Claims for Patent: 6,100,061

Summary for Patent: 6,100,061

Title:	Recombinant cell clone having increased stability in serum- and protein-free medium and a method of recovering the stable cell clone and the production of recombinant proteins by using a stable cell clone
Abstract:	Disclosed are a stable recombinant cell clone which is stable in serum- and protein-free medium for at least 40 generations, a biomass obtained by multiplying the stable cell clone under serum- and protein-free culturing conditions, and a method of preparing recombinant proteins by means of the biomass. Furthermore, the invention relates to a method of recovering stable recombinant cell clones.
Inventor(s):	Reiter; Manfred (Vienna, AT), Mundt; Wolfgang (Vienna, AT), Dorner; Friedrich (Vienna, AT)
Assignee:	Immuno Aktiengesellschaft (Vienna, AT)
Application Number:	09/100,253
Patent Claims:	1. A recombinant CHO cell clone stable in serum- and protein-free medium for at least 40 generations and expressing a recombinant product. 2. A recombinant CHO cell clone as set forth in claim 1, said cell clone being stable for at least 50 generations. 3. A recombinant CHO cell clone as set forth in claim 1, said cell clone being present in isolated form. 4. A recombinant CHO cell clone as set forth in claim 1, wherein said recombinant product is a recombinant polypeptide or a recombinant protein and said cell clone contains sequences encoding said recombinant polypetide or said recombinant protein. 5. A recombinant CHO cell clone as set forth in claim 1, wherein said recombinant CHO cell clone expresses von Willebrand factor as said recombinant product. 6. A recombinant CHO cell clone as set forth in claim 1, wherein said recombinant CHO cell clone expresses factor VIII as said recombinant product. 7. A recombinant CHO cell clone as set forth in claim 1, wherein said recombinant CHO cell clone co-expresses factor VIII and vWF as said recombinant product. 8. A recombinant CHO cell clone as set forth in claim 1, wherein said recombinant CHO cell clone expresses factor IX as said recombinant product. 9. A recombinant CHO cell clone as set forth in claim 1, wherein said recombinant CHO cell clone expresses factor II as said recombinant product. 10. A recombinant CHO cell clone as set forth in claim 1, obtainable by culturing a recombinant original cell clone on serum-containing medium so as to provide cells of a starting culture, and re-adapting said cells of said starting culture to serum- and protein free medium so as to provide a re-adapted cell culture, testing said re-adapted cell culture for stable product producer cells, and cloning a stable product producer cell clone under serum- and protein-free conditions from said stable product producer cells. 11. A recombinant CHO cell clone as set forth in claim 6, wherein said recombinant protein is a blood factor selected from the group consisting of factor II, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, protein S, protein C, an activated form of any one of these factors, and vWF. 12. A recombinant CHO cell clone as set forth in claim 5, having ECACC Deposit No. 98012206. 13. A CHO cell culture comprising at least 90% of stable CHO recombinant cells, said stable recombinant cells being stable for more than 50 generations under serum- and protein-free conditions expressing recombinant product. 14. A cell culture as set forth in claim 13 obtainable by culturing a recombinant cell clone stable in serum- and protein-free medium for at least 40 generations, said stable recombinant cell clone expressing a recombinant product. 15. A cell culture as set forth in claim 13, obtainable by the steps of multiplying a recombinant original cell clone in serum-containing medium so as to obtain cells, culturing said cells under serum- and protein-free conditions so as to obtain a cell culture, assaying said cell culture under serum- and protein-free conditions for producers of said recombinant product, cloning stable cell clones under serum- and protein-free conditions, multiplying stable cell clones under serum- and protein-free conditions. 16. A cell culture as set forth in claim 13, wherein said recombinant CHO cells are CHO-DHFR.sup.- cells or CHO-K1 cells. 17. A cell culture as set forth in claim 13, wherein said stable cells contain a sequence encoding a recombinant polypeptide or recombinant protein. 18. A cell culture as set forth in claim 13, wherein said cells are immobilized on a microcarrier. 19. A method of producing a recombinant product under serum- and protein-free conditions on a large technical scale comprising the steps of providing an isolated, stable recombinant original cell clone, said original cell clone being stable in serum- and protein-free medium for at least 40 generations and expressing a recombinant product, multiplying said original stable cell clone in serum- and protein-free medium so as to obtain a cell culture, culturing said cell culture containing stable cells in a bioreactor, thereby obtaining said recombinant product, and harvesting said recombinant product from a supernatant of said cell culture. 20. A method as set forth in claim 19, wherein said original cell clone is stable in serum- and protein-free medium for at least 50 generations. 21. A method as set forth in claim 19, wherein said serum- and protein-free medium is a synthetic mimimum medium. 22. A method as set forth in claim 19, wherein said serum- and protein-free medium contains a protease inhibitor. 23. A method of claim 19, wherein the stable cell clone is cultivated on a microcarrier. 24. A method as set forth in claim 21, wherein said synthetic minimum medium contains a yeast extract. 25. A method as set forth in claim 21, wherein said synthetic minimum medium contains a soybean extract. 26. A method of claim 21, wherein the serum- and protein-free medium further comprises peptone, L-Glutamine, NaHCO.sub.3, Ascorbic acid, Ethanol amine, and Na-selenite. 27. A cell culture comprising more than 95% of stable recombinant cells, said stable recombinant cells being stable for more than 50 generations under serum- and protein-free conditions and expressing recombinant product. 28. A method for recovering a stable recombinant cell clone comprising the steps of multiplying a recombinant original cell clone in serum-containing medium so as to obtain a cell culture, culturing the cells of said cell culture under serum- and protein-free conditions, assaying said cell culture under serum- and protein-free conditions for producers of said recombinant product, cloning recombinant cell clones that are stable under serum and protein-free conditions, and multiplying said stable recombinant cell clones under serum- and protein-free conditions. 29. A method of claim 28, wherein the serum- and protein-free medium comprises minimum medium and peptone. 30. A method of claim 28, wherein the cells are cultivated on a microcarrier. 31. A method of recovering a stable recombinant cell clone comprising the steps of: multiplying a non-recombinant original cell under serum- and protein-free conditions, cloning a stable, non-recombinant cell clone under serum- and protein-free conditions, transfecting said stable cell clone with a recombinant nucleic acid and isolating stable transfectants, culturing said isolated stable transfectants in serum- and protein-free medium, and assaying said cultured transfectants for production stability. 32. A method of claim 31, wherein the serum- and protein-free medium comprises minimum medium and peptone. 33. A serum- and protein-free synthetic medium comprised of minimum medium, soybean peptone, glutamine, sodium hydrogencarbonate, ascorbic acid, ethanol amine and sodium selenite. 34. A medium as set forth in claim 33, wherein said minimum medium comprises inorganic salts, amino acids, vitamins and a carbohydrate source. 35. A cell culture in a serum- and protein-free synthetic medium, said serum- and protein-free synthetic medium being comprised of minimum medium, soybean peptone, glutamine, sodium hydrogencarbonate, ascorbic acid, ethanol amine and sodium selenite. 36. A method of producing a recombinant polypeptide in a cell culture comprising the steps of multiplying a stable starting cell clone expressing a recombinant polypeptide serum- and protein-free medium so as to obtain a cell culture, culturing said cell culture containing stable cells in a bioreactor so as to obtain a cell culture, and harvesting said recombinant polypeptide from a supernatant of said cell culture. 37. A method as set forth in claim 36, wherein said recombinant polypeptide is a blood factor selected from the group consisting of factor II, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, protein S, protein C and vWF or wherein said recombinant polypeptide is a polypeptide having the activity of said factors or an activated form of one of said factors. 38. A method as set forth in claim 36, wherein said stable starting cell clone is a recombinant CHO cell clone expressing von Willebrand factor as said recombinant polypeptide or expressing a polypeptide having von Willebrand factor activity. 39. A method as set forth in claim 36, wherein said stable starting cell clone is a recombinant CHO cell clone expressing factor VIII as said recombinant polypeptide or expressing a polypeptide having factor VIII activity. 40. A method as set forth in claim 36, wherein said stable starting cell clone is a recombinant CHO cell clone co-expressing factor VIII and vWF as said recombinant polypeptide. 41. A method as set forth in claim 36, wherein said stable starting cell clone is a recombinant CHO cell clone expressing factor IX as said recombinant polypeptide. 42. A method as set forth in claim 36, wherein said stable starting cell clone is a recombinant CHO cell clone expressing factor II as said recombinant polypeptide. 43. A method of claim 36, wherein the serum- and protein-free medium comprises minimum medium and peptone. 44. A recombinant CHO cell clone which has been cultivated in serum- and protein-free medium for at least 40 generations and which expresses a recombinant product.

Details for Patent 6,100,061

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Takeda Pharmaceuticals U.s.a., Inc.	ADVATE	antihemophilic factor (recombinant), plasma/albumin free method	For Injection	125063	July 25, 2003		2018-06-19
Takeda Pharmaceuticals U.s.a., Inc.	ADVATE	antihemophilic factor (recombinant), plasma/albumin free method	For Injection	125063	April 12, 2006		2018-06-19
Takeda Pharmaceuticals U.s.a., Inc.	ADVATE	antihemophilic factor (recombinant), plasma/albumin free method	For Injection	125063	July 03, 2007		2018-06-19
Takeda Pharmaceuticals U.s.a., Inc.	ADVATE	antihemophilic factor (recombinant), plasma/albumin free method	For Injection	125063	July 12, 2012		2018-06-19