Anion exchange chromatography method for the purification of recombinant adeno-associated viruses

Abstract

A method is provided for separating full and empty capsid particles in a viral capsid preparation using an anion exchange medium. The method may involve using a washing solution containing a quaternary ammonium salt. The method includes applying the viral capsid preparation to an anion exchange medium in combination with a number of quaternary ammonium salt washing solutions. The present disclosure is based in part on the insight that separation of full and empty capsid particles in a viral capsid preparation (e.g., a rAAV preparation) using anion exchange (AEX) chromatography can be improved when a quaternary ammonium (QA) chloride (e.g., tetraethylammonium chloride [TEAC]) is included in one or more of the solutions used for separation; further improvement is particularly observed when used in conjunction with a magnesium salt (e.g., magnesium chloride [MgCl2]).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 234,059, filed August 17, 2021, the entire contents of which are incorporated by reference herein in their entirety for all purposes. [Background technology]

[0002] background A variety of gene therapy products are currently under development to treat human diseases. Many of these therapeutic agents use recombinant adeno-associated viruses (rAAVs), which have been engineered to deliver heterologous nucleic acids of interest (e.g., genes encoding therapeutic proteins, antisense nucleic acid molecules, ribozymes, miRNAs, siRNAs, nucleic acids encoding CRISPR / Cas systems, etc.) to diseased target cells in patients.

[0003] These rAAVs are engineered by deleting, in whole or in part, the internal portion of the AAV genome and inserting the heterologous nucleic acid of interest between the inverted terminal repeats (ITRs). The ITRs remain functional in such vectors, allowing the replication and packaging of AAV particles containing the nucleic acid cargo enclosed within the AAV capsid. Typically, the heterologous nucleic acid is operably linked to a regulatory sequence (e.g., a promoter or enhancer) that can drive the expression of the cargo in the target cell of the patient.

[0004] Various methods have been developed to produce large quantities of these rAAVs. These methods typically involve an "upstream" operation in which the rAAVs are produced in host cells (e.g., mammalian or insect cells) and a "downstream" operation in which the rAAVs are harvested and purified. Typically, inefficiencies in packaging the nucleic acid cargo during the upstream steps result in rAAV preparations that contain a mixture of "complete" AAV particles (i.e., particles that contain the nucleic acid of interest) and "empty" AAV particles (i.e., AAV particles that are completely or partially devoid of the nucleic acid cargo).

[0005] The presence of empty AAV particles in a gene therapy product can increase the overall dose required to achieve therapeutic efficacy and can promote an immune response (e.g., the generation of neutralizing antibodies or T cell activation) upon administration to a patient.

[0006] Thus, there is a need in the art for downstream purification methods that can be used to separate full from empty AAV particles in rAAV preparations produced by current upstream processes. Summary of the Invention [Means for solving the problem]

[0007] Abstract The present disclosure is based in part on the insight that separation of full and empty capsid particles in viral capsid preparations (e.g., rAAV preparations) using anion exchange (AEX) chromatography can be improved when a quaternary ammonium (QA) chloride (e.g., tetraethylammonium chloride [TEAC]) is included in one or more of the solutions used for the separation; further improvement is particularly seen when used in conjunction with a magnesium salt (e.g., magnesium chloride [MgCl2]).

[0008] In one aspect, there is provided a method for separating full and empty capsid particles in a viral capsid preparation, the method comprising: a) applying the viral capsid preparation to an anion exchange medium; b) passing a first wash solution containing a quaternary ammonium salt through the anion exchange medium to obtain a wash fraction containing empty viral capsid particles; c) passing a second wash solution through the anion exchange medium to obtain a second wash fraction comprising the quaternary ammonium salt; d) passing an elution solution through the anion exchange medium to elute the intact capsid particles; and e) collecting the elution fraction containing intact capsid particles; thereby separating full and empty capsid particles, Methods are provided wherein (i) the concentration of the quaternary ammonium salt in the first wash solution remains constant throughout step (b), and / or (ii) the salt composition of the elution solution remains constant throughout step (d).

[0009] In some embodiments, the method includes repeating one or more of steps b), c), and d) one or more times.

[0010] In one aspect, a method of elution, the method comprising: (a) contacting an anion exchange medium with a viral capsid preparation comprising full and empty capsid particles; (b) applying a first wash solution comprising a quaternary ammonium salt to the anion exchange medium to obtain a first wash fraction; (c) applying a second wash solution to the anion exchange medium to obtain a second wash fraction; and (d) applying an elution solution to the anion exchange medium and collecting the elution fraction; Inclusive of Methods are provided wherein (i) the concentration of the quaternary ammonium salt in the first wash solution remains constant throughout step (b), and / or (ii) the salt composition of the elution solution remains constant throughout step (d).

[0011] In some embodiments, the method includes repeating one or more of steps b), c), and d) one or more times.

[0012] In some embodiments, the second wash solution and the elution solution do not include a quaternary ammonium salt.

[0013] In some embodiments, apart from the first wash solution, no other solutions containing the quaternary ammonium salt are passed through or applied to the anion exchange medium.

[0014] In some embodiments, apart from the first wash solution, no other solutions containing any quaternary ammonium salts are passed through or applied to the anion exchange medium.

[0015] In some embodiments, the first wash solution contains a quaternary ammonium salt at a concentration of about 90 mM to about 130 mM, for example, about 110 mM.

[0016] In some embodiments, the quaternary ammonium salt is a tetraalkylammonium chloride. For example, in some embodiments, the tetraalkylammonium chloride is selected from the group consisting of tetramethylammonium chloride (TMAC), tetraethylammonium chloride (TEAC), tetrapropylammonium chloride (TPAC), tetrabutylammonium chloride (TBAC), benzyltributylammonium chloride (BTBAC), and any combination thereof. In some embodiments, the tetraalkylammonium chloride is TEAC. In some embodiments, the quaternary ammonium salt is a tetraalkylammonium acetate.

[0017] In some embodiments, the quaternary ammonium salt is selected from the group consisting of tetramethylammonium acetate, tetraethylammonium acetate (TEA-Ac), tetrapropylammonium acetate, tetrabutylammonium acetate, and any combination thereof. For example, in some embodiments, the quaternary ammonium salt is TEA-Ac.

[0018] In some embodiments, the quaternary ammonium salt is choline chloride.

[0019] In some embodiments, any one or more of the first wash solution, the second wash solution, and / or the elution solution further comprises a divalent salt.

[0020] In some embodiments, the first wash solution further comprises a divalent salt, such as MgCl2. In some embodiments, the first wash solution comprises MgCl2 at a concentration of about 1 mM to about 10 mM, such as about 2 mM.

[0021] In some embodiments, the first wash solution comprises TEAC at a concentration of about 30 mM to about 200 mM, e.g., about 110 mM. In some such embodiments, the first wash solution further comprises a divalent salt, e.g., MgCl2. In some embodiments, the first wash solution comprises MgCl2 at a concentration of about 1 mM to about 10 mM, e.g., about 2 mM.

[0022] In some embodiments, the second washing solution and / or the elution solution includes NaCl, Na2SO4, MgSO4, or any combination thereof. In some embodiments, the second washing solution includes NaCl at a concentration of, for example, about 25 mM to about 375 mM, about 50 mM to about 250 mM, about 70 mM to about 200 mM, about 70 mM to about 140 mM, or about 90 mM to about 140 mM. In some embodiments, the elution solution includes NaCl at a concentration of, for example, about 25 mM to about 375 mM, about 50 mM to about 250 mM, about 70 mM to about 200 mM, about 70 mM to about 140 mM, or about 90 mM to about 140 mM.

[0023] In some embodiments, the elution solution comprises an anionic species, for example, the anionic species is selected from the group consisting of tetrafluoroborate (BF4), bromide (Br), and acetate (Ac).

[0024] In some embodiments, the elution solution comprises MgCl2, for example, at a concentration of at least 1 mM, about 1 mM to about 5 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM.

[0025] In some embodiments, the first wash solution, the second wash solution, and / or the elution solution each comprise a buffer system at about pH 9.

[0026] In some embodiments, the first wash solution elutes at least 60% or at least 90% (e.g., 90%-100%, e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the empty capsid particles present in the virus capsid preparation. In some embodiments, the first wash solution does not substantially elute the full capsid particles present in the virus capsid preparation. In some embodiments, the first wash solution elutes 30% or less (e.g., 0-30%, e.g., about 0, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%) or 1% or less of the complete capsid particles present in the virus capsid preparation.

[0027] In some embodiments, the second wash fraction comprises the quaternary ammonium salt. In some embodiments, the second wash solution elutes at least 50%, at least 90% (e.g., 90%-100%, e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%), or at least 99% of the quaternary ammonium salt present in the first wash solution.

[0028] In some embodiments, the second wash solution does not substantially elute the complete capsid particles present in the virus capsid preparation. In some embodiments, the second wash solution elutes 20% or less (e.g., 0-20%, e.g., about 0, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%) or 1% or less of the complete capsid particles present in the virus capsid preparation.

[0029] In some embodiments, the elution solution substantially elutes the complete capsid particles present in the virus capsid preparation, hi some embodiments, the elution solution elutes at least 50%, at least 90%, or at least 99% of the complete capsid particles present in the virus capsid preparation.

[0030] In some embodiments, the elution fraction comprises at least 50% (e.g., 50%-100%, e.g., about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%), at least 90% (e.g., 90%-100%, e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%), or at least 99% of the complete capsid particles present in the virus capsid preparation.

[0031] In some embodiments, the elution fraction contains 50% or less, 10% or less, or 1% or less of the empty capsid particles present in the virus capsid preparation, thereby substantially purifying the full capsid particles from the virus capsid preparation.

[0032] In some embodiments, at least 35% (e.g., 35% to 100%, e.g., about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of the capsid particles present in the elution fraction are complete capsid particles.

[0033] In some embodiments, at least 40% (e.g., 40% to 100%, e.g., about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of the capsid particles present in the elution fraction are complete capsid particles.

[0034] In some embodiments, at least 45% (e.g., 45% to 100%, e.g., about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of the capsid particles present in the elution fraction are complete capsid particles.

[0035] In some embodiments, at least 50% (e.g., 50% to 100%, e.g., about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of the capsid particles present in the elution fraction are complete capsid particles.

[0036] In some embodiments, more than 50% (e.g., more than 50% and up to 100%, e.g., about 51%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of the capsid particles present in the elution fraction are complete capsid particles.

[0037] In certain embodiments, the concentration of the quaternary ammonium salt in the first wash solution remains constant throughout step (b). In some embodiments, the salt composition of the first wash solution remains constant throughout step (b). In some embodiments, the salt composition of the second wash solution remains constant throughout step (c).

[0038] In some embodiments, the salt composition of the elution solution remains constant throughout step (d). In some embodiments, between 1 and 20 total column volumes, for example between 1 and 10 total column volumes, are eluted.

[0039] In some embodiments, the salt composition of the first wash solution, the salt composition of the second wash solution, and the salt composition of the elution solution remain constant throughout each individual wash or elution step.

[0040] In some embodiments, the composition of the first wash solution is varied during step (b).

[0041] In some embodiments, the composition of the second wash solution is varied during step (c). For example, in some embodiments, the concentration of salt in the second wash solution is increased continuously over time during step (c). In some embodiments, this increase is linear over time during step (c).

[0042] In some embodiments, the composition of the elution solution is varied during step (d). For example, in some embodiments, the concentration of salt in the elution solution is increased continuously over time during step (d). In some embodiments, this continuous increase is linear over time during step (d).

[0043] In some embodiments, between 0 and 200 total column volumes are eluted, such as between 50 and 150 total column volumes, for example about 90 total column volumes.

[0044] In some embodiments, the capsid is derived from AAV8 or a variant thereof.

[0045] In some embodiments, the anion exchange medium is a monolithic column. For example, in some embodiments, the monolithic column is a CIMmultus® QA column.

[0046] In some embodiments, the method does not include using an isocratic elution gradient of MgCl2.

[0047] In some embodiments, if one or more of the first wash solutions contains MgCl, and the concentration of MgCl in the first wash solutions is constant, then the concentration of MgCl is only constant throughout step (b).

[0048] In one aspect, there is provided a method for separating full and empty capsid particles in a viral capsid preparation, the method comprising: (a) applying the viral capsid preparation to an anion exchange medium; (b) passing a first wash solution containing a quaternary ammonium salt through the anion exchange medium to obtain a wash fraction containing empty viral capsid particles; (c) passing a second wash solution through the anion exchange medium to obtain a second wash fraction comprising the quaternary ammonium salt; (d) passing an elution solution through the anion exchange medium to elute the intact capsid particles; and (e) collecting the elution fraction containing intact capsid particles; Inclusive of Thereby, full and empty capsid particles are separated, wherein (i) the concentration of the quaternary ammonium salt in the first wash solution remains constant throughout step (b); wherein the capsid is derived from AAV8 or a variant thereof, Herein, a method is provided, wherein at least 35% (e.g., 35% to 100%, e.g., about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of the capsid particles present in the elution fraction are complete capsid particles.

[0049] In some embodiments, the quaternary ammonium salt is a tetraalkylammonium chloride, for example, the tetraalkylammonium chloride is selected from the group consisting of tetramethylammonium chloride (TMAC), tetraethylammonium chloride (TEAC), tetrapropylammonium chloride (TPAC), tetrabutylammonium chloride (TBAC), benzyltributylammonium chloride (BTBAC), and any combination thereof. In some embodiments, the tetraalkylammonium chloride is TEAC.

[0050] In some embodiments, the quaternary ammonium salt is a tetraalkylammonium acetate, for example, the tetraalkylammonium acetate is selected from the group consisting of tetramethylammonium acetate, tetraethylammonium acetate (TEA-Ac), tetrapropylammonium acetate, tetrabutylammonium acetate, and any combination thereof. In some embodiments, the quaternary ammonium salt is TEA-Ac.

[0051] In some embodiments, the quaternary ammonium salt is choline chloride.

[0052] In one aspect, a method for producing a complete recombinant adeno-associated virus (rAAV) capsid particle, the method comprising: (a) producing a viral capsid preparation comprising full and empty rAAV capsid particles from cultured mammalian or insect cells; (b) applying the viral capsid preparation to an anion exchange medium; (c) passing a first wash solution containing a quaternary ammonium salt through the anion exchanger to obtain a wash fraction containing empty rAAV viral capsid particles; (d) passing a second wash solution through the anion exchanger to obtain a second wash fraction comprising the quaternary ammonium salt; (e) passing an elution solution through the anion exchanger to elute the intact rAAV capsid particles; and (f) collecting the elution fraction containing intact rAAV capsid particles; Includes; Methods are provided wherein (i) the concentration of the quaternary ammonium salt in the first wash solution remains constant throughout step (c), and / or (ii) the salt composition of the elution solution remains constant throughout step (e).

[0053] In one aspect, a method for producing a complete recombinant adeno-associated virus (rAAV) capsid particle, the method comprising: (a) producing a viral capsid preparation comprising full and empty rAAV capsid particles from cultured mammalian or insect cells; (b) contacting the viral capsid preparation with an anion exchange medium; (c) applying a first washing solution containing a quaternary ammonium salt to the anion exchanger to obtain a first washing fraction; (d) applying a second wash solution to the anion exchanger to obtain a second wash fraction; and (e) applying an elution solution to the anion exchanger and collecting the eluted fraction; Inclusive of wherein (i) the concentration of the quaternary ammonium salt in the first wash solution remains constant throughout step (c); and / or (ii) the salt composition of the elution solution remains constant throughout step (e). Methods are provided herein, wherein the elution fraction contains complete rAAV capsid particles.

[0054] In some embodiments, the concentration of the quaternary ammonium salt in the first wash solution remains constant throughout step (c).

[0055] In some embodiments, the rAAV capsid particle is a rAAV8 capsid particle.

[0056] In some embodiments, the quaternary ammonium salt is a tetraalkylammonium chloride, for example, the tetraalkylammonium chloride is selected from the group consisting of tetramethylammonium chloride (TMAC), tetraethylammonium chloride (TEAC), tetrapropylammonium chloride (TPAC), tetrabutylammonium chloride (TBAC), benzyltributylammonium chloride (BTBAC), and any combination thereof. In some embodiments, the quaternary ammonium salt is TEAC.

[0057] In some embodiments, the quaternary ammonium salt is a tetraalkylammonium acetate, for example, the tetraalkylammonium acetate is selected from the group consisting of tetramethylammonium acetate, tetraethylammonium acetate (TEA-Ac), tetrapropylammonium acetate, tetrabutylammonium acetate, and any combination thereof. In some embodiments, the quaternary ammonium salt is TEA-Ac.

[0058] In some embodiments, the quaternary ammonium salt is choline chloride.

[0059] In some embodiments, at least 35% (e.g., 35% to 100%, e.g., about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of the rAAV capsid particles in the elution fraction are complete rAAV capsid particles.

[0060] In one aspect, a composition is provided that includes complete recombinant adeno-associated virus (rAAV) capsid particles produced by a method as disclosed herein. In some embodiments, at least 35% (e.g., 35% to 100%, e.g., about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of the rAAV capsid particles in the composition are complete rAAV capsid particles. [Brief description of the drawings]

[0061] [Figure 1A-B] 1A and 1B show exemplary chromatograms illustrating the separation of empty AAV particles from full AAV8 particles using NaCl gradients for various types of stationary phases. FIG. 1A shows a chromatogram from an experiment on AAV8-1 particles using either a POROS™ 50 HQ column or a CIM-Q column. FIG. 1B shows a chromatogram from an experiment on AAV8-2 particles using either a Nuvia™ Q column or a CIM-Q column. "E" indicates fractions corresponding to high relative amounts of empty capsids, and "F" indicates fractions corresponding to high relative amounts of full capsids. The ascending lines show the increase in solution conductivity caused by the corresponding increase in NaCl concentration (see Example 1).

[0062] [Figure 2A-B]Figures 2A and 2B show results from separation experiments performed on AAV8-2 material against a NaCl gradient and on other Nuvia™ Q columns or CIMmultus® QA columns (see Example 1). Figure 2A shows the step yield, i.e., the amount of genome of interest in the pooled fractions relative to the amount in the load material, as determined by quantitative polymerase chain reaction (qPCR). Figure 2B shows the percentage of complete capsid particles present in the eluate, as determined by analytical ultracentrifugation (AUC). Values ​​in both Figures 2A and 2B are normalized to those observed on the Nuvia™ Q column.

[0063] [Figure 3A-E] 3A-3E show exemplary chromatograms from separation experiments performed with human embryonic kidney (HEK)-produced AAV8-1 (FIG. 3A), AAV8-2 (FIG. 3B), AAV8-3 (FIG. 3C), and hu37 (FIG. 3D), and with HeLa-produced AAV9 (FIG. 3E). Chromatography was performed on a CIMmultus® QA column using a NaCl gradient. The ascending line indicates the increase in solution conductivity caused by the corresponding increase in NaCl concentration (see Example 2).

[0064] [Figure 4] 4 shows a graph comparing results from experiments performed with HEK-produced AAV8-1 and AAV8-2. The normalized percentage of complete capsid particles in the eluate (y-axis) is shown compared to the percentage of complete capsid particles in the load material (x-axis) (see Example 2).

[0065] [Diagram 5]Figure 5 shows a flow chart (left side of Figure 5) outlining downstream processing of AAV material generated from HeLa cells from experiments in which samples containing no MgCl2, 67 mM MgCl2 ("no dilution in load"), or 6-7 mM MgCl2 ("dilution in load") were subjected to NaCl gradient elution conditions on a POROS™ XQ column, and representative chromatograms (right side of Figure 5). "E" indicates fractions corresponding to high relative amounts of empty capsids, and "F" indicates fractions corresponding to high relative amounts of full capsids (see Example 3).

[0066] [Figure 6] 6 shows exemplary chromatograms demonstrating increased separation of empty versus full AAV particles (lower chromatogram) for a quaternary ammonium (QA) salt gradient (in this example, tetramethylammonium chloride (TMAC)) compared to a NaCl gradient (upper chromatogram). The chromatogram shows A254 and A280 measurements of the eluent during gradient elution. The ascending line indicates the increase in solution conductivity caused by the corresponding increase in eluent concentration (see Example 4).

[0067] [Figure 7A-D] 7A-7D show exemplary chromatograms demonstrating increasing empty versus full AAV particle separation for a QA salt gradient with increasing functional group size. From top to bottom, elution is obtained from sodium chloride (NaCl) (FIG. 7A), tetramethylammonium chloride (TMAC) (FIG. 7B), tetrabutylammonium chloride (TBAC) (FIG. 7C), and benzyltributylammonium chloride (BTBAC) (FIG. 7D) gradients. The chromatogram of the NaCl gradient is shown as a reference for comparing "empty" and "full" fraction separation. The chromatograms show A280 measurements of the eluent during the gradient elution. The ascending line indicates the increase in solution conductivity caused by the corresponding increase in eluent concentration (see Example 5).

[0068] [Figure 8] FIG. 8 shows a graph plotting the ion intensity (x-axis) of the first and second peaks as a function of cation size from a separation experiment described in Example 6 using a QA salt gradient with increasing functional group size (see Example 5).

[0069] [Figure 9] 9 shows an exemplary chromatogram illustrating the separation of empty and intact AAV particles using gradient elution with a tetraethylammonium chloride (TEAC) gradient from about 25 mM to about 337.5 mM on a CIMmultus® QA column over 90 column volumes at a constant magnesium chloride (MgCl) concentration of 2 mM at pH 9. The ascending line shows the increase in solution conductivity caused by a corresponding increase in TEAC concentration (see Example 6).

[0070] [Figure 10] 10 shows an exemplary chromatogram illustrating the separation of empty and intact AAV particles using gradient elution with an initial wash with tetraethylammonium acetate (TEA-Ac) and a constant MgCl2 concentration. The ascending line shows the increase in solution conductivity caused by a corresponding increase in TEA-Ac concentration (see Example 6).

[0071] [Figure 11] 11 shows an exemplary chromatogram illustrating the separation of empty and intact AAV particles using isocratic elution with an initial wash with tetraethylammonium chloride (TEAC) and magnesium chloride (MgCl2), followed by two successive step washes with sodium chloride (NaCl) and MgCl2, and then elution with NaCl and MgCl2. The dashed line corresponds to the volumetric output of pump "B" (i.e., the pump that supplies the wash and elution buffers) relative to pump "A" (i.e., the pump that supplies the equilibration buffer) (see Example 8).

[0072] [Figure 12A-E] Figures 12A-12E show exemplary chromatograms from experiments using various protocols using MgSO4 to separate empty and full AAV8-2 particles. From top to bottom, chromatograms are shown from experiments using a CIMmultus® QA column and 1) gradient elution (Figure 12A); 2) gradient wash followed by isocratic elution (Figure 12B); 3) one isocratic wash (Figure 12C); 4) isocratic elution with three washes (Figure 12D); or 5) isocratic elution with two washes (Figure 12E). The orange line in each chromatogram indicates the solution conductivity, which corresponds to the salt concentration (see Example 9).

[0073] [Figure 13A-B] Figures 13A and 13B show results from separation experiments performed on AAV8-1 material using gradient or isocratic elution protocols on a CIMmultus® QA column (see Example 10). Figure 13A shows the step yield, i.e., the amount of genome of interest in the pooled fractions relative to the amount of loaded material, as determined by quantitative polymerase chain reaction (qPCR). Figure 13B shows the percentage of complete capsid particles present in the eluate, as determined by analytical ultracentrifugation (AUC). Values ​​in both Figures 13A and 13B are normalized to those observed with the gradient elution protocol (see Example 9).

[0074] [Figure 14] 14 shows an exemplary chromatogram illustrating the separation of empty and intact AAV particles using gradient elution with an initial wash with choline chloride and a constant MgCl2 concentration. The ascending line shows the increase in solution conductivity caused by a corresponding increase in choline chloride concentration (see Example 10).

[0075] [Figure 15A] Figure 15A shows the parameters of the five different runs tested in the experimental set described in Example 11. The column was loaded with 1x, 6x, and 12x amounts of AAV8-2 material at 1x, (1 / 2)x, or (1 / 4)x residence times. Samples were run on a CIMmultus® QA column using a NaCl gradient. [Figure 15B] Figure 15B shows chromatograms from various runs, the conditions of which are shown in Figure 15A. "E" indicates fractions corresponding to high relative amounts of empty capsids, and "F" indicates fractions corresponding to high relative amounts of full capsids (see Example 11).

[0076] [Figure 15C] FIG. 15C shows plots from a desirability analysis using JMP® software for variables such as capsid loading, normalized host cell protein amount in the eluate, and normalized percentage of complete capsids in the eluate (see Example 11).

[0077] [Figure 16A-B] Figures 16A and 16B show the yield (of genome of interest), percentage of complete capsid, and host cell protein amount from a chromatography experiment using AAV8-1 material run on a CIMmultus® QA column using a NaCl gradient. Figure 16A shows the amount per fraction, and Figure 16B shows the cumulative amount. As can be seen in Figures 16A and 16B, fractions after the second peak have detectable host cell protein levels, and collecting additional fractions to increase the percentage of complete capsid protein collected also increases the cumulative amount of host cell protein.

[0078] [Figure 17]FIG. 17 shows representative chromatograms from experiments on samples of AAV8-1 and AAV8-2 material containing full and empty capsid particles run on a CIMmultus® QA column using either a NaCl gradient or a gradient with a mobile phase containing MgSO4 (see Example 12).

[0079] [Figure 18A-C] 18A-18C show representative chromatograms from experiments on samples of AAV8.1 material containing full and empty capsid particles run on a CIMmultus® QA column using one of three conditions: 1) NaCl with gradient elution (FIG. 18A); 2) tetraethylammonium chloride (TEAC) gradient and 2 mM MgCl2 (FIG. 18B); and 3) tetraethylammonium chloride (TEAC) and 2 mM MgCl2 with isocratic elution (FIG. 18C). "E" indicates fractions corresponding to high relative amounts of empty capsids and "F" indicates fractions corresponding to high relative amounts of full capsids (see Example 13).

[0080] [Figure 19A-B] Figures 19A and 19B show plots showing the step yields (genome of interest as measured by quantitative PCR) and percentage of complete capsid particles (as measured by analytical ultracentrifugation) from these experiments normalized to levels for the NaCl gradient experiments (see Example 13).

[0081] [Figure 20]Figure 20 shows representative chromatograms from an experiment on a sample of AAV8.1 material containing full and empty capsid particles run on a CIMmultus® QA column using one of several linear gradient elution solutions containing an anionic species: tetraethylammonium (TEA)-BF4 (top panel), TEA-Br (middle panel), or TEA-Ac (bottom panel). Within each chromatogram, the solid line represents absorbance at 280 nm (A280) and the dashed line represents ionic strength (see Example 14).

[0082] [Figure 21] Figure 21 shows representative chromatograms from an experiment on a sample of AAV8.1 material containing full and empty capsid particles run on an IMmultus® QA column using various concentrations of MgCl2 (0, 0.2 mM, 1 mM, and 5 mM) in the elution solution. The traces (solid lines) represent the absorbance at 280 nm (A280), and the asterisks indicate a third population that is not clearly visible in the traces from runs using elution solutions containing 1 mM or 5 mM MgCl2. Particle titers measured in the elution pool of the "empty" peak (the left-most peak in each chromatogram) are shown next to their corresponding elution profiles (see Example 15).

[0083] [Figure 22A-B]Figures 22A-22D show representative chromatograms from experiments on samples of AAV8.1 material containing full and empty capsid particles run on a CIMmultus® QA column using a gradient elution protocol (Figures 22A and 22B) or an isocratic elution protocol (Figures 22C and 22D). Figures 22B and 22D show magnified images of the peaks from Figures 22A and 22C, respectively. Within each chromatogram, the light solid line corresponds to the absorbance at 280 nm (A280) and the dark solid line corresponds to the absorbance at 254 nm (A254). The dashed lines in Figures 22A and 22C correspond to the volume fraction of running buffer B (20 mM bis-tris-propane, 0.001% (w / v) Pluronic® F-68, 2 mM MgCl2, and 200 mM NaCl at pH 9) pumped through the system. The dashed lines in Figures 22B and 22D correspond to the solution conductivity (see Example 16). [Fig. 22C-D] Figures 22A-22D show representative chromatograms from experiments on samples of AAV8.1 material containing full and empty capsid particles run on a CIMmultus® QA column using a gradient elution protocol (Figures 22A and 22B) or an isocratic elution protocol (Figures 22C and 22D). Figures 22B and 22D show magnified images of the peaks from Figures 22A and 22C, respectively. Within each chromatogram, the light solid line corresponds to the absorbance at 280 nm (A280) and the dark solid line corresponds to the absorbance at 254 nm (A254). The dashed lines in Figures 22A and 22C correspond to the volume fraction of running buffer B (20 mM bis-tris-propane, 0.001% (w / v) Pluronic® F-68, 2 mM MgCl2, and 200 mM NaCl at pH 9) pumped through the system. The dashed lines in Figures 22B and 22D correspond to the solution conductivity (see Example 16).

[0084] [Diagram 23]23 shows a regression plot of the trade-off between process yield (% genome recovery) (y-axis) versus percentage of intact capsid particles (x-axis) obtained from anion exchange chromatography runs using NaCl in the process buffer (open markers) or anion exchange chromatography runs using an isocratic TEA-Ac wash. Circles show data from runs using isocratic elution, while squares show data from runs using gradient elution (see Example 17). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0085] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS The present disclosure provides improved methods for separating populations within a viral capsid preparation, for example, for separating complete capsid particles from empty capsid particles. Methods for producing complete recombinant adeno-associated virus (rAAV) capsid particles are also provided.

[0086] definition When the term "about" is used before a quantitative value, the present invention also includes the particular quantitative value itself, unless specifically stated otherwise. As used herein, the term "about" refers to a ±10% variation from the nominal value, unless otherwise indicated or inferred.

[0087] As used herein, the term "adeno-associated virus" refers to a small, replication-deficient, non-enveloped virus that infects humans and some other primate species. AAV is not known to cause disease and elicits a very mild immune response. Gene therapy vectors utilizing AAV can infect both dividing and quiescent cells and can survive in an extrachromosomal state without integrating into the genome of the host cell. These characteristics make AAV an attractive viral vector for gene therapy. With regard to AAV serotypes, 13 are currently recognized (AAV1-13).

[0088] Unless otherwise noted, when the term "between" is used to refer to a numerical range, the range includes its specified endpoints. For example, the range "between 1 mM and 10 mM" includes 1 mM, 10 mM, and values ​​greater than 1 mM but less than 10 mM.

[0089] As used herein, the term "capsid particle" refers to a particle that (i) encapsidates a nucleic acid, e.g., a vector genome or a portion thereof, and / or (ii) comprises at least one viral capsid protein that forms a structure around a core. In the case of an empty capsid particle, the core may be empty or collapsed, or may contain only a portion of a vector genome, as described herein. In some embodiments, the capsid particle encapsidates a nucleic acid that is a vector genome and / or a gene of interest. In some embodiments, the capsid particle encapsidates a nucleic acid species that is not a vector genome or gene of interest, e.g., a plasmid or host cell DNA, or a portion thereof.

[0090] As used herein, the term "full capside particle" refers to a capsid particle that contains a complete vector genome, i.e., a vector genome that contains a heterologous nucleic acid of interest flanked on both sides by AAV ITRs.

[0091] As used herein, the term "empty capside particle" refers to a capsid particle that comprises at least one capsid protein and lacks a complete vector genome, e.g., lacking in whole or in part a heterologous nucleic acid of interest flanked on both sides by AAV ITRs, or lacking in whole or in part another portion of the vector genome.

[0092] Unless otherwise noted, the terms "from" and "to" are used to refer to a numerical range, which includes its particular endpoints. For example, the range "from 1 mM to 10 mM" includes 1 mM, 10 mM, and values ​​greater than 1 mM but less than 10 mM, as well as all integers between and including 1 mM and 10 mM.

[0093] As used herein, the term "gradient elution" or "gradient separation" refers to a mode of chromatographic separation in which the concentration of one or more salts in the elution solution applied to the separation medium is gradually changed during the separation.

[0094] As used herein, the term "inverted terminal repeat" (abbreviated as "ITR") refers to symmetrical nucleic acid sequences in the genome of adeno-associated virus that are required for efficient replication. ITR sequences are located at each end of the AAV DNA genome. The ITRs serve as replication origins for viral DNA synthesis and are essential cis elements for generating AAV integration vectors.

[0095] Use of the terms "include," "including," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including grammatical equivalents thereof, should generally be understood to be open-ended and non-limiting, e.g., not excluding additional, unrecited elements or steps unless specifically stated otherwise or understood otherwise from the context.

[0096] As used herein, the term "isocratic elution" or "isocratic separation" refers to a mode of chromatographic separation in which the concentrations of all salts in a solution are maintained constant during a defined period of separation (e.g., a "wash" solution during a "wash" step and an "elution" solution during an "elution" step). In some embodiments, isocratic elution uses a series of two or more separation solutions during the separation, each of which may have a different fixed concentration of one or more salts relative to another solution in the series.

[0097] As used herein, the phrase "isocratic elution gradient" refers to a gradient in which the composition of the mobile phase is changed among steps during a single chromatographic run. At each step of an isocratic elution gradient, the mobile phase is maintained at the same composition (e.g., constant concentration) until the subsequent step in the chromatographic run, at which time the composition of the mobile phase is changed such that the concentration of the mobile phase constituents is increased relative to the concentration of the constituents in the previous step. Thus, for example, an isocratic elution gradient of MgCl2 involves using various steps with a constant MgCl2 concentration at each individual step, but increasing MgCl2 concentration from one step to the subsequent step.

[0098] As used herein, "obtaining" or "to obtain" a fraction, e.g., a wash fraction following a step of passing or applying a solution to an anion exchange medium, means that the fraction is produced following that step. The "obtained" fraction may or may not be collected.

[0099] As used herein, the term "quaternary ammonium salt" refers to an ionic compound having a quaternary ammonium nitrogen, four groups (e.g., alkyl or aryl groups) attached to the ammonium nitrogen, and an anionic ion (e.g., acetate, bromide, or chloride).

[0100] As used herein, the term "recombinant" may be used to describe, for example, a nucleic acid molecule having a sequence that is not naturally occurring or that is made by the artificial combination of two otherwise isolated sequence segments, which may be accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid molecules (e.g., by genetic engineering techniques).

[0101] "Recombinant adeno-associated virus preparation" or "rAAV preparation" refers to a product resulting from a method of producing (e.g., manufacturing) recombinant AAV in a host cell (e.g., in a mammalian cell or an insect cell). In some embodiments, the recombinant AAV preparation comprises a mixture of full and empty AAV particles. In some embodiments, the recombinant AAV preparation has been subjected to one or more downstream steps after an initial upstream step, e.g., nuclease treatment, filtration to remove host cell impurities, and / or affinity purification using a ligand that binds the AAV capsid, as known to those of skill in the art.

[0102] As used herein, the term "salt composition," when used in reference to a solution, refers to the identities and amounts of all salts in that solution. Thus, when the "salt composition" of a solution is said to remain constant for a particular duration of time, it means that the identities and amounts of all salts in the solution remain constant for that particular duration of time.

[0103] As used herein, the term "separation chemistry" refers to the mixing matrix of an active ligand (eg, a quaternary amine) or separation medium and a support matrix.

[0104] As used herein, the term "separation medium" refers to a physical structure, such as a column packed with a resin or monolith or membrane, onto which an rAAV preparation is applied to achieve separation of a certain fraction of the preparation. For example, an rAAV preparation can be applied to a column, which is then washed with one or more solutions to separate empty and full AAV particles from each other (and the separated fractions are collected). In some embodiments, the separation medium is an anion exchange medium. In some embodiments, the separation medium is a mixed-modal medium that can act as an anion exchange medium. In some embodiments, the separation medium is a column (e.g., a monolithic column or particles in a packed column). In some embodiments, the separation medium is a membrane.

[0105] As used herein, the term "vector" refers to a nucleic acid molecule that allows the insertion of foreign nucleic acid without destroying the vector's ability to replicate and / or integrate in a host cell. A vector may contain a nucleic acid sequence (e.g., origin of replication) that allows it to replicate in a host cell. A vector may also contain one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences to allow the transcription and translation of the inserted gene. In some embodiments herein, the vector is an AAV vector.

[0106] Viral capsid preparation The methods of the disclosure are useful for separating full and empty capsid particles in a viral capsid preparation, for example, a viral capsid preparation resulting from a process intended to produce a recombinant virus that contains a heterologous nucleic acid.

[0107] Generation of viral capsid preparations Viral capsid preparations can be produced (e.g., "manufactured") from cultured host cells. A wide range of host cells can be used, such as bacterial, yeast, insect, or mammalian cells. In some embodiments, the cultured host cells are mammalian or insect host cells. In some embodiments, the host cells can be cells (or cell lines) suitable for recombinant AAV (rAAV) generation or production (e.g., HeLa, Cos-7, HEK293, A549, BHK, Vero, RD, HT-1080, ARPE-19, or MRC-5 cells). In some embodiments, the host cells are HeLa cells. In some embodiments, the host cells are HEK293 cells.

[0108] Recombinant nucleic acid molecule or vector (e.g., recombinant AAV vector) can be delivered to host cell culture using any suitable method known in the art. In some embodiments, a stable host cell line is generated that has the recombinant nucleic acid molecule or vector integrated into its genome. In some embodiments, a stable host cell line is generated that contains the AAV vector described herein. After transfection of the AAV vector into the host culture, integration of the rAAV into the host genome can be assayed by a variety of methods (e.g., antibiotic selection, fluorescence-activated cell sorting, Southern blot, PCR-based detection, fluorescent in situ hybridization) as described in Nakai et al., Nature Genetics (2003) 34, 297-302; Philpott et al., Journal of Virology (2002) 76(11):5411-5421, and Howden et al., J Gene Med 2008; 10:42-50. Additionally, stable cell lines can be established according to protocols well known in the art (e.g., those described in Clark, Kidney International Vol 61 (2002):S9-Sl5, and Yuan et al., Human Gene Therapy 2011 May; 22(5):613-24).

[0109] In producing a viral capsid preparation, the host cell can typically be provided with a viral vector. For example, to produce (e.g., manufacture) an AAV capsid preparation, the host cell can be provided with an AAV vector, Rep and Cap gene functions, and additional helper functions. The Rep and Cap gene functions can be provided to the host cell by various means (e.g., by electroporation of a plasmid or any type of vector containing wild-type AAV Rep and Cap genes, and Rep and Cap mRNA). The additional helper functions can be provided, for example, by adenovirus (AV) infection, by a plasmid carrying all of the required AV helper function genes, or by other viruses such as herpes simplex virus (HSV) or baculovirus. Any genes, gene functions, or other genetic material required for rAAV production by the host cell can be transiently present in the host cell or stably inserted into the host cell genome. Suitable rAAV production methods for generating viral capsid preparations include those disclosed in Clark et al., Human Gene Therapy 6:1329-1341 (1995), Martin et al., Human Gene Therapy Methods 24:253-269 (2013), Thorne et al., Human Gene Therapy 20:707-714 (2009), Fraser Wright, Human Gene Therapy 20:698-706 (2009), and Virag et al., Human Gene Therapy 20:807-817 (2009).

[0110] Various methods can be used to release viral capsids from host cells. For example, lysis of virus-infected cells (e.g., AAV-infected cells) can be achieved by chemical or enzymatic treatment of the cells to release infectious viral particles. These methods include the use of nucleases (e.g., benzonase or DNAse), proteases (e.g., trypsin), or detergents or surfactants. Physical disruption (e.g., homogenization or trituration), or application of pressure via a microfluidizer to pressurize the cells, or freeze-thaw cycles can also be used.

[0111] Alternatively, supernatant can be collected from AAV-infected cells without the need to lyse the cells.

[0112] After release of the viral capsids, the sample may be subjected to one or more processes (e.g., purification to remove cellular debris and / or helper virus particles, and / or heat inactivation of the helper virus) before being used with the methods of the present disclosure.

[0113] Characterization of viral capsid preparations Typically, the full and empty capsid particles in a given virus capsid preparation are of the same virus and the same serotype. In some embodiments, the capsid particles comprise capsids of adeno-associated virus (AAV) (e.g., recombinant AAV (rAAV)). Examples of AAV capsid serotypes include serotypes 8, 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, rh10, hu37, or any one of these variants. In some embodiments, the capsid is derived from AAV8 or a variant thereof. In some embodiments, the capsid is derived from AAV9 or a variant thereof. In some embodiments, the capsid is derived from AAVrhlO or a variant thereof. In some embodiments, the capsid is derived from AAVhu37 or a variant thereof.

[0114] Anion Exchange Media The disclosed methods are not limited to any particular column configuration or dimensions, type of separation medium, or type of separation chemistry. For example, in some embodiments, the anion exchange medium is a weak ion exchanger. In some embodiments, the anion exchange medium is a strong ion exchanger.

[0115] In some embodiments, the anion exchanger is in the form of a packed bed.

[0116] In some embodiments, the anion exchanger is a chromatographic monolithic column. As an example, a CIMmultus® monolithic column (e.g., a CIMmultus® monolithic QA column) can be used.

[0117] Wash solutions and fractions Wash solutions compatible for use in the methods of the present disclosure generally contain one or more salts and optionally a buffer (eg, a buffer described herein).

[0118] First Wash Solution The method of the present disclosure generally includes a step of passing or applying a first washing solution to an anion exchange medium, for example, to obtain a first washing fraction (e.g., a washing fraction containing empty virus capsid particles). Such a washing solution generally contains a quaternary ammonium salt. In many embodiments, (i) the concentration of the quaternary salt in the first washing solution remains constant during the process while the first washing solution is passed or applied to the anion exchange medium; and / or (ii) the salt composition of the elution solution remains constant during the process while the elution solution is passed or applied to the anion exchange medium.

[0119] In some embodiments, the concentration of the quaternary ammonium salt in the washing solution is 30 mM to 200 mM (e.g., 50 mM to 180 mM, 70 mM to 160 mM, 90 mM to 150 mM, 100 mM to 130 mM, or 100 mM to 120 mM and 30 mM to 200 mM, including all integers therebetween).

[0120] In some embodiments, the concentration of the quaternary ammonium salt in the wash solution is at least 30 mM, at least 50 mM, at least 70 mM, at least 90 mM, or at least 100 mM.

[0121] In some embodiments, the concentration of the quaternary ammonium salt in the wash solution is 200 mM or less, 180 mM or less, 160 mM or less, 150 mM or less, 140 mM or less, 130 mM or less, or 120 mM or less.

[0122] In some embodiments, the concentration of the quaternary ammonium salt in the washing solution is 30 mM to 200 mM (e.g., 50 mM to 180 mM, 70 mM to 160 mM, 90 mM to 150 mM, 100 mM to 140 mM, 100 mM to 130 mM, or 100 mM to 120 mM and 30 mM to 200 mM, including all integers therebetween).

[0123] In some embodiments, the concentration of the quaternary ammonium salt in the wash solution is about 110 mM.

[0124] In some embodiments, the quaternary ammonium salt is a tetraalkylammonium salt, such as a tetraalkylammonium chloride or a tetraalkylammonium acetate.In some embodiments, the quaternary ammonium salt is a tetraalkylammonium chloride selected from the group consisting of tetramethylammonium chloride (TMAC), tetraethylammonium chloride (TEAC), tetrapropylammonium chloride (TPAC), tetrabutylammonium chloride (TBAC), benzyltributylammonium chloride (BTBAC), or any combination thereof.In some embodiments, the quaternary ammonium salt is tetraethylammonium chloride (TEAC).

[0125] In some embodiments, the quaternary ammonium salt is a tetraalkylammonium acetate selected from the group consisting of tetramethylammonium acetate, tetraethylammonium acetate (TEA-Ac), tetrapropylammonium acetate, tetrabutylammonium acetate, and any combination thereof. In some embodiments, the quaternary ammonium salt is TEA-Ac.

[0126] In some embodiments, the quaternary ammonium salt is choline chloride.

[0127] In some embodiments, the first wash solution further comprises a divalent salt, e.g., MgCl2, as further described herein. For example, in some embodiments, the first wash solution comprises MgCl2 at a concentration of 1 mM to 10 mM, e.g., 1 mM to 5 mM (e.g., about 2 mM and all numbers in and between 1 mM and 10 mM).

[0128] In some embodiments, the first wash solution elutes empty capsid particles present in the viral capsid preparation, e.g., such that empty capsid particles present in the viral capsid preparation appear in the first wash fraction. For example, in some embodiments, the first wash solution comprises 50% or more (e.g., 50%-100%, including all numbers in and between 50%-100%), 60% or more (e.g., 60%-100%), 70% or more (e.g., 70%-100%), 75% or more (e.g., 75%-100), 80% or more (e.g., 80%-100%), 85% or more (e.g., 85%-100%), 90% or more (e.g., 90%-100%), 95% or more (e.g., 95%-100%), or 99% or more (e.g., 99%-100%) of the empty capsid particles present in the virus capsid preparation.

[0129] In some embodiments, the first wash fraction comprises 50% or more (e.g., 50%-100%, including all integers in and between 50%-100%), 60% or more (e.g., 60%-100%), 70% or more (e.g., 70%-100%), 75% or more (e.g., 75%-100%), 80% or more (e.g., 80%-100%), 85% or more (e.g., 85%-100%), 90% or more (e.g., 90%-100%), 95% or more (e.g., 95%-100%), or 99% or more (e.g., 99%-100%) of the empty capsid particles present in the virus capsid preparation.

[0130] In some embodiments, the first washing solution does not substantially elute the complete capsid particles present in the virus capsid preparation. For example, in some embodiments, the first washing solution elutes 50% or less (e.g., 0-50% (including all integers in and between 0-50%)), 45% or less (e.g., 0-40%), 40% or less (e.g., 0-40%), 35% or less (e.g., 0-35%), 30% or less (e.g., 0-30%), 25% or less (e.g., 0-25%), 20% or less (e.g., 0-20%), 15% or less (e.g., 0-15%), 10% or less (e.g., 0-10%), 5% or less (e.g., 0-5%), or 1% or less (e.g., 0-1%) of the complete capsid particles present in the virus capsid preparation.

[0131] In some embodiments, the first wash fraction comprises 50% or less, e.g., 0-50% (including all integers in and between 0-50%), 45% or less (e.g., 0-45%), 40% or less (e.g., 0-40%), 35% or less (e.g., 0-35%), 30% or less (e.g., 0-30%), 25% or less (e.g., 0-25%), 20% or less (e.g., 0-20%), 15% or less (e.g., 0-15%), 10% or less (e.g., 0-10%), 5% or less (e.g., 0-5%), or 1% or less (e.g., 0-1%) of the complete capsid particles present in the virus capsid preparation.

[0132] Second Wash Solution The disclosed methods generally include passing or applying a second wash solution to an anion exchange medium, for example, to obtain a second wash fraction (e.g., a wash fraction that includes the quaternary ammonium salt present in the first wash solution). In many embodiments, the second wash solution does not include a quaternary ammonium salt.

[0133] In some embodiments, the second washing solution includes NaCl, Na2SO4, MgSO4, or any combination thereof. For example, in some embodiments, the second washing solution includes NaCl at a concentration of, for example, 25 mM to 375 mM, 50 mM to 250 mM, 70 mM to 200 mM, 70 mM to 140 mM, or 90 mM to 140 mM (including all integers in and between 25 mM to 375 mM).

[0134] In some embodiments, the second wash solution contains at least 30% (e.g., 30% to 100% (including all integers in and between 30% to 100%)), at least 40% (e.g., 40% to 100%), at least 45% (e.g., 45% to 100%), at least 50% (e.g., 50% to 100%), at least 55% (e.g., 55% to 100%), at least 60% of the quaternary ammonium salt present in the first wash solution. (e.g., 60%-100%), at least 65% (e.g., 65%-100%), at least 70% (e.g., 70%-100%), at least 75% (e.g., 75%-100%), at least 80% (e.g., 80%-100%), at least 85% (e.g., 85%-100%), at least 90% (e.g., 90%-100%), at least 95% (e.g., 95%-100%), or at least 99% (e.g., 99%-100%).

[0135] In some embodiments, the second wash fraction comprises at least 30% (e.g., 30% to 100%, including all integers in and between 30% to 100%), at least 40% (e.g., 40% to 100%), at least 45% (e.g., 45% to 100%), at least 50% (e.g., 50% to 100%), at least 55% (e.g., 55% to 100%), at least 60% of the quaternary ammonium salts present in the first wash solution. (e.g., 60%-100%), at least 65% (e.g., 65%-100%), at least 70% (e.g., 70%-100%), at least 75% (e.g., 75%-100%), at least 80% (e.g., 85%-100%), at least 85% (e.g., 85%-100%), at least 90% (e.g., 90%-100%), at least 95% (e.g., 95%-100%), or at least 99% (e.g., 99%-100%).

[0136] In some embodiments, the second washing solution does not substantially elute the complete capsid particles present in the virus capsid preparation. For example, in some embodiments, the second washing solution elutes 30% or less (e.g., 0-30% and all integers in and between 0-30%), 25% or less (e.g., 0-25%), 20% or less (e.g., 0-30%), 15% or less (e.g., 0-15%), 10% (e.g., 0-10%), 8% or less (e.g., 0-8%), 5% (e.g., 0-5%), 3% or less (e.g., 0-3%), 2% (e.g., 0-2%), or 1% (e.g., 0-1%) of the complete capsid particles present in the virus capsid preparation.

[0137] In some embodiments, the second wash fraction comprises 30% or less (e.g., including 0-30% and all integers in and between 0-30%), 25% or less (e.g., 0-25%), 20% or less (e.g., 0-20%), 15% or less (e.g., 0-15%), 10% or less (e.g., 0-10%), 8% or less (e.g., 0-8%), 5% or less (e.g., 0-5%), 3% or less (e.g., 0-3%), 2% or less (e.g., 0-2%), or 1% or less (e.g., 0-1%) of the complete capsid particles present in the virus capsid preparation.

[0138] Elution solutions and fractions The disclosed methods generally involve passing or applying an elution solution to an anion exchange medium, e.g., to elute intact capsid particles in an elution fraction. In many embodiments, the elution solution does not include a quaternary ammonium salt.

[0139] In some embodiments, the elution solution includes NaCl, Na2SO4, MgSO4, or any combination thereof. For example, in some embodiments, the elution solution includes NaCl at a concentration of, for example, 25 mM to 375 mM (including all integers in and between 25 mM to 375 mM), 50 mM to 250 mM, 70 mM to 200 mM, or 70 mM to 140 mM, or 90 mM to 140 mM.

[0140] In some embodiments, the elution solution comprises an anionic species, tetrafluoroborate (BF4), bromide (Br), or acetate (Ac). In some embodiments, the elution solution comprises an anionic species associated with tetraethylammonium (TEA), such as TEA-BF4, TEA-Br, or TEA-Ac.

[0141] In some embodiments, the elution solution contains at least one salt in common with the second wash solution but at a different concentration, hi some embodiments, the elution solution contains a different salt than that present in the second wash solution.

[0142] In some embodiments, the elution solution substantially elutes the complete capsid particles present in the virus capsid preparation. For example, in some embodiments, the elution solution substantially elutes at least 15% (e.g., 15%-100% (including all integers in and between 15%-100%)), at least 20% (e.g., 20%-100%), at least 25% (e.g., 25%-100%), at least 30% (e.g., 30%-100%), at least 35% (e.g., 35%-100%), at least 40% (e.g., 40%-100%), at least 45% (e.g., 45%-100%), at least 50% (e.g., 50%-100%), at least 60% (e.g., 50%-100%), at least 70% (e.g., 50%-100%), at least 80% (e.g., 50%-100%), at least 90% (e.g., 50%-100%), at least 10 ... % (e.g., 50%-100%), at least 55% (e.g., 55%-100%), at least 60% (e.g., 60%-100%), at least 65% (e.g., 65%-100%), at least 70% (e.g., 70%-100%), at least 75% (e.g., 75%-100%), at least 80% (e.g., 80%-100%), at least 85% (e.g., 85%-100%), at least 90% (e.g., 90%-100%), at least 95% (e.g., 95%-100%), or at least 99% (e.g., 99%-100%). In some embodiments, the elution solution elutes 15%-75% of the complete capsid particles present in the virus capsid preparation.

[0143] In some embodiments, the elution fraction comprises at least 15% (e.g., 15%-100%, including all integers in and between 15%-100%), at least 20% (e.g., 20%-100%), at least 25% (e.g., 25%-100%), at least 30% (e.g., 30%-100%), at least 35% (e.g., 35%-100%), at least 40% (e.g., 40%-100%), at least 45% (e.g., 45%-100%), at least 50% of the complete capsid particles present in the virus capsid preparation. (e.g., 50%-100%), at least 55% (e.g., 55%-100%), at least 60% (e.g., 60%-100%), at least 65% (e.g., 65%-100%), at least 70% (e.g., 70%-100%), at least 75% (e.g., 75%-100%), at least 80% (e.g., 80%-100%), at least 85% (e.g., 85%-100%), at least 90% (e.g., 90%-100%), at least 95% (e.g., 95%-100%), or at least 99% (e.g., 99%-100%). In some embodiments, the elution fraction comprises 15%-75% of the complete capsid particles present in the virus capsid preparation.

[0144] In some embodiments, at least 35% (e.g., 35%-100% (including all integers in and between 35%-100%)), at least 40% (e.g., 40%-100%), at least 45% (e.g., 45%-100%), or at least 50% (e.g., 50%-100%) of the capsid particles present in the elution fraction are complete capsid particles. In some embodiments, at least 50% (e.g., 50%-100%) of the capsid particles present in the elution fraction are complete capsid particles.

[0145] In some embodiments, the elution fraction comprises 65% or less (e.g., 0 to 65% (including all integers in and between 0 to 65%)), 60% or less (e.g., 0 to 60%), 55% or less (e.g., 0 to 55%), 50% or less (e.g., 0 to 50%), 45% or less (e.g., 0 to 45%), 40% or less (e.g., 0 to 40%), 35% or less (e.g., 0 to 35%), 30% or less (e.g., 0 to 30%), 25% or less (e.g., 0 to 25%), 20% or less (e.g., 0 to 20%), 15% or less (e.g., 0 to 15), 10% or less (e.g., 0 to 10%), 5% or less (e.g., 0 to 5%), or 1% or less (e.g., 0 to 1%) of the empty capsid particles from the virus capsid preparation.

[0146] Buffer Systems and Other Solution Components In some embodiments, one or more of the first wash solution, the second wash solution, and the elution solution include a buffer system. For example, the buffer system may maintain a pH around a certain value or within a certain range (e.g., a pH of 6.0 to 10.0, e.g., 7.0 to 9.0). In some embodiments, the included buffer maintains the pH of the solution at about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, or about 10.0. In some embodiments, the included buffer maintains the pH of the solution at about pH 9.0.

[0147] Non-limiting examples of suitable buffer systems include bis-tris-propane based buffers (eg, 20 mM bis-tris-propane system).

[0148] In some embodiments, one or more of the first wash solution, the second wash solution, and the elution solution include a divalent salt, e.g., MgCl2. In some embodiments, one or more of the first wash solution, the second wash solution, and the elution solution include MgCl2 at a concentration of 0.5 mM to 10 mM, e.g., 1 mM to 10 mM, or 1 mM to 5 mM (e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM). In some embodiments, one or more of the first wash solution, the second wash solution, and the elution solution include MgCl2 at a concentration of about 2 mM. In some embodiments, one or more of the first wash solution, the second wash solution, and the elution solution contain MgCl2 at a concentration of at least 1 mM or at least 2 mM. In some embodiments, one or more of the first wash solution, the second wash solution, and the elution solution contain MgCl2 at a concentration of 5 mM or less.

[0149] In some embodiments, one or more of the first wash solution, the second wash solution, and the elution solution include a stabilizing agent or surfactant, such as a non-ionic surfactant. Non-limiting examples of suitable non-ionic surfactants include, for example, Pluronic® F-68. In some embodiments, the stabilizing agent or surfactant is present in the solution at a concentration of about 0.0001%, about 0.0005%, or about 0.001%. In some embodiments, the stabilizing agent or surfactant is present in the solution at a concentration of about 0.001% or less.

[0150] Separation method The disclosed method typically includes a washing step using a washing solution containing a quaternary ammonium salt, the concentration of which remains constant (i.e., is kept isocratic) throughout the washing step. In some embodiments, the concentrations of all salts in the washing solution containing the quaternary ammonium salt are kept isocratic throughout the washing step. In some embodiments, the concentrations of all components in the washing solution containing the quaternary ammonium salt are kept isocratic throughout the washing step.

[0151] Other steps of the disclosed methods, such as additional washing or elution steps, may involve isocratic or gradient separations, as further described herein.

[0152] Isocratic Separation As mentioned, in many embodiments, (i) the concentration of quaternary salt in the first washing solution remains constant throughout the process while the first washing solution is passed or applied to the anion exchanger, and / or (ii) the salt composition of the elution solution remains constant throughout the process while the elution solution is passed or applied to the anion exchanger.In some embodiments, the concentration of the additional components of the first washing solution remains constant throughout the process.For example, in some embodiments, the salt composition of the first washing solution remains constant throughout the process.

[0153] In some embodiments, one or both of the steps of (1) passing or applying the second wash solution to the anion exchanger and (2) passing or applying the elution solution to the anion exchanger involve an isocratic separation. In some embodiments, the salt composition of the second wash solution and / or the salt composition of the elution solution remain constant throughout the steps while the solutions are passed or applied to the anion exchanger.

[0154] In some embodiments, the salt composition of the first wash solution, the salt composition of the second wash solution, and the salt composition of the elution solution remain constant throughout each individual wash or elution step.

[0155] Gradient separation In certain embodiments, one or more steps (e.g., washing and / or elution steps) in the methods disclosed herein involve using a solution whose composition varies during the step or steps. For example, in a gradient separation step, the concentration of salt in the solution may be gradually and continuously (e.g., linearly) increased over time during the step.

[0156] Evaluation of viral capsid preparations and / or fractions In certain embodiments, the viral capsid preparation and / or the elution fraction, or a sample thereof, is evaluated. The first and second wash fractions may or may not be collected. In some embodiments, at least one or both wash fraction samples are collected and evaluated, for example, for quality control purposes.

[0157] Full and empty capsid particles In some embodiments, the presence and / or amount of full and / or empty capsid particles and / or one or more fractions (e.g., elution fractions) in the virus capsid preparation is evaluated. A variety of methods are known in the art to determine the presence of full or empty capsids; many of these methods can also be used to determine the amount, e.g., relative amount, of full and empty capsids. Examples of such methods include, but are not limited to, transmission electron microscopy (TEM), sedimentation velocity-analytical ultracentrifugation (SV-AUC), charge detection mass spectrometry (CDMS), anion exchange high performance liquid chromatography (AEX-HPLC), UV spectrophotometry, and measurement of capsid and genome copies, e.g., by ELISA and qPCR for quality control purposes.

[0158] In some embodiments, ultracentrifugation is used to evaluate viral capsid preparations and / or fractions (e.g., elution fractions). For example, SV-AUC is a solution-state method that measures the sedimentation rate of a molecule when subjected to a high spin speed that applies centrifugal force. Sedimentation rate is measured by the sedimentation coefficient s, which is related to the buoyancy mass, density, specific volume, and frictional forces of the molecule in the formulation matrix. The s value, when normalized to standard solution conditions (standard temperature and pressure) of water at 20°C, is s 20, w The sedimentation coefficient distribution is known as the area under the peak, which is a fundamental molecular parameter that defines the mass and shape as well as the conformation of a molecule. The sedimentation coefficient distribution can be quantified by the area under the peak, which is directly related to the amount of the molecule at that sedimentation coefficient. The accuracy with which the peak area represents the true population depends in part on the suitability of the detection system and the number of data points collected.

[0159] SV-AUC can be applied, for example, to separate different types of capsid particles, for example, complete capsid particles from empty capsid particles. The mass of virus particles with the same virus type and serotype can be different depending on the presence of complete vector genome (similar to complete capsid particles), or the presence of only a part of vector genome or the complete absence of vector genome (similar to empty capsid particles). For example, empty capsid particles have less DNA than complete capsids, are lighter than complete capsids, and sediment more slowly than complete capsids. Thus, the s value in the SV-AUC method reflects the size of the DNA packaged within the virus capsid particle.

[0160] In some embodiments, UV spectrophotometry is used to evaluate viral capsid preparations and / or fractions (e.g., elution fractions). For example, the amount of light at or about 254 nm and / or 260 nm wavelengths absorbed by a sample is roughly proportional to the concentration of nucleic acid in the sample. Additionally, proteins can be detected at 254 nm (A 254 ) or 260nm(A 260 The absorbance (A) at 280 nm is 280 ) is larger; the opposite is true for nucleic acids, where A 280 A than 254 Or A 260 Therefore, full capsid particles (which have a larger amount of DNA than empty capsid particles) have a larger A 254 / A 280 Or A 260 / A 280 This difference can be utilized to assess the relative amounts of full and empty particles in a sample. 254 , A 260 , A 280 , A 254 / A 280 Ratio, or A 260 / A 280 One or more of the ratios may be evaluated.

[0161] In addition, some impurities in the sample may absorb light at a wavelength of 230 nm; these contaminants typically outnumber those that absorb at 280 nm. 260 / A 230 The ratio may provide some indication of the purity of the sample. 230 and / or A 260 / A 230 The value is evaluated.

[0162] For example, in some embodiments, empty capsid particles are preferentially released from the AEX medium before full capsid particles are released. In these embodiments, the A of one fraction (where empty capsid particles are preferentially released) 254 / A 280 Or A 260 / A 280 The ratio is lower than that of subsequent fractions, where intact capsid particles are preferentially released (eg, as shown in FIG. 1).

[0163] In some embodiments, full capsid particles are preferentially released from the AEX medium before empty capsid particles are released. In these embodiments, the A of one fraction (where full capsid particles are preferentially released) 254 / A 280 Ratio or A 260 / A 280 The ratio is higher than that of the subsequent fractions, where empty capsid particles are preferentially released.

[0164] The order of preferential release may be related to one or more of a variety of aspects, including, but not limited to, a) the characteristics of the nucleic acid payload, b) the capsid serotype, c) the viral capsid (e.g., rAAV) preparation conditions, and d) the characteristics of the AEX medium.

[0165] Other evaluations In some embodiments, fraction or sample thereof is evaluated to determine the presence or amount of analyte, for example, a component of washing solution.For example, in some embodiments, the sample of the second washing fraction is evaluated to determine the presence or amount of quaternary ammonium salt in the sample.Methods for detecting or quantifying quaternary ammonium salt are known in the art, and include, for example, liquid chromatography-mass spectrometry (LC-MS) and reverse phase high performance liquid chromatography (RP-HPLC). EXAMPLES

[0166] Example 1: Effect of the stationary phase on the separation of empty and full capsid particles in a NaCl gradient To test the effect of stationary phase type on the separation of empty and full capsid particles, viral capsid preparations containing full and empty AAV capsid particles were subjected to NaCl gradient elution conditions with different types of stationary phases.

[0167] Both monolithic and bead-based anion exchange media were tested. CIMmultus® QA columns (BIA Separation) are monolithic poly(glycidyl methacrylate-co-ethylene dimethacrylate) columns functionalized with quaternary amines, which are strong anion exchangers. POROS TM The 50 HQ column (Thermo Scientific) is based on poly(styrene-divinylbenzene) beads functionalized with quaternized polyethyleneimine, a strong anion exchanger. TM Q (Bio-Rad) columns are based on poly(methacrylate) beads functionalized with quaternary amines, a strong anion exchanger.

[0168] AAV8.1 material, including empty and full capsid particles, was purified using POROS TM A NaCl gradient was applied to the 50 HQ column and, in a separate experiment, to a CIMmultus® QA column. AAV8.2 material containing empty and full capsid particles was purified using Nuvia TMA NaCl gradient was applied to the Q column and, in a separate experiment, to a CIMmultus® QA column.

[0169] FIG. 1A shows a representative chromatogram from a separation experiment for AAV8.1 material.

[0170] Figure 1B shows a representative chromatogram from a separation experiment for AAV8.2 material. Figure 2A shows the step yield, i.e., the amount of genome of interest in the pooled fractions relative to the amount in the load material, as determined by quantitative polymerase chain reaction (qPCR). Figure 2B shows the percentage of complete capsid particles present in the eluate, as determined by analytical ultracentrifugation (AUC). Values ​​in both Figures 2A and 2B are from Nuvia TM Normalized to that observed in the Q column.

[0171] For AAV8-1, the POROS TM A better separation of empty capsids (high amounts found in the fractions indicated by "E") from full capsids (high amounts found in the fractions indicated by "F") occurred using the Nuvia 50 HQ column. TM A better separation of empty capsids (high amounts found in the fractions indicated by "E") from full capsids (high amounts found in the fractions indicated by "F") occurred than using the Q column. Furthermore, for AAV8-2, both the step yield and the percentage of full capsid particles were higher in the experiments using the CIMmultus® Q column.

[0172] Example 2: Effect of producer cell line and AAV serotype on the isolation of empty and full capsid particles To test the effect of the load material (producer cell line and AAV serotype) on the separation of empty and full capsid particles, various viral capsid preparations containing full and empty viral particles were subjected to NaCl gradient elution conditions on a CIMmultus® QA column.

[0173] HEK-produced AAV of serotypes AAV8-1, AAV8-2, AAV8-3, and hu37, as well as HeLa-produced AAV9, were subjected to NaCl gradient elution conditions. The results are shown in Figure 3 and Table 1. Figure 3 shows representative chromatograms from experiments using various AAV materials. "E" indicates fractions corresponding to high relative amounts of empty capsids, and "F" indicates fractions corresponding to high relative amounts of full capsids.

[0174] [Table 1]

[0175] Figure 4 shows a graph comparing results from experiments performed on HEK-produced AAV8-1 and AAV8-2, showing the normalized percentage of complete capsid particles in the eluate (y-axis) compared to the percentage of complete capsid particles in the load material (x-axis).

[0176] Example 3: Effect of loading salt concentration on the separation of empty and full capsid particles To test the effect of loading salt concentration on the separation of empty and full capsid particles, viral capsid preparations containing full and empty HeLa-produced AAV8.2 capsid particles were subjected to downstream processing with various amounts of MgCl2.

[0177] After an initial chromatography process, the AAV material produced from HeLa cells was heat inactivated to inactivate any helper virus, and samples were spiked with 200 mM MgCl2 to a final concentration of either 67 mM ("no dilution in load") or 6-7 mM ("with dilution in load"). Another sample did not have any MgCl2 added ("no Mg in load"). The left side of Figure 5 shows an overview of the process.

[0178] The samples were then TM The XQ column was subjected to NaCl gradient elution conditions. A representative chromatogram from this experiment is shown on the right side of Figure 5. In Figure 5, "E" indicates fractions corresponding to high relative amounts of empty capsids and "F" indicates fractions corresponding to high relative amounts of full capsids.

[0179] As shown in FIG. 5, no elution peak was detected for the "no dilution in loading" (67 mM MgCl2) sample, whereas an elution peak was detected when the MgCl2 concentration was diluted down to 6-7 mM.

[0180] Example 4: Gradient elution with various tetramethylammonium chlorides (TMAC) In this example, viral capsid preparations containing full and empty viral capsid particles were subjected to gradient elution conditions using various tetramethylammonium chloride (TMAC) concentrations. As a comparison, similar elution experiments were performed using NaCl. The concentration of TMAC or NaCl was increased over time.

[0181] material and method AAV material was obtained from a pilot-scale 250L bioreactor run. The clarified harvest was affinity captured and an affinity eluate sample was retained for the laboratory-scale AEX run. 1.5 mL of the affinity eluate was diluted 8-fold (to 15 mL) with 20 mM Bis-Tris-Propane (BTP) adjusted to pH 9.0 to reduce the loading conductivity to approximately 4 mS / cm. 10 mL of the diluted affinity eluate was then loaded onto the AEX media pre-equilibrated with pH-matched equilibration buffer (NaCl buffer: 20 mM BTP, 25 mM NaCl, 0.001% (w / v) Pluronic® F-68, pH 9.0; TMAC buffer: 20 mM BTP, 25 mM TMAC, 0.001% (w / v) Pluronic® F-68, pH 9.0). After washing with 10 column volumes (CV) of equilibration buffer, a linear gradient (generated over 90 CV) from about 25 mM to about 182.5 mM NaCl and from about 25 mM to about 326.7 mM TMAC was applied for NaCl and TMAC, respectively. The eluent was collected in fractions, and fractions corresponding to the "empty" and "full" peaks were pooled accordingly.

[0182] The column was then washed with 10 CV of high salt strip solution (20 mM BTP, 2 M NaCl, 2 mM MgCl2, pH 9.0), followed by 10 CV of sanitation solution (3 M NaCl, 1 M NaOH), then 10 CV of column storage solution (50 mM Tris, 150 mM NaCl, 20% ethanol, pH 7.5).

[0183] The relative amount of complete versus empty viral capsid particles eluted in each peak was determined by the absorbance at 254 nm (A 254 ) and absorbance at 280 nm (A 280 ) was determined.

[0184] Results and Discussion FIG. 6 shows exemplary chromatograms from these experiments.

[0185] The chromatogram in Figure 1 shows the A of the eluent during gradient elution. 254 and A 280 As shown, the first fraction is enriched in "empty" AAV particles (A of the first elution peak). 254 / A 280 The ratio is usually less than 1.) On the other hand, the second, later elution fraction is enriched in “intact” AAV particles (A ratio of the second elution peak is usually less than 1.) 254 / A 280 (The ratio is usually greater than 1.) The ascending line indicates an increase in solution conductivity caused by a corresponding increase in eluent concentration.

[0186] This example demonstrates that gradient elution using TMAC achieves superior separation of empty and complete capsids than a comparable gradient elution using NaCl.

[0187] Example 5: Various tetramethylammonium chloride (TMAC), tetrabutylammonium chloride (TBAC), or benzyltributylammonium chloride (BTBAC) and constant MgCl 2 Gradient elution at concentration In this example, viral capsid preparations containing full and empty viral capsid particles were subjected to gradient elution conditions using a series of tetraalkylammonium salts and the yield of full viral capsid particles was assessed.

[0188] material and method AAV material was obtained as described in Example 1. 1.5 mL of the affinity eluate was diluted 8-fold (to 15 mL) with 20 mM Bis-Tris-Propane (BTP) adjusted to pH 9.0 to reduce the loading conductivity to approximately 4 mS / cm. 10 mL of the diluted affinity eluate was then loaded onto an AEX medium (in this case a CIMmultus® QA column) pre-equilibrated with pH-matched equilibration buffer (20 mM BTP, 25 mM NaCl, 2 mM MgCl2, pH 9.0). For elution with NaCl, a linear gradient (generated over 90 CV) from about 25 mM to about 182.5 mM NaCl was applied after washing with 10 column volumes (CV) of equilibration buffer. For elution with QA salts (TMAC, TBAC, or BTBAC), a linear gradient (generated for 90 CV) from about 25 mM to about 337.5 mM QA was applied after washing with 10 column volumes (CV) of equilibration buffer. The eluent was collected in fractions, and fractions corresponding to the "empty" and "full" peaks were pooled accordingly. The column was then washed with 10 CV of high salt strip solution (20 mM BTP, 2 M NaCl, 2 mM MgCl2, pH 9.0), followed by 10 CV of sanitation solution (3 M NaCl, 1 M NaOH), and then 10 CV of column storage solution (50 mM Tris, 150 mM NaCl, 20% ethanol, pH 7.5).

[0189] Results and Discussion Figures 7A-7D show exemplary chromatograms for empty to full AAV particle separation using gradient elution with NaCl or various QAs of increasing functional group size. From top to bottom, elution was obtained from sodium chloride (NaCl) (Figure 7A), tetramethylammonium chloride (TMAC) (Figure 7B), tetrabutylammonium chloride (TBAC) (Figure 7C), or benzyltributylammonium chloride (BTBAC) (Figure 7D) gradients. The chromatogram of the NaCl gradient is shown as a reference for comparing the empty and full fraction separations. The chromatograms show the A of the eluent during the gradient elution. 254 and A 280The measured values ​​are shown. The ascending line indicates the increase in solution conductivity caused by a corresponding increase in eluent concentration.

[0190] Figure 8 shows a graph plotting the ion intensity (x-axis) of the first and second peaks as a function of the size of the cation. As can be seen in Figures 7 and 8, an increasing difference between the peaks is observed with increasing size of the cation.

[0191] This example demonstrates increased separation of full and empty viral capsid particles as the size of the cation (Na or QA) added to the eluent is increased.

[0192] Example 6: Various tetraethylammonium chlorides (TEAC) and constant MgCl 2 Gradient elution at concentration In this example, viral capsid preparations containing empty and complete AAV particles were separated using gradient elution with a tetraethylammonium chloride (TEAC) gradient on a CIMmultus® QA column at a constant magnesium chloride (MgCl) concentration of 2 mM at pH 9.

[0193] material and method 1.5 mL of the affinity eluent was diluted 8-fold (to 15 mL) with 20 mM Bis-Tris-Propane (BTP) adjusted to pH 9.0 to reduce the loading conductivity to approximately 4 mS / cm. 10 mL of the diluted affinity eluent was then loaded onto the AEX medium pre-equilibrated with pH-matched equilibration buffer (20 mM BTP, 25 mM TEAC, 2 mM MgCl2, pH 9.0). After washing with 10 column volumes (CV) of equilibration buffer, a linear gradient (generated for 90 CV) from about 25 to about 337.5 mM TEAC (the concentration of MgCl2 was kept constant at 2 mM) was applied. The eluent was collected in fractions and fractions corresponding to the "empty" and "full" peaks were pooled accordingly. The column was then washed with 10 CV of high salt strip solution (20 mM BTP, 2 M NaCl, 2 mM MgCl2, pH 9.0), followed by 10 CV of sanitation solution (3 M NaCl, 1 M NaOH), and then 10 CV of column storage solution (50 mM Tris, 150 mM NaCl, 20% ethanol, pH 7.5).

[0194] Results and Discussion Figure 9 shows the chromatogram from this experiment. The chromatogram shows the A of the eluent during the gradient elution. 254 and A 280 The measured values ​​are shown. The ascending line indicates the increase in solution conductivity caused by a corresponding increase in TEAC concentration.

[0195] This example demonstrates the separation of empty and complete AAV particles using TEAC gradient elution.

[0196] Example 7: Various Tetraethylammonium Acetate (TEA-Ac) and Constant MgCl 2 Gradient elution at concentration In this example, viral capsid preparations containing full and empty viral capsid particles were subjected to gradient elution conditions using tetraethylammonium acetate (TEA-Ac), the concentration of TEA-Ac was increased over time, and the MgCl2 concentration remained constant at 2 mM MgCl2. material and method

[0197] 1.5 mL of the affinity eluent was diluted 8-fold (to 15 mL) with 20 mM Bis-Tris-Propane (BTP) adjusted to pH 9.0 to reduce the loading conductivity to approximately 4 mS / cm. 10 mL of the diluted affinity eluent was then loaded onto the AEX medium pre-equilibrated with pH-matched equilibration buffer (20 mM BTP, 25 mM NaCl, 2 mM MgCl2, pH 9.0). After washing with 10 column volumes (CV) of equilibration buffer, a linear gradient (generated over 90 CV) from about 25 mM to about 337.5 mM TEA-Ac was applied. The eluent was collected in fractions and fractions corresponding to the "empty" and "full" peaks were pooled accordingly. The column was then washed with 10 CV of high salt strip solution (20 mM BTP, 2 M NaCl, 2 mM MgCl2, pH 9.0), followed by 10 CV of sanitation solution (3 M NaCl, 1 M NaOH), then 10 CV of column storage solution (50 mM Tris, 150 mM NaCl, 20% ethanol, pH 7.5).

[0198] Results and Discussion Figure 10 shows the chromatogram from this experiment. The chromatogram shows the A of the eluent during the gradient elution. 254 and A 280 The measured values ​​are shown. The ascending line indicates the increase in solution conductivity caused by a corresponding increase in TEA-Ac concentration.

[0199] This example demonstrates the separation of full and empty AAV particles from a viral capsid preparation using a TEA-Ac gradient.

[0200] Example 8: Isocratic Elution with Tetraethylammonium Acetate (TEAC) Using a Wash Solution, a Second Wash Solution, and an Elution Solution In this example, viral capsid preparations containing full and empty viral capsid particles were subjected to an initial wash with tetraethylammonium chloride (TEAC) and magnesium chloride (MgCl2), followed by two successive step washes with sodium chloride (NaCl) and MgCl2, followed by isocratic elution with elution with NaCl and MgCl2.

[0201] material and method 1.5 mL of the affinity eluate was diluted 8-fold (to 15 mL) with 20 mM Bis-Tris-propane (BTP) adjusted to pH 9.0 to reduce the loading conductivity to approximately 4 mS / cm. 10 mL of the diluted affinity eluate was then loaded onto the AEX medium pre-equilibrated with pH-matched equilibration buffer (20 mM BTP, 25 mM NaCl, 2 mM MgCl2, pH 9.0). After washing with 10 column volumes (CV) of equilibration buffer, a first 10 CV isocratic wash with TEAC (20 mM BTP, 124.75 mM TEAC, 2 mM MgCl2, pH 9.0) was applied to wash the empty capsids out of the AEX medium. After the first wash, a second isocratic wash containing only NaCl (20 mM BTP, 68.75 mM NaCl, 2 mM MgCl2, pH 9.0) was applied to remove residual TEAC salt from the AEX medium. Finally, the AAV particles (full capsid enriched) were eluted from the AEX medium by elution buffer (20 mM BTP, 109.35 mM NaCl, 2 mM MgCl2, pH 9.0) and collected. The column was then washed with 10 CV of high salt strip solution (20 mM BTP, 2 M NaCl, 2 mM MgCl2, pH 9.0), followed by 10 CV of sanitation solution (3 M NaCl, 1 M NaOH), and then 10 CV of column storage solution (50 mM Tris, 150 mM NaCl, 20% ethanol, pH 7.5).

[0202] Results and Discussion Figure 11 shows chromatograms from these experiments. The peaks corresponding to full and empty AAV particles are shown in Figure 11 and are clearly separated. The dashed line corresponds to the volumetric output of pump "B" (i.e., the pump supplying the wash and elution buffers) relative to pump "A" (i.e., the pump supplying the equilibration buffer).

[0203] This example demonstrates the successful separation of empty and complete viral capsid particles using isocratic elution with TEAC.

[0204] Example 9: Comparison of various gradient and isocratic protocols for the separation of full and empty capsid particles To compare the various gradient and isocratic elution protocols for their ability to separate full and empty particles, AAV8-1 material containing full and empty capsid particles was subjected to the various protocols, all using MgSO4, on a CIMmultus® QA column.

[0205] The following protocols were tested: 1) gradient elution; 2) gradient wash followed by an isocratic elution step; 3) one isocratic wash; 4) three isocratic washes followed by isocratic elution; and 5) two isocratic washes followed by isocratic elution.

[0206] Figures 12A-12E show exemplary chromatograms from these experiments. As shown in Figures 12A-12E, the isocratic elution strategy resulted in a better percentage of collected intact capsid particles and better process yields (of genomes of interest) than the gradient elution method.

[0207] Figures 13A and 13B show graphs comparing the results from the gradient elution protocol compared to the results from the isocratic elution protocol. Figure 13A shows the step yield, i.e., the amount of genome of interest in the pooled fractions relative to the amount of loaded material, as determined by quantitative polymerase chain reaction (qPCR). Figure 13B shows the percentage of complete capsid particles present in the eluate, as determined by analytical ultracentrifugation (AUC). The values ​​in both Figures 13A and 13B are normalized to those observed with the gradient elution protocol.

[0208] Example 10: Various Choline Chloride Concentrations and Constant MgCl 2 Gradient elution at concentration In this example, a viral capsid preparation containing full and empty viral capsid particles was subjected to gradient elution conditions using choline chloride, the concentration of which was increased over time while the MgCl2 concentration remained constant at 2 mM MgCl2.

[0209] material and method 1.5 mL of the affinity eluent was diluted 8-fold (to 15 mL) with 20 mM Bis-Tris-Propane (BTP) adjusted to pH 9.0 to reduce the loading conductivity to approximately 4 mS / cm. 10 mL of the diluted affinity eluent was then loaded onto the AEX medium pre-equilibrated with pH-matched equilibration buffer (20 mM BTP, 25 mM NaCl, 2 mM MgCl2, pH 9.0). After washing with 10 column volumes (CV) of equilibration buffer, a linear gradient (generated over 90 CV) from about 25 mM to about 340 mM choline chloride was applied. The eluent was collected in fractions and fractions corresponding to the "empty" and "full" peaks were pooled accordingly. The column was then washed with 10 CV of high salt strip solution (20 mM BTP, 2 M NaCl, 2 mM MgCl2, pH 9.0), followed by 10 CV of sanitation solution (3 M NaCl, 1 M NaOH), and then 10 CV of column storage solution (50 mM Tris, 150 mM NaCl, 20% ethanol, pH 7.5).

[0210] result Figure 14 shows an exemplary chromatogram from this experiment. As shown in Figure 14, gradient separation using choline chloride resulted in the separation of full and empty AAV capsids from the viral capsid preparation.

[0211] Example 11: Optimization for elution of host cell proteins and intact capsids To examine the effect of column loading and load residence time on the amount of host cell proteins and of full capsid particles in the eluent, different run conditions were tested with AAV material containing full and empty capsid particles.

[0212] Figure 15A shows the parameters for the five different runs tested in this set of experiments. The column was loaded with 1x, 6x, and 12x amounts of AAV8-2 material at 1x, 1 / 2x, or 1 / 4x residence times. Samples were run on a CIMmultus® QA column using a NaCl gradient. Figure 15B shows chromatograms from the various runs, the conditions of which are shown in Figure 15A. "E" indicates fractions corresponding to high relative amounts of empty capsids, and "F" indicates fractions corresponding to high relative amounts of full capsids.

[0213] FIG. 15C shows plots from a desirability analysis using JMP® software for variables such as capsid loading, normalized host cell protein amount in the eluate, and normalized percentage of complete capsids in the eluate.

[0214] As shown in FIG. 15C, increasing capsid loading resulted in an increased percentage of intact capsids in the eluate, but also in an increased amount of host cell protein in the eluate.

[0215] Figures 16A and 16B show the yield (of genome of interest), percentage of complete capsid, and host cell protein amount from a similar experiment using AAV8-1 material run on a CIMmultus® QA column using a NaCl gradient. Figure 16A shows the amount per fraction, and Figure 16B shows the cumulative amount. As can be seen in Figures 16A and 16B, fractions after the second peak have detectable host cell protein levels, and collecting additional fractions to increase the percentage of complete capsid protein collected also increases the cumulative amount of host cell protein.

[0216] Example 12: Use of divalent ions in the mobile phase in the separation of empty and full capsid particles To test the effect of using a mobile phase with divalent ions on the separation of full and empty capsid particles, samples of AAV8-1 and AAV8-2 material containing full and empty capsid particles were run on a CIMmultus® QA column using either a NaCl gradient or a gradient with a mobile phase containing MgSO4.

[0217] Representative chromatograms from these experiments are shown in Figure 17. As shown in Figure 17, the use of a mobile phase containing divalent ions enhanced the separation of full and empty capsid particles for both AAV8-1 and AAV8-2.

[0218] Example 13: Integrated optimization of mobile phase and isocratic elution To test the effect of combining the use of divalent ions and isocratic elution on the separation of full and empty capsid particles, samples of AAV8.1 material containing full and empty capsid particles were run on a CIMmultus® QA column using one of three conditions: 1) NaCl with gradient elution; 2) tetraethylammonium chloride (TEAC) gradient and 2 mM MgCl2 with gradient elution; and 3) tetraethylammonium chloride (TEAC) and 2 mM MgCl2 with isocratic elution.

[0219] Figures 18A-18C show representative chromatograms from these experiments, where "E" indicates fractions corresponding to high relative amounts of empty capsids and "F" indicates fractions corresponding to high relative amounts of full capsids.

[0220] Figures 19A and 19B show plots showing the step yields (genome of interest as measured by quantitative PCR) and percentage of complete capsid particles (as measured by analytical ultracentrifugation) from these experiments, normalized to levels in the NaCl gradient experiments.

[0221] As shown in Figure 18A, the use of TEAC and MgCl2 in the gradient elution method improved the step yield by 2.3-fold, but as shown in Figure 18B, the use of TEAC and MgCl2 in the isocratic elution method improved the percentage of intact capsid particles by 1.8-fold while maintaining a step yield comparable to that obtained with the NaCl gradient.

[0222] Example 14: Use of anionic species in the mobile phase To test the effect of using an elution solution containing an anionic species on the separation of full and empty capsid particles, samples of AAV8.1 material containing full and empty capsid particles were run on a CIMmultus® QA column using one of several linear gradient elution solutions.

[0223] 1.5 mL of the affinity eluent was diluted 8-fold (to 15 mL) with 20 mM Bis-Tris-Propane (BTP) adjusted to pH 9.0 to reduce the loading conductivity to approximately 4 mS / cm. 10 mL of the diluted affinity eluent was then loaded onto the AEX medium pre-equilibrated with pH-matched equilibration buffer (20 mM BTP, 25 mM NaCl, 2 mM MgCl2, pH 9.0). The AAV particles were eluted from the AEX medium by a linear gradient elution containing 20 mM BTP, 0.001% (w / v) Pluronic® F-68, 25-340 mM TEA-X (pH 9.0) over 90 column volumes (CV). where X was one of the following counterions associated with tetraethylammonium (TEA): tetrafluoroborate (BF4), bromide (Br), and acetate (Ac). The column was then washed with 10 CV of high salt strip solution (20 mM BTP, 2 M NaCl, 2 mM MgCl2, pH 9.0), followed by 10 CV of sanitation solution (3 M NaCl, 1 M NaOH), and then 10 CV of column storage solution (50 mM Tris, 150 mM NaCl, 20% ethanol, pH 7.5).

[0224] Representative results from these experiments are shown in Figure 20. For all anion species tested, two elution peaks were observed, corresponding to primarily empty capsid particles (the left-most peak in each chromatogram) and primarily full capsid particles (the right-most peak in each chromatogram). Compared to the elution peak obtained when TEA-BF4 was used in the elution solution, the elution peak obtained when TEA-Br was used in the elution solution was clearer, and the elution peak obtained when TEA-Ac was used in the elution solution was even clearer.

[0225] Thus, this example demonstrates that an elution solution containing anionic species can be used in a method to separate empty capsid particles from full capsid particles.

[0226] Example 15: MgCl in gradient elution 2 Concentration effect To test the effect of MgCl2 concentration in the elution solution on the separation of full and empty capsid particles, a sample of AAV8.1 material containing full and empty capsid particles was run on a CIMmultus® QA column.

[0227] 1.5 mL of the affinity eluent was diluted 8-fold (to 15 mL) with 20 mM Bis-Tris-propane (BTP) adjusted to pH 9.0 to reduce the loading conductivity to approximately 4 mS / cm. 10 mL of the diluted affinity eluent was then loaded onto the AEX media pre-equilibrated with pH-matched equilibration buffer (20 mM BTP, 25 mM NaCl, 2 mM MgCl, pH 9.0). The AAV particles were eluted from the AEX medium by linear gradient elution containing 20 mM BTP, 0.001% (w / v) Pluronic F-68®, 25-340 mM TEA-Ac (pH 9) over 90 column volumes (CV) with or without MgCl2 (the MgCl2 concentration in the elution solution was held constant at 0, 0.2 mM, 1 mM, or 5 mM). The column was then washed with 10 CV of high salt strip solution (20 mM BTP, 2 M NaCl, 2 mM MgCl2, pH 9.0), followed by 10 CV of sanitation solution (3 M NaCl, 1 M NaOH), and then 10 CV of column storage solution (50 mM Tris, 150 mM NaCl, 20% ethanol, pH 7.5).

[0228] Representative chromatograms obtained are shown in FIG. 21. At all concentrations tested, two elution peaks were observed. These corresponded to mainly empty capsid particles (the left-most peak in each chromatogram) and mainly full capsid particles (the right-most peak in each chromatogram). However, at 0.2 mM MgCl2 and without MgCl2, a shoulder peak was also detectable. This shoulder peak disappeared in the 1 mM and 5 mM MgCl2 conditions tested. While the distance between the peaks decreased as the concentration of MgCl2 increased from 1 mM to 5 mM, the two peaks were still distinguishable at 5 mM MgCl2.

[0229] Example 16: Comparison of isocratic vs. gradient elution methods for separating empty and full capsid particles In this example, two methods were used to separate empty and full capsid particles: one using gradient elution and the other using isocratic elution, both of which used at least one isocratic wash step with a solution containing a quaternary ammonium salt.

[0230] Samples of AAV8.1 material containing full and empty capsid particles were run over a CIMmultus® QA column using either Protocol 1 or Protocol 2 below.

[0231] Protocol 1 (Gradient Elution): - an isocratic wash using a solution containing 20 mM BTP, 0.001% (w / v) Pluronic® F-68, and 206.5 mM TEA-Ac (pH 9) over 10 column volumes - Linear gradient elution using a solution containing 20 mM BTP, 0.001% (w / v) Pluronic® F-68, 2 mM MgCl2, and 25-182.5 mM NaCl (pH 9) over 30 column volumes.

[0232] Protocol 2 (Isocratic Elution): - an isocratic wash using a solution containing 20 mM BTP, 0.001% (w / v) Pluronic® F-68, and 206.5 mM TEA-Ac (pH 9) over 10 column volumes - A second isocratic wash using a solution containing 20 mM BTP, 0.001% (w / v) Pluronic® F-68, 2 mM MgCl2, and 25 mM NaCl (pH 9) over 10 column volumes. - Isocratic elution using a solution containing 20 mM BTP, 0.001% (w / v) Pluronic® F-68, 2 mM MgCl2, and 116.7 mM NaCl (pH 9) over 10 column volumes.

[0233] Figures 22A-22D show representative results from these experiments, with Figures 22A and 22C showing chromatograms from experiments using Protocol 1 (gradient elution) and Protocol 2 (isocratic elution), respectively. Figures 22B and 22D show enlarged images of the left peak (left panels of Figures 22B and 22D) and right peak (right panels of Figures 22B and 22D) of the peaks from Figures 22A and 22C, respectively. The leftmost peaks in Figures 22A and 22C correspond primarily to empty capsid particles, while the rightmost peaks in Figures 22A and 22C correspond primarily to full capsid particles.

[0234] Both Protocol 1 and Protocol 2 yield clearly distinguishable peaks, allowing separation of empty capsid particles from full capsid particles.

[0235] Example 17: Trade-off between percentage of eluted intact capsid particles and process yield The experiments described in this example evaluate the trade-off between the percentage of eluted intact capsid particles and process yield (% genome recovery) and compare the trade-off for a method using an isocratic wash with a quaternary ammonium salt to a method using NaCl in the process buffer.

[0236] Samples of AAV8.1 material containing full and empty capsid particles were run on a CIMmultus® QA column using either (1) NaCl process buffer or (2) an isocratic wash using a solution containing TEA-Ac. For both the NaCl process and the isocratic TEA-Ac wash process, parallel sets of experiments were run using either isocratic or gradient elution with a solution containing NaCl and MgCl2. For the NaCl process experiments with isocratic elution, NaCl was included in both the isocratic and wash steps. For the NaCl process experiments with gradient elution, the elution was run with a gradient of NaCl and no wash step was used.

[0237] The percentage of intact capsid particles present in the eluate was calculated using analytical ultracentrifugation. The process yield, i.e., the amount of genome of interest in the pooled fractions relative to the amount in the load, was determined by quantitative polymerase chain reaction (qPCR) methods.

[0238] Figure 23 shows a plot of step yield (% genome recovery) (y-axis) against the percentage of intact capsid particles (x-axis) obtained from these experiments. Open markers show data from anion exchange chromatography runs using NaCl process buffer, and filled markers show data from anion exchange chromatography runs using an isocratic TEA-Ac wash. Circles show data from runs using isocratic elution, while squares show data from runs using gradient elution.

[0239] For both the NaCl process runs and the isocratic TEA-Ac wash process runs, the general trends that emerged indicated a trade-off between the percentage of intact capsid particles recovered in the eluent and the process yield, however both the process yield and the percentage of intact capsid particles in the eluent were better for the runs used with the isocratic TEA-Ac wash (see the shift to the right in the trend lines for the filled circles and filled squares compared to the trend lines for the open circles and open squares in Figure 23).

[0240] Thus, the methods of the present disclosure represent an improvement over previously disclosed methods of separating empty capsid particles from full capsid particles.

Claims

1. 1. A method for separating full and empty capsid particles in a viral capsid preparation, the method comprising: (a) applying the viral capsid preparation to an anion exchange medium; (b) passing a first wash solution containing a quaternary ammonium salt through the anion exchange medium to obtain a wash fraction containing empty viral capsid particles; (c) passing a second wash solution through the anion exchange medium to obtain a second wash fraction comprising the quaternary ammonium salt; (d) passing an elution solution through the anion exchange medium to elute the intact capsid particles; and (e) collecting the elution fraction containing intact capsid particles; thereby separating full and empty capsid particles, wherein (i) the concentration of the quaternary ammonium salt in the first wash solution remains constant throughout step (b), and / or (ii) the salt composition of the elution solution remains constant throughout step (d). method.

2. (i) step (b); (ii) Step (c); (iii) step (d); or (iv) any combination thereof 2. The method of claim 1, comprising repeating one or more times.

3. The first cleaning solution comprises the quaternary ammonium salt at a concentration of about 90 mM to about 130 mM, wherein the quaternary ammonium salt is (i) a tetraalkylammonium chloride selected from the group consisting of tetramethylammonium chloride (TMAC), tetraethylammonium chloride (TEAC), tetrapropylammonium chloride (TPAC), tetrabutylammonium chloride (TBAC), benzyltributylammonium chloride (BTBAC), and any combination thereof; (ii) tetramethylammonium acetate, tetraethylammonium acetate (TEA-Ac), tetrapropylammonium acetate, tetrabutylammonium acetate, and any combination thereof; and (iii) choline chloride and The method of claim 1 , wherein the second wash solution and the elution solution are free of quaternary ammonium salts.

4. 4. The method of claim 3, wherein, apart from the first wash solution, no other solutions containing the quaternary ammonium salt are passed through or applied to the anion exchange medium.

5. 5. The method of claim 4, wherein, apart from the first wash solution, no other solutions containing any quaternary ammonium salts are passed through or applied to the anion exchange medium.

6. 10. The method of claim 1, wherein any one or more of the first wash solution, the second wash solution, and / or the elution solution further comprises a divalent salt.

7. The divalent salt is MgCl ２ The method of claim 6, wherein

8. The first wash solution contains MgCl at a concentration of about 1 mM to about 10 mM. ２ The method of claim 7, comprising:

9. 4. The method of claim 3, wherein the first wash solution comprises TEAC at a concentration of about 30 mM to about 200 mM.

10. The second wash solution and / or the elution solution may contain NaCl, Na ２ SO ４ , MgSO ４ or any combination thereof.

11. (a) the second wash solution comprises NaCl at a concentration of about 25 mM to about 375 mM; (b) the elution solution comprises NaCl at a concentration of about 25 mM to about 375 mM; (c) the elution solution comprises MgCl 2 at a concentration of about 1 mM to about 5 mM; or (d) any combination thereof.

12. 10. The method of claim 1, wherein the elution solution comprises an anionic species selected from the group consisting of tetrafluoroborate (BF4), bromide (Br), and acetate (Ac).

13. 13. The method of claim 12, wherein the anionic species is associated with tetraethylammonium (TEA).

14. 10. The method of claim 1, wherein the first wash solution, the second wash solution, and / or the elution solution each comprise a buffer system at a pH of about 9.

15. (a) the first wash solution elutes at least 60% of the empty capsid particles present in the viral capsid preparation; or 2. The method of claim 1, wherein (b) the first wash solution elutes 30% or less of the complete capsid particles present in the viral capsid preparation.

16. The second wash fraction comprises the quaternary ammonium salt, wherein: (i) the second wash solution elutes at least 50% of the quaternary ammonium salts present in the first wash solution; (ii) the second wash solution elutes 20% or less of the intact capsid particles present in the viral capsid preparation; (iii) the elution fraction comprises at least 50% of the intact capsid particles present in the viral capsid preparation; (iv) at least 35% of the capsid particles present in the elution fraction are complete capsid particles; (v) the elution fraction contains 50% or less of the empty capsid particles present in the viral capsid preparation; or (vi) any combination thereof.

17. (i) the concentration of the salt in the first wash solution remains constant throughout step (b); (ii) the salt composition of the first wash solution remains constant throughout step (b); (iii) the salt composition of the second wash solution remains constant throughout step (c); (iv) the salt composition of the elution solution remains constant throughout step (d); or (v) any combination thereof.

18. 10. The method of claim 1, wherein the salt composition of the first wash solution, the salt composition of the second wash solution, and the salt composition of the elution solution remain constant throughout each individual wash or elution step.

19. (i) The composition of the first cleaning solution varies during step (b); (ii) the composition of the second wash solution is varied during step (c); (iii) the composition of the elution solution is varied during step (d); or (iv) any combination thereof.

20. (i) the concentration of salt in the second wash solution increases continuously over time during step (c); (ii) the concentration of salt in the elution solution is continuously increased over time during step (d); or (iii) any combination thereof.

21. (i) the concentration of salt in the second wash solution increases linearly with time during step (c); (ii) the concentration of salt in the elution solution increases linearly with time during step (d); or (iii) a combination thereof.

22. The method of claim 21, wherein 1 to 200 total column volumes are eluted.

23. 2. The method of claim 1, wherein the capsid is derived from AAV8 or a variant thereof.

24. 10. The method of claim 1, wherein the anion exchange medium is a monolithic column.

25. The method of claim 25, wherein the concentration of the quaternary ammonium salt in the first cleaning solution remains constant throughout step (b); wherein the capsid is derived from AAV8 or a variant thereof; 2. The method of claim 1, wherein at least 35% of the capsid particles present in the elution fraction are complete capsid particles.

26. 1. A method for producing complete recombinant adeno-associated virus (rAAV) capsid particles, the method comprising: (a) producing a viral capsid preparation comprising full and empty rAAV capsid particles from cultured mammalian or insect cells; (b) applying the viral capsid preparation to an anion exchange medium; (c) passing a first wash solution containing a quaternary ammonium salt through the anion exchange medium to obtain a wash fraction containing empty rAAV viral capsid particles; (d) passing a second wash solution through the anion exchange medium to obtain a second wash fraction comprising the quaternary ammonium salt; (e) passing an elution solution through the anion exchange medium to elute the intact rAAV capsid particles; and (f) collecting the elution fraction containing intact rAAV capsid particles; including; wherein (i) the concentration of the quaternary ammonium salt in the first wash solution remains constant throughout step (c), and / or (ii) the salt composition of the elution solution remains constant throughout step (e).

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