homologous recombine with the first DNA sequence. 42. The method of claim 40, wherein the nucleic acid construct cannot homologous recombine with the locus. 43. The method of claim 40, wherein the locus is on a different chromosome than the first DNA sequence or on the same chromosome but at least 3,000 bps from the 5' or 3' terminus of the first DNA sequence. 44. A method of producing a polypeptide, comprising (a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein (i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence, wherein the introduction of the nucleic acid construct disrupts a gene encoding an oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, or regulatory or control sequences thereof, other than a gene encoding a protease which hydrolyzes the polypeptide under the conditions; and (ii) the mutant cell synthesizes more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and (b) recovering the polypeptide. 45. A method of producing a polypeptide, comprising (a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein (i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence, not within a second DNA sequence encoding a protein that negatively regulates secretion of the polypeptide, and not within a third DNA sequence encoding a protease which hydrolyzes the polypeptide under the conditions; and (ii) the mutant cell secretes more of the polypeptide than the parent cell when both cells are cultivated under the conditions; (b) recovering the polypeptide. 46. The method of claim 45, wherein the nucleic acid construct cannot homologous recombine with the first DNA sequence. 47. The method of claim 45, wherein the nucleic acid construct cannot homologous recombine with the locus. 48. The method of claim 45, wherein the locus is on a different chromosome than the first DNA sequence or on the same chromosome but at least 3,000 bps from the 5' or 3' terminus of the first DNA sequence. 49. A method of producing a polypeptide, comprising (a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein (i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence, wherein the introduction of the nucleic acid construct disrupts a gene encoding an oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, or regulatory or control sequences thereof, other than a gene encoding a protease which hydrolyzes the polypeptide under the conditions; and (ii) the mutant cell secretes more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and (b) recovering the polypeptide. 50. A method of producing a polypeptide, comprising (a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein (i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the random integration of a nucleic acid construct into the genome of the parent cell at a locus wherein the nucleic acid construct is not homologous with the locus and wherein the locus is not within the first DNA sequence nor within a second DNA sequence encoding a protease which hydrolyzes the polypeptide under the conditions; and (ii) the mutant cell produces more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and (b) recovering the polypeptide. 51. A method of producing a metabolite, comprising (A) cultivating a mutant cell under conditions conducive for production of the metabolite, wherein (i) the mutant cell is related to a parent cell, which comprises one or more first DNA sequences encoding first polypeptides in the biosynthetic pathway of the metabolite, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within (a) the first DNA sequences, (b) a second DNA sequence encoding a protein that negatively regulates transcription, translation or secretion of the first polypeptides, (c) a third DNA sequence encoding a protease which hydrolyzes any of the first polypeptides under the conditions, and (d) one or more fourth DNA sequences encoding a second polypeptide in the second biosynthetic pathway of a second metabolite wherein the biosynthetic pathway and the second biosynthetic pathway involve the production of the same intermediate and the second polypeptide catalyzes a step after the production of the intermediate; and (ii) the mutant cell produces more of the metabolite than the parent cell when both cells are cultivated under the conditions; and (B) recovering the metabolite. 52. A method of producing a first polypeptide, comprising (a) forming a mutant cell by introducing a nucleic acid construct into the genome of the parent cell, which comprises a first DNA sequence encoding the polypeptide, at a locus which is not within the first DNA sequence, a second DNA sequence encoding a protein that negatively regulates transcription, translation or secretion of a second polypeptide, and a third DNA sequence encoding a protease which hydrolyzes the polypeptide under conditions conducive to the production of the first polypeptide; (b) isolating the mutant cell which produces more of the polypeptide than the parent cell when both cells are cultivated under the conditions; (c) identifying the locus wherein the nucleic acid construct has been integrated; (d) producing a cell in which a corresponding locus has been disrupted; (e) culturing the cell under the conditions; and (f) recovering the first polypeptide. 53. A method of producing a polypeptide, comprising (a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein (i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence and a second DNA sequence encoding a protease which hydrolyzes the polypeptide under the conditions, wherein the introduction of the nucleic acid construct specifically enhances transcription, translation or secretion of the polypeptide; and (ii) the mutant cell produces more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and (b) recovering the polypeptide.

Details for Patent 5,958,727

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2016-09-13
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2016-09-13
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2016-09-13