INDUSTRIAL PRODUCTION OF VIRAL PARTICLES

Claims

1. A method for obtaining a lentivirus composition, including (i) filtering the suspension mixture containing the host cell population and lentiviral particles to remove contaminants, wherein the filtration comprises: (a) filtering the mixture using a first filter, wherein the first filter is a depth filter, thereby producing a first filtrate, (b) filtering the first filtrate using a second filter, wherein the second filter has a retention threshold lower than the first filter, thereby obtaining a second filtrate, and (c) filtering the second filtrate using a third filter, wherein the third filter has a retention threshold lower than the second filter, thereby obtaining a filtered lentiviral particle composition; and (ii) concentration of the filtered composition of lentiviral particles, including chromatography and ultrafiltration.

2. A method for obtaining a lentivirus composition, comprising (i) contacting a population of host cells in suspension with at least one plasmid encoding a lentiviral protein; (ii) culturing the population of host cells from step (i) for a period of time sufficient to obtain a suspension mixture containing the population of host cells and lentiviral particles; (iii) filtration of the suspension mixture to remove contaminants, including: (a) contacting the mixture with an endonuclease, (b) filtering the mixture using a first filter, wherein the first filter is a depth filter, thereby producing a first filtrate, (c) filtering the first filtrate using a second filter, wherein the second filter has a retention threshold lower than the first filter, thereby obtaining a second filtrate, and (d) filtering the second filtrate using a third filter, wherein the third filter has a retention threshold lower than the second filter, thereby obtaining a filtered lentiviral particle composition; and (iv) concentration of the filtered composition of lentiviral particles, including chromatography and ultrafiltration.

3. The method according to claim 1 or 2, wherein the host cell comprises a human cell.

4. The method according to claim 3, wherein the human cell comprises a HEK293 cell, a HEK293T cell, a HEK293F cell, a HEK293FT cell, a Te671 cell, an HT1080 cell, or a CEM cell.

5. The method according to claim 3 or 4, wherein the cell comprises a HEK293 cell.

6. The method according to claim 3 or 4, wherein the cell comprises a HEK293T cell.

7. The method according to any one of paragraphs 1-6, wherein the first filter has a retention threshold of 1-60 μm.

8. The method according to claim 7, wherein the first filter has a retention threshold of 60 µm.

9. The method according to any one of paragraphs 1-8, wherein the second filter has a retention threshold of 0.4-4 µm.

10. The method according to claim 9, wherein the second filter has a retention threshold of 0.45 µm.

11. The method according to any one of paragraphs. 1-10, wherein the third filter has a retention threshold of 0.45 μm ± 0.2 μm.

12. The method according to any one of paragraphs. 1-10, wherein the third filter has a retention threshold of 0.2-0.3 µm.

13. The method according to claim 12, wherein the third filter has a retention threshold of 0.2 µm.

14. The method according to any one of paragraphs 1-10, 12 and 13, wherein the first filter has a retention threshold of 60 μm, the second filter has a retention threshold of 0.45 μm, and the third filter has a retention threshold of 0.2 μm.

15. The method according to any one of claims 1 and 3-14, wherein the endonuclease is present during steps (i)(a)-(i)(c).

16. The method according to any one of claims 2 to 15, wherein the endonuclease is present during steps (iii)(b) to (iii)(d).

17. The method according to any one of paragraphs. 1-16, wherein the second filter and the third filter are two layers within a two-layer filter component.

18. The method according to any one of claims 1-16, wherein the third filter is a two-layer filter comprising a first layer filter and a second layer filter, wherein the second layer filter has a retention threshold lower than the first layer filter.

19. The method according to claim 18, wherein the retention threshold of the first filter is 60 μm, the retention threshold of the second filter is 0.45 μm, the retention threshold of the first layer filter is 0.45 μm, and the retention threshold of the second layer filter is 0.2 μm.

20. The method according to any one of claims 1 to 19, wherein the chromatography is anion exchange chromatography (AEX).

21. The method according to claim 20, wherein the AEX chromatography comprises elution of lentiviral particles using a saline buffer.

22. The method according to claim 21, wherein the salt buffer comprises NaCl.

23. The method of claim 22, wherein NaCl is present in a concentration of from about 0.5 M to 3 M.

24. The method of claim 22, wherein NaCl is present in a concentration of from about 0.5 M to 1 M.

25. The method according to any one of claims 22-24, wherein NaCl is exactly or approximately 0.75 M.

26. The method of claim 22, wherein NaCl is present in a concentration of from about 1 M to 3 M.

27. The method of claim 22, wherein NaCl is present in a concentration of from about 1.5 M to 2.5 M.

28. The method according to any one of claims 22, 26 or 27, wherein NaCl is approximately 2 M.

29. The method according to any one of claims 1-28, wherein the chromatography is carried out before ultrafiltration.

30. The method according to any one of claims 1-29, wherein the ultrafiltration is ultrafiltration / diafiltration (UF / DF).

31. The method of claim 30, wherein UF / DF is carried out by means of one or more stages of flow filtration along the flow (TFF).

32. The method of claim 31, wherein the filter for one or more TFFs is a hollow fiber filter.

33. The method of claim 32, wherein the nominal molecular weight cutoff (NMWC) of the hollow fiber filter is exactly or approximately 500 kDa.

34. The method according to any one of claims 31-33, wherein the TFF comprises a first flow filtration (TFF) stage and a second flow filtration (TFF) stage.

35. The method of claim 33, wherein the first TFF is performed using a first hollow fiber filter, and the second TFF is performed using a second hollow fiber filter.

36. The method of claim 35, wherein the first and second hollow fiber filters have the same nominal molecular weight cutoff value (NMWC).

37. The method of claim 36, wherein the NMWC is 500 kDa.

38. The method of claim 35, wherein the first hollow fiber filter has a higher nominal molecular weight cutoff (NMWC) than the second hollow fiber filter.

39. The method of claim 38, wherein the NMWC of the first hollow fiber filter is 500 kDa.

40. The method according to any one of paragraphs. 35-39, wherein the first hollow fiber filter has a larger surface area than the second hollow fiber filter.

41. The method of claim 40, wherein the surface area of the first hollow fiber filter is between 790 cm 2 and 1600 cm 2 .

42. The method according to any one of paragraphs. 35-41, wherein the first hollow fiber filter holds a greater volume than the second hollow fiber filter.

43. The method according to claim 42, wherein the volume of the first hollow fiber filter is 300 ml.

44. The method according to any one of claims 1 to 43, comprising sterilizing by filtration the filtered composition after concentration, thereby obtaining a sterilized composition.

45. The method according to claim 44, wherein sterilization by filtration comprises filtering the filtered composition using a fourth filter.

46. The method according to claim 45, wherein the fourth filter has a retention threshold of 0.2 μm.

47. The method according to any one of claims 44-46, comprising formulating the sterilized composition in a buffer, thereby obtaining a drug substance.

48. The method according to any one of paragraphs 1-47, wherein the method is carried out at a pH of 6-8.

49. The method according to any one of claims 1-48, wherein the lentivirus composition is intended for administration to a subject in vivo.

50. The method according to any one of claims 1-49, wherein the amount of contaminants in the filtered composition is reduced, compared to the amount of contaminants in the second filtrate.

51. The method according to any one of claims 44-50, wherein the amount of contaminants in the sterilized composition is at a level acceptable for administration to a subject in vivo.

52. The method according to any one of claims 1-51, wherein the contaminants comprise host cells, host cell DNA (hcDNA) and / or host cell proteins (HCPs).

53. The method of claim 52, wherein the amount of hcDNA is less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% of the sterilized composition.

54. The method of claim 52, wherein the amount of hcDNA is reduced by more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% in the sterilized composition.

55. The method of claim 52, wherein the amount of NSR is less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% of the sterilized composition.

56. The method of claim 52, wherein the amount of NSR is reduced by more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% in the sterilized composition.

57. The method of any one of claims 52-56, wherein the amount of hcDNA in the filtered composition is less than about 2500 ng / 1E9 TU.

58. The method of any one of claims 52-57, wherein the amount of hcDNA in the filtered composition is at least about 80 times lower than the amount of hcDNA in the suspension mixture.

59. The method of any one of claims 52-58, wherein the amount of hcDNA in the filtered composition is at least about 5 times lower than the amount of hcDNA in the second filtrate.

60. The method of any one of claims 52-59, wherein the amount of HCP after chromatography is less than about 3000 μg / 1E9 TU.

61. The method according to any one of claims 52-60, wherein the amount of HCP after chromatography is at least about 40 times lower than the amount of HCP before chromatography.

62. The method of any one of claims 52-61, wherein the amount of HCP after chromatography is at least about 99% lower than the amount of HCP before chromatography.

63. The method of any one of claims 52-62, wherein the amount of HCP is less than about 1500 μg / 1E9 TU after the first UF / DF step.

64. The method according to any one of claims 52-63, wherein the amount of HSR is not detectable after the second UF / DF step.

65. The method according to any one of claims 1-64, wherein the suspension mixture comprises a medium, wherein the medium does not contain serum and / or animal by-products.

66. The method according to any one of claims 2 to 65, wherein the culturing from step (ii) is carried out for 40-48 hours.

67. The method according to any one of claims 1-66, wherein the filtration and concentration are carried out over a period of time of 5-8 hours.

68. The method according to any one of paragraphs. 1-67, wherein the suspension mixture has a volume of 3-50 liters.

69. The method according to any one of paragraphs. 1-67, wherein the suspension mixture has a volume of 5 l - 200 l.

70. The method according to any one of paragraphs. 1-67, wherein the suspension mixture has a volume of 100 l - 200 l.

71. The method according to any one of claims 1-67, wherein the suspension mixture has a volume of from exactly or approximately 180 L to exactly or approximately 200 L.

72. The method according to any one of claims 1-71, wherein the lentiviral particle comprises at least one payload.

73. The method of claim 72, wherein at least one load comprises a non-coding nucleic acid, optionally wherein the non-coding nucleic acid is a miRNA, miRNA or shRNA.

74. The method of claim 72, wherein at least one load is a polynucleotide encoding a polypeptide of interest.

75. The method according to any one of claims 2-74, wherein at least one plasmid is a polynucleotide encoding a polypeptide of interest.

76. The method of claim 74 or 75, wherein the polypeptide of interest is a chimeric antigen receptor (CAR).

77. The method of claim 76, wherein the CAR is specific for a tumor-associated antigen.

78. The method of claim 77, wherein the tumor-associated antigen is CD19, BCMA, GPRC5D, ROR1, FcRL5, alpha-fetoprotein, or Her2.

79. The method of claim 76, wherein the CAR is a universal CAR.

80. The method of claim 79, wherein the universal CAR comprises an extracellular domain comprising a label-binding domain.

81. The method according to claim 80, wherein the label is fluorescein.

82. The method of claim 76, wherein the CAR comprises an extracellular domain comprising a hapten binding domain.

83. The method of any one of claims 1-82, wherein the lentiviral particle comprises a surface-engineered fusion protein exposed on the surface of the lentiviral particle, optionally wherein the surface-engineered protein is embedded in a lipid bilayer.

84. The method of claim 83, wherein the surface engineered protein consists of a protein with a single binding domain that binds to a target molecule on a target cell.

85. The method of claim 84, wherein the surface engineered protein comprises a protein with multiple binding domains, wherein each binding domain binds to a target molecule on a target cell, optionally wherein each binding domain binds to a different target molecule.

86. The method according to claim 84 or 85, wherein the protein with a single binding domain or the protein with multiple binding domains is a transduction enhancer protein.

87. The method according to claims 83-86, wherein the protein with an engineered surface is a fusion protein containing a transduction enhancer and a viral envelope protein.

88. The method according to any one of claims 1-87, wherein the lentiviral particle comprises a viral envelope containing a transduction enhancer protein and a viral envelope protein.

89. The method according to any one of claims 2-88, wherein at least one plasmid is a plasmid encoding a fusion protein comprising a transduction enhancer protein and a viral envelope protein, optionally wherein the transduction enhancer comprises an immunocyte activating protein and / or a costimulatory molecule.

90. The method according to any one of claims 2-89, wherein at least one plasmid is a plasmid encoding a fusion protein comprising an immunocyte activating protein and a viral envelope protein.

91. The method of claim 89 or 90, wherein the immunocyte activating protein comprises at least one binding domain that specifically binds CD2, CD3, CD28H, LFA-1, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244, TCR α chain, TCR β chain, TCR ζ chain, TCR γ chain, TCR δ chain, CD3 ε TCR subunit, CD3 γ TCR subunit, CD3 δ TCR subunit, or NKp80, or combinations thereof.

92. The method according to any one of claims 2-89, wherein at least one plasmid is a plasmid encoding a fusion protein comprising at least one costimulatory molecule and a viral envelope protein.

93. The method of claims 89 and 92, wherein the costimulatory molecule is CD45, CD2, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, OX40, 4-1BB, CD40L, or any combination thereof.

94. The method according to any one of claims 86-90, wherein the transduction enhancer comprises at least one binding domain that binds a target molecule selected from the group consisting of an immunocyte activating receptor, a T cell costimulatory receptor, or an adhesion molecule.

95. The method according to any of paragraphs 86-94, where: the transduction enhancer comprises one binding domain that binds one target molecule selected from the group consisting of an immunocyte activating receptor, a T cell costimulatory receptor, or an adhesion molecule; the transduction enhancer comprises a plurality of binding domains that bind two or more target molecules selected from the group consisting of an immunocyte activating receptor, a T cell costimulatory receptor, and an adhesion molecule; or The transduction enhancer contains multiple binding domains, each of which binds a different target molecule, including an immunocyte activating receptor, a T cell costimulatory receptor, and an adhesion molecule.

96. The method according to any of paragraphs 94, 95, where: The activating receptor of immune cells is CD3; the costimulatory molecule is CD28, CD137, or CD134; and / or The adhesion molecule is CD58 or CD2.

97. The method according to any one of claims 84-96, wherein each of the at least one binding domain is independently selected from an antibody or antigen-binding fragment, or an ectodomain of a native ligand of a target molecule.

98. The method according to any one of claims 87-97, wherein the viral envelope protein is VSV-G envelope protein, measles virus envelope protein, Nipah virus envelope protein, or Cocal virus G protein.

99. The method according to any one of claims 2 to 98, wherein at least one plasmid is a plasmid encoding a viral helper protein.

100. The method according to claim 99, wherein the viral helper protein is rev and / or gagpol.

101. The method according to any one of claims 2 to 100, wherein the population of host cells is contacted with a mixture of plasmids comprising (i) a plasmid encoding a gene of interest; (ii) a plasmid encoding a viral protein rev; (iii) a plasmid encoding a viral protein gagpol; and (iv) a plasmid encoding a viral envelope protein.

102. The method of claim 101, wherein the mixture of plasmids comprises (v) a plasmid encoding an immune cell activating protein, (vi) a plasmid encoding a costimulatory molecule, or (vii) any combination of (v)-(vi).

103. A lentiviral composition obtained by the method according to any one of claims 1-102.

104. The lentiviral composition according to item 103, wherein the composition has an infectious titer of 2.0 TU / ml - 6×10 8 TU / ml.

105. A lentiviral composition according to item 103 or 104, wherein the composition has an infectious titer of 2.5 TU / ml - 4.7×10 8 TU / ml.

106. A lentiviral composition according to item 104 or 105, wherein the total number of infectious units in the composition is 4×10 10 TU - 8x10 10 TU.

107. A lentiviral composition according to any one of paragraphs 104-106, wherein the total number of infectious units in the composition is 5×10 10 TU - 7x10 10 TU, optional, exactly or approximately 6×10 10 TU.

108. The lentiviral composition of any one of claims 104-107, wherein the composition comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% HCP.

109. The lentiviral composition of any one of claims 104-108, wherein the composition comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% hcDNA.

110. The lentiviral composition of any one of claims 104-109, wherein the composition has a reduction in the amount of HSR of greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%.

111. The lentiviral formulation of any one of claims 104-110, wherein the formulation has a reduction in the amount of hcDNA of greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%, optionally reduced, compared to a filtered formulation prior to concentration.

112. The lentiviral formulation of any one of claims 104-111, wherein the formulation has a greater than 99% reduction in hcDNA and a greater than 99% reduction in HCP, optionally reduced compared to a filtered formulation prior to concentration.

113. A lentiviral composition according to any one of claims 104-112, wherein the composition comprises less than 1% hcDNA and less than 1% HCP.

114. Lentiviral composition containing a lentiviral vector with a titer of 2.5-4.7×10 8 TU / ml, where the composition contains less than 1% hcDNA and less than 1% HCP.

115. A lentiviral composition according to any one of paragraphs 103-114, wherein the volume of the composition is 1 ml - 500 ml, optionally 10 ml - 100 ml.