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1. WO2013013193 - POLYPEPTIDE SEPARATION METHODS

Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

[ EN ]

We claim:

1 . A method of separating polypeptide glycoforms in a load fluid, the method

comprising:

(a) providing a medium comprising an immunoglobulin Fc receptor;

(b) contacting the medium with a load fluid comprising a polypeptide under conditions in which the polypeptide binds to the immunoglobulin Fc receptor, wherein the polypeptide comprises an immunoglobulin Fc receptor binding moiety, wherein the load fluid comprises a plurality of glycoforms of the polypeptide, and wherein the Fc receptor preferentially binds one or more of the glycoforms;

(c) contacting the medium with an elution solution under conditions in which bound polypeptide elutes from the medium; and

(d) recovering bound polypeptide that elutes from the medium, thereby producing an eluate.

2. The method of claim 1 , wherein the Fc receptor binds to a first glycoform with an affinity which is at least 2 fold, 5 fold, 10 fold, 20 fold, 30 fold greater, 40 fold, 50 fold, 100 fold, or 150 fold greater than the affinity with it binds to another glycoform.

3. The method of claim 1 , wherein the method includes contacting the medium with one or more wash solutions, prior to contacting the medium with the elution solution.

4. The method of claim 1 , wherein the method includes recovering polypeptide that flows through the medium.

5. The method of claim 4, wherein the method includes loading the medium at 5-1900% of its polypeptide capacity.

6. The method of claim 1 , wherein the load fluid comprises cell culture medium.

7. The method of claim 1 , wherein the load fluid comprises a fluid that has been purified by one or more of ion exchange chromatography.

8. The method of claim 1 , wherein the load fluid comprises a pharmaceutical drug product or drug substance.

9. The method of claim 1 , wherein the immunoglobulin Fc receptor binding moiety comprises an immunoglobulin Fc region.

10. The method of claim 1 , wherein the polypeptide comprises an antibody.

1 1 . The method of claim 1 , wherein the polypeptide comprises an Fc fusion protein.

12. The method of claim 1 , wherein the polypeptide comprises one or more polypeptides selected from single domain antibodies, maxibodies, minibodies, intrabodies, small modular immunopharmaceuticals (SMIPs), IgG-scFv bispecific antibodies, antibody-peptide conjugates, antibody-drug conjugates, and Fc receptor binding polypeptides on a virus or virus capsid.

13. The method of claim 1 , wherein the polypeptide is produced in a mammalian cell.

14. The method of claim 1 , wherein the polypeptide is produced in a fungal cell.

15. The method of claim 1 , wherein the polypeptide is produced in an insect cell.

16. The method of claim 1 , wherein the polypeptide is produced in a plant cell.

17. The method of claim 13, wherein the polypeptide is produced in a CHO cell.

18. The method of claim 13, wherein the polypeptide is produced in an NS0 cell.

19. The method of claim 13, wherein the polypeptide is produced in an Sp2/0 cell.

20. The method of claim 1 , wherein the Fc receptor preferentially binds to a glycoform having reduced fucose.

21 . The method of claim 20, wherein the Fc receptor preferentially binds to polypeptide glycoforms having reduced core N-fucose.

22. The method of claim 1 , wherein the Fc receptor preferentially binds to polypeptide glycoforms having increased high mannose oligosaccharides.

23. The method of claim 1 , wherein the Fc receptor is glycosylated.

24. The method of claim 1 , wherein the Fc receptor comprises an Fc binding portion of an Fc gamma Rill polypeptide or an Fc gamma RIV polypeptide.

25. The method of claim 21 , wherein the Fc receptor comprises an extracellular domain of an Fc gamma RIM polypeptide or an Fc gamma RIV polypeptide.

26. The method of claim 24, wherein the Fc receptor comprises a sequence at least 85% identical to amino acid residues 21 -209 of SEQ ID NO:1 .

27. The method of claim 25, wherein the extracellular domain of the Fc gamma RIM polypeptide is a V176 allotype.

28. The method of claim 25, wherein the Fc receptor comprises a full length Fc gamma RIM polypeptide.

29. The method of claim 1 , wherein the load fluid comprises a first glycoform that preferentially binds to the Fc receptor, and wherein the percentage of the first glycoform in the eluate is increased by at least 20%, relative to the load fluid.

30. The method of claim 29, wherein the percentage of the first glycoform in the eluate is increased by at least 50%, 100%, 2-fold, 5-fold, 10-fold, or 20-fold, relative to the load fluid.

31 . The method of claim 29, wherein the percentage of the first glycoform in the eluate is at least 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%.

32. The method of claim 29, wherein the first glycoform is an afucosylated glycoform.

33. The method of claim 29, wherein the first glycoform is a glycoform lacking or having reduced sialylation.

34. The method of claim 29, wherein the first glycoform is a galactosylated glycoform.

35. The method of claim 29, wherein the first glycoform is a glycoform having high mannose oligosaccharides.

36. The method of claim 1 , wherein the eluate comprises one or more fractions of bound polypeptide eluted from the medium.

37. The method of claim 1 , further comprising fractionating the eluate.

38. The method of claim 1 , wherein the load fluid comprises a second glycoform that does not preferentially bind to the Fc receptor, and wherein the percentage of the second glycoform in the eluate is decreased, relative to the load fluid.

39. The method of claim 1 , wherein a biological activity of polypeptide in the eluate is altered relative to the activity of the polypeptide in the load fluid.

40. The method of claim 39, wherein the biological activity comprises antibody dependent cell mediated cytotoxicity (ADCC), and wherein ADCC is increased.

41 . The method of claim 4, wherein a biological activity of polypeptide that has flowed through the medium is altered, relative to the activity of the polypeptide in the load fluid.

42. The method of claim 1 , wherein the medium comprises beads, membranes, monoliths, a fiber matrix, porous media, or a gel.

43. The method of claim 1 , wherein the medium comprises agarose, cellulose, or dextran, ceramic, metal, glass, nylon, Teflon, nylon, polycarbonate, polyacrylamide, polystyrene, polypropylene, polyether sulfone, polyamide, polytetrafluoroethylene,

polysulfone, polyester, polyvinylidene fluoride, a fluorocarbon, polyethylene, polyacrylate, or poly(azolactone).

44. The method of claim 1 , wherein the Fc receptor is linked to the medium via a crosslinker or enzyme-mediated coupling.

45. The method of claim 1 , wherein the Fc receptor is linked to the medium via a disulfide bond, metal chelation, cyanogen bromide, an NHS linkage, a histidine tag, a glycidyl ether, an epoxy, a tresyl chloride linkage, a tosyl chloride linkage, an EAH linkage, an ECH linkage, an activated thiol linkage, or a thiopropyl linkage.

46. The method of claim 1 , wherein the medium comprises the Fc receptor at a concentration of 0.1 to 15 mg/mL.

47. The method of claim 1 , wherein the medium is equilibrated with an equilibration solution comprising a buffer between 1 and 500 mM, and a salt between 0 and 2000 mM, at a pH between 3.5 and 10, prior to contacting the medium with the load fluid.

48. The method of claim 1 , wherein the load fluid comprises the polypeptide at a concentration between 0.001 and 100 mg/mL.

49. The method of claim 1 , wherein the amount of polypeptide contacted with the medium ranges from 0.1 to 25,000 mg polypeptide/mL medium.

50. The method of claim 3, wherein the one or more wash solutions comprises a buffer between 1 and 500 mM, and a salt between 0 and 2000 mM, and/or an additive and/or a solvent at a pH between 3.5 and 10.

51 . The method of claim 1 ,wherein the elution solution has a pH between 2 and 5 and or a salt between 0 and 2000 mM, and/or an additive and/or a solvent.

52. The method of claim 1 , wherein the medium is contacted with one or more elution solutions under conditions in which a pH gradient is applied.

53. The method of claim 1 , further comprising neutralizing the eluate.

54. The method of claim 1 , further comprising contacting the eluate with a second medium, and recovering polypeptide that flows through, or is eluted from, the second medium.

55. The method of claim 54, wherein the second medium comprises an ion exchange medium, a hydroxyapatite medium, a protein A medium, a hydrophobic interaction medium, an immobilized metal affinity medium, a synthetic medium (biomimetic), a lectin, or a combination thereof.

54. The method of claim 1 , further comprising producing a pharmaceutical composition from polypeptide in the eluate.

55. The method of claim 4, further comprising producing a pharmaceutical composition from polypeptide that has flowed through the medium.

56. The method of claim 1 , further comprising analyzing a characteristic of polypeptide eluted from the medium.

57. The method of claim 56, wherein oligosaccharides from the polypeptide are analyzed.

58. The method of claim 56, wherein a biological activity of polypeptide is analyzed.

59. The method of claim 56, wherein one or more of toxicity, stability, or efficacy are analyzed.

60. The method of claim 56, wherein the analyzing further comprises analyzing polypeptide in the load fluid and/or polypeptide that has flowed through the medium.

61 . The method of claim 4, wherein the method comprises analyzing polypeptide that has flowed through the medium.

62. The method of claim 61 , wherein oligosaccharides on the polypeptide are analyzed.

63. The method of claim 61 , wherein one or more of toxicity, stability, or efficacy are analyzed.

64. The method of claim 61 , wherein a biological activity of polypeptide is analyzed.

65. A composition comprising a polypeptide recovered by the method of claim 1 .

66. A method comprising:

(a) providing a medium comprising an Fc receptor, wherein the Fc receptor comprises an Fc binding portion of an Fc gamma RIM polypeptide;

(b) contacting the medium with a load fluid comprising a polypeptide under conditions in which the polypeptide binds to the medium, wherein the polypeptide comprises an immunoglobulin Fc region;

(c) contacting the medium with an elution solution under conditions in which bound polypeptide elutes from the medium; and

(d) recovering bound polypeptide that elutes from the medium, thereby producing an eluate.

67. The method of claim 66, wherein the Fc receptor comprises an extracellular domain of an Fc gamma RIM polypeptide.

68. A medium comprising an Fc receptor linked to a solid support, wherein the Fc receptor comprises an Fc binding portion of an Fc gamma RIM polypeptide.

69. The medium of claim 68, wherein the solid support comprises agarose, cellulose, or dextran, ceramic, metal, glass, nylon, Teflon, nylon, polycarbonate, polyacrylamide, polystyrene, polypropylene, polyether sulfone, polyamide, polytetrafluoroethylene, polysulfone, polyester, polyvinylidene fluoride, a fluorocarbon (e.g.

poly(tetrafluoroethylene-co-perfluoro(alkyl vinyl ether)), polyethylene, polyacrylate, or poly(azolactone).

70. The medium of claim 68, wherein the solid support comprises beads, membranes, monoliths, a fiber matrix, porous media, or a gel.

71 . The method of claim 1 or 66, wherein the load fluid comprises serum IgG.

72. The method of claim 1 , wherein the immunoglobulin Fc receptor is selected from the group consisting of: Fc gamma Rllla V176, Fc gamma Rllla F176, Fc gamma Rlllb NA1 , Fc gamma Rlllb NA2, Fc gamma Rlla H131 , Fc gamma Rlla R131 , Fc gamma Rllb I232, and Fc gamma Rllb T232.

73. A kit, comprising (a) a medium comprising an immunoglobulin Fc receptor linked to a solid support, wherein the immunoglobulin Fc receptor comprises an Fc binding portion selected from the group consisting of: an Fc binding portion of an Fc gamma Rl polypeptide, an Fc gamma Rll polypeptide, an Fc gamma RIM polypeptide, and an Fc gamma RIV polypeptide; and (b) instructions for use according to the method of claim 1 .

74. The kit of claim 73, wherein the solid support comprises agarose, cellulose, or dextran, ceramic, metal, glass, nylon, Teflon, nylon, polycarbonate, polyacrylamide, polystyrene, polypropylene, polyether sulfone, polyamide, polytetrafluoroethylene, polysulfone, polyester, polyvinylidene fluoride, a fluorocarbon (e.g.

poly(tetrafluoroethylene-co-perfluoro(alkyl vinyl ether)), polyethylene, polyacrylate, or poly(azolactone).

75. The kit of claim 73, wherein the solid support comprises beads, membranes, monoliths, a fiber matrix, porous media, or a gel.