Arenavirus formulations, methods and uses thereof

Abstract

The present disclosure relates to improved liquid and lyophilized arenavirus formulations, as well as pharmaceutical compositions derived therefrom. The disclosure also relates to methods of making such arenavirus formulations, and methods of treating or preventing diseases or conditions (e.g., cancer and infectious diseases) using such arenavirus formulations and pharmaceutical compositions derived therefrom.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480058994.4 (22) Application Date 2024.09.13 (30) Priority Data 63 / 582,913 2023.09.15 US (85) PCT International Application Entering National Phase Date 2026.03.13 (86) PCT International Application Application Data PCT / EP2024 / 075683 2024.09.13 (87) PCT International Application Publication Data WO2025 / 056782 EN 2025.03.20 (71) Applicant Gilead Sciences, Inc. Address California, USA (72) Inventors G. Forman, B. Brim, C. Renishch (74) Patent Agency King & Wood Mallesons, Beijing 11256 Patent Attorney Ma Hui (51) Int.Cl. A61K 9 / 00 (2006.01) A61K 9 / 08 (2006.01) A61K 9 / 19 (2006.01) A61K 39 / 12 (2006.01) A61K 47 / 12 (2006.01) A61P 35 / 00 (2006.01) (54) Invention Title: Sand Virus Preparations, Methods and Uses Thereof (57) Abstract: This disclosure relates to improved liquid and lyophilized sand virus preparations, and pharmaceutical compositions derived therefrom. This disclosure also relates to methods for preparing such sand virus preparations, and methods for using such sand virus preparations and pharmaceutical compositions derived therefrom to treat or prevent diseases or conditions (e.g., cancer and infectious diseases). Claims (10 pages), Description (63 pages), Sequence List (electronic publication), Drawings (20 pages), CN 121866044 A, 2026.04.14, CN 1 21 86 60 44 A. 1. A liquid isovirus preparation comprising: (a) isovirus; and (b) a polyvalent anion at a concentration between about 5 mM and about 150 mM, optionally wherein the concentration of the polyvalent anion is between about 10 mM and about 60 mM. 2. The liquid isovirus preparation of claim 1, wherein the concentration of the polyvalent anion is about 50 mM or about 25 mM. 3. The liquid isovirus preparation of claim 1, wherein the liquid isovirus preparation is suitable for lyophilization, and the concentration of the polyvalent anion is at most about 50 mM or between about 20 mM and about 50 mM. 4. A liquid isovirus formulation comprising: (a) isovirus; and (b) a polyvalent anion, wherein the concentration of the polyvalent anion (i) reduces the formation of subvisible particles during manufacturing, andAnd / or (ii) reduce the loss of virus titer during filtration, optionally wherein the filtration includes bioburden reduction filtration or sterilization filtration, and / or the manufacturing includes a purification process. 5. The liquid isovirus preparation of claim 4, wherein the concentration of the polyvalent anion is between about 5 mM and about 150 mM, optionally wherein the concentration of the polyvalent anion is between about 10 mM and about 60 mM. 6. The liquid isovirus preparation of claim 5, wherein the concentration of the polyvalent anion is about 50 mM or about 25 mM. 7. The liquid isovirus preparation of claim 5, wherein the liquid isovirus preparation is suitable for lyophilization, and the concentration of the polyvalent anion is at most about 50 mM or between about 20 mM and about 50 mM. 8. The liquid isoflavone virus preparation according to any one of claims 1 to 7, wherein the polyvalent anion is selected from the group consisting of citrate, tetrasodium pyrophosphate (TSPP), malate, succinate, tartrate, fumarate, maleate, and combinations thereof. 9. The liquid isoflavone virus preparation according to claim 8, wherein the polyvalent anion is citrate. 10. The liquid isoflavone virus preparation according to claim 9, wherein the concentration of citrate is about 50 mM or about 25 mM. 11. The liquid isoflavone virus preparation according to claim 9, wherein the liquid isoflavone virus preparation is suitable for lyophilization, and the concentration of citrate is at most about 50 mM or between about 20 mM and about 50 mM. 12. The liquid sand virus preparation according to any one of claims 1 to 11, wherein the liquid sand virus preparation further comprises at least one of: (c) carbohydrates; (d) salts; (e) buffers; (f) surfactants; and (g) stabilizers. 13. The liquid sand-like virus preparation according to claim 12, wherein: (c) the concentration of the carbohydrate is between about 0.1% and about 20% (w / v), optionally wherein the concentration of the carbohydrate is between about 3% and about 10% (w / v); (d) the concentration of the salt is at most about 450 mM, optionally wherein the concentration of the salt is between about 50 mM and about 250 mM; (e) the concentration of the buffer is between about 1 mM and about 50 mM, optionally wherein the concentration of the buffer is between about 10 mM and about 20 mM; (f) the concentration of the surfactant is between about 0.001% and about 1% (w / v), optionally wherein the concentration of the surfactant is between about 0.01% and about 0.05% (w / v); and / or (g)The concentration of the stabilizer is between about 0.001% (w / v) and about 3% (w / v), optionally wherein the concentration of the stabilizer is between about 0.05% (w / v) and 0.15% (w / v). 14. The liquid sand-like virus preparation according to claim 12 or 13, wherein (c) the concentration of the carbohydrate is about 5% (w / v); (d) the concentration of the salt is about 125 mM or about 75 mM; (e) the concentration of the buffer is about 10 mM; (f) the concentration of the surfactant is about 0.03% (w / v); and / or (g) the concentration of the stabilizer is about 0.1% (w / v). 15. The liquid sand virus preparation according to claim 12 or 13, wherein the liquid sand virus preparation is suitable for lyophilization, and (c) the concentration of the carbohydrate is about 5% (w / v); (d) the concentration of the salt is at most about 125 mM or between about 50 mM and about 125 mM; (e) the concentration of the buffer is about 10 mM; (f) the concentration of the surfactant is about 0.03% (w / v); and / or (g) the concentration of the stabilizer is about 0.1% (w / v). 16. The liquid sand virus preparation according to any one of claims 12 to 15, wherein: (c) the carbohydrate is a sugar or polyol; optionally, the sugar or polyol is selected from the group consisting of sucrose, trehalose, sorbitol, mannitol, dextran, and combinations thereof; further optionally, the carbohydrate is sucrose, trehalose, or combinations thereof; (d) the salt is selected from the group consisting of sodium chloride, sodium phosphate, potassium phosphate, potassium chloride, magnesium chloride, sodium sulfate, potassium sulfate, ammonium sulfate, and combinations thereof; optionally, the salt is sodium chloride; (e) the buffer is selected from the group consisting of phosphate, citrate, TRIS, HEPES, bicarbonate, histidine, and combinations thereof; optionally, the buffer is TRIS; (f) the surfactant is selected from the group consisting of poloxamer 188, poloxamer 407, polysorbate 20, polysorbate 80, and combinations thereof; optionally, the surfactant is poloxamer 188; and / or (g) The stabilizer is selected from the group consisting of albumin, recombinant protein, plasma protein, gelatin, and combinations thereof; optionally, the albumin is lactalbumin, human serum albumin, recombinant human serum albumin (rHSA), or combinations thereof; further optionally, the stabilizer is rHSA. 17. The liquid sand virus preparation according to claim 16, wherein: (c) the carbohydrate is sucrose or trehalose at a concentration of about 5% (w / v); (d) the salt is sodium chloride at a concentration of about 125 mM or about 75 mM; (e)The buffer is TRIS at a concentration of about 10 mM; (f) the surfactant is poloxamer 188 at a concentration of about 0.03% (w / v); and / or (g) the stabilizer is rHSA at a concentration of about 0.1%. Claims 2 / 10 pages 3 CN 121866044 A 18. The liquid isovirus preparation according to claim 16, wherein the liquid isovirus preparation is suitable for lyophilization, and: (c) the carbohydrate is trehalose at a concentration of about 5% (w / v); (d) the salt is sodium chloride at a concentration of up to about 125 mM or between about 50 mM and about 125 mM; (e) the buffer is TRIS at a concentration of about 10 mM; (f) the surfactant is poloxamer 188 at a concentration of about 0.03% (w / v); and / or (g) the stabilizer is rHSA at a concentration of about 0.1%. 19. The liquid isovirus preparation according to any one of claims 1 to 18, wherein the pH of the liquid isovirus preparation is between about 7.0 and about 9.0. 20. The liquid isovirus preparation according to claim 19, wherein the pH is about 8.0. 21. The liquid isovirus preparation according to any one of claims 1 to 20, wherein the osmotic concentration of the liquid isovirus preparation is between about 200 mOsm / kg and about 1000 mOsm / kg. 22. The liquid isovirus preparation according to claim 21, wherein the osmotic concentration of the liquid isovirus preparation is between about 500 mOsm / kg and about 550 mOsm / kg, optionally wherein the osmotic concentration of the liquid isovirus preparation is about 530 mOsm / kg. 23. The liquid isovirus preparation of claim 21, wherein the permeation concentration of the liquid isovirus preparation is between about 350 mOsm / kg and about 450 mOsm / kg, optionally wherein the permeation concentration of the liquid isovirus preparation is about 400 mOsm / kg. 24. The liquid isovirus preparation of any one of claims 21, wherein the liquid isovirus preparation is suitable for lyophilization and the permeation concentration is between about 300 mOsm / kg and about 600 mOsm / kg. 25. The liquid isovirus preparation of any one of claims 12 to 24, wherein (c) to (g) are present to reduce viral titer loss during filtration, freeze-thaw cycles, and / or storage; and / or to reduce sub-visible particle formation during manufacturing, freeze-thaw cycles, and / or storage. 26. The liquid sand-like virus preparation according to claim 25, wherein the storage temperature is above about 2°C, between about 20°C and about 25°C, between about 2°C and about 8°C, above about 15°C, about -20°C or below about -20°C, or...Approximately -65°C or below, or approximately -80°C. 27. A liquid isovirus preparation comprising: (a) isovirus; (b) citrate at a concentration of approximately 50 mM; (c) sucrose at a concentration of approximately 5% (w / v); (d) NaCl at a concentration of approximately 125 mM; (e) TRIS at a concentration of approximately 10 mM; and (f) poloxamer 188 at a concentration of approximately 0.03% (w / v); and (g) rHSA at a concentration of approximately 0.1% (w / v), wherein the pH of the liquid isovirus preparation is approximately 8.0, and optionally wherein the osmotic concentration of the liquid isovirus preparation is approximately 530 mOsm / kg. 28. A liquid isovirus preparation comprising: (a) isovirus; (b) citrate at a concentration of about 25 mM; (c) trehalose at a concentration of about 5% (w / v); (d) NaCl at a concentration of about 75 mM; (e) TRIS at a concentration of about 10 mM; and (f) poloxamer 188 at a concentration of about 0.03% (w / v); and (g) rHSA at a concentration of about 0.1% (w / v), wherein the pH of the liquid isovirus preparation is about 8.0, and optionally wherein the osmotic concentration of the liquid isovirus preparation is about 400 mOsm / kg. 29. A liquid isovirus preparation suitable for freeze-drying, the liquid isovirus preparation comprising: (a) isovirus; (b) citrate at a concentration between about 20 mM and about 50 mM; (c) trehalose at a concentration of about 5% (w / v); (d) sodium chloride at a concentration of up to about 125 mM or between about 50 mM and about 125 mM; (e) TRIS at a concentration of about 10 mM; (f) poloxamer 188 at a concentration of about 0.03% (w / v); and (g) rHSA at a concentration of about 0.1% (w / v), wherein the pH of the liquid isovirus preparation is about 8.0, optionally wherein the osmotic concentration of the liquid isovirus preparation suitable for freeze-drying is between about 300 mOsm / kg and about 600 mOsm / kg. 30. The liquid isovirus preparation according to any one of claims 1 to 29, wherein the concentration of the isovirus is between about 10² RCV-FFU / ml and about 10¹⁰ RCV-FFU / ml, or between about 2 × 10⁵ RCV-FFU / ml and about 5 × 10⁸ RCV-FFU / ml. 31. The liquid isovirus preparation according to any one of claims 1 to 30, wherein the isovirus is a lymphocyte-derived virus.32. A liquid isovirus preparation according to any one of claims 1 to 31, wherein the isovirus is a genetically engineered isovirus. 33. A liquid isovirus preparation according to any one of claims 1 to 32, wherein the isovirus expresses an antigen, optionally wherein the antigen is selected from the group consisting of tumor antigens, tumor-associated antigens, antigens of pathogens capable of causing infectious diseases, testicular cancer antigen (CTA), and antigenic fragments of any of the foregoing. 34. A liquid isovirus preparation according to claim 33, wherein the antigen comprises cytomegalovirus (CMV) antigen, optionally wherein the CMV antigen is selected from the group consisting of gB(dCt), pp65, and combinations thereof. 35. A liquid isovirus preparation according to claim 33, wherein the antigen comprises human papillomavirus (HPV) antigen, optionally wherein the HPV antigen is an HPV E7 / E6 fusion protein. 36. The liquid isovirus formulation of claim 33, wherein the antigen comprises a prostate cancer antigen, optionally wherein the prostate cancer antigen is selected from the group consisting of PSA, PSMA, PAP, and combinations thereof. 37. The liquid isovirus formulation of claim 33, wherein the antigen comprises a hepatitis B virus (HBV) antigen, optionally wherein the HBV antigen is selected from the group consisting of a truncated HBV polymerase polypeptide comprising an inactivated reverse transcriptase domain and an inactivated RNase H, a fusion protein comprising an HBV core polypeptide and an HBV small surface antigen (sAg) polypeptide, and combinations thereof. 38. The liquid isovirus formulation of claim 33, wherein the antigen comprises a human immunodeficiency virus (HIV) antigen, optionally wherein the HIV antigen is selected from the group consisting of gag, pol, nef, and combinations thereof, further comprising: Optionally wherein the HIV antigen is a fusion polypeptide comprising multiple polypeptide segments of one or more HIV-1 proteins encoded by two or more HIV genes selected from gag, pol, and nef. 39. The liquid arena virus formulation of claim 33, wherein the antigen comprises at least one antigenic fragment of mutant KRAS, optionally wherein the at least one antigenic fragment comprises mutant G13D, G12V, G12C, G12D, and G12R from the N-terminus to the C-terminus, respectively. 40. The liquid arena virus formulation of claim 33, wherein the antigen comprises two or more testicular cancer antigen (CTA) fragments, optionally wherein the two or more CTA fragments are selected from antigens preferentially expressed in melanoma.The group consisting of epitopes or antigenic fragments of PRAME, melanoma-associated antigen 3 (MAGE-A3), melanoma-associated antigen 4 (MAGE-A4), and melanoma-associated antigen 6 (MAGE-A6). 41. The liquid isovirus formulation according to any one of claims 1 to 40, wherein the isovirus is a non-replicating isovirus, optionally wherein the isovirus ORF has been removed or functionally inactivated such that the resulting isovirus cannot produce further infectious progeny viral particles, further optionally wherein the ORF encoding the GP has been removed and replaced with a nucleotide sequence encoding the antigen. 42. The liquid isovirus formulation according to any one of claims 1 to 40, wherein the isovirus is a replicating trisegmented isovirus. 43. The liquid isovirus formulation according to claim 42, wherein the trisegmented isovirus comprises an isovirus ORF located outside the wild-type position of the ORF. 44. The liquid isovirus formulation of claim 43, wherein the three-segmented isovirus comprises an L segment and two S segments, wherein one of the two S segments is an S segment, and wherein the ORF encoding the isovirus glycoprotein (GP) is under the control of the isovirus 3' UTR. 45. The liquid isovirus formulation of claim 42, wherein the three-segmented isovirus is engineered such that the isovirus ORF is distributed on two or more mRNA transcripts. 46. A lyophilized isovirus formulation prepared from the liquid isovirus formulation of any one of claims 1 to 45. 47. A method for (i) reducing the formation of subvisible particles during manufacturing and / or (ii) reducing virus titer loss during filtration, the method comprising mixing a sand-like virus with a solution to form a liquid sand-like virus formulation, wherein the liquid sand-like virus formulation comprises a polyvalent anion at a concentration between about 5 mM and about 150 mM, optionally wherein the concentration of the polyvalent anion is between about 10 mM and about 60 mM. 48. The method of claim 47, wherein the concentration of the polyvalent anion is about 50 mM or about 25 mM. 49. The method of claim 47, wherein the liquid sand-like virus formulation is suitable for lyophilization, and the concentration of the polyvalent anion is between about 20 mM and about 50 mM. 50. A method for (i) reducing the formation of subvisible particles during manufacturing and / or (ii) reducing viral titer loss during filtration, the method comprising mixing a sand-like virus with a solution to form a liquid sand-like virus formulation, wherein the liquid sand-like virus formulation comprises a polyvalent anion, the concentration of which reduces the formation of subvisible particles during manufacturing and / or reduces viral titer loss during filtration. 51. Claim 51.50. The method of claim 51, wherein the concentration of the polyvalent anion is between about 5 mM and about 150 mM. 52. The method of claim 51, wherein the concentration of the polyvalent anion is about 50 mM or about 25 mM. 53. The method of claim 51, wherein the liquid sand virus preparation is suitable for lyophilization, and the concentration of the polyvalent anion is between about 20 mM and about 50 mM. 54. The method of any one of claims 47 to 53, wherein the polyvalent anion is selected from the group consisting of citrate, tetrasodium pyrophosphate (TSPP), malate, succinate, tartrate, fumarate, maleate, and combinations thereof. 55. The method of claim 54, wherein the polyvalent anion is citrate. 56. The method of claim 55, wherein the concentration of citrate is about 50 mM or about 25 mM. 57. The method of claim 55, wherein the concentration of citrate is between about 10 mM and about 60 mM. 58. The method of any one of claims 47 to 57, wherein the liquid sand virus preparation further comprises at least one of: (b) a carbohydrate; (c) a salt; (d) a buffer; (e) a surfactant; and (f) a stabilizer. 59. The method of claim 58, wherein: (b) the concentration of the carbohydrate is between about 0.1% and about 20% (w / v), optionally wherein the concentration of the carbohydrate is between about 3% and about 10% (w / v); (c) the concentration of the salt is at most about 450 mM, optionally wherein the concentration of the salt is between about 50 mM and about 250 mM; (d) the concentration of the buffer is between about 1 mM and about 50 mM, optionally wherein the concentration of the buffer is between about 10 mM and about 20 mM; (e) the concentration of the surfactant is between about 0.001% and about 1% (w / v), optionally wherein the concentration of the surfactant is between about 0.01% and about 0.05% (w / v); and / or (f) The concentration of the stabilizer is between about 0.001% (w / v) and about 3% (w / v), optionally wherein the concentration of the stabilizer is between about 0.05% (w / v) and 0.15% (w / v). 60. The method according to claim 58 or 59, wherein (b) the concentration of the carbohydrate is about 5% (w / v); (c) the concentration of the salt is about 125 mM or about 75 mM; (d) the concentration of the buffer is about 10 mM; (e) the concentration of the surfactant is about 0.03% (w / v); and / or (f)The concentration of the stabilizer is about 0.1% (w / v). 61. The method according to claim 58 or 59, wherein the liquid sand virus preparation is suitable for lyophilization, and (b) the concentration of the carbohydrate is about 5% (w / v); (c) the concentration of the salt is at most about 125 mM or between about 50 mM and about 125 mM; (d) the concentration of the buffer is about 10 mM; (e) the concentration of the surfactant is about 0.03% (w / v); and / or (f) the concentration of the stabilizer is about 0.1% (w / v). 62. The method according to any one of claims 58 to 61, wherein: (b) the carbohydrate is a sugar or a polyol; optionally, the sugar or polyol is selected from the group consisting of sucrose, trehalose, sorbitol, mannitol, dextran, and combinations thereof; further optionally, the carbohydrate is sucrose, trehalose, or combinations thereof; (c) the salt is selected from the group consisting of sodium chloride, sodium phosphate, potassium phosphate, potassium chloride, magnesium chloride, sodium sulfate, potassium sulfate, ammonium sulfate, and combinations thereof; optionally, the salt is sodium chloride; (d) the buffer is selected from the group consisting of phosphate, citrate, TRIS, HEPES, bicarbonate, histidine, and combinations thereof; optionally, the buffer is TRIS; (e) The surfactant is selected from the group consisting of poloxamer 188, poloxamer 407, polysorbate 20, polysorbate 80, and combinations thereof; optionally, the surfactant is poloxamer 188; and / or (f) the stabilizer is a protein selected from the group consisting of albumin, recombinant protein, plasma protein, gelatin, and combinations thereof; optionally, the albumin is lactalbumin, human serum albumin, recombinant human serum albumin (rHSA), or combinations thereof; further optionally, the protein is rHSA. 63. The method of claim 62, wherein: (b) the carbohydrate is sucrose or trehalose at a concentration of about 5% (w / v); (c) the salt is sodium chloride at a concentration of about 125 mM or about 75 mM; (d) the buffer is TRIS at a concentration of about 10 mM; (e) the surfactant is poloxamer 188 at a concentration of about 0.03% (w / v); and / or (f) the stabilizer is rHSA at a concentration of about 0.1%. 64. The method of claim 62, wherein the liquid sand virus preparation is suitable for lyophilization, and: (b) the carbohydrate is trehalose at a concentration of about 5% (w / v); (c) the salt is sodium chloride at a concentration of up to about 125 mM or between about 50 mM and about 125 mM; (d)The buffer is TRIS at a concentration of about 10 mM; (e) the surfactant is poloxamer 188 at a concentration of about 0.03% (w / v); and / or (f) the stabilizer is rHSA at a concentration of about 0.1%. 65. The method according to any one of claims 47 to 64, wherein the pH of the liquid isovirus preparation is between about 7.0 and about 9.0, optionally wherein the pH is about 8.0. 66. The method according to any one of claims 47 to 65, wherein the osmotic concentration of the liquid isovirus preparation is between about 200 mOsm / kg and about 1000 mOsm / kg. 67. The method according to claim 66, wherein the osmotic concentration of the liquid isovirus preparation is between about 500 mOsm / kg and about 550 mOsm / kg, optionally wherein the osmotic concentration of the liquid isovirus preparation is about 530 mOsm / kg. 68. The method of claim 66, wherein the permeation concentration of the liquid isovirus preparation is between about 350 mOsm / kg and about 450 mOsm / kg, optionally wherein the permeation concentration of the liquid isovirus preparation is about 400 mOsm / kg. 69. The method of claim 66, wherein the liquid isovirus preparation is suitable for lyophilization and the permeation concentration is between about 300 mOsm / kg and about 600 mOsm / kg. 70. The method of any one of claims 58 to 69, wherein (b) to (f) are present at concentrations that reduce virus titer loss during filtration, reduce subvisible particle formation, maintain the biophysical stability of the isovirus during freeze-thaw processes, and / or maintain the biophysical stability of the isovirus during storage. Claims 7 / 10, page 8, CN 121866044 A 71. The method according to claim 70, wherein the storage temperature is higher than about 2°C, between about 20°C and about 25°C, between about 2°C and about 8°C, higher than about 15°C, about -20°C or lower than about -20°C, about -65°C or lower than about -65°C, or about -80°C. 72. A method for (i) reducing the formation of subvisible particles during manufacturing and / or (ii) reducing virus titer loss during filtration, the method comprising mixing a sand-like virus with a solution to form a liquid sand-like virus formulation, wherein the liquid sand-like virus formulation comprises: (a) citrate at a concentration of about 50 mM; (b) sucrose at a concentration of about 5% (w / v); (c) sodium chloride at a concentration of about 125 mM; (d) TRIS at a concentration of about 10 mM; (e) poloxamer 188 at a concentration of about 0.03% (w / v); and (f) rHSA at a concentration of about 0.1%.The liquid sand virus preparation has a pH of about 8.0, and optionally, the permeation concentration of the liquid sand virus preparation is about 530 mOsm / kg. 73. A method for (i) reducing the formation of subvisible particles during manufacturing and / or (ii) reducing virus titer loss during filtration, the method comprising mixing a sand virus with a solution to form a liquid sand virus preparation, wherein the liquid sand virus preparation comprises: (a) citrate at a concentration of about 25 mM; (b) trehalose at a concentration of about 5% (w / v); (c) sodium chloride at a concentration of about 75 mM; (d) TRIS at a concentration of about 10 mM; (e) poloxamer 188 at a concentration of about 0.03% (w / v); and (f) rHSA at a concentration of about 0.1%, wherein the liquid sand virus preparation has a pH of about 8.0, and optionally, the permeation concentration of the liquid sand virus preparation is about 400 mOsm / kg. 74. A method for (i) reducing the formation of subvisible particles during manufacturing and / or (ii) reducing virus titer loss during filtration, the method comprising mixing a sand virus with a solution to form a liquid sand virus formulation, wherein the liquid sand virus formulation is suitable for lyophilization and comprises: (a) citrate at a concentration between 10 mM and about 60 mM; (b) trehalose at a concentration of about 5% (w / v); (c) sodium chloride at a concentration of up to about 125 mM or between about 50 mM and about 125 mM; (d) TRIS at a concentration of about 10 mM; (e) poloxamer 188 at a concentration of about 0.03% (w / v); and (f) rHSA at a concentration of about 0.1% (w / v), wherein the liquid sand virus formulation has a pH of about 8.0. Optionally, the filtration includes bioburden reduction filtration or sterilization filtration, and / or the manufacturing includes a purification process, wherein the permeation concentration of the liquid isovirus preparation suitable for lyophilization is between about 300 mOsm / kg and about 600 mOsm / kg. 75. The method according to any one of claims 47 to 74, wherein the concentration of the isovirus is between about 102 RCV-FFU / ml and about 1010 RCV-FFU / ml, or between about 2 × 10⁵ RCV-FFU / ml and about 5 × 10⁸ RCV-FFU / ml. 76. The method according to any one of claims 47 to 75, wherein the isovirus is lymphocytic choriomeningitis virus (LCMV), Piccind virus, Tamiyami virus, or Tacalib virus. 77. The method according to any one of claims 47 to 76, wherein the sand virus is a genetically engineered sand virus.78. The method of any one of claims 47 to 77, wherein the arenavirus expresses an antigen, optionally wherein the antigen is selected from the group consisting of tumor antigens, tumor-associated antigens, antigens of pathogens capable of causing infectious diseases, testicular cancer antigen (CTA), and antigenic fragments of any of the foregoing. 79. The method of claim 78, wherein the antigen comprises at least one cytomegalovirus (CMV) antigen, optionally wherein the CMV antigen is selected from the group consisting of gB(dCt), pp65, and combinations thereof. 80. The method of claim 78, wherein the antigen comprises at least one human papillomavirus (HPV) antigen, optionally wherein the HPV antigen is an HPV E7 / E6 fusion protein. 81. The method of claim 78, wherein the antigen comprises at least one prostate cancer antigen, optionally wherein the prostate cancer antigen is selected from the group consisting of PSA, PSMA, PAP, and combinations thereof. 82. The method of claim 78, wherein the antigen comprises hepatitis B virus (HBV) antigen, optionally wherein the HBV antigen is selected from the group consisting of a truncated HBV polymerase polypeptide comprising an inactivated reverse transcriptase domain and an inactivated RNase H, a fusion protein comprising an HBV core polypeptide and an HBV small surface antigen (sAg) polypeptide, and combinations thereof. 83. The method of claim 78, wherein the antigen comprises human immunodeficiency virus (HIV) antigen, optionally wherein the HIV antigen is selected from the group consisting of gag, pol, nef, and combinations thereof, further optionally wherein the HIV antigen is a fusion polypeptide comprising a plurality of polypeptide segments of one or more HIV-1 proteins encoded by two or more HIV genes selected from gag, pol, and nef. 84. The method of claim 78, wherein the antigen comprises at least one antigenic fragment of mutant KRAS, optionally wherein the antigenic fragment of mutant KRAS comprises mutant G13D, G12V, G12C, G12D, and G12R from the N-terminus to the C-terminus, respectively. 85. The method of claim 78, wherein the antigen comprises two or more testicular cancer antigen (CTA) fragments, optionally wherein the two or more CTA fragments are selected from the group consisting of epitopes or antigenic fragments of melanoma-preferred antigens (PRAME), melanoma-associated antigen 3 (MAGE-A3), melanoma-associated antigen 4 (MAGE-A4), and melanoma-associated antigen 6 (MAGE-A6). 86. The method of any one of claims 47 to 85, wherein the isanvirus is a non-replicating isanvirus, optionally wherein the isanvirus ORF has been removed or functionally inactivated such that the resulting isanvirus cannot produce further...The method further optionally includes an infectious progeny viral particle, wherein the ORF encoding the GP has been removed and replaced with a nucleotide sequence encoding an antigen. 87. The method of any one of claims 47 to 85, wherein the isovirus is a replicating trisegmented isovirus. 88. The method of claim 87, wherein the trisegmented isovirus comprises an isovirus ORF located outside the wild-type position of the ORF. 89. The method of claim 88, wherein the trisegmented isovirus comprises an L segment and two S segments, wherein one of the two S segments is an S segment, wherein the ORF encoding the GP is under the control of isovirus 3' UTR. 90. The method of claim 87, wherein the trisegmented isovirus is engineered such that the isovirus ORF is distributed on two or more mRNA transcripts. 91. The method according to any one of claims 47 to 90, further comprising lyophilizing the liquid isovirus preparation. 92. A method for producing a lyophilized isovirus preparation, the method comprising lyophilizing the liquid isovirus preparation according to any one of claims 1 to 45. 93. A method for treating or preventing a disease, the method comprising administering to a subject in need the liquid isovirus preparation according to any one of claims 1 to 45, optionally wherein the liquid isovirus preparation is further diluted prior to administration. 94. A method for treating or preventing a disease, the method comprising administering to a subject in need a liquid isovirus preparation reconstituted from a lyophilized preparation according to claim 46, wherein the lyophilized preparation is reconstituted prior to administration. 95. The method according to claim 93 or 94, wherein the liquid isovirus preparation is administered parenterally, optionally wherein the liquid isovirus preparation is administered intravenously, intramuscularly, intratumorally, or subcutaneously. 96. The method according to claims 93 to 95, wherein the disease is a neoplastic disease or an infectious disease. Claims 10 / 10 Page 11 CN 121866044 A Cross-Reference to Related Applications Regarding Sand Virus Preparations, Methods, and Uses Thereof

[0001] This application claims priority to U.S. Provisional Application No. 63 / 582,913, filed September 15, 2023, the entire contents of which are incorporated herein by reference.

[0002] Sequence List This application contains a computer-readable sequence list, which has been submitted with this application in XML file format, the entire contents of which are incorporated herein by reference. The sequence list XML file submitted with this application is named"13194-086-228_SEQLISTING.xml", created on September 11, 2024, is 54,146 bytes in size.

[0003] 1. Technical Field This disclosure relates to improved liquid and lyophilized arenavirus formulations, and pharmaceutical compositions comprising the formulations. This disclosure also relates to methods of treating or preventing diseases using said arenavirus formulations and pharmaceutical compositions comprising the formulations.

[0004] 2. Background Art A major challenge in developing therapies based on engineered arenavirus vectors is the design of the viral formulation to ensure optimal vector safety, efficacy, and stability. To maintain the activity of the arenavirus vector during purification and storage, the conditions and excipients of the formulation need to be carefully selected to stabilize the viral particles against mechanical or thermal stress (e.g., filtration steps or freeze-thaw cycles) and to avoid loss of vector infectivity due to inactivation or aggregation of viral particles and / or nonspecific binding to contact materials used during the purification process. Buffer conditions must be carefully adjusted throughout the manufacturing process to provide a stable environment with minimal interactions between viral particles.

[0005] Therefore, there is a need for improved viral formulations that maintain stability, infectivity, and viral titer during manufacturing, purification, filtration, and storage.

[0006] 3. Summary of the Invention This disclosure provides an improved sand-borne virus formulation that significantly increases the stability of sand-borne virus-based viral vectors in frozen and liquid states and greatly reduces the formation of vector-derived aggregates and subvisible particles, thereby promoting filterability and increasing virus recovery during the final filtration step. In particular, the sand-borne virus formulation disclosed in this invention advantageously keeps the viral vector dispersed compared to previously known formulations, thereby significantly reducing the significant filtration losses seen with many enveloped vectors (especially during sterile filtration). Furthermore, since particle formation is common in the field of viral vectors, minimizing particle (aggregate) formation with the formulation disclosed in this invention is considered advantageous in terms of product safety. Finally, the formulation disclosed in this invention improves the stability of the formulation when stored under refrigerated conditions (2°C–8°C) and environmental conditions (e.g., 20°C–25°C), enabling wider use and intermediate storage of arenavirus in hospitals and regions lacking cryogenic freezing equipment. Stability at -20°C can also be improved by using suitable carbohydrates disclosed herein. Furthermore, the liquid formulation disclosed in this invention can use excipients compatible with freeze-drying, which further improves storage stability at elevated temperatures.

[0007] In one aspect, this disclosure provides a liquid arenavirus formulation comprising: (a) arenavirus; and (b) a polyvalent anion at a concentration between about 5 mM and about 150 mM, optionally wherein the concentration of the polyvalent anion is between about 10 mM and about 60 mM.

[0008] In some embodiments, the concentration of the polyvalent anion is about 50 mM or about 25 mM. In some embodiments, the liquid sand virus preparation is suitable for lyophilization, and the concentration of the polyvalent anion is at most about 50 mM or between about 20 mM and about 50 mM.

[0009] In one aspect, this disclosure provides a liquid sand virus preparation comprising: (a) sand virus; and (b) a polyvalent anion, wherein the concentration of the polyvalent anion (i) reduces the formation of subvisible particles during manufacturing, and / or (ii) reduces the loss of virus titer during filtration, optionally wherein the filtration includes bioburden reduction filtration or sterilization filtration, and / or the manufacturing includes a purification process.

[0010] In some embodiments, the concentration of the polyvalent anion is between about 5 mM and about 150 mM, optionally wherein the concentration of the polyvalent anion is between about 10 mM and about 60 mM.

[0011] In some embodiments, the concentration of the polyvalent anion is about 50 mM or about 25 mM. In some embodiments, the liquid isovirus preparation is suitable for lyophilization, and the concentration of the polyvalent anion is at most about 50 mM or between about 20 mM and about 50 mM.

[0012] In some embodiments, the polyvalent anion is selected from the group consisting of citrate, tetrasodium pyrophosphate (TSPP), malate, succinate, tartrate, fumarate, maleate, and combinations thereof.

[0013] In some embodiments, the polyvalent anion is citrate. In some embodiments, the concentration of citrate is about 50 mM or about 25 mM. In some embodiments, the liquid isovirus preparation is suitable for lyophilization, and the concentration of citrate is at most about 50 mM or between about 20 mM and about 50 mM.

[0014] In some embodiments, the liquid sand-like virus preparation further comprises at least one of the following: (c) carbohydrates; (d) salts; (e) buffers; (f) surfactants; and (g) stabilizers.

[0015] In some embodiments, (c) the concentration of carbohydrates is between about 0.1% and about 20% (w / v), optionally wherein the concentration of carbohydrates is between about 3% and about 10% (w / v); (d) the concentration of salt is at most about 450 mM, optionally wherein the concentration of salt is between about 50 mM and about 250 mM; (e) the concentration of buffers is between about 1 mM and about 50 mM, optionally wherein the concentration of buffers is between about 10 mM and about 20 mM; (f) the concentration of surfactants is between about 0.001% and about 1% (w / v), optionally wherein the concentration of surfactants is between about 0.01% and about 0.05% (w / v); and / or(g) The concentration of the stabilizer is between about 0.001% (w / v) and about 3% (w / v), optionally wherein the concentration of the stabilizer is between about 0.05% (w / v) and 0.15% (w / v).

[0016] In some embodiments, (c) the concentration of carbohydrates is about 5% (w / v); (d) the concentration of salt is about 125 mM or about 75 mM; (e) the concentration of buffer is about 10 mM; (f) the concentration of surfactant is about 0.03% (w / v); and / or (g) the concentration of stabilizer is about 0.1% (w / v).

[0017] In some embodiments, the liquid sand virus formulation is suitable for lyophilization, and (c) the concentration of carbohydrates is about 5% (w / v); (d) the concentration of salt is at most about 125 mM or between about 50 mM and about 125 mM; (e) the concentration of buffer is about 10 mM; (f) the concentration of surfactant is about 0.03% (w / v); and / or (g) the concentration of stabilizer is about 0.1% (w / v).

[0018] In some embodiments, (c) the carbohydrate is a sugar or a polyol; optionally, the sugar or polyol is selected from the group consisting of sucrose, trehalose, sorbitol, mannitol, dextran, and combinations thereof; further optionally, the carbohydrate is sucrose, trehalose, or combinations thereof; (d) the salt is selected from the group consisting of sodium chloride, sodium phosphate, potassium phosphate, potassium chloride, magnesium chloride, sodium sulfate, potassium sulfate, ammonium sulfate, and combinations thereof; optionally, the salt is sodium chloride; (e) the buffer is selected from the group consisting of phosphate, citrate, TRIS, HEPES, bicarbonate, histidine, and combinations thereof; optionally, the buffer is TRIS; (f) the surfactant is selected from the group consisting of poloxamer 188, poloxamer 407, polysorbate 20, polysorbate 80, and combinations thereof; optionally, the surfactant is poloxamer 188; and / or (g) the stabilizer is selected from the group consisting of albumin, recombinant protein, plasma protein, gelatin, and combinations thereof; optionally, albumin specification 2 / 63 Page 13 CN 121866044 A is lactalbumin, human serum albumin, recombinant human serum albumin (rHSA), or a combination thereof; further optionally, wherein the stabilizer is rHSA.

[0019] In some embodiments, (c) the carbohydrate is sucrose or trehalose at a concentration of about 5% (w / v); (d) the salt is sodium chloride at a concentration of about 125 mM or about 75 mM; (e) the buffer is TRIS at a concentration of about 10 mM; (f) the surfactant is poloxamer 188 at a concentration of about 0.03% (w / v); and / or (g) the stabilizer is rHSA at a concentration of about 0.1%.

[0020] In some embodiments, the liquid sand virus preparation is suitable for lyophilization, and: (c) the carbohydrate is at a concentration ofThe following are the components of the liquid isovirus preparation: (a) trehalose at approximately 5% (w / v); (b) sodium chloride at a concentration of up to approximately 125 mM or between approximately 50 mM and approximately 125 mM; (c) a buffer at a concentration of approximately 10 mM TRIS; (d) a surfactant at a concentration of approximately 0.03% (w / v) poloxamer 188; and / or (g) a stabilizer at a concentration of approximately 0.1% rHSA.

[0021] In some embodiments, the pH of the liquid isovirus preparation is between approximately 7.0 and approximately 9.0. In some embodiments, the pH is approximately 8.0.

[0022] In some embodiments, the osmotic concentration of the liquid isovirus preparation is between approximately 200 mOsm / kg and approximately 1000 mOsm / kg. In some embodiments, the osmotic concentration of the liquid isovirus preparation is between approximately 500 mOsm / kg and approximately 550 mOsm / kg. In some embodiments, the permeation concentration of the liquid isovirus preparation is about 530 mOsm / kg. In some embodiments, the permeation concentration of the liquid isovirus preparation is between about 350 mOsm / kg and about 450 mOsm / kg. In some embodiments, the permeation concentration of the liquid isovirus preparation is about 400 mOsm / kg. In some embodiments, the liquid isovirus preparation is suitable for lyophilization and the permeation concentration is between about 300 mOsm / kg and about 600 mOsm / kg.

[0023] In some embodiments, (c) to (g) are present to reduce virus titer loss during filtration, freeze-thaw, and / or storage; and / or to reduce the concentration of subvisible particle formation during manufacturing, freeze-thaw, and / or storage.

[0024] In some embodiments, the storage temperature is above about 2°C, between about 20°C and about 25°C, between about 2°C and about 8°C, above about 15°C, about -20°C or below about -20°C, about -65°C or below about -65°C, or about -80°C.

[0025] In one aspect, this disclosure provides a liquid isovirus preparation comprising: (a) isovirus; (b) citrate at a concentration of about 50 mM; (c) sucrose at a concentration of about 5% (w / v); (d) NaCl at a concentration of about 125 mM; (e) TRIS at a concentration of about 10 mM; (f) poloxamer 188 at a concentration of about 0.03% (w / v); and (g) rHSA at a concentration of about 0.1% (w / v), wherein the pH of the liquid isovirus preparation is about 8.0. In some embodiments, the permeation concentration of the liquid sand virus preparation is about 530 mOsm / kg.

[0026] In one aspect, this disclosure provides a liquid sand virus preparation comprising: (a) sand virus; (b) citrate at a concentration of about 25 mM; (c) trehalose at a concentration of about 5% (w / v); and (d) a concentration of about...(e) 75 mM NaCl; (f) TRIS at a concentration of about 10 mM; (g) poloxamer 188 at a concentration of about 0.03% (w / v); and (g) rHSA at a concentration of about 0.1% (w / v), wherein the pH of the liquid isovirus preparation is about 8.0. In some embodiments, the permeation concentration of the liquid isovirus preparation is about 400 mOsm / kg.

[0027] In one aspect, this disclosure provides a liquid isovirus formulation suitable for freeze-drying, the liquid isovirus formulation comprising: (a) isovirus; (b) citrate at a concentration between about 20 mM and about 50 mM; (c) trehalose at a concentration of about 5% (w / v); (d) sodium chloride at a concentration of up to about 125 mM or between about 50 mM and about 125 mM; (e) TRIS at a concentration of about 10 mM; (f) poloxamer 188 at a concentration of about 0.03% (w / v); and (g) rHSA at a concentration of about 0.1% (w / v), wherein the pH of the liquid isovirus formulation is about 8.0. In some embodiments, the liquid isovirus formulation is suitable for freeze-drying and the permeation concentration is between about 300 mOsm / kg and about 600 mOsm / kg.

[0028] In some embodiments, the concentration of the isopyrvirus is between about 10² RCV-FFU / ml and about 10¹⁰ RCV-FFU / ml (see page 3 / 63 of the specification, CN 121866044 A), or between about 2 × 10⁵ RCV-FFU / ml and about 5 × 10⁸ RCV-FFU / ml.

[0029] In some embodiments, the isopyrvirus is lymphocytic choriomeningitis virus (LCMV), Piccind virus, Tamiyami virus, or Tacalib virus.

[0030] In some embodiments, the isopyrvirus is a genetically engineered isopyrvirus.

[0031] In some embodiments, the isopyrvirus expresses an antigen, optionally selected from the group consisting of tumor antigens, tumor-associated antigens, antigens of pathogens causing infectious diseases, or antigenic fragments of any of the foregoing. In some embodiments, the antigen is selected from the group consisting of tumor antigens, tumor-associated antigens, antigens of pathogens capable of causing infectious diseases, testicular cancer antigen (CTA), and antigenic fragments of any of the foregoing.

[0032] In some embodiments, the antigen includes a cytomegalovirus (CMV) antigen, optionally wherein the CMV antigen is selected from the group consisting of CMV glycoprotein gB (i.e., gB(dCt)) having a carboxyl-terminus truncated, CMV membrane protein pp65, and combinations thereof. In some embodiments, the antigen includes a human papillomavirus (HPV) antigen, optionally wherein the HPV antigen is an HPV E7 / E6 fusion protein. In some embodiments, the antigen includes a prostate cancer antigen, optionally wherein the prostate cancer antigen is a prostate cancer antigen.The cancer antigen is selected from the group consisting of PSA, PSMA, PAP, and combinations thereof. In some embodiments, the antigen includes a hepatitis B virus (HBV) antigen, optionally wherein the HBV antigen is selected from the group consisting of a truncated HBV polymerase polypeptide comprising an inactivated reverse transcriptase domain and an inactivated RNase H, a fusion protein comprising an HBV core polypeptide and an HBV small surface antigen (sAg) polypeptide, and combinations thereof. In some embodiments, the antigen includes a human immunodeficiency virus (HIV) antigen, optionally wherein the HIV antigen is selected from the group consisting of gag, pol, nef, and combinations thereof, and further optionally wherein the HIV antigen is a fusion polypeptide comprising multiple polypeptide segments of one or more HIV-1 proteins encoded by two or more HIV genes selected from gag, pol, and nef. In some embodiments, the antigen comprises at least one antigenic fragment of a mutant KRAS, optionally wherein the at least one antigenic fragment comprises mutant G13D, G12V, G12C, G12D, and G12R from the N-terminus to the C-terminus, respectively. In some embodiments, the antigen comprises two or more testicular cancer antigen (CTA) fragments (e.g., antigenic fragments or epitopes). In some embodiments, the two or more CTA fragments are selected from the group consisting of epitopes or antigenic fragments of melanoma-preferred antigens (PRAME), melanoma-associated antigen 3 (MAGE-A3), melanoma-associated antigen 4 (MAGE-A4), and melanoma-associated antigen 6 (MAGE-A6).

[0033] In some embodiments, the isanvirus is a non-replicating isanvirus, optionally wherein the isanvirus ORF has been removed or functionally inactivated such that the resulting isanvirus cannot produce further infectious progeny viral particles. In some embodiments, the isanvirus is a non-replicating isanvirus, and the ORF encoding GP has been removed and replaced with a nucleotide sequence encoding the antigen. In some embodiments, the isanvirus is a replicating trisegmented isanvirus. In some embodiments, the trisegmented isanvirus comprises an isanvirus ORF located outside the wild-type position of the ORF. In some embodiments, the trisegmented isavirus comprises an L segment and two S segments, wherein one of the two S segments is an S segment, and the ORF encoding GP is under the control of the isavirus 3' UTR. In some embodiments, the trisegmented isavirus is engineered such that the isavirus ORF is distributed on two or more mRNA transcripts.

[0034] In one aspect, this disclosure provides a lyophilized isavirus formulation prepared from the liquid isavirus formulation disclosed in this invention.

[0035] In one aspect, this disclosure provides a method for (i) reducing the formation of subvisible particles during manufacturing, and / or(ii) A method for reducing viral titer loss during filtration, the method comprising mixing sand-like virus with a solution to form a liquid sand-like virus preparation, wherein the liquid sand-like virus preparation contains a polyvalent anion at a concentration between about 5 mM and about 150 mM, optionally wherein the concentration of the polyvalent anion is between about 10 mM and about 60 mM.

[0036] In some embodiments, the concentration of the polyvalent anion is about 50 mM or about 25 mM. Specification 4 / 63 pages 15 CN 121866044 A

[0037] In some embodiments, the liquid sand-like virus preparation is suitable for lyophilization, and the concentration of the polyvalent anion is between about 20 mM and about 50 mM.

[0038] In one aspect, this disclosure provides a method for (i) reducing the formation of subvisible particles during manufacturing and / or (ii) reducing viral titer loss during filtration, the method comprising mixing a sand-like virus with a solution to form a liquid sand-like virus formulation, wherein the liquid sand-like virus formulation comprises a polyvalent anion, the concentration of which reduces the formation of subvisible particles during manufacturing and / or reduces viral titer loss during filtration.

[0039] In some embodiments, the concentration of the polyvalent anion is between about 5 mM and about 150 mM. In some embodiments, the concentration of the polyvalent anion is about 50 mM or about 25 mM.

[0040] In some embodiments, the liquid sand-like virus formulation is suitable for lyophilization, and the concentration of the polyvalent anion is between about 20 mM and about 50 mM.

[0041] In some embodiments, the polyvalent anion is selected from the group consisting of citrate, tetrasodium pyrophosphate (TSPP), malate, succinate, tartrate, fumarate, maleate, and combinations thereof.

[0042] In some embodiments, the polyvalent anion is citrate. In some embodiments, the concentration of citrate is about 50 mM or about 25 mM. In some embodiments, the concentration of citrate is between about 10 mM and about 60 mM.

[0043] In some embodiments, the liquid sand virus preparation further comprises at least one of the following: (b) carbohydrates; (c) salts; (d) buffers; (e) surfactants; and (f) stabilizers.

[0044] In some embodiments, (b) the concentration of carbohydrates is between about 0.1% and about 20% (w / v), optionally wherein the concentration of carbohydrates is between about 3% and about 10% (w / v); (c) the concentration of salt is at most about 450 mM, optionally wherein the concentration of salt is between about 50 mM and about 250 mM; (d) the concentration of buffer is between about 1 mM and about 50 mM, optionally wherein the concentration of buffer is between about 10 mM and about 20 mM; and (e) the concentration of surfactant is between about 0.001% and aboutThe concentration of the surfactant is between 1% (w / v), optionally between about 0.01% and about 0.05% (w / v); and / or (f) the concentration of the stabilizer is between about 0.001% (w / v) and about 3% (w / v), optionally between about 0.05% (w / v) and 0.15% (w / v).

[0045] In some embodiments, (b) the concentration of carbohydrate is about 5% (w / v); (c) the concentration of salt is about 125 mM or about 75 mM; (d) the concentration of buffer is about 10 mM; (e) the concentration of surfactant is about 0.03% (w / v); and / or (f) the concentration of stabilizer is about 0.1% (w / v).

[0046] In some embodiments, the liquid sand virus preparation is suitable for lyophilization, and (b) the concentration of carbohydrates is about 5% (w / v); (c) the concentration of salt is at most about 125 mM or between about 50 mM and about 125 mM; (d) the concentration of buffer is about 10 mM; (e) the concentration of surfactant is about 0.03% (w / v); and / or (f) the concentration of stabilizer is about 0.1% (w / v).

[0047] In some embodiments, (b) the carbohydrate is a sugar or a polyol; optionally, the sugar or polyol is selected from the group consisting of sucrose, trehalose, sorbitol, mannitol, dextran, and combinations thereof; further optionally, the carbohydrate is sucrose, trehalose, or combinations thereof; (c) the salt is selected from the group consisting of sodium chloride, sodium phosphate, potassium phosphate, potassium chloride, magnesium chloride, sodium sulfate, potassium sulfate, ammonium sulfate, and combinations thereof; optionally, the salt is sodium chloride; (d) the buffer is selected from the group consisting of phosphate, citrate, TRIS, HEPES, bicarbonate, histidine, and combinations thereof; optionally, the buffer is TRIS; (e) the surfactant is selected from the group consisting of poloxamer 188, poloxamer 407, polysorbate 20, polysorbate 80, and combinations thereof; optionally, the surfactant is poloxamer 188; and / or (f) the stabilizer is a protein selected from the group consisting of albumin, recombinant protein, plasma protein, gelatin, and combinations thereof; Optionally, the albumin is lactalbumin, human serum albumin, recombinant human serum albumin (rHSA), or a combination thereof; Specification 5 / 63 pages 16 CN 121866044 A Further optionally, the protein is rHSA.

[0048] In some embodiments, (b) the carbohydrate is sucrose or trehalose at a concentration of about 5% (w / v); (c) the salt is sodium chloride at a concentration of about 125 mM or about 75 mM; (d) the buffer is TRIS at a concentration of about 10 mM; (e) the surfactant is a concentrated...The concentration of poloxamer 188 is about 0.03% (w / v); and / or (f) the stabilizer is rHSA at a concentration of about 0.1%.

[0049] In some embodiments, the liquid isovirus formulation is suitable for lyophilization, and: (b) the carbohydrate is trehalose at a concentration of about 5% (w / v); (c) the salt is sodium chloride at a concentration of up to about 125 mM or between about 50 mM and about 125 mM; (d) the buffer is TRIS at a concentration of about 10 mM; (e) the surfactant is poloxamer 188 at a concentration of about 0.03% (w / v); and / or (f) the stabilizer is rHSA at a concentration of about 0.1%.

[0050] In some embodiments, the pH of the liquid isovirus formulation is between about 7.0 and about 9.0, optionally wherein the pH is about 8.0.

[0051] In some embodiments, the permeation concentration of the liquid isovirus preparation is between about 200 mOsm / kg and about 1000 mOsm / kg. In some embodiments, the permeation concentration of the liquid isovirus preparation is between about 500 mOsm / kg and about 550 mOsm / kg. In some embodiments, the permeation concentration of the liquid isovirus preparation is about 530 mOsm / kg. In some embodiments, the permeation concentration of the liquid isovirus preparation is between about 350 mOsm / kg and about 450 mOsm / kg. In some embodiments, the permeation concentration of the liquid isovirus preparation is about 400 mOsm / kg. In some embodiments, the liquid isovirus preparation is suitable for lyophilization and the permeation concentration is between about 300 mOsm / kg and about 600 mOsm / kg.

[0052] In some embodiments, (b) through (f) are present at concentrations that reduce viral titer loss during filtration, reduce subvisible particle formation, maintain the biophysical stability of the sand virus during freeze-thaw processes, and / or maintain the biophysical stability of the sand virus during storage.

[0053] In some embodiments, the storage temperature is above about 2°C, between about 20°C and about 25°C, between about 2°C and about 8°C, above about 15°C, about -20°C or below about -20°C, about -65°C or below about -65°C, or about -80°C.

[0054] In one aspect, this disclosure provides a method for (i) reducing the formation of subvisible particles during manufacturing and / or (ii) reducing virus titer loss during filtration, the method comprising mixing a sand-like virus with a solution to form a liquid sand-like virus formulation, wherein the liquid sand-like virus formulation comprises: (a) citrate at a concentration of about 50 mM; (b) sucrose at a concentration of about 5% (w / v); (c) sodium chloride at a concentration of about 125 mM; (d) TRIS at a concentration of about 10 mM; and (e) a solution of TRIS at a concentration of about 0.03%.(w / v) poloxamer 188; and (f) rHSA at a concentration of about 0.1%, wherein the pH of the liquid isovirus preparation is about 8.0. In some embodiments, the permeation concentration of the liquid isovirus preparation is about 530 mOsm / kg.

[0055] In one aspect, this disclosure provides a method for (i) reducing the formation of subvisible particles during manufacturing and / or (ii) reducing viral titer loss during filtration, the method comprising mixing a sand virus with a solution to form a liquid sand virus formulation, wherein the liquid sand virus formulation comprises: (a) citrate at a concentration of about 25 mM; (b) trehalose at a concentration of about 5% (w / v); (c) sodium chloride at a concentration of about 75 mM; (d) TRIS at a concentration of about 10 mM; (e) poloxamer 188 at a concentration of about 0.03% (w / v); and (f) rHSA at a concentration of about 0.1%, wherein the pH of the liquid sand virus formulation is about 8.0. In some embodiments, the permeation concentration of the liquid sand virus formulation is about 400 mOsm / kg.

[0056] In one aspect, this disclosure provides a method for (i) reducing the formation of subvisible particles during manufacturing and / or (ii) reducing viral titer loss during filtration, the method comprising mixing a sand-like virus with a solution to form a liquid sand-like virus formulation, wherein the liquid sand-like virus formulation is suitable for lyophilization and comprises: (a) citrate at a concentration between 10 mM and about 60 mM; (b) trehalose at a concentration of about 5% (w / v); (c) sodium chloride at a concentration of up to about 125 mM or between about 50 mM and about 125 mM; (d) TRIS at a concentration of about 10 mM; and (e) poloxamer 188 at a concentration of about 0.03% (w / v); Specification 6 / 63 pages 17 CN 121866044 A (f) a concentration of about 0.1% (w / v) of rHSA, wherein the pH of the liquid isovirus preparation is about 8.0, optionally wherein filtration includes bioburden reduction filtration or sterilization filtration, and / or the manufacturing process includes a purification process. In some embodiments, the permeation concentration of the liquid isovirus preparation suitable for lyophilization is between about 300 mOsm / kg and about 600 mOsm / kg.

[0057] In some embodiments, the concentration of isovirus is between about 102 RCV-FFU / ml and about 1010 RCV-FFU / ml, or between about 2 × 105 RCV-FFU / ml and about 5 × 108 RCV-FFU / ml.

[0058] In some embodiments, the isovirus is lymphocytic choriomeningitis virus (LCMV), Piccind virus, Tamiyami virus, or Tacalib virus.

[0059] In some embodiments, the isovirus is a genetically engineered isovirus.

[0060] In some embodiments, the isanoxic virus expresses an antigen, optionally wherein the antigen is selected from the group consisting of tumor antigens, tumor-associated antigens, antigens of pathogens causing infectious diseases, or antigenic fragments of any of the foregoing. In some embodiments, the antigen is selected from the group consisting of tumor antigens, tumor-associated antigens, antigens of pathogens capable of causing infectious diseases, testicular cancer antigen (CTA), and antigenic fragments of any of the foregoing.

[0061] In some embodiments, the antigen includes at least one cytomegalovirus (CMV) antigen, optionally wherein the CMV antigen is selected from the group consisting of gB(dCt), pp65, and combinations thereof. In some embodiments, the antigen includes at least one human papillomavirus (HPV) antigen, optionally wherein the HPV antigen is an HPV E7 / E6 fusion protein. In some embodiments, the antigen includes at least one prostate cancer antigen, optionally wherein the prostate cancer antigen is selected from the group consisting of PSA, PSMA, PAP, and combinations thereof. In some embodiments, the antigen comprises a hepatitis B virus (HBV) antigen, optionally wherein the HBV antigen is selected from the group consisting of a truncated HBV polymerase polypeptide comprising an inactivated reverse transcriptase domain and an inactivated RNase H, a fusion protein comprising an HBV core polypeptide and an HBV small surface antigen (sAg) polypeptide, and combinations thereof. In some embodiments, the antigen comprises a human immunodeficiency virus (HIV) antigen, optionally wherein the HIV antigen is selected from the group consisting of gag, pol, nef, and combinations thereof, further optionally wherein the HIV antigen is a fusion polypeptide comprising multiple polypeptide segments encoding one or more HIV-1 proteins selected from two or more HIV genes selected from gag, pol, and nef. In some embodiments, the antigen comprises at least one antigenic fragment of a mutant KRAS, optionally wherein the antigenic fragment of the mutant KRAS comprises mutant G13D, G12V, G12C, G12D, and G12R from the N-terminus to the C-terminus, respectively. In some embodiments, the antigen comprises two or more CTA fragments (e.g., antigenic fragments or epitopes). In some embodiments, the two or more CTA fragments are selected from the group consisting of epitopes or antigenic fragments of PRAME, MAGE-A3, MAGE-A4, and MAGE-A6.

[0062] In some embodiments, the isanvirus is a non-replicating isanvirus, optionally wherein the isanvirus ORF has been removed or functionally inactivated such that the resulting isanvirus cannot produce further infectious progeny viral particles. In some embodiments, the isanvirus is a non-replicating isanvirus, and the ORF encoding GP has been removed and replaced with a nucleotide sequence encoding the antigen.

[0063] In some embodiments, the isanvirus is a replicating trisegmented isanvirus.

[0064] In some embodiments, the trisegmented isavirus comprises an isavirus ORF located outside the wild-type position of the ORF.

[0065] In some embodiments, the trisegmented isavirus comprises an L segment and two S segments, wherein one of the two S segments is an S segment, wherein the ORF encoding GP is under the control of the isavirus 3' UTR.

[0066] In some embodiments, the trisegmented isavirus is engineered such that the isavirus ORF is distributed on two or more mRNA transcripts.

[0067] In some embodiments, the method further includes lyophilizing a liquid isavirus preparation. Specification 7 / 63 pages 18 CN 121866044 A

[0068] In one aspect, this disclosure provides a method for producing a lyophilized isavirus preparation, the method comprising lyophilizing the liquid isavirus preparation disclosed herein.

[0069] In one aspect, this disclosure provides a method for treating or preventing a disease, the method comprising administering the liquid isavirus preparation disclosed herein to a subject in need, optionally wherein the liquid isavirus preparation is further diluted prior to administration.

[0070] In one aspect, this disclosure provides a method of treating or preventing a disease, the method comprising administering to a subject in need a liquid isovirus preparation reconstituted from a lyophilized preparation disclosed herein, wherein the lyophilized preparation is reconstituted prior to administration.

[0071] In some embodiments, the liquid isovirus preparation is administered parenterally, optionally wherein the liquid isovirus preparation is administered intravenously, intramuscularly, intratumorally, or subcutaneously.

[0072] In some embodiments, the disease is a neoplastic disease or an infectious disease.

[0073] 3.1 Exemplary Embodiments Exemplary embodiments of this disclosure are provided in the following paragraphs.

[0074] 1. A liquid isovirus preparation comprising: (a) isovirus; and (b) a polyvalent anion at a concentration between about 5 mM and about 150 mM, optionally wherein the concentration of the polyvalent anion is between about 10 mM and about 60 mM.

[0075] 2. The liquid sand virus preparation according to embodiment 1, wherein the concentration of the polyvalent anion is about 50 mM or about 25 mM.

[0076] 3. The liquid sand virus preparation according to embodiment 1, wherein the liquid sand virus preparation is suitable for lyophilization, and the concentration of the polyvalent anion is at most about 50 mM or between about 20 mM and about 50 mM.

[0077] 4. A liquid sand virus preparation comprising: (a) sand virus; and (b) polyvalent anion, wherein the concentration of the polyvalent anion (i) reduces the formation of subvisible particles during manufacturing and / or (ii) reduces the loss of virus titer during filtration.Optionally, the filtration includes bioburden reduction filtration or sterilization filtration, and / or the manufacturing includes a purification process.

[0078] 5. The liquid isovirus preparation according to embodiment 4, wherein the concentration of the polyvalent anion is between about 5 mM and about 150 mM, optionally wherein the concentration of the polyvalent anion is between about 10 mM and about 60 mM.

[0079] 6. The liquid isovirus preparation according to embodiment 5, wherein the concentration of the polyvalent anion is about 50 mM or about 25 mM.

[0080] 7. The liquid isovirus preparation according to embodiment 5, wherein the liquid isovirus preparation is suitable for lyophilization, and the concentration of the polyvalent anion is at most about 50 mM or between about 20 mM and about 50 mM.

[0081] 8. The liquid isovirus preparation according to any one of embodiments 1 to 7, wherein the polyvalent anion is selected from the group consisting of citrate, tetrasodium pyrophosphate (TSPP), malate, succinate, tartrate, fumarate, maleate, and combinations thereof.

[0082] 9. The liquid isovirus preparation according to embodiment 8, wherein the polyvalent anion is citrate.

[0083] 10. The liquid isovirus preparation according to embodiment 9, wherein the concentration of citrate is about 50 mM or about 25 mM. Specification 8 / 63 pages 19 CN 121866044 A

[0084] 11. The liquid isovirus preparation according to embodiment 9, wherein the liquid isovirus preparation is suitable for lyophilization, and the concentration of citrate is at most about 50 mM or between about 20 mM and about 50 mM.

[0085] 12. The liquid sand virus preparation according to any one of embodiments 1 to 11, wherein the liquid sand virus preparation further comprises at least one of the following: (c) carbohydrates; (d) salts; (e) buffers; (f) surfactants; and (g) stabilizers.

[0086] 13. The liquid sand-like virus preparation according to embodiment 12, wherein: (c) the concentration of the carbohydrate is between about 0.1% and about 20% (w / v), optionally wherein the concentration of the carbohydrate is between about 3% and about 10% (w / v); (d) the concentration of the salt is at most about about 450 mM, optionally wherein the concentration of the salt is between about 50 mM and about 250 mM; (e) the concentration of the buffer is between about 1 mM and about 50 mM, optionally wherein the concentration of the buffer is between about 10 mM and about 20 mM; (f) the concentration of the surfactant is between about 0.001% and about 1% (w / v), optionally wherein ...The concentration of the surfactant is between about 0.01% and about 0.05% (w / v); and / or (g) the concentration of the stabilizer is between about 0.001% (w / v) and about 3% (w / v), optionally wherein the concentration of the stabilizer is between about 0.05% (w / v) and 0.15% (w / v).

[0087] 14. The liquid sand virus preparation according to embodiment 12 or 13, wherein (c) the concentration of the carbohydrate is about 5% (w / v); (d) the concentration of the salt is about 125 mM or about 75 mM; (e) the concentration of the buffer is about 10 mM; (f) the concentration of the surfactant is about 0.03% (w / v); and / or (g) the concentration of the stabilizer is about 0.1% (w / v).

[0088] 15. The liquid sand virus preparation according to embodiment 12 or 13, wherein the liquid sand virus preparation is suitable for lyophilization, and (c) the concentration of the carbohydrate is about 5% (w / v); (d) the concentration of the salt is at most about 125 mM or between about 50 mM and about 125 mM; (e) the concentration of the buffer is about 10 mM; (f) the concentration of the surfactant is about 0.03% (w / v); and / or (g) the concentration of the stabilizer is about 0.1% (w / v).

[0089] 16. A liquid sand virus preparation according to any one of embodiments 12 to 15, wherein: (c) the carbohydrate is a sugar or a polyol; optionally, the sugar or polyol is selected from the group consisting of sucrose, trehalose, sorbitol, mannitol, dextran, and combinations thereof; further optionally, the carbohydrate is sucrose, trehalose, or combinations thereof; (d) the salt is selected from the group consisting of sodium chloride, sodium phosphate, potassium phosphate, potassium chloride, magnesium chloride, sodium sulfate, potassium sulfate, ammonium sulfate, and combinations thereof; optionally, the salt is sodium chloride; (e) the buffer is selected from the group consisting of phosphate, citrate, TRIS, HEPES, bicarbonate, histidine, and combinations thereof; optionally, the buffer is TRIS; (f) the surfactant is selected from the group consisting of poloxamer 188, poloxamer 407, polysorbate 20, and polysorbate 80. The group consisting of, and combinations thereof; optionally, the surfactant is poloxamer 188; and / or (g) the stabilizer is selected from, the group consisting of, albumin, recombinant protein, plasma protein, gelatin, and combinations thereof; optionally, the albumin is lactalbumin, human serum albumin, recombinant human serum albumin (rHSA), or combinations thereof; further optionally, the stabilizer is rHSA.

[0090] 17. According to the embodimentThe liquid sand virus formulation of 16, wherein: (c) the carbohydrate is sucrose or trehalose at a concentration of about 5% (w / v); (d) the salt is sodium chloride at a concentration of about 125 mM or about 75 mM; (e) the buffer is TRIS at a concentration of about 10 mM; (f) the surfactant is poloxamer 188 at a concentration of about 0.03% (w / v); and / or (g) the stabilizer is rHSA at a concentration of about 0.1%.

[0091] 18. The liquid isovirus preparation according to embodiment 16, wherein the liquid isovirus preparation is suitable for lyophilization, and: (c) the carbohydrate is trehalose at a concentration of about 5% (w / v); (d) the salt is sodium chloride at a concentration of up to about 125 mM or between about 50 mM and about 125 mM; (e) the buffer is TRIS at a concentration of about 10 mM; (f) the surfactant is poloxamer 188 at a concentration of about 0.03% (w / v); and / or (g) the stabilizer is rHSA at a concentration of about 0.1%.

[0092] 19. The liquid isovirus preparation according to any one of embodiments 1 to 18, wherein the pH of the liquid isovirus preparation is between about 7.0 and about 9.0.

[0093] 20. The liquid isovirus preparation according to embodiment 19, wherein the pH is about 8.0.

[0094] 21. The liquid isovirus preparation according to any one of embodiments 1 to 20, wherein the permeation concentration of the liquid isovirus preparation is between about 200 mOsm / kg and about 1000 mOsm / kg.

[0095] 22. The liquid isovirus preparation according to embodiment 21, wherein the permeation concentration of the liquid isovirus preparation is between about 500 mOsm / kg and about 550 mOsm / kg, optionally wherein the permeation concentration of the liquid isovirus preparation is about 530 mOsm / kg.

[0096] 23. The liquid isovirus preparation according to embodiment 21, wherein the permeation concentration of the liquid isovirus preparation is between about 350 mOsm / kg and about 450 mOsm / kg, optionally wherein the permeation concentration of the liquid isovirus preparation is about 400 mOsm / kg.

[0097] 24. A liquid isovirus preparation according to any one of embodiments 21, wherein the liquid isovirus preparation is suitable for lyophilization and has an osmotic concentration between about 300 mOsm / kg and about 600 mOsm / kg.

[0098] 25. A liquid isovirus preparation according to any one of embodiments 12 to 24, wherein (c) to (g) is...Reduce virus titer loss during filtration, freeze-thaw cycles, and / or storage; and / or reduce the concentration of subvisible particle formation during manufacturing, freeze-thaw cycles, and / or storage.

[0099] 26. The liquid sand-like virus preparation according to embodiment 25, wherein the storage temperature is above about 2°C, between about 20°C and about 25°C, between about 2°C and about 8°C, above about 15°C, about -20°C or below about -20°C, about -65°C or below about -65°C, or about -80°C. Instructions for Use, Page 10 / 63, 21 CN 121866044 A

[0100] 27. A liquid isovirus preparation comprising: (a) isovirus; (b) citrate at a concentration of about 50 mM; (c) sucrose at a concentration of about 5% (w / v); (d) NaCl at a concentration of about 125 mM; (e) TRIS at a concentration of about 10 mM; and (f) poloxamer 188 at a concentration of about 0.03% (w / v); and (g) rHSA at a concentration of about 0.1% (w / v), wherein the pH of the liquid isovirus preparation is about 8.0, and optionally wherein the osmotic concentration of the liquid isovirus preparation is about 530 mOsm / kg.

[0101] 28. A liquid isovirus preparation comprising: (a) isovirus; (b) citrate at a concentration of about 25 mM; (c) trehalose at a concentration of about 5% (w / v); (d) NaCl at a concentration of about 75 mM; (e) TRIS at a concentration of about 10 mM; and (f) poloxamer 188 at a concentration of about 0.03% (w / v); and (g) rHSA at a concentration of about 0.1% (w / v), wherein the pH of the liquid isovirus preparation is about 8.0, and optionally wherein the osmotic concentration of the liquid isovirus preparation is about 400 mOsm / kg.

[0102] 29. A liquid isovirus preparation suitable for freeze-drying, the liquid isovirus preparation comprising: (a) isovirus; (b) citrate at a concentration between about 20 mM and about 50 mM; (c) trehalose at a concentration of about 5% (w / v); (d) sodium chloride at a concentration of up to about 125 mM or between about 50 mM and about 125 mM; (e) TRIS at a concentration of about 10 mM; (f) poloxamer 188 at a concentration of about 0.03% (w / v); and (g) rHSA at a concentration of about 0.1% (w / v), wherein the pH of the liquid isovirus preparation is about 8.0, optionally wherein the osmotic concentration of the liquid isovirus preparation suitable for freeze-drying is between about 300 mOsm / kg and about 600 mOsm / kg.

[0103] 30. According to implementation schemes 1 to 30...The liquid isovirus preparation according to any one of embodiments 29, wherein the concentration of the isovirus is between about 102 RCV-FFU / ml and about 1010 RCV-FFU / ml, or between about 2 × 105 RCV-FFU / ml and about 5 × 108 RCV-FFU / ml.

[0104] 31. The liquid isovirus preparation according to any one of embodiments 1 to 30, wherein the isovirus is lymphocytic choriomeningitis virus (LCMV), Piccind virus, Tamiyami virus, or Tacalib virus.

[0105] 32. The liquid isovirus preparation according to any one of embodiments 1 to 31, wherein the isovirus is a genetically engineered isovirus.

[0106] 33. A liquid isovirus preparation according to any one of embodiments 1 to 32, wherein the isovirus expresses an antigen, optionally wherein the antigen is selected from the group consisting of tumor antigens, tumor-associated antigens, antigens of pathogens capable of causing infectious diseases (as described in the specification, page 11 / 63, 22 CN 121866044 A), testicular cancer antigen (CTA), and antigenic fragments of any of the foregoing.

[0107] 34. A liquid isovirus preparation according to embodiment 33, wherein the antigen comprises cytomegalovirus (CMV) antigen, optionally wherein the CMV antigen is selected from the group consisting of gB(dCt), pp65, and combinations thereof.

[0108] 35. A liquid isovirus preparation according to embodiment 33, wherein the antigen comprises human papillomavirus (HPV) antigen, optionally wherein the HPV antigen is an HPV E7 / E6 fusion protein.

[0109] 36. The liquid isovirus preparation according to embodiment 33, wherein the antigen comprises a prostate cancer antigen, optionally wherein the prostate cancer antigen is selected from the group consisting of PSA, PSMA, PAP, and combinations thereof.

[0110] 37. The liquid isovirus preparation according to embodiment 33, wherein the antigen comprises a hepatitis B virus (HBV) antigen, optionally wherein the HBV antigen is selected from the group consisting of a truncated HBV polymerase polypeptide comprising an inactivated reverse transcriptase domain and an inactivated RNase H, a fusion protein comprising an HBV core polypeptide and an HBV small surface antigen (sAg) polypeptide, and combinations thereof.

[0111] 38. The liquid isovirus preparation according to embodiment 33, wherein the antigen comprises a human immunodeficiency virus (HIV) antigen, optionally wherein the HIV antigen is selected from the group consisting of gag, pol, nef, and combinations thereof, further optionally wherein the HIV antigen comprises a protein encoded by two or more HIV genes selected from gag, pol, and nef.A fusion polypeptide of multiple polypeptide segments of one or more HIV-1 proteins.

[0112] 39. The liquid isovirus formulation according to embodiment 33, wherein the antigen comprises at least one antigenic fragment of mutant KRAS, optionally wherein the at least one antigenic fragment comprises mutant G13D, G12V, G12C, G12D and G12R from the N-terminus to the C-terminus, respectively.

[0113] 40. The liquid isovirus formulation according to embodiment 33, wherein the antigen comprises two or more testicular cancer antigen (CTA) fragments, optionally wherein the two or more CTA fragments are selected from the group consisting of epitopes or antigenic fragments of melanoma-preferred antigen (PRAME), melanoma-associated antigen 3 (MAGE-A3), melanoma-associated antigen 4 (MAGE-A4) and melanoma-associated antigen 6 (MAGE-A6).

[0114] 41. A liquid isovirus preparation according to any one of embodiments 1 to 40, wherein the isovirus is a non-replicating isovirus, optionally wherein the isovirus ORF has been removed or functionally inactivated such that the resulting isovirus cannot produce further infectious progeny viral particles, and further optionally wherein the ORF encoding the GP has been removed and replaced with a nucleotide sequence encoding an antigen.

[0115] 42. A liquid isovirus preparation according to any one of embodiments 1 to 40, wherein the isovirus is a replicating trisegmented isovirus.

[0116] 43. A liquid isovirus preparation according to embodiment 42, wherein the trisegmented isovirus comprises an isovirus ORF located outside the wild-type position of the ORF.

[0117] 44. The liquid isovirus formulation according to embodiment 43, wherein the three-segment isovirus comprises an L segment and two S segments, wherein one of the two S segments is an S segment, wherein the ORF encoding the isovirus glycoprotein (GP) is under the control of the isovirus 3' UTR.

[0118] 45. The liquid isovirus formulation according to embodiment 42, wherein the three-segment isovirus is engineered such that the isovirus ORF is distributed on two or more mRNA transcripts.

[0119] 46. A lyophilized isovirus formulation prepared from the liquid isovirus formulation of any one of embodiments 1 to 45.

[0120] 47. A method for (i) reducing the formation of subvisible particles during manufacturing and / or (ii) reducing viral titer loss during filtration, the method comprising mixing sand-like virus with a solution to form a liquid sand-like virus formulation, wherein the specification is on pages 12 / 63 of CN 121866044 A.The liquid sand virus preparation comprises a polyvalent anion at a concentration between about 5 mM and about 150 mM, optionally wherein the concentration of the polyvalent anion is between about 10 mM and about 60 mM.

[0121] 48. The method according to embodiment 47, wherein the concentration of the polyvalent anion is about 50 mM or about 25 mM.

[0122] 49. The method according to embodiment 47, wherein the liquid sand virus preparation is suitable for lyophilization, and the concentration of the polyvalent anion is between about 20 mM and about 50 mM.

[0123] 50. A method for (i) reducing the formation of subvisible particles during manufacturing and / or (ii) reducing virus titer loss during filtration, the method comprising mixing a sand virus with a solution to form a liquid sand virus preparation, wherein the liquid sand virus preparation comprises a polyvalent anion, the concentration of which reduces the formation of subvisible particles during manufacturing and / or reduces virus titer loss during filtration.

[0124] 51. The method according to embodiment 50, wherein the concentration of the polyvalent anion is between about 5 mM and about 150 mM.

[0125] 52. The method according to embodiment 51, wherein the concentration of the polyvalent anion is about 50 mM or about 25 mM.

[0126] 53. The method according to embodiment 51, wherein the liquid sand virus preparation is suitable for lyophilization, and the concentration of the polyvalent anion is between about 20 mM and about 50 mM.

[0127] 54. The method according to any one of embodiments 47 to 53, wherein the polyvalent anion is selected from the group consisting of citrate, tetrasodium pyrophosphate (TSPP), malate, succinate, tartrate, fumarate, maleate, and combinations thereof.

[0128] 55. The method according to embodiment 54, wherein the polyvalent anion is citrate.

[0129] 56. The method according to embodiment 55, wherein the concentration of citrate is about 50 mM or about 25 mM.

[0130] 57. The method according to embodiment 55, wherein the concentration of citrate is between about 10 mM and about 60 mM.

[0131] 58. The method according to any one of embodiments 47 to 57, wherein the liquid sand virus preparation further comprises at least one of: (b) carbohydrates; (c) salts; (d) buffers; (e) surfactants; and (f) stabilizers.

[0132] 59. The method according to embodiment 58, wherein: (b) the concentration of the carbohydrates is between about 0.1% and about 20% (w / v), optionally wherein the carbohydrates(c) The concentration of the compound is between about 3% and about 10% (w / v); (d) The concentration of the salt is at most about 450 mM, optionally the concentration of the salt is between about 50 mM and about 250 mM; (e) The concentration of the buffer is between about 1 mM and about 50 mM, optionally the concentration of the buffer is between about 10 mM and about 20 mM; (f) The concentration of the surfactant is between about 0.001% and about 1% (w / v), optionally the concentration of the surfactant is between about 0.01% and about 0.05% (w / v); and / or the specification 13 / 63 pages 24 CN 121866044 A (f) The concentration of the stabilizer is between about 0.001% (w / v) and about 3% (w / v), optionally the concentration of the stabilizer is between about 0.05% (w / v) and 0.15% (w / v).

[0133] 60. The method according to embodiment 58 or 59, wherein (b) the concentration of the carbohydrate is about 5% (w / v); (c) the concentration of the salt is about 125 mM or about 75 mM; (d) the concentration of the buffer is about 10 mM; (e) the concentration of the surfactant is about 0.03% (w / v); and / or (f) the concentration of the stabilizer is about 0.1% (w / v).

[0134] 61. The method according to embodiment 58 or 59, wherein the liquid sand virus preparation is suitable for lyophilization, and (b) the concentration of the carbohydrate is about 5% (w / v); (c) the concentration of the salt is at most about 125 mM or between about 50 mM and about 125 mM; (d) the concentration of the buffer is about 10 mM; (e) the concentration of the surfactant is about 0.03% (w / v); and / or (f) the concentration of the stabilizer is about 0.1% (w / v).

[0135] 62. The method according to any one of embodiments 58 to 61, wherein: (b) the carbohydrate is a sugar or a polyol; optionally, the sugar or polyol is selected from the group consisting of sucrose, trehalose, sorbitol, mannitol, dextran, and combinations thereof; further optionally, the carbohydrate is sucrose, trehalose, or combinations thereof; (c) the salt is selected from the group consisting of sodium chloride, sodium phosphate, potassium phosphate, potassium chloride, magnesium chloride, sodium sulfate, potassium sulfate, ammonium sulfate, and combinations thereof; optionally, the salt is sodium chloride; (d) the buffer is selected from the group consisting of phosphate, citrate, TRIS, HEPES, bicarbonate, histidine, and combinations thereof; optionally, the buffer is TRIS; (e) the surfactant is selected from the group consisting of poloxamer 188, poloxamer 407, polysorbate 20, and polysorbate 80.The group consisting of, and combinations thereof; optionally, the surfactant is poloxamer 188; and / or (f) the stabilizer is a protein selected from the group consisting of albumin, recombinant protein, plasma protein, gelatin, and combinations thereof; optionally, the albumin is lactalbumin, human serum albumin, recombinant human serum albumin (rHSA), or combinations thereof; further optionally, the protein is rHSA.

[0136] 63. The method according to embodiment 62, wherein: (b) the carbohydrate is sucrose or trehalose at a concentration of about 5% (w / v); (c) the salt is sodium chloride at a concentration of about 125 mM or about 75 mM; (d) the buffer is TRIS at a concentration of about 10 mM; (e) the surfactant is poloxamer 188 at a concentration of about 0.03% (w / v); and / or (f) the stabilizer is rHSA at a concentration of about 0.1%.

[0137] 64. The method according to embodiment 62, wherein the liquid sand virus preparation is suitable for lyophilization, and: (b) the carbohydrate is trehalose at a concentration of about 5% (w / v); (c) the salt is sodium chloride at a concentration of up to about 125 mM or between about 50 mM and about 125 mM; (d) the buffer is TRIS at a concentration of about 10 mM; (e) the surfactant is poloxamer 188 at a concentration of about 0.03% (w / v); and / or (f) the stabilizer is rHSA at a concentration of about 0.1%.

[0138] 65. The method according to any one of embodiments 47 to 64, wherein the pH of the liquid sand virus preparation is between about 7.0 and about 9.0, optionally wherein the pH is about 8.0.

[0139] 66. The method according to any one of embodiments 47 to 65, wherein the permeation concentration of the liquid isovirus preparation is between about 200 mOsm / kg and about 1000 mOsm / kg.

[0140] 67. The method according to embodiment 66, wherein the permeation concentration of the liquid isovirus preparation is between about 500 mOsm / kg and about 550 mOsm / kg, optionally wherein the permeation concentration of the liquid isovirus preparation is about 530 mOsm / kg.

[0141] 68. The method according to embodiment 66, wherein the permeation concentration of the liquid isovirus preparation is between about 350 mOsm / kg and about 450 mOsm / kg, optionally wherein the permeation concentration of the liquid isovirus preparation is about 400 mOsm / kg.

[0142] 69. According to embodiment...The method of embodiment 66, wherein the liquid sand virus preparation is suitable for lyophilization and the permeation concentration is between about 300 mOsm / kg and about 600 mOsm / kg.

[0143] 70. The method of any one of embodiments 58 to 69, wherein (b) to (f) are present at concentrations that reduce virus titer loss during filtration, reduce subvisible particle formation, maintain the biophysical stability of the sand virus during freeze-thaw processes, and / or maintain the biophysical stability of the sand virus during storage.

[0144] 71. The method of embodiment 70, wherein the storage temperature is above about 2°C, between about 20°C and about 25°C, between about 2°C and about 8°C, above about 15°C, about -20°C or below about -20°C, about -65°C or below about -65°C, or about -80°C.

[0145] 72. A method for (i) reducing the formation of subvisible particles during manufacturing and / or (ii) reducing virus titer loss during filtration, the method comprising mixing a sand virus with a solution to form a liquid sand virus formulation, wherein the liquid sand virus formulation comprises: (a) citrate at a concentration of about 50 mM; (b) sucrose at a concentration of about 5% (w / v); (c) sodium chloride at a concentration of about 125 mM; (d) TRIS at a concentration of about 10 mM; (e) poloxamer 188 at a concentration of about 0.03% (w / v); and (f) rHSA at a concentration of about 0.1%, wherein the pH of the liquid sand virus formulation is about 8.0, and optionally wherein the permeation concentration of the liquid sand virus formulation is about 530 mOsm / kg.

[0146] 73. A method for (i) reducing the formation of subvisible particles during manufacturing and / or (ii) reducing virus titer loss during filtration, the method comprising mixing a sand virus with a solution to form a liquid sand virus formulation, wherein the liquid sand virus formulation comprises: (a) citrate at a concentration of about 25 mM; (b) trehalose at a concentration of about 5% (w / v); (c) sodium chloride at a concentration of about 75 mM; (d) TRIS at a concentration of about 10 mM; (e) poloxamer 188 at a concentration of about 0.03% (w / v); and (f) rHSA at a concentration of about 0.1%, wherein the pH of the liquid sand virus formulation is about 8.0, and optionally wherein the permeation concentration of the liquid sand virus formulation is about 400 mOsm / kg.

[0147] 74. A method for (i) reducing the formation of subvisible particles during manufacturing and / or (ii) reducing viral titer loss during filtration, the method comprising mixing sand-like virus with a solution to form a liquid sand-like virus formulation, whereinThe liquid isovirus preparation is suitable for lyophilization and comprises: (a) citrate at a concentration between 10 mM and about 60 mM; (b) trehalose at a concentration of about 5% (w / v); (c) sodium chloride at a concentration of up to about 125 mM or between about 50 mM and about 125 mM; (d) TRIS at a concentration of about 10 mM; (e) poloxamer 188 at a concentration of about 0.03% (w / v); and (f) rHSA at a concentration of about 0.1% (w / v), wherein the pH of the liquid isovirus preparation is about 8.0, optionally wherein the filtration comprises bioburden reduction filtration or sterilization filtration, and / or the manufacturing comprises a purification process, optionally wherein the permeate concentration of the liquid isovirus preparation suitable for lyophilization is between about 300 mOsm / kg and about 600 mOsm / kg.

[0148] 75. The method according to any one of embodiments 47 to 74, wherein the concentration of the isovirus is between about 10² RCV-FFU / ml and about 10¹⁰ RCV-FFU / ml, or between about 2 × 10⁵ RCV-FFU / ml and about 5 × 10⁸ RCV-FFU / ml.

[0149] 76. The method according to any one of embodiments 47 to 75, wherein the isovirus is lymphocytic choriomeningitis virus (LCMV), Piccind virus, Tamiyami virus, or Tacalib virus.

[0150] 77. The method according to any one of embodiments 47 to 76, wherein the isovirus is a genetically engineered isovirus.

[0151] 78. The method according to any one of embodiments 47 to 77, wherein the isopyrvirus expresses an antigen, optionally wherein the antigen is selected from the group consisting of tumor antigens, tumor-associated antigens, antigens of pathogens capable of causing infectious diseases, testicular cancer antigen (CTA), and antigenic fragments of any of the foregoing.

[0152] 79. The method according to embodiment 78, wherein the antigen comprises at least one cytomegalovirus (CMV) antigen, optionally wherein the CMV antigen is selected from the group consisting of gB(dCt), pp65, and combinations thereof.

[0153] 80. The method according to embodiment 78, wherein the antigen comprises at least one human papillomavirus (HPV) antigen, optionally wherein the HPV antigen is an HPV E7 / E6 fusion protein.

[0154] 81. The method according to embodiment 78, wherein the antigen comprises at least one prostate cancer antigen, optionally wherein the prostate cancer antigen is selected from the group consisting of PSA, PSMA, PAP, and combinations thereof.

[0155] 82. According to embodiment...78. The method thereof, wherein the antigen comprises hepatitis B virus (HBV) antigen, optionally wherein the HBV antigen is selected from the group consisting of a truncated HBV polymerase polypeptide comprising an inactivated reverse transcriptase domain and an inactivated RNase H, a fusion protein comprising an HBV core polypeptide and an HBV small surface antigen (sAg) polypeptide, and combinations thereof.

[0156] 83. The method thereof according to embodiment 78, wherein the antigen comprises human immunodeficiency virus (HIV) antigen, optionally wherein the HIV antigen is selected from the group consisting of gag, pol, nef, and combinations thereof, further optionally wherein the HIV antigen is a fusion polypeptide comprising a plurality of polypeptide segments encoded by two or more HIV genes selected from gag, pol, and nef.

[0157] 84. The method according to embodiment 78, wherein the antigen comprises at least one antigenic fragment of a mutant KRAS, optionally wherein the antigenic fragment of the mutant KRAS comprises mutant G13D, G12V, G12C, G12D and G12R from the N-terminus to the C-terminus, respectively.

[0158] 85. The method according to embodiment 78, wherein the antigen comprises two or more testicular cancer antigen (CTA) fragments, optionally wherein the two or more CTA fragments are selected from the group consisting of epitopes or antigenic fragments of melanoma-preferred antigens (PRAME), melanoma-associated antigen 3 (MAGE-A3), melanoma-associated antigen 4 (MAGE-A4) and melanoma-associated antigen 6 (MAGE-A6).

[0159] 86. The method according to any one of embodiments 47 to 85, wherein the isovirus is a non-replicating isovirus, optionally wherein the isovirus ORF has been removed or functionally inactivated such that the resulting isovirus cannot produce further infectious progeny viral particles, further optionally wherein the ORF encoding the GP has been removed and replaced with a nucleotide sequence encoding an antigen.

[0160] 87. The method according to any one of embodiments 47 to 85, wherein the isovirus is a replicating trisegmented isovirus.

[0161] 88. The method according to embodiment 87, wherein the trisegmented isovirus comprises an isovirus ORF located outside the wild-type position of the ORF.

[0162] 89. The method according to embodiment 88, wherein the trisegmented isovirus comprises an L segment and two S segments, wherein one of the two S segments is an S segment, wherein the ORF encoding the GP is under the control of the isovirus 3' UTR.

[0163] 90. According to the implementation plan87. The method wherein the three-segmented isavirus is engineered such that the isavirus ORF is distributed on two or more mRNA transcripts.

[0164] 91. The method according to any one of embodiments 47 to 90, the method further comprising lyophilizing the liquid isavirus preparation.

[0165] 92. A method for producing a lyophilized isavirus preparation, the method comprising lyophilizing the liquid isavirus preparation according to any one of embodiments 1 to 45.

[0166] 93. A method for treating or preventing a disease, the method comprising administering to a subject in need the liquid isavirus preparation according to any one of embodiments 1 to 45, optionally wherein the liquid isavirus preparation is further diluted prior to administration.

[0167] 94. A method for treating or preventing a disease, the method comprising administering to a subject in need a liquid isavirus preparation reconstituted from the lyophilized preparation of embodiment 46, wherein the lyophilized preparation is reconstituted prior to administration.

[0168] 95. The method according to embodiment 93 or 94, wherein the liquid isovirus preparation is administered parenterally, optionally wherein the liquid isovirus preparation is administered intravenously, intramuscularly, intratumorally, or subcutaneously.

[0169] 96. The method according to embodiments 93 to 95, wherein the disease is a neoplastic disease or an infectious disease.

[0170] 4. Brief Description of the Drawings Figure 1 depicts the effect of polyvalent anions on the 0.22µm sterile filtration recovery rate of virus particles from an isovirus preparation having a TRIS-based formulation (calculated as two-fold multiple linear regression (MLR) coefficients). Error bars represent 95% confidence levels. TSP = TSPP; Cit = citrate; Mal = malate; Suc = succinate; Tar = tartrate. Evaluation was performed using MODDE Pro software on pages 17 / 63 of the specification, and the data were fitted to a multiple linear regression (MLR) model.

[0171] Figure 2 depicts the relationship between the virus particle recovery rate [%] before and after sterilization filtration, as determined by FFU and RCV-FFU, and the concentrations of sodium chloride (NaCl) and citrate in the sand virus formulation. Evaluation was performed using MODDE Pro software, and data were fitted to a multiple linear regression (MLR) model.

[0172] Figures 3A and 3B depict the filtration recovery rate after filtration with reduced virus titer and bioburden, determined during the manufacturing process, for sand virus formulation batches treated with a HEPES-based formulation (10 mM HEPES, 150 mM NaCl, 20 mM glycine, 1% rHSA; pH 7.4). Virus titer was analyzed by quantitative RCV-FFU (Figure 3A) and FFU (Figure 3B). HEPESI-III correspond to different 20L laboratory-scale arenavirus production batches. Processing steps (PS) 1: Harvesting; PS2: Nuclease treatment; PS3: Chromatography 1; PS4: Chromatography 2; PS5: Final formulation; PS6: Bioburden reduction filtration.

[0173] Figures 4A and 4B depict the virus titers and filtration recoveries after bioburden reduction filtration (PS6) determined during the manufacturing process for arenavirus formulation batches treated with a TRIS-based formulation (10mM Tris, 50mM citrate, 125mM NaCl, 5% sucrose, 0.03% poloxamer 188, 0.1% rHSA; pH 8.0). Virus titers were analyzed by quantitative RCV-FFU (Figure 4A) and FFU (Figure 4B). TRIS / citrate I-V correspond to different 20L laboratory-scale arenavirus production batches. PS1: Harvest; PS2: Nuclease treatment; PS3: Chromatography 1; PS4: Chromatography 2; PS5: Final formulation; PS6: Bioburden reduction filtration.

[0174] Figure 5 depicts the subvisible particle concentration of the arenavirus formulation prepared in TRIS buffer formulations containing different concentrations (25 mM (first column) and 50 mM (second column)) of polyanions (TSPP, succinate, tartrate, and malate), as measured by flow imaging microscopy (FIM).

[0175] Figure 6 depicts the subvisible particle concentration of the arenavirus formulation in arenavirus formulations containing different concentrations of NaCl, citrate, and poloxamer 188, as measured by FIM. Evaluation was performed using MODDE Pro software, and the data were fitted to an MLR model.

[0176] Figures 7A to 7C depict the subvisible particle concentrations of total subvisible particles (i.e., particles ≥ 1 µm) (Figure 7A), particles ≥ 10 µm (Figure 7B), and particles ≥ 25 µm (Figure 7C) determined during the manufacturing process and bioburden reduction filtration (PS6) of arenavirus preparations prepared in either a TRIS-based formulation (10 mM Tris, 50 mM citrate, 125 mM NaCl, 5% (w / v) sucrose, 0.03% poloxamer 188, 0.1% rHSA; pH 8.0) or a HEPES-based formulation (10 mM HEPES, 150 mM NaCl, 20 mM glycine, 1% rHSA; pH 7.4). HEPES I–III correspond to different HEPES-based arenavirus purification batches; citrate I–V correspond to different TRIS-based arenavirus purification batches. For each intermediate obtained after PS3-PS6 in the figure, the bar chart from left to right represents HEPES I, HEPES II, HEPES III, citrate I, citrate II, citrate III, citrate IV, and citrate V. All samples underwent a [missing information - likely a specific process or process] before subvisible particle measurement.Three freeze-thaw cycles. PS3: Chromatography 1; PS4: Chromatography 2; PS5: Final formulation; PS6: Bioburden reduction filtration.

[0177] Figure 8 depicts the virus titers of sand virus formulations prepared in a TRIS-based formulation (10 mM Tris, 125 mM NaCl, 0.03% poloxamer 188, 0.1% rHSA; pH 8.0) containing different polyanions (20 mM TSPP; 50 mM succinate; or 50 mM citrate) and different carbohydrates (sucrose, trehalose, or sorbitol, each at 5% (w / v)). Virus titers were measured by RCV-FFU assay before freezing (without F / T), after three (3) freeze-thaw cycles (3 × F / T), and after subsequent sterile filtration. As a reference, virus titers of a sand-like virus formulation lacking polyanions and carbohydrates, prepared in a HEPES-based formulation (10 mM HEPES, 20 mM glycine, 150 mM NaCl, 1% rHSA; pH 7.4), were also measured before freezing the reference formulation (without F / T), after three (3) freeze-thaw cycles (3 × F / T), and after subsequent sterile filtration. For each test combination, the bar charts from left to right represent no F / T, 3 × F / T, and sterile filtration.

[0178] Figure 9 depicts the virus titers of sand virus formulations with different concentrations of sucrose (0% – 10% (w / v)) prepared in a TRIS-based formulation (10 mM Tris, 50 mM citrate, 125 mM NaCl, 0.03% polosulfate, 18 / 63 pages, CN 121866044 A, Sham 188, 0.1% rHSA; pH 8.0). The virus titers were measured by RCV-FFU assay before and after five (5) freeze-thaw cycles, i.e., before sterile filtration (RCV-FFU before 0.22 µm filtration), after sterile filtration (RCV-FFU after 0.22 µm filtration), and after five freeze-thaw cycles (5 × F / T). For each test sucrose concentration, the bar chart from left to right shows RCV-FFU before 0.22µm filtration, RCV-FFU after 0.22µm filtration, and 5×F / T.

[0179] Figure 10 depicts the virus titer recovery of sand virus formulations with different sucrose concentrations (0% – 10% (w / v)) prepared in a TRIS-based formulation (10mM Tris, 50mM citrate, 125mM NaCl, 0.03% poloxamer 188, 0.1% rHSA; pH 8.0) after five (5) freeze-thaw cycles and filtration (0.22µm).

[0180] Figure 11 depicts the virus titer recovery of sand virus formulations with different sucrose concentrations (0% – 10% (w / v)) prepared in a TRIS-based formulation (10mM Tris, 50mM citrate, 125mM NaCl, 0.03% poloxamer 188, 0.1% rHSA; pH 8.0) after five (5) freeze-thaw cycles and filtration (0.22µm).Subvisible particle concentrations of ≥1µm, ≥10µm, and ≥25µm particles in arenavirus formulations prepared in NaCl, 5% sucrose, 0.03% poloxamer 188, and 0.1% rHSA (pH 8.0). Particle concentrations in the formulations were measured by FIM before three (3) consecutive freeze-thaw cycles (0×FT) and after each cycle (1×FT, 2×FT, and 3×FT). For each test F / T condition, the bars are from left to right for particles ≥1µm, particles ≥10µm, and particles ≥25µm.

[0181] Figure 12 depicts the subvisible particle concentrations (≥1µm) in arenavirus formulations prepared in 10mM Tris, 20mM TSPP, 250mM NaCl, and 0.03% poloxamer 188 (pH 8.0), with and without 5% sucrose. The particle concentration in the formulation was measured by FIM after each of five (5) consecutive freeze-thaw cycles.

[0182] Figure 13 depicts the virus titers of sand virus formulations with different rHSA concentrations (0% – 1% (w / v)) prepared in a TRIS-based formulation (10 mM Tris, 20 mM TSPP, 125 mM NaCl, 5% sucrose, 0.015% poloxamer 188; pH 8.0) after the formulations were stored in 15 mL polypropylene (PP) tubes at 2 °C – 8 °C for five weeks. The virus titers were analyzed by quantitative RCV-FFU.

[0183] Figures 14A to 14B depict the viral titers of a sand virus formulation prepared in a TRIS-based formulation (10 mM Tris, 50 mM citrate, 125 mM NaCl, 0.03% (w / v) poloxamer 188, 0.1% (w / v) rHSA; pH 8.0), without carbohydrate additives (sugar-free), and containing 5% sucrose, 5% trehalose, or 5% sorbitol, before and after storage at -20°C for one (1) to eighteen (18) months. Viral titers were analyzed by quantitative RCV-FFU (Figure 14A) and FFU (Figure 14B).

[0184] Figure 15 depicts the subvisible particle concentration (≥1 µm) of a sand virus formulation prepared in a carbohydrate additive-free (sugar-free) formulation containing 5% sucrose, 5% trehalose, or 5% sorbitol before and after storage at -20°C for one (1) to eighteen (18) months. Subvisible particle concentration in the formulation was analyzed by FIM before freezing and after 1, 2, 3, and 6 months of storage. For each storage duration, the bar charts from left to right represent sugar-free, trehalose, sorbitol, and sucrose conditions.

[0185] Figure 16 depicts the virus particle diameter of a sand-like virus formulation with 5% sucrose, 5% trehalose, or 5% sorbitol without carbohydrate additives (sugar-free) before and after storage at -20°C for one (1) to three (3) months.After one, two, and three months, the diameter of the virus particles was analyzed by nanoparticle tracking analysis (NTA). For each storage duration, the bar charts from left to right show the sugar-free, trehalose, sorbitol, and sucrose conditions.

[0186] Figures 17A and 17B depict the diameter of sand virus particles after storage at -20°C for three (3) months for sand virus formulations without carbohydrate additives (Figure 17A) or supplemented with 5% trehalose (Figure 17B).

[0187] 5. Detailed Description This disclosure provides improved sand virus formulations (e.g., liquid formulations and lyophilized formulations). In some embodiments, the sand virus formulation can maintain biophysical stability and viral titer during the manufacture, purification, and storage of the formulation containing sand virus. In particular, the sand-particle virus formulations disclosed herein can minimize virus titer loss during purification processes (e.g., sterile filtration), minimize the formation of aggregates and sub-visible particles, maintain the biophysical stability of virus particles during freeze-thaw processes, and / or maintain the biophysical stability of virus particles during storage.

[0188] This disclosure is partly based on the finding that certain sand-particle virus formulations significantly increase the stability of sand-particle-based viral vectors in frozen and liquid states and greatly reduce the formation of vector-derived aggregates and sub-visible particles, thereby promoting filterability and increasing virus recovery during the final filtration step. Compared to previously known formulations, the sand-particle virus formulations disclosed herein advantageously keep the viral vector dispersed, thereby significantly reducing the significant filtration losses seen in many enveloped vectors (especially during sterile filtration). Furthermore, since particle (aggregate) formation is common in the field of viral vectors, minimizing particle formation with the formulations disclosed herein is considered to have an advantage in terms of product safety. Finally, the formulation disclosed in this invention improves the stability of the formulation when stored under refrigerated conditions (2°C–8°C) and environmental conditions (e.g., 20°C–25°C), enabling wider use and intermediate storage of arenavirus in hospitals and regions lacking cryogenic freezing equipment. Stability at -20°C can also be improved by using suitable carbohydrates disclosed herein. Furthermore, the liquid formulation disclosed in this invention can use excipients compatible with freeze-drying, which further improves storage stability at elevated temperatures.

[0189] In some embodiments, the arenavirus formulation disclosed herein is a liquid arenavirus formulation (see Section 5.1). In some embodiments, the liquid arenavirus formulation comprises arenavirus (see Section 5.3) and a polyvalent anion (e.g., citrate) (see Section 5.1.1). In some embodiments, the liquid arenavirus formulation also comprises carbohydrates (e.g., sucrose) (see Section 5.1).The formulation contains at least one of the following: a salt (e.g., sodium chloride) (see Section 5.1.3), a buffer (e.g., TRIS) (see Section 5.1.4), a surfactant (e.g., poloxamer 188) (see Section 5.1.5), and a stabilizer (e.g., rHSA) (see Section 5.1.6). In some embodiments, the liquid isovirus formulation is suitable for lyophilization.

[0190] In some embodiments, the isovirus formulation disclosed herein is a lyophilized isovirus formulation (see Section 5.2). In some embodiments, the lyophilized isovirus formulation comprises isovirus (see Section 5.3) and a polyvalent anion (e.g., citrate) (see Section 5.1.1). In some embodiments, the lyophilized arenavirus formulation further comprises at least one of a carbohydrate (e.g., trehalose) (see Section 5.1.2), a salt (e.g., sodium chloride) (see Section 5.1.3), a buffer (e.g., TRIS) (see Section 5.1.4), a surfactant (e.g., poloxamer 188) (see Section 5.1.5), and a stabilizer (e.g., rHSA) (see Section 5.1.6). In some embodiments, the lyophilized arenavirus formulation is prepared from the liquid arenavirus formulation disclosed herein (see Section 5.1).

[0191] This disclosure also provides pharmaceutical compositions (see Section 5.4) comprising either the liquid arenavirus formulation disclosed herein (see Section 5.1) or the lyophilized arenavirus formulation (see Section 5.2).

[0192] This disclosure also provides a method for reducing subvisible particle formation and / or reducing viral titer loss by mixing sand-like viruses with a solution to form the liquid sand-like virus formulation disclosed herein (see Section 5.5).

[0193] This disclosure also provides a method for treating or preventing disease (see Section 5.6), the method comprising administering to a subject in need the liquid sand-like virus formulation disclosed herein (see Section 5.1) or the lyophilized sand-like virus formulation disclosed herein (see Section 5.2).

[0194] The term “about” means a variation within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of a given value or range.

[0195] For purposes of clarity rather than limitation, the specific embodiments are divided into the following subsections: 5.1 Liquid sand virus preparation; 5.2 Lyophilized sand virus preparation; 5.3 Sand virus; Specification 20 / 63 pages 31 CN 121866044 A 5.4 Pharmaceutical composition; 5.5 Method for reducing subvisible particle formation or viral titer loss; and 5.6 Treatment method.

[0196] 5.1 Liquid Arenavirus Formulation This disclosure provides liquid arenavirus formulations having various improved properties. In some embodiments, the liquid arenavirus formulations disclosed herein can facilitate the manufacture, purification, and storage of formulations containing arenaviruses, wherein these formulations can be used to treat or prevent disease.

[0197] In some embodiments, the liquid arenavirus formulation comprises arenaviruses and a polyvalent anion. In some embodiments, the liquid arenavirus formulation further comprises at least one of a stabilizer, a carbohydrate, a salt, a buffer, and a surfactant.

[0198] In some embodiments, the liquid arenavirus formulations disclosed herein are suitable for lyophilization. In some embodiments, the liquid arenavirus formulation is suitable for preparing the lyophilized arenavirus formulation disclosed in Section 5.2.

[0199] In some embodiments, the liquid arenavirus formulation is reconstituted from the lyophilized arenavirus formulation disclosed herein (see Section 5.2) before being administered to a subject, and thus may contain the same components but at different concentrations as the liquid arenavirus formulation used to prepare the lyophilized arenavirus formulation.

[0200] In some embodiments, the liquid isovirus preparation is diluted from the liquid isovirus preparation disclosed herein (see Section 5.1) before being administered to a subject, and may therefore contain the same components as the liquid isovirus preparation disclosed herein but at different concentrations.

[0201] 5.1.1 Polyvalent Anions In some embodiments, the liquid isovirus preparation disclosed herein contains polyvalent anions. Without being bound by theory, polyvalent anions can reduce the aggregation of virus particles, thereby minimizing the formation of aggregates and subvisible particles. Such properties allow filtration (e.g., bioburden reduction filtration or sterilization filtration) to be performed without significant loss of virus titers.

[0202] In some embodiments, the polyvalent anion is selected from the group consisting of citrate, tetrasodium pyrophosphate (TSPP), malate, succinate, tartrate, fumarate, maleate, and combinations thereof. In some embodiments, the polyvalent anion is selected from the group consisting of citrate, TSPP, malate, succinate, tartrate, and combinations thereof. In some embodiments, the polyvalent anion is citrate.

[0203] Citrate (C6H5O7 3−), malate (C4H4O5 2−), succinate (C4H4O4 2−), tartrate (C4H4O6 2−), fumarate (C4H2O4 2−), and maleate (C4H2O4 2−) refer to any salt or ester of the corresponding dicarboxylic acid and tricarboxylic acid. Pyrophosphate (P2O7 4−) refers to any salt of pyrophosphate, such as disodium pyrophosphate or tetrasodium pyrophosphate (TSPP).

[0204] In some embodiments, the liquid isovirus preparation comprises a polyvalent anion as disclosed herein at concentrations of up to about 150 mM, up to about 125 mM, up to about 100 mM, up to about 80 mM, up to about 70 mM, up to about 60 mM, up to about 50 mM, up to about 40 mM, up to about 30 mM, up to about 20 mM, or up to about 10 mM. In some embodiments, the liquid isovirus preparation comprises a polyvalent anion at concentrations of at least about 5 mM, at least about 10 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, at least about 50 mM, at least about 60 mM, at least about 70 mM, at least about 80 mM, at least about 100 mM, or at least about 125 mM. In some embodiments, the liquid sand-like virus preparation comprises polyvalent anions at concentrations between about 5 mM and about 150 mM, between about 10 mM and about 150 mM, between about 20 mM and about 150 mM, between about 30 mM and about 150 mM, between about 40 mM and about 150 mM, between about 50 mM and about 150 mM, between about 60 mM and about 150 mM, between about 70 mM and about 150 mM, between about 80 mM and about 150 mM, between about 100 mM and about 150 mM, between about 125 mM, and (as per page 21 / 63 of the specification, CN 121866044 A) Between approximately 150 mm, between approximately 5 mm and approximately 125 mm, between approximately 10 mm and approximately 125 mm, between approximately 20 mm and approximately 125 mm, between approximately 30 mm and approximately 125 mm, between approximately 40 mm and approximately 125 mm, between approximately 50 mm and approximately 125 mm, between approximately 60 mm and approximately 125 mm, between approximately 70 mm and approximately 125 mm, between approximately 80 mm and approximately 125 mm. Between approximately 100mm and approximately 125mm, between approximately 5mm and approximately 100mm, between approximately 10mm and approximately 100mm, between approximately 20mm and approximately 100mm, between approximately 30mm and approximately 100mm, between approximately 40mm and approximately 100mm, between approximately 50mm and approximately 100mm, between approximately 60mm and approximately 100mm, between approximately 70mm and approximately 100mm, between approximately 80mm and approximately 100mm, between approximately 5mm and approximately 80mm, between approximately 10mm and approximately 80mm, between approximately 20mm and approximately 80mm Between approximately 30mm and approximately 80mm, between approximately 40mm and approximately 80mm, between approximately 50mm and approximately 80mm, between approximately 60mm and approximately 80mm, between approximately 70mm and approximately 80mm, between approximately 5mm and approximately 70mm, between approximately 10mmBetween approximately 70 mm, between approximately 20 mm and approximately 70 mm, between approximately 30 mm and approximately 70 mm, between approximately 40 mm and approximately 70 mm, between approximately 50 mm and approximately 70 mm, between approximately 60 mm and approximately 70 mm, between approximately 5 mm and approximately 60 mm, between approximately 10 mm and approximately 60 mm, between approximately 20 mm and approximately 60 mm, between approximately 30 mm and approximately 60 mm, between approximately 40 mm and approximately 60 mm, between approximately 50 mm and approximately 60 mm, between approximately 5 mm and approximately 50 mm, between approximately 10 mm and approximately 50 mm, between approximately 20 mm and approximately 50 mm, between approximately 30 mm and approximately 40 mm and approximately 60 mm, between approximately 50 mm and approximately 60 mm, between approximately 50 mm and approximately 50 mm, between approximately 10 mm and approximately 50 mm, between approximately 20 mm and approximately 50 mm, between approximately 30 mm and approximately 50 mm. Between M and approximately 50mM, between approximately 40mM and approximately 50mM, between approximately 5mM and approximately 40mM, between approximately 10mM and approximately 40mM, between approximately 20mM and approximately 40mM, between approximately 30mM and approximately 40mM, between approximately 5mM and approximately 30mM, between approximately 10mM and approximately 30mM, between approximately 20mM and approximately 30mM, between approximately 5mM and approximately 20mM, between approximately 10mM and approximately 20mM, or between approximately 5mM and approximately 10mM. In some embodiments, the liquid isovirus preparation comprises polyvalent anions as disclosed herein at concentrations of about 150 mM, about 125 mM, about 100 mM, about 80 mM, about 70 mM, about 60 mM, about 50 mM, about 40 mM, about 30 mM, about 20 mM, or about 10 mM.

[0205] In some embodiments, the liquid isovirus preparation comprises polyvalent anions at concentrations between about 10 mM and about 60 mM. In some embodiments, the liquid isovirus preparation comprises polyvalent anions at concentrations between about 40 mM and about 60 mM. In some embodiments, the liquid isovirus preparation comprises polyvalent anions at a concentration of about 50 mM. In some embodiments, the liquid isovirus preparation comprises polyvalent anions at concentrations between about 20 mM and about 30 mM. In some embodiments, the liquid isovirus preparation comprises polyvalent anions at a concentration of about 25 mM.

[0206] In some embodiments, the liquid sand virus preparation comprises citrate at concentrations of up to about 150 mM, up to about 125 mM, up to about 100 mM, up to about 80 mM, up to about 70 mM, up to about 60 mM, up to about 50 mM, up to about 40 mM, up to about 30 mM, up to about 20 mM, or up to about 10 mM. In some embodiments, the liquid sand virus preparation comprises citrate at concentrations of at least about 5 mM, at least about 10 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, at least about 50 mM, at least about 60 mM, at least about 70 mM, at least about 80 mM, at least about 100 mM, or at least about 125 mM. In some embodiments, the liquid sand...The granular virus preparation contains citrate at the following concentrations: between approximately 5 mM and approximately 150 mM, between approximately 10 mM and approximately 150 mM, between approximately 20 mM and approximately 150 mM, between approximately 30 mM and approximately 150 mM, between approximately 40 mM and approximately 150 mM, between approximately 50 mM and approximately 150 mM, between approximately 60 mM and approximately 150 mM, between approximately 70 mM and approximately 150 mM, between approximately 80 mM and approximately 150 mM, between approximately 100 mM and approximately 150 mM, between approximately 125 mM and approximately 150 mM, and between approximately 5 mM. Between approximately 125 mm, between approximately 10 mm and approximately 125 mm, between approximately 20 mm and approximately 125 mm, between approximately 30 mm and approximately 125 mm, between approximately 40 mm and approximately 125 mm, between approximately 50 mm and approximately 125 mm, between approximately 60 mm and approximately 125 mm Between, between approximately 70mm and approximately 125mm, between approximately 80mm and approximately 125mm, between approximately 100mm and approximately 125mm, between approximately 5mm and approximately 100mm, between approximately 10mm and approximately 100mm, between approximately 20mm and approximately 100mm, between approximately 30mm and approximately 100mm, between approximately 40mm and approximately 100mm, between approximately 50mm and approximately 100mm, between approximately 60mm and approximately 100mm, between approximately 70mm and approximately 100mm, between approximately 80mm and approximately 100mm, between approximately 5mm and the instruction manual, page 22 / 63, 33 CN 121866044 A Between approximately 80 mm, between approximately 10 mm and approximately 80 mm, between approximately 20 mm and approximately 80 mm, between approximately 30 mm and approximately 80 mm, between approximately 40 mm and approximately 80 mm, between approximately 50 mm and approximately 80 mm, between approximately 60 mm and approximately 80 mm, between approximately 70 mm and approximately 80 mm, between approximately 5 mm and approximately 70 mm, between approximately 10 mm and approximately 70 mm, between approximately 20 mm and approximately 70 mm, between approximately 30 mm and approximately 70 mm, between approximately... Between 40mm and approximately 70mm, between approximately 50mm and approximately 70mm, between approximately 60mm and approximately 70mm, between approximately 5mm and approximately 60mm, between approximately 10mm and approximately 60mm, between approximately 20mm and approximately 60mm, between approximately 30mm and approximately 60mm, between approximately 40mm and approximately 60mm, between approximately 50mm and approximately 60mm, between approximately 5mm and approximately 50mm, between approximately 10mm and approximately 50mm, between approximately 20mm and approximately 50mmBetween approximately 30mM and approximately 50mM, between approximately 40mM and approximately 50mM, between approximately 5mM and approximately 40mM, between approximately 10mM and approximately 40mM, between approximately 20mM and approximately 40mM, between approximately 30mM and approximately 40mM, between approximately 5mM and approximately 30mM, between approximately 10mM and approximately 30mM, between approximately 20mM and approximately 30mM, between approximately 5mM and approximately 20mM, between approximately 10mM and approximately 20mM, or between approximately 5mM and approximately 10mM. In some embodiments, the liquid isoflavone virus preparation comprises citrate at concentrations of about 150 mM, about 125 mM, about 100 mM, about 80 mM, about 70 mM, about 60 mM, about 50 mM, about 40 mM, about 30 mM, about 20 mM, or about 10 mM as disclosed herein.

[0207] In some embodiments, the liquid isoflavone virus preparation comprises citrate at concentrations between about 10 mM and about 60 mM. In some embodiments, the liquid isoflavone virus preparation comprises citrate at concentrations between about 40 mM and about 60 mM. In some embodiments, the liquid isoflavone virus preparation comprises citrate at a concentration of about 50 mM. In some embodiments, the liquid isoflavone virus preparation comprises citrate at a concentration between about 20 mM and about 30 mM. In some embodiments, the liquid isoflavone virus preparation comprises citrate at a concentration of about 25 mM.

[0208] In some embodiments, the liquid sand-like virus formulation comprises the polyvalent anion disclosed herein, wherein the concentration of the polyvalent anion (e.g., citrate) (i) reduces the formation of subvisible particles during manufacturing and / or (ii) reduces the loss of virus titer during filtration. In some embodiments, filtration includes bioburden reduction filtration (e.g., 0.45 µm filtration), sterilization filtration (e.g., 0.2 µm or 0.22 µm filtration), or a combination thereof. In some embodiments, manufacturing includes a purification process. In some embodiments, manufacturing includes at least one chromatographic step.

[0209] Subvisible particles are particles that are too large to be analyzed by size exclusion chromatography (SEC) (e.g., at least about 0.3 µm), but too small to be seen with the naked eye (e.g., no more than about 100 µm). In some embodiments, the concentration of the polyvalent anion (e.g., citrate) reduces the formation of subvisible particles during manufacturing compared to a control formulation that does not contain the polyvalent anion. In some embodiments, the size of the subvisible particles is at least about 0.3 μm, at least about 0.5 μm, at least about 1 μm, at least about 5 μm, at least about 10 μm, at least about 15 μm, at least about 20 μm, at least about 25 μm, at least about 30 μm, at least about 35 μm,At least about 40 μm, at least about 45 μm, at least about 50 μm, at least about 55 μm, at least about 60 μm, at least about 65 μm, at least about 70 μm, at least about 75 μm, at least about 80 μm, at least about 85 μm, at least about 90 μm, or at least about 95 μm. In some embodiments, the size of the subvisible particles is less than 100 μm. In some embodiments, the size of the subvisible particles is at least about 1 μm. In some embodiments, the size of the subvisible particles is at least about 10 μm. In some embodiments, the size of the subvisible particles is at least about 25 μm.

[0210] The size distribution, shape, and concentration of the subvisible particles can be measured by any suitable method known in the art, such as optical obscuration particle counting techniques and microscopic particle counting techniques. In some embodiments, the size distribution, shape, and concentration of the subvisible particles can be measured by flow cytometry (FIM). For example, flow cytometry (FIM) can be used to measure the size distribution, shape, and concentration of particles ranging in size from about 1 µm to several hundred µm. Sample analysis was performed on a FlowCam 8100 (Fluid Imaging Technologies, Inc.) equipped with a 10x objective lens, an FOV80 flow cell, and a 1mL precision syringe. The flow rate was set to 0.15mL / min, and the automatic imaging rate was 19 images per second, resulting in an efficiency of approximately 71%. The effective spherical diameter (ESD), a calculated value based on particle volume, was measured. Evaluation was performed using MODDE Pro (Satorius Stedim Data Analytics AB). Data were fitted using a multiple linear regression (MLR) model.

[0211] In some embodiments, the concentration of polyvalent anions (e.g., citrate) reduces the formation of subvisible particles during manufacturing, wherein, as measured by FIM, the concentration of subvisible particles in the formulation is at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, at most about 50%, at most about 60%, at most about 70%, or at most about 80% of the concentration of subvisible particles in a control formulation without polyvalent anions. In some embodiments, the concentration of polyvalent anions (e.g., citrate) reduces the formation of subvisible particles during manufacturing, wherein, as measured by FIM, the concentration of subvisible particles in the formulation is approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 60%, approximately 70%, or approximately 80% of the subvisible particle concentration in a control formulation without polyvalent anions. In some embodiments, the control formulation, in addition to lacking polyvalent anions, is compared to the liquid sand virus formulation disclosed herein.Or may contain different polyvalent anions, otherwise identical.

[0212] Viral titer can be measured by any method known in the art. In some embodiments, viral titer can be measured by a replication capacity viral focus-forming unit (RCV-FFU) assay. In some embodiments, viral titer can be measured by a focus-forming unit (FFU) assay.

[0213] Any assay known to those skilled in the art can be used to measure the infectivity of a sand-particle viral vector formulation. For example, the virus / vector titer can be determined by a focus-forming unit assay (FFU assay). Briefly, complementary cells, such as HEK293-TVL cells, are plated and inoculated with different dilutions of a viral vector sample. After incubation for a period of time, a monolayer of cells forms, and the virus attaches to the cells. The monolayer is then covered with methylcellulose. After further incubation, the initially infected cells release viral progeny. Due to the presence of the methylcellulose covering layer, the spread of new virus is restricted to neighboring cells. Thus, each infectious particle produces a circular area of ​​infected cells, called a focus. These lesions can be made visible and countable using anti-LCMV-NP or anti-PICV-NP antibodies, or another protein expressed by arenavirus particles or trisegmented arenavirus particles, along with an HRP-based colorimetric reaction. The virus / vector titer can be calculated per lesion-forming unit per milliliter (FFU / mL). Similarly, the proportion of trisegmented replicating virus particles can be determined using non-complementary cells (e.g., HEK293). This allows only the trisegmented virus particles to infect neighboring cells. The titer of replicating virus / vector (RCV) can be calculated per lesion-forming unit per milliliter (RCV FFU / mL).

[0214] In some embodiments, the concentration of the polyvalent anion (e.g., citrate) reduces the loss of viral titer during filtration, wherein, as measured by RCV-FFU or FFU assay, the viral titer in the filtered formulation is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the viral titer before filtration. In some embodiments, the concentration of the polyvalent anion (e.g., citrate) reduces the loss of viral titer during filtration, wherein, as measured by RCV-FFU or FFU assay, the viral titer in the filtered formulation is at about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the viral titer before filtration.

[0215] In some embodiments, the concentration of polyvalent anions (e.g., citrate) reduces the virus titer during filtration.The loss, if measured by RCV-FFU or FFU assay, is such that the viral titer in the filtered formulation is at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, or at least about 500%. In some embodiments, the concentration of the polyvalent anion (e.g., citrate) reduces the loss of virus titer during filtration, wherein, as measured by RCV-FFU or FFU assay, the virus titer in the filtered formulation is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 400%, or about 500% higher than that of a control formulation without the polyvalent anion. In some embodiments, the control formulation is identical to the liquid sand virus formulation disclosed herein except that it does not contain the polyvalent anion or contains a different polyvalent anion.

[0216] In some embodiments, the liquid sand virus formulation is suitable for lyophilization. In some embodiments, the liquid isovirus preparation suitable for lyophilization comprises a polyvalent anion disclosed herein at a concentration of up to about 50 mM, up to about 40 mM, up to about 30 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, between about 20 mM and about 50 mM, between about 20 mM and about 40 mM, between about 20 mM and about 30 mM, between about 30 mM and about 50 mM, between about 30 mM and about 40 mM, or between about 40 mM and about 50 mM. In some embodiments, the liquid isovirus preparation suitable for lyophilization comprises a polyvalent anion disclosed herein at a concentration of about 20 mM, about 30 mM, about 40 mM, or about 50 mM. In some embodiments, the liquid isovirus preparation suitable for lyophilization comprises a polyvalent anion disclosed herein at a concentration of up to about 50 mM. In some embodiments, the liquid isovirus preparation suitable for lyophilization contains the polyvalent anions disclosed herein at concentrations between about 20 mM and about 50 mM.

[0217] In some embodiments, the liquid isovirus preparation suitable for lyophilization contains concentrations of up to about 50 mM, up to about 40 mM, up to about 30 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, between about 20 mM and about 50 mM, between about 20 mM and about 40 mM, between about 20 mM and about 30 mM, between about 30 mM and about 50 mM, between about 30 mM and about 50 mM.The concentration of citrate is between about 40 mM and about 50 mM. In some embodiments, the liquid isovirus preparation suitable for freeze-drying contains citrate at a concentration of about 20 mM, about 30 mM, about 40 mM, or about 50 mM. In some embodiments, the liquid isovirus preparation suitable for freeze-drying contains citrate at a concentration of up to about 50 mM. In some embodiments, the liquid isovirus preparation suitable for freeze-drying contains citrate at a concentration between about 20 mM and about 50 mM.

[0218] 5.1.2 Carbohydrates In some embodiments, the liquid isovirus preparations disclosed herein also contain carbohydrates. Without being bound by theory, the addition of one or more carbohydrates to the liquid isovirus preparations disclosed herein can improve the stability of isovirus particles during freeze-thaw cycles. Without being bound by any particular theory, the addition of one or more carbohydrates to the preparations described herein can improve the stability of isovirus particles during freeze-thaw cycles by reducing the aggregation and / or inactivation of isovirus particles.

[0219] In some embodiments, the carbohydrates used in the formulations and methods disclosed herein are sugars or polyols. Suitable sugars that can be used with this disclosure include, but are not limited to, monosaccharides (e.g., glucose, galactose, ribose, mannose, rhamnose, tarose, xylose, allose, and arabinose), disaccharides (e.g., trehalose, sucrose, maltose, isomaltose, cellobiose, gentiobiose, laminarin, xylobiose, manniobiose, lactose, and fructose), trisaccharides (e.g., acarbose, raffinose, pinotriose, panose, and cellotriose), and sugar polymers (e.g., dextran, xanthan gum, pullulan, cyclodextrin, amylose, amylopectin, starch, cellooligosaccharides, cellulose, maltooligosaccharides, glycogen, chitosan, and chitin). Polyols, also known as sugar alcohols, are a class of low molecular weight polyols typically obtained by the hydrogenation of sugars. Suitable polyols that can be used with this disclosure include, but are not limited to, mannitol, sorbitol, arabinitol, erythritol, maltitol, xylitol, glycol, ethylene glycol, polyglycol, polyethylene glycol, polypropylene glycol, isomaltitol, hydrogenated starch hydrolysate, and glycerol.

[0220] In some embodiments, the liquid sand virus formulation disclosed herein further comprises a carbohydrate selected from the group consisting of sucrose, trehalose, mannitol, sorbitol, dextran, and combinations thereof. In some embodiments, the carbohydrate is sucrose, trehalose, or combinations thereof. In some embodiments, the carbohydrate is sucrose. In some embodiments, the carbohydrate is trehalose.

[0221] In some embodiments, the liquid sand virus formulation comprises at a concentration of up to about 20% (w / v) and up to about 15% (w / v). [Specification 25 / 63 pages 36 CN 121866044 A]The liquid sand virus preparation contains at least about 0.1% (w / v), at least about 0.5% (w / v), at least about 1% (w / v), at least about 1% (w / v), at least about 0.5% (w / v), or at least about 0.1% (w / v) of carbohydrates. In some embodiments, the liquid sand virus preparation contains at least about 0.1% (w / v), at least about 0.5% (w / v), at least about 1% (w / v), at least about 5% (w / v), at least about 10% (w / v), at least about 15% (w / v), or at least about 20% (w / v) of carbohydrates. In some embodiments, the liquid sand-like virus preparation comprises carbohydrates at the following concentrations: between about 0.1% (w / v) and about 20% (w / v), between about 0.5% (w / v) and about 20% (w / v), between about 1% (w / v) and about 20% (w / v), between about 5% (w / v) and about 20% (w / v), between about 10% (w / v) and about 20% (w / v), between about 15% (w / v) and about 20% (w / v), between about 0.1% (w / v) and about 15% (w / v), between about 0.5% (w / v) and about 15% (w / v), between about 1% (w / v) and about 15% (w / v), and between about 5% (w / v) and about 15%. Between (w / v), between about 10% (w / v) and about 15% (w / v), between about 0.1% (w / v) and about 10% (w / v), between about 0.5% (w / v) and about 10% (w / v), between about 1% (w / v) and about 10% (w / v), between about 5% (w / v) and about 10% (w / v), between about 0.1% (w / v) and about 5% (w / v), between about 0.5% (w / v) and about 5% (w / v), between about 1% (w / v) and about 5% (w / v), between about 0.1% (w / v) and about 1% (w / v), or between about 0.5% (w / v) and about 1% (w / v). In some embodiments, the liquid isovirus preparation comprises carbohydrates at concentrations of about 20% (w / v), about 15% (w / v), about 10% (w / v), about 5% (w / v), about 1% (w / v), about 0.5% (w / v), or about 0.1% (w / v).

[0222] In some embodiments, the liquid isovirus preparation comprises carbohydrates at concentrations between about 3% and about 10% (w / v). In some embodiments, the liquid isovirus preparation comprises carbohydrates at concentrations between about 4% (w / v) and about 6% (w / v). In some embodiments, the liquid isovirus preparation comprises carbohydrates at a concentration of about 5% (w / v).

[0223] In some embodiments, the liquid isovirus preparation comprises carbohydrates at concentrations of up to about 20% (w / v), up to about 15% (w / v), or about 0.1% (w / v).(v), up to about 10% (w / v), up to about 5% (w / v), up to about 1% (w / v), up to about 0.5% (w / v), or up to about 0.1% (w / v). In some embodiments, the liquid sand virus preparation disclosed herein contains sucrose at a concentration of at least about 0.1% (w / v), at least about 0.5% (w / v), at least about 1% (w / v), at least about 5% (w / v), at least about 10% (w / v), at least about 15% (w / v), or at least about 20% (w / v). In some embodiments, the liquid sand virus preparation comprises sucrose at the following concentrations: between about 0.1% (w / v) and about 20% (w / v), between about 0.5% (w / v) and about 20% (w / v), between about 1% (w / v) and about 20% (w / v), between about 5% (w / v) and about 20% (w / v), between about 10% (w / v) and about 20% (w / v), between about 15% (w / v) and about 20% (w / v), between about 0.1% (w / v) and about 15% (w / v), and between about 0.5% (w / v) and about 20% (w / v). Between approximately 15% (w / v), between approximately 1% (w / v) and approximately 15% (w / v), between approximately 5% (w / v) and approximately 15% (w / v), between approximately 10% (w / v) and approximately 15% (w / v), between approximately 0.1% (w / v) and approximately 10% (w / v), between approximately 0.5% (w / v) and approximately 10% (w / v), between approximately 1% (w / v) and approximately 10% (w / v), between approximately 0.1% (w / v) and approximately 5% (w / v), between approximately 0.5% (w / v) and approximately 5% (w / v), between approximately 1% (w / v) and approximately 10% (w / v), between approximately 0.1% (w / v) and approximately 5% (w / v), between approximately 0.5% (w / v) and approximately 5% (w / v), between approximately 1% (w / v) and approximately 10% (w / v). The concentration of sucrose is between about 5% (w / v), between about 0.1% (w / v) and about 1% (w / v), or between about 0.5% (w / v) and about 1% (w / v). In some embodiments, the liquid isovirus preparation comprises sucrose at concentrations of about 20% (w / v), about 15% (w / v), about 10% (w / v), about 5% (w / v), about 1% (w / v), about 0.5% (w / v), or about 0.1% (w / v).

[0224] In some embodiments, the liquid isovirus preparation comprises sucrose at concentrations between about 3% and about 10% (w / v). In some embodiments, the liquid isovirus preparation comprises sucrose at concentrations between about 4% (w / v) and about 6% (w / v). In some embodiments, the liquid isovirus preparation disclosed herein comprises sucrose at a concentration of about 5% (w / v).

[0225] In some embodiments, the liquid sand-like virus preparation contains a concentration of up to about 20% (w / v) and up to about 15% (w / v).(v), up to about 10% (w / v), up to about 5% (w / v), up to about 1% (w / v), up to about 0.5% (w / v), or up to about 0.1% (w / v). In some embodiments, the liquid sand virus preparation disclosed herein contains at least about 0.1% (w / v), at least about 0.5% (w / v), at least about 1% (w / v), at least about 5% (w / v), at least about 10% (w / v), at least about 15% (w / v), or at least about 20% (w / v) of trehalose. In some embodiments, the liquid sand-virus preparation comprises trehalose at the following concentrations: between about 0.1% (w / v) and about 20% (w / v), between about 0.5% (w / v) and about 20% (w / v), between about 1% (w / v) and about 20% (w / v), between about 5% (w / v) and about 20% (w / v), between about 10% (w / v) and about 20% (w / v), between about 15% (w / v) and about 20% (w / v), between about 0.1% (w / v) and about 15% (w / v), between about 0.5% (w / v) and about 15% (w / v), between about 1% (w / v) and about 15% (w / v), and between about 5% (w / v) and about 15%. Between (w / v), between about 10% (w / v) and about 15% (w / v), between about 0.1% (w / v) and about 10% (w / v), between about 0.5% (w / v) and about 10% (w / v), between about 1% (w / v) and about 10% (w / v), between about 5% (w / v) and about 10% (w / v), between about 0.1% (w / v) and about 5% (w / v), between about 0.5% (w / v) and about 5% (w / v), between about 1% (w / v) and about 5% (w / v), between about 0.1% (w / v) and about 1% (w / v), or between about 0.5% (w / v) and about 1% (w / v). In some embodiments, the liquid isovirus formulation comprises trehalose at concentrations of about 20% (w / v), about 15% (w / v), about 10% (w / v), about 5% (w / v), about 1% (w / v), about 0.5% (w / v), or about 0.1% (w / v).

[0226] In some embodiments, the liquid isovirus formulation comprises trehalose at concentrations between about 3% and about 10% (w / v). In some embodiments, the liquid isovirus formulation comprises trehalose at concentrations between about 4% (w / v) and about 6% (w / v). In some embodiments, the liquid isovirus formulation disclosed herein comprises trehalose at a concentration of about 5% (w / v).

[0227] In some embodiments, the concentration of carbohydrates (e.g., sucrose, trehalose) reduces the formation of subvisible particles during freeze-thaw cycles. In some embodiments, the concentration of carbohydrates (e.g., sucrose, trehalose) reduces the loss of viral titers during freeze-thaw cycles.

[0228] In some embodiments, the concentration of carbohydrates (e.g., sucrose, trehalose) reduces the formation of subvisible particles during freeze-thaw cycles, wherein, as measured by FIM, the concentration of subvisible particles in the freeze-thawed formulation is at most about 10%, at most about 20%, at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 70%, or at most about 80% of the concentration of subvisible particles in a control formulation without the carbohydrate. In some embodiments, the concentration of carbohydrates (e.g., sucrose, trehalose) reduces the formation of subvisible particles during freeze-thaw cycles, wherein, as measured by FIM, the concentration of subvisible particles in the freeze-thawed formulation is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% of the concentration of subvisible particles in a control formulation that does not contain the carbohydrate. In some embodiments, the control formulation is identical to the formulation disclosed herein except that it does not contain carbohydrates or contains different carbohydrates.

[0229] In some embodiments, the concentration of carbohydrates (e.g., sucrose, trehalose) reduces the loss of viral titer during freeze-thaw cycles, wherein, as measured by RCV-FFU or FFU assay, the viral titer in the freeze-thawed formulation is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the viral titer in the formulation before freeze-thaw. In some embodiments, the concentration of carbohydrates (e.g., sucrose, trehalose) reduces the loss of viral titer during freeze-thaw cycles, wherein, as measured by RCV-FFU or FFU assay, the viral titer in the freeze-thawed formulation is approximately 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the viral titer in the formulation before freeze-thaw.

[0230] In some embodiments, the concentration of carbohydrates (e.g., sucrose, trehalose) reduces the loss of virus titer during freeze-thaw, wherein, as measured by RCV-FFU or FFU assay, the virus titer in the freeze-thawed formulation is at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, or at least about 500%. In some embodiments, the concentration of carbohydrates (e.g., sucrose, trehalose) reduces the loss of virus titer during freeze-thaw, wherein, as measured by RCV-FFU or FFU assay, the virus titer in the freeze-thawed formulation is at least about 20%, at least about 30%, at least about 400%, or at least about 500% higher than the virus titer in the control formulation without the carbohydrate.The concentration of sugar (trehalose) reduces the loss of virus titer during freeze-thaw cycles, wherein, as measured by RCV-FFU or FFU assay, the virus titer in the freeze-thawed formulation is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 400%, or about 500% higher than that of the control formulation without the carbohydrate. In some embodiments, the control formulation is identical to the liquid sand virus formulation disclosed herein except that it does not contain carbohydrates or contains different carbohydrates.

[0231] In some embodiments, freeze-thaw cycles include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more freeze-thaw (F / T) cycles. In some embodiments, the freeze-thaw cycle comprises 3 F / T cycles. In some embodiments, the freeze-thaw cycle comprises 5 F / T cycles. In some embodiments, the freezing conditions are at or below -20°C, or below -20°C, or below -65°C, or about -80°C.

[0232] In some embodiments, the concentration of carbohydrates (e.g., sucrose, trehalose) reduces the formation of subvisible particles during storage. In some embodiments, the concentration of carbohydrates (e.g., sucrose, trehalose) reduces the loss of virus titers during storage. In some embodiments, the sand-borne virus preparation is subjected to bioburden reduction filtration and / or sterilization filtration before storage.

[0233] In some embodiments, the concentration of carbohydrates (e.g., sucrose, trehalose) reduces the formation of subvisible particles during storage, wherein, as measured by FIM, the concentration of subvisible particles in the formulation after storage is at most about 10%, at most about 20%, at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 70%, or at most about 80% of the subvisible particle concentration of a control formulation without the carbohydrate after storage. In some embodiments, the concentration of carbohydrates (e.g., sucrose, trehalose) reduces the formation of subvisible particles during storage, wherein, as measured by FIM, the concentration of subvisible particles in the formulation after storage is at most about 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, or at most 80% of the subvisible particle concentration of a control formulation without the carbohydrate after storage. In some embodiments, the control formulation is identical to the formulation disclosed herein except that it does not contain carbohydrates or contains different carbohydrates.

[0234] In some embodiments, the concentration of carbohydrates (e.g., sucrose, trehalose) reduces viral droplets during storage.The loss of viral titer during storage, wherein, as measured by RCV-FFU or FFU assay, the viral titer in the formulation after storage is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the viral titer in the formulation before storage. In some embodiments, the concentration of carbohydrates (e.g., sucrose, trehalose) reduces the loss of viral titer during storage, wherein, as measured by RCV-FFU or FFU assay, the viral titer in the formulation after storage is at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the viral titer in the formulation before storage.

[0235] In some embodiments, the concentration of carbohydrates (e.g., sucrose, trehalose) reduces the loss of viral titer during storage, wherein, as measured by RCV-FFU or FFU assay, the viral titer in the formulation after storage is at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, or at least about 500%. In some embodiments, the concentration of carbohydrates (e.g., sucrose, trehalose) reduces the loss of viral titer during storage, wherein, as measured by RCV-FFU or FFU assay, the viral titer in the formulation after storage is approximately 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, or 500% higher than that of a control formulation without the carbohydrate. In some embodiments, the control formulation is identical to the formulation disclosed herein except that it contains no carbohydrates or contains different carbohydrates. Specification page 28 / 63 39 CN 121866044 A

[0236] In some embodiments, the storage temperature is above about 2°C, is room temperature (e.g., 20°C–25°C), is between about 2°C and about 8°C, is above about 15°C, is about -20°C or below about -20°C, is about -65°C or below about -65°C, or is about -80°C. In some embodiments, the storage temperature is about -20°C. In some embodiments, the storage temperature is about -65°C. In some embodiments, the storage temperature is about -80°C. In some embodiments, the storage lasts for at least about 3 months.The storage period is approximately 1 month, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 3 years, at least about 4 years, at least about 5 years, or longer. In some embodiments, the storage lasts for at least about 6 months. In some embodiments, the storage lasts for at least about 9 months. In some embodiments, the storage lasts for at least about 18 months.

[0237] In some embodiments, the liquid sand virus preparation disclosed herein is suitable for lyophilization. In some embodiments, the liquid sand virus preparation suitable for freeze-drying contains carbohydrates disclosed herein at concentrations of up to about 10% (w / v), up to about 8% (w / v), up to about 6% (w / v), up to about 4% (w / v), at least about 3% (w / v), at least about 6% (w / v), at least about 8% (w / v), between about 3% and about 10% (w / v), between about 3% (w / v) and about 8% (w / v), between about 3% and about 6% (w / v), between about 4% and about 10% (w / v), between about 4% and about 8% (w / v), between about 4% and about 6% (w / v), between about 6% and about 10% (w / v), between about 6% and about 8% (w / v), or between about 8% and about 10% (w / v). In some embodiments, the liquid isovirus preparation suitable for lyophilization comprises about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of the carbohydrates disclosed herein. In some embodiments, the liquid isovirus preparation suitable for lyophilization comprises about 5% (w / v) of the carbohydrates disclosed herein.

[0238] In some embodiments, the liquid sand virus preparation suitable for freeze drying contains trehalose at concentrations of up to about 10% (w / v), up to about 8% (w / v), up to about 6% (w / v), up to about 4% (w / v), at least about 3% (w / v), at least about 6% (w / v), at least about 8% (w / v), between about 3% and about 10% (w / v), between about 3% (w / v) and about 8% (w / v), between about 3% and about 6% (w / v), between about 4% and about 10% (w / v), between about 4% and about 8% (w / v), between about 4% and about 6% (w / v), between about 6% and about 10% (w / v), between about 6% and about 8% (w / v), or between about 8% and about 10% (w / v). In some embodiments, the liquid sand virus preparation suitable for freeze-drying contains trehalose at concentrations of about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In some embodiments, the liquid...The sand-borne virus preparation contains trehalose at a concentration of about 5% (w / v).

[0239] 5.1.3 Salt In some embodiments, the liquid sand-borne virus preparation contains salt. Without being bound by any theory, adding one or more salts to the liquid sand-borne virus preparation disclosed herein can improve the stability of sand-borne virus particles during freeze-thaw cycles. Without being bound by any particular theory, adding salts to the preparations described herein increases ionic strength to enhance the stability of virus particles and prevents virus particles from forming aggregates, thereby improving filterability during the manufacturing process of the sand-borne virus preparation disclosed herein.

[0240] In some embodiments, the liquid sand-borne virus preparation disclosed herein contains a salt selected from the group consisting of sodium chloride (NaCl), sodium phosphate (Na3PO4), potassium phosphate (K3PO4), potassium chloride (KCl), magnesium chloride (MgCl2), sodium sulfate (Na2SO4), potassium sulfate (K2SO4), ammonium sulfate ((NH4)2SO4), and combinations thereof. In some embodiments, the liquid sand-borne virus preparation disclosed herein contains sodium chloride.

[0241] In some embodiments, the liquid sand virus preparation comprises salts at concentrations of up to about 450 mM, up to about 400 mM, up to about 350 mM, up to about 300 mM, up to about 250 mM, up to about 225 mM, up to about 200 mM, up to about 175 mM, up to about 150 mM, up to about 125 mM, up to about 100 mM, up to about 75 mM, or up to about 50 mM. In some embodiments, the liquid sand virus preparation contains salts at concentrations of at least about 50 mM, at least about 75 mM, at least about 100 mM, at least about 125 mM, at least about 150 mM, at least about 175 mM, at least about 200 mM, at least about 225 mM, at least about 250 mM, at least about 300 mM, at least about 350 mM, at least about 400 mM, or at least about 450 mM. In some embodiments, the liquid sand-like virus preparation comprises salts at concentrations between approximately 50 mM and approximately 450 mM, between approximately 50 mM and approximately 400 mM, between approximately 50 mM and approximately 350 mM, between approximately 50 mM and approximately 300 mM, between approximately 50 mM and approximately 250 mM, between approximately 50 mM and approximately 225 mM, between approximately 50 mM and approximately 200 mM, between approximately 50 mM and approximately 175 mM, between approximately 50 mM and approximately 150 mM, between approximately 50 mM and approximately 125 mM, between approximately 50 mM and approximately 100 mM, between approximately 50 mM and approximately 75 mM, and between approximately 75 mM. Between approximately 450mM, between approximately 75mM and approximately 400mM, between approximately 75mM and approximately 350mM, between approximately 75mM and approximately...Between 300mM, between approximately 75mM and approximately 250mM, between approximately 75mM and approximately 225mM, between approximately 75mM and approximately 200mM, between approximately 75mM and approximately 175mM, between approximately 75mM and approximately 150mM, between approximately 75mM and approximately 125mM Between approximately 75mm and approximately 100mm, between approximately 100mm and approximately 450mm, between approximately 100mm and approximately 400mm, between approximately 100mm and approximately 350mm, between approximately 100mm and approximately 300mm, between approximately 100mm and approximately 250mm, between approximately 100mm and approximately 225mm, between approximately 100mm and approximately 200mm, between approximately 100mm and approximately 175mm, between approximately 100mm and approximately 150mm, between approximately 100mm and approximately 125mm, between approximately 125mm and approximately 450mm, etc. Between approximately 125 mm and approximately 400 mm, between approximately 125 mm and approximately 350 mm, between approximately 125 mm and approximately 300 mm, between approximately 125 mm and approximately 250 mm, between approximately 125 mm and approximately 225 mm, between approximately 125 mm and approximately 200 mm, between approximately 125 mm and approximately 175 mm, between approximately 125 mm and approximately 150 mm, between approximately 150 mm and approximately 450 mm, between approximately 150 mm and approximately 400 mm, between approximately 150 mm and approximately 350 mm, between approximately 150 mm and approximately 300 mm, between... Between approximately 150mm and approximately 250mm, between approximately 150mm and approximately 225mm, between approximately 150mm and approximately 200mm, between approximately 150mm and approximately 175mm, between approximately 175mm and approximately 450mm, between approximately 175mm and approximately 400mm, between approximately 175mm and approximately 350mm, between approximately 175mm and approximately 300mm, between approximately 175mm and approximately 250mm, between approximately 175mm and approximately 225mm, between approximately 175mm and approximately 200mm, between approximately 200mm and approximately 450mm, between Between approximately 200mm and approximately 400mm, between approximately 200mm and approximately 350mm, between approximately 200mm and approximately 300mm, between approximately 200mm and approximately 250mm, between approximately 200mm and approximately 225mm, between approximately 225mm and approximately 450mm, between approximately 225mm and approximately 400mm, between approximately 225mm and approximately 350mm, between approximately 225mm and approximately 300mm, between approximately 225mm and approximately 250mm, between approximately 250mm and approximately 450mm, between approximately 250mm and approximately 400mm, between approximatelyThe concentrations are between 250 mM and about 350 mM, between about 250 mM and about 300 mM, between about 300 mM and about 450 mM, between about 300 mM and about 400 mM, between about 300 mM and about 350 mM, between about 350 mM and about 450 mM, between about 350 mM and about 400 mM, or between about 400 mM and about 450 mM. In some embodiments, the liquid sand-like virus preparation comprises salts at concentrations of about 450 mM, about 400 mM, about 350 mM, about 300 mM, about 250 mM, about 225 mM, about 200 mM, about 175 mM, about 150 mM, about 125 mM, about 100 mM, about 75 mM, about 50 mM, or 0 mM.

[0242] In some embodiments, the liquid isovirus preparation comprises a salt at a concentration between about 50 mM and about 250 mM. In some embodiments, the liquid isovirus preparation comprises a salt at a concentration between about 100 mM and about 150 mM. In some embodiments, the liquid isovirus preparation comprises a salt at a concentration of about 125 mM. In some embodiments, the liquid isovirus preparation comprises a salt at a concentration between about 50 mM and about 100 mM. In some embodiments, the liquid isovirus preparation comprises a salt at a concentration of about 75 mM.

[0243] In some embodiments, the liquid sand virus preparation contains sodium chloride at concentrations of up to about 450 mM, up to about 400 mM, up to about 350 mM, up to about 300 mM, up to about 250 mM, up to about 225 mM, up to about 200 mM, up to about 175 mM, up to about 150 mM, up to about 125 mM, up to about 100 mM, up to about 75 mM, or up to about 50 mM. In some embodiments, the liquid sand virus preparation contains sodium chloride at concentrations of at least about 50 mM, at least about 75 mM, at least about 100 mM, at least about 125 mM, at least about 150 mM, at least about 175 mM, at least about 200 mM, at least about 225 mM, at least about 250 mM, at least about 300 mM, at least about 350 mM, at least about 400 mM, or at least about 450 mM. In some embodiments, the liquid sand-like virus preparation comprises sodium chloride at concentrations between approximately 50 mM and approximately 450 mM, between approximately 50 mM and approximately 400 mM, between approximately 50 mM and approximately 350 mM, between approximately 50 mM and approximately 300 mM, between approximately 50 mM and approximately 250 mM, between approximately 50 mM and approximately 225 mM, between approximately 50 mM and approximately 200 mM, between approximately 50 mM and approximately 175 mM, and between approximately 50 mM and approximately 150 mM.Between approximately 50mm and approximately 125mm, between approximately 50mm and approximately 100mm, between approximately 50mm and approximately 75mm, between approximately 75mm and approximately 450mm, between approximately 75mm and approximately 400mm, between approximately 75mm and approximately 350mm, between approximately 75mm and approximately 300mm, between approximately 75mm and approximately 250mm, between approximately 75mm and approximately 225mm, between approximately 75mm and approximately 200mm, between approximately 75mm and approximately 175mm, between approximately 75mm and approximately 150mm, between approximately 75mm Between approximately 125 mm, between approximately 75 mm and approximately 100 mm, between approximately 100 mm and approximately 450 mm, between approximately 100 mm and approximately 400 mm, between approximately 100 mm and approximately 350 mm, between approximately 100 mm and approximately 300 mm, between approximately 100 mm and approximately 250 mm, between approximately 100 mm and approximately 225 mm, between approximately 100 mm and approximately 200 mm, between approximately 100 mm and approximately 175 mm, between approximately 100 mm and approximately 150 mm. Between approximately 100mm and approximately 125mm, between approximately 125mm and approximately 450mm, between approximately 125mm and approximately 400mm, between approximately 125mm and approximately 350mm, between approximately 125mm and approximately 300mm, between approximately 125mm and approximately 250mm, between approximately 125mm and approximately 225mm, between approximately 125mm and approximately 200mm, between approximately 125mm and approximately 175mm, between approximately 125mm and approximately 150mm, between approximately 150mm and... Between approximately 450 mm, between approximately 150 mm and approximately 400 mm, between approximately 150 mm and approximately 350 mm, between approximately 150 mm and approximately 300 mm, between approximately 150 mm and approximately 250 mm, between approximately 150 mm and approximately 225 mm, between approximately 150 mm and approximately 200 mm, between approximately 150 mm and approximately 175 mm, between approximately 175 mm and approximately 450 mm, between approximately 175 mm and approximately 400 mm, between approximately 175 mm and approximately 350 mm. Between approximately 175mm and approximately 300mm, between approximately 175mm and approximately 250mm, between approximately 175mm and approximately 225mm, between approximately 175mm and approximately 200mm, between approximately 200mm and approximately 450mm, between approximately 200mm and approximately 400mm, between approximately 200mm and approximately 350mm, between approximately 200mm and approximately 300mm, between approximately 200mm and approximately 250mm, between approximately 200mm and approximately 225mm, between approximately 225mm and approximately...Between 450mM, between approximately 225mM and approximately 400mM, between approximately 225mM and approximately 350mM, between approximately 225mM and approximately 300mM, between approximately 225mM and approximately 250mM, between approximately 250mM and approximately 450mM, between approximately 250mM and approximately 400mM, between approximately 250mM and approximately 350mM, between approximately 250mM and approximately 300mM, between approximately 300mM and approximately 450mM, between approximately 300mM and approximately 400mM, between approximately 300mM and approximately 350mM, between approximately 350mM and approximately 450mM, between approximately 350mM and approximately 400mM, or between approximately 400mM and approximately 450mM. In some embodiments, the liquid isovirus preparation comprises sodium chloride at concentrations of about 450 mM, about 400 mM, about 350 mM, about 300 mM, about 250 mM, about 225 mM, about 200 mM, about 175 mM, about 150 mM, about 125 mM, about 100 mM, about 75 mM, about 50 mM, or 0 mM.

[0244] In some embodiments, the liquid isovirus preparation comprises sodium chloride at a concentration between about 50 mM and about 250 mM. In some embodiments, the liquid isovirus preparation comprises sodium chloride at a concentration between about 100 mM and about 150 mM. In some embodiments, the liquid isovirus preparation comprises sodium chloride at a concentration of about 125 mM. In some embodiments, the liquid isovirus preparation comprises sodium chloride at a concentration between about 50 mM and about 100 mM. In some embodiments, the liquid isovirus preparation comprises sodium chloride at a concentration of about 75 mM.

[0245] In some embodiments, the concentration of salt (e.g., sodium chloride) reduces the formation of subvisible particles during manufacturing. In some embodiments, manufacturing includes a purification process. In some embodiments, manufacturing includes at least one chromatographic step. In some embodiments, the concentration of salt (e.g., sodium chloride) reduces the loss of viral titers during filtration. In some embodiments, filtration includes bioburden reduction filtration, sterilization filtration, or a combination thereof. Specification 31 / 63 pages 42 CN 121866044 A

[0246] In some embodiments, the concentration of salt (e.g., sodium chloride) reduces the formation of subvisible particles during manufacturing, wherein, as measured by FIM, the concentration of subvisible particles in the formulation is at most about 10%, at most about 20%, at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 70%, or at most about 80% of the concentration of subvisible particles in a control formulation without the salt. In some embodiments, the concentration of a salt (e.g., sodium chloride) reduces the formation of subvisible particles during manufacturing, wherein, as measured by FIM, the concentration of subvisible particles in the formulation is a fraction of the concentration of subvisible particles in a control formulation without the salt.About 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%. In some embodiments, the control formulation is identical to the formulation disclosed in this invention except that it does not contain salt or contains a different salt.

[0247] In some embodiments, the concentration of salt (e.g., sodium chloride) reduces the loss of viral titer during filtration, wherein, as measured by RCV-FFU or FFU assay, the viral titer in the filtered formulation is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the viral titer in the unfiltered formulation. In some embodiments, the concentration of salt (e.g., sodium chloride) reduces the loss of viral titer during filtration, wherein, as measured by RCV-FFU or FFU assay, the viral titer in the filtered formulation is approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% of the viral titer in the unfiltered formulation.

[0248] In some embodiments, the concentration of salt (e.g., sodium chloride) reduces the loss of viral titer during filtration, wherein, as measured by RCV-FFU or FFU assay, the viral titer in the filtered formulation is at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, or at least about 500%. In some embodiments, the concentration of salt (e.g., sodium chloride) reduces the loss of viral titer during filtration, wherein, as measured by RCV-FFU or FFU assay, the viral titer in the filtered formulation is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 400%, or about 500% higher than that of a control formulation without the salt. In some embodiments, the control formulation is identical to the formulation disclosed herein except that it is salt-free or contains a different salt.

[0249] In some embodiments, the liquid sand-like virus formulation disclosed herein is suitable for lyophilization. In some embodiments, the liquid sand-like virus preparation suitable for freeze-drying comprises concentrations of up to about 125 mM, up to about 100 mM, up to about 75 mM, at least about 50 mM, at least about 75 mM, at least about 100 mM, between about 50 mM and about 125 mM, and between about 50 mM and about 100 mM.The salts disclosed herein are present in concentrations between about 50 mM and about 75 mM, between about 75 mM and about 125 mM, between about 75 mM and about 100 mM, or between about 100 mM and about 125 mM. In some embodiments, the liquid isovirus preparation suitable for lyophilization contains salts disclosed herein at concentrations of about 50 mM, about 75 mM, about 100 mM, or about 125 mM. In some embodiments, the liquid isovirus preparation suitable for lyophilization contains salts disclosed herein at concentrations of up to about 125 mM or between about 50 mM and about 125 mM.

[0250] In some embodiments, the liquid isovirus preparation suitable for freeze-drying comprises sodium chloride at concentrations of up to about 125 mM, up to about 100 mM, up to about 75 mM, at least about 50 mM, at least about 75 mM, at least about 100 mM, between about 50 mM and about 125 mM, between about 50 mM and about 100 mM, between about 50 mM and about 75 mM, between about 75 mM and about 125 mM, between about 75 mM and about 100 mM, or between about 100 mM and about 125 mM. In some embodiments, the liquid isovirus preparation suitable for freeze-drying comprises sodium chloride at concentrations of about 50 mM, about 75 mM, about 100 mM, or about 125 mM. In some embodiments, the liquid isovirus formulation suitable for lyophilization contains sodium chloride at a concentration of up to about 125 mM or between about 50 mM and about 125 mM.

[0251] 5.1.4 Buffer Specification 32 / 63 pages 43 CN 121866044 A In some embodiments, the liquid isovirus formulation contains a buffer. A buffer is a buffer solution that resists pH changes through the action of its acid-base conjugate components.

[0252] The pH of the buffers used herein is generally within the physiological pH range (e.g., between about 6 and about 8); however, those skilled in the art will understand that a wider pH range may be required for the stability of the isovirus particles and / or other components present in the particular isovirus formulations disclosed herein. In some embodiments, the pH of the buffer disclosed herein may be at most about 6.5, at most about 7.0, at most about 7.5, at most about 8.0, at most about 8.5, at most about 9.0, at least about 6.5, at least about 7.0, at least about 7.5, at least about 8.0, at least about 8.5, at least about 9.0, between about 6.5 and about 9.0, between about 6.5 and about 8.5, between about 6.5 and about 8.0, between about 6.5 and about 7.5, between about 6.5 and about 7.0, between about 7.0 and about 7.5, between about 7.5 and about 8.0, between about 8.0 and about 8.5, or between about 8.5.The pH of the buffer disclosed herein may be between about 7.0 and about 9.0. In some embodiments, the pH of the buffer disclosed herein may be about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0. In some embodiments, the pH of the buffer disclosed herein may be about 8.0.

[0253] Examples of suitable buffers that can be used with this disclosure include, but are not limited to, phosphates (e.g., potassium phosphate, sodium phosphate), citrates (e.g., sodium citrate), sodium acetate, sodium succinate, histidine, imidazole, bicarbonate, carbonate, HEPES, TRIS, etc. In some embodiments, the liquid sand virus formulations disclosed herein may comprise buffers selected from the group consisting of phosphates, citrates, TRIS, HEPES, bicarbonate, histidine, and combinations thereof. HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (C8H18N2O4S)) buffers as used herein refer to zwitterionic N-substituted aminosulfonic acid buffers that may have a pKa of 7.5 at 25°C and may be useful in the pH range of 6.8–8.2. TRIS (C4H11NO3) buffers as used herein may have a pKa of 8.1 at 25°C and may be useful in the pH range of 7.0–9.0. In some embodiments, the liquid isovirus formulation disclosed herein comprises a HEPES buffer. In some embodiments, the liquid isovirus formulation disclosed herein comprises a TRIS buffer.

[0254] In some embodiments, the liquid isovirus formulation comprises a buffer at a concentration of up to about 50 mM, up to about 45 mM, up to about 40 mM, up to about 35 mM, up to about 30 mM, up to about 25 mM, up to about 20 mM, up to about 15 mM, up to about 10 mM, up to about 5 mM, or up to about 1 mM. In some embodiments, the liquid isovirus formulation comprises a buffer at a concentration of at least about 1 mM, at least about 5 mM, at least about 10 mM, at least about 15 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, or at least about 50 mM. In some embodiments, the liquid sand-like virus preparation contains buffer concentrations between approximately 1 mM and approximately 50 mM, between approximately 1 mM and approximately 45 mM, and between approximately 1 mM and approximately...Between 40mm, between approximately 1mm and approximately 35mm, between approximately 1mm and approximately 30mm, between approximately 1mm and approximately 25mm, between approximately 1mm and approximately 20mm, between approximately 1mm and approximately 15mm, between approximately 1mm and approximately 10mm, between approximately 1mm and approximately 5mm, between approximately 5mm and approximately 50mm, between approximately 5mm and approximately 45mm, between approximately 5mm and approximately 40mm, between approximately 5mm and approximately 35mm, between approximately 5mm and approximately 30mm, between approximately 5mm and approximately 25mm, between approximately 5mm and approximately 20mm, between approximately 5mm and approximately 15mm, between approximately 5mm and approximately 10mm, between approximately 10mm and approximately 50mm Between approximately 10mm and approximately 45mm, between approximately 10mm and approximately 40mm, between approximately 10mm and approximately 35mm, between approximately 10mm and approximately 30mm, between approximately 10mm and approximately 25mm, between approximately 10mm and approximately 20mm, between approximately 10mm and approximately 15mm, between approximately 15mm and approximately 50mm, between approximately 15mm and approximately 45mm, between approximately 15mm and approximately 40mm, between approximately 15mm and approximately 35mm, between approximately 15mm and approximately 30mm, between approximately 15mm and approximately 25mm, between approximately 15mm and approximately 20mm, between approximately 20mm and approximately 50mm, between approximately 20mm and approximately 45mm, between approximately 20mm and the instruction manual, page 33 / 63, 44 CN. 121866044 A Approximately 40 mm, between approximately 20 mm and approximately 35 mm, between approximately 20 mm and approximately 30 mm, between approximately 20 mm and approximately 25 mm, between approximately 25 mm and approximately 50 mm, between approximately 25 mm and approximately 45 mm, between approximately 25 mm and approximately 40 mm, between approximately 25 mm and approximately 35 mm, between approximately 25 mm and approximately 30 mm, between approximately 30 mm and approximately 50 mm Between M, between about 30 mM and about 45 mM, between about 30 mM and about 40 mM, between about 30 mM and about 35 mM, between about 35 mM and about 50 mM, between about 35 mM and about 45 mM, between about 35 mM and about 40 mM, between about 40 mM and about 50 mM, between about 40 mM and about 45 mM, or between about 45 mM and about 50 mM. In some embodiments, the liquid sand-like virus preparation comprises a buffer at concentrations of about 50 mM, about 45 mM, about 40 mM, about 35 mM, about 30 mM, about 25 mM, about 20 mM, about 15 mM, about 10 mM, about 5 mM, or about 1 mM.

[0255] In some embodiments, the liquid isovirus preparation comprises a buffer at a concentration between about 1 mM and about 50 mM. In some embodiments, the liquid isovirus preparation comprises a buffer at a concentration between about 10 mM and about 20 mM. In some embodiments, the liquid isovirus preparation comprises a buffer at a concentration between about 5 mM and about 15 mM. In some embodiments, the liquid isovirus preparation disclosed herein comprises a buffer at a concentration of about 10 mM.

[0256] In some embodiments, the liquid isovirus preparation comprises TRIS at concentrations of up to about 50 mM, up to about 45 mM, up to about 40 mM, up to about 35 mM, up to about 30 mM, up to about 25 mM, up to about 20 mM, up to about 15 mM, up to about 10 mM, up to about 5 mM, or up to about 1 mM. In some embodiments, the liquid sand virus preparation contains TRIS at concentrations of at least about 1 mM, at least about 5 mM, at least about 10 mM, at least about 15 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, or at least about 50 mM. In some embodiments, the liquid sand-like virus preparation comprises the following concentrations of TRIS: between about 1 mM and about 50 mM, between about 1 mM and about 45 mM, between about 1 mM and about 40 mM, between about 1 mM and about 35 mM, between about 1 mM and about 30 mM, between about 1 mM and about 25 mM, between about 1 mM and about 20 mM, between about 1 mM and about 15 mM, between about 1 mM and about 10 mM, between about 1 mM and about 5 mM, between about 5 mM and about 50 mM, between about 5 mM and about 45 mM, between about 5 mM and about 40 mM, between about 5 mM and about 35 mM, between about 5 mM and about 30 mM, between about 5 mM and about 25 mM, between about 5 mM and about 20 mM. Between approximately 5mm and approximately 15mm, Between approximately 5mm and approximately 10mm, Between approximately 10mm and approximately 50mm, Between approximately 10mm and approximately 45mm, Between approximately 10mm and approximately 40mm, Between approximately 10mm and approximately 35mm, Between approximately 10mm and approximately 30mm, Between approximately 10mm and approximately 25mm, Between approximately 10mm and approximately 20mm, Between approximately 10mm and approximately 15mm, Between approximately 15mm and approximately 50mm, Between approximately 15mm and approximately 45mm, Between approximately 15mm and approximately 40mm, Between approximately 15mm and approximately 35mm, Between approximately 15mm and approximately 30mm, Between approximately 15mm and approximately 25mm, Between approximately 15mm and approximately 20mmBetween approximately 20mm and approximately 50mm, Between approximately 20mm and approximately 45mm, Between approximately 20mm and approximately 40mm, Between approximately 20mm and approximately 35mm, Between approximately 20mm and approximately 30mm, Between approximately 20mm and approximately 25mm, Between approximately 25mm and approximately 50mm, Between approximately 25mm and approximately 45mm, Between approximately 25mm and approximately 40mm, Between approximately 25mm and approximately 35mm, Between approximately 25mm and approximately 30mm, Between approximately 30mm and approximately 50mm, Between approximately 30mm and approximately 45mm, Between approximately 30mm and approximately 40mm, Between approximately 30mm and approximately 35mm, Between approximately 35mm and approximately 50mm, Between approximately 35mm The concentrations are between about 45 mM, between about 35 mM and about 40 mM, between about 40 mM and about 50 mM, between about 40 mM and about 45 mM, or between about 45 mM and about 50 mM. In some embodiments, the liquid isovirus preparation contains TRIS at concentrations of about 50 mM, about 45 mM, about 40 mM, about 35 mM, about 30 mM, about 25 mM, about 20 mM, about 15 mM, about 10 mM, about 5 mM, or about 1 mM.

[0257] In some embodiments, the liquid isovirus preparation contains TRIS at concentrations between about 1 mM and about 50 mM. In some embodiments, the liquid isovirus preparation contains TRIS at concentrations between about 10 mM and about 20 mM. In some embodiments, the liquid isovirus preparation contains TRIS at concentrations between about 5 mM and about 15 mM. In some embodiments, as described on pages 34 / 63 of CN 121866044 A, the liquid sand virus preparation disclosed herein contains TRIS at a concentration of about 10 mM.

[0258] In some embodiments, the liquid sand virus preparation disclosed herein is suitable for lyophilization. In some embodiments, the liquid sand virus preparation suitable for lyophilization contains a buffer disclosed herein at a concentration of up to about 20 mM, up to about 18 mM, up to about 15 mM, at least about 10 mM, at least about 15 mM, at least about 18 mM, between about 10 mM and about 20 mM, between about 10 mM and about 18 mM, between about 10 mM and about 15 mM, between about 15 mM and about 20 mM, between about 15 mM and about 18 mM, or between about 18 mM and about 20 mM. In some embodiments, the liquid isovirus formulation suitable for lyophilization comprises a buffer disclosed herein at a concentration of about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, or about 20 mM. In some embodiments, the liquid isovirus formulation suitable for lyophilization comprises a buffer disclosed herein at a concentration of about 10 mM.

[0259] In some embodiments, the liquid isovirus preparation suitable for lyophilization contains TRIS at concentrations of up to about 20 mM, up to about 18 mM, up to about 15 mM, at least about 10 mM, at least about 15 mM, at least about 18 mM, between about 10 mM and about 20 mM, between about 10 mM and about 18 mM, between about 10 mM and about 15 mM, between about 15 mM and about 20 mM, between about 15 mM and about 18 mM, or between about 18 mM and about 20 mM. In some embodiments, the liquid isovirus preparation suitable for lyophilization contains TRIS at concentrations of about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, or about 20 mM. In some embodiments, the liquid isovirus preparation suitable for lyophilization contains TRIS at a concentration of about 10 mM.

[0260] 5.1.5 Surfactants In some embodiments, the liquid sand virus formulation comprises a surfactant. Without being bound by any theory, the addition of one or more surfactants to the liquid sand virus formulation disclosed herein can prevent or reduce the formation of subvisible particles in the sand virus formulation.

[0261] Suitable surfactants that can be used with this disclosure include, but are not limited to, alkylphenyl alkoxylates, alcohol alkoxylates, fatty amine alkoxylates, polyoxyethylene glycerol fatty acid esters, castor oil alkoxylates, fatty acid alkoxylates, fatty acid amide alkoxylates, fatty acid polydiethanolamides, lanolin ethoxylates, fatty acid polyethylene glycol esters, isotrigine alcohol, fatty acid amides, methylcellulose, fatty acid esters, silicone oil, alkyl polyglycosides, glycerol fatty acid esters, polyethylene glycol, polypropylene glycol, polyethylene glycol / polypropylene glycol block copolymers, polyethylene glycol alkyl ethers, polypropylene glycol alkyl ethers, Polyethylene glycol / polypropylene glycol ether block copolymers, polyacrylates, acrylic acid graft copolymers, alkyl aryl sulfonates, phenyl sulfonates, alkyl sulfates, alkyl sulfonates, alkyl ether sulfates, alkyl aryl ether sulfates, alkyl polyethylene glycol ether phosphates, polyarylphenyl ether phosphates, alkyl sulfosuccinates, olefin sulfonates, paraffin sulfonates, petroleum sulfonates, taurine salts, sarcosinates, fatty acids, alkyl naphthalene sulfonic acid, naphthalene sulfonic acid, lignin sulfonic acid, sulfonated naphthalene condensates, sulfite lignin waste liquid, alkyl phosphates, quaternary ammonium compounds, amine oxides, betaine, etc. In some embodiments, the surfactant is a nonionic surfactant, an ionic surfactant, or a combination thereof. In some embodiments, the surfactant is polysorbate, polyethylene glycol dehydrated sorbitan monolaurate (Tween® 20), polyoxyethylene dehydrated sorbitan monooleate (Tween® 80), a block copolymer of polyethylene and polypropylene glycol (e.g., poloxamer or Pluronic®), or a combination thereof.

[0262] In some embodiments, the surfactant is poloxamer. Poloxamer is a nonionic triblock copolymer consisting of a central hydrophobic polyoxypropylene (poly(propylene oxide)) chain and two flanking hydrophilic polyoxyethylene (poly(ethylene oxide)) chains. Because the lengths of the polymer blocks can be customized, many different poloxamers with slightly different properties can be used herein. For general terminology, poloxamer is typically named with the letter "P" (representing poloxamer) followed by three digits: the first two digits multiplied by 100 give the approximate molecular weight of the polyoxypropylene core, and the last digit multiplied by 10 gives the percentage of polyoxyethylene content (e.g., P407 = poloxamer with a polyoxypropylene molecular weight of 4000 g / mol and a polyoxyethylene content of 70%). In some embodiments, such as page 35 / 63 of CN 121866044 A, the surfactant used in this disclosure is selected from the group consisting of poloxamer 188, poloxamer 407, and combinations thereof.

[0263] In some embodiments, the surfactant used in this disclosure is a polysorbate. A polysorbate is an oily liquid derived from ethoxylated dehydrated sorbitol (a derivative of sorbitol) esterified with fatty acids. There are many examples of polysorbates, wherein the number following "polysorbate" is associated with the predominant fatty acid type related to the molecule. For example, monolaurate is represented as "20" in "polysorbate 20," and monooleate is represented as "80" in "polysorbate 80." In some embodiments, the surfactant used in this disclosure is selected from the group consisting of polysorbate 20, polysorbate 80, and combinations thereof.

[0264] In some embodiments, the surfactant is poloxamer, polysorbate, or a combination thereof. In some embodiments, the surfactant is selected from the group consisting of poloxamer 188, poloxamer 407, polysorbate 20, polysorbate 80, and combinations thereof. In some embodiments, the surfactant is poloxamer 188.

[0265] In some embodiments, the liquid sand-like virus preparation comprises concentrations of up to about 0.9% (w / v), up to about 0.8% (w / v), up to about 0.7% (w / v), up to about 0.6% (w / v), up to about 0.5% (w / v), up to about 0.4% (w / v), up to about 0.3% (w / v), up to about 0.2% (w / v), up to about 0.1% (w / v), up to about 0.09% (w / v), up to about 0.08% (w / v), up to about 0.07% (w / v), up to about 0.06% (w / v), up to about 0.05% (w / v), and up to about 0. 0.04% (w / v), up to about 0.03% (w / v), up to about 0.02% (w / v), up to about 0.01% (w / v), up to about 0.009% (w / v), up to about 0.008%Surfactants in amounts of up to about 0.007% (w / v), up to about 0.006% (w / v), up to about 0.005% (w / v), up to about 0.004% (w / v), up to about 0.003% (w / v), up to about 0.002% (w / v), or up to about 0.001% (w / v). In some embodiments, the liquid sand-like virus preparation comprises concentrations of at least about 0.001% (w / v), at least about 0.002% (w / v), at least about 0.003% (w / v), at least about 0.004% (w / v), at least about 0.005% (w / v), at least about 0.006% (w / v), at least about 0.007% (w / v), at least about 0.008% (w / v), at least about 0.009% (w / v), at least about 0.01% (w / v), and at least about 0.02%. (w / v), at least about 0.03% (w / v), at least about 0.04% (w / v), at least about 0.05% (w / v), at least about 0.06% (w / v), at least about 0.07% (w / v), at least about 0.08% (w / v), at least about 0.09% (w / v), at least about 0.1% (w / v), at least about 0.2% (w / v), at least about 0.3% (w / v), at least about 0.4% (w / v), at least about 0.5% (w / v), at least about 0.6% (w / v), at least about 0.7% (w / v), at least about 0.8% (w / v), or at least about 0.9% (w / v) surfactant. In some embodiments, the liquid sand-like virus preparation comprises concentrations between about 0.001% (w / v) and about 0.9% (w / v), between about 0.01% (w / v) and about 0.9% (w / v), between about 0.05% (w / v) and about 0.9% (w / v), between about 0.1% (w / v) and about 0.9% (w / v), between about 0.5% (w / v) and about 0.9% (w / v), between about 0.001% (w / v) and about 0.5% (w / v), between about 0.01% (w / v) and about 0.5% (w / v), between about 0.05% (w / v) and about 0.5% (w / v), and between about 0.1% (w / v) and about 0.1% (w / v). Between approximately 0.001% (w / v) and approximately 0.1% (w / v), between approximately 0.01% (w / v) and approximately 0.1% (w / v), between approximately 0.05% (w / v) and approximately 0.1% (w / v), between approximately 0.001% (w / v) and approximately 0.05% (w / v), between approximately 0.01% (w / v) and approximately 0.05% (w / v), between approximately 0.01% (w / v) and approximately 0.04% (w / v), and between approximately 0.05% (w / v) and approximately 0.05% (w / v).A surfactant at concentrations between about 0.02% (w / v) and about 0.04% (w / v), or between about 0.001% (w / v) and about 0.01% (w / v). In some embodiments, the liquid sand-like virus formulation comprises concentrations of about 0.9% (w / v), about 0.8% (w / v), about 0.7% (w / v), about 0.6% (w / v), about 0.5% (w / v), about 0.4% (w / v), about 0.3% (w / v), about 0.2% (w / v), about 0.1% (w / v), about 0.09% (w / v), about 0.08% (w / v), about 0.07% (w / v), about 0.06% (w / v), about 0.05% (w / v), and about 0.04%. (w / v), about 0.03% (w / v), about 0.02% (w / v), about 0.01% (w / v), about 0.009% (w / v), about 0.008% (w / v), about 0.007% (w / v), about 0.006% (w / v), about 0.005% (w / v), about 0.004% (w / v), about 0.003% (w / v), about 0.002% (w / v), or about 0.001% (w / v).

[0266] In some embodiments, the liquid sand virus formulation comprises a surfactant at a concentration between about 0.01% (w / v) and about 0.05% (w / v). In some embodiments, the liquid sand virus formulation comprises a surfactant at a concentration of about 0.03% (w / v).

[0267] In some embodiments, the liquid sand-like virus preparation comprises concentrations of up to about 0.9% (w / v), up to about 0.8% (w / v), up to about 0.7% (w / v), up to about 0.6% (w / v), up to about 0.5% (w / v), up to about 0.4% (w / v), up to about 0.3% (w / v), up to about 0.2% (w / v), up to about 0.1% (w / v), up to about 0.09% (w / v), up to about 0.08% (w / v), up to about 0.07% (w / v), up to about 0.06% (w / v), up to about 0.05% (w / v), and up to about 0. Poloxamer 188 at up to about 0.04% (w / v), up to about 0.03% (w / v), up to about 0.02% (w / v), up to about 0.01% (w / v), up to about 0.009% (w / v), up to about 0.008% (w / v), up to about 0.007% (w / v), up to about 0.006% (w / v), up to about 0.005% (w / v), up to about 0.004% (w / v), up to about 0.003% (w / v), up to about 0.002% (w / v), or up to about 0.001% (w / v). In someIn the implementation plan, the liquid sand-like virus preparation comprises concentrations of at least about 0.001% (w / v), at least about 0.002% (w / v), at least about 0.003% (w / v), at least about 0.004% (w / v), at least about 0.005% (w / v), at least about 0.006% (w / v), at least about 0.007% (w / v), at least about 0.008% (w / v), at least about 0.009% (w / v), at least about 0.01% (w / v), and at least about 0.02%. Poloxamer 188 at least about 0.03% (w / v), at least about 0.04% (w / v), at least about 0.05% (w / v), at least about 0.06% (w / v), at least about 0.07% (w / v), at least about 0.08% (w / v), at least about 0.09% (w / v), at least about 0.1% (w / v), at least about 0.2% (w / v), at least about 0.3% (w / v), at least about 0.4% (w / v), at least about 0.5% (w / v), at least about 0.6% (w / v), at least about 0.7% (w / v), at least about 0.8% (w / v), or at least about 0.9% (w / v). In some embodiments, the liquid sand-like virus preparation comprises concentrations between about 0.001% (w / v) and about 0.9% (w / v), between about 0.01% (w / v) and about 0.9% (w / v), between about 0.05% (w / v) and about 0.9% (w / v), between about 0.1% (w / v) and about 0.9% (w / v), between about 0.5% (w / v) and about 0.9% (w / v), between about 0.001% (w / v) and about 0.5% (w / v), between about 0.01% (w / v) and about 0.5% (w / v), between about 0.05% (w / v) and about 0.5% (w / v), and between about 0.1% (w / v) and about 0.1% (w / v). Poloxamer 188, between about 0.001% (w / v) and about 0.5% (w / v), between about 0.001% (w / v) and about 0.1% (w / v), between about 0.01% (w / v) and about 0.1% (w / v), between about 0.05% (w / v) and about 0.1% (w / v), between about 0.001% (w / v) and about 0.05% (w / v), between about 0.01% (w / v) and about 0.05% (w / v), between about 0.01% (w / v) and about 0.04% (w / v), between about 0.02% (w / v) and about 0.04% (w / v), or between about 0.001% (w / v) and about 0.01% (w / v). In some embodiments, the liquid sand-like virus preparation comprises concentrations of approximately 0.9% (w / v), approximately 0.8% (w / v), and approximately 0.7%.(w / v), approximately 0.6% (w / v), approximately 0.5% (w / v), approximately 0.4% (w / v), approximately 0.3% (w / v), approximately 0.2% (w / v), approximately 0.1% (w / v), approximately 0.09% (w / v), approximately 0.08% (w / v), approximately 0.07% (w / v), approximately 0.06% (w / v), approximately 0.05% (w / v), approximately 0.04% Poloxamer 188 at concentrations between about 0.01% (w / v), about 0.03% (w / v), about 0.02% (w / v), about 0.01% (w / v), about 0.009% (w / v), about 0.008% (w / v), about 0.007% (w / v), about 0.006% (w / v), about 0.005% (w / v), about 0.004% (w / v), about 0.003% (w / v), about 0.002% (w / v), or about 0.001% (w / v).

[0268] In some embodiments, the liquid isovirus preparation comprises poloxamer 188 at concentrations between about 0.01% (w / v) and about 0.05% (w / v). In some embodiments, the liquid isovirus preparation comprises poloxamer 188 at a concentration of about 0.03% (w / v).

[0269] In some embodiments, the concentration of a surfactant (e.g., poloxamer 188) reduces the formation of subvisible particles during manufacturing. In some embodiments, manufacturing includes a purification process. In some embodiments, manufacturing includes at least one chromatographic step.

[0270] In some embodiments, the concentration of a surfactant (e.g., poloxamer 188) reduces the formation of subvisible particles during manufacturing, wherein, as measured by FIM, the concentration of subvisible particles in the formulation is at most about 10%, at most about 20%, at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 70%, or at most about 80% of the concentration of subvisible particles in a control formulation without the surfactant. In some embodiments, the concentration of a surfactant (e.g., poloxamer 188) reduces the formation of subvisible particles during manufacturing, wherein, as measured by FIM, the concentration of subvisible particles in the formulation is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% of the concentration of subvisible particles in a control formulation without the surfactant. In some embodiments, the control formulation is identical to the formulation disclosed herein except that it does not contain a surfactant or contains a different surfactant.

[0271] In some embodiments, the liquid sand virus formulation disclosed herein is suitable for lyophilization. In some embodiments, the liquid sand virus formulation suitable for lyophilization contains at a concentration of up to about 0.05% (w / v), up to about 0.04% (w / v), and up toMore than about 0.03% (w / v), up to about 0.02% (w / v), at least about 0.01% (w / v), at least about 0.02% (w / v), at least about 0.03% (w / v), at least about 0.04% (w / v), between about 0.01% and about 0.05% (w / v), between about 0.01% (w / v) and about 0.04% (w / v), between about 0.01% and about 0.03% (w / v), between about 0.01% and about 0.02% (w / v), between about 0.02% and about 0.05% (w / v), between about 0.02% and about 0.04% (w / v), between about 0.03% (w / v), between about 0.02% and about 0.04% (w / v), between about 0.03% (w / v), between about 0.03% (w / v), between about 0.02% (w / v) and about 0.04% (w / v), between about 0.03% (w / v) and about 0.04% (w / v). The surfactant disclosed herein is present in concentrations between 0.02% and about 0.03% (w / v), between about 0.03% and about 0.05% (w / v), between about 0.03% and about 0.04% (w / v), or between about 0.04% and about 0.05% (w / v). In some embodiments, the liquid isovirus formulation suitable for lyophilization comprises a concentration of about 0.01%, about 0.02%, about 0.03%, about 0.04%, or about 0.05% of the surfactant disclosed herein. In some embodiments, the liquid isovirus formulation suitable for lyophilization comprises a concentration of about 0.03% (w / v) of the surfactant disclosed herein.

[0272] In some embodiments, the liquid isovirus formulation suitable for lyophilization comprises a concentration of up to about 0.05% (w / v). v), up to about 0.04% (w / v), up to about 0.03% (w / v), up to about 0.02% (w / v), at least about 0.01% (w / v), at least about 0.02% (w / v), at least about 0.03% (w / v), at least about 0.04% (w / v), between about 0.01% and about 0.05% (w / v), between about 0.01% and about 0.04% (w / v), between about 0.01% and about 0.03% (w / v), between about 0.01% and about 0.02% (w / v), between about 0.02% and about 0.05% (w / v), between about 0.01% and about 0.03% (w / v), between about 0.01% and about 0.02% (w / v), between about 0.02% and about 0.05% (w / v), between about 0.02% and about 0.04% (w / v), The concentration of poloxamer 188 is between about 0.02% and about 0.03% (w / v), between about 0.03% and about 0.05% (w / v), between about 0.03% and about 0.04% (w / v), or between about 0.04% and about 0.05% (w / v). In some embodiments, the liquid isovirus preparation suitable for lyophilization contains poloxamer 188 at a concentration of about 0.01%, about 0.02%, about 0.03%, about 0.04%, or about 0.05%. In some embodiments, the liquid isovirus preparation suitable for lyophilization contains poloxamer 188 at a concentration of about 0.03% (w / v).

[0273] 5.1.6 Stabilizer In some embodiments, the liquid isovirus formulation includes a stabilizer. Without being bound by any theory, adding a stabilizer to the liquid isovirus formulation disclosed herein inhibits nonspecific adsorption of virus particles throughout the manufacturing, formulation, and / or storage process, and / or stabilizes the isovirus formulation during freeze-thaw and storage processes.

[0274] In some embodiments, the stabilizer is a protein. In some embodiments, the protein is selected from the group consisting of albumin, recombinant proteins, plasma proteins, gelatin, and combinations thereof. In some embodiments, the stabilizer is albumin. In some embodiments, the albumin is lactalbumin, human serum albumin, recombinant human serum albumin (rHSA), or combinations thereof. In some embodiments, the stabilizer is rHSA.

[0275] In some embodiments, the liquid sand virus formulation comprises a stabilizer at concentrations of up to about 3% (w / v), up to about 2% (w / v), up to about 1% (w / v), up to about 0.5% (w / v), up to about 0.1% (w / v), up to about 0.05% (w / v), up to about 0.01% (w / v), up to about 0.005% (w / v), or up to about 0.001% (w / v). In some embodiments, the liquid sand virus preparation comprises a stabilizer at a concentration of at least about 3% (w / v), at least about 2% (w / v), at least about 1% (w / v), at least about 0.5% (w / v), at least about 0.1% (w / v), at least about 0.05% (w / v), at least about 0.01% (w / v), at least about 0.005% (w / v), or at least about 0.001% (w / v) of the product specification (page 38 / 63, 49 CN 121866044 A). In some embodiments, the liquid sand-like virus preparation comprises stabilizers at concentrations between about 0.001% (w / v) and about 3% (w / v), between about 0.005% (w / v) and about 3% (w / v), between about 0.01% (w / v) and about 3% (w / v), between about 0.05% (w / v) and about 3% (w / v), between about 0.1% (w / v) and about 3% (w / v), between about 0.5% (w / v) and about 3% (w / v), between about 1% (w / v) and about 3% (w / v), between about 2% (w / v) and about 3% (w / v), between about 0.001% (w / v) and about 2% (w / v), and between about 0.005% (w / v) and about 2% (w / v). Between, between about 0.01% (w / v) and about 2% (w / v), between about 0.05% (w / v) and about 2% (w / v), between aboutBetween 0.1% (w / v) and about 2% (w / v), between about 0.5% (w / v) and about 2% (w / v), between about 1% (w / v) and about 2% (w / v), between about 0.001% (w / v) and about 1% (w / v), between about 0.005% (w / v) and about 1% (w / v), between about 0.01% (w / v) and about 1% (w / v), between about 0.05% (w / v) and about 1% (w / v), between about 0.1% (w / v) and about 1% (w / v), between about 0.5% (w / v) and about 1% (w / v), between about 0.001% (w / v) and about 0.5% (w / v) Between approximately 0.005% (w / v) and approximately 0.5% (w / v), between approximately 0.01% (w / v) and approximately 0.5% (w / v), between approximately 0.05% (w / v) and approximately 0.5% (w / v), between approximately 0.1% (w / v) and approximately 0.5% (w / v), between approximately 0.05% (w / v) and approximately 0.3% (w / v), between approximately 0.05% (w / v) and approximately 0.2% (w / v), between approximately 0.05% (w / v) and approximately 0.2% (w / v). Between approximately 0.001% (w / v) and approximately 0.1% (w / v), between approximately 0.005% (w / v) and approximately 0.1% (w / v), between approximately 0.01% (w / v) and approximately 0.1% (w / v), between approximately 0.05% (w / v) and approximately 0.1% (w / v), between approximately 0.001% (w / v) and approximately 0.05% (w / v), between approximately 0.005% (w / v) and approximately 0.05% (w / v), The concentration is between about 0.01% (w / v) and about 0.05% (w / v), between about 0.001% (w / v) and about 0.01% (w / v), between about 0.005% (w / v) and about 0.01% (w / v), or between about 0.001% (w / v) and about 0.005% (w / v). In some embodiments, the liquid sand virus preparation comprises a stabilizer at concentrations of about 3% (w / v), about 2% (w / v), about 1% (w / v), about 0.5% (w / v), about 0.1% (w / v), about 0.05% (w / v), about 0.01% (w / v), about 0.005% (w / v), or about 0.001% (w / v).

[0276] In some embodiments, the liquid isovirus formulation comprises a stabilizer at a concentration between about 0.05% and about 0.15% (w / v). In some embodiments, the liquid isovirus formulation comprises a stabilizer at a concentration of about 0.1% (w / v).

[0277] In some embodiments, the liquid isovirus preparation contains rHSA at concentrations of up to about 3% (w / v), up to about 2% (w / v), up to about 1% (w / v), up to about 0.5% (w / v), up to about 0.1% (w / v), up to about 0.05% (w / v), up to about 0.01% (w / v), up to about 0.005% (w / v), or up to about 0.001% (w / v). In some embodiments, the liquid isovirus preparation contains rHSA at concentrations of at least about 3% (w / v), at least about 2% (w / v), at least about 1% (w / v), at least about 0.5% (w / v), at least about 0.1% (w / v), at least about 0.05% (w / v), at least about 0.01% (w / v), at least about 0.005% (w / v), or at least about 0.001% (w / v). In some embodiments, the liquid sand-like virus preparation comprises rHSA at concentrations between about 0.001% (w / v) and about 3% (w / v), between about 0.005% (w / v) and about 3% (w / v), between about 0.01% (w / v) and about 3% (w / v), between about 0.05% (w / v) and about 3% (w / v), between about 0.1% (w / v) and about 3% (w / v), between about 0.5% (w / v) and about 3% (w / v), between about 1% (w / v) and about 3% (w / v), between about 2% (w / v) and about 3% (w / v), between about 0.001% (w / v) and about 2% (w / v), and between about 0.005% (w / v) and about 2% (w / v). Between approximately 0.01% (w / v) and approximately 2% (w / v), between approximately 0.05% (w / v) and approximately 2% (w / v), between approximately 0.1% (w / v) and approximately 2% (w / v), between approximately 0.5% (w / v) and approximately 2% (w / v), between approximately 1% (w / v) and approximately 2% (w / v), between approximately 0.001% (w / v) and approximately 1% (w / v), between approximately 0.005% (w / v) and approximately 1% (w / v), between approximately 0.01% (w / v) and approximately 1% (w / v), between approximately 0.05% (w / v) and approximately 1% (w / v), between approximately 0.1% (w / v) and approximately 1% (w / v). Between (w / v), between about 0.5% (w / v) and about 1% (w / v), between about 0.001% (w / v) and about 0.5% (w / v), between about 0.005% (w / v) and about 0.5% (w / v), between about 0.01% (w / v) and about 0.5% (w / v), between about 0.05% (w / v) and about 0.5% (w / v), between about 0.1% (w / v) and about 0.5% (w / v), between about 0.05% (w / v) and about 0.5% (w / v) (Note)Page 39 / 63, CN 121866044, A v) and about 0.3% (w / v), between about 0.05% (w / v) and about 0.2% (w / v), between about 0.05% (w / v) and about 0.15% (w / v), between about 0.001% (w / v) and about 0.1% (w / v), between about 0.005% (w / v) and about 0.1% Between (w / v), between about 0.01% (w / v) and about 0.1% (w / v), between about 0.05% (w / v) and about 0.1% (w / v), between about 0.001% (w / v) and about 0.05% (w / v), between about 0.005% (w / v) and about 0.05% (w / v), between about 0.01% (w / v) and about 0.05% (w / v), between about 0.001% (w / v) and about 0.01% (w / v), between about 0.005% (w / v) and about 0.01% (w / v), or between about 0.001% (w / v) and about 0.005% (w / v). In some embodiments, the liquid isovirus formulation comprises rHSA at concentrations of about 3% (w / v), about 2% (w / v), about 1% (w / v), about 0.5% (w / v), about 0.1% (w / v), about 0.05% (w / v), about 0.01% (w / v), about 0.005% (w / v), or about 0.001% (w / v).

[0278] In some embodiments, the liquid isovirus formulation comprises rHSA at concentrations between about 0.05% and about 0.15% (w / v). In some embodiments, the liquid isovirus formulation comprises rHSA at a concentration of about 0.1% (w / v).

[0279] In some embodiments, the concentration of the stabilizer (e.g., rHSA) reduces the loss of virus titer during freeze-thaw processes and / or storage.

[0280] In some embodiments, the concentration of the stabilizer (e.g., rHSA) reduces the loss of viral titer during freeze-thaw processes and / or storage, wherein, as measured by RCV-FFU or FFU assay, the viral titer in the formulation after freeze-thaw processes and / or storage is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the viral titer in the formulation before freeze-thaw processes and / or storage. In some embodiments, the concentration of the stabilizer (e.g., rHSA) reduces the loss of viral titer during freeze-thaw processes and / or storage, wherein, as measured by RCV-FFU or FFU assay, the viral titer in the formulation after freeze-thaw processes and / or storage is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the viral titer in the formulation before freeze-thaw processes and / or storage.The viral titers in the pre-existing formulation are approximately 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0281] In some embodiments, the concentration of the stabilizer (e.g., rHSA) reduces the loss of viral titer during freeze-thaw processes and / or storage, wherein, as measured by RCV-FFU or FFU assay, the viral titer in the formulation after freeze-thaw processes and / or storage is at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, or at least about 500%. In some embodiments, the concentration of the stabilizer (e.g., rHSA) reduces the loss of viral titer during freeze-thaw cycles and / or storage, wherein, as measured by RCV-FFU or FFU assay, the viral titer in the formulation after freeze-thaw cycles and / or storage is approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 100%, approximately 150%, approximately 200%, approximately 250%, approximately 300%, approximately 400%, or approximately 500% higher than that of a control formulation without the stabilizer after freeze-thaw cycles and / or storage. In some embodiments, the control formulation is identical to the formulation disclosed herein except that it does not contain a stabilizer or contains a different stabilizer.

[0282] In some embodiments, the storage temperature is above about 2°C, is room temperature (e.g., 20°C–25°C), is between about 2°C and about 8°C, is above about 15°C, is about -20°C or below about -20°C, is about -65°C or below about -65°C, or is about -80°C. In some embodiments, the storage temperature is between about 2°C and about 8°C. In some embodiments, the storage lasts for at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 3 years, at least about 4 years, at least about 5 years, or longer. In some embodiments, the storage lasts for at least about 1 month.

[0283] In some embodiments, the liquid sand virus preparation disclosed herein is suitable for lyophilization. In some embodiments, the liquid sand-like virus preparation suitable for lyophilization contains concentrations of up to about 0.15% (w / v), up to about 0.1% (w / v), and up to...At least about 0.05% (w / v), at least about 0.1% (w / v), between about 0.05% and about 0.15% (w / v), between about 0.05% (w / v) and about 0.1% (w / v), or between about 0.1% and about 0.15% (w / v) of the stabilizer disclosed herein. In some embodiments, the liquid isovirus formulation suitable for lyophilization comprises a concentration of about 0.05%, about 0.08%, about 0.1%, about 0.12%, or about 0.15% of the stabilizer disclosed herein. In some embodiments, the liquid isovirus formulation suitable for lyophilization comprises a concentration of about 0.1% (w / v) of the stabilizer disclosed herein.

[0284] In some embodiments, the liquid isovirus formulation disclosed herein is suitable for lyophilization. In some embodiments, the liquid isovirus formulation suitable for lyophilization contains rHSA at concentrations of up to about 0.15% (w / v), up to about 0.1% (w / v), at least about 0.05% (w / v), at least about 0.1% (w / v), between about 0.05% and about 0.15% (w / v), between about 0.05% (w / v) and about 0.1% (w / v), or between about 0.1% and about 0.15% (w / v). In some embodiments, the liquid isovirus formulation suitable for lyophilization contains rHSA at concentrations of about 0.05%, about 0.08%, about 0.1%, about 0.12%, or about 0.15%. In some embodiments, the liquid isovirus formulation suitable for lyophilization contains rHSA at a concentration of about 0.1% (w / v).

[0285] 5.1.7 Osmotic Concentration and pH In some embodiments, the osmotic concentration of the liquid sand-like virus preparation disclosed herein is at least about 200 mOsm / kg, at least about 300 mOsm / kg, at least about 400 mOsm / kg, at least about 500 mOsm / kg, at least about 600 mOsm / kg. kg, at least about 700 mOsm / kg, at least about 800 mOsm / kg, at least about 900 mOsm / kg, at least about 1000 mOsm / kg, at most about 200 mOsm / kg, at most about 300 mOsm / kg, at most about 400 mOsm / kg, at most about 500 mOsm / kg, at most about 600 mOsm / kg, at most about 700 mOsm / kg, at most about 800 mOsm / kg, at most about 900 mOsm / kg, at most about 1000 mOsm / kg, between about 200 mOsm / kg and about 1000 mOsm / kg, between about 300 mOsm / kg and about 1000 mOsm / kg, betweenBetween approximately 400 mOsm / kg and approximately 1000 mOsm / kg, between approximately 500 mOsm / kg and approximately 1000 mOsm / kg, between approximately 600 mOsm / kg and approximately 1000 mOsm / kg, between approximately 700 mOsm / kg and approximately 1000 mOsm / kg, between approximately 800 mOsm / kg and approximately 1000 mOsm / kg, between approximately 900 mOsm / kg and approximately 1000 mOsm / kg, between approximately 200 mOsm / kg and approximately 900 mOsm / kg, between approximately 300 mOsm / kg and approximately 900 mOsm / kg, between approximately 400 mOsm / kg / kg and approximately 900 mOsm / kg, between approximately 500 mOsm / kg and approximately 900 mOsm / kg, between approximately 600 mOsm / kg and approximately 900 mOsm / kg, between approximately 700 mOsm / kg and approximately 900 mOsm / kg, between approximately 800 mOsm / kg and approximately 900 mOsm / kg, between approximately 200 mOsm / kg and approximately 800 mOsm / kg, between approximately 300 mOsm / kg and approximately 800 mOsm / kg, between approximately 400 mOsm / kg and approximately 800 mOsm / kg, between approximately 500 mOsm / kg and approximately 800 mOsm / kg Between approximately 600 mOsm / kg and approximately 800 mOsm / kg, between approximately 700 mOsm / kg and approximately 800 mOsm / kg, between approximately 200 mOsm / kg and approximately 700 mOsm / kg, between approximately 300 mOsm / kg and approximately 700 mOsm / kg, between approximately 400 mOsm / kg and approximately 700 mOsm / kg, between approximately 500 mOsm / kg and approximately 700 mOsm / kg, between approximately 600 mOsm / kg and approximately 700 mOsm / kg, between approximately 200 mOsm / kg and approximately 600 mOsm / kg, between approximately 300 mOsm / kg Between approximately 600 mOsm / kg, between approximately 400 mOsm / kg and approximately 600 mOsm / kg, between approximately 500 mOsm / kg and approximately 600 mOsm / kg, between approximately 200 mOsm / kg and approximately 500 mOsm / kg, between approximately 300 mOsm / kg and approximately 500 mOsm / kg, between approximately 400 mOsm / kg and approximately 500 mOsm / kg, between approximately 200 mOsm / kg and approximately 400 mOsm / kg, between approximately 300 mOsm / kg and approximately 400 mOsm / kg, or between approximately 200 mOsm / kg and approximately 300 mOsm / kg. In some implementationsIn this specification, the penetrating concentration of the liquid isovirus preparation disclosed herein is approximately 200 mOsm / kg, approximately 300 mOsm / kg, approximately 400 mOsm / kg, approximately 500 mOsm / kg, approximately 600 mOsm / kg, approximately 700 mOsm / kg, approximately 800 mOsm / kg, approximately 900 mOsm / kg, or approximately 1000 mOsm / kg. In some embodiments, the penetrating concentration of the liquid isovirus preparation disclosed herein is between approximately 500 mOsm / kg and approximately 600 mOsm / kg. In some embodiments, the penetrating concentration of the liquid isovirus preparation disclosed herein (pages 41 / 63, 52 CN 121866044 A) is between approximately 500 mOsm / kg and approximately 550 mOsm / kg. In some embodiments, the osmotic concentration of the liquid isovirus preparation disclosed herein is about 530 mOsm / kg. In some embodiments, the osmotic concentration of the liquid isovirus preparation disclosed herein is between about 350 mOsm / kg and about 450 mOsm / kg. In some embodiments, the osmotic concentration of the liquid isovirus preparation disclosed herein is about 400 mOsm / kg.

[0286] In some embodiments, the liquid isovirus preparation disclosed herein is suitable for lyophilization and has an osmotic concentration between about 300 mOsm / kg and about 600 mOsm / kg.

[0287] In some embodiments, the pH of the liquid isovirus preparation disclosed herein is between about 3.0 and about 9. Between 0.0, between approximately 4.0 and approximately 9.0, between approximately 5.0 and approximately 9.0, between approximately 6.0 and approximately 9.0, between approximately 7.0 and approximately 9.0, between approximately 8.0 and approximately 9.0, between approximately 3.0 and approximately 8.0, between approximately 4.0 and approximately 8.0, between approximately 5.0 and approximately 8.0, between approximately 6.0 and approximately 8.0, between approximately 7.0 and approximately 8.0, between approximately 3.0 and approximately 7.0, between approximately 4.0 and approximately 7.0, between approximately 5.0 and approximately 7.0, between approximately 6.0 and approximately 7.0, between approximately 3.0 and approximately 6.0, between approximately 4.0 and approximately 6.0, between approximately 5.0 and approximately 6.0, between approximately 3.0 and approximately 5.0, between approximately 4.0 and approximately 5.0, or between approximately 3.0 and approximately 4.0. In some embodiments, the pH of the liquid sand-like virus preparation disclosed herein is about 3.0, about 4.0, about 5.0, about 6.0, or about 7.0.

[0288] In some embodiments, the pH of the liquid sand-like virus preparation disclosed herein is between about 7.0 and about 9.0. In some embodiments, the pH of the liquid sand-like virus preparation disclosed herein is about 8.0.

[0289] 5.1.8 Exemplary Liquid Sand-like Virus PreparationIn some embodiments, the liquid sand virus formulation disclosed herein comprises (a) sand virus (see Section 5.3); (b) a polyvalent anion at a concentration between about 10 mM and about 60 mM (see Section 5.1.1); (c) a carbohydrate at a concentration between about 3% and about 10% (w / v) (see Section 5.1.2); (d) a salt at a concentration between about 50 mM and about 250 mM (see Section 5.1.3); (e) a buffer at a concentration between about 10 mM and about 20 mM (see Section 5.1.4); (f) a surfactant at a concentration between about 0.01% and about 0.05% (w / v) (see Section 5.1.5); and (g) a stabilizer at a concentration between about 0.05% (w / v) and 0.15% (w / v) (see Section 5.1.5). (Section 5.1.6); wherein the pH of the liquid isovirus preparation is about 8.0. In some embodiments, the permeation concentration of the liquid isovirus preparation is between about 500 mOsm / kg and about 600 mOsm / kg. In some embodiments, the permeation concentration of the liquid isovirus preparation is between about 350 mOsm / kg and about 450 mOsm / kg. In some embodiments, the liquid isovirus preparation disclosed herein is suitable for lyophilization. In some embodiments, the permeation concentration of the liquid isovirus preparation suitable for lyophilization is between about 300 mOsm / kg and about 600 mOsm / kg.

[0290] In some embodiments, the liquid isovirus formulation disclosed herein comprises (a) isovirus (see Section 5.3); (b) a polyvalent anion at a concentration of about 50 mM (see Section 5.1.1); (c) a carbohydrate at a concentration of about 5% (w / v) (see Section 5.1.2); (d) a salt at a concentration of about 125 mM (see Section 5.1.3); (e) a buffer at a concentration of about 10 mM (see Section 5.1.4); (f) a surfactant at a concentration of about 0.03% (w / v) (see Section 5.1.5); and (g) a stabilizer at a concentration of about 0.1% (w / v) (see Section 5.1.6); wherein the pH of the liquid isovirus formulation is about 8.0. In some embodiments, the osmotic concentration of the liquid isovirus formulation is about 530 mOsm / kg.

[0291] In some embodiments, the liquid sand virus formulation disclosed herein comprises (a) sand virus (see Section 5.3); (b) a polyvalent anion at a concentration of about 25 mM (see Section 5.1.1); (c) a carbohydrate at a concentration of about 5% (w / v) (see Section 5.1.2); (d) a salt at a concentration of about 75 mM (see Section 5.1.3); and (e) a buffer at a concentration of about 10 mM (see Section 5.1.3).(5.1.4); (f) a surfactant at a concentration of about 0.03% (w / v) (see Section 5.1.5), and (g) a stabilizer at a concentration of about 0.1% (w / v) (see Section 5.1.6); wherein the pH of the liquid sand-particle virus preparation is about 8.0. In some embodiments, as described on pages 42 / 63 of the specification, 53 CN 121866044 A, the permeation concentration of the liquid sand-particle virus preparation is about 400 mOsm / kg.

[0292] In some embodiments, the liquid isovirus formulation disclosed herein comprises (a) isovirus (see Section 5.3); (b) citrate at a concentration between about 10 mM and about 60 mM; (c) sucrose at a concentration between about 3% and about 10% (w / v); (d) sodium chloride at a concentration between about 50 mM and about 250 mM; (e) TRIS at a concentration between about 10 mM and about 20 mM; (f) poloxamer 188 at a concentration between about 0.01% and about 0.05% (w / v); and (g) rHSA at a concentration between about 0.05% (w / v) and 0.15% (w / v), wherein the pH of the liquid isovirus formulation is about 8.0. In some embodiments, the osmotic concentration of the liquid isovirus formulation is between about 500 mOsm / kg and about 600 mOsm / kg.

[0293] In some embodiments, the liquid isovirus formulation disclosed herein comprises (a) isovirus; (b) citrate at a concentration of about 50 mM; (c) sucrose at a concentration of about 5% (w / v); (d) sodium chloride at a concentration of about 125 mM; (e) TRIS at a concentration of about 10 mM; (f) poloxamer 188 at a concentration of about 0.03% (w / v); and (g) rHSA at a concentration of about 0.1% (w / v), wherein the pH of the liquid isovirus formulation is about 8.0. In some embodiments, the permeation concentration of the liquid isovirus formulation is about 530 mOsm / kg.

[0294] In some embodiments, the liquid isovirus formulation disclosed herein comprises (a) isovirus (see Section 5.3); (b) citrate at a concentration between about 10 mM and about 60 mM; (c) trehalose at a concentration between about 3% and about 10% (w / v); (d) sodium chloride at a concentration between about 50 mM and about 250 mM; (e) TRIS at a concentration between about 10 mM and about 20 mM; (f) poloxamer 188 at a concentration between about 0.01% and about 0.05% (w / v); and (g) rHSA at a concentration between about 0.05% (w / v) and 0.15% (w / v), wherein the pH of the liquid isovirus formulation is about 8.0. In some embodiments, the osmotic concentration of the liquid isovirus formulation is between about 350 mOsm / kg and about 450 mOsm / kg.

[0295] In some embodiments, the liquid isovirus formulation disclosed herein comprises (a) isovirus; (b) citrate at a concentration of about 25 mM; (c) trehalose at a concentration of about 5% (w / v); (d) sodium chloride at a concentration of about 75 mM; (e) TRIS at a concentration of about 10 mM; (f) poloxamer 188 at a concentration of about 0.03% (w / v); and (g) rHSA at a concentration of about 0.1% (w / v), wherein the pH of the liquid isovirus formulation is about 8.0. In some embodiments, the permeation concentration of the liquid isovirus formulation is about 400 mOsm / kg.

[0296] In some embodiments, the liquid sand virus formulation disclosed herein is suitable for lyophilization and comprises (a) sand virus (see Section 5.3); (b) a polyvalent anion at a concentration of up to about 50 mM or between about 20 mM and 50 mM (see Section 5.1.1); (c) a carbohydrate at a concentration of about 5% (w / v) (see Section 5.1.2); (d) a salt at a concentration of up to about 125 mM or between 50 mM and about 125 mM (see Section 5.1.3); (e) a buffer at a concentration of about 10 mM (see Section 5.1.4); (f) a surfactant at a concentration of about 0.03% (w / v) (see Section 5.1.5); and (g) a stabilizer at a concentration of about 0.1% (w / v) (see Section 5.1.5). (Section 5.1.6); wherein the pH of the liquid sand virus preparation is about 8.0. In some embodiments, the permeation concentration of the liquid sand virus preparation suitable for lyophilization is between about 300 mOsm / kg and about 600 mOsm / kg.

[0297] In some embodiments, the liquid isovirus formulation suitable for lyophilization disclosed herein comprises (a) isovirus; (b) citrate at a concentration of up to about 50 mM or between about 20 mM and 50 mM; (c) trehalose at a concentration of about 5% (w / v); (d) sodium chloride at a concentration of up to about 125 mM or between about 50 mM and about 125 mM; (e) TRIS at a concentration of about 10 mM; (f) poloxamer 188 at a concentration of about 0.03% (w / v); and (g) rHSA at a concentration of about 0.1% (w / v), wherein the pH of the liquid isovirus formulation is about 8.0. In some embodiments, the permeation concentration of the liquid isovirus formulation suitable for lyophilization is between about 300 mOsm / kg and about 600 mOsm / kg.

[0298] 5.2 Freeze-dried Sand-like Virus Formulation This disclosure also provides freeze-dried sand-like virus formulations. In some embodiments, the freeze-dried sand-like virus formulation is prepared from the liquid sand-like virus formulation disclosed herein (see Section 5.1). In some embodiments, the freeze-dried sand-like virus formulation is prepared from the liquid sand-like virus formulation disclosed herein (see Section 5.1).The agent is prepared from the liquid isovirus formulation disclosed herein (see Section 5.1), wherein the liquid isovirus formulation is suitable for lyophilization.

[0299] In some embodiments, the lyophilized isovirus formulation comprises isovirus (see Section 5.3) and a polyvalent anion (see Section 5.1.1). In some embodiments, the lyophilized isovirus formulation comprises at least one of a carbohydrate (see Section 5.1.2), a salt (see Section 5.1.3), a buffer (see Section 5.1.4), a surfactant (see Section 5.1.5), and a stabilizer (see Section 5.1.6).

[0300] In some embodiments, the lyophilized arenavirus preparation is prepared from a liquid arenavirus preparation suitable for lyophilization, wherein the liquid arenavirus preparation suitable for lyophilization contains a polyvalent anion disclosed herein at a concentration of up to about 50 mM, up to about 40 mM, up to about 30 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, between about 20 mM and about 50 mM, between about 20 mM and about 40 mM, between about 20 mM and about 30 mM, between about 30 mM and about 50 mM, between about 30 mM and about 40 mM, or between about 40 mM and about 50 mM. In some embodiments, the liquid arenavirus preparation suitable for lyophilization contains a polyvalent anion disclosed herein at a concentration of about 20 mM, about 30 mM, about 40 mM, or about 50 mM. In some embodiments, the liquid isovirus preparation suitable for lyophilization contains up to about 50 mM of the polyvalent anions disclosed herein. In some embodiments, the liquid isovirus preparation suitable for lyophilization contains polyvalent anions disclosed herein at concentrations between about 20 mM and about 50 mM.

[0301] In some embodiments, the lyophilized isovirus preparation is prepared from a liquid isovirus preparation suitable for lyophilization, wherein the liquid isovirus preparation suitable for lyophilization contains citrate at concentrations of up to about 50 mM, up to about 40 mM, up to about 30 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, between about 20 mM and about 50 mM, between about 20 mM and about 40 mM, between about 20 mM and about 30 mM, between about 30 mM and about 50 mM, between about 30 mM and about 40 mM, or between about 40 mM and about 50 mM. In some embodiments, the liquid isovirus preparation suitable for lyophilization contains citrate at a concentration of about 20 mM, about 30 mM, about 40 mM, or about 50 mM. In some embodiments, the liquid isovirus preparation suitable for lyophilization contains citrate at a concentration of up to about 50 mM. In some embodiments, the liquid isovirus preparation suitable for lyophilization contains citrate at a concentration between about 20 mM and about 50 mM.

[0302] In some embodiments, the lyophilized isan virus preparation is prepared from a liquid isan virus preparation suitable for lyophilization, wherein the liquid isan virus preparation suitable for lyophilization comprises concentrations of up to about 10% (w / v), up to about 8% (w / v), up to about 6% (w / v), up to about 4% (w / v), at least about 3% (w / v), at least about 6% (w / v), at least about 8% (w / v), between about 3% and about 10% (w / v), between about 3% (w / v) and about 8% (w / v), between about 3% and about 6% (w / v), between about 4% and about 10% (w / v), between about 4% and about 8% (w / v), between about 4% and about 6% (w / v), and between about 6% and about 10%. The carbohydrates disclosed herein are present at concentrations between 6% and 8% (w / v), or between 8% and 10% (w / v). In some embodiments, the liquid isovirus formulation suitable for lyophilization comprises the carbohydrates disclosed herein at concentrations of about 3% (w / v), about 4% (w / v), about 5% (w / v), about 6% (w / v), about 7% (w / v), about 8% (w / v), about 9% (w / v), or about 10% (w / v). In some embodiments, the liquid isovirus formulation suitable for lyophilization comprises the carbohydrates disclosed herein at a concentration of about 5% (w / v).

[0303] In some embodiments, the freeze-dried isovirus preparation is prepared from a liquid isovirus preparation suitable for freeze-drying, wherein the liquid isovirus preparation suitable for freeze-drying comprises concentrations of up to about 10% (w / v), up to about 8% (w / v), up to about 6% (w / v), up to about 4% (w / v), at least about 3% (w / v), at least about 6% (w / v), at least about 8% (w / v), between about 3% and about 10% (w / v), between about 3% (w / v) and about 8% (w / v), between about 3% and about 6% (w / v), between about 4% and about 10% (w / v), between about 4% and about 8% (w / v), between about 4% and about 6% (w / v), and between about 6% and about 10%. The concentration of trehalose is between 6% and 8% (w / v), or between 8% and 10% (w / v). In some embodiments, the liquid isovirus preparation suitable for lyophilization contains trehalose at concentrations of about 3% (w / v), about 4% (w / v), about 5% (w / v), about 6% (w / v), about 7% (w / v), about 8% (w / v), about 9% (w / v), or about 10% (w / v). In some embodiments, the liquid isovirus preparation suitable for lyophilization contains trehalose at a concentration of about 5% (w / v).

[0304] In some embodiments, the lyophilized arenavirus preparation is prepared from a liquid arenavirus preparation suitable for lyophilization, wherein the liquid arenavirus preparation suitable for lyophilization comprises salts disclosed herein at concentrations of up to about 125 mM, up to about 100 mM, up to about 75 mM, at least about 50 mM, at least about 75 mM, at least about 100 mM, between about 50 mM and about 125 mM, between about 50 mM and about 100 mM, between about 50 mM and about 75 mM, between about 75 mM and about 125 mM, between about 75 mM and about 100 mM, or between about 100 mM and about 125 mM. In some embodiments, the liquid arenavirus preparation suitable for lyophilization comprises salts disclosed herein at concentrations of about 50 mM, about 75 mM, about 100 mM, or about 125 mM. In some embodiments, the liquid isovirus preparation suitable for lyophilization comprises a salt disclosed herein at a concentration of up to about 125 mM. In some embodiments, the liquid isovirus preparation suitable for lyophilization comprises a salt disclosed herein at a concentration between about 50 mM and about 125 mM.

[0305] In some embodiments, the freeze-dried isovirus preparation is prepared from a liquid isovirus preparation suitable for freeze-drying, wherein the liquid isovirus preparation suitable for freeze-drying contains sodium chloride at concentrations of up to about 125 mM, up to about 100 mM, up to about 75 mM, at least about 50 mM, at least about 75 mM, at least about 100 mM, between about 50 mM and about 125 mM, between about 50 mM and about 100 mM, between about 50 mM and about 75 mM, between about 75 mM and about 125 mM, between about 75 mM and about 100 mM, or between about 100 mM and about 125 mM. In some embodiments, the liquid isovirus preparation suitable for freeze-drying contains sodium chloride at concentrations of about 50 mM, about 75 mM, about 100 mM, or about 125 mM. In some embodiments, the liquid isovirus preparation suitable for lyophilization contains sodium chloride at a concentration of up to about 125 mM. In some embodiments, the liquid isovirus preparation suitable for lyophilization contains sodium chloride at a concentration between about 50 mM and about 125 mM.

[0306] In some embodiments, the lyophilized isovirus preparation is prepared from a liquid isovirus preparation suitable for lyophilization, wherein the liquid isovirus preparation suitable for lyophilization contains a buffer disclosed herein at a concentration of up to about 20 mM, up to about 18 mM, up to about 15 mM, at least about 10 mM, at least about 15 mM, at least about 18 mM, between about 10 mM and about 20 mM, between about 10 mM and about 18 mM, between about 10 mM and about 15 mM, between about 15 mM and about 20 mM, between about 15 mM and about 18 mM, or between about 18 mM and about 20 mM. In some implementations, liquid sand virus suitable for freeze-dryingThe preparation contains a buffer disclosed herein at a concentration of about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, or about 20 mM. In some embodiments, the liquid sand virus preparation suitable for lyophilization contains a buffer disclosed herein at a concentration of about 10 mM.

[0307] In some embodiments, the freeze-dried isovirus preparation is prepared from a liquid isovirus preparation suitable for freeze-drying, wherein the liquid isovirus preparation suitable for freeze-drying contains TRIS at concentrations of up to about 20 mM, up to about 18 mM, up to about 15 mM, at least about 10 mM, at least about 15 mM, at least about 18 mM, or between about 10 mM and about 20 mM, between about 10 mM and about 18 mM, between about 10 mM and about 15 mM, between about 15 mM and about 20 mM, between about 15 mM and about 18 mM, or between about 18 mM and about 20 mM. In some embodiments, the liquid isovirus preparation suitable for freeze-drying contains TRIS at concentrations of about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, or about 20 mM. In some embodiments, the liquid isovirus preparation suitable for lyophilization contains TRIS at a concentration of about 10 mM.

[0308] In some embodiments, the lyophilized isovirus preparation is prepared from a liquid isovirus preparation suitable for lyophilization, wherein the liquid isovirus preparation suitable for lyophilization contains at a concentration of up to about 0.05% (w / v), up to about 0.04% (w / v), up to about 0.03% (w / v), up to about 0.02% (w / v), at least about 0.01% (w / v), at least about 0.02% (w / v), at least about 0.03% (w / v), at least about 0.04% (w / v), between about 0.01% and about 0.05% (w / v), between about 0.01% (w / v) and about 0.04% (w / v), between about 0.01% and about 0.05% (w / v), between about 0.01% (w / v) and about 0.05% (w / v). (See page 45 / 63 of the specification, 56 CN 121866044 A) The surfactants disclosed herein are present in amounts between 0.04% (w / v), between about 0.01% and about 0.03% (w / v), between about 0.01% and about 0.02% (w / v), between about 0.02% and about 0.05% (w / v), between about 0.02% and about 0.04% (w / v), between about 0.02% and about 0.03% (w / v), between about 0.03% and about 0.05% (w / v), between about 0.03% and about 0.04% (w / v), or between about 0.04% and about 0.05% (w / v). In some embodiments, the liquid sand-like virus preparation suitable for freeze-drying comprises concentrations of about 0.01% (w / v), about 0.02% (w / v), about 0.03% (w / v), and about...0.04% (w / v) or about 0.05% (w / v) of the surfactant disclosed herein. In some embodiments, the liquid sand virus formulation suitable for lyophilization contains about 0.03% (w / v) of the surfactant disclosed herein.

[0309] In some embodiments, the freeze-dried sand virus preparation is prepared from a liquid sand virus preparation suitable for freeze-drying, wherein the liquid sand virus preparation suitable for freeze-drying comprises concentrations of up to about 0.05% (w / v), up to about 0.04% (w / v), up to about 0.03% (w / v), up to about 0.02% (w / v), at least about 0.01% (w / v), at least about 0.02% (w / v), at least about 0.03% (w / v), at least about 0.04% (w / v), between about 0.01% and about 0.05% (w / v), between about 0.01% (w / v) and about 0.04% (w / v), between about 0.01% and about 0.03% (w / v), between about 0.01% and about 0.02% (w / v), between about 0.01% and about 0.02% (w / v), between about 0.01% and about 0.03% (w / v), between about 0.01% and about 0.02% (w / v), between about 0.01% and about 0.03% (w / v), between about 0.01% and about 0.02% (w / v), between about 0.01% and about 0.04 ... The concentrations of poloxamer 188 are between 0.02% and about 0.05% (w / v), between about 0.02% and about 0.04% (w / v), between about 0.02% and about 0.03% (w / v), between about 0.03% and about 0.05% (w / v), between about 0.03% and about 0.04% (w / v), or between about 0.04% and about 0.05% (w / v). In some embodiments, the liquid isovirus preparation suitable for lyophilization comprises poloxamer 188 at concentrations of about 0.01% (w / v), about 0.02% (w / v), about 0.03% (w / v), about 0.04% (w / v), or about 0.05% (w / v). In some embodiments, the liquid isovirus preparation suitable for lyophilization comprises poloxamer 188 at a concentration of about 0.03% (w / v).

[0310] In some embodiments, the lyophilized isovirus preparation is prepared from a liquid isovirus preparation suitable for lyophilization, wherein the liquid isovirus preparation suitable for lyophilization comprises a stabilizer disclosed herein at a concentration of up to about 0.15% (w / v), up to about 0.1% (w / v), at least about 0.05% (w / v), at least about 0.1% (w / v), between about 0.05% and about 0.15% (w / v), between about 0.05% (w / v) and about 0.1% (w / v), or between about 0.1% and about 0.15% (w / v). In some embodiments, the liquid sand-like virus formulation suitable for lyophilization comprises a stabilizer disclosed herein at a concentration of about 0.05% (w / v), about 0.08% (w / v), about 0.1% (w / v), about 0.12% (w / v), or about 0.15% (w / v). In some embodiments, suitable for...The lyophilized liquid isovirus preparation contains a stabilizer disclosed herein at a concentration of about 0.1% (w / v).

[0311] In some embodiments, the lyophilized isovirus preparation is prepared from a liquid isovirus preparation suitable for lyophilization, wherein the liquid isovirus preparation suitable for lyophilization contains rHSA at a concentration of up to about 0.15% (w / v), up to about 0.1% (w / v), at least about 0.05% (w / v), at least about 0.1% (w / v), between about 0.05% and about 0.15% (w / v), between about 0.05% (w / v) and about 0.1% (w / v), or between about 0.1% and about 0.15% (w / v). In some embodiments, the liquid isovirus formulation suitable for lyophilization contains rHSA at a concentration of about 0.05% (w / v), about 0.08% (w / v), about 0.1% (w / v), about 0.12% (w / v), or about 0.15% (w / v). In some embodiments, the liquid isovirus formulation suitable for lyophilization contains rHSA at a concentration of about 0.1% (w / v).

[0312] In some embodiments, the freeze-dried sand virus preparation is prepared from a liquid sand virus preparation suitable for freeze-drying, wherein the liquid sand virus preparation suitable for freeze-drying comprises (a) sand virus (see Section 5.3); (b) a polyvalent anion at a concentration of up to about 50 mM or between about 20 mM and 50 mM (see Section 5.1.1); (c) a carbohydrate at a concentration of about 5% (w / v) (see Section 5.1.2); (d) a salt at a concentration of up to about 125 mM or between 50 mM and about 125 mM (see Section 5.1.3); (e) a buffer at a concentration of about 10 mM (see Section 5.1.4); (f) a surfactant at a concentration of about 0.03% (w / v) (see Section 5.1.5); and (g) a stabilizer at a concentration of about 0.1% (w / v) (see Section 5.1.5). Section 5.1.6); wherein the pH of the liquid isovirus preparation is about 8.0. Specification 46 / 63 pages 57 CN 121866044 A

[0313] In some embodiments, the lyophilized isovirus preparation is prepared from a liquid isovirus preparation suitable for lyophilization, wherein the liquid isovirus preparation suitable for lyophilization comprises: (a) isovirus; (b) citrate at a concentration of up to about 50 mM or between about 20 mM and 50 mM; (c) trehalose at a concentration of about 5% (w / v); (d) sodium chloride at a concentration of up to about 125 mM or between 50 mM and about 125 mM; (e) TRIS at a concentration of about 10 mM; (f) poloxamer 188 at a concentration of about 0.03% (w / v); and (g) rHSA at a concentration of about 0.1% (w / v), wherein the pH of the liquid isovirus preparation is about 8.0.

[0314] Methods for preparing lyophilized formulations from liquid formulations are well known in the art. Examples of methods applicable to this document for preparing lyophilized formulations from liquid formulations are described, for example, in Hansen et al., Freeze-drying of live virus vaccines: a review, TRM; Vaccine 33, 2015 (5507–5519). Viruses (especially attenuated live viruses) are generally unstable in aqueous media without freezing. This instability is primarily mediated by water, and the purpose of lyophilization is to remove the significant amount of water present in the formulation. This document provides an exemplary method. After sterile filtration at 0.22 μm, the active pharmaceutical ingredient is filled into glass vials and partially stoppered (both the vials and stoppers must be suitable for lyophilization). The applied lyophilization cycle includes a freezing step, a preliminary drying process to remove most of the water by sublimation, and a secondary drying process to remove bound water. The freezing rate is performed at the fastest possible rate to avoid crystallization of the formulation components, which could damage the virus. The initial drying (between -10°C and -40°C) and the secondary drying (between +10°C and +40°C) are carried out at the highest possible temperature and pressure (>0.03 mbar) without causing the cake to collapse or the risk of viral structural or infectivity instability. The duration of the respective drying steps will ultimately result in a residual moisture content in the dried material between 0.5% and 2%. Freeze-drying and low residual moisture content are beneficial for storage at elevated temperatures (refrigerated or room temperature) without loss of infectivity over extended periods.

[0315] In some embodiments, the freeze-dried areca virus formulation disclosed herein is reconstituted before administration to a subject (see Section 5.6).

[0316] In some embodiments, the freeze-dried areca virus formulation provided herein retains viral titer after the storage period. In some embodiments, the freeze-dried sand-like virus preparations provided herein retain at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the viral titer after a storage period. In some embodiments, the storage period is up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months, up to about 12 months, up to about 18 months, up to about 24 months, up to about 3 years, up to about 4 years, up to about 5 years, or longer. In some embodiments, the storage period is about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 18 months, about 24 months, about 3 years, or about 4 years.For approximately 5 years or longer. In some embodiments, the storage temperature is above about 2°C, at room temperature (e.g., 20°C–25°C), between about 2°C and about 8°C, above about 15°C, at about -20°C or below about -20°C, at about -65°C or below about -65°C, or at about -80°C. In some embodiments, the viral titer is measured by RCV-FFU or FFU assay.

[0317] 5.3 Sandworm Virus Any sandworm virus known in the art may be included in the liquid and lyophilized formulations disclosed herein. In some embodiments, the sandworm virus is a genetically engineered sandworm virus.

[0318] In some embodiments, the sandworm virus is lymphocytic choriomeningitis virus (LCMV). In some embodiments, the sandworm virus is Pichind virus. In some embodiments, the sandworm virus is Tamiyami virus. In some embodiments, the sandworm virus is Tacalib virus.

[0319] In some embodiments, the isovirus is a non-replicating isovirus. In some embodiments, the isovirus is a non-replicating isovirus in which the isovirus ORF has been removed or functionally inactivated such that the resulting isovirus cannot produce further infectious progeny viral particles. In some embodiments, the isovirus is a non-replicating isovirus, and the ORF encoding GP has been removed and replaced with a nucleotide sequence encoding an antigen. In some embodiments, the isovirus is a replicating isovirus. In some embodiments, the isovirus is a replicating trisegmented isovirus. In some embodiments, the replicating trisegmented isovirus is engineered such that the isovirus contains an isovirus ORF located outside the wild-type position of the ORF. In some embodiments, the replicating trisegmented isovirus contains an L segment and two S segments, wherein one of the two S segments is an S segment, and the ORF encoding GP is under the control of the isovirus 3' UTR. In some embodiments, the isanox virus is a replicating trisegmented isanox virus engineered such that the isanox virus ORF is distributed on two or more mRNA transcripts. Non-limiting examples of non-replicating and replicating isanox viruses are described in International Publications WO 2009 / 083210, WO 2016 / 075250, WO 2021 / 089853, and U.S. Publications US20100297172A1, US20170319673A1, and US20220380805A1, the contents of each of which are incorporated herein by reference.

[0320] In some embodiments, the isanox virus expresses an antigen. In some embodiments, the isanox virus expression is selected from...At least one antigen is selected from the group consisting of tumor antigens, tumor-associated antigens, antigens of pathogens causing infectious diseases, or antigenic fragments of any of the foregoing. In some embodiments, the isanoplasmic virus expresses at least one antigen selected from the group consisting of tumor antigens, tumor-associated antigens, antigens of pathogens capable of causing infectious diseases, testicular cancer antigen (CTA), and antigenic fragments of any of the foregoing.

[0321] In some embodiments, the isanoplasmic virus expresses at least one cytomegalovirus (CMV) antigen. In some embodiments, at least one CMV antigen is selected from the group consisting of CMV glycoprotein gB or an antigenic fragment thereof, CMV envelope protein pp65 or an antigenic fragment thereof, and combinations thereof. In some embodiments, at least one CMV antigen is selected from the group consisting of CMV glycoprotein gB having a carboxyl-terminus truncated (i.e., gB(dCt)), pp65, and combinations thereof. In some embodiments, the isanoplasmic virus expressing at least one CMV antigen is a non-replicating isanoplasmic virus. Non-limiting examples of arenaviruses expressing CMV antigens are described in International Publication No. WO 2015 / 082570 and US Publication No. US20160296619A1, the contents of each of which are incorporated herein by reference.

[0322] In some embodiments, the arenavirus expresses at least one human papillomavirus (HPV) antigen. In some embodiments, the arenavirus expresses two HPV antigens fused together. In some embodiments, the arenavirus expresses the HPV E7 / E6 fusion protein. In some embodiments, the arenavirus expressing at least one HPV antigen is a non-replicating arenavirus or a replicating trisegmented arenavirus. In some embodiments, the arenavirus expressing at least one HPV antigen is LCMV. In some embodiments, the arenavirus expressing at least one HPV antigen is Pitchind virus. Non-limiting examples of arenaviruses expressing HPV antigens are described in International Publications WO 2016 / 198531 and WO2021 / 239471, and U.S. Publications US20180179257A1 and US20230346906A1, the contents of each of which are incorporated herein by reference.

[0323] In some embodiments, the arenavirus expresses at least one prostate cancer antigen. In some embodiments, the at least one prostate cancer antigen is selected from the group consisting of prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), prostate acid phosphatase (PAP), and combinations thereof. In some embodiments, the arenavirus expressing at least one prostate cancer antigen is a replicating trisegmental arenavirus. In some embodiments, the arenavirus expressing at least one prostate cancer antigen is LCMV. In some embodiments, the arenavirus expressing at least one prostate cancer antigen is a Pichind virus.Viruses. Non-limiting examples of arenaviruses expressing prostate cancer antigens are described in International Publication No. WO2022 / 200373 and US Publication No. US20240174724A1, the contents of each of which are incorporated herein by reference.

[0324] In some embodiments, the arenavirus expresses at least one hepatitis B virus (HBV) antigen. In some embodiments, page 48 / 63 of CN 121866044 A, the at least one HBV antigen is selected from the group consisting of a truncated HBV polymerase polypeptide comprising an inactivated reverse transcriptase domain and an inactivated RNase H, a fusion protein comprising an HBV core polypeptide and an HBV small surface antigen (sAg) polypeptide, and combinations thereof. In some embodiments, the arenavirus expressing at least one HBV antigen is a non-replicating arenavirus or a replicating trisegmented arenavirus. In some embodiments, the arenavirus expressing at least one HBV antigen is LCMV. In some embodiments, the arenavirus expressing at least one HBV antigen is Pichind virus. Non-limiting examples of arenaviruses expressing HBV antigens are described in International Publications WO 2017 / 076988 and WO 2021 / 067181, and U.S. Publications US20180319845A1 and US20210093712A1, the contents of each of which are incorporated herein by reference.

[0325] In some embodiments, the arenavirus expresses at least one human immunodeficiency virus (HIV) antigen. In some embodiments, the at least one HIV antigen is selected from the group consisting of gag, pol, nef, and combinations thereof, and optionally wherein the HIV antigen is a fusion polypeptide comprising multiple polypeptide segments of one or more HIV-1 proteins encoded by two or more HIV genes selected from gag, pol, and nef. In some embodiments, the arenavirus expressing at least one HIV antigen is a non-replicating arenavirus or a replicating trisegmented arenavirus. In some embodiments, the arenavirus expressing at least one HIV antigen is LCMV. In some embodiments, the isanoplasmic virus expressing at least one HIV antigen is a Pichind virus. Non-limiting examples of isanoplasmic viruses expressing HIV antigens are described in International Publication No. WO 2022 / 155258 and US Publication No. US20220218813A1, the contents of each of which are incorporated herein by reference.

[0326] In some embodiments, the isanoplasmic virus expresses at least one antigenic fragment of a mutant KRAS. In some embodiments, the isanoplasmic virus encodes antigenic fragments of the mutant KRAS from the N-terminus to the C-terminus, comprising mutant G13D, G12V, G12C, G12D, and G12R, respectively. In some embodiments, the isanoplasmic virus expressing an antigenic fragment of the mutant KRAS is a non-replicating isanoplasmic virus.Typed isovirus or replicating trisegmented isovirus. In some embodiments, the isovirus expressing an antigenic fragment of mutant KRAS is a “split-type” isovirus particle. In some embodiments, the isovirus expressing an antigenic fragment of mutant KRAS is LCMV. In some embodiments, the isovirus expressing an antigenic fragment of mutant KRAS is Pitchind virus. Non-limiting examples of isoviruses expressing antigenic fragments of mutant KRAS are described in International Patent Publication No. WO2023079153, the contents of which are incorporated herein by reference.

[0327] In some embodiments, the isovirus expresses two or more CTA fragments (e.g., antigenic fragments or epitopes). In some embodiments, the isovirus expresses at least one fusion protein comprising two or more CTA fragments (e.g., antigenic fragments or epitopes). In some embodiments, the isovirus encodes a first fusion protein comprising two or more CTA fragments and a second fusion protein comprising two or more CTA fragments. In some embodiments, two or more CTA fragments of at least one fusion protein, the first fusion protein, and / or the second fusion protein are independently selected from the group consisting of epitopes or antigenic fragments of PRAME, MAGE-A3, MAGE-A4, and MAGE-A6. In some embodiments, the two or more CTA fragments of the first fusion protein comprise at least one PRAME fragment, at least one MAGE-A3 fragment, at least one MAGE-A4 fragment, and at least one MAGE-A6 fragment. In some embodiments, at least one PRAME fragment of the first fusion protein is selected from the group consisting of PRAME3-13, PRAME7-23, PRAME73-82, PRAME84-95, PRAME123-141, PRAME150-161, PRAME166-178, PRAME210-230, PRAME224-232, PRAME237-250, PRAME248-256, PRAME267-277, PRAME278-292, PRAME315-327, PRAME325-334, PRAME425-433, PRAME435-443, PRAME445-458, PRAME472-491, PRAME485-509, and combinations thereof. In some embodiments, at least one MAGE-A3 fragment of the first fusion protein comprises MAGE-A3135-149. In some embodiments, at least one MAGE-A4 fragment of the first fusion protein comprises MAGE-A4112-121 and / or MAGE-A4286-305. In some embodiments, at least one MAGE-A6 fragment of the first fusion protein comprises MAGE-A6167-184. In some embodiments...In the specification, pages 49 / 63, 60 CN 121866044 A, the two or more CTA fragments of the second fusion protein comprise at least one MAGE-A3 fragment, at least one MAGE-A4 fragment, and at least one MAGE-A6 fragment. In some embodiments, the at least one MAGE-A3 fragment of the second fusion protein is selected from the group consisting of MAGE-A3243-255, MAGE-A3171-184, MAGE-A3284-296, MAGE-A3167-176, MAGE-A3192-202, MAGE-A3302-311, MAGE-A370-81, MAGE-A3160-171, MAGE-A3143-151, and combinations thereof. In some embodiments, at least one MAGE-A4 fragment of the second fusion protein is selected from the group consisting of MAGE-A4136-155, MAGE-A4168-179, MAGE-A4190-199, MAGE-A477-89, MAGE-A4300-308, MAGE-A4175-183, MAGE-A4262-272, MAGE-A4222-246, MAGE-A4109-117, MAGE-A4205-218, MAGE-A4205-217, MAGE-A4155-168, MAGE-A4266-274, MAGE-A473-83, and combinations thereof. In some embodiments, at least one MAGE-A6 fragment of the second fusion protein is selected from the group consisting of MAGE-A6196-204, MAGE-A6194-202, MAGE-A6231-241, MAGE-A6243-255, and combinations thereof. Sequences of exemplary CTA fragments are disclosed in Section 6 herein and are listed with SEQ ID NO: 1-51. In some embodiments, the isanoplasmic virus expressing a first fusion protein comprising two or more CTA fragments and a second fusion protein is a non-replicating isanoplasmic virus or a replicating trisegmented isanoplasmic virus. In some embodiments, the isanoplasmic virus expressing a first fusion protein comprising two or more CTA fragments and a second fusion protein is a "split-type" isanoplasmic virus particle. In some embodiments, the isanoplasmic virus expressing a first fusion protein comprising two or more CTA fragments and a second fusion protein is an LCMV. In some embodiments, the isanoplasmic virus expressing a first fusion protein comprising two or more CTA fragments and a second fusion protein is a Pichind virus. A non-limiting example of a sand virus expressing a first fusion protein and a second fusion protein comprising two or more CTA fragments is described in U.S. Provisional Application No. 63 / 565,812, filed March 15, 2024, the contents of which are incorporated herein by reference.

[0328] In some embodiments, the concentration of sand virus in the liquid sand virus formulation disclosed herein (see Section 5.1) is between about 102 RCV-FFU / ml and about 1010 RCV-FFU / ml. In some embodiments, the concentration of isovirus in the liquid isovirus formulation disclosed herein (see Section 5.1) is between approximately 10² RCV-FFU / ml and approximately 10⁹ RCV-FFU / ml, between approximately 10² RCV-FFU / ml and approximately 10⁸ RCV-FFU / ml, between approximately 10² RCV-FFU / ml and approximately 10⁷ RCV-FFU / ml, between approximately 10² RCV-FFU / ml and approximately 10⁶ RCV-FFU / ml, between approximately 10² RCV-FFU / ml and approximately 10⁵ RCV-FFU / ml, between approximately 10² RCV-FFU / ml and approximately 10⁴ RCV-FFU / ml, and between approximately 10² RCV-FFU / ml and approximately 10⁴ RCV-FFU / ml. FFU / ml between approximately 103 RCV-FFU / ml, between approximately 103 RCV-FFU / ml and approximately 1010 RCV-FFU / ml, between approximately 103 RCV-FFU / ml and approximately 109 RCV-FFU / ml, between approximately 103 RCV-FFU / ml and approximately 108 RCV-FFU / ml, between approximately 103 RCV-FFU / ml and approximately 107 RCV-FFU / ml, between approximately 103 RCV-FFU / ml and approximately 106 RCV-FFU / ml, between approximately 103 RCV-FFU / ml and approximately 105 RCV-FFU / ml, between approximately 103 RCV-FFU / ml and approximately 104 RCV-FFU / ml Between approximately 10⁴ RCV-FFU / ml and approximately 10¹⁰ RCV-FFU / ml, between approximately 10⁴ RCV-FFU / ml and approximately 10⁹ RCV-FFU / ml, between approximately 10⁴ RCV-FFU / ml and approximately 10⁸ RCV-FFU / ml, between approximately 10⁴ RCV-FFU / ml and approximately 10⁷ RCV-FFU / ml, between approximately 10⁴ RCV-FFU / ml and approximately 10⁶ RCV-FFU / ml, between approximately 10⁴ RCV-FFU / ml and approximately 10⁵ RCV-FFU / ml, between approximately 10⁵ RCV-FFU / ml and approximately 10¹⁰ ...¹⁰ RCV-FFU / ml, between approximately 10⁵ RCV-FFU / ml and approximately 10¹⁰ RCV-FFU / ml, between approximately 10⁵ RCV-FFU / ml and approximately 10¹⁰ RCV-FFU / ml, between approximately 10⁵ RCV-FFU / ml and approximately 10¹⁰ RCV-FFU / ml, between approximately 10� FFU / ml between approximately 10⁹ RCV-FFU / ml, between approximately 10⁵ RCV-FFU / ml and approximately 10⁸ RCV-FFU / ml, between approximately 10⁵ RCV-FFU / ml and approximately 10⁷ RCV-FFU / ml, between approximately 10⁵ RCV-FFU / ml and approximately 10⁶ RCV-FFU / ml, and so on.Between approximately 10⁶ RCV-FFU / ml and approximately 10¹⁰ RCV-FFU / ml, between approximately 10⁶ RCV-FFU / ml and approximately 10⁹ RCV-FFU / ml, between approximately 10⁶ RCV-FFU / ml and approximately 10⁸ RCV-FFU / ml, between approximately 10⁶ RCV-FFU / ml and approximately 10⁷ RCV-FFU / ml, between approximately 10⁷ RCV-FFU / ml and approximately 10¹⁰ RCV-FFU / ml, between approximately 10⁷ RCV-FFU / ml and approximately 10⁹ RCV-FFU / ml Between FFU / ml, between approximately 107 RCV-FFU / ml and approximately 108 RCV-FFU / ml, between approximately 108 RCV-FFU / ml and approximately 1010 RCV-FFU / ml, between approximately 108 RCV-FFU / ml and approximately 109 RCV-FFU / ml, or between approximately 109 RCV-FFU / ml and approximately 1010 RCV-FFU / ml. (Instructions for Use, pages 50 / 63, 61 CN 121866044 A) In some embodiments, the concentration of isovirus in the liquid isovirus formulation disclosed herein (see Section 5.1) is between about 10⁵ RCV-FFU / ml and about 10⁹ RCV-FFU / ml, between about 10⁵ RCV-FFU / ml and about 8 × 10⁸ RCV-FFU / ml, between about 10⁵ RCV-FFU / ml and about 6 × 10⁸ RCV-FFU / ml, between about 10⁵ RCV-FFU / ml and about 5 × 10⁸ RCV-FFU / ml, between about 2 × 10⁵ RCV-FFU / ml and about 10⁹ RCV-FFU / ml, between about 2 × 10⁵ RCV-FFU / ml and about 8 × 10⁸ RCV-FFU / ml, or between about 2 × 10⁵ RCV-FFU / ml and about 6 × 10⁸ RCV-FFU / ml. In some embodiments, the concentration of isovirus in the liquid isovirus formulation disclosed herein (see Section 5.1) is between about 2 × 10⁵ RCV-FFU / ml and about 5 × 10⁸ RCV-FFU / ml.

[0329] 5.4 Pharmaceutical Compositions This document provides pharmaceutical compositions comprising the liquid isovirus formulation disclosed herein (see Section 5.1). In some embodiments, the pharmaceutical composition comprises a liquid isovirus formulation diluted from the liquid isovirus formulation disclosed herein (see Section 5.1). Therefore, the concentration of certain components in the pharmaceutical composition may differ from the concentration of these components in the liquid isovirus formulation from which the dilution is derived.

[0330] In some embodiments, the pharmaceutical composition comprises a lyophilized formulation disclosed herein (see Section 5.2).Section 5.2). In some embodiments, the pharmaceutical composition comprises a liquid isopyrvirus preparation reconstituted from a lyophilized formulation disclosed herein (see Section 5.2).

[0331] In some embodiments, the pharmaceutical composition further comprises an adjuvant. In some embodiments, the adjuvant is administered before, simultaneously with, or after the pharmaceutical composition. An adjuvant is a compound that, when administered in combination with or as part of the pharmaceutical composition described herein, enhances, elevates, and / or promotes an immune response induced by isopyrvirus; but when administered alone, does not produce an immune response against isopyrvirus or antigens expressed by isopyrvirus. Adjuvants that may be used with this disclosure include, but are not limited to, mineral salt adjuvants or mineral salt gel adjuvants, particulate adjuvants, microparticle adjuvants, mucosal adjuvants, and immunostimulatory adjuvants. Examples of adjuvants suitable for use herein are described in Rushit N et al., Overview of vaccine adjuvants, edited by Kolhe and Ohtake, Academic Press, 2022 (9–25). Additional examples of adjuvants include, but are not limited to, aluminum salts (alum) (such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate), 3-de-O-acylated monophospholipid A (MPL) (see GB 2220211), MF59® (Novartis), AS03 (GlaxoSmithKline), AS04 (GlaxoSmithKline), VacciGrade™ (AS03-like adjuvant, InvivoGen), Montanide ISA™VG (Seppic), AF03 (Sanofi), GLA-SE (Infectious Disease Research Institute (IDRI)), polysorbate 80 (Tween® 80; ICL Americas, Inc.), imidazopyridine compounds (see International Application No. PCT / US2007 / 064857, published as International Publication No. WO2007 / 109812), imidazoquinoxaline compounds (see International Application No. PCT / US2007 / 064858, published as International Publication No. WO2007 / 109813), and saponins, such as QS21 (see Kensil et al., published in, edited by Powell and Newman, Plenum Press, NY, 1995; US Patent No. 5,057,540). In some embodiments, the adjuvant is Freund's adjuvant (complete or incomplete). Other adjuvants are oil-in-water emulsions (such as squalene or peanut oil), optionally combined with an immunostimulant such as monophospholipid A (see Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)).

[0332] In some embodiments, the pharmaceutical compositions disclosed herein may also comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers, excipients, or diluents include those approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other recognized pharmacopoeia for use in animals, and more specifically, in humans. The term "carrier" refers to a diluent, excipient, or medium for administering a therapeutic agent. Pharmaceutically acceptable carriers, excipients, or diluents include any and all solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic agents, and absorption delay agents, etc. [Prescription 51 / 63 pages 62 CN 121866044 A] Acceptable carriers, excipients, and / or diluents for therapeutic use are well known in the pharmaceutical industry and are described, for example, in Mack Publishing Co. (edited by AR Gennaro, 1995). As those skilled in the art will understand, the selection of pharmaceutically acceptable carriers, excipients, and / or diluents can be made in accordance with the intended route of administration and standard pharmaceutical practice.

[0333] In some embodiments, the pharmaceutical composition comprises isopyrvirus between about 102 RCV-FFU / ml and about 1010 RCV-FFU / ml. In some embodiments, the pharmaceutical composition comprises between about 102 RCV-FFU / ml and about 109 RCV-FFU / ml, between about 102 RCV-FFU / ml and about 108 RCV-FFU / ml, between about 102 RCV-FFU / ml and about 107 RCV-FFU / ml, between about 102 RCV-FFU / ml and about 106 RCV-FFU / ml, between about 102 RCV-FFU / ml and about 105 RCV-FFU / ml, between about 102 RCV-FFU / ml and about 104 RCV-FFU / ml, between about 102 RCV-FFU / ml and about 103 RCV-FFU / ml, between about 103 RCV-FFU / ml and about 1010 RCV-FFU / ml, and between about 103 RCV-FFU / ml and about 1010 RCV-FFU / ml. FFU / ml between approximately 10⁹ RCV-FFU / ml, between approximately 10³ RCV-FFU / ml and approximately 10⁸ RCV-FFU / ml, between approximately 10³ RCV-FFU / ml and approximately 10⁷ RCV-FFU / ml, between approximately 10³ RCV-FFU / ml and approximately 10⁶ RCV-FFU / ml, between approximately 10³ RCV-FFU / ml and approximately 10⁵ RCV-FFU / ml, between approximately 10³ RCV-FFU / ml and approximately 10⁴ RCV-FFU / mlBetween approximately 10⁴ RCV-FFU / ml and approximately 10¹⁰ RCV-FFU / ml, between approximately 10⁴ RCV-FFU / ml and approximately 10⁹ RCV-FFU / ml, between approximately 10⁴ RCV-FFU / ml and approximately 10⁸ RCV-FFU / ml, between approximately 10⁴ RCV-FFU / ml and approximately 10⁷ RCV-FFU / ml, between approximately 10⁴ RCV-FFU / ml and approximately 10⁶ RCV-FFU / ml, between approximately 10⁴ RCV-FFU / ml and approximately 10⁵ RCV-FFU / ml, between approximately 10⁵ RCV-FFU / ml and approximately 10¹⁰ RCV-FFU / ml, between approximately 10⁵ RCV-FFU / ml Between approximately 10⁹ RCV-FFU / ml, between approximately 10⁵ RCV-FFU / ml and approximately 10⁸ RCV-FFU / ml, and between approximately 10⁵ RCV-FFU / ml. FFU / ml between approximately 10⁷ RCV-FFU / ml, between approximately 10⁵ RCV-FFU / ml and approximately 10⁶ RCV-FFU / ml, between approximately 10⁶ RCV-FFU / ml and approximately 10¹⁰ RCV-FFU / ml, between approximately 10⁶ RCV-FFU / ml and approximately 10⁹ RCV-FFU / ml, between approximately 10⁶ RCV-FFU / ml and approximately 10⁸ RCV-FFU / ml, between approximately 10⁶ RCV-FFU / ml and approximately 10⁷ RCV-FFU / ml, between approximately 10⁷ RCV-FFU / ml and approximately 10¹⁰ RCV-FFU / ml, between approximately 10⁷ RCV-FFU / ml and approximately 10⁹ RCV-FFU / ml Sand virus between approximately 10⁷ RCV-FFU / ml and approximately 10⁸ RCV-FFU / ml, between approximately 10⁸ RCV-FFU / ml and approximately 10¹⁰ RCV-FFU / ml, between approximately 10⁸ RCV-FFU / ml and approximately 10⁹ RCV-FFU / ml, or between approximately 10⁹ RCV-FFU / ml and approximately 10¹⁰ RCV-FFU / ml. In some embodiments, the pharmaceutical composition comprises between about 10⁵ RCV-FFU / ml and about 10⁹ RCV-FFU / ml, between about 10⁵ RCV-FFU / ml and about 8 × 10⁸ RCV-FFU / ml, between about 10⁵ RCV-FFU / ml and about 6 × 10⁸ RCV-FFU / ml, between about 10⁵ RCV-FFU / ml and about 5 × 10⁸ RCV-FFU / ml, between about 2 × 10⁵ RCV-FFU / ml and about 8 × 10⁸ RCV-FFU / ml, and between about 2 × 10⁵ RCV-FFU / ml and about 8 × 10⁸ RCV-FFU / ml.The concentration of isaminifera is between 10⁸ RCV-FFU / ml or between about 2 × 10⁵ RCV-FFU / ml and about 6 × 10⁸ RCV-FFU / ml. In some embodiments, the pharmaceutical composition comprises isaminifera between about 2 × 10⁵ RCV-FFU / ml and about 5 × 10⁸ RCV-FFU / ml.

[0334] 5.5 Method for Reducing Subvisible Particle Formation or Virus Titer Loss This disclosure provides a method for reducing the formation of subvisible particles in isaminifera formulations. This disclosure also provides a method for reducing virus titer loss (e.g., during the manufacture, filtration, freeze-thaw process, and / or storage of the isaminifera formulation). In some embodiments, the method comprises mixing isaminifera with a solution to form a liquid isaminifera formulation disclosed in Section 5.1. In some embodiments, the liquid isaminifera formulation comprises isaminifera (see Section 5.3) and a polyvalent anion (e.g., citrate) (see Section 5.1.1). In some embodiments, the liquid isovirus formulation further comprises at least one of a carbohydrate (e.g., sucrose or trehalose) (see Section 5.1.2), a salt (e.g., sodium chloride) (see Section 5.1.3), a buffer (e.g., TRIS) (see Section 5.1.4), a surfactant (e.g., poloxamer 188) (see Section 5.1.5, pages 52 / 63 of the specification, CN 121866044 A), and a stabilizer (e.g., rHSA) (see Section 5.1.6). In some embodiments, the liquid isovirus formulation is suitable for lyophilization.

[0335] In some embodiments, the methods disclosed herein reduce the formation of subvisible particles during manufacturing. In some embodiments, manufacturing includes a purification process. In some embodiments, manufacturing includes at least one chromatographic step. In some embodiments, the method includes mixing sand-like virus with a solution to form a liquid sand-like virus formulation disclosed in Section 5.1, wherein the liquid sand-like virus formulation comprises sand-like virus (see Section 5.3) and a polyvalent anion (see Section 5.1.1). In some embodiments, the liquid sand-like virus formulation further comprises a salt (see Section 5.1.3) and / or a surfactant (see Section 5.1.5). In some embodiments, the concentrations of the polyvalent anion, salt, and / or surfactant in the liquid sand-like virus formulation reduce the formation of subvisible particles during manufacturing. In some embodiments, the liquid sand-like virus formulation further comprises carbohydrates (see Section 5.1.2), buffers (see Section 5.1.4), and / or stabilizers (see Section 5.1.6).

[0336] In some embodiments, the methods disclosed herein reduce the loss of virus titers during filtration. In some embodimentsIn this implementation, filtration includes bioburden reduction filtration, sterilization filtration, or a combination thereof. In some embodiments, the method includes mixing isovirus with a solution to form a liquid isovirus preparation disclosed in Section 5.1, wherein the liquid isovirus preparation comprises isovirus (see Section 5.3) and a polyvalent anion (see Section 5.1.1). In some embodiments, the liquid isovirus preparation also comprises a salt (see Section 5.1.3). In some embodiments, the concentration of the polyvalent anion and / or salt in the liquid isovirus preparation reduces the loss of virus titer during filtration. In some embodiments, the liquid isovirus preparation also comprises carbohydrates (see Section 5.1.2), buffers (see Section 5.1.4), surfactants (see Section 5.1.5), and / or stabilizers (see Section 5.1.6).

[0337] In some embodiments, the size of the subvisible particles reduced by the method disclosed in this invention is at least about 0.1 μm, at least about 0.5 μm, at least about 1 μm, at least about 5 μm, at least about 10 μm, at least about 15 μm, at least about 20 μm, at least about 25 μm, at least about 30 μm, at least about 35 μm, at least about 40 μm, at least about 45 μm, at least about 50 μm, at least about 55 μm, at least about 60 μm, at least about 65 μm, at least about 70 μm, at least about 75 μm, at least about 80 μm, at least about 85 μm, at least about 90 μm, at least about 95 μm, or at least about 100 μm. In some embodiments, the size of the subvisible particles reduced by the method disclosed in this invention is at least about 1 μm. In some embodiments, the size of the subvisible particles reduced by the method disclosed in this invention is at least about 10 μm. In some embodiments, the size of the subvisible particles reduced by the method disclosed herein is at least about 25 μm.

[0338] In some embodiments, the method disclosed herein reduces the formation of subvisible particles during storage. In some embodiments, the method disclosed herein reduces the loss of viral titer during storage. In some embodiments, the method includes mixing isovirus with a solution to form a liquid isovirus preparation disclosed in Section 5.1, wherein the liquid isovirus preparation comprises isovirus (see Section 5.3) and a polyvalent anion (see Section 5.1.1), and also comprises carbohydrates (see Section 5.1.2) and / or stabilizers (see Section 5.1.6). In some embodiments, the concentration of carbohydrates and / or stabilizers in the liquid isovirus preparation reduces the formation of subvisible particles during storage. In some embodiments, the liquid sand-like virus preparation also contains salt (see Section 5.1.3), buffer (see Section 5.1.4), and / or surfactant (see Section 5.1.5).

[0339] In some embodiments, the storage temperature is above about 2°C, is room temperature (e.g., 20°C–25°C), is between about 2°C and about 8°C, is above about 15°C, is about -20°C or below about -20°C, is about -65°C or below about -65°C, or is about -80°C. In some embodiments, the storage temperature is about -20°C. In some embodiments, the storage temperature is about -80°C. In some embodiments, the storage lasts for at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 3 years, at least about 4 years, at least about 5 years, or longer. In some embodiments, the storage lasts for at least about 6 months. In some embodiments, the storage lasts for at least about 9 months. In some embodiments, storage lasts for at least about 18 months.

[0340] In some embodiments, the methods disclosed herein reduce the formation of sub-visible particles after freeze-thaw cycles. In some embodiments, the methods disclosed herein reduce the loss of virus titer after freeze-thaw cycles. In some embodiments, the method includes mixing isovirus with a solution to form a liquid isovirus preparation disclosed in Section 5.1, wherein the liquid isovirus preparation comprises isovirus (see Section 5.3) and a polyvalent anion (see Section 5.1.1), and also comprises carbohydrates (see Section 5.1.2) and / or salts (see Section 5.1.3). In some embodiments, the concentration of carbohydrates and / or salts in the liquid isovirus preparation reduces the formation of sub-visible particles after freeze-thaw cycles. In some embodiments, the liquid isovirus preparation also comprises a buffer (see Section 5.1.4), a surfactant (see Section 5.1.5), and / or a stabilizer (see Section 5.1.6).

[0341] In some embodiments, the freeze-thaw cycle includes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 6, at least 7, at least 8, at least 9, at least 10 or more freeze-thaw (F / T) cycles. In some embodiments, the freeze-thaw cycle includes 3 F / T cycles. In some embodiments, the freeze-thaw cycle includes 5 F / T cycles. In some embodiments, the freezing conditions are at a temperature of about -20°C or below, about -65°C or below, or about -80°C.

[0342] 5.6 Treatment Methods This document provides methods for treating or preventing disease using the arenavirus preparations disclosed herein (see Sections 5.1 and 5.2) and pharmaceutical compositions thereof (see Section 5.4).

[0343] In some embodiments, the method includes administering the liquid isovirus preparation disclosed herein (see Section 5.1) to a subject in need. In some embodiments, the method includes administering a liquid isovirus preparation, obtained by diluting the liquid isovirus preparation disclosed herein (see Section 5.1), to a subject in need.

[0344] In some embodiments, the method includes first diluting the liquid isovirus preparation disclosed herein (see Section 5.1) before administering it to a subject. In some embodiments, dilution includes adding a known volume of buffer (see Section 5.1.4) or a suitable diluent (e.g., sterile water, water for injection, or saline) to the liquid isovirus preparation disclosed herein (see Section 5.1) to obtain a final isovirus concentration suitable for administration to a subject. Dilution may be performed according to and in accordance with pharmacy manuals, applicable regulations, guidelines, good clinical practice (GCP), aseptic preparation techniques, and / or standard operating procedures (SOPs).

[0345] In some embodiments, the liquid isoflavone virus formulation comprises a therapeutically effective amount of isoflavone virus.

[0346] In some embodiments, the method includes administering the lyophilized isoflavone virus formulation disclosed herein (see Section 5.2) to a subject in need. In some embodiments, the method includes administering a liquid isoflavone virus formulation reconstituted from the lyophilized isoflavone virus formulation disclosed herein (see Section 5.2) to a subject in need. In some embodiments, the method includes reconstituted the lyophilized isoflavone virus formulation before administering it to the subject.

[0347] Reconstitution of lyophilized formulations is a skill well known in the art. In some embodiments, reconstitution includes adding a known volume of liquid (e.g., sterile water, water for injection, buffer, or saline) to the lyophilized formulation. In some embodiments, reconstitution also includes rotating or stirring (e.g., gently) the components to mix. In some embodiments, reconstitution also includes confirming that all solids are substantially completely dissolved or suspended in the liquid component, for example, by visual inspection. In some embodiments, the reconstituted composition may be administered immediately or may be stored and / or incubated for a period of time prior to use. In some embodiments, the reconstituted compositions disclosed herein may be stored at room temperature (e.g., about 20°C to 25°C) or refrigerated (e.g., about 4°C) and / or incubated for a period of time. In some embodiments, the reconstituted compositions disclosed herein may be stored for up to about 48 hours, up to about 36 hours, up to about 24 hours, up to about 12 hours, up to about 6 hours, up to about 2 hours, up to about 1 hour, or up to about 30 minutes prior to administration to a subject.

[0348] In some embodiments, the method includes administering the liquid sand virus preparation or lyophilized preparation via intratumoral injection,The drug can be administered to a subject orally, intradermally, intramuscularly, intraperitoneally, intravenously, locally, subcutaneously, percutaneously, intranasally, or by inhalation, or via scratch inoculation (scraping the surface of the skin, e.g., using a bifurcated needle). Specifically, subcutaneous, intramuscular, or intravenous routes may be used.

[0349] In some embodiments, the liquid isovirus preparation or lyophilized preparation is administered to a subject intravenously, intramuscularly, intratumorally, or subcutaneously.

[0350] In some embodiments, the method includes administering the pharmaceutical composition disclosed herein (see Section 5.4) to a subject in need.

[0351] Diseases that can be treated or prevented by the methods disclosed herein include neoplastic diseases and infectious diseases.

[0352] Oncological diseases that can be treated or prevented by the methods disclosed herein include acute lymphoblastic leukemia; acute lymphoblastic lymphoma; acute lymphoblastic leukemia; acute myeloid leukemia; acute myeloid leukemia (adult / child); adrenocortical carcinoma; AIDS-related cancer; AIDS-related lymphoma; anal cancer; appendix cancer; astrocytoma; atypical teratoid / rhabdoid tumor; basal cell carcinoma; extrahepatic bile duct carcinoma (cholangiocarcinoma); bladder cancer; osteosarcoma / Malignant fibrous histiocytoma; Brain cancer (adult / child); Brain tumor, cerebellar astrocytoma (adult / child); Brain tumor, cerebral astrocytoma / malignant glioma; Brain tumor, ependymoma; Brain tumor, medulloblastoma; Brain tumor, supratentorial primitive neuroectodermal tumor; Brain tumor, visual pathway and hypothalamic glioma; Brainstem glioma; Breast cancer; Bronchial adenoma / carcinoid; Bronchial tumor; Burkitt lymphoma; Childhood cancer; Gastrointestinal carcinoid tumors; Carcinoid tumors; Cancer of unknown primary site in adults; Cancer of unknown primary site; Embryonic tumors of the central nervous system; Primary central nervous system lymphoma; Cervical cancer; Childhood adrenocortical carcinoma; Childhood cancer; Childhood cerebral astrocytoma; Childhood chordoma; Chronic lymphocytic leukemia; Chronic myeloid leukemia; Chronic myeloid leukemia; Chronic myeloproliferative disorders; Colon cancer; Colorectal cancer; Craniopharyngioma; Skin T-cell tumors Cellular lymphoma; small round cell tumors that promote connective tissue proliferation; emphysema; endometrial cancer; ependymoblastoma; ependymoma; esophageal cancer; Ewing sarcoma in the Ewing family of tumors; extracranial germ cell tumors; extragonadal germ cell tumors; extrahepatic bile duct cancer; gallbladder cancer; gastric cancer; gastric carcinoid; gastrointestinal carcinoid tumors; gastrointestinal stromal tumors; germ cell tumors: extracranial, extragonadal, or ovarian gestational trophoblastic tumors; gestational trophoblastic tumors of unknown primary site; glioma; brainstem glioma; visual pathway and hypothalamic glioma in children; hairy cell leukemia; head and neck cancer; cardiac cancer; hepatocellular carcinoma; Hodgkin lymphoma; hypopharyngeal cancer; hypothalamic and visual pathway glioma; intraocular melanoma; islet cell carcinoma (endocrine pancreas); Kaposi's sarcomaTumors; Renal cell carcinoma; Langerhans cell histiocytosis; Laryngeal cancer; Lip and oral cancer; Liposarcoma; Primary liver cancer; Non-small cell lung cancer; Small cell lung cancer; Lymphoma, primary central nervous system cancer; Macroglobulinemia, Waldenström macroglobulinemia; Male breast cancer; Malignant fibrous histiocytoma / osteosarcoma of bone; Medulloblastoma; Medullary epithelioma; Melanoma; Intraocular (ocular) melanoma; Merkel cell carcinoma; Merkel cell skin carcinoma; mesothelioma; adult malignant mesothelioma; metastatic squamous neck carcinoma of unknown primary origin; oral cancer; multiple endocrine neoplasia syndrome; multiple myeloma / plasma cell tumor; mycosis fungoides, myelodysplastic syndrome; myelodysplastic / myeloproliferative disorders; myeloid leukemia, chronic; myeloid leukemia, acute in adults; myeloid leukemia, acute in children; multiple myeloma (myeloma); myeloproliferative disorders, chronic; nasal cavity and paranasal sinus carcinoma; nasopharyngeal carcinoma; neuroblastoma, non-small cell lung cancer; non-Hodgkin lymphoma; oligodendroglioma; oral cancer; oral cavity cancer; oropharyngeal cancer; osteosarcoma / malignant fibrous histiocytoma of bone; ovarian cancer; ovarian epithelial carcinoma (surface epithelial-stromal tumor); ovarian germ cell tumor; low-grade malignant potential ovarian tumor; pancreatic cancer; pancreatic cancer, islet cell... (Instructions for use 55 / 63 pages 66 CN) 121866044 A cell; papilloma; paranasal sinus and nasal cavity carcinoma; parathyroid carcinoma; penile cancer; pharyngeal carcinoma; pheochromocytoma; pineal astrocytoma; pineal germ cell tumor; moderately differentiated pineal parenchymal tumor; pineal blastoma and supratentorial primitive neuroectodermal tumor; pituitary tumor; pituitary adenoma; plasma cell tumor / multiple myeloma; pleural pulmonary blastoma; primary central nervous system lymphoma; prostate cancer; rectal cancer; renal cell carcinoma (kidney cancer); transitional cell carcinoma of the renal pelvis and ureter; respiratory tract cancer involving the NUT gene on chromosome 15; retinoblastoma Rhabdomyosarcoma, in children; salivary gland cancer; sarcoma, Ewing family tumors; Cézari syndrome; skin cancer (melanoma); skin cancer (non-melanoma); small cell lung cancer; small intestinal cancer soft tissue sarcoma; soft tissue sarcoma; spinal cord tumors; squamous cell carcinoma; metastatic squamous neck cancer of unknown primary origin; gastric cancer; supratentorial primitive neuroectodermal tumors; T-cell lymphoma, skin (mycosis fungoides and Cézari syndrome); testicular cancer; laryngeal cancer; thymoma; thymoma and thymic carcinoma; thyroid cancer; pediatric thyroid cancer; transitional cell carcinoma of the renal pelvis and ureter; urethral cancer; uterine cancer, endometrium; uterine sarcoma; vaginal cancer; vulvar cancer; and nephroblastoma.

[0353] Infectious diseases that can be treated or prevented by the methods disclosed herein include acute flaccid myelitis, anaplasmosis, anthrax, babesiosis, brucellosis, campylobacteriosis, carbapenem-resistant infections, chancroid, and chikungunya.Viral infections, chlamydia infections, Clostridium difficile infections, coccidioidomycosis, fungal infections, Covid-19, cryptosporidiosis, cyclosporidiosis, dengue fever, diphtheria, Escherichia coli infections, eastern equine encephalitis, Ebola hemorrhagic fever, ehrlichiosis, arbovirus encephalitis, parainfectious encephalitis, enterovirus infections, giardiasis, glanders, gonorrhea, granuloma inguinale, Haemophilus influenzae type b infection, Hantavirus pulmonary syndrome, hemolytic uremic syndrome, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes, herpes zoster, histoplasmosis, acquired immunodeficiency syndrome, human papillomavirus infection, influenza, Legionnaires' disease, leprosy, leptospirosis, listeriosis, Lyme disease, lymphogranuloma venereum infection, malaria, measles, glanders, viral meningitis. Viral meningitis, Middle East Respiratory Syndrome (MERS), Multisystem Inflammatory Syndrome (MISS), mumps, norovirus infection, lice, pelvic inflammatory disease, pertussis, plague, pneumococcal disease, poliomyelitis, Poivasan virus infection, psittacosis, pubic lice, impetigo, Q fever, rabies, rickettsial disease, rubella, salmonellosis gastroenteritis, scabies infection, sepsis, severe acute respiratory syndrome, Shigella gastroenteritis, smallpox, staphylococcal infection, streptococcal infection, streptococcal toxic shock syndrome, syphilis, tetanus infection, trichomoniasis, trichinosis infection, tuberculosis, tularemia, typhus, spotted typhus, chickenpox, Vibrio cholerae, vibriosis, viral hemorrhagic fever, West Nile virus infection, coronavirus infection, yeast infection, yellow fever, Yersiniasis, and Zika virus infection.

[0354] This document also provides kits that can be used to perform the methods described herein. In some embodiments, the kits provided herein comprise one or more containers. These containers may store the formulations or compositions disclosed herein (e.g., liquid isovirus formulations, lyophilized formulations, pharmaceutical compositions). In some embodiments, the kits also comprise liquids (e.g., sterile water, water for injection, buffers, or saline) for diluting the liquid isovirus formulation or reconstituted lyophilized isovirus formulation. Instructions for use are also included in the kits. These instructions describe in sufficient detail a treatment regimen using the formulations and compositions contained therein. For example, the instructions may include dosage and administration instructions for methods of treating neoplastic or infectious diseases as provided herein.

[0355] 6. Sequence Specification 56 / 63 pages 67 CN 121866044 A Specification 57 / 63 pages 68 CN 121866044 A

[0356] 7. Examples The following is a description of various methods and materials used in the studies. This content is presented to provide those skilled in the art with a complete disclosure and description of how the invention can be made and used, and is not intended to limit the inventors' view.The scope of the invention is not intended to imply that all the following experiments have been performed or that all possible experiments have been conducted. It should be understood that the exemplary descriptions written in the present tense are not necessarily actually performed, but these descriptions can be performed to generate data, etc., in connection with the teachings of the invention. Efforts have been made to ensure the accuracy of the figures used (e.g., amounts, percentages, etc.), but some experimental errors and biases should be taken into account.

[0357] 7.1 Example 1: Minimizing Viral Titer Loss During Filtration Asepticity is required when administering a viral preparation (e.g., a pharmaceutical composition) for therapeutic use via parenteral administration. This is typically achieved by sterile filtration of the preparation. Therefore, a preparation is needed that allows for adequate filterability (e.g., sterile filtration using 0.2 µm or 0.22 µm pore sizes). The excipients of the preparation need to be carefully selected and optimized to minimize the aggregation of viral particles and the formation of sub-visible particles during manufacturing, purification, and storage. Furthermore, the infectivity of the viral vector in the preparation needs to be maintained during storage. Therefore, an ideal isovirus formulation has the following characteristics: (a) minimal virus titer loss during purification, (b) minimal formation of sub-visible particles, (c) preservation of the biophysical stability of virus particles during freeze-thaw cycles, and (d) preservation of the biophysical stability of virus particles during storage.

[0358] Isoviruses are enveloped, bisegmented, bisense, single-stranded RNA (ssRNA) viruses. They exhibit significant polydispersity (polymorphism / typically spherical), with a diameter of approximately 50 nm to 300 nm. Virus particles tend to aggregate and / or form sub-visible particles during manufacturing and storage. The formation of sub-visible particles can lead to significant loss of virus particles during sterile filtration (i.e., through 0.2 µm or 0.22 µm pore size membranes). Therefore, the main challenge for formulations containing isovirus particles is to select a formulation that allows for sterile filtration through 0.2 µm or 0.22 µm pore size filters, avoids significant loss of virus titer, and maintains the biophysical stability of virus particles during subsequent storage.

[0359] In addition, the sand-particle virus preparation should be able to be stored at -20°C, or ideally 2°C–8°C, for ease of handling. Furthermore, the sand-particle virus preparation may also be able to be stored at room temperature (e.g., 20°C–25°C) for a defined short period of time.

[0360] A closed system described in U.S. Patent No. 10,662,412 has been developed to allow the preparation of viruses under completely sterile conditions, thereby eliminating the sterile filtration step, reducing virus loss during purification, and increasing virus recovery. However, this closed system has the disadvantages of higher production costs and more complex operating conditions compared to manufacturing processes using terminal 0.2µm or 0.22µm sterile filtration.

[0361] 7.1.1 The presence of polyvalent anions in the sand-particle virus preparation reduces virus particle loss during sterile filtration.The effects of polyvalent anions (e.g., TSPP, citrate, malate, succinate, and tartrate) on the filtration recovery rate of bacteria other than those listed on pages 58 / 63 of the specification, CN 121866044 A, were evaluated. The corresponding viral titers were analyzed before and after filtration using the Replication Capacity Virus Focal Formation Unit (RCV-FFU) assay. The percentage of viral vector particles recovered after filtration was determined. The effect of polyanions on filtration recovery rate was calculated (see Figure 1). The evaluation was performed using MODDE Pro (Satorius Stedim Data Analytics AB) software. A D-optimal design was applied, and the data were fitted using a multiple linear regression (MLR) model. The effect plot shows the effect calculated as twice the MLR coefficient, arranged in descending order of absolute value (95% confidence level). Excipients with a positive effect on filterability are indicated by positive values.

[0362] As shown in Figure 1, all tested polyanions showed a positive effect on filtration recovery rate, with TSPP and citrate showing the strongest positive effects.

[0363] 7.1.2 Analysis of the relationship between the recovery rate of virus particles after sterile filtration and the increase in the concentrations of sodium chloride (NaCl) and citrate in the isovirus preparation. Virus titers were determined by foci-forming units (FFU) and RCV-FFU assays before and after sterile filtration. Evaluation was performed using MODDE Pro (Satorius Stedim Data Analytics AB). A D-optimal design was applied, and the data were fitted using a multiple linear regression (MLR) model.

[0364] The results showed that increasing the concentrations of NaCl and citrate in the isovirus preparation significantly improved the recovery rate of virus particles after sterile filtration (see Figure 2).

[0365] 7.1.3 Reducing Bioburden and Virus Titer Loss During Filtration with TRIS / Citrate-Based Formulations A small-scale purification run was conducted to compare virus particle recovery during the manufacture of sand-borne virus formulations using either a HEPES-based formulation (10 mM HEPES, 150 mM NaCl, 20 mM glycine, 1% rHSA, pH 7.4) or a TRIS-based formulation (10 mM Tris, 50 mM citrate, 125 mM NaCl, 5% (w / v) sucrose, 0.03% poloxamer 188, 0.1% rHSA; pH 8.0). The effects of different formulations (HEPES-based vs. TRIS / citrate-based) on virus titer and filtration recovery during the manufacturing process were compared.

[0366] Three batches of sand-borne virus formulations were treated with the HEPES-based formulation (see Figures 3A and 3B). The TRIS / citrate-based formulation was used...Five batches of sand virus formulation were treated with acid salts (see Figures 4A and 4B). Virus titers and filtration recoveries were analyzed by quantitative RCV-FFU and FFU after each manufacturing step (PS1–PS6). Table 1 shows ...

Claims

1. A liquid isovirus preparation, said liquid isovirus preparation comprising: (a) Sand virus; and (b) A polyvalent anion with a concentration between about 5 mM and about 150 mM, optionally wherein the concentration of the polyvalent anion is between about 10 mM and about 60 mM.

2. The liquid sand-like virus preparation according to claim 1, wherein the concentration of the polyvalent anion is about 50 mM or about 25 mM.

3. The liquid sand virus preparation according to claim 1, wherein the liquid sand virus preparation is suitable for lyophilization, and the concentration of the polyvalent anion is at most about 50 mM or between about 20 mM and about 50 mM.

4. A liquid isovirus preparation, said liquid isovirus preparation comprising: (a) Sand virus; and (b) Multivalent anions, wherein the concentration of said multivalent anions (i) reduces the formation of subvisible particles during manufacturing and / or (ii) reduces the loss of virus titers during filtration. Optionally, the filtration includes bioburden reduction filtration or sterilization filtration, and / or the manufacturing includes a purification process.

5. The liquid sand-like virus preparation according to claim 4, wherein the concentration of the polyvalent anion is between about 5 mM and about 150 mM, optionally wherein the concentration of the polyvalent anion is between about 10 mM and about 60 mM.

6. The liquid sand-like virus preparation according to claim 5, wherein the concentration of the polyvalent anion is about 50 mM or about 25 mM.

7. The liquid sand virus preparation according to claim 5, wherein the liquid sand virus preparation is suitable for lyophilization, and the concentration of the polyvalent anion is at most about 50 mM or between about 20 mM and about 50 mM.

8. The liquid sand virus preparation according to any one of claims 1 to 7, wherein the polyvalent anion is selected from the group consisting of citrate, tetrasodium pyrophosphate (TSPP), malate, succinate, tartrate, fumarate, maleate, and combinations thereof.

9. The liquid sand-like virus preparation according to claim 8, wherein the polyvalent anion is citrate.

10. The liquid sand virus preparation according to claim 9, wherein the concentration of citrate is about 50 mM or about 25 mM.

11. The liquid sand virus preparation according to claim 9, wherein the liquid sand virus preparation is suitable for freeze drying, and the concentration of citrate is at most about 50 mM or between about 20 mM and about 50 mM.

12. The liquid isovirus preparation according to any one of claims 1 to 11, wherein the liquid isovirus preparation further comprises at least one of the following: (c) Carbohydrates; (d) Salt; (e) Buffer; (f) Surfactants; and (g) Stabilizer.

13. The liquid sand-like virus preparation according to claim 12, wherein: (c) The concentration of the carbohydrate is between about 0.1% and about 20% (w / v), and optionally the concentration of the carbohydrate is between about 3% and about 10% (w / v); (d) The concentration of the salt is at most about 450 mM, and optionally the concentration of the salt is between about 50 mM and about 250 mM; (e) The concentration of the buffer is between about 1 mM and about 50 mM, optionally wherein the concentration of the buffer is between about 10 mM and about 20 mM; (f) The concentration of the surfactant is between about 0.001% and about 1% (w / v), optionally wherein the concentration of the surfactant is between about 0.01% and about 0.05% (w / v); and / or (g) The concentration of the stabilizer is between about 0.001% (w / v) and about 3% (w / v), and optionally the concentration of the stabilizer is between about 0.05% (w / v) and 0.15% (w / v).

14. The liquid sand-like virus preparation according to claim 12 or 13, wherein... (c) The concentration of the carbohydrate is about 5% (w / v); (d) The concentration of the salt is about 125 mM or about 75 mM; (e) The concentration of the buffer is approximately 10 mM; (f) The concentration of the surfactant is approximately 0.03% (w / v); and / or (g) The concentration of the stabilizer is about 0.1% (w / v).

15. The liquid isovirus preparation according to claim 12 or 13, wherein the liquid isovirus preparation is suitable for lyophilization, and (c) The concentration of the carbohydrate is about 5% (w / v); (d) The concentration of the salt is at most about 125 mM or between about 50 mM and about 125 mM; (e) The concentration of the buffer is approximately 10 mM; (f) The concentration of the surfactant is approximately 0.03% (w / v); and / or (g) The concentration of the stabilizer is about 0.1% (w / v).

16. The liquid sand-like virus preparation according to any one of claims 12 to 15, wherein: (c) The carbohydrate is a sugar or a polyol; optionally, the sugar or polyol is selected from the group consisting of sucrose, trehalose, sorbitol, mannitol, dextran, and combinations thereof; further optionally, the carbohydrate is sucrose, trehalose, or combinations thereof; (d) The salt is selected from the group consisting of sodium chloride, sodium phosphate, potassium phosphate, potassium chloride, magnesium chloride, sodium sulfate, potassium sulfate, ammonium sulfate, and combinations thereof; optionally, the salt is sodium chloride; (e) The buffer is selected from the group consisting of phosphate, citrate, TIS, HEPES, bicarbonate, histidine, and combinations thereof; optionally, the buffer is TIS. (f) The surfactant is selected from the group consisting of poloxamer 188, poloxamer 407, polysorbate 20, polysorbate 80, and combinations thereof; optionally, the surfactant is poloxamer 188; and / or (g) The stabilizer is selected from the group consisting of albumin, recombinant protein, plasma protein, gelatin, and combinations thereof; optionally, the albumin is lactalbumin, human serum albumin, recombinant human serum albumin (rHSA), or combinations thereof; further optionally, the stabilizer is rHSA.

17. The liquid sand-like virus preparation according to claim 16, wherein: (c) The carbohydrate is sucrose or trehalose at a concentration of about 5% (w / v); (d) The salt is sodium chloride with a concentration of about 125 mM or about 75 mM; (e) The buffer is TRIS with a concentration of about 10 mM; (f) The surfactant is poloxamer 188 at a concentration of about 0.03% (w / v); and / or (g) The stabilizer is rHSA at a concentration of about 0.1%.

18. The liquid isovirus preparation of claim 16, wherein the liquid isovirus preparation is suitable for lyophilization, and: (c) The carbohydrate is trehalose at a concentration of about 5% (w / v); (d) The salt is sodium chloride with a concentration of up to about 125 mM or between about 50 mM and about 125 mM; (e) The buffer is TRIS with a concentration of about 10 mM; (f) The surfactant is poloxamer 188 at a concentration of about 0.03% (w / v); and / or (g) The stabilizer is rHSA at a concentration of about 0.1%.

19. The liquid sand virus preparation according to any one of claims 1 to 18, wherein the pH of the liquid sand virus preparation is between about 7.0 and about 9.

0.

20. The liquid sand-like virus preparation according to claim 19, wherein the pH is about 8.

0.

21. The liquid sand-like virus preparation according to any one of claims 1 to 20, wherein the permeation concentration of the liquid sand-like virus preparation is between about 200 mOsm / kg and about 1000 mOsm / kg.

22. The liquid sand-like virus preparation according to claim 21, wherein the osmotic concentration of the liquid sand-like virus preparation is between about 500 mOsm / kg and about 550 mOsm / kg, optionally wherein the osmotic concentration of the liquid sand-like virus preparation is about 530 mOsm / kg.

23. The liquid sand-like virus preparation according to claim 21, wherein the osmotic concentration of the liquid sand-like virus preparation is between about 350 mOsm / kg and about 450 mOsm / kg, optionally wherein the osmotic concentration of the liquid sand-like virus preparation is about 400 mOsm / kg.

24. The liquid sand virus preparation according to any one of claims 21, wherein the liquid sand virus preparation is suitable for lyophilization and has an osmotic concentration between about 300 mOsm / kg and about 600 mOsm / kg.

25. The liquid sand-like virus preparation according to any one of claims 12 to 24, wherein (c) to (g) are present to reduce virus titer loss during filtration, freeze-thaw cycles and / or storage; and / or to reduce the concentration of subvisible particle formation during manufacturing, freeze-thaw cycles and / or storage.

26. The liquid sand-like virus preparation according to claim 25, wherein the storage temperature is above about 2°C, between about 20°C and about 25°C, between about 2°C and about 8°C, above about 15°C, about -20°C or below about -20°C, about -65°C or below about -65°C, or about -80°C.

27. A liquid isovirus preparation, said liquid isovirus preparation comprising: (a) Sand virus; (b) Citrate at a concentration of approximately 50 mM; (c) Sucrose at a concentration of approximately 5% (w / v); (d) NaCl with a concentration of approximately 125 mM; (e) TRIS at a concentration of approximately 10 mM; and (f) Poloxamer 188 at a concentration of approximately 0.03% (w / v); and (g) rHSA at a concentration of approximately 0.1% (w / v), The liquid sand-like virus preparation has a pH of about 8.0, and optionally the osmotic concentration of the liquid sand-like virus preparation is about 530 mOsm / kg.

28. A liquid isovirus preparation, said liquid isovirus preparation comprising: (a) Sand virus; (b) Citrate at a concentration of approximately 25 mM; (c) Trehalose at a concentration of approximately 5% (w / v); (d) NaCl with a concentration of approximately 75 mM; (e) TRIS at a concentration of approximately 10 mM; and (f) Poloxamer 188 at a concentration of approximately 0.03% (w / v); and (g) rHSA at a concentration of approximately 0.1% (w / v), The liquid sand-like virus preparation has a pH of about 8.0, and optionally the osmotic concentration of the liquid sand-like virus preparation is about 400 mOsm / kg.

29. A liquid isovirus preparation suitable for freeze-drying, said liquid isovirus preparation comprising: (a) Sand virus; (b) Citrate ions with concentrations between approximately 20 mM and approximately 50 mM; (c) Trehalose at a concentration of approximately 5% (w / v); (d) Sodium chloride with a concentration of up to about 125 mM or between about 50 mM and about 125 mM; (e) TRIS at a concentration of approximately 10 mM; (f) Poloxamer 188 at a concentration of approximately 0.03% (w / v); and (g) rHSA at a concentration of approximately 0.1% (w / v), The liquid sand virus preparation has a pH of about 8.0, and optionally the liquid sand virus preparation suitable for freeze drying has an osmotic concentration between about 300 mOsm / kg and about 600 mOsm / kg.

30. The liquid isovirus preparation according to any one of claims 1 to 29, wherein the concentration of said isovirus is between about 10... 2 RCV-FFU / ml and approximately 10 10 RCV-FFU / ml is between, or between, approximately 2×10 5 RCV-FFU / ml and approximately 5×10 8 RCV-FFU / ml is between.

31. The liquid sand virus preparation according to any one of claims 1 to 30, wherein the sand virus is lymphocytic choriomeningitis virus (LCMV), Piccind virus, Tamiyami virus, or Tacalib virus.

32. The liquid sand virus preparation according to any one of claims 1 to 31, wherein the sand virus is a genetically engineered sand virus.

33. The liquid isovirus preparation according to any one of claims 1 to 32, wherein the isovirus expresses an antigen, optionally wherein the antigen is selected from the group consisting of tumor antigens, tumor-associated antigens, antigens of pathogens capable of causing infectious diseases, testicular cancer antigen (CTA), and antigenic fragments of any one of the foregoing.

34. The liquid sand-like virus preparation of claim 33, wherein the antigen comprises cytomegalovirus (CMV) antigen, optionally wherein the CMV antigen is selected from the group consisting of gB(dCt), pp65, and combinations thereof.

35. The liquid sand-like virus preparation of claim 33, wherein the antigen comprises human papillomavirus (HPV) antigen, optionally wherein the HPV antigen is an HPV E7 / E6 fusion protein.

36. The liquid sand-like virus preparation of claim 33, wherein the antigen comprises a prostate cancer antigen, optionally wherein the prostate cancer antigen is selected from the group consisting of PSA, PSMA, PAP, and combinations thereof.

37. The liquid sand-like virus preparation of claim 33, wherein the antigen comprises hepatitis B virus (HBV) antigen, optionally wherein the HBV antigen is selected from the group consisting of a truncated HBV polymerase polypeptide comprising an inactivated reverse transcriptase domain and an inactivated RNase H, a fusion protein comprising an HBV core polypeptide and an HBV small surface antigen (sAg) polypeptide, and combinations thereof.

38. The liquid sand-like virus preparation of claim 33, wherein the antigen comprises human immunodeficiency virus (HIV) antigen, optionally wherein the HIV antigen is selected from the group consisting of gag, pol, nef and combinations thereof, and further optionally wherein the HIV antigen is a fusion polypeptide comprising a plurality of polypeptide segments of one or more HIV-1 proteins encoded by two or more HIV genes selected from gag, pol and nef.

39. The liquid sand virus preparation of claim 33, wherein the antigen comprises at least one antigenic fragment of mutant KRAS, optionally wherein the at least one antigenic fragment comprises mutants G13D, G12V, G12C, G12D and G12R from the N-terminus to the C-terminus, respectively.

40. The liquid sand-like virus preparation of claim 33, wherein the antigen comprises two or more testicular cancer antigen (CTA) fragments, optionally wherein the two or more CTA fragments are selected from the group consisting of epitopes or antigenic fragments of melanoma-preferred antigens (PRAME), melanoma-associated antigen 3 (MAGE-A3), melanoma-associated antigen 4 (MAGE-A4), and melanoma-associated antigen 6 (MAGE-A6).

41. The liquid isovirus preparation according to any one of claims 1 to 40, wherein the isovirus is a non-replicating isovirus, optionally wherein the isovirus ORF has been removed or functionally inactivated such that the resulting isovirus cannot produce further infectious progeny virus particles, and further optionally wherein the ORF encoding the GP has been removed and replaced with a nucleotide sequence encoding an antigen.

42. The liquid sand virus preparation according to any one of claims 1 to 40, wherein the sand virus is a replicating trisegmented sand virus.

43. The liquid isovirus preparation of claim 42, wherein the three-segment isovirus comprises isovirus ORF located outside the wild-type position of the ORF.

44. The liquid isovirus preparation of claim 43, wherein the three-segment isovirus comprises an L segment and two S segments, wherein one of the two S segments is an S segment, wherein the ORF encoding the isovirus glycoprotein (GP) is under the control of the isovirus 3' UTR.

45. The liquid isovirus formulation of claim 42, wherein the three-segmented isovirus is engineered such that the isovirus ORF is distributed on two or more mRNA transcripts.

46. ​​A freeze-dried sand virus preparation, said freeze-dried sand virus preparation being prepared from any one of the liquid sand virus preparations according to claims 1 to 45.

47. A method for (i) reducing the formation of subvisible particles during manufacturing and / or (ii) reducing viral titer loss during filtration, the method comprising mixing a sand-like virus with a solution to form a liquid sand-like virus preparation, wherein the liquid sand-like virus preparation comprises a polyvalent anion at a concentration between about 5 mM and about 150 mM, optionally wherein the concentration of the polyvalent anion is between about 10 mM and about 60 mM.

48. The method of claim 47, wherein the concentration of the polyvalent anion is about 50 mM or about 25 mM.

49. The method of claim 47, wherein the liquid sand virus preparation is suitable for lyophilization, and the concentration of the polyvalent anion is between about 20 mM and about 50 mM.

50. A method for (i) reducing the formation of subvisible particles during manufacturing and / or (ii) reducing viral titer loss during filtration, the method comprising mixing a sand-like virus with a solution to form a liquid sand-like virus formulation, wherein the liquid sand-like virus formulation comprises a polyvalent anion, the concentration of which reduces the formation of subvisible particles during manufacturing and / or reduces viral titer loss during filtration.

51. The method of claim 50, wherein the concentration of the polyvalent anion is between about 5 mM and about 150 mM.

52. The method of claim 51, wherein the concentration of the polyvalent anion is about 50 mM or about 25 mM.

53. The method of claim 51, wherein the liquid sand virus preparation is suitable for lyophilization, and the concentration of the polyvalent anion is between about 20 mM and about 50 mM.

54. The method according to any one of claims 47 to 53, wherein the polyvalent anion is selected from the group consisting of citrate, tetrasodium pyrophosphate (TSPP), malate, succinate, tartrate, fumarate, maleate, and combinations thereof.

55. The method of claim 54, wherein the polyvalent anion is citrate.

56. The method of claim 55, wherein the concentration of citrate is about 50 mM or about 25 mM.

57. The method of claim 55, wherein the concentration of citrate is between about 10 mM and about 60 mM.

58. The method according to any one of claims 47 to 57, wherein the liquid sand-like virus preparation further comprises at least one of the following: (b) Carbohydrates; (c) Salt; (d) Buffer; (e) Surfactants; and (f) Stabilizers.

59. The method according to claim 58, wherein: (b) The concentration of the carbohydrate is between about 0.1% and about 20% (w / v), and optionally the concentration of the carbohydrate is between about 3% and about 10% (w / v); (c) The concentration of the salt is at most about 450 mM, and optionally the concentration of the salt is between about 50 mM and about 250 mM; (d) The concentration of the buffer is between about 1 mM and about 50 mM, optionally wherein the concentration of the buffer is between about 10 mM and about 20 mM; (e) The concentration of the surfactant is between about 0.001% and about 1% (w / v), optionally wherein the concentration of the surfactant is between about 0.01% and about 0.05% (w / v); and / or (f) The concentration of the stabilizer is between about 0.001% (w / v) and about 3% (w / v), and optionally the concentration of the stabilizer is between about 0.05% (w / v) and 0.15% (w / v).

60. The method according to claim 58 or 59, wherein (b) The concentration of the carbohydrate is about 5% (w / v); (c) The concentration of the salt is about 125 mM or about 75 mM; (d) The concentration of the buffer is approximately 10 mM; (e) The concentration of the surfactant is approximately 0.03% (w / v); and / or (f) The concentration of the stabilizer is about 0.1% (w / v).

61. The method according to claim 58 or 59, wherein the liquid sand-like virus preparation is suitable for lyophilization, and (b) The concentration of the carbohydrate is about 5% (w / v); (c) The concentration of the salt is at most about 125 mM or between about 50 mM and about 125 mM; (d) The concentration of the buffer is approximately 10 mM; (e) The concentration of the surfactant is approximately 0.03% (w / v); and / or (f) The concentration of the stabilizer is about 0.1% (w / v).

62. The method according to any one of claims 58 to 61, wherein: (b) The carbohydrate is a sugar or a polyol; optionally, the sugar or polyol is selected from the group consisting of sucrose, trehalose, sorbitol, mannitol, dextran, and combinations thereof; further optionally, the carbohydrate is sucrose, trehalose, or combinations thereof; (c) The salt is selected from the group consisting of sodium chloride, sodium phosphate, potassium phosphate, potassium chloride, magnesium chloride, sodium sulfate, potassium sulfate, ammonium sulfate, and combinations thereof; optionally, the salt is sodium chloride; (d) The buffer is selected from the group consisting of phosphate, citrate, TRIS, HEPES, bicarbonate, histidine, and combinations thereof; optionally, the buffer is TRIS. (e) The surfactant is selected from the group consisting of poloxamer 188, poloxamer 407, polysorbate 20, polysorbate 80, and combinations thereof; optionally, the surfactant is poloxamer 188; and / or (f) The stabilizer is a protein selected from the group consisting of albumin, recombinant protein, plasma protein, gelatin, and combinations thereof; optionally, the albumin is lactalbumin, human serum albumin, recombinant human serum albumin (rHSA), or combinations thereof; further optionally, the protein is rHSA.

63. The method according to claim 62, wherein: (b) The carbohydrate is sucrose or trehalose at a concentration of about 5% (w / v); (c) The salt is sodium chloride with a concentration of about 125 mM or about 75 mM; (d) The buffer is TRIS with a concentration of approximately 10 mM; (e) The surfactant is poloxamer 188 at a concentration of about 0.03% (w / v); and / or (f) The stabilizer is rHSA at a concentration of about 0.1%.

64. The method of claim 62, wherein the liquid sand-like virus preparation is suitable for lyophilization, and: (b) The carbohydrate is trehalose at a concentration of about 5% (w / v); (c) The salt is sodium chloride with a concentration of up to about 125 mM or between about 50 mM and about 125 mM; (d) The buffer is TRIS with a concentration of approximately 10 mM; (e) The surfactant is poloxamer 188 at a concentration of about 0.03% (w / v); and / or (f) The stabilizer is rHSA at a concentration of about 0.1%.

65. The method according to any one of claims 47 to 64, wherein the pH of the liquid sand-like virus preparation is between about 7.0 and about 9.0, optionally wherein the pH is about 8.

0.

66. The method according to any one of claims 47 to 65, wherein the permeation concentration of the liquid sand-like virus preparation is between about 200 mOsm / kg and about 1000 mOsm / kg.

67. The method of claim 66, wherein the osmotic concentration of the liquid sand-like virus preparation is between about 500 mOsm / kg and about 550 mOsm / kg, optionally wherein the osmotic concentration of the liquid sand-like virus preparation is about 530 mOsm / kg.

68. The method of claim 66, wherein the osmotic concentration of the liquid sand-like virus preparation is between about 350 mOsm / kg and about 450 mOsm / kg, optionally wherein the osmotic concentration of the liquid sand-like virus preparation is about 400 mOsm / kg.

69. The method of claim 66, wherein the liquid sand-like virus preparation is suitable for freeze-drying and has a permeation concentration between about 300 mOsm / kg and about 600 mOsm / kg.

70. The method according to any one of claims 58 to 69, wherein (b) to (f) are present at concentrations that reduce viral titer loss during filtration, reduce subvisible particle formation, maintain the biophysical stability of the sand virus during freeze-thaw processes, and / or maintain the biophysical stability of the sand virus during storage.

71. The method of claim 70, wherein the storage temperature is above about 2°C, between about 20°C and about 25°C, between about 2°C and about 8°C, above about 15°C, about -20°C or below about -20°C, about -65°C or below about -65°C, or about -80°C.

72. A method for (i) reducing the formation of subvisible particles during manufacturing and / or (ii) reducing viral titer loss during filtration, the method comprising mixing sand-like virus with a solution to form a liquid sand-like virus formulation, wherein the liquid sand-like virus formulation comprises: (a) Citrate at a concentration of approximately 50 mM; (b) Sucrose at a concentration of approximately 5% (w / v); (c) Sodium chloride at a concentration of approximately 125 mM; (d) TRIS at a concentration of approximately 10 mM; (e) Poloxamer 188 at a concentration of about 0.03% (w / v); and (f) rHSA at a concentration of approximately 0.1%, The liquid sand-like virus preparation has a pH of about 8.0, and optionally the osmotic concentration of the liquid sand-like virus preparation is about 530 mOsm / kg.

73. A method for (i) reducing the formation of subvisible particles during manufacturing and / or (ii) reducing viral titer loss during filtration, the method comprising mixing sand-like virus with a solution to form a liquid sand-like virus formulation, wherein the liquid sand-like virus formulation comprises: (a) Citrate at a concentration of approximately 25 mM; (b) Trehalose at a concentration of approximately 5% (w / v); (c) Sodium chloride at a concentration of approximately 75 mM; (d) TRIS at a concentration of approximately 10 mM; (e) Poloxamer 188 at a concentration of about 0.03% (w / v); and (f) rHSA at a concentration of approximately 0.1%, The liquid sand-like virus preparation has a pH of about 8.0, and optionally the osmotic concentration of the liquid sand-like virus preparation is about 400 mOsm / kg.

74. A method for (i) reducing subvisible particle formation during manufacturing and / or (ii) reducing virus titer loss during filtration, the method comprising mixing sand-like virus with a solution to form a liquid sand-like virus formulation, wherein the liquid sand-like virus formulation is suitable for lyophilization and comprises: (a) Citrate ions with concentrations between 10 mM and approximately 60 mM; (b) Trehalose at a concentration of approximately 5% (w / v); (c) Sodium chloride with a concentration of up to about 125 mM or between about 50 mM and about 125 mM; (d) TRIS at a concentration of approximately 10 mM; (e) Poloxamer 188 at a concentration of about 0.03% (w / v); and (f) rHSA at a concentration of approximately 0.1% (w / v), The pH of the liquid sand-like virus preparation is approximately 8.

0. Optionally, the filtration includes bioburden reduction filtration or sterilization filtration, and / or the manufacturing includes a purification process, wherein the permeation concentration of the liquid sand virus preparation suitable for lyophilization is between about 300 mOsm / kg and about 600 mOsm / kg.

75. The method according to any one of claims 47 to 74, wherein the concentration of the sand-like virus is between about 10. 2 RCV-FFU / ml and approximately 10 10 RCV-FFU / ml is between, or between, approximately 2×10 5 RCV-FFU / ml and approximately 5×10 8 RCV-FFU / ml is between.

76. The method according to any one of claims 47 to 75, wherein the sand virus is lymphocytic choriomeningitis virus (LCMV), Piccind virus, Tamiyami virus, or Tacalib virus.

77. The method according to any one of claims 47 to 76, wherein the sand virus is a genetically engineered sand virus.

78. The method according to any one of claims 47 to 77, wherein the arenavirus expresses an antigen, optionally wherein the antigen is selected from the group consisting of tumor antigens, tumor-associated antigens, antigens of pathogens capable of causing infectious diseases, testicular cancer antigen (CTA), and antigenic fragments of any of the foregoing.

79. The method of claim 78, wherein the antigen comprises at least one cytomegalovirus (CMV) antigen, optionally wherein the CMV antigen is selected from the group consisting of gB(dCt), pp65, and combinations thereof.

80. The method of claim 78, wherein the antigen comprises at least one human papillomavirus (HPV) antigen, optionally wherein the HPV antigen is an HPV E7 / E6 fusion protein.

81. The method of claim 78, wherein the antigen comprises at least one prostate cancer antigen, optionally wherein the prostate cancer antigen is selected from the group consisting of PSA, PSMA, PAP, and combinations thereof.

82. The method of claim 78, wherein the antigen comprises hepatitis B virus (HBV) antigen, optionally wherein the HBV antigen is selected from the group consisting of a truncated HBV polymerase polypeptide comprising an inactivated reverse transcriptase domain and an inactivated RNase H, a fusion protein comprising an HBV core polypeptide and an HBV small surface antigen (sAg) polypeptide, and combinations thereof.

83. The method of claim 78, wherein the antigen comprises human immunodeficiency virus (HIV) antigen, optionally wherein the HIV antigen is selected from the group consisting of gag, pol, nef, and combinations thereof, and further optionally wherein the HIV antigen is a fusion polypeptide comprising a plurality of polypeptide segments of one or more HIV-1 proteins encoded by two or more HIV genes selected from gag, pol, and nef.

84. The method of claim 78, wherein the antigen comprises at least one antigenic fragment of a mutant KRAS, optionally wherein the antigenic fragment of the mutant KRAS comprises mutant G13D, G12V, G12C, G12D and G12R from the N-terminus to the C-terminus, respectively.

85. The method of claim 78, wherein the antigen comprises two or more testicular cancer antigen (CTA) fragments, optionally wherein the two or more CTA fragments are selected from the group consisting of epitopes or antigenic fragments of melanoma preferentially expressed antigens (PRAME), melanoma-associated antigen 3 (MAGE-A3), melanoma-associated antigen 4 (MAGE-A4), and melanoma-associated antigen 6 (MAGE-A6).

86. The method according to any one of claims 47 to 85, wherein the isovirus is a non-replicating isovirus, optionally wherein the isovirus ORF has been removed or functionally inactivated such that the resulting isovirus cannot produce further infectious progeny virus particles, and further optionally wherein the ORF encoding the GP has been removed and replaced with a nucleotide sequence encoding an antigen.

87. The method according to any one of claims 47 to 85, wherein the sand virus is a replicating trisegmental sand virus.

88. The method of claim 87, wherein the three-segmented sand virus comprises a sand virus ORF located outside the wild-type position of the ORF.

89. The method of claim 88, wherein the three-segmented sand virus comprises an L segment and two S segments, wherein one of the two S segments is an S segment, wherein the ORF encoding the GP is under the control of the sand virus 3' UTR.

90. The method of claim 87, wherein the three-segmented isavirus is engineered such that the isavirus ORF is distributed on two or more mRNA transcripts.

91. The method according to any one of claims 47 to 90, the method further comprising freeze-drying the liquid sand-virus preparation.

92. A method for producing a freeze-dried isovirus preparation, the method comprising freeze-drying a liquid isovirus preparation according to any one of claims 1 to 45.

93. A method of treating or preventing a disease, the method comprising administering to a subject in need a liquid sand-like virus preparation according to any one of claims 1 to 45, optionally wherein the liquid sand-like virus preparation is further diluted prior to administration.

94. A method of treating or preventing a disease, the method comprising administering to a subject in need a liquid sand virus preparation reconstituted from a lyophilized preparation according to claim 46, wherein the lyophilized preparation is reconstituted prior to administration.

95. The method according to claim 93 or 94, wherein the liquid sand virus preparation is administered parenterally, optionally wherein the liquid sand virus preparation is administered intravenously, intramuscularly, intratumorally, or subcutaneously.

96. The method according to claims 93 to 95, wherein the disease is a neoplastic disease or an infectious disease.

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