Methods of purifying compositions comprising group b adenoviruses

Abstract

A method of purifying a composition comprising a Group B adenovirus, for example comprising the following purification step: diafiltration of a composition comprising the Group B adenovirus using a diafiltration buffer having a conductivity of at least 180 mS cm ‑1 , for example a conductivity of 190, 200, 210, 220, 230, 240, 250, 260, 270, 280 or 290 mS cm ‑1 . Also provided are compositions obtained using the purification methods disclosed herein.

Description

[0001] The present disclosure relates to methods of purifying compositions comprising Group B adenoviruses, and purified compositions obtainable from the methods. BACKGROUND

[0002] Currently, the pharmaceutical field is approaching the realization of the potential of viruses as therapeutic agents for use in humans. To date, a virus derived from ONXY-15 (ONYX Pharmaceuticals, and obtained by Shanghai Sunway Biotech) is approved for use in head and neck cancer in a limited number of countries. However, there are currently many viruses in the clinic, which should lead to some of these viruses being registered for use in humans.

[0003] One or more therapies are based on Group B adenovirus EnAd (formerly known as ColoAdl), a chimeric oncolytic adenovirus derived from Adl l (WO 2005 / 118825, and armed versions of which are disclosed in WO2015 / 059303 and WO2016 / 174200, each incorporated herein by reference). EnAd is currently in clinical trials treating colorectal cancer. As part of the manufacturing process, the virus is propagated in vitro in mammalian cells, for example in cell suspension cultures. The virus is recovered from these cells by cell lysis and subsequent purification. These adenovirus-based therapeutics need to be manufactured at purity levels free of host cell proteins and in compliance with good manufacturing practice (GMP).

[0004] WO00 / 32754 discloses a process for preparing highly purified adenovirus. The disclosure in this PCT application, Figure 23 and page 164, can be summarised as follows:

[0005] • Ad5 (adenovirus Group C) is released from HEK293 cells by lysis buffer;

[0006] • The crude cell lysate containing Ad5 is clarified by filtration through two 5 micron filters;

[0007] • The supernatant is then concentrated by about 10-fold by diafiltration using buffer 0.5M Tris, 1 mM MgCl2at pH 8;

[0008] • It is then treated with benzonase in 0.5M Tris / HCl, 1 mM MgCl2at pH 8 and filtered through a 0.2 micron filter;

[0009] • The resulting composition is subjected to strong anion exchange chromatography using Source 15Q resin, with elution buffer 20mM Tris, 1 mM MgCl2, 250mM (0.25M) NaCl at pH 8;

[0010] • The purified composition is concentrated and put into the final isotonic buffer using diafiltration.

[0011] Anion exchange chromatography is a process that separates substances according to their charge using ion exchange resins containing positively charged groups, such as diethylaminoethyl (DEAE). In the case of adenovirus production, anion exchange chromatography is used to purify adenovirus from proteins in the host cell (host cell proteins or HCPs) that are negatively charged at higher pH levels. Secondary ion exchange chromatography is known from Brument et al., Molecular Therapy Vol. 6, No. 5, November 2002.

[0012] However, the inventors have found that group B adenoviruses, such as Adl 1, cannot be sufficiently separated from host cell proteins by anion exchange chromatography. Figure 1A Retention times of Adl 1 virus and Ad5 virus are shown when analyzed by anion exchange chromatography. These viruses have very different retention times on the x-axis of approximately 10 and 15. Figure 1B It is shown that Adl 1 type viruses, such as EnAd, are eluted with host cell proteins using anion exchange chromatography. Thus, while ion exchange chromatography is currently the gold standard for adenovirus purification, it is not effective for group B viruses (e.g. Adl 1 type viruses, such as EnAd) because these viruses behave differently than group C viruses, such as Ad5.

[0013] The prior art in the field of GMP manufacturing for adenoviruses is mainly performed on Ad5, i.e. group C adenoviruses.

[0014] The inventors have found that the optimal conditions and processes for purifying adenoviruses differ depending on the group of adenoviruses. Adenoviruses are grouped based on DNA homology and / or their hexon, fiber and capsid characteristics in chromatographic analysis.

[0015] Developing a successful purification process for recombinant adenoviruses requires a detailed understanding of the recombinant virus, e.g. the interactions between the host cell line and the virus. The process essentially needs to be adjusted depending on the specific group of viruses.

[0016] Surprisingly, the inventors have found that group B adenoviruses (e.g. Adl 1 type adenoviruses, such as EnAd) can be purified from host cell proteins essentially using one diafiltration step with a high concentration of salt in the buffer. This is not possible with standard prior art processes. In the examples, ion exchange chromatography can be completely omitted from the process.

[0017] Therefore, there is a need for an improved purification process specifically adapted to produce group B adenoviruses. SUMMARY

[0018] Surprisingly, the inventors have determined that group B adenovirus vectors can be purified through a process that significantly reduces the level of host cell proteins contaminating the final product. This disclosure is described in the following paragraphs:

[0019] 1. A method for purifying a group B adenovirus with replication capability from host cell proteins, the method comprising the following purification steps:

[0020] Using at least 180 mScm -1 The electrical conductivity, for example, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280 or 290 mS / cm. -1 The composition containing the group B adenovirus was percolated with a percolation buffer having a conductivity of [insert conductivity value here].

[0021] 2. According to the method in paragraph 1, the conductivity is provided by a strong electrolyte.

[0022] 3. According to the method in paragraph 2, the electrolyte is a salt, such as an ionic salt (especially a salt that is completely soluble and highly dissociable in water).

[0023] 4. A method for purifying a group B adenovirus with replication capability from host cell proteins, the method comprising the following purification steps:

[0024] Using, for example, having at least 180 mScm -1 The electrical conductivity, such as 190, 200, 210, 220, 230, 240, 250, 260, 270, 280 or 290 mS / cm -1 The composition containing the group B adenovirus is percolated with a percolation buffer having a high conductivity and a high salt concentration, wherein the salt concentration is at least 2M, for example in the range of 2.5M to 5.5M, such as 3M, 3.5M, 4M, 4.5M or 5M, particularly 4M, 4.1M, 4.2M, 4.3M, 4.4M, 4.5M, 4.6M, 4.7M, 4.8M or 4.9M, more specifically 4.3M.

[0025] 5. The method according to any one of paragraphs 3 or 4, wherein the buffer solution comprises a salt selected from chloride salts (e.g., having cations selected from Li, Na, Mg, K, Ca, Cs and NH4), sulfates, and any combination that is completely soluble and dissociable in water.

[0026] 6. The method according to any one of paragraphs 3 or 5, wherein the salt in the dialysis buffer comprises one or more of the following: alkaline earth metal salts (such as NaCl, KCl and MgCl2), sodium acetate, Tris, Bis-Tris, NaH2PO4, such as NaCl or KCl, especially NaCl.

[0027] 7. The method according to any one of paragraphs 1 to 6, wherein the dialysis buffer is selected from: meglumine buffer, Gly-NaCl buffer, and TRIS buffer.

[0028] 8. The method according to paragraph 7, wherein the percolation buffer contains HEPES, for example at least 10, 20, 30, 40, 50, 60 or 70 mM HEPES, particularly 50 mM HEPES.

[0029] 9. The method according to any of the preceding paragraphs, wherein the pH of the percolation filtration buffer is in the range of 7 to 9.8, for example 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, such as pH 7.5.

[0030] 10. The method according to any one of paragraphs 1 to 10, wherein the percolation uses a 500 kDa MWCO ultrafiltration membrane, for example at least 300 kDa or greater.

[0031] 11. The method according to any one of paragraphs 1 to 10, wherein the percolation has a depth of 1 to 3 m 2 Flow rates per second, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 m / s. 2 / s.

[0032] 12. The method according to any one of paragraphs 1 to 11, wherein the percolation is a pressure-independent method.

[0033] 13. The method according to any of the preceding paragraphs, wherein the percolation is performed using a hollow fiber cartridge or a flat membrane box filter.

[0034] 14. The method in paragraph 13 is used, where the uniform volume method is used for TFF.

[0035] 15. The method according to any of the preceding paragraphs, wherein at least 8 perfiltration volumes, such as 11, 12, 13, 14, 15, 16, 17, or 18 perfiltration volumes, for example 11, 12, 13, 14, or 15 perfiltration volumes, such as 12 perfiltration volumes of high-salt perfiltration buffer, are used for perfiltration.

[0036] 16. The method according to any of the preceding paragraphs, wherein the percolation process comprises two steps (i.e., first and second steps).

[0037] 17. According to the method in paragraph 16, the first step of the process is percolation with a high-conductivity percolation buffer.

[0038] 18. According to the method in paragraph 16 or 17, the second step of the process is percolation with the final formulation buffer.

[0039] 19. The method according to paragraph 18, wherein the final formulation buffer comprises meglumine buffer, glycine buffer, TRIS buffer, and HEPES.

[0040] 20. The method according to paragraph 19, wherein the final formulation buffer contains HEPES, for example 5 mM HEPES.

[0041] 21. The method according to any one of paragraphs 18 to 20, wherein the final formulation buffer contains glycerol, for example 20% m / v glycerol.

[0042] 22. The method according to paragraph 20 or 21, wherein the final formulation buffer consists of 5 mM HEPES and 20% m / V glycerol.

[0043] 23. The method according to any one of paragraphs 16 to 22, wherein the second percolation step is performed using at least 8 percolation volumes, such as 11, 12, 13, 14, 15, 16, 17, or 18 percolation volumes of final formulation buffer, for example, 15 percolation volumes.

[0044] 24. The method according to any one of paragraphs 1 to 23, wherein only one percolation buffer is used.

[0045] 25. The method according to any one of paragraphs 16 to 24, wherein the first percolation step sequentially uses a plurality of percolation buffers.

[0046] 26. The method in paragraph 25, wherein two, three or four percolation buffers are used, such as two percolation buffers.

[0047] 27. According to the method in paragraph 26, one of the various percolation buffers used is 1M NaCl at pH 7.5, 50M m HEPES, 1.0% m / V Tween 20, and 1.0% m / V glycerol.

[0048] 28. The method according to any one of paragraphs 18 to 27, wherein the pH of the final formulation buffer is in the range of 7 to 9.8, for example 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, such as pH 7.5.

[0049] 29. The method according to any of the preceding paragraphs, further comprising a purification step, said step comprising chromatographic purification of the adenovirus composition.

[0050] 30. The method according to paragraph 29, wherein the chromatographic purification step is performed prior to percolation.

[0051] 31. According to the method of paragraph 29, the chromatographic purification step is performed after the percolation step.

[0052] 32. The method according to any one of paragraphs 29 to 31, wherein the chromatographic step uses ion exchange chromatography, such as anion exchange chromatography.

[0053] 33. The method according to paragraph 32, wherein the anion exchange chromatography utilizes DEAE, TMAE, QAE or PEI.

[0054] 34. The method according to any one of paragraphs 29 to 33, wherein the chromatography uses IEX membrane absorber capsules.

[0055] 35. According to the method in paragraph 34, the elution buffer used is 450 mM NaCl, 50 mM HEPES, and 1.0% m / V Tween 20 at pH 7.5.

[0056] 36. The method according to any one of paragraphs 29 to 35, wherein high performance liquid chromatography, such as CIMQAIEX2, is used.

[0057] 37. According to the method in paragraph 36, the elution buffer used is 400 mM NaCl, 50 mM Tris, 2 M MgCl2, and 5% glycerol at pH 7.8.

[0058] 38. According to any of the methods in paragraphs 1 to 37, all adenovirus purification steps in the preparation of the final adenovirus formulation are filtration steps.

[0059] 39. The method according to any one of paragraphs 1 to 28 and 38, wherein the adenovirus purification step does not use chromatography.

[0060] 40. The method according to any one of paragraphs 1 to 39, which includes a preliminary step of lysing a host cell in which an adenovirus has been replicated to obtain a crude cell lysate.

[0061] 41. The method of claim 40, wherein the lysis step uses a lysis buffer.

[0062] 42. The method of claim 41, wherein the lysis buffer contains at least 10% surfactant.

[0063] 43. According to the method of paragraph 42, the surfactant is a nonionic surfactant, such as Tween-20.

[0064] 44. The method according to any one of paragraphs 41 to 43 further comprises a salt with a concentration range of 10 to 50 mM, such as 20, 30 or 40 mM, particularly 20 mM.

[0065] 45. The method according to any one of paragraphs 41 to 44, wherein the lysis buffer comprises meglumine buffer, glycine buffer, TRIS buffer, or HEPES.

[0066] 46. ​​The method according to paragraph 45, wherein the lysis buffer comprises HEPES.

[0067] 47. The method according to paragraph 46, wherein the HEPES concentration is in the range of 4.5M to 5.5M, such as 5M.

[0068] 48. The method according to any one of paragraphs 41 to 47, wherein the pH range of the lysis buffer is 7.75 to 8.25, for example, pH 8.

[0069] 49. The method according to any one of paragraphs 40 to 48, wherein an endonuclease, such as Benzonase, is added to the crude cell lysate.

[0070] 50. The method described in paragraph 49, wherein the adenovirus is transferred to an inactivation buffer.

[0071] 51. According to the method of paragraph 50, the inactivation buffer contains a high salt content, for example, ranging from 0.75 to 1.25 M, such as 1 M.

[0072] 52. According to the method in paragraph 50 or 51, wherein the pH range of the inactivation buffer is 7.25 to 7.75, for example, pH 7.5.

[0073] 53. The method according to any one of paragraphs 40 to 52, wherein the crude cell lysate after the addition of a nuclease is filtered to clarify the adenovirus composition.

[0074] 54. According to the method in paragraph 53, the filter is a deep filter.

[0075] 55. The method according to paragraph 53 or 54, wherein the depth filter used has a specification of 4 to 2 μm, such as CE35 (from Merck Millipore).

[0076] 56. The method according to any one of paragraphs 53 to 55, wherein a second filter is used in the clarification.

[0077] 57. According to the method in paragraph 56, the second filter is a deep filter.

[0078] 58. The method according to paragraph 57, wherein the depth filter used has a specification of 1 to 0.4 μm.

[0079] 59. The method according to any one of paragraphs 1 to 58, comprising a filtration step, said filtration step comprising passing the adenovirus composition through a 0.2 μm filter.

[0080] 60. According to the method of paragraph 59, the filtration step is performed before the percolation step.

[0081] 61. The method according to any of the preceding paragraphs, wherein the group B adenovirus includes the sequence of formula (I):

[0082] 5'ITR-B1-B A -B2-B X -B B -B Y -B3-3'ITR

[0083] in:

[0084] B1 is a key or contains: E1A, E1B, or E1A-E1B;

[0085] B A Includes -E2B-L1-L2-L3-E2A-L4;

[0086] B2 is a key or containment: E3;

[0087] B X It is a bond or DNA sequence that contains: restriction sites, one or more transgenes, or both;

[0088] B B Includes L5;

[0089] B Y It is a bond or DNA sequence that contains: restriction sites, one or more transgenes, or both;

[0090] B3 is a key or containment: E4;

[0091] Among them B X Or B Y At least one of them is not a key.

[0092] 62. According to the method in paragraph 61, where B X Contains genetically modified organisms (GMOs) or GMO boxes.

[0093] 63. According to the method in paragraph 61, where B X It is a key.

[0094] 64. According to any one of the methods in paragraphs 61 to 63, where B Y Contains genetically modified organisms (GMOs) or GMO boxes.

[0095] 65. The method according to any one of paragraphs 61 to 64, wherein the one or more transgenes or transgene cassettes are controlled by an endogenous or exogenous promoter, such as an endogenous promoter.

[0096] 66. According to the method of paragraph 65, the transgenic cassette is controlled by an endogenous promoter selected from the group consisting of E4 and major late promoters, such as major late promoters.

[0097] 67. The method according to any one of paragraphs 61 to 66, wherein the transgenic box further comprises an adjustment element independently selected from:

[0098] a. Clipping the acceptor sequence,

[0099] b. Internal ribosome entry sequence or highly autocleaved 2A peptide

[0100] c. Kozak sequence, and

[0101] d. Its combination.

[0102] 68. According to the method of paragraph 67, the transgenic cassette contains a Kozak sequence located at the beginning of a protein-coding sequence.

[0103] 69. The method according to any one of paragraphs 61 to 68, wherein the transgenic cassette encodes a highly autocleavage-efficient 2A peptide.

[0104] 70. The method according to any one of paragraphs 61 to 69, wherein the transgenic cassette further comprises a polyadenylated sequence.

[0105] 71. The method according to any one of paragraphs 61 to 70, wherein the transgenic cassette further comprises a restriction site located at the 3' end and / or the 5' end of the DNA sequence.

[0106] 72. The method according to any one of paragraphs 61 to 71, wherein at least one transgenic cassette encodes a monocistronic mRNA.

[0107] 73. The method according to any one of paragraphs 61 to 72, wherein at least one transgenic cassette encodes a polycistronic mRNA.

[0108] 74. The method according to any one of paragraphs 61 to 73, wherein the transgene encodes an RNAi sequence, peptide, or protein.

[0109] 75. The method according to paragraph 74, wherein the transgene encodes an antibody or a binding fragment thereof.

[0110] 76. The method according to paragraph 75, wherein the antibody or its binding fragment is specific to the following: OX40, OX40 ligand, CD27, CD28, CD30, CD40, CD40 ligand, CD70, CD137, GITR, 4-1BB, ICOS, ICOS ligand, CTLA-4, PD-1, PD-L1, PD-L2, VISTA, B7-H3, B7-H4, HVEM, ILT-2, ILT-3, ILT-4, TIM-3, LAG-3, BTLA, LIGHT, CD160, CTLA-4, PD-1, PD-L1, PD-L2, such as CD40 and CD40 ligand.

[0111] 77. The method according to any one of paragraphs 61 to 76, wherein the transgene encodes a cytokine independently selected from the group comprising: IL-1α, IL-1β, IL-6, IL-9, IL-12, IL-13, IL-17, IL-18, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-33, IL-35, IL-2, IL-4, IL-5, IL-7, IL-10, IL-15, IL-21, IL-25, IL-1RA, IFNα, IFNβ, IFNγ, TNFα, TGFβ, lymphotoxin α (LTA), and GM-CSF, such as IL-12, IL-18, IL-22, IL-7, IL-15, IL-21, IFNγ, TNFα, TGFβ, and lymphotoxin α (LTA).

[0112] 78. The method according to any one of paragraphs 61 to 77, wherein the transgene encodes a chemokine independently selected from the group comprising: IL-8, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10, CXCL11, CXCL13, CXCL12, CCL2, CCL19, CCL21, CXCR2, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CXCR3, CXCR4, CXCR5 and CRTH2, such as CCL5, CXCL9, CXCL12, CCL2, CCL19, CCL21, CXCR2, CCR2, CCR4 and CXCR4 or their receptors.

[0113] 79. The method according to any one of paragraphs 61 to 78, wherein said transgene is a reporter gene, such as a sodium iodide cotransporter, an intracellular metalloprotein, HSV1-tk, GFP, luciferase, or an estrogen receptor, such as a sodium iodide cotransporter.

[0114] 80. The method according to any one of paragraphs 1 to 79, wherein the E4orf4 region of the adenovirus is nonfunctional, such as completely deleted, partially deleted, or truncated.

[0115] 81. The method according to any one of paragraphs 1 to 80, wherein the E2B region of the adenovirus is chimeric, for example, wherein the E2B region comprises a nucleic acid sequence derived from a first adenovirus serotype and a nucleic acid sequence derived from a second different adenovirus serotype; wherein the first serotype and the second serotype are each selected from adenovirus subgroups B, C, D, E or F.

[0116] 82. The method according to any one of paragraphs 1 to 81, wherein the adenovirus is Ad11.

[0117] 83. The method according to any one of paragraphs 1 to 81, wherein the adenovirus is a chimeric EnAd.

[0118] 84. The method according to any one of paragraphs 1 to 83, wherein the adenovirus is replicable, for example, has the ability to replicate.

[0119] 85. The method according to any one of paragraphs 1 to 83, wherein the adenovirus is a replication-defective type.

[0120] 86. An adenovirus composition which is obtained or can be obtained from the method according to any one of paragraphs 1 to 85.

[0121] 87. The adenovirus composition according to paragraph 86, which is used for treatment, particularly for the treatment of cancer.

[0122] 88. The adenovirus composition according to paragraph 86, used to prepare a medicament for treating cancer.

[0123] 89. A treatment method comprising the step of administering a therapeutically effective amount of the adenovirus composition as defined in paragraph 86. Attached Figure Description

[0124] Figure 1A This is a chromatogram showing the analysis and separation of adenovirus 5 (Ad5) and adenovirus 11 (Ad11) by anion exchange chromatography.

[0125] Figure 1B This is a chromatogram showing that Ad11 cannot be separated from host cell proteins by anion exchange chromatography alone.

[0126] Figure 2(A) is a flowchart depicting the standard purification process for adenovirus, and (B) is a flowchart depicting the improved purification process for group B adenovirus vectors disclosed herein.

[0127] Figure 3 Showing Figure 2B The technical details of the improvement process are shown.

[0128] Figure 4 A flowchart depicting a one-step purification process for adenovirus vectors disclosed herein is shown.

[0129] Figure 5 Showing Figure 4 The technical details of the one-step purification process described in the text. Detailed Implementation

[0130] By reference Figure 2B The process, as defined in Example 2, can be performed in any suitable order and may include or consist of the following steps:

[0131] Steps 1, 2, and 5; or Steps 1, 2, 5, and 4a; or

[0132] Steps 1, 2, 5, and 4b; or Steps 1, 2, 5, 4a, and 4b; or

[0133] Steps 1, 2, 3, 4a, and 5; or Steps 1, 2, 3, 4b, and 5; or

[0134] Step 1, Step 2, Step 3, Step 4a, Step 4b and Step 5.

[0135] As used herein, ultrafiltration refers to a separation process that uses a membrane to separate components in a liquid composition based on particle size differences. This method uses pressure and / or concentration gradients to separate components. By controlling the pore size of the membrane, components in the composition can be retained or allowed to pass through the membrane.

[0136] Suitable membranes include 500 kDa MWCO ultrafiltration membranes, for example, which retain molecules of at least 300 kDa and larger.

[0137] As used herein, perfiltration or buffer exchange refers to the ultrafiltration process commonly used for protein desalting and solvent exchange. In the context of this disclosure, perfiltration is used to wash microspecies, such as host cell proteins and other unwanted contaminants, from a culture medium used to produce adenoviruses, thereby producing a purified solution of the retained species, i.e., the adenovirus.

[0138] Percolation can be performed using either continuous percolation (also known as the uniform volume method) or discontinuous percolation. In the uniform volume method, percolation buffer is added to the sample feed reservoir at the same rate as the filtrate is generated. This means that the volume of solution in the sample feed reservoir remains constant, but molecules small enough to cross the membrane, such as host cell proteins, are washed away. In contrast, in the discontinuous method, the sample solution is first diluted and then concentrated back to the starting volume. This process is repeated until the remaining small molecules in the reservoir reach the desired concentration, i.e., until the desired sample solution purity is achieved. Continuous percolation typically requires a smaller filtrate volume to achieve the same degree of reduction in the concentration of the "drug" molecules in the starting solution compared to discontinuous percolation.

[0139] As used herein, tangential flow filtration (TFF) or cross-flow filtration refers to an ultrafiltration technique in which the feed stream passes parallel to the membrane surface as a portion (permeate) permeates through the membrane, while the remainder (retention) is recycled back to the feed reservoir. This contrasts with direct flow filtration (DFF), in which the feed stream is fed perpendicular to the membrane surface and attempts to allow all fluid to pass through the membrane. In TFF, the sample solution flows across the membrane surface, sweeping away aggregated molecules that could form membrane-clogging gels, while allowing molecules smaller than the membrane pores to migrate toward and pass through the membrane. Therefore, for size separation, TFF methods tend to be faster and more efficient than DFF methods.

[0140] As used herein, percolation volume is a measure of the degree of washing performed during the percolation step. It is based on a comparison of the volume of percolation buffer introduced into the unit operation with the volume of retentate.

[0141] As used in this article, percolation buffer refers to a biological buffer solution used in the percolation process.

[0142] Unless the context otherwise requires, elution buffer refers to the buffer solution used in the chromatographic procedure.

[0143] As used in this article, lysis buffer refers to a buffer solution suitable for lysing host cells in which viruses grow, and it typically contains surfactants.

[0144] As used herein, final formulation buffer refers to a buffer solution suitable for storing adenovirus under appropriate conditions and / or suitable for administration to humans.

[0145] The concentration factor used here refers to the factor or multiple that increases the concentration by reducing the volume of a given solute.

[0146] As used herein, biological buffers (also simply buffers) are buffer solutions suitable for suspending or storing viruses without negatively affecting the structural integrity of the adenovirus or its replication ability. Most biological buffers currently in use were developed by NE Good and his research team (Good et al., 1966; Good and Izawa, 1972; Ferguson et al., 1980; "Good buffers") and contain N-substituted taurine or glycine buffers. Table 1 below lists some commonly used biological buffers. This list is not exhaustive, and other buffers will be known to those skilled in the art.

[0147] Table 1 - List of Common Biological Buffers

[0148]

[0149]

[0150] As used in this article, a strong electrolyte is a substance that, when dissolved in water, breaks down into cations and anions. Strong electrolytes are completely ionized and are classified into three categories: strong acids, strong bases, and salts.

[0151] Strong acids include HCl, HBr, HI, HNO3, HClO3, and H2SO4.

[0152] Strong bases include NaOH, KOH, LiOH, Ba(OH)2, and Ca(OH)2.

[0153] As used herein, salt refers to any salt suitable for use as a dialysis buffer, and is therefore suitable for biological applications, particularly as a biological buffer. Examples of such salts are known to those skilled in the art and include, but are not limited to, NaCl, Tris, Bis-Tris, and NaH2PO4.

[0154] Electrical conductivity is typically measured by determining the resistance of a liquid between two electrodes spaced at a fixed distance. Conductivity meters are available from Omega and Baumer.

[0155] Unless the context otherwise requires, the term adenovirus as used herein generally refers to a replicating adenovirus or a replication-defective adenovirus, such as group B viruses, particularly Ad11, such as Ad11p (including viruses derived from it). In some cases, it may be used to refer only to a replicating virus, and this will be clear from the context.

[0156] As used herein, subgroup B (group B or type B) refers to a virus having at least fibrils and hexapods from group B adenoviruses, such as fibrils, hexapods and pentads, or, for example, the entire capsid from group B viruses, such as a virus with essentially the entire genome from group B viruses.

[0157] Enadenotucirev (EnAd) is a chimeric oncolytic adenovirus formerly known as ColoAd1 (WO2005 / 118825) with fibers, pentagons, and hexagons from Ad11p, thus it is a group B virus derived from Ad11p. It possesses a chimeric E2B region containing DNA from both Ad11p and Ad3. Almost the entire E3 region and a portion of the E4 region (E4orf4) are missing in EnAd.

[0158] As used in this article, EnAd also includes viruses that encode one or more genetically modified organisms.

[0159] As used herein, the process for manufacturing adenovirus refers to the process in which the virus is replicated and thus the number of viral particles increases. Specifically, manufacturing involves providing a sufficient number of viral particles to formulate a therapeutic product, for example, producing a range of 1-9 x 102 5 Up to 1-9x10 20 More or more particles, such as those ranging from 1 to 9 x 10⁻⁶ 8 Up to 1-9x10 15 Virus particles, especially those produced by 10L batches, can generate 1 to 9 x 10^10 virus particles. 10 Or 1-9x10 15 Virus particles.

[0160] The purification process for group B adenovirus used herein requires that the process be suitable for the intended purpose. In one embodiment, the virus purified by this process is Ad11, such as EnAd.

[0161] As used herein, partial deletion of the E3 region (partial deletion in the E3 region) refers to at least a portion of the E3 region, for example, ranging from 1% to 99%, such as a deletion of 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% in the coding and / or non-coding regions of a gene.

[0162] A complete deletion of the E3 region (also referred to herein as a total deletion) means that the coding portion of a gene is completely deleted. In one embodiment, both the coding and non-coding portions of a gene are completely deleted.

[0163] As used herein, E3 refers to the DNA sequence encoding a portion or the entire E3 region (i.e., protein / peptide) of an adenovirus, which can be mutated to give the protein encoded by the E3 gene conserved or non-conserved amino acid changes, such as giving it the same function as the wild-type (corresponding unmutated protein); increased function compared to the wild-type protein; or decreased function, such as no function compared to the wild-type protein or a new function compared to the wild-type protein or a combination thereof, as the case may be.

[0164] The virus disclosed herein has no missing parts in area E4.

[0165] In one embodiment, Eorf4 is missing.

[0166] As used herein, partial deletion of E4 region (partial deletion in E4 region) means that at least a portion of E4 region is missing, for example, in the range of 1% to 99%, such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% deletion.

[0167] The complete presence of the E4 region means that E4 is 100% present, i.e., nothing has been removed. That said, the gene can be: mutated, in which up to 10% of the base pairs are replaced (but not deleted); or interrupted, for example, the E4 region may be interrupted by a transgene. Therefore, as used in this article, 100% complete means that the relevant location in the genome is 100% present; however, many genes are either continuous or discontinuous.

[0168] A complete deletion of the E4 region (also referred to herein as a total deletion) means that the coding portion of a gene is completely missing. In one embodiment, both the coding and non-coding portions of a gene are missing.

[0169] As used herein, E4 refers to the DNA sequence encoding the E4 region (i.e., the polypeptide / protein region) of an adenovirus, which can be mutated to give the protein encoded by the E4 gene conserved or non-conserved amino acid changes and give it the same function as the wild-type (corresponding non-mutated protein); increased function compared to the wild-type protein; decreased function, such as no function compared to the wild-type protein or new function compared to the wild-type protein or a combination thereof, as the case may be.

[0170] The E4 region can have one or more functions related to viral replication, and therefore modifications such as the deletion of the E4 region may affect the viral life cycle and replication, for example, making it possible to package cells for replication.

[0171] As used herein, “derived from” means, for example, that the DNA fragment is derived from an adenovirus or corresponds to a sequence originally found in an adenovirus. This language is not intended to limit how the sequence was obtained, for example, the sequence that can be synthesized for use in the virus according to this disclosure.

[0172] In one embodiment, the derivative has 100% sequence identity with the original DNA sequence along its full length, meaning the original DNA sequence may be part of all associated adenoviruses. In one instance, the DNA sequence encodes fibrils and hexagonal proteins, such as capsid proteins.

[0173] In one embodiment, the derivative has 95%, 96%, 97%, 98%, or 99% identity or similarity to the original DNA sequence.

[0174] In one embodiment, the derivative hybridizes with the original DNA sequence under stringent conditions.

[0175] As used herein, “strict” generally occurs in the range of about Tm (melting temperature) - 50°C (5° lower than the probe’s Tm) to about 20°C to 25°C below Tm. As those skilled in the art will understand, strict hybridization can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences. As used herein, the term “strict condition” means that hybridization will generally occur if there is at least 95%, such as at least 97%, identity between sequences.

[0176] As used herein, “hybridization” should include any process by which a polynucleotide chain is linked (aligned) with a complementary chain through base pairing (Coombs, J., Dictionary of Biotechnology, Stockton Press, New York, NY, 1994).

[0177] In one embodiment, the virus disclosed herein further comprises genetically modified organisms.

[0178] In one embodiment, the lack of cell adhesion may be related to viral hexapods and fibers.

[0179] In one embodiment, the adenovirus used in this disclosure is an oncolytic adenovirus.

[0180] Oncolytic viruses are viruses that preferentially infect cancer cells and accelerate cell death, for example, by lysing cancer cells or selectively replicating within them. Viruses that preferentially infect cancer cells are those that exhibit a higher rate of cancer cell infection compared to normal healthy cells.

[0181] In one embodiment, the virus of this disclosure is chimeric, for example, comprising genomic sequences from at least two adenovirus subgroups (excluding subgroup A, which is considered oncogenic). In one embodiment, the chimeric adenovirus of this disclosure is not chimeric in the E2B region.

[0182] Adenoviruses, such as replicating group B adenoviruses, can be used to evaluate their preference for specific tumor types by examining their lytic potential in a subset of tumor cell lines, such as colon tumor cell lines including HT-29, DLD-1, LS174T, LS1034, SW403, HCT116, SW48, and Colo320DM. Any available colon tumor cell line is equally useful for this type of evaluation.

[0183] Prostate cell lines include DU145 and PC-3 cells. Pancreatic cell lines include Panc-1 cells. Breast tumor cell lines include the MDA231 cell line, and ovarian cell lines include the OVCAR-3 cell line. Hematopoietic cell lines include, but are not limited to: Raji and Daudi B lymphocytes, K562 erythroblast-like cells, U937 bone marrow cells, and HSB2 T lymphocytes. Other available tumor cell lines are also useful.

[0184] In one embodiment, the virus disclosed herein is an oncolytic virus. Oncolytic viruses include non-chimeric ones (i.e., oncolytic viruses can be chimeric or non-chimeric), such as Ad11, such as Ad11p, which can be similarly evaluated in these cell lines and have oncolytic activity.

[0185] Viruses that selectively replicate in cancer cells are those that require upregulated genes or proteins in cancer cells to replicate, such as the p53 gene.

[0186] In one embodiment, the oncolytic virus of this disclosure is apoptotic, i.e., accelerates programmed cell death. In another embodiment, the oncolytic virus of this disclosure is cytolytic. The cytolytic activity of the chimeric oncolytic adenovirus of this disclosure can be determined in representative tumor cell lines, and the data are converted into a titer measurement, for example using an adenovirus belonging to subgroup C, preferably Ad5, as a standard (i.e., titer 1). A suitable method for determining cytolytic activity is the MTS assay (see Example 4 of WO2005 / 118825, Figure 2, which is incorporated herein by reference). In one embodiment, the oncolytic adenovirus of this disclosure induces cell necrosis.

[0187] In one embodiment, the chimeric oncolytic virus has an enhanced therapeutic index against cancer cells. The "therapeutic index" or "therapeutic window" refers to a number indicating the oncolytic potential of a given adenovirus, which can be determined by dividing the potency of the oncolytic adenovirus of this disclosure in the relevant cancer cell line by the potency of the same adenovirus in a normal (i.e., non-cancerous) cell line. In one embodiment, the oncolytic virus has an enhanced therapeutic index in one or more cancer cells selected from the group comprising: colon cancer cells, breast cancer cells, head and neck cancer cells, pancreatic cancer cells, ovarian cancer cells, hematopoietic tumor cells, leukemia cells, glioma cells, prostate cancer cells, lung cancer cells, melanoma cells, sarcoma cells, liver cancer cells, kidney cancer cells, bladder cancer cells, and metastatic cancer cells.

[0188] Group B viruses include Ad3, 7, 11, 14, 16, 21, 34, 35, 50, and 55.

[0189] The E2B region is a known region in adenoviruses and represents approximately 18% of the viral genome. It is thought to encode the protein IVa2, DNA polymerase, and terminal proteins. In the Slobitski strain of Ad11 (called Ad11p), these proteins are encoded at positions 5588–3964, 8435–5067, and 10342–8438 of the genome sequence, respectively, with the E2B region extending from position 10342–3950. The exact location of the E2B region may vary in other serotypes, but its function has been preserved in all human adenovirus genomes examined to date, as they all share the same general organization.

[0190] In one embodiment, the virus disclosed herein, such as an oncolytic virus, has a subgroup B hexane.

[0191] In one embodiment, the virus has hexagonal proteins and fibrils from group B adenoviruses such as Ad11. In one embodiment, the virus of this disclosure, such as an oncolytic virus, has Ad11 hexagonal proteins, such as A11p hexagonal proteins. In one embodiment, the virus of this disclosure, such as an oncolytic virus, has subgroup B fibrils. In one embodiment, the virus of this disclosure, such as an oncolytic virus, has Ad11 fibrils, such as A11p fibrils. In one embodiment, the virus of this disclosure, such as an oncolytic virus, has fibrils and hexagonal proteins from the same serotype, such as subgroup B adenoviruses, such as Ad11, particularly Ad11p.

[0192] In one embodiment, the virus of this disclosure, such as an oncolytic virus, has fibrillary, hexagonal, and pentagonal proteins from the same serotype, such as Ad11, particularly Ad11p, found, for example, at positions 30811-31788, 18254-21100, and 13682-15367 in the latter's genomic sequence.

[0193] In one embodiment, the virus of this disclosure has an Ad11 capsid, such as an Ad11p capsid.

[0194] The mammalian cells in which the virus is cultured (and, for example, replicated) are derived from mammals. In one embodiment, the mammalian cells are selected from the group comprising HEK, CHO, COS-7, HeLa, Viro, A549, PerC6, and GMK, particularly HEK293.

[0195] In one embodiment, the adenovirus is replicable, for example, it has the ability to replicate.

[0196] As used herein, reproducible viruses are adenoviruses that can replicate in host cells. In one embodiment, reproducible viruses include viruses with replication capability and replication selectivity.

[0197] As used herein, "replicating capability" refers to an adenovirus capable of replicating in human cells such as cancer cells without any additional supplementation required by wild-type viruses, such as dependence on defective cellular mechanisms. Replicating viruses can be produced in complementary cell lines that do not encode essential viral proteins (such as complementary cell lines encoded by the E1 region, also known as packaging cell lines), and the viruses are capable of replicating without the assistance of helper viruses.

[0198] As used herein, replication selectivity or selective replication is intended to refer to the ability to replicate in cancer cells using elements that are specific to or upregulated therein (e.g., defective cellular mechanisms such as p53 mutations), thereby allowing oncolytic viruses to have a degree of selectivity for healthy / normal cells.

[0199] In one embodiment, the adenovirus of this disclosure is capable of replication.

[0200] In one embodiment, the adenovirus disclosed herein is a replication-defective type.

[0201] Replication-defective viruses require packaging cell lines to replicate. Packaging cell lines contain one or more genes to supplement the genes that are defective in the virus.

[0202] In one embodiment, the cells are grown in adherent or suspension cultures, particularly suspension cultures.

[0203] As used herein, culturing mammalian cells refers to the process of growing cells under controlled in vitro conditions. Suitable conditions are known to those skilled in the art and may include temperatures such as 37°C. It may be necessary to control CO2 levels, for example, maintaining them at 5%. The same details are given in the textbooks: *Culture of Animal Cells: A Manual of Basic Techniques and Specialized Applications*, 6th revised edition, by Ian Freshney, and *Basic Cell Culture (Practical Approach)*, 2nd edition, edited by JMDavis.

[0204] Typically, cells are cultured to produce a sufficient number before being infected with adenovirus. These methods are known to those skilled in the art or are readily available in published protocols or literature.

[0205] Cells are typically cultured on a commercial scale, such as 5L, 10L, 15L, 20L, 25L, 30L, 35L, 40L, 45L, 50L, 100L, 200L, 300L, 400L, 500L, 600L, 700L, 800L, 900L, 1000L, or similar.

[0206] Suitable culture media for culturing mammalian cells include, but are not limited to, those from Sigma-Aldrich. Culture media, such as those used for HEK293 cells 293 Serum-free culture medium for CHO cells ACF CHO medium, a serum-free medium for CHO cells. 302 serum-free medium, EX-CELL CD hydrolysate fusion medium supplements from Lonza RMPI (such as RMPI1640 with HEPES and L-glutamine, RMPI1640 with or without L-glutamine, RMPI1640 with UltraGlutamine), MEM and DMEM, and SFMII medium.

[0207] In one embodiment, the culture medium does not contain serum. This is advantageous because it facilitates registration of the manufacturing process with regulatory authorities.

[0208] The viruses disclosed herein, such as oncolytic viruses, possess properties that differ from those of adenoviruses used as vectors, such as Ad5. These properties include the fact that they can be recovered from the culture medium without cell lysis. Therefore, while not wishing to be bound by theory, viruses appear to possess mechanisms for exiting the cell.

[0209] In one embodiment, the incubation period ranges from 30 to 100 hours post-infection, for example, 35 to 70 hours, such as 40, 45, 50, 55, 60, or 65 hours.

[0210] In one embodiment, the incubation period is 65, 70, 75, 80, 85, 90, 95 hours or longer.

[0211] In one embodiment, the incubation period ranges from 60 to 96 hours.

[0212] In one embodiment, maximum total viral production is achieved approximately 60 to 96 hours post-infection, for example, 70 to 90 hours.

[0213] Cells can be cultured using perfusion culture, fed-batch culture, batch culture, steady-state culture, continuous culture, or, where technically appropriate, one or more combinations thereof, especially perfusion culture.

[0214] In one embodiment, the process is a perfusion process, such as a continuous perfusion process.

[0215] In one embodiment, the culture process includes one or more culture medium replacements. This can be beneficial for optimizing cell growth, yield, etc. When using culture medium replacements, it may be necessary to recover viral particles from the medium being replaced. These particles can be conjugated with the master batch of virus to ensure optimized virus yield. Similar techniques can also be used with the culture medium in the perfusion process to optimize virus recovery.

[0216] In one embodiment, the culture process does not include a culture medium replacement step. This can be advantageous because no virus particles are lost, thus allowing for optimized yield.

[0217] In one embodiment, the culture process includes one or more cell additions or replacements. As used herein, cell addition means replenishing some or all of the cells, and replacement means removing dead cells and adding new cells (not necessarily in that order).

[0218] In one embodiment, the adenovirus concentration during culture ranges from 20 to 150 particles per cell (ppc), such as 40 to 100 ppc, particularly 50 ppc.

[0219] Lower virus concentration values, such as less than 100 ppc, especially 50 ppc, may be advantageous because this can lead to increased cell viability compared to cultures with higher virus concentrations, particularly when cell viability is measured before harvest.

[0220] Low cell viability can lead to cell lysis, which may expose cells to enzymes that could attack viruses over time. However, in dynamic processes such as cell culture, a certain percentage, usually a small percentage, of cells may fail to survive. This typically does not cause significant problems in practice.

[0221] In one embodiment, during the process, for example at a time point of 96 hours after viral infection (i.e., 96 hours post-infection), cell viability is approximately 80% to 95%, for example, 83% to 90%.

[0222] In one embodiment, during this process, for example at a time point of 96 hours after infection with Ad11 (i.e., 96 hours post-infection), cell survival is approximately 80% to 90%. For example, the survival rate is 85%.

[0223] In one embodiment, the culture medium and / or cells are supplements or are replenished periodically.

[0224] In one embodiment, cells are harvested during the process, for example, at a discrete point in time or at multiple points in time or continuously.

[0225] In one embodiment, virus harvesting is performed at a time point selected from approximately 40, 46, 49, 64, 70, 73, 89, or 96 hours post-infection, or combinations thereof.

[0226] In one embodiment of this process, mammalian cells are used with 1-9x10 4 Infection with an initial viral concentration of vp / ml or higher, such as 1-9x10 5 1-9x10 6 1-9x10 7 1-9x10 8 1-9x10 9 Especially 1-5x10 6 vp / ml or 2.5-5x108 vp / ml.

[0227] In one embodiment of this process, mammalian cells are used at a ratio of 1x10 6 The initial concentration is approximately 1 to 200 ppc, for example 40 to 120 ppc, such as 50 ppc for infection.

[0228] As used in this article, Ppc refers to the number of viral particles per cell.

[0229] In one embodiment, the method is operated at approximately 35 to 39°C, for example, 37°C.

[0230] In one embodiment, the process is operated at approximately 4-6% CO2, for example, 5% CO2.

[0231] In one embodiment, the culture medium containing the virus, such as chimeric oncolytic virus particles, is filtered to remove cells and provide a crude supernatant for further downstream processing. In another embodiment, a tangential flow filter is used.

[0232] In one embodiment, Millipore is used. The POD single-use deep filtration system filters culture media. It is ideal for a wide range of primary and secondary clarification applications, including cell culture.

[0233] Pod filters are available in three different media grade series to meet specific application requirements. DE, CE, and HC dielectrics offer optimal performance through gradient density matrix and positive surface charge properties.

[0234] If necessary, the virus can also be formulated into the final buffer solution for this step.

[0235] Therefore, in one embodiment of the filtration step, the concentrated and regulated adenovirus material is provided in the form of the final or near-final formulation.

[0236] In one embodiment, the process includes two or more filtering steps.

[0237] In one embodiment, downstream processing includes Millistak+POD system 35CE and 50CE cartridges, followed by tandem Opticap XL 10express 0.5 / 0.2µm membrane filters.

[0238] Ion exchange chromatography (IEX) binds DNA very strongly and is typically used to remove any residual DNA. Ion exchange resins / membranes bind viruses and DNA; during salt gradient elution, viruses usually elute from the column first (low salt gradient), while DNA elutes at much higher salt concentrations because the interaction between DNA and the resin is stronger than that between the virus and the resin.

[0239] In one embodiment, one or more chromatographic steps utilize a monolithic technique, such as that available from BIASeparations. The monolithic column contains a highly cross-linked porous polymethacrylate material with a well-defined channel size distribution.

[0240] In one embodiment, the chromatography is ion exchange, such as two-stage ion exchange. Exchanges can be obtained from, for example, GE HealthBioSciences AB, Cytiva, and Sartorius.

[0241] Strong ion exchange (such as Q, S, and SP) can occur over a wide pH range. Q binds to “proteins” at its isoelectric point at pH 7.

[0242] The exchange capacity of weak ion exchangers (such as DEAE, ANX, and CM) varies with pH.

[0243] Sartobind Q is a strong ion exchanger suitable for purifying adenoviruses.

[0244] Source 15Q (from Cytiva) is a polymeric strong anion exchanger designed for polishing processes and suitable for industrial applications.

[0245] In one embodiment, at least two chromatographic steps are performed, such as at least one of which is ion exchange.

[0246] In one embodiment, at least two ion exchange steps are performed.

[0247] In one embodiment, at least two chromatographic steps include an ion exchange step and a liquid chromatography step.

[0248] In one embodiment, the purified virus contains less than 80 ng / mL (e.g., 60 ng / mL to 10 ng / mL) of contaminating DNA.

[0249] In one embodiment, virtually all contaminating DNA fragments are 700 base pairs or less, such as 500 bp or less, such as 200 bp or less.

[0250] In one embodiment, the residual benzonase content in the purified viral product is 1 ng / mL or less, such as 0.5 ng / mL or less.

[0251] In one embodiment, the residual host cell protein content in the purified viral product is 20 ng / mL or less, for example, 15 ng / mL or less, particularly when measured by ELISA.

[0252] In one embodiment, the residual Tween in the purified virus product is 0.1 mg / mL or less, such as 0.05 mg / mL or less.

[0253] In one embodiment, a group B adenovirus isolated and purified according to the present disclosure is provided, wherein the contamination DNA content is less than 80 ng / mL.

[0254] In one embodiment, the virus of this disclosure, such as the oncolytic virus of this disclosure, comprises one or more transgenes, such as one or more transgenes encoding one or more therapeutic peptides or one or more protein sequences.

[0255] In one embodiment, the virus encodes a therapeutic polynucleotide, such as a therapeutic RNA molecule.

[0256] In one embodiment, the virus, such as an oncolytic virus, encodes at least one transgene. Suitable transgenes include so-called suicide genes, such as p53; polynucleotide sequences encoding cytokines such as IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, GM-CSF or G-CSF, interferons (e.g., interferon I such as IFN-α or β, interferon II such as IFN-γ), TNF (e.g., TNF-α or TNF-β), TGF-β, CD22, CD27, CD30, CD40, CD120; and polynucleotide sequences encoding monoclonal antibodies such as trastuzumab, cetuximab, panitumumab, pertuzumab, epazolizumab, anti-EGF antibodies, anti-VEGF antibodies, anti-PDGF antibodies, and anti-FGF antibodies.

[0257] A range of different types of transgenes and their combinations are envisioned, encoding molecules that themselves regulate tumor or immune responses and exert therapeutic effects, or agents that directly or indirectly inhibit, activate, or enhance the activity of such molecules. These molecules include protein ligands or active binding fragments of ligands, antibodies (full-length or fragment-specific binding fragments, such as Fv, ScFv, Fab, F(ab)'2, or smaller), or other target-specific binding proteins or peptides (e.g., selectable via techniques such as phage display), natural or synthetic binding receptors, ligands or fragments, and specific molecules that regulate the transcription or translation of genes encoding targets (e.g., siRNA or shRNA molecules, transcription factors). The molecule can be a fusion protein form with other peptide sequences to enhance its activity, stability, specificity, etc. (For example, ligands can fuse with the Fc region of immunoglobulins to form dimers and enhance stability, or fuse with antibodies or antibody fragments that are specific to antigen-presenting cells such as dendritic cells (e.g., anti-DEC-205, anti-mannose receptor, anti-dectin). Transgenic molecules can also encode reporter genes that can be used, for example, to detect cells infected with "adenovirus with inserts," for tumor imaging, or for draining lymph nodes.

[0258] In one embodiment, cancer cells infected with an oncolytic virus are lysed, releasing the cell contents, which may include proteins encoded by genetically modified organisms.

[0259] In one embodiment, the process is a GMP manufacturing process, such as a cGMP manufacturing process. In one embodiment, the process further includes the step of preparing the virus in a storage-appropriate buffer. In one embodiment, this disclosure extends to viruses or viral preparations obtained or obtainable from this method.

[0260] Known cell lysis methods include the use of lysis buffers, such as lysis buffer containing 1% Tween-20. Multiple freeze-thaw cycles are also a common method for cell lysis. Pulmozyme can also be used for cell lysis. An alternative method for cell lysis involves centrifuging the cell suspension at 1000 x g for 10 minutes at 4°C. The cell clumps are resuspended in 1 ml of Ex-Cell medium containing 5% glycerol, and the virus is released from the cells via freeze-thaw by freezing the tubes containing the responding cells from the clumps in liquid nitrogen for 3–5 minutes and then thawing them in a +37°C water bath until thawed. Typically, the freezing and thawing steps are repeated more than twice. This cycle releases the virus from the cells. After the final thawing step, cell debris is removed by centrifugation, for example, at 1936 x g for 20 minutes at +4°C, and host cell DNA is removed by digestion with benzonase.

[0261] In the context of this application, the terms "medium" and "media" are used interchangeably. In the context of this specification, "comprising" should be interpreted as "including".

[0262] The aspects of the invention that include certain elements are also intended to be extended to alternative embodiments of the related elements that are “consisting” or “consisting essentially”. Where technically appropriate, embodiments of the invention may be combined.

[0263] Technical references such as patents and applications are incorporated herein by reference.

[0264] Any embodiment specifically and explicitly described herein may form the basis of this disclaimer, either alone or in combination with one or more other embodiments.

[0265] This application claims priority to GB1909081.0, filed on June 25, 2019, which is incorporated herein by reference. The priority claim may serve as the basis for correcting this specification.

[0266] The invention is further described by way of the following examples only.

[0267] Example

[0268] Example 1 - Evaluation of a standard purification procedure for purifying group B adenovirus

[0269] Figure 2A The standard, known purification procedure for adenovirus vectors is shown. The EnAd virus is then subjected to this standard purification procedure.

[0270] Ad11 / 3 vector Vector #1 Vector #1 Vector #1 Vector #2 Drug substance (ng HCP / 2x10 12 vp) 11,692 7,869 2,991 1,298

[0271] Even after purification using standard processes, a significant amount of host cell proteins remain in the final product.

[0272] Example 2 - Improved purification process for group B adenovirus

[0273] Figure 2B An improved purification procedure of this disclosure for EnAd encoding the transgene between the L5 and E4 regions is shown. A new step 5 is added after step 4 of the known procedure. The new step 5 is a percolation step using a percolation buffer with a high salt content. Figure 3 Listed Figure 2B Technical details of the process.

[0274] Step 1: Lyse the virus-infected HEK293 cells using lysis buffer.

[0275] Benzonase (a low-salt buffer is required during Benzonase treatment because high concentrations of salt will inactivate the enzyme);

[0276] Then, benzonase was inactivated using an inactivation buffer: 4.3M NaCl, 0.05M HEPES, pH 7.5;

[0277] Step 2 involves clarification using two depth filters. Filter 1 is a Merck Millipore pod depth filter CE35 (4 to 2 μm), followed by filtration through a Merck Millipore pod depth filter CE50 (1 to 0.4 μm). The final stage of clarification is achieved using a Millipore filter. SHC 0.5 / 0.2μM hydrophilic membrane XL disposable capsule filter composition;

[0278] Step 3: Tangential tracking ultrafiltration / difiltration (UF / DF) in Biomax V sieve box, using a concentration factor (CF) of 8, a difiltration volume (DV) of 12, and a difiltration buffer of 1M NaCl, 0.05M HEPES, 1.0% m / V Tween 20, 1.0% glycerol, pH 7.5;

[0279] Step 4 The IEX1 system was used to perform ion exchange chromatography on the composition obtained from step 3 using the following elution buffer: 0.45 M NaCl, 0.05 M HEPES, 1.0% m / V Tween 20, pH 7.5 (step 4a), and then the CIMQA IEX2 system was used to perform ion exchange chromatography using the following elution buffer: 0.4 M NaCl, 0.05 M Tris, 0.002 M MgCl2, 5% glycerol, pH 7.8 (step 4b).

[0280] Step 5 utilizes a concentration factor of 1.5 to filter the composition from Step 4 using tangential flow ultrafiltration / percolation in a hollow fiber cartridge with 12 percolation volumes of the following high-salt concentration percolation buffer: 3M NaCl, 0.05M HEPES, kJ 1.0% m / V Tween 20, 1.0% m / V glycerol, pH 7.5 (Step 5a); and

[0281] Buffer exchange was performed using 15 percolation volumes of final formulation buffer (FFB) (0.005 M HEPES, 20% m / V glycerol, pH 7.8) (step 5b).

[0282] At the end of the improved purification process, the adenovirus and host cell proteins were quantified again using the method described in Example 1 above. The host protein was below the limit of quantification after purification. Therefore, due to the inclusion of an additional percolation process, the amount of contaminating host cell protein in the final product was drastically reduced below the quantification level.

[0283] Table 2 shows the viral particle and host cell protein content obtained at different points in the purification process with an additional percolation step.

[0284]

[0285] Example 3 - One-step purification process of group B adenovirus

[0286] The possibility of completely omitting the chromatographic steps was investigated. Figure 4 The purification process design is shown, which consists of only a diafiltration step after steps 1 and 2 (lysis, endonuclease treatment, inactivation, and clarification). Technical details of this process are as follows... Figure 5 As shown. This process utilizes the EnAd virus, which encodes a genetically modified organism.

[0287] Steps 1 and 2 are detailed in Example 2 above. Then, after step 2, percolation is performed as described in step 5 above. The results are shown in Table 2 below.

[0288] Table 3

[0289] Process stage vp / mL Total vp % recovery HCP (ng / ml) HCP (ng / 2E12 vp) After step 2 6.39E+10 3.26E+14 na 2.31E+04 7.23E+05 Final formulation 4.69E+11 9.85E+13 30 Below LOQ na

[0290] It can be seen that the host cell protein levels obtained using only the percolation step were below the quantification level. Therefore, a similar level of purity was achieved using a one-step purification process compared to an improved purification process involving three different purification steps. This provides strong evidence that chromatographic steps can be omitted or performed together with the percolation step to produce a high-purity final group B adenovirus product.

Claims

1. A method for purifying a replication-competent Group B adenovirus from host cell proteins, the method comprising: lysing a host cell comprising the replication-competent Group B adenovirus to obtain a crude cell lysate; contacting the crude cell lysate with an endonuclease to obtain an endonuclease-treated cell lysate; filtering the endonuclease-treated cell lysate to obtain a clarified cell lysate comprising the replication-competent Group B adenovirus; and diafiltering the clarified cell lysate comprising the Group B adenovirus using a diafiltration buffer with a high salt concentration, wherein the high salt concentration is at least 2 M.

2. The method of claim 1, wherein the salt concentration ranges from 2.5 M to 5.5 M.

3. The method of claim 1 or 2, wherein the salt concentration is 4.3 M.

4. The method of claim 1 or 2, wherein the buffer comprises a salt selected from the group consisting of any salt that is fully soluble and dissociated in water and combinations thereof.

5. The method of claim 4, wherein the buffer comprises a salt selected from the group consisting of a chloride salt, a sulfate salt, and combinations thereof.

6. The method of claim 1 or 2, wherein the salt in the diafiltration buffer comprises one or more of: an alkaline earth metal salt, sodium acetate, Tris, Bis-Tris, and NaH2PO4.

7. The method of claim 6, wherein the alkaline earth metal salt is selected from the group consisting of NaCl, KC1, and MgCl2.

8. The method of claim 1 or 2, wherein the diafiltration buffer is selected from the group consisting of: meglumine buffer, Gly-NaCl buffer, TRIS buffer.

9. The method of claim 8, wherein the diafiltration buffer comprises HEPES.

10. The method of claim 9, wherein the diafiltration buffer comprises 50 mM HEPES.

11. The method of claim 1 or 2, wherein the pH of the diafiltration buffer ranges from 7 to 9.

8.

12. The method of claim 11, wherein the pH is 7.

5.

13. The method of claim 1 or 2, wherein the diafiltration uses a MWCO ultrafiltration membrane of at least 300 KDa or greater.

14. The method of claim 1 or 2, wherein the diafiltration has a flow rate of 1 to 3 m 2 / s.

15. The method of claim 1 or 2, wherein the diafiltration is performed using a hollow fiber cartridge or a flat membrane cassette filter.

16. The method of claim 15, wherein the diafiltration is performed using a consistent volume method.

17. The method of claim 1 or 2, wherein the diafiltration is performed using at least 8 diafiltration volumes of high salt diafiltration buffer.

18. The method of claim 17, wherein the diafiltration is performed using 12 diafiltration volumes.

19. The method of claim 1 or 2, wherein the first step of the diafiltration process is diafiltration with a high salt diafiltration buffer.

20. The method of claim 19, wherein the second step of the diafiltration process is diafiltration with a final formulation buffer.

21. The method of claim 1 or 2, wherein only one diafiltration buffer is used.

22. The method of claim 1 or 2, comprising a further purification step comprising chromatography purification of the adenovirus composition.

23. The method of claim 22, wherein the chromatography purification comprises two chromatography steps.

24. The method of claim 23, wherein the chromatography steps use ion exchange chromatography.

25. The method of claim 24, wherein the chromatography steps use anion exchange chromatography.

26. The method of claim 24 or 25, wherein the chromatography steps use anion exchange chromatography with DEAE, TMAE, QAE or PEI.

27. The method of claim 1 or 2, wherein the adenovirus purification steps do not use chromatography.

28. The method of claim 1 or 2, wherein the filtration is performed with a depth filter.

29. The method of claim 28, wherein the depth filter has a 4 to 2 pm rating.

30. The method of claim 28, wherein the depth filter has a 1 to 0.4 pm rating.

31. The method of claim 1 or 2, wherein the filtration comprises passing the adenovirus composition through a 0.2 pm filter.

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