AAV Refining Method

Abstract

Provided herein are methods for obtaining AAV full capsid from AAV starting preparations containing full capsid and empty capsid. In some embodiments, the AAV starting preparation is injected into an anion exchange chromatography (AEX) column, an elution buffer containing a divalent cation salt is injected into the AEX column, and an elution fraction containing AAV full capsid in a larger proportion than AAV empty capsid is recovered from the anion exchange chromatography column. In certain embodiments, a wash buffer containing a monovalent cation salt is injected into the AEX column before the divalent cation salt elution buffer, and a wash fraction containing AAV empty capsid in a larger proportion than AAV full capsid is eluted from the AEX column.

Description

[Technical Field]

[0001] This disclosure provides compositions and methods for obtaining AAV flucapsid, including a method for isolating AAV flucapsid from an AAV starting preparation containing adeno-associated virus (AAV) flucapsid, such as by using column chromatography techniques.

[0002] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 468,965 filed on 25 May 2023, which is incorporated in its entirety by reference.

[0003] Sequence List This application includes a sequence listing submitted electronically in XML format, the entire listing of which is incorporated herein by reference. The XML copy was created on 30 April 2024, named 01277-0039-00PCT, and has a size of 6,592 bytes.

[0004] Introduction The use of adeno-associated viruses (AAVs), such as recombinant adeno-associated viruses (rAAVs), for diverse gene therapy and vaccine approaches has been documented. However, efficient and scalable methods for purifying AAVs are lacking.

[0005] Adeno-associated viruses (AAVs), members of the Parvoviridae family, are small, non-enveloped viruses. AAV particles contain an AAV capsid composed of 60 capsid protein subunits, each consisting of VP1, VP2, and VP3 proteins. The VP1, VP2, and VP3 proteins are present in a predicted ratio of approximately 1:1:10 and possess icosahedral symmetry. The AAV capsid encapsulates a small, single-stranded DNA (ssDNA) genome of approximately 4.8 kilobases (kb). The ssDNA AAV genome contains two open reading frames, Rep and Cap, flanked by two 145-base inverted terminal repeat sequences (ITRs). These ITR bases pair up, enabling the synthesis of complementary DNA strands. Rep and Cap are translated to produce several distinct proteins (Rep78, Rep68, Rep52, Rep40; and VP1, VP2, and VP3 capsid proteins, which play important roles in the life cycle of AAV).

[0006] Generally, individual AAV particles package only one DNA molecular strand (either the positive or negative strand). The AAV life cycle includes an incubation period and an infection period, and particles containing either strand can be infectious. Replication occurs through the conversion of a linear single-stranded DNA genome to a double-stranded form and subsequent amplification. The single strand of the offspring is then rescued, replicated, and packaged into a capsid in the presence of a helper molecule. Its non-pathogenicity, infectivity to a wide range of hosts (including non-dividing cells), and integration properties make AAV an attractive delivery medium for therapeutic applications and other uses.

[0007] Recombinant AAV particles are produced in a permissive (packaging) host cell culture. Replication of the recombinant genome and packaging into viral particles require co-expression of helper virus AAV replication (Rep) genes and AAV capsid (Cap) genes derived from a transfer plasmid, etc. When constructing an AAV transfer plasmid, the transgene may be placed between two ITRs, and Rep and Cap may be given in trans. The genes necessary for genome replication, capsid formation, and genome packaging can be expressed from the transfected plasmid and integrated into the host cell genome, or introduced into the host cell using a recombinant virus. For example, a transfer plasmid containing Rep / Cap, and a helper plasmid containing E4, E2a, and VA, may be transfected into a host cell containing the adenovirus gene E1+ to produce infectious AAV particles.

[0008] Generally, host cells are lysed to release infectious AAV particles, such as infectious rAAV particles, and to maximize recovery. However, cell lysates contain various cellular components, such as host cell DNA, host cell proteins, culture medium components, and in some cases, helper viruses or helper virus plasmid DNA, which must be separated from the AAV vector before they can be used, such as for the delivery of therapeutic nucleic acids of interest in vivo. Advances in AAV production include the use of non-adherent cell suspension processes in agitated tank bioreactors and production conditions in which AAV particles (such as rAAV) are released into the culture medium or supernatant, reducing the concentration of host cellular components present in the production material, but still resulting in a considerable amount of in-process impurities. Therefore, AAV particles (such as rAAV) may be recovered from the culture medium and / or cell lysates and further purified.

[0009] However, industrially standardized purification methods, such as standard chromatographic purification techniques, rarely achieve efficient separation of pharmacologically active (full) AAV capsids from empty capsids, partially empty capsids, and / or other impurities present in culture media and / or cell lysates. Therefore, this disclosure presents an improved method for obtaining AAV full capsids, such as AAV (e.g., rAAV) full capsids containing the therapeutic nucleic acid of interest, from AAV starting preparations containing AAV full capsids, genome-deficient intermediates (i.e., AAV empty capsids and / or AAV partially empty capsids), and / or other impurities. [Overview of the project]

[0010] A method is provided for obtaining AAV full capsid from an AAV starting preparation containing AAV full capsid and AAV empty capsid (and / or AAV partially empty capsid). In some embodiments, the AAV starting preparation is injected into an anion exchange chromatography (AEX) column. In such embodiments, an elution buffer containing a divalent cation salt (i.e., a divalent cation salt elution buffer) is injected into the column, and an elution fraction containing AAV full capsid in a larger proportion than AAV empty capsid is recovered from the AEX column. In some embodiments, the elution buffer does not contain a monovalent cation salt (such as NaCl) before being injected into the column.

[0011] In some embodiments, a wash buffer containing a monovalent cation salt (i.e., a monovalent cation salt wash buffer) is injected into the AEX column before the divalent cation salt elution buffer, and the injection of the monovalent cation salt wash buffer elutes a wash fraction containing AAV empty capsids in a larger proportion than AAV full capsids from the AEX column. In certain embodiments, the monovalent cation salt wash buffer does not contain divalent cation salts.

[0012] The following exemplary embodiments are provided.

[0013] Embodiment 1 is a method for obtaining AAV full capsids from an AAV starting preparation containing adeno-associated virus (AAV) full capsids and AAV empty capsids, the method comprising: (a) injecting the AAV starting preparation into an anion exchange chromatography column; and (b) injecting a divalent cation salt elution buffer containing a divalent cation salt into the anion exchange chromatography column, wherein the divalent cation salt elution buffer does not contain a monovalent cation salt, and injecting the divalent cation salt elution buffer elutes an elution fraction containing AAV full capsids at a higher proportion than AAV empty capsids from the anion exchange chromatography column.

[0014] Embodiment 2 is the method according to any one of the preceding embodiments, wherein the elution fraction contains at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% AAV full capsids relative to the AAV starting preparation.

[0015] Embodiment 3 is the method according to any one of the preceding embodiments, wherein the proportion of AAV full capsids compared to all AAV capsids in the elution fraction is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60%.

[0016] Embodiment 4 is the method according to any one of the preceding embodiments, wherein the ratio of AAV full capsids to AAV empty capsids in the elution fraction is at least 1.5:1, at least 1.6:1, at least 1.7:1, at least 1.8:1, at least 1.9:1, at least 2:1, at least 2.25:1, at least 2.5:1, at least 2.75:1, at least 3:1, at least 4:1, at least 5:1, at least 10:1, at least 20:1, at least 30:1, at least 40:1, at least 50:1, or at least 100:1.

[0017] Embodiment 5 is any one of the methods of the preceding embodiments, wherein at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or substantially all of the AAV full capsids are eluted from the anion exchange chromatography column before the AAV empty capsids.

[0018] Embodiment 6 is any one of the methods of the preceding embodiments, wherein the elution fraction contains 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, or 2% or less, or 1% or less AAV empty capsids.

[0019] Embodiment 7 is any one of the methods of the preceding embodiments, wherein the divalent cation salt elution buffer is injected into the anion exchange chromatography column under the condition that at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or substantially all of the AAV full capsids are eluted from the column and at least 60%, at least 70%, at least 80%, at least 90%, or substantially all of the AAV empty capsids remain bound to the column.

[0020] Embodiment 8 is any one of the methods of Embodiments 1-7, wherein the divalent cation salt elution buffer is injected at a constant concentration.

[0021] Embodiment 9 is any one of the methods of Embodiments 1-7, wherein the divalent cation salt elution buffer is injected in a linear gradient.

[0022] Embodiment 10 is any one of the methods of Embodiments 1-9, wherein the elution fraction is the first elution fraction and the second elution fraction is eluted after the first elution fraction.

[0023] Embodiment 11 is the method of Embodiment 10, wherein the second elution fraction contains a larger proportion of AAV empty capsid than the first elution fraction.

[0024] Embodiment 12 is a method of either Embodiment 10 or Embodiment 11, wherein the second eluted fraction contains 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, or 2% or less, or 1% or less of AAV flucapsid.

[0025] Embodiment 13 further comprises injecting a monovalent cation wash buffer containing a monovalent cation into the column before injecting a divalent cation wash buffer, wherein the monovalent cation wash buffer does not contain a divalent cation, and injecting the monovalent cation wash buffer is one of the methods of the preceding embodiments in which a wash fraction containing AAV empty capsids in a larger proportion than AAV full capsids is eluted from the anion exchange chromatography column.

[0026] Embodiment 14 is the method of Embodiment 13, wherein a monovalent cation wash buffer is injected at a constant concentration.

[0027] Embodiment 15 is the method of Embodiment 13, wherein the monovalent cation wash buffer is injected in a linear gradient.

[0028] Embodiment 16 is a method for obtaining an AAV flucapsid from an AAV starting preparation containing an adeno-associated virus (AAV) flucapsid and an AAV empty capsid, wherein the method is (a) Inject the AAV starting preparation into an anion exchange chromatography column; (b) Inject monovalent cation wash buffer into the column, thereby (c) Elute the washing fraction containing AAV empty capsids in a larger proportion than AAV full capsids from the anion exchange chromatography column; and (d) Inject a divalent cation salt elution buffer into an anion exchange chromatography column, thereby eluting a fraction containing AAV full capsid in a larger proportion than AAV empty capsid from the anion exchange chromatography column. This method includes [something].

[0029] Embodiment 17 is the method of Embodiment 16, wherein the eluted fraction contains at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the AAV fulcapsid relative to the AAV starting preparation.

[0030] Embodiment 18 is the method of Embodiment 16 or Embodiment 17, wherein the proportion of AAV full capsid in the eluted fraction compared to the total AAV capsid is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60%.

[0031] Embodiment 19 is one of the methods of Embodiments 16 to 18, wherein the ratio of AAV full capsid to AAV empty capsid in the eluted fraction is at least 1.5:1, at least 1.6:1, at least 1.7:1, at least 1.8:1, at least 1.9:1, at least 2:1, at least 2.25:1, at least 2.5:1, at least 2.75:1, at least 3:1, at least 4:1, at least 5:1, at least 10:1, at least 20:1, at least 30:1, at least 40:1, at least 50:1, or at least 100:1.

[0032] Embodiment 20 is one of the methods of Embodiments 16 to 19, wherein the washing fraction comprises at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or substantially 100% empty capsid.

[0033] Embodiment 21 is one of the methods of Embodiments 16 to 20, wherein the eluted fraction contains 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, or 2% or less, or 1% or less of AAV empty capsid.

[0034] Embodiment 22 is any one of Embodiments 13 to 21, wherein at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or substantially all of the AAV empty capsid is eluted from the anion exchange chromatography column before the AAV full capsid.

[0035] Embodiment 23 is one of the methods of Embodiments 13 to 22, wherein a monovalent cation wash buffer is injected into an anion exchange chromatography column under conditions that at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or substantially all AAV empty capsids are eluted from the column, and at least 60%, at least 70%, at least 80%, at least 90%, or substantially all AAV full capsids remain bound to the column.

[0036] Embodiment 24 is one of the methods of the preceding embodiments, wherein the divalent cation salt elution buffer does not contain NaCl.

[0037] Embodiment 25 is one of the methods of the preceding embodiments, wherein the divalent cation salt elution buffer comprises a 50-150 mM, 50-100 mM, 70-120 mM, 80-110 mM, or 100 mM divalent cation salt.

[0038] Embodiment 26 is one of the methods of the preceding embodiments, wherein the divalent cation salt comprises a divalent cation selected from Mg2+, Ca2+, or Sr2+.

[0039] Embodiment 27 is one of the methods of the preceding embodiments, wherein the divalent cation salt is magnesium chloride (MgCl2), calcium chloride (CaCl2), magnesium sulfate (MgSO4), magnesium phosphate (MgPO4), calcium acetate (Ca(C2H3O2)2), calcium sulfate (CaSO4), magnesium acetate (Mg(C2H3O2)2), or magnesium citrate (MgC6H6O7).

[0040] Embodiment 28 is one of the methods of the preceding embodiments, wherein the divalent cation salt is MgCl2, CaCl2, or MgSO4.

[0041] Embodiment 29 is a divalent cation salt elution buffer, (a) 25-175 mM, 25-150 mM, 25-100 mM, 25-75 mM, or 50 mM MgCl2; (b) 25-175 mM, 25-150 mM, 25-100 mM, 25-75 mM, or 50 mM CaCl2; or (c) 25-175 mM, 25-150 mM, 25-100 mM, 25-75 mM, or 50 mM MgSO4 This is one of the methods of the prior embodiments, including the method described above.

[0042] Embodiment 30 is one of the methods of the prior embodiments, wherein the divalent cation salt elution buffer contains a buffering agent.

[0043] Embodiment 31 is one of the methods of the preceding embodiments, wherein the divalent cation salt elution buffer comprises a buffer of 5-100 mM, 10-100 mM, 10-50 mM, 10-40 mM, 10-30 mM, 20-30 mM, or 25 mM.

[0044] Embodiment 32 is the method of Embodiment 30 or Embodiment 31, wherein the buffering agent is selected from Tris, bis-trispropane, CHES (N-cyclohexyl-2-aminoethanesulfonic acid), or AMPSO (N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid).

[0045] Embodiment 33 is one of the methods of the prior embodiments, wherein the divalent cation salt elution buffer contains a stabilizing substance.

[0046] Embodiment 34 is the method of Embodiment 33, wherein the stabilizing substance is selected from poloxamer, polysorbate 80 (PS-80), polysorbate 20 (PS-20), sorbitol, sucrose, or trehalose.

[0047] Embodiment 35 is one of the methods of the preceding embodiments, wherein the divalent cation salt elution buffer is approximately pH 7-10, approximately 8.5-9.5, or approximately pH 9.

[0048] Embodiment 36 is a divalent cation salt elution buffer, (a) Contains 25 mM Tris, 100 mM divalent cation salt, and 0.001% poloxamer, with a pH of approximately 9; (b) A solution containing 25 mM Tris, 38 mM divalent cation salt, and 0.001% poloxamer, with a pH of approximately 9; or (c) Contains 25 mM Tris, 33 mM divalent cation salt, and 0.001% poloxamer, with a pH of approximately 9. This is one of the methods of the prior embodiments.

[0049] Embodiment 37 is one of the methods from Embodiments 13 to 36, wherein the monovalent cation salt washing buffer comprises a monovalent cation salt in a concentration of 5-300 mM, 5-250 mM, 5-200 mM, 50-150 mM, 50-100 mM, 70-120 mM, 80-110 mM, or 100 mM.

[0050] Embodiment 38 is one of the methods from Embodiments 13 to 37, wherein the monovalent cation salt is NaCl, Na2SO4, Na3PO4, or CH3COONa.

[0051] Embodiment 39 is a monovalent cation salt washing buffer, (a) 50-200 mM, 50-150 mM, 100-200 mM, 125-175 mM, or 150 mM NaCl; (b) 15-125 mM, 15-100 mM, 25-100 mM, 25-75 mM, or 50 mM Na2SO4; (c) 10-225 mM, 10-200 mM, 25-175 mM, 25-150 mM, 50-125 mM, 100-200 mM, 125-175 mM, 15-125 mM, 15-100 mM, 25-100 mM, or 25-75 mM Na3PO4; or (d) 10-225mM, 10-200mM, 25-175mM, 25-150mM, 50-125mM, 100-200mM, 125-175mM, 15-125mM, 15-100mM, 25-100mM, or 25-75mM CH3COONa This is one of the methods from Embodiments 13 to 38, including the method described above.

[0052] Embodiment 40 is one of the methods from Embodiments 13 to 39, wherein the monovalent cation salt washing buffer contains a buffering agent.

[0053] Embodiment 41 is one of the methods from Embodiments 13 to 40, wherein the monovalent cation salt washing buffer contains a buffer of 5-100 mM, 10-100 mM, 10-50 mM, 10-40 mM, 10-30 mM, 20-30 mM, or 25 mM.

[0054] Embodiment 42 is the method of Embodiment 40 or 41, wherein the buffering agent is selected from Tris, bis-trispropane, CHES (N-cyclohexyl-2-aminoethanesulfonic acid), or AMPSO (N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid).

[0055] Embodiment 43 is the method of Embodiments 13 to 42, wherein the monovalent cation salt washing buffer contains a stabilizing substance.

[0056] Embodiment 44 is the method of Embodiment 43, wherein the stabilizing substance is selected from poloxamer, polysorbate 80 (PS-80), polysorbate 20 (PS-20), sorbitol, sucrose, or trehalose.

[0057] Embodiment 45 is one of the methods of Embodiments 13 to 44, wherein the monovalent cation wash buffer is approximately pH 7.0-10.0, approximately pH 8.5-9.5, or approximately pH 9.

[0058] Embodiment 46 is one of the methods of Embodiments 13 to 45, wherein the monovalent cation wash buffer comprises 25 mM Tris, 150 mM NaCl, and 0.001% poloxamer, and has a pH of approximately 9.

[0059] Embodiment 47 is one of the methods of Embodiments 13 to 45, wherein the monovalent cation wash buffer comprises 25 mM Tris, 50 mM Na2SO4, and 0.001% poloxamer, and has a pH of approximately 9.

[0060] Embodiment 48 is one of the methods of Embodiments 9 to 47, wherein the linear gradient is 0%-100% divalent cation salt elution buffer.

[0061] Embodiment 49 is one of the methods of the preceding embodiments, wherein the anion exchange chromatography column is washed with a flash buffer before the divalent cation salt elution buffer is injected into the anion exchange chromatography column, and the flash buffer is monovalent or divalent cation salt-free.

[0062] Embodiment 50 is one of the methods of Embodiments 9 to 49, wherein the linear gradient is formed with a flash buffer and a divalent cation salt elution buffer, and the flash buffer is either monovalent or divalent cation salt-free.

[0063] Embodiment 51 is the method of Embodiment 50, wherein the linear gradient is 100%-0% flash buffer.

[0064] Embodiment 52 is one of the methods of Embodiments 49 to 51, wherein the flash buffer comprises a buffer selected from Tris, bis-trispropane, CHES (N-cyclohexyl-2-aminoethanesulfonic acid), or AMPSO (N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid).

[0065] Embodiment 53 is one of the methods of Embodiments 49 to 52, wherein the flash buffer comprises a buffer material of 5-100 mM, 10-100 mM, 10-50 mM, 10-40 mM, 10-30 mM, 20-30 mM, or 25 mM.

[0066] Embodiment 54 is one of the methods of Embodiments 49 to 53, wherein the flash buffer contains a stabilizing substance.

[0067] Embodiment 55 is the method of Embodiment 54, wherein the stabilizing substance is selected from poloxamer, polysorbate 80 (PS-80), polysorbate 20 (PS-20), sorbitol, sucrose, or trehalose.

[0068] Embodiment 56 is one of the methods of Embodiments 49 to 55, wherein the flash buffer is approximately pH 7.0–10.0, approximately pH 8.5–9.5, or approximately pH 9.

[0069] Embodiment 57 is one of the methods of Embodiments 49 to 56, wherein the flash buffer contains 25 mM Tris and 0.001% poloxamer and has a pH of approximately 9.

[0070] Embodiment 58 is one of the methods of the preceding embodiments, wherein the anion exchange chromatography column is washed with an equilibration buffer containing a 10-100 mM monovalent cation before the divalent cation salt elution buffer is injected into the anion exchange chromatography column.

[0071] Embodiment 59 is the method of Embodiment 58, wherein the equilibration buffer is injected into the anion exchange chromatography column after the monovalent cation salt washing buffer has been injected into the anion exchange chromatography column.

[0072] Embodiment 60 is the method of Embodiment 58 or Embodiment 59, wherein the equilibration buffer contains a monovalent cation salt at a concentration of 10-80 mM, 20-80 mM, 25-75 mM, 30-70 mM, 40-60 mM, 50 mM, or 60 mM.

[0073] Embodiment 61 is one of the methods of Embodiments 58 to 60, wherein the monovalent cation salt is selected from NaCl, Na2SO4, Na3PO4, CH3COONa, or sodium citrate.

[0074] Embodiment 62 is the method of Embodiment 61, wherein the sodium citrate is monosodium citrate (NaC6H7O7), disodium citrate (Na2C6H6O7), or trisodium citrate (Na3C6H5O7).

[0075] Embodiment 63 is one of the methods of Embodiments 58 to 62, wherein the equilibration buffer includes a buffering material.

[0076] Embodiment 64 is the method of Embodiment 63, wherein the equilibration buffer contains a buffering material of 5-100 mM, 10-100 mM, 10-50 mM, 10-40 mM, 10-30 mM, 20-30 mM, or 25 mM.

[0077] Embodiment 65 is the method of Embodiment 63 or Embodiment 64, wherein the buffering agent is selected from Tris, bis-trispropane, CHES (N-cyclohexyl-2-aminoethanesulfonic acid), or AMPSO (N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid).

[0078] Embodiment 66 is one of the methods of Embodiments 58 to 65, wherein the equilibration buffer contains a stabilizing substance.

[0079] Embodiment 67 is the method of Embodiment 66, wherein the stabilizing substance is selected from poloxamer, polysorbate 80 (PS-80), polysorbate 20 (PS-20), sorbitol, sucrose, or trehalose.

[0080] Embodiment 68 is one of the methods of Embodiments 58 to 68, wherein the equilibration buffer is approximately pH 7.0–10.0, approximately pH 8.5–9.5, or approximately pH 9.

[0081] Embodiment 69 is one of the methods of Embodiments 58 to 70, wherein the equilibration buffer comprises 25 mM Tris, 60 mM NaCl, and 0.001% poloxamer, and has a pH of approximately 9.

[0082] Embodiment 70 is one of the methods of Embodiments 58 to 70, wherein the equilibration buffer comprises 25 mM Tris, 20 mM Na2SO4, and 0.001% poloxamer, and is approximately pH 9.

[0083] Embodiment 71 is one of the methods of the preceding embodiments, wherein the anion exchange chromatography column contains a substrate comprising a functional ligand selected from mixed amines, quaternary amines, trimethylammonium ethyl (TMAE), dimethylaminopropyl, diethylaminoethyl (DEAE), dimethylaminoethyl (DMAE), polyethyleneimine (PI), or guanidium.

[0084] Embodiment 72 is the method of Embodiment 71, wherein the mixed amine contains polyethyleneimine.

[0085] Embodiment 73 is the method of Embodiment 72, wherein the quaternary amine comprises a quaternized polyethyleneimine.

[0086] Embodiment 74 is one of the methods of the preceding embodiments, in which an AAV starting preparation is injected into an anion exchange chromatography column under conditions in which AAV full capsids and AAV empty capsids are bound to the column.

[0087] Embodiment 75 is one of the methods from Embodiments 9 to 74, wherein the AAV full capsid is eluted from an anion exchange chromatography column in a linear gradient of divalent cation salt elution buffer between 10% and 90%, 15% and 85%, 20% and 80%, or 30% and 70%.

[0088] Embodiment 76 is one of the methods of the preceding embodiments, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12.

[0089] Embodiment 77 is one of the methods of the prior embodiments, wherein the AAV is AAV6.

[0090] Embodiment 78 is one of the methods of the preceding embodiments, wherein the AAV capsid comprises an AAV capsid protein, and the AAV capsid protein is a chimeric capsid, an engineered capsid, or a native capsid.

[0091] Embodiment 79 is one of the methods of the prior embodiments, wherein the AAV is a recombinant AAV.

[0092] Embodiment 80 is one of the methods of the prior embodiments, wherein the AAV is a pseudotyped AAV.

[0093] Embodiment 81 is the method of Embodiment 80, wherein the pseudotyped AAV is AAV2 / 5, AAV2 / 8, or AAV2 / 7.

[0094] Embodiment 82 is one of the methods of the prior embodiments, wherein the AAV is a self-complementary AAV.

[0095] Embodiment 83 is one of the methods of the prior embodiments, wherein the AAV full capsid contains the nucleic acid molecule of interest.

[0096] Embodiment 84 is the method of the immediately preceding embodiment, wherein the nucleic acid molecule of interest encodes a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0097] Embodiment 85 further comprises culturing AAV packaging cells in a medium capable of producing AAV particles, wherein the AAV particles comprise an AAV capsid protein and a nucleic acid molecule comprising an AAV 5' inverted terminal repeat (ITR), a nucleic acid molecule of interest to be packaged in the AAV capsid, and a nucleic acid molecule comprising a 3' ITR, wherein the cell comprises (i) a nucleic acid of interest to be packaged in at least one AAV capsid, (ii) a nucleic acid molecule encoding an AAV capsid protein under the control of one or more sequences directing its expression in the packaging cell, (iii) a nucleic acid molecule encoding an AAV rep protein that expresses an AAV rep protein in the cell to enable packaging of the nucleic acid of interest into the AAV capsid, and (iv) one or more helper functions necessary for packaging the nucleic acid molecule of interest into the AAV capsid.

[0098] Embodiment 86 is a method of Embodiment 85, further comprising collecting AAV particles from AAV packaging cells and / or from a culture medium, wherein the collection involves cell disruption or the collection does not substantially involve cell disruption, thereby providing an AAV starting preparation.

[0099] Embodiment 87 is the method of Embodiment 86, wherein cell disruption includes cell lysis, thereby providing a cell lysate.

[0100] Embodiment 88 is any one of Embodiments 85 to 87, wherein the AAV starting preparation comprises a culture medium and / or cell lysate.

[0101] Embodiment 89 is any one of Embodiments 85-88, wherein collecting AAV particles from cells and / or culture medium includes recovering the culture medium without substantially disrupting the cells.

[0102] Embodiment 90 is one of the methods described in Embodiments 85 to 89, wherein AAV packaging cells are stably transformed with one or more nucleic acid molecules encoding one or more helper functions.

[0103] Embodiment 91 is one of the methods described in Embodiments 86 to 91, wherein one or more helper functions are expressed under an activatable or inducible promoter.

[0104] Embodiment 92 is one of the methods of Embodiments 85 to 91, wherein AAV packaging cells are stably transformed with a nucleic acid molecule encoding an AAV rep protein and / or a nucleic acid molecule encoding an AAV capsid protein.

[0105] Embodiment 93 is one of the methods described in Embodiments 85 to 92, wherein the AAV rep protein and / or AAV capsid protein are expressed under the direction of an activatable or inducible promoter.

[0106] Embodiment 94 is one of the methods described in Embodiments 85 to 93, wherein AAV packaging cells are stably transformed with the nucleic acid molecule of the choice. [Brief explanation of the drawing]

[0107] [Figure 1] Figure 1 shows the chromatograms of AAV6 preparations separated using industrially standard methods as described in Example 1. A260 (the line extending at the lowest position in the empty vector), A280 (the line extending at the second highest position in the empty vector), and conductivity (the line extending from approximately 3.5 mAU on the Y-coordinate) profiles are shown. Absorbance (in milliabsorbance units, mAU) is shown on the y-coordinate (far left). Run volume (mL) is shown as a solid line below the x-coordinate, while buffer is shown on the x-axis above the run volume. Major peaks (labeled "empty" and "full") are shown. Peak fraction pool boundaries are indicated by vertical dotted lines on the x-coordinate, and the percentage of AAV full capsid recovery in each peak fraction pool is shown separately in Table 2.

[0108] [Figure 2]Figure 2 shows chromatograms of exemplary AAV6 preparations separated using a MgCl2 gradient, as described in Example 2. A260 (the line extending at the lowest position in the empty vector), A280 (the line extending at the second highest position in the empty vector), and conductivity (the line extending from approximately 5 mAU on the Y-coordinate) profiles are shown. Absorbance (mAU) is shown on the y-coordinate. Run volume (mL) is shown as a solid line below the x-coordinate, while buffer is shown on the x-axis above the run volume. Major peaks (labeled "Full Vector," "Empty + Full Vector," and "Empty Vector") are shown. Peak fraction pool boundaries are indicated by vertical dotted lines on the x-coordinate, and the percentage of AAV full capsid recovery in each peak fraction pool is shown separately in Table 4.

[0109] [Figure 3] Figure 3 shows chromatograms of exemplary AAV6 preparations separated using a CaCl2 gradient, as described in Example 3. A260 (the line extending at the lowest position in the empty vector), A280 (the line extending at the second highest position in the empty vector), and conductivity (the line extending from approximately -12 mAU on the Y-coordinate) profiles are shown. Absorbance (mAU) is shown on the y-coordinate. Run volume (mL) is shown as a solid line below the x-coordinate, while buffer is shown on the x-axis above the run volume. Major peaks (labeled "full vector" and "empty vector") are shown. Peak fraction pool boundaries are indicated by vertical dotted lines on the x-coordinate, and the percentage of AAV full capsid recovery in each peak fraction pool is shown separately in Table 6.

[0110] [Figure 4]Figure 4 shows chromatograms of exemplary AAV6 preparations separated using the NaCl washing step and MgCl2 gradient as described in Example 4. A260 (the line extending from the lowest position in the empty vector), A280 (the line extending from the second highest position in the empty vector), and conductivity (the line extending from approximately 12 mAU on the Y-coordinate) profiles are shown. Absorbance (mAU) is shown on the y-coordinate. Run volume (mL) is shown as a solid line below the x-coordinate, while buffer is shown on the x-axis above the run volume. Major peaks (labeled "empty vector," "full vector," and "second 'full' peak") are shown. Peak fraction pool boundaries are indicated by vertical dotted lines on the x-coordinate, and the percentage of AAV full capsid recovery in each peak fraction pool is shown separately in Table 8.

[0111] [Figure 5] Figure 5 shows chromatograms of exemplary AAV6 preparations separated using a Na2SO4 washing step and a MgSO4 gradient, as described in Example 5. A260 (the line extending from the lowest position in the empty vector), A280 (the line extending from the second highest position in the empty vector), and conductivity (the line extending from approximately 12 mAU on the Y-coordinate) profiles are shown. Absorbance (mAU) is shown on the y-coordinate. Run volume (mL) is shown as a solid line below the x-coordinate, while buffer is shown on the x-axis above the run volume. Major peaks (labeled "empty capsid," "full capsid," and "secondary full capsid peak") are shown. Peak fraction pool boundaries are indicated by vertical dotted lines on the x-coordinate, and the percentage of AAV full capsid recovery in each peak fraction pool is shown separately in Table 10.

[0112] [Figure 6]Figure 6 shows a chromatogram of an exemplary AAV6 preparation containing exemplary transgene A, separated using the Na2SO4 washing step and MgSO4 isocratic elution as described in Example 6. The A260 (line extending from the lowest position of the left peak), A280 (line extending from the highest position of the left peak), and conductivity (line extending from approximately 50 mAU on the y-coordinate) profiles are shown. Absorbance (mAU) is shown on the left y-axis, and conductivity (mS / cm) is shown on the right y-axis. Column volume (CV) is shown on the x-axis. Major peaks (labeled "full vector" and "empty vector") are shown. Peak fraction pool boundaries are indicated by vertical dotted lines on the x-coordinate, and the percentage of AAV full capsid recovery in each peak fraction pool is shown separately in Table 12.

[0113] [Figure 7] Figure 7 shows a chromatogram of an exemplary AAV6 preparation containing exemplary transgene B, separated using the Na2SO4 washing step and MgSO4 isocratic elution as described in Example 7. The A260 (line extending from the lowest point of the left peak), A280 (line extending from the highest point of the left peak), and conductivity (line extending from approximately 50 mAU on the y-coordinate) profiles are shown. Absorbance (mAU) is shown on the left y-axis, and conductivity (mS / cm) is shown on the right y-axis. Column volume (CV) is shown on the x-axis. Major peaks (labeled "full vector" and "empty vector") are shown. Peak fraction pool boundaries are indicated by vertical dotted lines on the x-coordinate, and the percentage of AAV full capsid recovery in each peak fraction pool is shown separately in Table 14.

[0114] [Figure 8]Figure 8 shows a chromatogram of an exemplary AAV6 preparation containing exemplary transgene B, separated using a Na2SO4 washing step and MgSO4 isocratic elution with a smaller volume column (4 mL column compared to the 40 mL column in Figures 6 and 7) as described in Example 8. The A260 (line extending from the lowest position of the left peak), A280 (line extending from the highest position of the left peak), and conductivity (line extending from approximately 50 mAU on the y-coordinate) profiles are shown. Absorbance (mAU) is shown on the left y-axis, and conductivity (mS / cm) is shown on the right y-axis. Column volume (CV) is shown on the x-axis. Major peaks (labeled "full vector" and "empty vector") are shown. Peak fraction pool boundaries are indicated by vertical dotted lines on the x-coordinate, and the percentage of AAV full capsid recovery in each peak fraction pool is shown separately in Table 16. [Modes for carrying out the invention]

[0115] Specific embodiments of the present invention will now be described in detail. The present invention is described in conjunction with such embodiments, but it is understood that the invention is not intended to be limited to those embodiments. On the contrary, the present invention is intended to encompass all substitutes, modifications, and equivalents, which may be included in the invention as defined by the appended claims.

[0116] Before describing this instruction in detail, it should be understood that this disclosure is not limited to any particular composition or process and may be modified. It should be noted that, unless the context clearly indicates otherwise, the singular forms "a," "an," and "the," as used in this specification and the appended claims, include references to the plural. Thus, for example, a reference to "an AAV full capsid" includes multiple AAV full capsids, and a reference to "a packaging cell" includes multiple packaging cells, and so on.

[0117] Numerical ranges include all numbers defined within the range. Measured and measurable values ​​should be understood as approximate, and significant figures and measurement errors should be taken into consideration. Furthermore, the use of “comprise,” “comprises,” “comprising,” “contain,” “contains,” “containing,” “include,” “includes,” and “including” is not intended to be limiting. Both the general and detailed statements above should be understood as illustrative and descriptive only, and not binding teachings.

[0118] Unless otherwise specified in the above specification, embodiments in this specification that describe "comprise" various components may also be interpreted as "consisting of" or "consisting essentially of" the described components; embodiments in this specification that describe "consisting of" various components may also be interpreted as "comprising" or "consisting essentially of" the described components; embodiments in this specification that describe "essentially" "consisting of" various components may also be interpreted as "consisting of" or "comprising" the described components (this interchangeability does not apply to the use of these terms in the claims).

[0119] The section headings used herein are for structuring purposes only and should never be construed as limiting the scope of disclosures. In the event of any conflict between any document or other material incorporated by reference and any information expressly contained herein, including definitions, this specification shall prevail.

[0120] I. Definition As used herein, "or" is used in an inclusive sense, i.e., equivalent to "and / or" unless the context requires the opposite meaning.

[0121] As used herein, “adeno-associated virus” or “AAV” refers to an adeno-associated virus vector and includes, but is not limited to, any AAV serotype or variant, including, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh10 (e.g., see SEQ ID NO: 81 in US 9,790,472), AAVrh74 (e.g., see SEQ ID NO: 1 in US 2015 / 0111955), AAV9, AAV9P (e.g., see Weinmann et al., Nature Communication, 2020, 11:5432), AAV11, AAV12, and Myo-AAV (e.g., Tabebordbar et al., 2021, Cell, This includes AAV vectors as described in 184:1-20 (e.g., MyoAAV1A, 2A, 3A, 4A, 4C, or 4E) and their chimeras (such as those listed in Viney et. Al, J Virol. 2021, 95(7):e02023-20), where the number following AAV indicates the AAV serotype. The term "AAV" may also refer to any known AAV(vector) system. In some embodiments, the AAV vector is a single-stranded AAV (ssAAV). In some embodiments, the AAV vector is a double-stranded AAV (dsAAV). Any variant of an AAV vector or its serotype, such as a self-complementary AAV (scAAV) vector, is included in the general terms AAV vector, AAV6 vector, etc. For example, see McCarty et al., Gene Ther. 2001; 8:1248-54, Naso et al., BioDrugs. 2017; 31:317-334, and the references cited therein for a detailed discussion of various AAV vectors. Structurally, AAV is a small (~25 nm), single-stranded DNA, icosahedral capsid non-enveloped virus. As used herein, “AAV” may refer to naturally occurring or engineered AAV serotypes and recombinant AAV (rAAV) as well as variants whose capsid proteins may differ in composition and structure, potentially exhibiting alternating tropism, i.e., the ability to transduce different cell types.When combined with an activation promoter, this tropism determines the location of gene expression, for example, within the host.

[0122] As used herein, “recombinant AAV” refers to an AAV having a capsid in which a heterogeneous nucleic acid molecule is packaged, containing an expression cassette for a desired product, such as a gene product. Such an expression cassette may contain AAV 5' and / or 3' inverted terminal repeat sequences adjacent to the nucleic acid of interest, such as a gene sequence, and the nucleic acid of interest is operably ligated to the expression regulatory sequence.

[0123] As used herein, “AAV6 capsid” refers to an AAV capsid having an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the AAV capsid encoded by the exemplary AAV6 genome of GenBank Accession No:AF028704.1, which is incorporated herein by reference and replicated as Sequence ID No. 1.

[0124] Sequence ID 1:

[0125] In some embodiments, the methods provided herein are used to obtain AAV6 or another AAV having a capsid highly related to the AAV6 capsid. In some embodiments, an AAV having a capsid sequence having about 95%, about 96%, about 97%, about 98%, or about 99% identity with the amino acid sequence of SEQ ID NO: 1 may be purified using the methods provided herein. In some embodiments, the methods provided herein may be used to purify an AAV having a capsid sequence that is a variant of the capsid encoded by the AAV6 genome of SEQ ID NO: 1. Methods for generating a capsid, sequences encoding a capsid, and methods for producing an rAAV viral vector have been described. See, for example, Gao, et al, Proc. Natl. Acad. Sci. USA 100 (10), 6081-6086 (2003), U.S. Patent Nos. 6,759,237, 7,105,345, 7,186,552, and U.S. Patent Application No. 2013 / 0045186A1.

[0126] As used herein, “adeno-associated virus flucapsid” or “AAV flucapsid” refers to a capsid in which a nucleic acid molecule is packaged, containing an expression cassette that includes the nucleic acid molecule of interest (e.g., a nucleic acid molecule encoding a desired gene product, such as a chimeric antigen receptor (CAR) or T cell receptor (TCR)). Such an expression cassette may contain AAV 5' and / or 3' inverted terminal repeats (ITRs) adjacent to the gene sequence, and the gene sequence is operably linked to one or more expression regulatory sequences. Suitable elements of these and other expression cassettes, such as the elements described herein, may instead be referred to herein as transgene genome sequences. Such an AAV virus particle is referred to as “pharmacologically active” when it can deliver the transgene to a host cell capable of expressing the desired gene product carried by the expression cassette. An AAV flucapsid may be, for example, an rAAV flucapsid.

[0127] As used herein, “AAV empty capsid” refers to a constructed AAV capsid that lacks the genomic sequence to be packaged within it. “AAV partial empty capsid” as used herein contains only a partial genomic sequence insufficient to express a complete gene product. Neither AAV empty capsids nor AAV partial empty capsids function to deliver the complete gene of interest to a host cell. Both AAV empty capsids and AAV partial empty capsids are also referred herein as “AAV intermediates” or “AAV intermediate capsids.”

[0128] As used herein, “adeno-associated virus starting preparation” or “AAV starting preparation” refers to a mixture comprising AAV full capsids (such as rAAV full capsids) and genome-deficient AAV intermediates such as AAV empty capsids and / or AAV partially empty capsids. AAV starting preparations may also contain components of the culture medium and / or packaging cell lysates recovered from AAV packaging cell cultures. Therefore, AAV starting preparations may further contain impurities present in the packaging cell medium and / or packaging cell lysates, such as packaging cell DNA, packaging cell proteins, culture medium components, and, in some examples, various cellular components such as helper viruses or helper virus plasmid DNA.

[0129] As used herein, “purify,” “purified,” or “purifying” means separating a biological component (such as AAV fulcapsid) from some or all of the other components of a mixture (AAV starting preparations containing AAV fulcapsid, AAV partially empty capsid, AAV empty capsid, cell culture medium, whole cells, cell material, and / or cell lysates). The biological component (such as AAV fulcapsid) does not need to be completely separated from the other components of the mixture in order to be “purified,” but may be separated from at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the other components of the mixture (such as the other components of the AAV starting preparation).

[0130] As used herein, “substantially all” means at least 90%. “Substantially” does not have to be 100% (such as exactly 100% of the AAV flucapsid in an AAV starting preparation), but may include amounts greater than 90% (such as 90%–100% of the AAV flucapsid in an AAV starting preparation), such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%.

[0131] II. Exemplary Method for Obtaining AAV Flucapsid A. Overview A method is provided for obtaining AAVs containing a full capsid (AAV full capsid) for use in various gene transfer and / or other applications. The disclosed method isolates (purifies) the AAV full capsid from other components of AAV starting preparations such as AAV empty capsids and / or AAV partially empty capsids, production culture contaminants such as helper viruses, helper virus proteins, plasmids, cell proteins and nucleic acids, culture medium components, serum proteins, AAV rep proteins, unconstructed AAV VP1, VP2, and VP3 proteins, etc. In some embodiments of the disclosed method, the AAV is AAV6 such as rAAV6. In certain embodiments, the AAV full capsid contains a nucleic acid molecule encoding a chimeric antigen receptor (CAR) or T cell receptor (TCR).

[0132] Some embodiments of the disclosed method for obtaining AAV full capsids from AAV starting preparations, including AAV full capsids and AAV empty capsids, involve injecting the AAV starting preparations into an anion exchange chromatography (AEX) column using a suitable liquid chromatography method, such as HPLC, but not limited to AEX. Anion exchange chromatography is a form of ion exchange chromatography in which negatively charged molecules (such as AAV full capsids) bind to a positively charged resin. More specifically, AEX uses a positively charged ion exchange resin that has an affinity for molecules with a net negative surface charge. The net surface charge of a given protein (such as an AAV capsid protein) changes with pH in relation to the protein isoelectric point (pI). A protein has no net charge at a pI equal to its pH, a net positive charge at pH below its pI, and a net negative charge at pH above its pI. The pI of a protein may be calculated from its primary amino acid sequence. Next, a buffer can be selected such that the protein provides a known net charge. Thus, when the protein has a net negative charge at the working pH, a positively charged anion exchange resin can be used. Proteins with different pI values ​​have a charge that changes to a certain extent at a given pH and therefore have different affinities to the particulate positively charged surface groups of the anion exchange medium. Thus, different proteins bind to a given AEX resin with different strengths, facilitating their separation by methods such as those disclosed herein.

[0133] Generally, at a given packing buffer pH, most (at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) or virtually all properly charged proteins will bind to the resin. For example, if an anion exchange resin is used at pH 7.5, generally, proteins with pI < 7.5 will have a net negative charge and will bind to the positively charged resin. Subsequently, a salt-containing buffer (such as a salt gradient) can be used to separate the target protein (such as AAV full capsid) from other bound proteins. When the salt-containing buffer is injected, proteins are eluted from the AEX column in an order typically dependent on their net surface charge. In the example above, proteins with pI values ​​close to 7.5 will elute at lower ionic strengths, while proteins with very low pI values ​​will elute at higher salt concentrations.

[0134] In standard AEX AAV purification methods, such as those involving a buffer containing NaCl, empty AAV capsids elute before full AAV capsids (as shown in Figure 1 and Example 1, etc.). Surprisingly, we have found that replacing the monovalent cation salt in the elution buffer with a divalent cation salt can reverse the order of eluted chemical species. In other words, using the same AAV and column resin at the same pH, but using a divalent cation salt instead of a monovalent cation salt in the elution buffer, full AAV capsids may elute from the column before empty AAV capsids.

[0135] In some embodiments, an AAV starting preparation is injected into an anion exchange chromatography (AEX) column, followed by the injection of elution buffer, which elutes a fraction containing AAV full capsids from the AEX column in a larger proportion than AAV empty capsids. In certain embodiments, the AAV starting preparation further comprises AAV partially empty capsids. The AAV starting preparation, containing AAV full, empty, and / or partially empty capsids, may be suspended in a suitable packing buffer and injected into an AEX resin. AAV particles and intermediates bind to the resin, while other components are carried out into the buffer. In one embodiment, the total flow rate of the buffer is kept constant, while in another embodiment, the proportion of the elution buffer gradually increases from 0% to 100% according to a programmed concentration gradient.

[0136] In certain embodiments, the AAV starting preparation is subjected to one or more processing steps disclosed herein before being filled into the AEX resin, such as clarification (e.g., filtration and / or centrifugation), one or more nucleases and / or proteases (to digest contaminating nucleic acids and / or proteins), additional chromatography steps (e.g., affinity chromatography), and / or concentration steps). In some embodiments, the AAV starting preparation, comprising AAV full capsids and AAV empty capsids (and / or AAV partially empty capsids), contains less than about 10% contamination from non-AAV viral and cellular protein and nucleic acid material, or less than about 5% contaminants, or less than 1% contaminating viral and cellular protein and nucleic acid material. Thus, in some embodiments, the AAV starting preparation filled into the AEX resin is free of contaminants from about 80%, about 85%, about 95%, and from about 99%.

[0137] In some embodiments, the elution buffer is a divalent cation salt elution buffer. In some specific embodiments, the elution buffer is injected into the AEX column at a constant concentration ("isocratic" elution). In other specific embodiments, the elution buffer is injected into the AEX column in a linear gradient ranging from 0% to 100% elution buffer. In some embodiments, isocratic cation salt elution is superior to gradient divalent cation salt elution in that isocratic divalent cation salt elution elutes the AAV full capsid without empty capsids, or substantially without them (e.g., empty capsids are observed in later peaks in gradient elution, as shown in the examples provided herein). Thus, monovalent salt isocratic washing followed by divalent salt isocratic elution allows for the elution of the full capsid, which first removes a population of empty capsids and avoids (or substantially avoids) co-purification of the full capsid with any remaining empty capsids not removed by the first wash. The AAV starting preparation is injected into an AEX column under conditions that all or part of the AAV full capsid present in the AAV starting preparation binds to the column. In certain embodiments, the AAV full capsid is eluted from the AEX column in a linear gradient of elution buffer (such as a divalent cation salt elution buffer) between 10% and 90%, 15% and 85%, 20% and 80%, or 30% and 70%.

[0138] In some embodiments, AEX column-bound AAV full capsids and AAV empty capsids (and / or AAV partially empty capsids) are subjected to a monovalent cation wash buffer before a divalent cation elution buffer, and the injection of the monovalent cation buffer elutes a wash fraction from the AEX column containing AAV empty capsids in a larger proportion than AAV full capsids. In certain embodiments, the monovalent cation wash buffer does not contain divalent cations.

[0139] In some embodiments, a method for obtaining an AAV full capsid from an AAV starting preparation comprising an AAV full capsid and an AAV empty capsid (and / or an AAV partially empty capsid) comprises subjecting the AAV starting preparation to a suitable liquid chromatography process such as described herein (e.g., high-performance liquid chromatography (HPLC)), wherein the AAV full capsid and AAV empty capsid (and / or an AAV partially empty capsid) are bound to an anion exchange resin and subjected to a cation salt elution buffer (such as a divalent cation salt buffer), during which the eluate is monitored for ultraviolet absorbance at approximately 260 nm and approximately 280 nm, for example, by using a detector.

[0140] As provided above, some embodiments of the disclosed method include a divalent cation elution buffer and optionally a monovalent cation wash buffer, such as the divalent and monovalent cation salts specified herein. However, it will be understood that other divalent or monovalent cation salts of equivalent ionic strength; other divalent or monovalent cation salts having different ionic strengths but prepared to have equivalent ionic strengths; or combinations of such salts may be substituted. The formula for ionic strength is well known to those skilled in the art:

number

[0141] At different points in time in the methods disclosed herein, AEX resin-bound AAV6 full, empty, and / or partially empty capsids dissociate from the resin and appear in the column effluent. The effluent passes through a detector such as one that measures salt concentration (by conductivity) and protein concentration (by absorption of ultraviolet light at a predetermined wavelength). However, other suitable detection means may be used. Since each AAV particle is eluted, it appears in the effluent as a "peak" of protein concentration and can be recovered for further use.

[0142] As described herein, the fraction containing the AAV full capsid is recovered at the 260 nm elution peak and processed for further use. In one embodiment, the resulting AAV stock has a particle-to-vector genome ratio of 1. Optionally, the recovered AAV is placed in a suspension having a near-neutral pH for long-term storage and / or delivery to the target. Such a pH may be in the range of about 6.5 to about 8, or about 7 to about 7.5.

[0143] Those skilled in the art will understand that pH values ​​are approximate and may be rounded up or down to the nearest significant figure, and may vary slightly depending on the measuring instrument used for the measurement and its calibration. In some examples, the provided pH value is an average value obtained by taking one or more pH measurements and then averaging them. In some examples, the pH value is rounded to the nearest significant figure.

[0144] In one embodiment, the average yield of AAV full capsid from AAV starting preparations packed into a column is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%. This may be calculated by determining the titer (genomic copy) in the preparations packed into the column and the amount present in the final eluate. Furthermore, these may be determined based on quantitative PCR (qPCR) analysis, droplet digital PCR analysis, SDS-PAGE technology, light scattering, a combination of static or dynamic light scattering with UV, analytical ultracentrifugation, HPLC, CryoTEM, nanotracking analysis, enzyme-linked immunosorbent assay (ELISA), or any other assay capable of quantifying viral titer and / or distinguishing between empty AAV and full AAV, such as methods described herein or in the art.

[0145] Methods for analyzing empty and full AAV capsids are generally known in this field. See, for example, Grimm et al., Gene Therapy (1999) 6:1322-1330; Sommer et al., Molec. Ther. (2003) 7:122-128. A method for testing capsids involves subjecting an AAV-containing preparation to SDS-polyacrylamide gel electrophoresis, using any gel capable of separating three capsid proteins, for example, a gradient gel containing 3-8% trisacetic acid in buffer, followed by electrophoresis of the gel until the sample material is separated, and finally blotting the gel onto a nylon or nitrocellulose membrane, preferably nylon. Subsequently, an anti-AAV capsid antibody is used as a primary antibody that binds to the denatured capsid protein. Subsequently, a secondary antibody is used that binds to the primary antibody and contains means for detecting binding to the primary antibody. Methods for detecting binding, such as chemiluminescence detection kits that can detect radioactive isotope emission, electromagnetic radiation, or colorimetric changes, are used to semi-quantitatively determine the binding between primary and secondary antibodies.

[0146] In one embodiment, the concentration of the AAV vector genome (vg) in the eluted fraction can be measured by qPCR. The sample can be diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA, and optionally further treated with proteinase K (or another suitable proteinase). After nuclease inactivation, the sample is further diluted and treated with a probe specific to the DNA sequence between primers (e.g., TagMan). (登録商標)The AAV can be amplified using a fluorescence-generating probe. The number of cycles required to reach a predetermined level of fluorescence (threshold cycles, Ct) is measured for each sample using a suitable detection system. Plasmid DNA containing the same sequence as that contained in the AAV vector is used to generate a standard curve for the qPCR reaction. The Ct values ​​obtained from the sample are used to determine the vector genome titer by normalizing them to the Ct values ​​of the plasmid standard curve. Endpoint assays based on digital PCR can also be used. In some embodiments, qPCR may be combined with ELISA for AAV analysis.

[0147] Furthermore, or alternatively, droplet digital PCR (ddPCR) may be used. For example, methods for determining single-stranded and self-complementary AAV vector genome titers using ddPCR have been described. See, for example, M. Lock et al, Hu Gene Therapy Methods, 2014, 25(2): 15-25.

[0148] In another embodiment, a combination of UV absorbance and light scattering is used to determine the percentage of AAV full capsid in a chromatogram peak (e.g., in the elution fraction), such as the percentage of AAV full capsid in the chromatogram peak shown in Figure 1-5. In a specific embodiment, a Stunner instrument (Unchained Labs) is used in this method, and capsid particle counts are obtained by dynamic and static light scattering (DLS and SLS) followed by UV absorbance measurements at 260 nm and 280 nm wavelengths. In addition to the sequence of the gene of interest, these measurements are used to determine which portion of the particle population contains the gene of interest.

[0149] B. Anion exchange chromatography method Embodiments of the disclosed method for obtaining AAV full capsid from an AAV starting preparation containing AAV full capsid and AAV empty capsid include, but are not limited to, injecting the AAV starting preparation into an anion exchange chromatography (AEX) column using a suitable liquid chromatography method such as HPLC. In some embodiments, the AAV starting preparation also includes AAV partially empty capsid. In some embodiments, the AAV starting preparation is injected into an AEX column, followed by injection of an elution buffer, and the injection of the elution buffer elutes an elution fraction containing AAV full capsid from the AEX column in a larger proportion than AAV empty capsid. In some embodiments, the elution buffer is a divalent cation salt elution buffer that does not contain monovalent cation salts. In some embodiments, the elution buffer is injected at a constant divalent cation salt concentration (also referred to herein as "isocratic" elution). In some embodiments, the elution buffer is injected in a linear gradient of divalent cation salt concentrations.

[0150] In some embodiments, following the loading of the column with the AAV starting preparation, a wash buffer is injected into the column before the elution buffer, and injecting the wash buffer elutes a wash fraction from the AEX column containing AAV empty capsids (and / or partially empty AAV capsids) in a greater proportion than AAV full capsids. In certain embodiments, the wash buffer is a monovalent cation wash buffer that does not contain divalent cations. In some embodiments, the wash buffer is injected at a constant monovalent cation concentration. In some embodiments, the wash buffer is injected in a linear gradient of monovalent cation concentrations. In certain, non-limiting embodiments, a monovalent cation wash buffer is injected into the column before the divalent cation elution buffer, and the monovalent cation wash buffer does not contain divalent cations, and injecting the monovalent cation buffer elutes a wash fraction from the AEX column containing AAV empty capsids in a greater proportion than AAV full capsids.

[0151] In a particular embodiment, a method for obtaining AAV full capsid from an AAV starting preparation containing AAV full capsid and AAV empty capsid (and / or AAV partially empty capsid) comprises (a) injecting the AAV starting preparation into an AEX column; (b) injecting a monovalent cation wash buffer into the column to elute from the AEX column a wash fraction containing AAV empty capsid in a larger proportion than AAV full capsid; and (c) injecting a divalent cation elution buffer into the AEX column to elute from the AEX column an ​​elution fraction containing AAV full capsid in a larger proportion than AAV empty capsid (and / or AAV partially empty capsid). In some embodiments, the wash buffer is injected at a constant monovalent cation concentration. In some embodiments, the wash buffer is injected in a linear gradient of monovalent cation concentration. In some embodiments, the elution buffer is injected at a constant divalent cation concentration. In some embodiments, the elution buffer is injected in a linear gradient of divalent cation salt concentration. In some embodiments, the washing buffer is injected at a constant monovalent cation salt concentration, and the elution buffer is injected at a constant divalent cation salt concentration. In some embodiments, the washing buffer is injected at a constant monovalent cation salt concentration, and the elution buffer is injected in a linear gradient of divalent cation salt concentration. In some embodiments, the washing buffer is injected in a linear gradient of monovalent cation salt concentration, and the elution buffer is injected at a constant divalent cation salt concentration. In some embodiments, the washing buffer is injected in a linear gradient of monovalent cation salt concentration, and the elution buffer is injected in a linear gradient of divalent cation salt concentration.

[0152] In some embodiments, the eluted fraction contains at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% AAV fulcapsid relative to the AAV starting preparation. In some embodiments, the proportion of AAV fulcapsid in the eluted fraction compared to the total AAV capsid is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60%. In certain embodiments, the ratio of AAV full capsid to AAV empty capsid (and / or AAV partially empty capsid) in the eluted fraction is at least 1.5:1, at least 1.6:1, at least 1.7:1, at least 1.8:1, at least 1.9:1, at least 2:1, at least 2.25:1, at least 2.5:1, at least 2.75:1, at least 3:1, at least 4:1, at least 5:1, at least 10:1, at least 20:1, at least 30:1, at least 40:1, at least 50:1, or at least 100:1. In some embodiments, the eluted fraction comprises 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, or 2% or less, or 1% or less of AAV empty capsid (and / or AAV partially empty capsid).

[0153] In some embodiments where the washing buffer (such as a monovalent cation wash buffer) is not injected into the column before the elution buffer (such as a divalent cation elution buffer), at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or substantially all of the AAV full capsid is eluted from the AEX column before the AAV empty capsid (and / or AAV partially empty capsid). In certain embodiments, the elution buffer (such as a divalent cation elution buffer) is injected into the AEX column under conditions that at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or substantially all of the AAV full capsid is eluted from the column, and at least 60%, at least 70%, at least 80%, at least 90%, or substantially all of the AAV empty capsid (and / or AAV partially empty capsid) remains bound to the column.

[0154] In some embodiments, where a wash buffer (such as a monovalent cation wash buffer) is injected into the column before an elution buffer (such as a divalent cation elution buffer), at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or substantially all AAV empty capsids (and / or partially empty AAV capsids) are eluted from the AEX column before the AAV full capsids. In certain embodiments, the wash buffer (such as a monovalent cation wash buffer) is injected into the AEX column under conditions that at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or substantially all AAV empty capsids (and / or partially empty AAV capsids) are eluted from the column, and at least 60%, at least 70%, at least 80%, at least 90%, or substantially all AAV full capsids remain bound to the column. In some embodiments, the washing fraction comprises at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or substantially 100% AAV empty capsid (and / or AAV partially empty capsid).

[0155] In certain embodiments, the eluted fraction is a first eluted fraction, and a second eluted fraction is eluted after the first eluted fraction. In such embodiments, the second eluted fraction may contain a larger proportion of AAV empty capsid (and / or AAV partially empty capsid) than the first eluted fraction. In certain embodiments, the second eluted fraction contains 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, or 2% or less, or 1% or less of AAV full capsid. In certain embodiments, the second eluted fraction contains at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% AAV full capsid.

[0156] 1. Dissolution buffer In some embodiments of the disclosed methods, the divalent cation salt dissolution buffer contains 5 - 300 mM, 5 - 250 mM, 5 - 200 mM, 25 - 175 mM, 25 - 150 mM, 25 - 100 mM, 25 - 75 mM, 50 - 150 mM, 50 - 100 mM, 70 - 120 mM, 80 - 120 mM, 80 - 110 mM, 100 mM, or 50 mM divalent cation salt. In some embodiments, the divalent cation salt dissolution buffer is injected at a constant concentration. In other embodiments, the divalent cation salt dissolution buffer is injected with a linear gradient. In certain embodiments, the range of the linear gradient is from 0% to 100% divalent cation salt dissolution buffer. In certain specific embodiments, the AAV full capsid elutes from an AEX column with a linear gradient of the dissolution buffer with a divalent cation salt dissolution buffer between 10% and 90%, 15% and 85%, 20% and 80%, or 30% and 70%.

[0157] In certain embodiments, the divalent cation salt contains a divalent cation selected from 2+ Mg 2+ Ca 2+ or Sr 2+ In one embodiment, the divalent cation is Mg 2+ In another embodiment, the divalent cation is Ca 2+In some embodiments, the divalent cation salt is magnesium chloride (MgCl2), calcium chloride (CaCl2), magnesium sulfate (MgSO4), magnesium phosphate (MgPO4), calcium acetate (Ca(C2H3O2)2), calcium sulfate (CaSO4), magnesium acetate (Mg(C2H3O2)2), magnesium citrate (C6H6MgO7), strontium chloride (SrCl2), or strontium acetate (C4H6O4Sr). In certain embodiments, the divalent cation salt is MgCl2, CaCl2, or MgSO4. In some embodiments, the divalent cation salt elution buffer contains 5-300 mM, 5-250 mM, 5-200 mM, 25-175 mM, 25-150 mM, 25-100 mM, 25-75 mM, 50-150 mM, 50-100 mM, 70-120 mM, 80-120 mM, 80-110 mM, 100 mM, or 50 mM MgCl2. In some embodiments, the divalent cation salt elution buffer includes 5-300 mM, 5-250 mM, 5-200 mM, 25-175 mM, 25-150 mM, 25-100 mM, 25-75 mM, 50-150 mM, 50-100 mM, 70-120 mM, 80-120 mM, 80-110 mM, 100 mM, or 50 mM CaCl2. In some embodiments, the divalent cation salt elution buffer contains 5-300 mM, 5-250 mM, 5-200 mM, 25-175 mM, 25-150 mM, 25-100 mM, 25-75 mM, 50-150 mM, 50-100 mM, 70-120 mM, 80-120 mM, 80-110 mM, 100 mM, or 50 mM MgSO4.

[0158] In certain embodiments, the divalent cation elution buffer does not contain a monovalent cation. In certain embodiments, the divalent cation elution buffer does not contain NaCl. In other embodiments, the divalent cation elution buffer contains a monovalent cation at concentrations of 100 mM or less, 90 mM or less, 80 mM or less, 70 mM or less, 60 mM or less, 50 mM or less, 40 mM or less, 30 mM or less, 25 mM or less, 20 mM or less, 15 mM or less, 10 mM or less, or 5 mM or less.

[0159] In some embodiments, the divalent cation salt elution buffer includes a buffering agent. In some embodiments, the divalent cation salt elution buffer includes 5-100 mM, 10-100 mM, 10-50 mM, 10-40 mM, 10-30 mM, 20-30 mM, or 25 mM buffering agents. Suitable buffers include, for example, N-methylpiperazine; piperazine; bis-tris; bis-trispropane; CHES (N-cyclohexyl-2-aminoethanesulfonic acid); AMPSO (N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid); MES; Hepes; N-methyldiethanolamine; 1,3-diaminopropane; ethanolamine; acetic acid, etc. Such buffers may be used at a pH between 8.5 and 10.0 (e.g., approximately pH 8.5 to approximately pH 9.5, approximately pH 9.0 to approximately pH 10.0, approximately pH 9.0 to approximately pH 9.5, or approximately pH 9.0). In certain embodiments, the buffering agent of the divalent cation salt elution buffer is Tris, bis-trispropane, CHES (N-cyclohexyl-2-aminoethanesulfonic acid), or AMPSO (N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid).

[0160] In certain embodiments, the divalent cation salt elution buffer includes a stabilizing substance such as a surfactant. In certain embodiments, the stabilizing substance is poloxamer, polysorbate 80 (PS-80), polysorbate 20 (PS-20), sorbitol, sucrose, or trehalose.

[0161] In some embodiments, the divalent cation salt elution buffer has a pH of about 7–10, about 8.5–9.5, or about pH 9. In certain, non-limiting embodiments, the divalent cation salt elution buffer comprises 25 mM Tris, 100 mM divalent cation salt, and 0.001% poloxamer, and has a pH of about 9. In another certain, non-limiting embodiment, the divalent cation salt elution buffer comprises 25 mM Tris, 100 mM MgCl2, and 0.001% poloxamer, and has a pH of about 9. In yet another certain, non-limiting embodiment, the divalent cation salt elution buffer comprises 25 mM Tris, 100 mM MgSO4, and 0.001% poloxamer, and has a pH of about 9. In another specific, non-limiting embodiment, the divalent cation salt elution buffer comprises 25 mM Tris, 38 mM MgSO4, and 0.001% poloxamer, and has a pH of approximately 9.

[0162] 2. Washing buffer In some embodiments of the disclosed method, the monovalent cation wash buffer comprises monovalent cations of 5-300 mM, 5-250 mM, 5-200 mM, 10-225 mM, 10-200 mM, 15-125 mM, 15-100 mM, 25-175 mM, 25-150 mM, 25-100 mM, 25-75 mM, 50-200 mM, 50-150 mM, 50-100 mM, 50-125 mM, 70-120 mM, 80-110 mM, 100-200 mM, 125-175 mM, 150 mM, or 100 mM. In some embodiments, the monovalent cation wash buffer is injected at a constant concentration. In other embodiments, the monovalent cation wash buffer is injected in a linear gradient. In certain embodiments, the linear gradient range is 0% to 100% monovalent cation salt washing buffer.

[0163] In some embodiments, the monovalent cation wash buffer comprises NaCl, Na2SO4, Na3PO4, or CH3COONa. In specific embodiments, the monovalent cation wash buffer comprises about 50–250 mM NaCl, about 15–150 mM Na2SO4, about 10–250 mM Na3PO4, or about 10–250 mM CH3COONa. In some embodiments, the monovalent cation salt washing buffer includes 5-300 mM, 5-250 mM, 5-200 mM, 10-225 mM, 10-200 mM, 15-125 mM, 15-100 mM, 25-175 mM, 25-150 mM, 25-100 mM, 25-75 mM, 50-200 mM, 50-150 mM, 50-100 mM, 50-125 mM, 70-120 mM, 80-110 mM, 100-200 mM, 125-175 mM, 150 mM, or 100 mM NaCl. In some embodiments, the monovalent cation salt washing buffer includes 5-300 mM, 5-250 mM, 5-200 mM, 10-225 mM, 10-200 mM, 15-125 mM, 15-100 mM, 25-175 mM, 25-150 mM, 25-100 mM, 25-75 mM, 50-200 mM, 50-150 mM, 50-100 mM, 50-125 mM, 70-120 mM, 80-110 mM, 100-200 mM, 125-175 mM, 150 mM, or 100 mM Na2SO4. In some embodiments, the monovalent cation salt washing buffer includes 5-300 mM, 5-250 mM, 5-200 mM, 10-225 mM, 10-200 mM, 15-125 mM, 15-100 mM, 25-175 mM, 25-150 mM, 25-100 mM, 25-75 mM, 50-200 mM, 50-150 mM, 50-100 mM, 50-125 mM, 70-120 mM, 80-110 mM, 100-200 mM, 125-175 mM, 150 mM, or 100 mM Na3PO4.In some embodiments, the monovalent cation salt washing buffer comprises 5-300 mM, 5-250 mM, 5-200 mM, 10-225 mM, 10-200 mM, 15-125 mM, 15-100 mM, 25-175 mM, 25-150 mM, 25-100 mM, 25-75 mM, 50-200 mM, 50-150 mM, 50-100 mM, 50-125 mM, 70-120 mM, 80-110 mM, 100-200 mM, 125-175 mM, 150 mM, or 100 mM CH3COONa. In certain, non-limiting embodiments, the monovalent cation salt washing buffer comprises 75-250 mM NaCl or 25-150 mM Na2SO4.

[0164] In certain embodiments, the monovalent cation wash buffer does not contain divalent cations. In other embodiments, the monovalent cation wash buffer contains divalent cations at concentrations of 100 mM or less, 90 mM or less, 80 mM or less, 70 mM or less, 60 mM or less, 50 mM or less, 40 mM or less, 30 mM or less, 25 mM or less, 20 mM or less, 15 mM or less, 10 mM or less, or 5 mM or less.

[0165] In some embodiments, the monovalent cation wash buffer includes a buffering agent. In some embodiments, the monovalent cation wash buffer includes 5-100 mM, 10-100 mM, 10-50 mM, 10-40 mM, 10-30 mM, 20-30 mM, or 25 mM buffering agents. Suitable buffers may include N-methylpiperazine; piperazine; bis-tris; bis-trispropane; CHES (N-cyclohexyl-2-aminoethanesulfonic acid); AMPSO (N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid); MES; Hepes; N-methyldiethanolamine; 1,3-diaminopropane; ethanolamine; acetic acid, etc. Such buffers may be used at a pH between 8.5 and 10.0 (e.g., approximately pH 8.5 to approximately pH 9.5, approximately pH 9.0 to approximately pH 10.0, approximately pH 9.0 to approximately pH 9.5, or approximately pH 9.0). In certain embodiments, the buffering agent of the monovalent cation salt washing buffer is Tris, bis-trispropane, CHES (N-cyclohexyl-2-aminoethanesulfonic acid), or AMPSO (N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid).

[0166] In certain embodiments, the monovalent cation salt washing buffer includes a stabilizing substance such as a surfactant. In certain embodiments, the stabilizing substance is poloxamer, polysorbate 80 (PS-80), polysorbate 20 (PS-20), sorbitol, sucrose, or trehalose.

[0167] In some embodiments, the monovalent cation wash buffer has a pH of 7.0–10.0, about pH 8.5–9.5, or about pH 9. In certain, non-limiting embodiments, the monovalent cation wash buffer comprises 25 mM Tris, 150 mM NaCl, and 0.001% poloxamer, and has a pH of about 9. In another certain, non-limiting embodiment, the monovalent cation wash buffer comprises 25 mM Tris, 53 mM Na2SO4, and 0.001% poloxamer, and has a pH of about 9.

[0168] In certain, non-limiting embodiments, the monovalent cation wash buffer contains 10-100 mM Tris (e.g., 25 mM Tris), 150 mM NaCl, and 0.001% poloxamer, and is approximately pH 9. In another certain, non-limiting embodiment, the monovalent cation wash buffer contains 25 mM Tris, 53 mM Na2SO4, and 0.001% poloxamer, and is approximately pH 9. In certain, non-limiting embodiments including both a divalent cation elution buffer and a monovalent cation wash buffer, the divalent cation elution buffer contains 10-100 mM Tris (e.g., 25 mM Tris), 100 mM MgCl2, and 0.001% poloxamer, and is approximately pH 9, while the monovalent cation wash buffer contains 10-100 mM Tris (e.g., 25 mM Tris), 150 mM NaCl, and 0.001% poloxamer, and is approximately pH 9. In another specific, non-limiting embodiment, which includes both a divalent cation elution buffer and a monovalent cation wash buffer, the divalent cation elution buffer comprises 25 mM Tris, 100 mM MgSO4, and 0.001% poloxamer and has a pH of approximately 9, and the monovalent cation wash buffer comprises 25 mM Tris, 50 mM Na2SO4, and 0.001% poloxamer and has a pH of approximately 9.

[0169] 3. Flash buffer In some embodiments, the column is washed with a flash buffer before the elution buffer is injected. In certain embodiments, the flash buffer does not contain a monovalent or divalent cation. In other specific embodiments, the flash buffer contains a monovalent cation. In certain embodiments, where the elution buffer is injected into the column in a linear gradient, the linear gradient is formed of the flash buffer and the elution buffer. In some embodiments, the linear gradient is 100%-0% flash buffer.

[0170] In some embodiments, the monovalent cation salt of the flash buffer is NaCl, Na2SO4, Na3PO4, CH3COONa, or sodium citrate (monosodium citrate (NaC6H7O7), disodium citrate (Na2C6H6O7), or trisodium citrate (Na3C6H5O7)).

[0171] In some embodiments, the flash buffer includes a buffering agent. In some embodiments, the flash buffer includes 5-100 mM, 10-100 mM, 10-50 mM, 10-40 mM, 10-30 mM, 20-30 mM, or 25 mM buffering agents. Suitable buffers may include, for example, N-methylpiperazine; piperazine; bis-tris; bis-trispropane; CHES (N-cyclohexyl-2-aminoethanesulfonic acid); AMPSO (N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid MES; Hepes; N-methyldiethanolamine; 1,3-diaminopropane; ethanolamine; acetic acid, etc. Such buffers are 8.5 and 10.0 It may be used at a pH between (for example, approximately pH 8.5 to approximately pH 9.5, approximately pH 9.0 to approximately pH 10.0, approximately pH 9.0 to approximately pH 9.5, or approximately pH 9.0). In certain embodiments, the buffering agent of the monovalent cation salt washing buffer is Tris, bis-trispropane, CHES (N-cyclohexyl-2-aminoethanesulfonic acid), or AMPSO (N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid).

[0172] In certain embodiments, the flash buffer includes a stabilizing substance such as a surfactant. In certain embodiments, the stabilizing substance is poloxamer, polysorbate 80 (PS-80), polysorbate 20 (PS-20), sorbitol, sucrose, or trehalose.

[0173] In some embodiments, the flash buffer has a pH of 7.0–10.0, approximately pH 8.5–9.5, or approximately pH 9. In certain, non-limiting embodiments, the flash buffer comprises 25 mM Tris, 60 mM NaCl, and 0.001% poloxamer, and is approximately pH 9. In another certain, non-limiting embodiment, the flash buffer comprises 25 mM Tris, 20 mM Na2SO4, and 0.001% poloxamer, and is approximately pH 9.

[0174] In some embodiments, a second flash buffer is injected into the AEX column after the injection of the monovalent cation wash buffer disclosed herein and before the injection of the divalent cation elution buffer. In some embodiments, the second flash buffer does not contain a monovalent or divalent cation. In certain embodiments, the second flash buffer contains a buffering agent such as any suitable buffering agent such as Tris (as disclosed herein). In some embodiments, the flash buffer contains a 5-100 mM, 10-100 mM, 10-50 mM, 10-40 mM, 10-30 mM, 20-30 mM, or 25 mM buffering agent. In some embodiments, the second flash buffer contains a stabilizing agent such as a surfactant. In certain embodiments, the stabilizing agent is poloxamer, polysorbate 80 (PS-80), polysorbate 20 (PS-20), sorbitol, sucrose, or trehalose. In certain, non-limiting embodiments, the second flash buffer comprises 25 mM Tris and 0.001% poloxamer and has a pH of approximately 9.

[0175] 4. Equilibrium buffer In some embodiments, the anion exchange chromatography column is washed with an equilibration buffer before the divalent cation salt elution buffer is injected. In some embodiments, the equilibration buffer is injected into the column after the monovalent cation salt wash buffer. In some embodiments, the equilibration buffer is injected into the column after the flash buffer. In certain embodiments, the equilibration buffer contains 10–100 mM monovalent cation salts, such as 10–80 mM, 20–80 mM, 25–75 mM, 30–70 mM, 40–60 mM, 50 mM, or 60 mM monovalent cation salts. In certain embodiments, a pre-equilibrium solution is injected into the column after the flash buffer is injected and before the equilibration buffer is injected. In some embodiments, the pre-equilibrium solution contains a monovalent cation salt such as a 1000-3000 mM monovalent cation salt, such as 1250-2750 mM, 1500-2500 mM, 1750-2250 mM, 1900-2100 mM, or 2000 mM monovalent cation salt. In certain embodiments, the monovalent cation salt of the pre-equilibrium buffer is NaCl.

[0176] In some embodiments, the monovalent cation salt of the equilibration buffer is NaCl, Na2SO4, Na3PO4, CH3COONa, or sodium citrate (such as monosodium citrate (NaC6H7O7), disodium citrate (Na2C6H6O7), or trisodium citrate (Na3C6H5O7)).

[0177] In some embodiments, the equilibration buffer includes a buffering agent. In some embodiments, the equilibration buffer includes 5-100 mM, 10-100 mM, 10-50 mM, 10-40 mM, 10-30 mM, 20-30 mM, or 25 mM buffering agents. Suitable buffers may include, for example, N-methylpiperazine; piperazine; bis-tris; bis-trispropane; CHES (N-cyclohexyl-2-aminoethanesulfonic acid); AMPSO (N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid); MES; Hepes; N-methyldiethanolamine; 1,3-diaminopropane; ethanolamine; acetic acid, etc. Such buffers may be used at a pH between 8.5 and 10.0 (e.g., approximately pH 8.5 to approximately pH 9.5, approximately pH 9.0 to approximately pH 10.0, approximately pH 9.0 to approximately pH 9.5, or approximately pH 9.0). In certain embodiments, the buffering agent of the monovalent cation salt washing buffer is Tris, bis-trispropane, CHES (N-cyclohexyl-2-aminoethanesulfonic acid), or AMPSO (N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid).

[0178] In certain embodiments, the equilibration buffer includes a stabilizing substance such as a surfactant. In certain embodiments, the stabilizing substance is poloxamer, polysorbate 80 (PS-80), polysorbate 20 (PS-20), sorbitol, sucrose, or trehalose.

[0179] In some embodiments, the equilibration buffer has a pH of 7.0–10.0, approximately pH 8.5–9.5, or approximately pH 9. In certain, non-limiting embodiments, the equilibration buffer comprises 25 mM Tris, 60 mM NaCl, and 0.001% poloxamer, and is approximately pH 9. In another certain, non-limiting embodiment, the equilibration buffer comprises 25 mM Tris, 20 mM Na2SO4, and 0.001% poloxamer, and is approximately pH 9.

[0180] 5. AEX column, column volume, flow rate Any suitable AEX column and resin known in the art (such as commercially available AEX columns and resins) may be used in embodiments of the disclosed method. The net charge of the protein to be separated (such as the AAV full capsid to be separated from other components of the AAV starting preparation) determines the choice of resin. Proteins are zwitterionic and can therefore have either a net positive or net negative charge. Given a buffer pH, the net charge of the protein (such as the net surface charge of the AAV full capsid) is determined, and theoretically, the protein can be purified using either cation exchange or anion exchange. However, proteins are not generally stable at all pH levels, and pH levels that would positively charge the protein may also denature it. Therefore, the stability of the protein and the choice of buffer can determine the choice of resin for ion exchange chromatography-based protein separation. In certain embodiments of the disclosed method, a positively charged anion exchange resin is selected to capture a negatively charged AAV full capsid.

[0181] The anion exchange resins used herein are insoluble substrates or solid supports (e.g., beads) capable of surface ionization over a pH range of about 1 to about 14. In one embodiment, the strong anion exchange resin is a solid support having a surface coated with a functionalized ligand such as a mixed amine, a quaternary amine, trimethylammonium ethyl (TMAE), dimethylaminopropyl, diethylaminoethyl (DEAE), dimethylaminoethyl (DMAE), polyethyleneimine (PI), or guanidium. In some embodiments, the anion exchange resin is a mixed amine ion exchange resin such as polyethyleneimine. In some embodiments, the anion exchange resin is a quaternary amine ion exchange resin such as quaternized polyethyleneimine. In further embodiments, the anion exchange resin comprises a support substrate containing trimethylamine and poly(glycidyl methacrylate-co-ethylenedimethacrylate). However, other suitable anion exchange resins may be selected. An example of a strong anion exchange resin is CIMultus QA (商標) Columns and POROS HQ (商標) These are for the columns. These resins and other suitable columns are commercially available from suppliers such as Amersham / Pharmacia (Piscataway, NJ), PerSeptive Biosystems (Foster City, Calif.), TosoHaas (Montgomeryville, Pa.), and others.

[0182] The anion exchange material may be in the form of an integrated column, a traditional bead-based column, a membrane, a filter, a fiber (such as a nanofiber), or another convective medium. In some embodiments, the ion exchange material is present in a column having a volume of 0 to 0.5 mL or 1 mL. In some embodiments, the ion exchange material is present in a column having a volume of 1 mL to 8 mL, such as 1, 2, 3, 4, 5, 6, 7, or 8 mL. In some embodiments, the ion exchange material is present in a column having a volume of 8 mL to 140 L, such as 8-100 mL, 8-250 mL, 8-500 mL, 8-750 mL, 8-1000 mL (1 L), 1-140 L, 1-120 L, 1-100 L, 1-80 L, 1-60 L, 1-40 L, 1-20 L, 1-10 L, or 1-5 L. In some embodiments, the column is at least an 8 mL column, at least a 10 mL column, at least a 20 mL column, at least a 30 mL column, at least a 40 mL column, at least a 50 mL column, at least a 100 mL column, at least a 200 mL column, at least a 300 mL column, at least a 400 mL column, at least a 500 mL column, at least a 600 mL column, at least a 700 mL column, at least a 800 mL column, at least a 900 mL column, at least a 1000 mL (1 L) column, at least a 2000 mL (2 L) column, at least a 10 L column, at least a 20 L column, at least a 30 L column, at least a 40 L column, at least a 50 L column, at least a 60 L column, at least a 70 L column, at least a 80 L column, at least a 90 L column, at least a 100 L column, at least a 140 L column, or a column with a capacity greater than 140 L, as well as any other column with a capacity between the volumes listed above. Alternatively, another type of container may be used to contain the anion exchange resin solid carrier.

[0183] Suitable liquid chromatography methods used herein include, but are not limited to, high-performance liquid chromatography (HPLC) and high-performance protein liquid chromatography (FPLC), which are known in the art. HPLC and FPLC are forms of liquid chromatography that can be used to analyze or purify mixtures of proteins. HPLC systems are known in the art and generally include a pump, injector, column, detector, and computer. As with other forms of chromatography, separation is possible by having different components of a mixture have different affinities to two materials: a fluid to which different components move ("mobile phase") and a porous solid (stationary phase). In the present invention, the mobile phase is an aqueous solution or a "buffer." The buffer flow rate may be controlled by gravity or a pump (e.g., a positive-displacement pump) and may be kept constant or varied. The composition of the buffer may be varied by drawing fluid in different proportions from two or more external reservoirs as appropriate. In certain embodiments of the disclosed methods, the stationary phase is an anion exchange resin (such as a strong anion exchange resin) typically composed of beads. These beads may be packed into a container, such as a cylindrical glass or plastic column, or another suitable container.

[0184] HPLC UV-Vis light detectors (such as tunable detectors and photodiode array (PDA) detectors) work by detecting the passage of visible and UV light through a sample (e.g., in a flow cell) and measuring the absorption of different wavelengths that have passed through the sample. The amount of light absorbed provides information about certain properties of the sample. AAV capsids that have some genomic sequences packaged within them (partially empty) or no genomic sequences (empty) have a 260 / 280 absorbance ratio of less than 1. Peaks where the area under the curve at 280 nm is larger than the corresponding area under the curve at 260 nm indicate a fraction more concentrated with empty AAV capsids. Similarly, peaks with an area under the curve at 260 nm larger than the corresponding area under the curve at 280 nm (where the A260 / 280 ratio is >1) generally indicate a fraction more concentrated with full AAV capsids. AAV flucapsid can be recovered from the fraction eluted when the A260 peak crosses and surpasses the A280 peak (i.e., reaches a bend).

[0185] Where provided herein, the volume of the mobile phase (e.g., a packing buffer containing the AAV starting preparations described herein, or an elution buffer, wash buffer, flash buffer, or equilibration buffer) is described as “column volume” (CV). These volumes may be extrapolated to other vessel shapes and designs. In some embodiments, the disclosed method includes injecting 1-20 CV of the flash buffer described herein into an AEX column, such as 1-15, 5-20, 5-15, 8-12, or 10 CV of flash buffer. In some embodiments, the disclosed method includes injecting 1-20 CV of the pre-equilibrium buffer described herein into an AEX column, such as 1-15, 5-20, 5-15, 8-12, or 10 CV of pre-equilibrium buffer. In some embodiments, the disclosed method includes injecting 1-20 CV of the equilibration buffer described herein into an AEX column, such as 1-15, 5-20, 5-15, 8-12, or 10 CV of equilibration buffer. In some embodiments, the disclosed method includes injecting a monovalent cation wash buffer described herein for 1-20CV, such as 1-15, 5-20, 5-15, 8-12, or 10CV, into an AEX column. In some embodiments, the disclosed method includes injecting a monovalent cation wash buffer described herein for 1-20CV, such as 1-15, 5-20, 5-15, 8-12, 7, 10, or 12CV, into an AEX column. In some embodiments, the disclosed method includes injecting a second flash buffer described herein for 1-20CV, such as 1-15, 5-20, 5-15, 8-12, or 10CV, into an AEX column.In some embodiments, the disclosed method includes injecting a 2-100 CV of divalent cation salt elution buffer described herein, such as 2-90, 2-80, 2-70, 2-60, 2-50, 2-40, 2-30, 2-20, 2-10, 2-9, 2-8, 2-7, 3-10, 3-9, 3-8, 3-7, 4-8, 5-7, 10-90, 20-100, 20-90, 20-80, 30-70, 40-80, 50-70, 55-65, or 60 CV of divalent cation salt elution buffer, into an AEX column. In some embodiments, the AAV starting preparation is packed into the column to a total of 5-60 CV, such as 5-50, 5-40, 5-30, 5-20, 5-10, 10-50, 20-60, 10-45, 15-45, 20-40, 25-35, 30, 33, 33.33, 8, 8.03, 16, 16.3, 22, or 22.8 CV, after injection of equilibration buffer and before injection of divalent cation elution buffer (and optionally before injection of monovalent cation wash buffer), etc.

[0186] Adjusting the packing rate and flow rate through the column (such as the mobile phase), including the flow rate of any buffer or AAV starting preparation described herein, can enhance the separation of AAV full capsids and empty capsids. In one embodiment, the sample packing flow rate is less than or equal to the elution flow rate. For example, the packing flow rate (such as the packing flow rate of the AAV starting preparation into the column) may be in the range of approximately 10 mL / min to approximately 60 mL / min, approximately 15 mL / min to approximately 50 mL / min, approximately 30 mL to approximately 50 mL, approximately 35 mL to approximately 45 mL, or approximately 20 mL / min to approximately 45 mL / min for an 8 mL integrated column, or approximately 10 mL / min, approximately 20 mL / min, approximately 30 mL / min, approximately 40 mL / min, or approximately 50 mL / min, or for a 1 mL integrated column, it may be in the range of approximately 3 mL / min to approximately 10 mL / min, approximately 3 mL / min to approximately 8 mL / min, approximately 3 mL / min to approximately 7 mL / min, or approximately 4 mL / min to approximately 6 mL / min, or approximately 3 mL / min, approximately 4 mL / min, approximately 5 mL / min, approximately 6 mL / min, or approximately 7 mL / min, or within the range of approximately 2 mL / min to approximately 15 mL / min. The appropriate flow rate may be extrapolated to the non-integrated column.

[0187] C.AAV Departure Preparations In some embodiments, obtaining an AAV starting preparation involves culturing AAV packaging cells in a medium capable of producing AAV. Thus, in some embodiments, the AAV starting preparation includes an AAV full capsid (such as AAV6 full capsid) and other components such as components of the medium and / or packaging cell lysates recovered from the packaging cell culture. The AAV starting preparation may include impurities present in the packaging cell medium and / or packaging cell lysates, such as various cellular components including packaging cell DNA, packaging cell proteins, and medium components, and in some examples, helper viruses or helper virus plasmid DNA.

[0188] Various suitable cells and cell lines have been described for use in the production of AAV. Packaging cells useful in this disclosure may be eukaryotic cells, fungal cells, insect cells, prokaryotic cells (e.g., bacterial or archaeal cells), or cells derived from multicellular organisms cultured as unicellular bodies (e.g., cell lines), and if such cells have been transformed with nucleic acids, they may include offspring of the original cells. In some embodiments, the cells are eukaryotic cells such as 293T cells (e.g., HEK293T cells). Exemplary mammalian cells include, but are not limited to, A549, WEHI, 3T3, 10T1 / 2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, HEK 293 (expressing functional adenovirus E1), Saos, C2C12, L cells, HT1080, HepG2 cells, and primary fibroblasts, hepatocytes, and myoblasts derived from mammals including humans, monkeys, mice, rats, rabbits, and hamsters. In certain embodiments, the cells are suspension-adapted cells. The selection of mammalian species providing the cells is not limiting to the invention, nor is the type of mammalian cell, i.e., fibroblasts, hepatocytes, tumor cells, etc., limiting to the invention.

[0189] In other embodiments, the cells are fungal cells, such as yeast cells of the genus Saccharomyces (e.g., Saccharomyces cerevisiae). In other embodiments, the cells are insect cells, such as Sf9 cells (for use, for example, in baculovirus-based AAV production systems). Recombinant packaging cells (also called genetically modified packaging cells) are packaging cells into which heterologous nucleic acids, such as expression vectors, have been introduced. For example, bacterial packaging cells are genetically modified bacterial packaging cells into which exogenous nucleic acids (e.g., plasmids or recombinant expression vectors) have been introduced into suitable bacterial packaging cells, and eukaryotic packaging cells are genetically modified eukaryotic packaging cells (e.g., mammalian cells) into which exogenous nucleic acids have been introduced into suitable eukaryotic packaging cells.

[0190] In certain embodiments, the disclosed method includes culturing AAV-packaging cells in a medium capable of producing AAV. Methods for culturing AAV-packaging cells to produce AAV are known in the art. In certain embodiments, the disclosed method includes culturing AAV-packaging cells in a medium capable of producing AAV. Methods for culturing AAV-packaging cells to produce AAV are known in the art, and exemplary methods are discussed herein. Suitable media known in the art may be used for AAV production, but are not limited to, custom formulations such as Modified Eagle Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), media described in U.S. Patent No. 6,566,118, and media manufactured by Hydrone Laboratories and JRH, including Sf-900 II SFM medium described in U.S. Patent No. 6,723,551.

[0191] The AAV production culture medium may contain serum or serum-derived recombinant protein, for example, at levels of 0.5%–20% (v / v or w / v). Alternatively, as is known in the art, AAV vectors may be produced under serum-free conditions, which may be called animal-derived product-free media. To increase the titer or yield of AAV in production culture, one or more cell culture components known in the art, including glucose, vitamins, amino acids, and / or growth factors, may be added, but are not limited to commercial or custom media designed to support AAV production.

[0192] AAV production cultures can be grown under a variety of conditions suitable for the specific host cells being used (such as a wide temperature range and length of time of change). As is known in this field, AAV production cultures include adhesion-dependent cultures that can be cultured in suitable adhesion-dependent containers such as roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluidized-bed bioreactors. AAV vector production cultures may also include suspension-adapted host cells such as HeLa, 293, and SF-9 cells that can be cultured in a variety of adhesion-dependent containers, including, for example, spinner flasks, agitated tank bioreactors, batch bioreactors, fed-batch bioreactors, continuous culture bioreactors (e.g., perfusion bioreactors), and disposable systems such as wave bag systems.

[0193] In some embodiments, AAV is collected from AAV packaging cells and / or from culture media to provide an AAV starting preparation. Collection may involve cell disruption (e.g., cell lysis) or may not substantially involve cell disruption (e.g., in embodiments where AAV is collected from cell culture media (e.g., supernatant)). Thus, the AAV of the present invention may be collected from an AAV production culture by lysis of packaging cells in the production culture or by collection of culture media derived from the production culture ("spent" medium) if the cells are cultured under conditions known in the art that release of AAV particles from intact cells into the culture medium occurs (as fully described by U.S. Patent No. 6,566,118, etc.). Suitable methods of cell lysis are also known in the art and include, for example, multiple freeze / thaw cycles, sonication, micro-solution, and treatment with chemicals such as surfactants and / or proteases.

[0194] AAV, by itself, lacks the ability to efficiently replicate its genetic material and thus typically requires the presence of a helper virus. Three helper viruses are commonly used for AAV production: adenovirus (Ad), herpes simplex virus (HSV), and baculovirus (Bac). Ad and HSV helper AAV production methods use mammalian cell lines, while the Bac system uses insect cells. Unlike the HSV and Bac systems, the Ad-based system genes that require helper function can be identified and expressed via plasmids, thereby eliminating the need for actual Ad to be produced. An exemplary "helper-free" system can use three plasmids: the first containing the Ad E2A, E4, and VA RNA helper genes; the second expressing the AAV rep and cap genes; and the third the nucleic acid of interest flanked by the AAV inverted terminal repeat (ITR) (such as those described elsewhere in this specification). Thus, an AAV production system useful in the disclosed method does not need to contain a helper.

[0195] In some embodiments of the disclosed method, the AAV full capsid (e.g., AAV6 full capsid) obtained from the AAV starting preparation comprises an AAV capsid protein and a nucleic acid molecule containing an AAV 5' inverted terminal repeat (ITR), a nucleic acid molecule of interest to be packaged in the AAV capsid, and a nucleic acid molecule containing a 3' ITR. Thus, in such embodiments, the packaging cell may include (i) a nucleic acid of interest to be packaged in at least one AAV capsid, (ii) a nucleic acid molecule encoding an AAV capsid protein under the control of one or more sequences directing its expression in the packaging cell, (iii) a nucleic acid molecule encoding an AAV Rep protein that expresses the AAV Rep protein in the cell to enable the packaging of the nucleic acid of interest into the AAV capsid, and / or (iv) one or more helper functions necessary for packaging the nucleic acid molecule of interest into the AAV capsid. In certain embodiments, the nucleic acid molecule of interest encodes a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0196] Preparation of packaging cells suitable for use in the disclosed method involves techniques such as the assembly of selected DNA sequences. This assembly may be achieved using prior art. Such techniques include cDNA and gene cloning, which are well known and include polymerase chain reactions, synthesis methods, and any other suitable methods for providing the desired nucleotide sequence, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY.

[0197] When DNA is introduced into a cell, the cell (such as an AAV packaging cell) is transformed by exogenous DNA, for example, a recombinant expression vector. The presence of exogenous DNA can result in persistent or transient genetic changes. The transformed DNA may or may not be incorporated (covalently) into the cell's genome. Cells in which transformed DNA is incorporated into the cell's genome are "stable transformed". In certain embodiments, AAV packaging cells are stable transformed with one or more (i) one or more nucleic acid molecules encoding one or more helper functions, (ii) a nucleic acid molecule encoding an AAV Rep protein, (iii) a nucleic acid molecule encoding an AAV capsid protein, and (iv) a nucleic acid molecule of interest. In some embodiments, AAV packaging cells are stable transformed with each of (i) one or more nucleic acid molecules encoding one or more helper functions, (ii) a nucleic acid molecule encoding an AAV Rep protein, (iii) a nucleic acid molecule encoding an AAV capsid protein, and (iv) a nucleic acid molecule of interest. In some embodiments, one or more helper functions, AAV Rep proteins, AAV capsid proteins, and / or the nucleic acid molecule of interest are expressed under an activatable or inducible promoter. In certain embodiments, the nucleic acid molecule of interest encodes a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0198] In any of the embodiments described herein, the nucleic acid of interest to be packaged in the AAV capsid may encode a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In certain embodiments, the CAR includes an intracellular signaling region comprising an antigen-binding domain and an intracellular signaling domain. In certain embodiments, the antigen-binding domain is an antibody or an antibody fragment, which is optionally a single-stranded fragment, or comprises them. In certain embodiments, the fragment comprises an scFv. The intracellular signaling domain may include a primary signaling domain, a signaling domain capable of inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain comprising an immunoreceptor tyrosine-based activation motif. In certain embodiments, the intracellular signaling domain is an intracellular signaling domain of a CD3 chain, optionally a CD3-zeta chain, or a signaling portion thereof, or comprises them. The CAR may include a transmembrane domain located between the extracellular domain and the intracellular signaling region, which may further include a co-stimulated signaling domain. The co-stimulation signaling domain may include the intracellular signaling domain or signaling moiety of a T cell co-stimulating molecule, such as the intracellular signaling domain or signaling moiety of CD28, 4-1BB, or ICOS. In certain embodiments, the co-stimulation signaling domain is located between the transmembrane domain and the intracellular signaling domain. In certain embodiments, the TCR includes an alpha chain containing a variable alpha (Vα) region and a beta chain containing a variable beta (Vβ) region, and the TCR can bind to or recognize peptide epitopes in the context of MHC molecules such as HLA-A2.

[0199] A useful AAV starting preparation in the disclosed method is either an unprocessed AAV production culture collection or may be processed before packing into an AEX column using one or more clarification steps (such as filtration and / or centrifugation), one or more nucleases and / or proteases (for digesting contaminating nucleic acids and / or proteins), an additional chromatography step (such as affinity chromatography), a concentration step, etc. At collection, the AAV starting preparation of this disclosure may contain one or more of the following: packaging cell material (such as packaging cell proteins and / or DNA); plasmid DNA; helper viruses; helper virus proteins; helper virus DNA; and culture medium components including, for example, serum proteins, amino acids, transferrin, and other low molecular weight proteins. In some embodiments, the AAV starting preparation is clarified and packaging cell debris is removed before separating the AAV from the other components of the AAV starting preparation. In some embodiments, the production culture recovery is clarified by filtration with a series of depth filters, including, for example, Grade DOHC Millipore Millistak+HC Pod filters, Grade A1HC Millipore Millistak+HC Pod filters, and / or 0.2 μm filter Opticap XL10 Millipore Express SHC Hydrophilic membrane filters. Clarification can also be achieved by various other standard techniques known in the art, such as centrifugation or filtration with any suitable filter known in the art (such as any suitable acetylcellulose filter with a pore size of 0.2 μm or larger). For example, other suitable depth filters in the range of about 0.045 μm to about 0.2 μm, or other filtration techniques may be used.

[0200] AAV starting preparations may be treated with a nuclease or a combination of nucleases to digest any contaminating high molecular weight nucleic acids present in the production culture. Suitable nucleases include, but are not limited to, DNAse, for example, benzonase, which is used under standard conditions known in this art.(登録商標) This includes digestion. For example, Benzonase with a final concentration of 1 unit / mL to 2.5 units / mL. (登録商標) However, it is used for a period of 30 minutes to several hours, or about 2 hours, at a temperature range of ambient temperature to 37°C. In another example, a turbonuclease is used. However, those skilled in the art may use another suitable nuclease or a mixture of nucleases. An exonuclease may also be used to remove contaminated nucleic acids. Such a nuclease may be selected to degrade single-stranded DNA and / or double-stranded DNA, and RNA. Such a step may involve a single nuclease or a mixture of nucleases for different targets, and may be an endonuclease or an exonuclease.

[0201] The AAV in the AAV starting preparation may be isolated or purified before being packed into the AEX resin using one or more of the following steps: tangential flow filtration (TFF) to concentrate AAV particles, thermal inactivation of helper viruses, AAV capture by hydrophobic interaction chromatography, affinity capture chromatography to remove production system contaminants, buffer exchange by size exclusion chromatography (SEC), and / or nanofiltration. These steps may be used individually, in various combinations, or in different orders. In some embodiments, the method includes all steps in the order described above.

[0202] In some embodiments, a nuclease (e.g., benzonase) is used. (登録商標)The processed mixture is concentrated by TFF. Large-scale enrichment of viruses using TFF ultrafiltration is described by R. Paul et al., Human Gene Therapy, 4:609-615 (1993). TTF enrichment of AAV starting preparations allows preparations of technically manageable volume to be subjected to the methods of the present disclosure and allows for more appropriate sizing of solid carriers (such as beads in a column). In some embodiments, the AAV starting preparation is enriched between at least 2x and at least 10x. In some embodiments, the AAV starting preparation is enriched between at least 10x and at least 20x, such as at least 10x, at least 11x, at least 12x, at least 13x, at least 14x, at least 15x, at least 16x, at least 17x, at least 18x, at least 19x, or at least 20x. In some embodiments, the AAV starting preparation is enriched between at least 20x and at least 50x. Those skilled in the art will also recognize that TFF can be used in any step in a disclosed method where it is desirable to replace the buffer before performing the next step in the method.

[0203] In one embodiment, the AAV in the AAV starting preparation is isolated from contaminants present in the production system (e.g., packaging cells, viral and other nucleic acid or protein materials present in or by-products of the production culture). In some embodiments, AAV starting preparations containing AAV full capsids and AAV empty capsids (and / or AAV partially empty capsids) contain less than 10% non-AAV viral and cellular protein and nucleic acid material, or less than 5% contaminants, or less than 1% viral and cellular protein and nucleic acid material. Thus, in some embodiments, the AAV starting preparations filled into AEX resin are about 95% to about 99% free of contaminants.

[0204] In some embodiments, affinity capture chromatography may be used to separate AAV from AAV starting preparations from production system contaminants. This affinity capture can be performed using an antibody-capture affinity resin. In one embodiment, the solid support is a cross-linked poly(styrene-divinylbenzene) with an average particle size of about 50 μm having an AAV-specific antibody. One example of such a commercially available affinity resin is POROS, commercially available from Thermo Fisher Scientific. (商標) This is a high-performance affinity resin. The resin contains a ligand produced by a proprietary technology based on a camel-derived single-domain antibody fragment that binds to the resin via carbonyldiimidazole (CDI). The ligand is a 13kDa single-domain fragment containing three CDRs that form an antigen-binding domain and are efficiently produced by the yeast Saccharomyces cerevisiae in an animal-component-free manufacturing process. Other suitable affinity resins containing AAV-specific antibodies, AAV6-specific antibodies, or other immunoglobulin constructs that are AAV-specific ligands may be selected or designed. Such solid carriers may be any suitable polymer substrate material, such as agarose, cepharose, cephadex, etc.

[0205] In some embodiments, the AAV starting preparation (such as an AAV starting preparation that has undergone one or more processing steps described herein) is diluted with a buffer or the like before being filled into the AEX resin. In certain embodiments, the AAV starting preparation (such as an AAV starting preparation that has undergone one or more processing steps described herein) is diluted with a buffer or the like between 2X and 50X, such as between 2X and 40X, between 2X and 30X, between 2X and 20X, between 2X and 10X, between 2X and 5X, between 3X and 7X, between 4X and 6X, or 5X, before being filled into the AEX resin. In certain embodiments, the buffer for diluting the AAV starting preparation includes Tris buffer or BTP buffer, such as 30-70 mM Tris or BTP, 35-65 mM Tris or BTP, 40-60 mM Tris or BTP, 45-55 mM Tris or BTP, or 50 mM Tris or BTP, at a pH of 9.0, between 8.5 and 9.5, or between 8 and 10. In other specific embodiments, the buffer for diluting the AAV starting preparation and then packing the column includes one or more of 20-100 mM Tris, 1-10 mM citrate, 10-100 mM NaCl, and 0.0001-0.01% poloxamer, at a pH of 9.0, between 8.5 and 9.5, or between 8 and 10. In certain, non-restrictive cases, the buffer used to dilute the AAV starting preparation and subsequently pack it into the column is pH 9.0 and contains 50 mM Tris, 4 mM citrate, 40 mM NaCl, and 0.001% poloxamer.

[0206] The appropriate filling volume is approximately 2 to 5 x 10 12 The GC / mL resin may be in the range of or less than that. Equivalent amounts may be calculated for columns or other containers of other sizes. Prior to the anion exchange resin separation described herein, “genome copy” refers to the fulcapsid in a mixture of both AAV fulcapsid and AAV6 intermediates.

[0207] AAV-packaged cell cultures may yield a mixture of AAV full capsids, AAV empty capsids, and / or AAV partially empty capsids. In some embodiments, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% of the AAV capsids recovered from the culture are empty and / or partially empty capsids. In other embodiments, more or less of the capsids are empty and / or partially empty capsids. In some embodiments, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% of the AAV capsids recovered from the culture are full capsids. In other embodiments, more or less of the capsids are full capsids.

[0208] In certain embodiments, the AAV starting preparation comprises at least 1%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 20%, at least 25%, or at least 30% AAV flucapsid (such as AAV6 flucapsid). In certain embodiments, the AAV starting preparation includes 1-40% AAV flucapsid, such as 1-30%, 1-20%, 1-19%, 1-18%, 1-17%, 1-16%, 1-15%, 1-14%, 1-13%, 1-12%, 2-40%, 3-40%, 4-40%, 5-40%, 5-30%, 5-20%, 5-15%, 10-40%, 10-30%, 10-20%, or 10-15% AAV flucapsid (e.g., AAV6 flucapsid).

[0209] III. Additional features of a particular method of disclosure A. Adeno-associated virus (AAV) In embodiments of this disclosure, the AAV full capsid is isolated (purified) from production culture contaminants such as AAV intermediates (AAV empty capsid and / or AAV partially empty capsid), helper viruses, helper virus proteins, plasmids, cellular proteins and nucleic acids, culture medium components, serum proteins, AAV rep proteins, and unconstructed AAV VP1, VP2, and VP3 proteins, which are also contained in the AAV starting preparation, which also includes AAV. In some embodiments, the AAV is recombinant or engineered AAV. In some embodiments, the AAV is pseudotyped AAV. The use of recombinant or engineered AAV allows for the insertion, deletion, or substitution of a target DNA sequence within the genome of a mammalian cell. The AAV contains a protein capsid that surrounds and protects a single-stranded DNA genome of approximately 4.8 kilobases (kb). Naso et al., BioDrugs. 2017; 31(4): 317-334. AAV belongs to the Parvoviridae family and relies on co-infection with other viruses, primarily adenoviruses, for replication. Its single-stranded genome contains three genes: Rep (replication), Cap (capsid), and aap (assembly). These three genes, through the use of three promoters, alternative translation start sites, and different splicing, produce at least nine gene products. These coding sequences are flanked by inverted terminal repeats (ITRs) necessary for genome replication and packaging. The Rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40) necessary for viral genome replication and packaging, while Cap expression results in viral capsid proteins (VP; VP1 / VP2 / VP3) that protect the viral genome and form the capsid shell involved in cell binding and internal migration. The viral capsid is composed of approximately 60 proteins arranged in an icosahedral structure with a molar ratio of 1:1:10 (VP1:VP2:VP3) of capsid proteins. The aap gene encodes the assembly activation protein (AAP) in an alternative reading frame that overlaps with the cap gene.This nucleoprotein is thought to provide a scaffold for capsid assembly, but it may not be essential in certain AAV serotypes.

[0210] Recombinant AAVs lacking viral DNA (rAAVs) are protein-based nanoparticles engineered to traverse the cell membrane, ultimately capable of transporting and delivering DNA cargo (contained within the viral capsid) to the cell nucleus. Naso et al., BioDrugs. 2017; 31(4): 317-334. In the absence of the Rep protein, the ITR-adjacent transgene encoded within the rAAV can form a circular concatemer that remains within the nucleus of the transduced cell as an episome. Since the recombinant episomal DNA is not integrated into the host genome, it will eventually be diluted over time as the cell replicates. This ultimately leads to the loss of transgene and transgene expression, with the rate of transgene loss depending on the turnover rate of the transduced cell. These properties make rAAVs attractive for certain gene therapy applications.

[0211] In some embodiments, the AAV purified using the disclosed method is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh10, AAVrh74, AAV9, AAV9P, AAV10, AAV11, AAV12, or Myo-AAV, or a novel chimera thereof. In certain embodiments, the AAV is AAV6. AAV6 has been shown to have enhanced capsid-associated tropism in tissues such as lung, cardiomyocyte, and skeletal muscle (Halbert et al., J Virol. 2001; 75(14):6615-24; Rengo et al., Circulation. 2009; 119(1):89-98; Bortolanza et al. Mol Ther. 2011; 19(11):2055-64). Like other AAVs, AAV6 can transduce non-dividing cells (Halbert et al., J Virol. 2001; 75(14):6615-24). In some embodiments, AAV6 has an ITR-to-ITR size of less than 5kb, including both ITRs. In certain embodiments, AAV6 has an ITR-to-ITR size of less than 4.9kb, including both ITRs. In further embodiments, AAV6 has an ITR-to-ITR size of less than 4.85kb, including both ITRs. In further embodiments, AAV6 has an ITR-to-ITR size of less than 4.8kb, including both ITRs. In further embodiments, AAV6 has an ITR-to-ITR size of less than 4.75kb, including both ITRs. In further embodiments, AAV6 has an ITR-to-ITR size of less than 4.7kb, including both ITRs.In some embodiments, AAV6, including both ITRs, has an ITR-to-ITR size of 3.9-5kb, 4-5kb, 4.2-5kb, 4.4-5kb, 4.6-5kb, 4.7-5kb, 3.9-4.9kb, 4.2-4.9kb, 4.4-4.9kb, 4.7-4.9kb, 3.9-4.85kb, 4.2-4.85kb, 4.4-4.85kb, 4.6-4.85kb, 4.7-4.85kb, 4.7-4.9kb, 3.9-4.8kb, 4.2-4.8kb, 4.4-4.8kb, or 4.6-4.8kb. In some embodiments, the vector, including both ITRs, has an ITR-to-ITR size of 4.4-4.85kb.

[0212] In some embodiments, AAV6 has a capsid containing an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of the AAV6 capsid encoded by the AAV6 genome of SEQ ID NO: 1. In some embodiments, the amino acid sequence of the AAV6 capsid protein is that of SEQ ID NO: 1. Furthermore, the methods provided herein may be used to purify other AAVs having capsids highly related to the AAV6 capsid. If the integrity of the peptide binding sites is maintained, for example, AAVs having sequences that are about 95%, about 96%, about 97%, about 98%, or about 99% identical to the reference amino acid sequence of SEQ ID NO: 1 may be obtained using the methods herein (e.g., by isolation). Methods for generating capsids, the coding sequences of capsids, and methods for producing rAAV viral vectors have been described. See, for example, Gao, et al., Proc. Natl. Acad. Sci. USA 100 (10), 6081-6086 (2003), U.S. Patents No. 6,759,237, No. 7,105,345 and No. 7,186,552 and U.S. Patent Application Publication No. 2013 / 0045186A1. In some embodiments, the AAV capsid is a chimeric capsid, an engineered capsid, or a natural capsid.

[0213] In some embodiments, the AAV comprises a nucleic acid molecule of interest (i.e., encapsulated within a fully constructed AAV capsid), such as a nucleic acid molecule encoding a therapeutic molecule, such as a therapeutic protein or RNA molecule. The nucleic acid molecule of interest can be operably ligated to one or more regulatory elements, such as a promoter, such as a tissue-specific promoter. The term “operably ligated” means that the nucleic acid molecule of interest is ligated to the regulatory elements in a manner that enables the expression of a nucleotide sequence. An AAV obtained using the disclosed method may include any AAV that includes a tissue-specific promoter to facilitate the administration of a gene therapy, which may include any gene editing system known in the art. Promoters described herein may also be “cell-specific,” meaning that a particular promoter selected for an AAV can direct the expression of a selected transgene / nucleotide sequence of interest in a particular cell or cell type.

[0214] Exemplary promoters useful in this disclosure may be constitutively active promoters (i.e., promoters that are constitutively active / "ON"), inducible promoters (i.e., promoters whose state, active / "ON" or inactive / "OFF") is controlled by an external stimulus, such as the presence of a specific temperature, compound, or protein), spatially restricted promoters (i.e., transcriptional regulatory elements, enhancers, etc., such as tissue-specific promoters, cell-type-specific promoters, etc.), or temporally restricted promoters (i.e., promoters that are "ON" or "OFF" at a specific stage of embryonic development or a specific stage of a biological process).

[0215] A suitable promoter may be derived from a virus (viral promoter) or from any organism, including prokaryotes or eukaryotes. A suitable promoter may be used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include, but are not limited to, the SV40 early promoter, the mouse mammary cancer virus long-term repeat (LTR) promoter; the adenovirus major late promoter (Ad MLP); the herpes simplex virus (HSV) promoter; cytomegalovirus (CMV) promoters such as the CMV immediate early promoter region (CMVIE); the Roussarcoma virus (RSV) promoter; the human U6 small nucleus promoter (U6) (Miyagishi et al., Nature Biotechnology. 20: 497-500; 2002); the enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. Sep 1(31): 17; 2003); and the human H1 promoter (H1).

[0216] Nucleic acids of interest as useful herein may also include other regulatory elements, namely transcriptional and translational regulatory sequences such as enhancers, polyadenylation signals, terminators, and proteolytic signals, which provide and / or regulate the transcription of non-coding sequences (e.g., guide RNA) or coding sequences (e.g., site-directed modified polypeptides or Cas9 polypeptides) and / or regulate the translation of encoded polypeptides. Exemplary regulatory sequences are known in the art and are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology. Vol. 185, Academic Press, San Diego, CA (1990).

[0217] All patents, patent applications, websites, other publications or documents, accession numbers, etc., referenced herein are incorporated by reference in whole for all purposes to the same extent as each individual item is indicated to be incorporated by reference specifically and individually. Where different versions of an array of documents are associated with an accession number at different points in time, it means the version associated with the accession number at the effective filing date of this application. The effective filing date means the earlier of the actual filing date or, where available, the filing date of the priority application referencing the accession number. Similarly, where different versions of a publication, website, etc., are published at different points in time, unless otherwise specified, it means the most recently published version at the effective filing date of this application. [Examples]

[0218] The following examples demonstrate remarkable progress in obtaining full AAV capsids by chromatography. Industrially standard monovalent cation salt washing under linear gradient conditions elutes the full capsid followed by the empty capsid. This was observed across different monovalent cations. Surprisingly, as shown in Examples 1-5 herein, divalent cation salts under gradient conditions were found to elute the empty capsid followed by the full capsid. This was observed across different divalent cations. While not bound by any particular theory, these results exhibit an ion-valence-dependent elution pattern. Examples 1-5 combine (1) isocratic monovalent cation salt washing, which utilizes the order in which the full capsid is eluted followed by the empty capsid, with (2) gradient divalent cation salt elution, which utilizes the order in which the empty capsid is eluted followed by the full capsid (only the full capsid is eluted at this stage, since the empty capsid was eluted during washing). Examples 6-8 combined (1) isocratic monovalent cation salt washing, which utilizes the order in which empty capsids are eluted followed by full capsids, and (2) isocratic divalent cation salt elution, which utilizes the order in which full capsids are eluted followed by empty capsids, in a completely isocratic process (since empty capsids were eluted during washing, only full capsids are eluted at this stage). These experiments demonstrated the use of remarkable ion valency dependence across a range of process operating modes. These approaches addressed the challenging issue of inherently low chromatographic separation between empty capsids and full capsids.

[0219] Example 1: Separation of AAV6 preparations using industrially standard methods This example describes the separation of AAV6 preparations using an industrially standard method. The workflow described in this example is shown in Table 1, and the results are shown in Figure 1.

[0220] The AAV6 preparation process involved lysis of HEK293-packaged cell cultures and subsequent clarification of the lysates using depth filtration. Additional AAV6 enrichment and purification methods included tangential flow filtration and affinity chromatography prior to the AEX chromatography method described. Separation was performed using CIMmultus. (登録商標) The procedure was performed on an AKTA Avant150 HPLC system (Cytiva, Massachusetts, USA) using a QA integrated anion exchange chromatography column. First, 10 CV of 15 mM HCl, 60 mM NaCl, and 25 mM bistrispropane (BTP) buffer (pH 9) containing 0.001% poloxamer was run through the column. The column was pre-equilibrated with 10 CV of 2000 mM NaCl, followed by equilibration with 10 CV of 15 mM HCl, 60 mM NaCl, and 25 mM bistrispropane (BTP) buffer (pH 9) containing 0.001% poloxamer. The AAV6 preparation was diluted 5X with 50 mM BTP (pH 9.0), then filtered through a 0.2 μm filter to obtain 3.33 CV (approximately 1.5 x 10⁻¹⁶). 12 Viral genome / mL and 1x10 13 The column was packed with capsid ( / mL). The column was again equilibrated with 10 CV of 15 mM HCl, 60 mM NaCl, and 25 mM BTP buffer (pH 9) containing 0.001% poloxamer (Buffer A). Buffer B contained 15 mM HCl, 382.5 mM NaCl, and 25 mM BTP buffer (pH 9) containing 0.001% poloxamer. The AAV full capsid was eluted using a 40 CV linear salt gradient with 0-100% Buffer B. The flow rate was maintained at 5 mL / min throughout the run. Table 1: AAV6 preparation separation using industrial standard conditions [Table 1]

[0221] As shown in Figure 1, the empty capsid peak appeared before the full capsid peak in the chromatogram, indicating that at least some of the empty capsid eluted before at least some of the full capsid. Smaller peaks appearing after the full capsid peak may contain AAV full capsid, AAV intermediates, or any combination thereof. This method yielded a recovery rate of approximately 59% of the AAV full capsid present in the AAV starting preparation packed into the column (Table 2). The values ​​in Table 2 were calculated based on capsid particle counts obtained by dynamic and static light scattering followed by UV absorbance measurements at wavelengths of 260 nm and 280 nm. These measurements showed that the AAV9 full capsid peak contained 20% AAV9 full capsid and 80% AAV9 empty / partially empty capsid, and further, that the AAV9 full capsid peak contained 59% of the total full capsid packed into the column in the AAV starting preparation. Notably, the additional peak following the flucapsid peak could not be completely separated (degraded), resulting in the simultaneous elution of AAV flucapsid and a certain amount of AAV intermediate. Table 2. AAV6 capsid recovery in Example 1. The 4% full vector in the empty vector peak is likely due to substandard performance of the industrial standard AEX method. [Table 2]

[0222] Example 2: Separation of AAV6 preparations using MgCl2 gradient This example describes the separation of AAV6 preparations using a MgCl2 gradient. The workflow described in this example is shown in Table 3, and the results are shown in Figure 2.

[0223] The AAV6 preparation process involved lysis of HEK293-packaged cell cultures and subsequent clarification of the lysates using depth filtration. Additional AAV6 enrichment and purification methods included tangential flow filtration and affinity chromatography prior to the AEX chromatography method described. Separation was performed using an AKTA Avant150 HPLC system (Cytiva, Massachusetts, USA). Separation was performed using CIMmultus. (登録商標) The procedure was performed on an AKTA Avant150 HPLC system (Cytiva, Massachusetts, USA) using a QA integrated anion exchange chromatography column. First, 10 CV of 60 mM NaCl and 25 mM Tris buffer (pH 9) containing 0.001% poloxamer were run through the column. The column was pre-equilibrated with 10 CV of 2000 mM NaCl, followed by 10 CV of 60 mM NaCl and 25 mM Tris buffer (pH 9) containing 0.001% poloxamer. The AAV6 preparation was diluted 5X with 50 mM Tris (pH 9.0), then filtered through a 0.2 μm filter to obtain 33.33 CV (approximately 1.5 x 10⁻¹⁶). 12 Viral genome / mL and 1x10 13 The column was packed with capsid ( / mL). The column was again equilibrated with 10 CV of 60 mM NaCl and 25 mM Tris buffer (pH 9) containing 0.001% poloxamer, followed by 2 CV of 25 mM Tris buffer (pH 9) containing 0.001% poloxamer (Buffer A). Buffer B contained 100 mM MgCl2 and 25 mM Tris buffer (pH 9) containing 0.001% poloxamer. The AAV full capsid was eluted using a 60 CV linear salt gradient with 0-100% Buffer B. The flow rate was maintained at 5 mL / min throughout the run. Table 3. AAV6 preparations using MgCl2 gradient [Table 3]

[0224] As shown in Figure 2, the chromatogram shows that the full capsid peak (containing approximately 37% AAV full capsid) appears before the empty + full capsid peak, indicating that at least a portion of the full capsid eluted before the peak containing at least a portion of the AAV full capsid and empty and / or partially empty capsids. This method yielded a recovery rate of approximately 87% of the AAV full capsid present in the AAV starting preparation packed into the column (Table 4). The values ​​in Table 4 were calculated based on capsid particle counts obtained by dynamic and static light scattering followed by UV absorbance measurements at wavelengths of 260 nm and 280 nm. These measurements showed that the AAV9 full capsid peak contained 27% AAV9 full capsid and 73% AAV9 empty / partially empty capsids, and further, that the AAV9 full capsid peak contained 87% of the total full capsid packed into the column in the AAV starting preparation. Notably, the full capsid peak and subsequent peaks were more separated (degraded) than the peaks in Example 1, resulting in the relatively reduced simultaneous elution of the AAV intermediate and AAV full capsid. Furthermore, the elution order of the AAV6 full capsid and AAV6 empty capsid peaks was reversed compared to Example 1. Thus, AAV empty capsids that were not washed away in the first wash (and therefore remained bound to the column resin) remained bound to the column resin and were not eluted in the elution step. Table 4. AAV6 capsid recovery in Example 2 [Table 4]

[0225] Example 3: Separation of AAV6 preparations using a CaCl2 gradient This example describes the separation of AAV6 preparations using a CaCl2 gradient. The workflow described in this example is shown in Table 5, and the results are shown in Figure 3.

[0226] The AAV6 preparation process involved lysis of HEK293-packaged cell cultures and subsequent clarification of the lysates using depth filtration. Additional AAV6 enrichment and purification methods included tangential flow filtration and affinity chromatography prior to the AEX chromatography method described. Separation was performed using CIMmultus. (登録商標) The procedure was performed on an AKTA Avant150 HPLC system using a QA integrated anion exchange chromatography column. First, 10 CV of 60 mM NaCl and 25 mM Tris buffer (pH 9) containing 0.001% poloxamer were passed through the column. The column was pre-equilibrated with 10 CV of 2000 mM NaCl, followed by 10 CV of 60 mM NaCl and 25 mM Tris buffer (pH 9) containing 0.001% poloxamer. The AAV6 preparation was diluted 5X with 50 mM Tris (pH 9.0), then filtered through a 0.2 μm filter to obtain 6.67 CV (approximately 1.5 x 10⁻¹⁶). 12 Viral genome / mL and 1x10 13 The column was packed with capsid ( / mL). The column was again equilibrated with 10 CV of 60 mM NaCl and 25 mM Tris buffer (pH 9) containing 0.001% poloxamer, followed by 2 CV of 25 mM Tris buffer (pH 9) containing 0.001% poloxamer (Buffer A). Buffer B contained 100 mM CaCl2 and 25 mM Tris buffer (pH 9) containing 0.001% poloxamer. The AAV full capsid was eluted using a 60 CV linear salt gradient with 0-100% Buffer B. The flow rate was maintained at 5 mL / min throughout the run. Table 5. Separation of AAV6 preparations using CaCl2 gradient. [Table 5]

[0227] As shown in Figure 3, the full capsid peak (containing approximately 30-35% AAV full capsid) appeared before the empty capsid peak in the chromatogram, indicating that at least a portion of the full capsid eluted before at least a portion of the peaks containing AAV full capsid and empty and / or partially empty capsids. This method yielded an estimated recovery rate of approximately 60-70% of the AAV full capsid present in the AAV starting preparation packed into the column (Table 6). The values ​​in Table 6 were calculated based on the area of ​​the full peak (260 nm UV absorbance) and the A260 nm to A280 nm ratio. These measurements showed that the AAV9 full-capsid peak contained 30-35% AAV9 full-capsid and 65-70% AAV9 empty / partially empty capsid, and furthermore, the AAV9 full-capsid peak contained 60-70% of the total full-capsid packed into the column in the AAV starting preparation. The full-capsid and empty-capsid peaks were more separated (degraded) than the full-capsid and empty-capsid peaks in Example 1, resulting in the relatively reduced simultaneous elution of AAV intermediates and AAV full-capsid. Notably, the elution order of the AAV6 full-capsid and AAV6 empty-capsid peaks was switched compared to Example 1. Thus, AAV empty capsids that were not washed away in the first wash (and thus remained bound to the column resin) remained bound to the column resin and were not eluted in the elution step. Table 6. AAV capsid recovery in Example 3. [Table 6]

[0228] Example 4: Separation of AAV6 preparation using NaCl washing step and MgCl2 gradient This example describes the separation of the AAV6 preparation using a NaCl washing step and a MgCl2 gradient. The workflow described in this example is shown in Table 7, and the results are shown in Figure 4.

[0229] The AAV6 preparation process involved lysis of HEK293-packaged cell cultures and subsequent clarification of the lysates using depth filtration. Additional AAV6 enrichment and purification methods included tangential flow filtration and affinity chromatography prior to the AEX chromatography method described. Separation was performed using CIMmultus. (登録商標) The procedure was performed on an AKTA Avant150 HPLC system (Cytiva, Massachusetts, USA) using a QA integrated anion exchange chromatography column. First, 10 CV of 60 mM NaCl and 25 mM Tris buffer (pH 9) containing 0.001% poloxamer were run through the column. The column was pre-equilibrated with 10 CV of 2000 mM NaCl, followed by 10 CV of 60 mM NaCl and 25 mM Tris buffer (pH 9) containing 0.001% poloxamer. The AAV6 preparation was diluted 5X with 50 mM Tris (pH 9.0), then filtered through a 0.2 μm filter to obtain 33.33 CV (approximately 1.5 x 10⁻¹⁶). 12 Viral genome / mL and 1x10 13 The column was packed with capsid ( / mL). The column was again equilibrated with 10 CV of 60 mM NaCl and 25 mM Tris buffer (pH 9) containing 0.001% poloxamer. Subsequently, the column was washed with 7 CV of 150 mM NaCl washing solution (50% buffer A1 and 50% buffer B1, where buffer A1 contained 25 mM Tris buffer (pH 9) containing 0.001% poloxamer, and buffer B1 contained 300 mM NaCl and 25 mM Tris buffer (pH 9) containing 0.001% poloxamer). The column was then run with 2 CV of 25 mM Tris buffer (pH 9) (buffer A2) containing 0.001% poloxamer. Buffer B2 contained 100 mM MgCl2 and 25 mM Tris buffer (pH 9) containing 0.001% poloxamer. AAV full capsids were eluted using a 60 CV linear salt gradient with 0-100% buffer B2. The flow rate was maintained at 5 mL / min throughout the run. Table 7. Separation of AAV6 preparations using NaCl washing step and MgCl2 gradient. [Table 7]

[0230] As shown in Figure 4, the majority of the AAV empty capsids eluted from the column with a 50% buffer B1 wash. During the 0-100% buffer B2 linear salt gradient, the first full-capsid peak (containing approximately 60% AAV full-capsids) appeared before the second full-capsid peak, indicating that at least a portion of the full-capsids eluted before at least a portion of the second peak containing full-capsids. This method yielded a recovery rate of approximately 46% (from the first full-capsid peak) of the AAV full-capsids present in the AAV starting preparation packed into the column (Table 8). The values ​​in Table 8 were calculated based on capsid particle counts obtained by dynamic and static light scattering followed by UV absorbance measurements at wavelengths of 260 nm and 280 nm. These measurements showed that the AAV9 full-capsid peak contained 46% AAV9 full-capsid and 54% AAV9 empty / partially empty-capsid, and furthermore, the AAV9 full-capsid peak contained 46% of the total full-capsid packed into the column in the AAV starting preparation. Notably, since the majority of the empty-capsid was washed from the column before the elution gradient, at least partially, the full-capsid and empty-capsid peaks were more separated (degraded) than the full-capsid and empty-capsid peaks in Example 1, resulting in the simultaneous elution of a relatively reduced amount of AAV intermediate and AAV full-capsid. In addition, the elution order of the AAV6 full-capsid and AAV6 empty-capsid peaks was switched compared to Example 1. Thus, AAV empty-capsid that was not washed away in the first wash (and thus remained bound to the column resin) remained bound to the column resin and was not eluted in the elution step. Table 8. AAV6 capsid recovery in Example 4 [Table 8]

[0231] Example 5: Separation of AAV6 preparation using Na2SO4 washing step and MgSO4 gradient This example describes the separation of the AAV6 preparation using a Na2SO4 washing step and a MgSO4 gradient. The workflow described in this example is shown in Table 9, and the results are shown in Figure 5.

[0232] The AAV6 preparation process involved lysis of the packaging cell culture and subsequent clarification of the lysate using depth filtration. Additional AAV6 enrichment and purification methods included tangential flow filtration and affinity chromatography prior to the AEX chromatography method described. Separation was performed using CIMmultus. (登録商標) The process was performed on an AKTA Avant150 HPLC system (Cytiva, Massachusetts, USA) using a QA integrated anion exchange chromatography column. First, 10 CV of 20 mM Na₂SO₄ and 25 mM Tris buffer (pH 9) containing 0.001% poloxamer were run through the column. The column was pre-equilibrated with 10 CV of 2000 mM NaCl, followed by equilibration with 10 CV of 20 mM Na₂SO₄ and 25 mM Tris buffer (pH 9) containing 0.001% poloxamer. The AAV6 preparation was diluted 5X with 50 mM Tris (pH 9.0), then filtered through a 0.2 μm filter to obtain 33.33 CV (approximately 1.5 x 10⁻¹⁶). 12 Viral genome / mL and 1x10 13The column was packed with capsid ( / mL) and chased with 10 CV of 20 mM Na2SO4 and 25 mM Tris buffer (pH 9) containing 0.001% poloxamer. The column was then washed with 12 CV of 53.2% buffer B1, where buffer A1 contained 25 mM Tris buffer (pH 9) containing 0.001% poloxamer, and buffer B1 contained 100 mM Na2SO4 and 25 mM Tris buffer (pH 9) containing 0.001% poloxamer. The column was then flushed with 10 CV of 25 mM Tris buffer (pH 9) (buffer A2) containing 0.001% poloxamer. Buffer B2 contained 100 mM MgSO4 and 25 mM Tris buffer (pH 9) containing 0.001% poloxamer. AAV full capsids were eluted using a 60 CV linear salt gradient with 0-100% buffer B2. The flow rate was maintained at 5 mL / min throughout the run. Table 9. Separation of AAV6 preparations using Na2SO4 washing step and MgSO4 gradient. [Table 9]

[0233] As shown in Figure 5, the majority of the AAV empty capsids eluted from the column with a 53.2% buffer B1 wash. During the 0-100% buffer B2 linear salt gradient, the first full-capsid peak (containing approximately 54% AAV full vector) appeared before the second full-capsid peak, indicating that at least a portion of the full-capsids eluted before at least a portion of the second peak containing the full-capsids. This method yielded a recovery rate of approximately 73% (from the first full-capsid peak) of the AAV full-capsids present in the AAV starting preparation packed into the column (Table 10). The values ​​in Table 10 were calculated based on capsid particle counts obtained by dynamic and static light scattering followed by UV absorbance measurements at wavelengths of 260 nm and 280 nm. These measurements showed that the AAV9 full-capsid peak contained 45% AAV9 full-capsid and 55% AAV9 empty / partially empty-capsid, and furthermore, the AAV9 full-capsid peak contained 73% of the total full-capsid packed into the column in the AAV starting preparation. Notably, since the majority of the empty-capsid was washed from the column before the elution gradient, at least partially, the full-capsid and empty-capsid peaks were more separated (degraded) than the full-capsid and empty-capsid peaks in Example 1, resulting in the simultaneous elution of a relatively reduced amount of AAV intermediate and AAV full-capsid. In addition, the elution order of the AAV6 full-capsid and AAV6 empty-capsid peaks was switched compared to Example 1. Thus, AAV empty-capsid that was not washed away in the first wash (and thus remained bound to the column resin) remained bound to the column resin and were not eluted in the elution step. Table 10. AAV6 capsid recovery in Example 5 [Table 10]

[0234] Example 6: Separation of AAV6 preparations using Na2SO4 washing step and MgSO4 isocratic elution This example describes the separation of AAV6 preparations using a Na2SO4 washing step and a MgSO4 elution buffer at a constant concentration. The workflow described in this example is shown in Table 11, and the results are shown in Figure 6. The AAV6 in this example contained transgene "A".

[0235] The AAV6 preparation process involved lysis of the packaging cell culture and subsequent clarification of the lysate using depth filtration. Additional AAV6 enrichment and purification methods included tangential flow filtration and affinity chromatography prior to the AEX chromatography method described. Separation was performed using CIMmultus. (登録商標) The analysis was performed on an AKTA Pilot 600 HPLC system (Cytiva, Massachusetts, USA) using a QA integrated anion exchange chromatography column (40 mL). First, 5 CV of 25 mM Tris buffer containing 20 mM Na2SO4 and 0.001% poloxamer (pH 9.0) was run. The column was pre-equilibrated with 5 CV of 2000 mM NaCl, followed by 5 CV of 25 mM Tris, 20 mM Na2SO4, and 0.001% poloxamer-containing buffer (pH 9.0). The AAV6 preparation was diluted 5X with 50 mM Tris (pH 9.0), then filtered through a 0.2 μm filter, and 8.03 CV was packed into the column. The column was then chased with 5 CV of 25 mM Tris, 20 mM Na2SO4, and 0.001% poloxamer-containing buffer (pH 9.0). Next, the column was washed with 5 CV of 25 mM Tris, 53 mM Na2SO4, and 0.001% poloxamer-containing buffer (pH 9.0). The column was then run with 5 CV of 25 mM Tris, 20 mM Na2SO4, and 0.001% poloxamer-containing buffer (pH 9.0). The AAV full capsid was eluted with 7 CV using a constant concentration of 25 mM Tris, 38 mM MgSO4, and 0.001% poloxamer-containing elution buffer (pH 9.0). Table 11. Separation of AAV6 preparations using Na2SO4 washing and MgSO4 isocratic elution. [Table 11]

[0236] As shown in Figure 6, isocratic washing with a monovalent cation salt followed by isocratic elution with a divalent cation salt produced an empty capsid peak that appeared before the full capsid peak in the chromatogram. First, the empty capsid was washed away by isocratic monovalent washing, and then the full capsid was eluted by divalent isocratic elution. In isocratic divalent elution, the full capsid was eluted without the additional empty capsid seen in the later peaks in gradient elution as shown in Figure 4, etc., demonstrating the superiority of isocratic divalent elution over gradient divalent elution. Monovalent isocratic washing followed by divalent isocratic elution enabled the removal of the initial population of empty capsids and subsequent elution of full capsid, avoiding co-purification of full capsid with the empty capsids remaining after the first wash.

[0237] The percentage of full capsid was measured before and after purification (i.e., the percentage of full capsid in the AAV starting preparation and the percentage of full capsid in the eluted AAV peak containing full AAV capsid), and is shown in Table 12. This method yielded an estimated recovery rate of approximately 80-91% of the AAV full capsid present in the AAV starting preparation packed into the column (Table 12). The values ​​in the second column of Table 12 were calculated based on capsid particle counts obtained using a Stunner instrument (Unchained Labs) by dynamic and static light scattering followed by UV absorbance measurements at wavelengths of 260 nm and 280 nm. These methods showed that the AAV full capsid peak contained 50% full capsid compared to the starting AAV preparation containing only 21% full capsid.

[0238] In parallel, as shown in columns 2 and 3 of Table 12, the concentration of the AAV vector genome in the eluted AAV full capsid fraction was measured using qPCR and ELISA. Samples were diluted, digested with DNase I (or another suitable nuclease) to remove exogenous DNA, and further treated with proteinase K (or another suitable protease). After inactivation of the nuclease, the samples were diluted and amplified using primers and a probe specific for the DNA sequence between the primers (e.g., TagMan (商標) fluorescent generating probe). The number of cycles (threshold cycle, Ct) required to reach a defined level of fluorescence was measured using a suitable detection system. Plasmid DNA containing the same sequence as that contained in the AAV vector was utilized to generate a standard curve in the qPCR reaction. The vector genome titer was determined by normalizing the Ct value obtained from the sample to the Ct value of the plasmid standard curve. AAV capsids containing transgene A were detected by combining qPCR with ELISA. By these methods, the AAV full capsid peak contained 100% full capsids, compared to the starting AAV preparation that contained only 40% full capsids. Table 12. AAV6 Capsid Recovery in Example 6 [Table 12]

[0239] Example 7: Separation of an AAV6 Preparation Using a Na2SO4 Washing Step and MgSO4 Isocratic Elution on a 40 mL Anion Exchange Chromatography Column This example describes the separation of an AAV6 preparation using a Na2SO4 washing step and an MgSO4 elution buffer at a constant concentration. The workflow described in this example is shown in Table 13, and the results are shown in Figure 7. The AAV6 in this example contained the transgene "B".

[0240] The AAV6 preparation process involved lysis of the packaging cell culture and clarification of the lysate using subsequent depth filtration. Additional AAV6 concentration and purification methods included tangential flow filtration and affinity chromatography prior to the described AEX chromatography method. Separation was performed on an AKTA Pilot 600 HPLC system (Cytiva, Massachusetts, USA) using a CIMmultus (登録商標) QA integrated anion exchange chromatography column (40 mL). First, it was run with a buffer containing 25 mM Tris, 20 mM Na2SO4, and 0.001% poloxamer (pH 9.0) for 5 column volumes (CV). The column was pre-equilibrated with 5 CV of 2000 mM NaCl and then equilibrated with 5 CV of a buffer containing 25 mM Tris, 20 mM Na2SO4, and 0.001% poloxamer (pH 9.0). The AAV6 preparation was diluted 5-fold with 50 mM Tris (pH 9.0), then filtered through a 0.2 µm filter, loaded onto the column at 16.3 CV, and chased with 5 CV of a buffer containing 25 mM Tris, 20 mM Na2SO4, and 0.001% poloxamer (pH 9.0). Subsequently, the column was washed with 5 CV of a buffer containing 25 mM Tris, 53 mM Na2SO4, and 0.001% poloxamer (pH 9.0). The column was run with 5 CV of a buffer containing 25 mM Tris, 20 mM Na2SO4, and 0.001% poloxamer (pH 9.0). An elution buffer containing 25 mM Tris, 38 mM MgSO4, and 0.001% poloxamer (pH 9.0) at a constant concentration was used to elute the AAV full capsid at 7 CV. Table 13. Separation of AAV6 preparations of transgene B using the Na2SO4 washing step and MgSO4 isocratic elution [Table 13]

[0241] As shown in Figure 7, isocratic washing with a monovalent cation salt followed by isocratic elution with a divalent cation salt produced an empty capsid peak that appeared before the full capsid peak in the chromatogram. Similar to Example 6, although it involved a different transgene, the empty capsid was first washed away by isocratic monovalent washing, followed by the elution of full capsid by divalent isocratic elution. In isocratic divalent elution, full capsid was eluted without the additional empty capsid seen in the later peaks in gradient elution as shown in Figure 4, demonstrating its superiority over gradient divalent elution. Monovalent isocratic washing followed by divalent isocratic elution enabled the removal of the initial empty capsid population and subsequent elution of full capsid, avoiding co-purification of full capsid with the remaining empty capsid not removed by the first wash.

[0242] The percentage of full capsid was measured before and after purification (i.e., the percentage of full capsid in the AAV starting preparation and the percentage of full capsid in the eluted AAV peak containing full AAV capsid), and is shown in Table 14. This method yielded an estimated recovery rate of approximately 54-59% of the AAV full capsid present in the AAV starting preparation packed into the column (Table 12). The values ​​in the second column of Table 14 were calculated based on capsid particle counts obtained using a Stunner instrument (Unchained Labs) by dynamic and static light scattering followed by UV absorbance measurements at wavelengths of 260 nm and 280 nm. These methods showed that the AAV full capsid peak contained 37% full capsid compared to the starting AAV preparation containing only 14% full capsid.

[0243] In parallel, as shown in Table 14, column 3, the concentration of the AAV vector genome in the eluted AAV full capsid fraction was measured using qPCR and ELISA. Samples were diluted, digested with DNase I (or another suitable nuclease) to remove exogenous DNA, and further treated with proteinase K (or another suitable proteinase). After nuclease inactivation, samples were diluted and treated with a probe specific to the DNA sequence between primers (e.g., TagMan). (商標) Amplification was performed using a fluorescence-generating probe. The number of cycles required to reach a predetermined level of fluorescence (threshold cycles, Ct) was measured using a suitable detection system. Plasmid DNA containing the same sequence as that contained in the AAV vector was used to generate a standard curve in the qPCR reaction. The vector genome titer was determined by normalizing the Ct values ​​obtained from the sample to the Ct values ​​of the plasmid standard curve. AAV capsids containing transgene A were detected by combining qPCR with ELISA. Using these methods, the AAV full-capsid peak contained 76% full-capsid compared to the starting AAV preparation which contained only 27% full-capsid. Table 14. AAV6 capsid recovery in Example 7 [Table 14]

[0244] Example 8: Separation of AAV6 preparations using Na2SO4 washing and MgSO4 isocratic elution on a 4 mL anion exchange chromatography column. This example describes the separation of AAV6 preparations using a Na2SO4 washing step and a MgSO4 elution buffer at a constant concentration. The workflow described in this example is shown in Table 15, and the results are shown in Figure 8. The AAV6 in this example contained transgene "B".

[0245] The AAV6 preparation process involved lysis of the packaging cell culture and subsequent clarification of the lysate using depth filtration. Additional AAV6 enrichment and purification methods included tangential flow filtration and affinity chromatography prior to the AEX chromatography method described. Separation was performed using CIMmultus. (登録商標) The analysis was performed on an AKTA Avant150 HPLC system (Cytiva, Massachusetts, USA) using a QA integrated anion exchange chromatography column (4 mL). First, 5 CV of 25 mM Tris, 20 mM Na₂SO₄, and 0.001% poloxamer-containing buffer (pH 9.0) was run. The column was pre-equilibrated with 5 CV of 2000 mM NaCl, followed by 5 CV of 25 mM Tris, 20 mM Na₂SO₄, and 0.001% poloxamer-containing buffer (pH 9.0). The AAV6 preparation was diluted 5X with 50 mM Tris (pH 9.0), then filtered through a 0.2 μm filter, and 16.3 CV was packed into the column. The column was then chased with 5 CV of 25 mM Tris, 20 mM Na₂SO₄, and 0.001% poloxamer-containing buffer (pH 9.0). Next, the column was washed with 5 CV of 25 mM Tris, 54 mM Na2SO4, and 0.001% poloxamer-containing buffer (pH 9.0). The column was then run with 5 CV of 25 mM Tris, 20 mM Na2SO4, and 0.001% poloxamer-containing buffer (pH 9.0). The AAV full capsid was eluted at 7 CV using a constant concentration of 25 mM Tris, 33 mM MgSO4, and 0.001% poloxamer-containing elution buffer (pH 9.0). Table 15. Separation of AAV6 preparations of transgene B using Na2SO4 washing and MgSO4 isocratic elution. [Table 15]

[0246] As shown in Figure 8, isocratic washing with a monovalent cation salt followed by isocratic elution with a divalent cation salt produced an empty capsid peak that appeared before the full capsid peak in the chromatogram. Similar to Example 7, albeit on a different scale, the empty capsid was first washed away by isocratic monovalent washing, followed by the elution of full capsid by divalent isocratic elution. In isocratic divalent elution, full capsid was eluted without the additional empty capsid seen in the later peaks in gradient elution, as shown in Figure 4, demonstrating its superiority over gradient divalent elution. Monovalent isocratic washing followed by divalent isocratic elution enabled the removal of the initial empty capsid population and subsequent elution of full capsid, avoiding co-purification of full capsid with the remaining empty capsid not removed by the first wash.

[0247] The percentage of full capsid was measured before and after purification (i.e., the percentage of full capsid in the AAV starting preparation and the percentage of full capsid in the eluted AAV peak containing full AAV capsid), and is shown in Table 16. This method yielded an estimated recovery rate of approximately 83% of the AAV full capsid present in the AAV starting preparation packed into the column (Table 16). The values ​​in Table 16 were calculated based on capsid particle counts obtained by dynamic and static light scattering and subsequent UV absorbance measurements at wavelengths of 260 nm and 280 nm using a Stunner instrument (Unchained Labs). These methods showed that the AAV full capsid peak contained 34% full capsid compared to the starting AAV preparation containing only 8% full capsid. Table 16. AAV6 capsid recovery in Example 8 [Table 16]

Claims

1. A method for obtaining an AAV flucapsid from an AAV starting preparation containing an adeno-associated virus (AAV) flucapsid and an AAV empty capsid, wherein the method is: a) Injecting the AAV starting preparation into an anion exchange chromatography column; and b) Injecting a divalent cation elution buffer containing a divalent cation into an anion exchange chromatography column, wherein the divalent cation elution buffer does not contain a monovalent cation, and the injection of the divalent cation elution buffer elutes from the anion exchange chromatography column a fraction containing a larger proportion of AAV full capsid than AAV empty capsid. Methods that include...

2. The method according to claim 1, wherein the eluted fraction comprises at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of AAV fullcapsid relative to the AAV starting preparation.

3. The method according to any one of the preceding claims, wherein the proportion of AAV fullcapsid in the eluted fraction compared to total AAV capsid is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60%.

4. The method according to any one of the preceding claims, wherein the ratio of AAV full capsid to AAV empty capsid in the eluted fraction is at least 1.5:1, at least 1.6:1, at least 1.7:1, at least 1.8:1, at least 1.9:1, at least 2:1, at least 2.25:1, at least 2.5:1, at least 2.75:1, at least 3:1, at least 4:1, at least 5:1, at least 10:1, at least 20:1, at least 30:1, at least 40:1, at least 50:1, or at least 100:

1.

5. The method according to any one of the preceding claims, wherein at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or substantially all of the AAV full capsid is eluted from the anion exchange chromatography column before the AAV empty capsid.

6. The method according to any one of the preceding claims, wherein the eluted fraction comprises 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, or 2% or less, or 1% or less of AAV empty capsid.

7. The method according to any one of the preceding claims, wherein a divalent cation salt elution buffer is injected into an anion exchange chromatography column under conditions that at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or substantially all AAV full capsids are eluted from the column, and at least 60%, at least 70%, at least 80%, at least 90%, or substantially all AAV empty capsids remain bound to the column.

8. The method according to any one of claims 1 to 7, wherein a divalent cation salt elution buffer is injected at a constant concentration.

9. The method according to any one of claims 1 to 7, wherein a divalent cation salt elution buffer is injected in a linear gradient.

10. The method according to any one of claims 1 to 9, wherein the eluted fraction is a first eluted fraction, and a second eluted fraction is eluted after the first eluted fraction.

11. The method according to claim 10, wherein the second elution fraction contains a larger proportion of AAV empty capsid than the first elution fraction.

12. The method according to any one of claims 10 or 11, wherein the second eluted fraction contains 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, or 2% or less, or 1% or less of AAV fullcapsid.

13. The method according to any one of the preceding claims, further comprising injecting a monovalent cation wash buffer containing a monovalent cation into the column before injecting a divalent cation wash buffer, wherein the monovalent cation wash buffer does not contain a divalent cation, and the injection of the monovalent cation wash buffer elutes a wash fraction containing AAV empty capsids in a larger proportion than AAV full capsids from the anion exchange chromatography column.

14. The method according to claim 13, wherein a monovalent cation salt washing buffer is injected at a constant concentration.

15. The method according to claim 13, wherein a monovalent cation wash buffer is injected in a linear gradient.

16. A method for obtaining an AAV flucapsid from an AAV starting preparation containing an adeno-associated virus (AAV) flucapsid and an AAV empty capsid, wherein the method is: a) Injecting the AAV starting preparation into an anion exchange chromatography column; b) Inject a monovalent cation wash buffer into the column to elute a wash fraction containing AAV empty capsids in a larger proportion than AAV full capsids from the anion exchange chromatography column; and c) Inject the divalent cation salt elution buffer into an anion exchange chromatography column, thereby eluting the elution fraction containing AAV full capsid in a larger proportion than AAV empty capsid from the anion exchange chromatography column. Methods that include...

17. The method according to claim 16, wherein the eluted fraction contains at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of AAV fullcapsid relative to the AAV starting preparation.

18. The method according to claim 16 or claim 17, wherein the proportion of AAV fullcapsid in the eluted fraction compared to total AAV capsid is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60%.

19. The method according to any one of claims 16 to 18, wherein the ratio of AAV full capsid to AAV empty capsid in the eluted fraction is at least 1.5:1, at least 1.6:1, at least 1.7:1, at least 1.8:1, at least 1.9:1, at least 2:1, at least 2.25:1, at least 2.5:1, at least 2.75:1, at least 3:1, at least 4:1, at least 5:1, at least 10:1, at least 20:1, at least 30:1, at least 40:1, at least 50:1, or at least 100:

1.

20. The method according to any one of claims 16 to 19, wherein the washing fraction comprises at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or substantially 100% empty capsid.

21. The method according to any one of claims 16 to 20, wherein the eluted fraction comprises AAV empty capsid in an amount of 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, or 2% or less, or 1% or less.

22. The method according to any one of claims 13 to 21, wherein at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or substantially all of the AAV empty capsid is eluted from the anion exchange chromatography column before the AAV full capsid.

23. The method according to any one of claims 13 to 22, wherein a monovalent cation wash buffer is injected into an anion exchange chromatography column under conditions that at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or substantially all AAV empty capsids are eluted from the column, and at least 60%, at least 70%, at least 80%, at least 90%, or substantially all AAV full capsids remain bound to the column.

24. The method according to any one of the preceding claims, wherein the divalent cation salt elution buffer does not contain NaCl.

25. The method according to any one of the preceding claims, wherein the divalent cation salt elution buffer comprises a 50-150 mM, 50-100 mM, 70-120 mM, 80-110 mM, or 100 mM divalent cation salt.

26. Divalent cation salts, Mg ２＋ Ca ２＋ , or Sr ２＋ The method according to any one of the preceding claims, comprising a divalent cation selected from.

27. The divalent cation salt is magnesium chloride (MgCl ２ ), calcium chloride (CaCl ２ ), magnesium sulfate (MgSO ４ ), magnesium phosphate (MgPO ４ ), calcium acetate (Ca(C ２ H ３ O ２ )), ２ calcium sulfate (CaSO ４ ), magnesium acetate (Mg(C ２ H ３ O ２ )), ２ or magnesium citrate (MgC ６ H ６ O ７ ), and the method according to any one of the preceding claims.

28. The divalent cation salt is MgCl ２ CaCl ２ , or MgSO ４ The method according to any one of the prior claims.

29. Divalent cation salt elution buffer, (a) 25-175 mM, 25-150 mM, 25-100 mM, 25-75 mM, or 50 mM MgCl ２ ; (b) 25-175 mM, 25-150 mM, 25-100 mM, 25-75 mM, or 50 mM CaCl ２ ;or (c) 25-175 mM, 25-150 mM, 25-100 mM, 25-75 mM, or 50 mM MgSO ４ The method according to any one of the prior claims, including the method described in any one of the prior claims.

30. The method according to any one of the preceding claims, wherein the divalent cation salt elution buffer comprises a buffering agent.

31. The method according to any one of the preceding claims, wherein the divalent cation salt elution buffer comprises a buffer in a concentration of 5-100 mM, 10-100 mM, 10-50 mM, 10-40 mM, 10-30 mM, 20-30 mM, or 25 mM.

32. The method according to claim 30 or claim 31, wherein the buffering agent is selected from Tris, bis-trispropane, CHES (N-cyclohexyl-2-aminoethanesulfonic acid), or AMPSO (N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid).

33. The method according to any one of the preceding claims, wherein the divalent cation salt elution buffer comprises a stabilizing substance.

34. The method according to claim 33, wherein the stabilizing substance is selected from poloxamer, polysorbate 80 (PS-80), polysorbate 20 (PS-20), sorbitol, sucrose, or trehalose.

35. The method according to any one of the preceding claims, wherein the divalent cation salt elution buffer has a pH of approximately 7–10, approximately 8.5–9.5, or approximately 9.

36. Divalent cation salt elution buffer, (a) A solution containing 25 mM Tris, 100 mM divalent cation salt, and 0.001% poloxamer, with a pH of approximately 9; (b) A solution comprising 25 mM Tris, 38 mM divalent cation salt, and 0.001% poloxamer, with a pH of approximately 9; or (c) A solution containing 25 mM Tris, 33 mM divalent cation salt, and 0.001% poloxamer, with a pH of approximately 9. The method according to any one of the prior claims.

37. The method according to any one of claims 13 to 36, wherein the monovalent cation salt washing buffer comprises a monovalent cation salt in a concentration of 5-300 mM, 5-250 mM, 5-200 mM, 50-150 mM, 50-100 mM, 70-120 mM, 80-110 mM, or 100 mM.

38. Monovalent cation salts, NaCl, Na ２ SO ４ Na ３ PO ４ , or CH ３ The method according to any one of claims 13 to 37, wherein COONa is used.

39. Monovalent cation wash buffer (a) 50-200 mM, 50-150 mM, 100-200 mM, 125-175 mM, or 150 mM NaCl; (b) 15-125 mM, 15-100 mM, 25-100 mM, 25-75 mM, or 50 mM Na ２ SO ４ ; (c) 10-225 mM, 10-200 mM, 25-175 mM, 25-150 mM, 50-125 mM, 100-200 mM, 125-175 mM, 15-125 mM, 15-100 mM, 25-100 mM, or 25-75 mM Na ３ PO ４ ;or (d) 10-225 mM, 10-200 mM, 25-175 mM, 25-150 mM, 50-125 mM, 100-200 mM, 125-175 mM, 15-125 mM, 15-100 mM, 25-100 mM, or 25-75 mM CH ３ COONa The method according to any one of claims 13 to 38, including the method described in any one of claims 13 to 38.

40. The method according to any one of claims 13 to 39, wherein the monovalent cation salt washing buffer contains a buffering substance.

41. The method according to any one of claims 13 to 40, wherein the monovalent cation salt washing buffer comprises a buffer in a concentration of 5-100 mM, 10-100 mM, 10-50 mM, 10-40 mM, 10-30 mM, 20-30 mM, or 25 mM.

42. The method according to claim 40 or 41, wherein the buffering agent is selected from Tris, bis-trispropane, CHES (N-cyclohexyl-2-aminoethanesulfonic acid), or AMPSO (N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid).

43. The method according to claims 13 to 42, wherein the monovalent cation salt washing buffer contains a stabilizing substance.

44. The method according to claim 43, wherein the stabilizing substance is selected from poloxamer, polysorbate 80 (PS-80), polysorbate 20 (PS-20), sorbitol, sucrose, or trehalose.

45. The method according to any one of claims 13 to 44, wherein the monovalent cation wash buffer has a pH of approximately 7.0–10.0, approximately 8.5–9.5, or approximately 9.

46. The method according to any one of claims 13 to 45, wherein the monovalent cation wash buffer comprises 25 mM Tris, 150 mM NaCl, and 0.001% poloxamer, and has a pH of approximately 9.

47. The monovalent cation wash buffer is 25 mM Tris, 50 mM Na ２ SO ４ The method according to any one of claims 13 to 45, comprising, and 0.001% poloxamer, and having a pH of about 9.

48. The method according to any one of claims 9 to 47, wherein the linear gradient is a 0%-100% divalent cation salt elution buffer.

49. The method according to any one of the preceding claims, wherein, before injecting the divalent cation salt elution buffer into the anion exchange chromatography column, the anion exchange chromatography column is washed with a flash buffer, and the flash buffer is monovalent or divalent cation salt-free.

50. The method according to any one of claims 9 to 49, wherein a linear gradient is formed with a flash buffer and a divalent cation salt elution buffer, and the flash buffer is monovalent or divalent cation salt-free.

51. The method according to claim 50, wherein the linear gradient is a 100%-0% flash buffer.

52. The method according to any one of claims 49 to 51, wherein the flash buffer comprises a buffer selected from Tris, bis-trispropane, CHES (N-cyclohexyl-2-aminoethanesulfonic acid), or AMPSO (N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid).

53. The method according to any one of claims 49 to 52, wherein the flash buffer comprises a buffering material in a concentration of 5-100 mM, 10-100 mM, 10-50 mM, 10-40 mM, 10-30 mM, 20-30 mM, or 25 mM.

54. The method according to any one of claims 49 to 53, wherein the flash buffer contains a stabilizing substance.

55. The method according to claim 54, wherein the stabilizing substance is selected from poloxamer, polysorbate 80 (PS-80), polysorbate 20 (PS-20), sorbitol, sucrose, or trehalose.

56. The method according to any one of claims 49 to 55, wherein the flash buffer is approximately pH 7.0–10.0, approximately pH 8.5–9.5, or approximately pH 9.

57. The method according to any one of claims 49 to 56, wherein the flash buffer comprises 25 mM Tris and 0.001% poloxamer and has a pH of approximately 9.

58. The method according to any one of the preceding claims, wherein the anion exchange chromatography column is washed with an equilibration buffer containing a 10-100 mM monovalent cation before the divalent cation salt elution buffer is injected into the anion exchange chromatography column.

59. The method according to claim 58, wherein the equilibration buffer is injected into the anion exchange chromatography column after the monovalent cation salt washing buffer has been injected into the anion exchange chromatography column.

60. The method according to claim 58 or claim 59, wherein the equilibration buffer contains a monovalent cation salt at a concentration of 10–80 mM, 20–80 mM, 25–75 mM, 30–70 mM, 40–60 mM, 50 mM, or 60 mM.

61. Monovalent cation salts, NaCl, Na ２ SO ４ Na ３ PO ４ ,CH ３ The method according to any one of claims 58 to 60, selected from COONa or sodium citrate.

62. Sodium citrate is monosodium citrate (NaC ６ H ７ O ７ ), or disodium citrate (Na ２ C ６ H ６ O ７ ), or trisodium citrate (Na ３ C ６ H ５ O ７ The method according to claim 61.

63. The method according to any one of claims 58 to 62, wherein the equilibration buffer comprises a buffering material.

64. The method according to claim 63, wherein the equilibration buffer comprises a buffering material of 5-100 mM, 10-100 mM, 10-50 mM, 10-40 mM, 10-30 mM, 20-30 mM, or 25 mM.

65. The method according to claim 63 or claim 64, wherein the buffering agent is selected from Tris, bis-trispropane, CHES (N-cyclohexyl-2-aminoethanesulfonic acid), or AMPSO (N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid).

66. The method according to any one of claims 58 to 65, wherein the equilibration buffer contains a stabilizing substance.

67. The method according to claim 66, wherein the stabilizing substance is selected from poloxamer, polysorbate 80 (PS-80), polysorbate 20 (PS-20), sorbitol, sucrose, or trehalose.

68. The method according to any one of claims 58 to 68, wherein the equilibration buffer is approximately pH 7.0–10.0, approximately pH 8.5–9.5, or approximately pH 9.

69. The method according to any one of claims 58 to 70, wherein the equilibration buffer comprises 25 mM Tris, 60 mM NaCl, and 0.001% poloxamer, and has a pH of approximately 9.

70. The equilibration buffer consists of 25 mM Tris and 20 mM Na. ２ SO ４ The method according to any one of claims 58 to 70, comprising, and 0.001% poloxamer, and having a pH of about 9.

71. The method according to any one of the preceding claims, wherein the anion exchange chromatography column comprises a substrate containing a functional ligand selected from a mixed amine, a quaternary amine, trimethylammonium ethyl (TMAE), dimethylaminopropyl, diethylaminoethyl (DEAE), dimethylaminoethyl (DMAE), polyethyleneimine (PI), or guanidium.

72. The method according to claim 71, wherein the mixed amine comprises polyethyleneimine.

73. The method according to claim 72, wherein the quaternary amine comprises quaternized polyethyleneimine.

74. The method according to any one of the preceding claims, wherein an AAV starting preparation is injected into an anion exchange chromatography column under conditions in which an AAV full capsid and an AAV empty capsid are bound to the column.

75. The method according to any one of claims 9 to 74, wherein the AAV full capsid is eluted from an anion exchange chromatography column in a linear gradient of divalent cation salt elution buffers in amounts between 10% and 90%, 15% and 85%, 20% and 80%, or 30% and 70%.

76. The method according to any one of the preceding claims, wherein AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12.

77. The method according to any one of the prior claims, wherein AAV is AAV6.

78. The method according to any one of the preceding claims, wherein the AAV capsid comprises an AAV capsid protein, and the AAV capsid protein is a chimeric capsid, an engineered capsid, or a natural capsid.

79. The method according to any one of the preceding claims, wherein AAV is recombinant AAV.

80. The method according to any one of the prior claims, wherein the AAV is a pseudotyped AAV.

81. The method according to claim 80, wherein the pseudotyped AAV is AAV2 / 5, AAV2 / 8, or AAV2 / 7.

82. The method according to any one of the preceding claims, wherein the AAV is a self-complementary AAV.

83. The method according to any one of the preceding claims, wherein the AAV full capsid comprises the nucleic acid molecule of interest.

84. The method according to the immediately preceding claim, wherein the target nucleic acid molecule encodes a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

85. The method according to any one of the preceding claims, further comprising culturing AAV packaging cells in a medium capable of producing AAV particles, wherein the AAV particles comprise an AAV capsid protein and a nucleic acid molecule comprising an AAV 5' inverted terminal repeat (ITR), a nucleic acid molecule of interest to be packaged in the AAV capsid, and a nucleic acid molecule comprising a 3' ITR, wherein the cell comprises (i) a nucleic acid of interest to be packaged in at least one AAV capsid, (ii) a nucleic acid molecule encoding an AAV capsid protein under the control of one or more sequences directing its expression in the packaging cell, (iii) a nucleic acid molecule encoding an AAV rep protein that expresses an AAV rep protein in the cell to enable packaging of the nucleic acid of interest into the AAV capsid, and (iv) one or more helper functions necessary for packaging the nucleic acid molecule of interest into the AAV capsid.

86. The method according to claim 85, further comprising collecting AAV particles from AAV packaging cells and / or from a culture medium, wherein the collection involves cell disruption or the collection does not substantially involve cell disruption, thereby providing an AAV starting preparation.

87. The method according to claim 86, wherein the cell disruption includes cell lysis, thereby providing a cell lysate.

88. The method according to any one of claims 85 to 87, wherein the AAV starting preparation comprises a culture medium and / or a cell lysate.

89. The method according to any one of claims 85 to 88, wherein collecting AAV particles from cells and / or culture media includes recovering the culture media without substantially disrupting the cells.

90. The method according to any one of claims 85 to 89, wherein AAV packaging cells are stably transformed with one or more nucleic acid molecules encoding one or more helper functions.

91. The method according to any one of claims 86 to 90, wherein one or more helper functions are expressed under an activatable or inducible promoter.

92. The method according to any one of claims 85 to 91, wherein AAV packaging cells are stably transformed with a nucleic acid molecule encoding an AAV rep protein and / or a nucleic acid molecule encoding an AAV capsid protein.

93. The method according to any one of claims 85 to 92, wherein the AAV rep protein and / or AAV capsid protein are expressed under the direction of an activatable or inducible promoter.

94. The method according to any one of claims 85 to 93, wherein AAV packaging cells are stably transformed with the target nucleic acid molecule.

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