Clarification of transgenic milk using depth filtration

• US 20070192878A1
• Filed: 02/16/2006
• Published: 08/16/2007
• Est. Priority Date: 02/16/2006
• Status: Active Grant

• Current Assignee: LFB USA, Inc. (Agence des Participations de l’Etat)
• Original Assignee: GTC Biotherapeutics, Inc.
• Inventors: Perreault, Mark
• Granted Patent: US 7,531,632 B2
• Time in Patent Office: Days
• US Class Current: 800/6
• CPC Class Codes: C07K 1/34 by filtration, ultrafiltration or reverse osmosis
• CPC Class Codes: C07K 14/765 Serum albumin, e.g. HSA

Abstract

Processes and apparati are provided for separating molecules of interest from a mixture by depth filtration (DF). The DF of the invention is useful in the clarification and processing of various feedstreams for the removal of a molecule of interest. According to a preferred embodiment, a transgenic milk feedstream is stabilized and particulate matter such as fat, casein miscelles and bacteria are removed. An aseptic filtration step was also developed to remove any bacteria remaining in a clarified transgenic milk feedstream.

Claims

2. The method of claim 1, wherein the filter media has or forms passages throughout said filter matrix which are capable of trapping and retaining very small particles, preferably in the range of about 1-5 microns.

11. The method of claim 2, wherein said filter medium is composed of one or more of the following materials:

metallic, polymeric, inorganic, or organic materials.

39. A method for separating a protein of interest from a transgenic milk feedstream, comprising:

(a) filtering said milk by a depth filtration process that separates said molecular species of interest from said feedstream on the basis of particulate size further comprising a filter surface and a filter matrix;

(b) performing a viral inactivation step that inactivates at least 90% of viral particles or fragments;

(c) providing aseptic filtration for the filtered feedstream that passes said protein of interest and retains accompanying contaminants; and

,

(d) clarifying the remaining material through downstream processing;

wherein particulates are trapped both upon said filter surface and within said filter matrix.

1. A method for separating a protein of interest from a feedstream, comprising:

(a) filtering said feedstream by a depth filtration process that separates said molecular species of interest from said feedstream on the basis of particulate size further comprising a filter surface and a filter matrix;

(b) performing a viral inactivation step that inactivates at least 90% of viral particles or fragments;

(c) providing aseptic filtration for the filtered feedstream that retains said protein of interest and any accompanying contaminants; and

,

(d) clarifying the remaining material through additional downstream processing.

wherein particulates are trapped both upon said filter surface and within said filter matrix.

3. The method of claim 1, wherein the minimum particulate size ranges from 5 microns to 50 microns.

4. The method of claim 1, wherein the minimum particulate size ranges from 10 microns to 25 microns.

5. The method of claim 1, wherein the feedstream containing the protein of interest is composed of the milk from a transgenic mammal.

6. The method of claim 5, wherein the casein proteins are disassociated from the rest of the mixture through the use of EDTA thereby clarifying cellular debris and micelles.

7. The method of claim 6, further comprising wherein the casein micelles are aggregated through the use of an ammonium sulfate solution.

8. The method of claim 1, further comprising wherein said viral inactivation step inactivates at least 99.9% of viral particles or fragments present in the feedstream.

9. The method of claim 1, further comprising wherein at least 3 liters/ft2 of feedstream containing a protein of interest can be processed in 2 hours at 60 LMH.

10. The method of claim 1, further comprising wherein 3 liters/ft2 of feedstream containing a protein of interest can be processed in 2-4 hours at 30-60 LMH.

12. The method of claim 1, wherein said protein of interest has a molecular weight of between 1 and 1000 kDa.

13. The method of claim 1, wherein said feedstream is diluted before depth filtration.

14. The method of claim 1, wherein all filtration stages are depth filtration.

15. The method of claim 1, wherein said feedstream is a cell lysate solution.

16. The method of claim 1, wherein said protein is bioactive.

17. The method of claim 5, wherein the condition of said milk is selected from one of the following states:

a) raw;

b) diluted;

c) treated with a buffer solution;

d) chemically treated; and

e) concentrated.

18. The method of claim 1, wherein the microfiltration process is carried out at a permeation flux of less than 30 lmh.

19. The method of claim 1, wherein the microfiltration process is carried out at a pH above that at which said protein of interest does not precipitate.

20. The method of claim 1, further comprising a fractionation step utilizing a cellulose filtration membrane.

21. The method of claim 1 wherein said protein of interest is a molecular species of interest are biological entities selected from the group consisting of proteins, immunoglobulins, polypeptides, peptides and glycoproteins.

22. The method of claim 1, wherein the optimal temperature range is from 4°

C., to 40°

C.

23. The method of claim 22, wherein the optimal temperature range is from 15°

C., to 30°

C.

24. The method of claim 23, wherein the optimal temperature range is from 20°

C., to 25°

C.

25. The method of claim 24, wherein the optimal temperature range is from 4°

C. to 40°

C.

26. The method of claim 1, wherein said viral inactivation step that inactivates at least 99% of viral particles or fragments

27. The method of claim 1, wherein said additional downstream processing comprises anion chromatography.

28. The method of claim 1, wherein said additional downstream processing comprises cation chromatography.

29. The method of claim 1, wherein said additional downstream processing comprises size exclusion chromatography.

30. The method of claim 1, wherein said additional downstream processing comprises reverse phase chromatography.

31. The method of claim 1, wherein the feedstream concentration is from 0.25×

to 4×

natural milk.

32. The method of claim 1, wherein the feedstream concentration is from 0.5×

to 3×

natural milk.

33. The method of claim 1, wherein the microfiltration process is carried out at the protein of interest'"'"'s isoelectric pH.

34. The method of claim 1, wherein the protein of interest is selected from the group consisting of alpha-1-antitrypsin, alkaline phosphatase, angiogenin, antithrombin, chitinase, extracellular superoxide dismutase, Factor VIII, Factor IX, Factor X, fibrinogen, glucocerebrosidase, glutamate decarboxylase, human serum albumin, insulin, myelin basic protein, lactoferrin, lactoglobulin, lysozyme, lactalbumin, proinsulin, soluble CD4, component and complex of soluble CD4, and tissue plaminogen activator.

35. The method of claim 1, wherein said feedstream is treated with a solution selected from the group consisting of:

a) water;

b) a buffered aqueous salt solution;

c) chelating agent;

d) acid solution; and

e) alkali solution.

36. The methods of claim 1 or 39 wherein said protein of interest is an antibody.

37. The methods of claim 1 or 39 wherein said protein of interest is recombinant human albumin.

38. The methods of claim 1 wherein the protein of interest is selected from the group consisting of hormone, enzyme, milk protein, cellular protein, and soluble receptor

40. The method of claim 39, wherein the filter media has or forms passages throughout said filter matrix which are capable of trapping and retaining very small particles, preferably in the range of about 1-5 microns.

41. The method of claim 39, wherein the minimum particulate size ranges from 1 microns to 50 microns.

42. The method of claim 39, wherein the minimum particulate size ranges from 5 microns to 25 microns.

43. The method of claim 39 wherein said milk feedstream is diluted with a 3.8M ammonium sulfate solution.

44. The method of claim 42, wherein the casein proteins are disassociated from the rest of the mixture through the use of EDTA thereby clarifying cellular debris and micelles.

45. The method of claim 44, further comprising wherein the casein micelles are aggregated through the use of an ammonium sulfate solution.

46. The method of claim 39, further comprising wherein said viral inactivation step inactivates at least 99% of viral particles or fragments present in the feedstream.

47. The method of claim 39, further comprising wherein at least 3 liters/ft2 of feedstream containing a protein of interest can be processed in 2 hours at 60 LMH.

48. The method of claim 39, further comprising wherein 3 liters/ft2 of feedstream containing a protein of interest can be processed in 2-4 hours at 30-80 LMH.

49. The method of claim 40, wherein said filter medium is composed of one or more of the following materials:

metallic, polymeric, inorganic, or organic materials.

50. The method of claim 39, wherein said protein of interest has a molecular weight of between 1 and 1000 kDa.

52. The method of claim 39, wherein all filtration stages are depth filtration.

53. The method of claim 39, wherein said feedstream is a cell lysate solution.

54. The method of claim 39, wherein said protein is bioactive.

55. The method of claim 39, wherein the condition of said milk is selected from one of the following states:

a) raw;

b) diluted;

c) treated with a buffer solution;

d) chemically treated; and

e) concentrated.

56. The method of claim 54, further comprising a fractionation step utilizing a ceramic filtration membrane.

57. The method of claim 54, further comprising a fractionation step utilizing a polymeric filtration membrane.

58. The method of claim 54, further comprising a fractionation step utilizing a cellulose filtration membrane.

59. The method of claim 39, wherein said protein of interest is a molecular species of interest are biological entities selected from the group consisting of proteins, immunoglobulins, polypeptides, peptides and glycoproteins.

60. The method of claim 39, wherein the optimal temperature range is from 15°

C. to 50°

C.

61. The method of claim 60, wherein the optimal temperature range is from 20°

C. to 35°

C.

62. The method of claim 61, wherein the optimal temperature range is from 25°

C. to 29°

C.

63. The method of claim 62, wherein the optimal temperature range is from 15°

C. to 50°

C.

64. The method of claim 39, wherein the feedstream transmembrane pressure is less than 30 psi.

65. The method of claim 39, wherein the feedstream transmembrane pressure is less than 15 psi.

66. The method of claim 65, wherein the feedstream transmembrane pressure is less than 30 psi.

67. The method of claim 39, wherein the feedstream concentration is from 0.25×

to 4×

natural milk.

69. The method of claim 39, wherein the feedstream concentration is from 1.0×

to 2×

natural milk.

70. The method of claim 6 or 39, further comprising wherein the casein micelles are aggregated through the use of an ammonium sulfate solution of a pH lower than 8.0.

71. The method of claim 6 or 39, further comprising wherein the casein micelles are aggregated through the use of an ammonium sulfate solution of a pH between the range of 6.0 and 7.0.

72. The method of claim 6 or 39, further comprising wherein the casein micelles are aggregated through the use of an ammonium sulfate solution of a pH at approximately 6.5.

51. The method of 39 wherein said feedstream is concentrated or diluted before depth filtration.

67-1. The method of claim 39, wherein the feedstream concentration is from 0.5×

to 3×

natural milk.

73. The method of claim 39, wherein said milk is treated with a solution selected from the group consisting of:

a) water;

b) a buffered aqueous salt solution;

c) chelating agent;

d) acid solution; and

e) alkali solution.

74. The method of claim 73, wherein the contaminates removed include micelles and caseins

75. The method of claim 73, wherein the feedstream is milk.

76. The method of claim 73, wherein the limit of separation of 5,000 to 80,000 daltons.

77. The method of claim 73, further comprising a coarse filter to remove large precipitates and cellular debris

78. The method of claim 73, further comprising a polishing second stage filtration utilizing filters with pore sizes from 0.2 micron to 1.2 micron.

79. The method of claim 73, further comprising a centrifugation step

80. The method of claim 73, further comprising a filter aid including diatomaceous earth.

81. The method of claim 73 wherein a polishing step after DF is carried out using a filter of hydrophilized polyvinylidene fluoride (PVDF) membrane.

73-2. A method for isolating a protein of interest from a feedstream comprising:

collecting a feedstream sample;

diluting the feedstream with ammonium sulfate sufficient to precipitate protein contaminates, wherein the ph of the collected feedstream is above ph 5.0

subjecting the collected feedstream to a microfiltration in a filtration unit with a mean pore size of from 1.5 um to 15 um;

subjecting the filtrate to tangential flow microfiltration to provide molecule separation with a limit of separation of 5,000 to 50,000 daltons.