A novel anion-exchange chromatography method for separating empty from intact recombinant adeno-associated virus particles.

Abstract

A method for separating full and empty capsid particles in a viral capsid preparation is provided by overloading the viral capsid preparation onto an anion exchange medium (AEX-WPC-MCC) integrated with weak partitioning chromatography and multi-column chromatography. Also provided is a loading range of viral capsids for overloading the AEX medium to facilitate separation of full and empty capsid particles.

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 / 397,668, filed August 12, 2022, and U.S. Provisional Patent Application No. 63 / 507,443, filed June 9, 2023, the disclosures of each of which are incorporated herein by reference in their entirety for all purposes. [Background technology]

[0002] Recombinant adeno-associated viruses (rAAVs) engineered to carry 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.) are increasingly being investigated as therapeutic agents for various diseases. These rAAVs are engineered by fully or partially deleting internal portions 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 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 regulatory sequences (e.g., promoter and / or enhancer sequences) capable of driving expression of the cargo in target cells of a patient.

[0003] Large-scale manufacturing of these rAAVs can suffer from inefficiencies in packaging nucleic acid cargo into viral capsids, resulting in rAAV preparations containing a mixture of "intact" AAV particles (i.e., particles containing the nucleic acid of interest) and "empty" AAV particles (i.e., AAV particles that lack the nucleic acid cargo in whole or in part). The presence of empty AAV particles in a gene therapy product can necessitate an increase in the overall dose of the rAAV preparation (e.g., in the form of a pharmaceutical composition containing such a rAAV preparation) administered to a patient to achieve therapeutic efficacy, and can enhance or exacerbate the immune response (e.g., development of neutralizing antibodies or T-cell activation) upon administration of the rAAV preparation to a patient.

[0004] In the downstream purification process of rAAV, anion exchange (AEX) chromatography has been utilized as the primary, scalable method for separating empty rAAV (empty rAAV) from complete rAAV (complete rAAV). Although several AEX factors, including the AEX stationary phase, AEX mobile phase, and AEX elution mode, have been evaluated to improve the percentage of complete rAAV, empty / complete separation challenges remain for rAAV viral products. This is thought to be primarily due to the minimal charge difference between empty and complete rAAV as well as the charge heterogeneity of both rAAV species. For example, when using salt linear gradient elution AEX chromatography, the empty rAAV peak elutes only a few milliseconds per centimeter (mS / cm) from the complete rAAV peak, making coelution of empty and complete rAAV in the AEX pool unavoidable. Summary of the Invention

[0005] There is a need in the art for new purification methods that can be used to separate complete AAV particles from empty AAV particles in rAAV preparations. The present disclosure addresses this need, in part, based on the insight that the separation of complete and empty capsid particles in a viral capsid preparation (e.g., a rAAV preparation) using anion exchange (AEX) chromatography can be improved by incorporating weak partitioning chromatography and multiple chromatography techniques into the AEX process, which involve overloading an AEX medium with an rAAV preparation (i.e., a loading material / rAAV capsid preparation), wherein the rAAV loading material contains a specific range of viral particles. In some embodiments, the present disclosure provides a method for incorporating weak partitioning chromatography (WPC) and multiple-column chromatography (MCC) into the AEX method, providing improved empty / complete rAAV separation, wherein empty rAAV preferentially flows through the AEX column during or as a result of column overloading. In certain embodiments, the present disclosure provides an AEX-WPC-MCC rAAV purification method that results in both enhanced viral genome recovery and % intact rAAV in the AEX pool, and reduces AEX column volume and buffer usage, contributing to improved efficiency and cost savings in downstream rAAV processing.

[0006] In some embodiments, the present disclosure provides a method for separating empty capsid particles in a viral capsid preparation comprising empty and full capsid particles, the method comprising the steps of: a) applying an equilibration solution to at least one anion exchange (AEX) medium; and b) applying a volume of the viral capsid preparation to the at least one AEX medium and passing the volume of the viral capsid preparation through the at least one AEX medium to generate a flow-through comprising empty capsid particles, wherein the volume of the viral capsid preparation is at least 1x10 per ml of AEX medium. 14 ~5.5x10 15 and (vp / ml) between 0.01 and 0.01.

[0007] In some embodiments, the present disclosure provides a method for separating empty capsid particles in a viral capsid preparation comprising empty and full capsid particles, the method comprising the steps of: a) applying an equilibration solution to at least one anion exchange (AEX) medium; and b) passing a volume of the viral capsid preparation through the at least one AEX medium to generate a flow-through comprising empty capsid particles, wherein the volume of the viral capsid preparation is at least 1x10 per ml of AEX medium to generate a flow-through comprising empty capsid particles. 14 ~5.5x10 15 The process includes steps including vp between steps 1 and 2.

[0008] In some embodiments, the present disclosure provides a method for separating full and empty capsid particles in a viral capsid preparation comprising empty and full capsid particles, the method comprising the steps of: a) applying an equilibration solution to at least one anion exchange (AEX) medium; b) applying a volume of the viral capsid preparation to the at least one AEX medium and passing the volume of the viral capsid preparation through the at least one AEX medium to generate a flow-through comprising empty capsid particles; and c) applying an elution solution comprising a salt to the at least one AEX medium and passing the elution solution through the at least one AEX medium to generate an eluate comprising full capsid particles, wherein the volume of the viral capsid preparation is at least 1x10 per ml of AEX medium. 14 ~5.5x10 15 The process includes steps including vp between steps 1 and 2.

[0009] In some embodiments, the present disclosure provides a method for separating full and empty capsid particles in a viral capsid preparation comprising empty and full capsid particles, the method comprising: a) applying an equilibration solution to at least one anion exchange (AEX) medium; b) passing a volume of the viral capsid preparation through the at least one AEX medium to generate a flow-through comprising empty capsid particles; and c) passing an elution solution comprising a salt through the at least one AEX medium to generate an eluate comprising full capsid particles, wherein the volume of the viral capsid preparation is at least 1x10 per ml of AEX medium. 14 ~5.5x10 15 Includes vp between.

[0010] The present disclosure also provides a method for increasing the percentage of complete capsid particles in a virus capsid preparation comprising empty and complete capsid particles, the method comprising: a) applying an equilibration solution to at least one anion exchange (AEX) medium; b) applying a volume of the virus capsid preparation to the at least one AEX medium and passing the volume of the virus preparation through the at least one AEX medium to generate a flow-through; c) applying an elution solution comprising a salt to the at least one AEX medium and passing the elution solution through the at least one AEX medium to generate an eluate; and d) collecting the eluate from step c), wherein the volume of the eluate from step d) comprises a higher percentage of complete capsid particles than the percentage of complete capsid particles in an equivalent volume of the virus capsid preparation, and wherein the volume of the virus capsid preparation is greater than or equal to 1x10 per ml of AEX medium. 14 ~5.5x10 15 Includes vp between.

[0011] The present disclosure provides a method for increasing the percentage of complete capsid particles in a viral capsid preparation comprising empty and complete capsid particles, the method comprising the steps of: a) applying an equilibration solution to at least one anion exchange (AEX) medium; b) passing a volume of the viral capsid preparation through the at least one AEX medium to generate a flow-through; c) passing an elution solution comprising a salt through the at least one AEX medium to generate an eluate; and d) collecting the eluate from step c), wherein the volume of the eluate from step d) contains a higher percentage of complete capsid particles than the percentage of complete capsid particles in an equivalent volume of the viral capsid preparation, and wherein the volume of the viral capsid preparation is greater than or equal to 1x10 per ml of AEX medium. 14 ~5.5x10 15 Includes vp between.

[0012] The disclosure also provides a method of elution comprising the steps of: a) contacting at least one anion exchange (AEX) medium with a volume of a viral capsid preparation comprising intact and empty capsid particles, and passing the volume of the viral capsid preparation through the AEX medium to generate a flow-through comprising empty capsid particles; and b) contacting the at least one AEX medium with an elution solution comprising a salt, and passing the elution solution through the at least one AEX medium to generate an eluate comprising intact capsid particles, wherein the volume of the viral capsid preparation is at least 1 x 10 per ml of AEX medium. 14 ~5.5x10 15 The present invention provides a method for detecting vp between the

[0013] The disclosure also provides a method of elution, comprising: a) passing a volume of a viral capsid preparation through at least one anion exchange (AEX) medium to produce a flow-through comprising empty capsid particles; and b) passing an elution solution comprising a salt through at least one AEX medium to produce an eluate comprising intact capsid particles, wherein the volume of the viral capsid preparation is greater than or equal to 1x10 per ml of AEX medium. 14 ~5.5x10 15 The present invention provides a method for detecting vp between the [Brief explanation of the drawings]

[0014] [Figure 1A] Figures 1A and 1B show the gradient elution profile of AEX chromatography for a normal batch load. Figure 1A shows a chromatogram illustrating the gradient elution profile of a normal batch load of rAAV-loaded material containing 4% intact rAAV using a 1 ml CIMmultus®-QA column. Traces of UV280 (mAU), UV254 (mAU), and conductivity (mS / cm) during the AEX salt linear gradient elution phase are shown by the thin solid line, thick solid line, and dashed line, respectively. With increasing conductivity, the empty rAAV peak (indicated as "E"), intact rAAV peak (indicated as "F"), and a third peak (indicated as "T") were sequentially resolved in the salt linear gradient. [Figure 1B] Figures 1A and 1B show the gradient elution profile of AEX chromatography for a regular batch load. Figure 1B shows a chromatogram illustrating the gradient elution profile of a regular batch load of rAAV packing material / capsid preparation containing 4% intact rAAV using a 0.1 ml CIMac®-QA column. The UV280 (mAU), UV254 (mAU), and conductivity (mS / cm) traces for the AEX salt linear gradient elution phase are shown by the thin solid line, thick solid line, and dashed line, respectively. With increasing conductivity, the empty rAAV peak (indicated as "E"), intact rAAV peak (indicated as "F"), and a third peak (indicated as "T") were sequentially resolved in the salt linear gradient. [Figure 2A]Figures 2A-2C show results from an AEX dynamic binding capacity (DBC) run experiment using 11% full rAAV packing material on a 1 ml CIMmultus®-QA column. Figure 2A shows the breakthrough curve for an AEX DBC run using 11% full rAAV packing material on a 1 ml CIMmultus®-QA column. The UV280 (mAU) and UV254 (mAU) traces during the AEX packing phase are shown as thin and thick solid lines, respectively. The viral particle concentrations (vp / ml) and viral genome concentrations (vg / ml) of selected flow-through fractions are shown as open and shaded bars, respectively. [Figure 2B] Figures 2A-2C show results from an AEX dynamic binding capacity (DBC) run experiment using 11% total rAAV packing material on a 1 ml CIMmultus®-QA column. Figure 2B shows a chromatogram illustrating the gradient elution profile of an AEX DBC run using 11% total rAAV packing material on a 1 ml CIMmultus®-QA column. The UV280 (mAU), UV254 (mAU), and conductivity (mS / cm) traces for the AEX salt linear gradient elution phase are shown by the thin solid line, thick solid line, and dashed line, respectively. With increasing conductivity, the empty rAAV peak (indicated as "E"), the total rAAV peak (indicated as "F"), and a third peak (indicated as "T") were sequentially resolved in the salt linear gradient. [Figure 2C]Figures 2A-2C show results from an AEX dynamic binding capacity (DBC) run experiment using 11% full rAAV packing material on a 1 ml CIMmultus®-QA column. Figure 2C shows a chromatogram illustrating the gradient elution profile of an AEX normal batch loading run using 11% full rAAV packing material on a 1 ml CIMmultus®-QA column. The UV280 (mAU), UV254 (mAU), and conductivity (mS / cm) traces for the AEX salt linear gradient elution phase are shown by the thin solid line, thick solid line, and dashed line, respectively. The UV280 (mAU), UV254 (mAU), and conductivity (mS / cm) traces for the AEX salt linear gradient elution phase are shown by the thin solid line, thick solid line, and dashed line, respectively. With increasing conductivity, the empty rAAV peak (designated "E"), the intact rAAV peak (designated "F"), and a third peak (designated "T") were sequentially resolved in the linear salt gradient. [Figure 3]Figures 3A-3F are schematic diagrams showing the operating scheme of the multicolumn chromatography for an AEX-WPC-MCC run. Collectively, all six operating schematics (Figures 3A-3F) show the sequential operation of one loop of a three-column MCC run. Each column is designated as column 1, 2, or 3. An equilibration phase (Eq), a packing phase (L), a gradient elution phase (G), a wash phase (C), and an idle phase (I) are performed sequentially for each designated column. Figure 3A shows the Eq phase on column 1 and the I phase on both columns 2 and 3. Figure 3B shows the packing phase on column 1 and the sequential Eq phases on columns 2 and 3. Figure 3C shows the gradient elution phase + wash phase + Eq phase on column 1, the L phase on column 2, and the I phase on column 3. Figure 3D shows the C phase on column 1 + the C phase on column 2 and the L phase on column 3. Figure 3E shows an L phase in column 1, an I phase in column 2, and a G phase plus a C phase in column 3, and Figure 3F shows a G phase plus a C phase in column 1 and an I phase in both columns 2 and 3. In all figures, a circled "B" with a black arrow represents the buffer pump delivering buffer to the column, and a circled "S" with a black arrow represents the sample pump delivering packing material to the column. [Figure 4A] Figures 4A-4C show graphs depicting breakthrough curves from AEX dynamic binding capacity (DBC) runs of rAAV-loaded material on a 0.1 ml CIMac®-QA column, indicating the loading range (lower and upper loading limits) of the viral load material. Figure 4A shows the breakthrough curve for an AEX DBC run using 11% full rAAV-loaded material on a 0.1 ml CIMac®-QA column. Breakthrough percentages for viral particle concentration (vp / ml) and viral genome concentration (vg / ml) are indicated by open squares connected by solid lines and open circles connected by solid lines, respectively. HCDNA concentration (ng / 1 + 12 vg) is indicated by open triangles. [Figure 4B]Figures 4A-4C show graphs depicting breakthrough curves from AEX dynamic binding capacity (DBC) runs of rAAV-loaded material on a 0.1 ml CIMac®-QA column, indicating the loading range (lower and upper loading limits) of the viral load material. Figure 4B shows the breakthrough curves for an AEX dynamic binding run using 4% full rAAV-loaded material on a 0.1 ml CIMac®-QA column. Breakthrough percentages for viral particle concentration (vp / ml) and viral genome concentration (vg / ml) are indicated by open squares connected by solid lines and open circles connected by solid lines, respectively. HCDNA concentration (ng / 1 + 12 vg) is indicated by open triangles. In FIG. 4B, the circled letter "L" represents the loading condition of the AEX-WPC-MCC run at low-end loading, and the circled letter "H" represents the loading condition of the AEX-WPC-MCC run at high-end loading. [Figure 4C] Figures 4A-4C show graphs depicting breakthrough curves from AEX dynamic binding capacity (DBC) runs of rAAV-loaded material on a 0.1 ml CIMac®-QA column, indicating the loading range (lower and upper loading limits) of the virus-loaded material. Figure 4C shows the relationship of % breakthrough in terms of viral particles (squares) and viral genomes (circles) with respect to column loading to determine the operating space for AEX-WPC-MCC overloaded runs. Breakthrough percentages for viral particle concentration (vp / ml) and viral genome concentration (vg / ml) are shown as open squares connected by solid lines and open circles connected by solid lines, respectively. HCDNA concentration (ng / 1+12vg) is shown as open triangles. [Figure 5A]Figures 5A-5F show the loading profiles, gradient elution profiles, and intact viral AAV capsid recovery for the lower and upper viral capsid preparation / loading materials in AEX-WPC-MCC runs. Figure 5A shows a chromatogram illustrating the loading profile of a lower-load AEX-WPC-MCC run using 11% fully loaded rAAV material on a 0.1 mL CIMac-QA column. Figures 5A and 5C show the gradient elution profiles of lower-load (Figure 5A) and upper-load (Figure 5C) AEX-WPC-MCC runs using 11% fully loaded rAAV material on a 0.1 mL CIMac-QA column, respectively. Loading phase UV280 (mAU) traces for four AEX runs within one MCC loop are indicated by the thin line (Run 1), thick line (Run 2), dashed line (Run 3), and dash-dotted line (Run 4), respectively. [Figure 5B] Figures 5A-5F show the loading profile, gradient elution profile, and recovery of intact viral AAV capsids for the lower and upper limits of viral capsid preparation / loading material in AEX-WPC-MCC runs. Figures 5B and 5D show chromatograms illustrating the gradient elution profile of the lower-load (Figure 5B) and upper-load (Figure 5D) AEX-WPC-MCC runs, respectively, using 11% intact rAAV load material on a 0.1 mL CIMac-QA column. Conductivity (mS / cm) traces are indicated by the long-dashed lines. With increasing conductivity, the empty rAAV peak (indicated as "E"), intact rAAV peak (indicated as "F"), and a third peak (indicated as "T") were sequentially resolved in the linear salt gradient. UV280 (mAU) traces of the gradient elution phase of four AEX runs within one MCC loop are shown in brown (run 1), blue (run 2), red (run 3), and purple (run 4), respectively. [Figure 5C]Figures 5A-5F show the loading profile, gradient elution profile, and recovery of intact viral AAV capsids for the lower and upper limits of viral capsid preparation / loading material in AEX-WPC-MCC runs. Figures 5A and 5C show chromatograms depicting the gradient elution profile of the lower-load (Figure 5A) and upper-load (Figure 5C) AEX-WPC-MCC runs, respectively, using 11% intact rAAV load material on a 0.1 mL CIMac-QA column. UV280 (mAU) traces for the loading phase of four AEX runs within one MCC loop are shown by the thin line (Run 1), thick line (Run 2), dashed line (Run 3), and dash-dotted line (Run 4), respectively. [Figure 5D] Figures 5A-5F show the loading profile, gradient elution profile, and recovery of intact viral AAV capsids for the lower and upper limits of viral capsid preparation / loading material in AEX-WPC-MCC runs. Figures 5B and 5D show chromatograms illustrating the gradient elution profile of the lower-load (Figure 5B) and upper-load (Figure 5D) AEX-WPC-MCC runs, respectively, using 11% intact rAAV load material on a 0.1 mL CIMac-QA column. Conductivity (mS / cm) traces are indicated by the long-dashed lines. With increasing conductivity, the empty rAAV peak (indicated as "E"), intact rAAV peak (indicated as "F"), and a third peak (indicated as "T") were sequentially resolved in the linear salt gradient. UV280 (mAU) traces of the gradient elution phase of four AEX runs within one MCC loop are shown in brown (run 1), blue (run 2), red (run 3), and purple (run 4), respectively. [Figure 5E]Figures 5A-5F show the loading profile, gradient elution profile, and intact viral AAV capsid recovery for the lower and upper limit viral capsid preparation / loading material in AEX-WPC-MCC runs. Figure 5E compares the gradient elution profile between lower and upper limit loading AEX-WPC-MCC runs using 11% intact rAAV load material on a 0.1 m CIMac-QA column. The UV280 (mAU) and UV254 (mAU) traces for the upper limit loading AEX-WPC-MCC run are shown as thick and thin solid lines, respectively. The UV280 (mAU) and UV254 (mAU) traces for the lower limit loading AEX-WPC-MCC run are shown as dashed and dotted lines, respectively. The conductivity (mS / cm) trace is shown as a long-dashed line. The conductivity (mS / cm) trace is shown by a long-dashed line. The empty rAAV peak (indicated as "E"), the full rAAV peak (indicated as "F"), and the third peak (indicated as "T") are indicated by black arrows. [Figure 5F] Figures 5A-5F show the loading profile, gradient elution profile, and intact viral AAV capsid recovery for the lower and upper viral capsid preparation / loading material in AEX-WPC-MCC runs. Figure 5F is a graph showing the relationship between viral genome recovery and % intact rAAV of the pool for AEX runs using 11% intact rAAV load material on a 0.1 mL CIMac-QA column. The normal batch load run, the lower-load AEX-WPC-MCC run, and the upper-load AEX-WPC-MCC run are shown as densely stippled, sparsely stippled, and open circles, respectively. [Figure 6]Figure 6A and 6B are bar graphs comparing the yield of complete viral capsids using regular AEX or AEX-WPC-MCC runs, as well as the separation of empty and complete AAV capsid particles from the capsid preparation / loading material. Figure 6A shows a graph comparing the AEX step yield of complete rAAV from two separate rAAV capsid preparations, AAV Prep 1 (top panel) and AAV Prep 2 (bottom panel), based on viral genome recovery (y-axis), between regular AEX runs and AEX-WPC-MCC runs (x-axis). Figure 6B shows a graph comparing the % AEX pool recoveries (y-axis) of empty rAAV capsids (E), intermediate rAAV capsids (I), and complete rAAV capsids (F) from two separate rAAV capsid preparations, rAAV prep 1 (top panel) and rAAV prep 2 (bottom panel), between regular AEX runs and AEX-WPC-MCC runs (x-axis). [Figure 7] Figure 7 is a chart comparing the projected process characteristics and yields of both the AEX-WPC-MCC run and the AEX regular run at a scale based on small-scale performance. Various process characteristics (total process time, column volume, total buffer usage, cycles, and load volume) and yields (viral genome recovery, pool load rAAV, and productivity) of both the AEX-WPC-MCC run and the AEX regular run are shown. Increases or decreases in process characteristic and yield values ​​between the AEX-WPC-MCC run and the AEX regular run are indicated by up and down arrows, respectively. [Figure 8] Figure 8 is a graph showing the distribution of host cell proteins (HCPs) in AEX salt gradient elution. The resolved and fractionated UV peaks (based on the UV254 / UV280 ratio) obtained from the separation of empty and intact AAV capsid particles from capsid preparations / loading materials using a combination of AEX-WPC-MCC runs and gradient salt elution are identified as the empty peak (denoted as E), the intact peak (denoted as F), and the third peak (denoted as T). The HCP concentration in each fraction is indicated by an open circle, and values ​​below the detection limit are indicated by a "<" symbol. DETAILED DESCRIPTION OF THE INVENTION

[0015] definition When the term "about" is used before a quantitative value, the present disclosure encompasses the specific 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.

[0016] 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 induces a very mild immune response. Gene therapy vectors utilizing AAV can infect both dividing and quiescent cells and can persist extrachromosomally without integrating into the host cell genome. These characteristics make AAV an attractive viral vector for gene therapy. Currently, 13 serotypes of AAV (AAV1-13) are recognized.

[0017] Unless otherwise specified, when the term "between" is used to refer to a range of numerical values, the range includes the 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.

[0018] 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 surrounding a core. In the case of an empty capsid particle, the core may be empty or folded, or may contain only a portion of the vector genome, or may contain a portion of host cell DNA, as described herein. In some embodiments, the capsid particle encapsidates a nucleic acid that is a vector genome and / or 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.

[0019] As used herein, the term "complete capsid 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 by AAV ITRs.

[0020] As used herein, the term "empty capsid particle" refers to a capsid particle that comprises at least one capsid protein and lacks a complete vector genome, e.g., lacks a heterologous nucleic acid of interest flanked on both sides by AAV ITRs, or lacks another portion of the vector genome, either in whole or in part.

[0021] 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 an elution solution applied to a separation medium is gradually changed during the separation.

[0022] As used herein, the term "inverted terminal repeat" (abbreviated as "ITR") refers to symmetrical nucleic acid sequences in the genome of adeno-associated viruses that are required for efficient replication. ITR sequences are located at each end of the AAV DNA genome. ITRs serve as origins of replication for viral DNA synthesis and are essential cis elements for generating AAV integrating vectors.

[0023] Use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing" should generally be understood as open-ended and non-limiting, including grammatical equivalents thereof, unless otherwise specifically stated or understood from context, and for example not excluding additional, unrecited elements or steps.

[0024] As used herein, the terms "isocratic elution" or "isocratic separation" refer to a mode of chromatographic separation in which the concentrations of all salts in solutions (e.g., the "wash" solution during the "wash" step and the "elution" solution during the "elution" step) are kept constant for a defined period of time during the separation. In some embodiments, isocratic elution uses a series of two or more separate solutions during the separation, each of which may have a different fixed concentration of one or more salts relative to the other solutions in the series.

[0025] As used herein, the phrase "isocratic elution gradient" refers to a gradient in which the composition of the mobile phase is changed from step to step 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 next step in the chromatographic run, at which point the composition of the mobile phase is changed such that the concentration of the mobile phase component is increased relative to the concentration of the component in the previous step. Thus, for example, an isocratic elution gradient of MgCl involves having a constant MgCl concentration at each individual step, but using various steps that increase the MgCl concentration from one step to the next.

[0026] As used herein, the terms "normal batch loading," "normal batch run," "normal batch loading run," and the like refer to an AEX process for separating empty and full viral capsid particles, the AEX process being carried out at a rate of approximately 1 x 10 14 vp / ml-column. As used herein, the term "batch loading" or "batch run" refers to an AEX process using an rAAV preparation (loading material) containing less than 1 x 10 vp / ml-column. As used herein, the term "batch loading" or "batch run" refers to an AEX process using an rAAV preparation (loading material) to separate empty and complete viral capsid particles, which includes passing the rAAV preparation (loading material) through a single AEX chromatography medium / column. Alternatively, terms such as "normal batch loading," "normal batch run," and "run of normal batch loading" refer to an AEX process using an rAAV preparation (loading material) to separate empty and complete viral capsid particles, which includes passing the rAAV preparation (loading material) through a single AEX chromatography column. 14vp / ml- refers to an AEX process that uses an rAAV preparation (loading material) containing less than a column and involves passing the rAAV preparation (loading material) through a single AEX chromatography medium / column. As used herein, the terms "normal batch loading," "normal batch run," "normal batch loading run," and the like refer to an AEX process that uses an rAAV preparation (loading material) to separate empty and complete viral capsid particles without the use of multiple-column chromatography methods.

[0027] As used herein, with respect to a "fraction," "eluate," "flow-through," or "wash fraction" (e.g., the eluate following a step of passing or applying an elution solution to an anion exchange medium), "obtaining" or "to obtain" means that the fraction or eluate is produced during and through the step. The "obtained" or "produced" fraction may or may not be collected.

[0028] 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). The quaternary ammonium salt may be 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. The quaternary ammonium salt may be a tetraalkylammonium acetate selected from the group consisting of tetramethylammonium acetate, tetraethylammonium acetate (TEA-Ac), tetrapropylammonium acetate, tetrabutylammonium acetate, and any combination thereof.

[0029] As used herein, the term "recombinant" may be used to describe, for example, a nucleic acid molecule having a sequence that is not found in nature or that is created by the artificial combination of two otherwise separated segments. This artificial combination can be accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid molecules, e.g., by genetic engineering techniques.

[0030] A "recombinant adeno-associated virus preparation" or "rAAV preparation" refers to a product resulting from a method of producing 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 complete and empty rAAV particles. In some embodiments, after an initial upstream operation, the recombinant AAV preparation is subjected to one or more downstream operations, such as, for example, nuclease treatment, filtration to remove host cell impurities, and / or affinity purification using a ligand that binds to the AAV capsid, as is well known to those skilled in the art.

[0031] As used herein, the term "salt composition," when used in reference to a solution, refers to the identity and amount of all salts in that solution. Thus, when the "salt composition" of a solution is said to be constant over a specified duration, it means that the identity and amount of all salts in that solution are constant over the specified duration.

[0032] As used herein, the term "separation chemistry" refers to an active ligand, such as a quaternary amine or a mixture and support matrix of a separation medium.

[0033] As used herein, terms such as "separation medium," "medium," "chromatography medium," and the like refer to a physical structure, such as a resin-packed column or monolith or membrane, to which an rAAV preparation is applied to achieve separation of a specific 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 collect the separated fractions). In some embodiments, the separation medium is an anion exchange medium. In some embodiments, the separation medium is a mixed-mode medium that can function as an anion exchange medium. In some embodiments, the separation medium is a column (e.g., particles in a monolithic column or a packed column). In some embodiments, the separation medium is a membrane.

[0034] As used herein, the term "vector" refers to a nucleic acid molecule that allows the insertion of foreign nucleic acid without impairing its ability to replicate and / or integrate in host cells. A vector may contain a nucleic acid sequence that allows it to replicate in host cells, such as an origin of replication. 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 inserted genes. In some embodiments herein, the vector is an AAV vector.

[0035] Separation method The present disclosure provides improved methods for separating complete virus capsid particles from empty capsid particles in a virus capsid preparation or population that contains both empty and complete capsid particles.

[0036] In some embodiments, the present disclosure provides a method for separating empty capsid particles from full capsid particles in a viral capsid preparation comprising empty and full capsid particles, the method comprising: a) applying an equilibration solution to at least one anion exchange (AEX) medium; and b) applying a volume of the viral capsid preparation to the at least one AEX medium and passing the volume of the viral capsid preparation through the at least one AEX medium to generate a flow-through comprising empty capsid particles. In some embodiments, the present disclosure provides a method for separating empty capsid particles from full capsid particles in a viral capsid preparation comprising empty and full capsid particles, the method comprising: a) applying an equilibration solution to at least one anion exchange (AEX) medium; and b) passing the volume of the viral capsid preparation through the at least one AEX medium to generate a flow-through comprising empty capsid particles. In some embodiments, the volume of the viral capsid preparation is 1 x 10 per milliliter of AEX medium. 14 ~5.5x10 15 Between (e.g., 1x10 14 ~1.5x10 14 , 1.5x1014 ~2x10 14 、2x10 14 ~2.5x10 14 、2.5x10 14 ~3x10 14 、3x10 14 ~3.5x10 14 、3.5x10 14 ~4x10 14 、4x10 14 ~4.5x10 14 、4.5x10 14 ~5x10 14 、5x10 14 ~5.5x10 14 、5.5x10 14 ~6.5x10 14 、6.5x10 14 ~7.5x10 14 、7.5x10 14 ~8.5x10 14 、8.5x10 14 ~9.5x10 14 、9.5x10 14 ~1x10 15 、1x10 15 ~1.5x10 15 、1.5x10 15 ~2x10 15 、2x10 15 ~2.5x10 15 、2.5x10 15 ~3x10 15 、3x10 15 ~3.5x10 15 、3.5x10 15 ~4x10 15 、4x10 15 ~4.5x10 15 、5x10 15 ~5.5x10 15 、1.5x10 14 x5x10 15 、2x10 14 ~4.5x10 15 、2.5x10 14 ~4x10 15 、3x10 14 ~3.5x10 15 、3.5x10 14 ~2.5x10 15 、4x1014 ~2x10 15 , 4.5x10 14 ~1.5x10 15 , 5x10 14 ~1x10 15 , 5.5x10 14 ~9.5x10 14 , 6.5x10 14 ~8.5x10 14 , 6.5x10 14 ~5x10 15 , 7.5x10 14 ~4.5x10 15 , 5.5x10 14 ~5.5x10 15 , 6.5x10 14 ~3.5x10 15 , 8.5x10 14 ~4x10 15 , 9.5x10 14 ~3.5x10 15 , 1x10 15 ~3x10 15 , or 1x10 15 ~2x10 15 , and 1x10 14 ~5.5x10 15 and all integers therebetween, inclusive) viral particles (referred to herein as "vp / mL" or "vp / ml-column").

[0037] In some embodiments, the present disclosure also provides a method for separating complete and empty capsid particles in a viral capsid preparation comprising empty and complete capsid particles, the method comprising: a) applying an equilibration solution to at least one anion exchange (AEX) medium; b) applying a volume of the viral capsid preparation to the at least one AEX medium and passing the volume of the viral capsid preparation through the at least one AEX medium to generate a flow-through comprising empty capsid particles; and c) applying an elution solution comprising a salt to the at least one AEX medium and passing the elution solution through the at least one AEX medium to generate an eluate comprising complete capsid particles. In some embodiments, the present disclosure provides a method for separating full and empty capsid particles in a viral capsid preparation comprising empty and full capsid particles, the method comprising: a) applying an equilibration solution to at least one anion exchange (AEX) medium; b) passing a volume of the viral capsid preparation through the at least one AEX medium to generate a flow-through comprising empty capsid particles; and c) passing an elution solution through the at least one AEX medium to generate an eluate comprising full capsid particles. In some embodiments, the volume of the viral capsid preparation is at least 1 x 10 per milliliter of AEX medium. 14 ~5.5x10 15 Between (e.g., 1x10 14 ~1.5x10 14 , 1.5x10 14 ~2x10 14 , 2x10 14 ~2.5x10 14 , 2.5x10 14 ~3x10 14 , 3x10 14 ~3.5x10 14 , 3.5x10 14 ~4x10 14 , 4x10 14 ~4.5x10 14 , 4.5x10 14 ~5x10 14 , 5x10 14 ~5.5x10 14 , 5.5x10 14 ~6.5x1014 、6.5x10 14 ~7.5x10 14 、7.5x10 14 ~8.5x10 14 、8.5x10 14 ~9.5x10 14 、9.5x10 14 ~1x10 15 、1x10 15 ~1.5x10 15 、1.5x10 15 ~2x10 15 、2x10 15 ~2.5x10 15 、2.5x10 15 ~3x10 15 、3x10 15 ~3.5x10 15 、3.5x10 15 ~4x10 15 、4x10 15 ~4.5x10 15 、5x10 15 ~5.5x10 15 、1.5x10 14 x5x10 15 、2x10 14 ~4.5x10 15 、2.5x10 14 ~4x10 15 、3x10 14 ~3.5x10 15 、3.5x10 14 ~2.5x10 15 、4x10 14 ~2x10 15 、4.5x10 14 ~1.5x10 15 、5x10 14 ~1x10 15 、5.5x10 14 ~9.5x10 14 、6.5x10 14 ~8.5x10 14 、6.5x10 14 ~5x10 15 、7.5x10 14 ~4.5x10 15 、5.5x10 14 ~5.5x10 15 、6.5x10 14~3.5x10 15 , 8.5x10 14 ~4x10 15 , 9.5x10 14 ~3.5x10 15 , 1x10 15 ~3x10 15 , or 1x10 15 ~2x10 15 , and 1x10 14 ~5.5x10 15 (all integers between, inclusive) virus particles.

[0038] In some embodiments, the present disclosure also provides a method for increasing the proportion of complete capsid particles in a virus capsid preparation comprising empty and complete capsid particles, the method comprising: a) applying an equilibration solution to at least one anion exchange (AEX) medium; b) applying a volume of the virus capsid preparation to the at least one AEX medium and passing the volume of the virus preparation through the at least one AEX medium to generate a flow-through; c) applying an elution solution comprising a salt to the at least one AEX medium and passing the elution solution through the at least one AEX medium to generate an eluate; and d) collecting the eluate from step c), wherein the volume of the eluate from step d) comprises a higher proportion of complete capsid particles than the proportion of complete capsid particles in an equivalent volume of the virus capsid preparation. In some embodiments, the present disclosure provides a method for increasing the proportion of complete capsid particles in a viral capsid preparation comprising empty and complete capsid particles, the method comprising: a) applying an equilibration solution to at least one anion exchange (AEX) medium; b) passing a volume of the viral capsid preparation through the at least one AEX medium to generate a flow-through; c) passing an elution solution comprising a salt through the at least one AEX medium to generate an eluate; and d) collecting the eluate from step c), wherein the volume of the eluate from step d) contains a higher proportion of complete capsid particles than the complete capsid particles in an equivalent volume of the viral capsid preparation. In some embodiments, the volume of the viral capsid preparation is 1x10 per ml of AEX medium. 14 ~5.5x1015 Between (e.g., 1x10 14 ~1.5x10 14 , 1.5x10 14 ~2x10 14 , 2x10 14 ~2.5x10 14 , 2.5x10 14 ~3x10 14 , 3x10 14 ~3.5x10 14 , 3.5x10 14 ~4x10 14 , 4x10 14 ~4.5x10 14 , 4.5x10 14 ~5x10 14 , 5x10 14 ~5.5x10 14 , 5.5x10 14 ~6.5x10 14 , 6.5x10 14 ~7.5x10 14 , 7.5x10 14 ~8.5x10 14 , 8.5x10 14 ~9.5x10 14 , 9.5x10 14 ~1x10 15 , 1x10 15 ~1.5x10 15 , 1.5x10 15 ~2x10 15 , 2x10 15 ~2.5x10 15 , 2.5x10 15 ~3x10 15 , 3x10 15 ~3.5x10 15 , 3.5x10 15 ~4x10 15 , 4x10 15 ~4.5x10 15 , 5x10 15 ~5.5x10 15 , 1.5x10 14 x5x10 15 , 2x10 14 ~4.5x10 15 , 2.5x10 14 ~4x10 15 , 3x10 14 ~3.5x1015 , 3.5x10 14 ~2.5x10 15 , 4x10 14 ~2x10 15 , 4.5x10 14 ~1.5x10 15 , 5x10 14 ~1x10 15 , 5.5x10 14 ~9.5x10 14 , 6.5x10 14 ~8.5x10 14 , 6.5x10 14 ~5x10 15 , 7.5x10 14 ~4.5x10 15 , 5.5x10 14 ~5.5x10 15 , 6.5x10 14 ~3.5x10 15 , 8.5x10 14 ~4x10 15 , 9.5x10 14 ~3.5x10 15 , 1x10 15 ~3x10 15 , or 1x10 15 ~2x10 15 , and 1x10 14 ~5.5x10 15 (all integers between , inclusive)

[0039] In some embodiments, step b) further comprises collecting at least a portion of the flow-through and analyzing the content of empty capsids and complete capsids in the collected portion of the flow-through, and the application or passing of the volume of the virus capsid preparation through at least one AEX medium in step b) is stopped if complete capsid particles are detected in the collected portion of the flow-through.

[0040] In some embodiments of the disclosed method, applying or passing a volume of the viral capsid preparation through at least one AEX medium in step b) is stopped when ≦5% (e.g., 5%, 4%, 3%, 2%, 1%, 0.5%, 0.25%, or 0%, and all percentage amounts between 0% and 5%, inclusive) intact capsid particles are detected in the collected portion of the flow-through. In some embodiments of the disclosed sequence, applying or passing a volume of the viral capsid preparation through at least one AEX medium in step b) is stopped when 5% intact capsid particles are detected in the collected portion of the flow-through.

[0041] In some embodiments of the disclosed methods, UV spectrophotometry is used to evaluate viral capsid preparations and / or fractions (e.g., elution fractions). For example, the amount of light absorbed by a sample at wavelengths around 254 nm and / or 260 nm is generally proportional to the concentration of nucleic acid in the sample. Furthermore, proteins have greater absorption at 280 nm (A280) than they do at 254 nm (A254) or 260 nm (A260), and the opposite is true for nucleic acids, which have greater absorption at A254 or A260 than at A280. Thus, intact capsid particles containing more DNA than empty capsid particles have a greater A254 / A280 or A260 / A280 ratio than empty capsid particles. This difference can be used to evaluate the relative amounts of intact and empty particles in a sample. In some embodiments, one or more of A254, A260, A280, the A254 / A280 ratio, or the A260 / A280 ratio are assessed.

[0042] In some embodiments of the disclosed methods, analyzing the content of empty and intact capsids includes determining the A254 / A280 ratio of a collected portion of the flow-through using UV spectrophotometry. In some embodiments, the A254 / A280 ratio is directly proportional to the abundance of intact capsid particles in the sample, e.g., the flow-through.

[0043] In some embodiments, the disclosed methods include using at least two, at least three, or at least four AEX media. In some embodiments, the disclosed methods include using two AEX media. In some embodiments, the disclosed methods include using three AEX media. In some embodiments, the disclosed methods include using four AEX media.

[0044] In some embodiments of the disclosed methods, the volume of the viral capsid preparation applied in step b) is constant among each of the at least two, at least three, or at least four AEX media.

[0045] In some embodiments, the disclosed methods involve the sequential or simultaneous use of at least two, at least three, or at least four AEX media, either simultaneously or sequentially. In some embodiments, the disclosed methods involve the sequential use of at least two, at least three, or at least four AEX media.

[0046] In some embodiments, sequential use involves completing each step using one AEX medium and then repeating each step starting from step a) using a subsequent AEX medium.

[0047] In some embodiments, sequential use involves completing at least one of the steps using one AEX medium, and then performing each of the steps starting with step a) using a subsequent AEX medium. In some embodiments, sequential use involves completing applying a volume of capsid preparation to at least one AEX medium, and then performing step a) using a subsequent AEX medium. In some embodiments, sequential use involves completing generating a flow-through from at least one AEX medium, and then performing step a) using a subsequent AEX medium.

[0048] In some embodiments, sequential use comprises completing application of elution solution to at least one AEX medium and then performing step a) using the subsequent AEX medium. In some embodiments, sequential use comprises completing generation of eluate from at least one AEX medium and then performing step a) using the subsequent AEX medium.

[0049] In some embodiments, the disclosed methods include repeating each of the steps at least once (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times), each time using a different AEX medium. In some embodiments, the disclosed methods include repeating each of the steps twice, each time using a different AEX medium. In some embodiments, the disclosed methods include repeating each of the steps three times, each time using a different AEX medium. In some embodiments, the disclosed methods include repeating each of the steps four times, each time using a different AEX medium.

[0050] In some embodiments, the volume of the viral capsid preparation applied to the at least one AEX medium remains constant between each repetition of the method (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repetitions). In some embodiments, the volume of the viral capsid preparation applied to the at least one AEX medium does not remain constant between each repetition. In some embodiments, the volume of the viral capsid preparation applied to the at least one AEX medium in each repetition is greater than the volume of the viral capsid preparation applied to the at least one AEX medium in the first or previous repetition.

[0051] In some embodiments, the volume of the viral capsid preparation applied to the at least one AEX medium in each repetition is smaller than the volume of the viral capsid preparation applied to the at least one AEX medium in the first or previous repetition. In some embodiments, the volume of the viral capsid preparation applied to the at least one AEX medium increases linearly with each repetition. In some embodiments, the volume of the viral capsid preparation applied to the at least one AEX medium increases stepwise with each repetition. In some embodiments, the volume of the viral capsid preparation applied to the at least one AEX medium decreases linearly with each repetition. In some embodiments, the volume of the viral capsid preparation applied to the at least one AEX medium decreases stepwise with each repetition.

[0052] In some embodiments in which the step is repeated, the method includes passing at least a first wash solution (e.g., at least one wash solution, at least two wash solutions, at least three wash solutions, at least four wash solutions, at least five wash solutions, or more wash solutions) through at least one AEX medium between each repetition.

[0053] In some embodiments, the present disclosure also provides elution methods comprising: a) contacting at least one anion exchange (AEX) medium with a volume of a viral capsid preparation comprising full and empty capsid particles, and passing the volume of the viral capsid preparation through the AEX medium to generate a flow-through comprising empty capsid particles; and b) contacting the at least one AEX medium with a salt-containing elution solution, and passing the elution solution through the at least one AEX medium to generate an eluate comprising full capsid particles. In some embodiments, the present disclosure also provides elution methods comprising: a) passing the volume of the viral capsid preparation comprising full and empty capsid particles through the AEX medium to generate a flow-through comprising empty capsid particles; and b) passing the salt-containing elution solution through the at least one AEX medium to generate an eluate comprising full capsid particles. In some embodiments, the volume of the viral capsid preparation is at least 1 x 10 per ml of AEX medium. 14 ~5.5x10 15 Between (e.g., 1x10 14 ~1.5x10 14 , 1.5x10 14 ~2x10 14 , 2x10 14 ~2.5x10 14 , 2.5x10 14 ~3x10 14 , 3x10 14 ~3.5x10 14 , 3.5x10 14 ~4x10 14 , 4x10 14 ~4.5x10 14 , 4.5x10 14 ~5x10 14 , 5x10 14 ~5.5x10 14 , 5.5x10 14 ~6.5x10 14 , 6.5x10 14 ~7.5x10 14 , 7.5x10 14 ~8.5x10 14 , 8.5x10 14 ~9.5x10 14 , 9.5x10 14~1x10 15 , 1x10 15 ~1.5x10 15 , 1.5x10 15 ~2x10 15 , 2x10 15 ~2.5x10 15 , 2.5x10 15 ~3x10 15 , 3x10 15 ~3.5x10 15 , 3.5x10 15 ~4x10 15 , 4x10 15 ~4.5x10 15 , 5x10 15 ~5.5x10 15 , 1.5x10 14 x5x10 15 , 2x10 14 ~4.5x10 15 , 2.5x10 14 ~4x10 15 , 3x10 14 ~3.5x10 15 , 3.5x10 14 ~2.5x10 15 , 4x10 14 ~2x10 15 , 4.5x10 14 ~1.5x10 15 , 5x10 14 ~1x10 15 , 5.5x10 14 ~9.5x10 14 , 6.5x10 14 ~8.5x10 14 , 6.5x10 14 ~5x10 15 , 7.5x10 14 ~4.5x10 15 , 5.5x10 14 ~5.5x10 15 , 6.5x10 14 ~3.5x10 15 , 8.5x10 14 ~4x10 15 , 9.5x10 14 ~3.5x10 15 , 1x10 15 ~3x10 15 , or 1x10 15~2x10 15 , and 1x10 14 ~5.5x10 15 (all integers between , inclusive)

[0054] In some embodiments of the elution method of the present disclosure, step a) further comprises collecting at least a portion of the flow-through and analyzing the content of empty capsids and complete capsids in the collected portion of the flow-through, and contacting or passing the volume of the virus capsid preparation with at least one AEX medium in step a) is stopped if complete capsid particles are detected in the collected portion of the flow-through.

[0055] In some embodiments of the elution method of the present disclosure, passing a volume of the viral capsid preparation through at least one AEX medium in step a) is stopped when ≦5% (e.g., 5%, 4%, 3%, 2%, 1%, 0.5%, 0.25%, or 0%, and all percentage amounts between 0.5 and 5%, inclusive) intact capsid particles are detected in the collected portion of the flow-through. In some embodiments, passing a volume of the viral capsid preparation through at least one AEX medium in step a) is stopped when 5% intact capsid particles are detected in the flow-through.

[0056] In some embodiments of the elution method of the present disclosure, UV spectrophotometry is used to evaluate viral capsid preparations and / or fractions (e.g., elution fractions). For example, the amount of light absorbed by a sample at wavelengths around 254 nm and / or 260 nm is generally proportional to the concentration of nucleic acid in the sample. Furthermore, proteins have greater absorption at 280 nm (A280) than they do at 254 nm (A254) or 260 nm (A260), and the opposite is true for nucleic acids, which have greater absorption at A254 or A260 than at A280. Thus, intact capsid particles containing more DNA than empty capsid particles will have a greater A254 / A280 or A260 / A280 ratio than capsid mass or empty capsid particles. This difference can be used to evaluate the relative amounts of intact and empty particles in a sample. In some embodiments, one or more of A254, A260, A280, the A254 / A280 ratio, or the A260 / A280 ratio are assessed.

[0057] In some embodiments of the elution methods of the present disclosure, analyzing the content of empty and complete capsids comprises determining the A254 / A280 ratio using UV spectrophotometry, where the ratio of OD at 254 nm / 280 nm is directly proportional to the abundance of complete capsid particles in the flow-through.

[0058] In some embodiments, the elution method of the present disclosure comprises using at least two, at least three, or at least four AEX media. In some embodiments, the elution method of the present disclosure comprises using two AEX media. In some embodiments, the elution method of the present disclosure comprises using three AEX media. In some embodiments, the elution method of the present disclosure comprises using four AEX media.

[0059] In some embodiments of the elution method of the present disclosure, the volume of the viral capsid preparation contacted with at least one AEX medium in step a) is constant among each of the at least two, at least three, or at least four AEX media.

[0060] In some embodiments, the elution methods of the present disclosure involve the simultaneous or sequential use of at least two, at least three, or at least four AEX media. In some embodiments, the elution methods of the present disclosure involve the sequential use of at least two, at least three, or at least four AEX media.

[0061] In some embodiments, sequential use involves completing each of the steps using one AEX medium and then repeating each of the steps starting from step a) using a subsequent AEX medium.

[0062] In some embodiments, sequential use involves completing at least one of the steps and then performing each of the steps starting with step a) using a subsequent AEX medium. In some embodiments, sequential use involves completing applying a volume of capsid preparation to at least one AEX medium and then performing step a) using a subsequent AEX medium. In some embodiments, sequential use involves completing generating a flow-through from at least one AEX medium and then performing step a) using a subsequent AEX medium.

[0063] In some embodiments, sequential use comprises completing application of elution solution to at least one AEX medium and then performing step a) using the subsequent AEX medium. In some embodiments, sequential use comprises completing generation of eluate from at least one AEX medium and then performing step a) using the subsequent AEX medium.

[0064] In some embodiments, the elution method of the present disclosure includes repeating each of the steps at least once (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times), each time using a different AEX medium. In some embodiments, the elution method of the present disclosure includes repeating each of the steps twice, each time using a different AEX medium. In some embodiments, the elution method of the present disclosure includes repeating each of the steps three times, each time using a different AEX medium. In some embodiments, the elution method of the present disclosure includes repeating each of the steps four times, each time using a different AEX medium.

[0065] In some embodiments of the elution method of the present disclosure, the volume of the viral capsid preparation applied to the at least one AEX medium remains constant between each repetition (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repetitions). In some embodiments of the elution method of the present disclosure, the volume of the viral capsid preparation applied to the at least one AEX medium does not remain constant between each repetition. In some embodiments of the elution method of the present disclosure, the volume of the viral capsid preparation applied to the at least one AEX medium in each repetition is greater than the volume of the viral capsid preparation applied to the at least one AEX medium in the first or previous repetition.

[0066] In some embodiments of the elution method of the present disclosure, the volume of the viral capsid preparation applied to the at least one AEX medium in each repetition is smaller than the volume of the viral capsid preparation applied to the at least one AEX medium in the first or previous repetition. In some embodiments of the elution method of the present disclosure, the volume of the viral capsid preparation applied to the at least one AEX medium increases linearly with each repetition. In some embodiments of the elution method of the present disclosure, the volume of the viral capsid preparation applied to the at least one AEX medium increases stepwise with each repetition. In some embodiments of the elution method of the present disclosure, the volume of the viral capsid preparation applied to the at least one AEX medium decreases linearly with each repetition. In some embodiments, the volume of the viral capsid preparation applied to the at least one AEX medium decreases stepwise with each repetition.

[0067] In some embodiments, the disclosed elution methods that include repeating each of the disclosed method steps include passing at least a first wash solution (e.g., at least one wash solution, at least two wash solutions, at least three wash solutions, at least four wash solutions, at least five wash solutions, or more wash solutions) through at least one AEX medium between each repetition.

[0068] Viral capsid preparation and flow-through The disclosed methods generally include applying or contacting a volume of a viral capsid preparation containing full and empty capsid particles to at least one anion exchange medium, and passing the viral capsid preparation through the anion exchange medium to generate a flow-through.

[0069] In some embodiments of the disclosed methods, the volume of viral capsid preparation applied to the AEX medium is 5.5x10 per ml of AEX medium. 14 ~5.5x10 15 Between (e.g., 5.5x10 14 ~6.5x10 14 , 6.5x1014 ~7.5x10 14 , 7.5x10 14 ~8.5x10 14 , 8.5x10 14 ~9.5x10 14 , 9.5x10 14 ~1x10 15 , 1x10 15 ~1.5x10 15 , 1.5x10 15 ~2x10 15 , 2x10 15~ 3x10 15 , 3x10 15~ 3.5x10 15 , 3.5x10 15~ 4x10 15 , 4x10 15~ 4.5x10 15 , 5x10 15~ 5.5x10 15 , 6.5x10 14 ~5x10 15 , 7.5x10 14 ~4.5x10 15 , 8.5x10 14 ~4x10 15 , 9.5x10 14 ~3.5x10 15 , 1x10 15 ~3x10 15 , or 1x10 15 ~2x10 15 , and 5.5x10 14 ~5.5x10 15 (all integers between inclusive) vp.

[0070] In some embodiments of the disclosed methods, the volume of viral capsid preparation applied to the AEX medium is 6.7x10 per ml of AEX medium. 14 ~3.1x10 15 Between (e.g., 6.7x10 14 ~7.4x10 14 , 7.4x10 14 ~8.1x10 14 , 8.1x10 14 ~8.5x10 14 , 8.5x10 14 ~9.2x1014 , 9.2x10 14 ~9.7x10 14 , 9.7x10 14 ~1x10 15 , 1x10 15 ~1.7x10 15 , 1.7x10 15 ~2.4x10 15 , or 2.4x10 15 ~3.1x10 15 ) including vp.

[0071] In some embodiments of the disclosed methods, the volume of viral capsid preparation applied to the AEX medium is 6.5x10 per ml of AEX medium. 14 ~5.5x10 15 Between (6.5x10 14 ~7x10 14 , 7x10 14 ~7.5x10 14 , 7.5x10 14 ~8x10 14 , 8x10 14 ~8.5x10 14 , 8.5x10 14 ~9x10 14 , 9x10 14 ~9.5x10 14 , 9.5x10 14 ~1x10 15 , 1x10 15 ~1.5x10 15 , 1.5x10 15 ~2x10 15 , 2x10 15 ~2.5x10 15 , 2.5x10 15 ~3.0x10 15 , 2.5x10 15 ~3x10 15 , 3x10 15 ~3.5x10 15 , 3.5x10 15 ~4x10 15 , 4x10 15 ~4.5x10 15 , 4.5x10 15 ~5x10 15 , 5x10 15 ~5.5x10 15, 7x10 14~ 5x10 15 , 7.5x10 14~ 4.5x10 15 , 8x10 14 ~4x10 15 , 8.5x10 14 ~3.5x10 15 , 9x10 14 ~2.5x10 15 , 9.5x10 14 ~2x10 15 , or 2x10 15 ~1.5x10 15 , and 6.5x10 14 ~5.5x10 15 (all integers between inclusive) vp.

[0072] In some embodiments of the disclosed methods, the volume of viral capsid preparation applied to the AEX medium is 5.5x10 per ml of AEX medium. 14 ~3.5x10 15 Between (e.g., 5.5x10 14 ~6x10 14 , 6x10 14 ~6.5x10 14 , 6.5x10 14 ~7x10 14 , 7x10 14 ~7.5x10 14 , 7.5x10 14 ~8x10 14 , 8x10 14 ~8.5x10 14 , 8.5x10 14 ~9x10 14 , 9x10 14 ~9.5x10 14 , 9.5x10 14 ~1x10 15 , 1x10 15 ~1.5x10 15 , 1.5x10 15 ~2x10 15 , 2x10 15 ~2.5x10 15 , 2.5x10 15 ~3x10 15 , 3x10 15 ~3.5x1015 , 6x10 14 ~3x10 15 , 6.5x10 14 ~2.5x10 15 , 7x10 14 ~2x10 15 , 7.5x10 14 ~1.5x10 15 , 8x10 14 ~1x10 15 , 8x10 14 ~9x10 14 , or 8.5x10 14 ~9.5x10 14 , and 5.5x10 14 ~3.5x10 15 In some embodiments of the disclosed methods, the volume of viral capsid preparation applied to the AEX medium is 7x10 per ml of AEX medium. 14 ~3x10 15 Between (7x10 14 ~7.5x10 14 , 7.5x10 14 ~8x10 14 , 8x10 14 ~8.5x10 14 , 8.5x10 14 ~9x10 14 , 9x10 14 ~9.5x10 14 , 9.5x10 14 ~1x10 15 , 1x10 15 ~1.5x10 15 , 1.5x10 15 ~2x10 15 , 2x10 15 ~2.5x10 15 , 2.5x10 15 ~3x10 15 , 7.5x10 14 ~2.5x10 15 , 8x10 14 ~2x10 15 , 8.5x10 14 ~1.5x10 15 , 9x10 14 ~1x10 15 , 9.5x10 14 ~1.5x1015 , and 7x10 14 ~3x10 15 (all integers between inclusive) vp.

[0073] In some embodiments of the disclosed methods, the viral capsid preparation comprises from about 1% to about 60% (e.g., from about 1% to about 5%, from about 5% to about 10%, from about 10% to about 15%, from about 15% to about 20%, from about 20% to about 25%, from about 25% to about 30%, from about 30% to about 35%, from about 35% to about 40%, from about 40% to about 45%, from about 45% to about 50%, from about 50% to about 55%, from about 55% to about 60%, from about 5% to about 55%, from about 10% to about 50%, from about 15% to about 45%, from about 20% to about 40%, from about 25 to about 35%, and all percentage amounts between and including about 1% to about 60%) intact capsid particles. In some embodiments of the disclosed methods, the viral capsid preparation contains about 1% to about 40% (e.g., about 1% to about 5%, about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 5% to about 35%, about 10% to about 30%, about 15% to about 25%, about 5% to about 15%, and all percentage amounts between and including about 1% to about 40%) intact capsids. In some embodiments of the disclosed elution methods, the virus capsid preparation contains about 4% to about 11% complete capsid particles (e.g., about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to about 10%, or about 10% to about 11%, about 5% to about 10%, about 6% to about 9%, about 7% to about 9%, about 5% to about 7%, about 4% to about 6%, about 6% to about 8%, about 8% to about 10%, or about 95% to about 11%, and all percentage amounts between and including about 4% to about 11%). In some embodiments of the disclosed methods, the virus capsid preparation contains about 4% complete capsid particles. In some embodiments of the disclosed methods or elution methods, the virus capsid preparation contains about 11% complete capsid particles. In some embodiments, the flow-through generated by passing a viral capsid preparation through at least one AEX medium may contain both "empty capsid particles" and "intermediate capsid particles." As used herein, the term "intermediate capsid particles" refers to viral capsid particles that are composed of fragments of host cell DNA (HCDNA), fragments or portions of the AAV genome, or a combination thereof.Typically, "empty capsid particles" and "intermediate capsid particles" may be released into the same flow-through fraction. In some embodiments, "intermediate capsid particles" comprise between 0% and 40% of the flow-through fraction containing empty and intermediate capsid particles.

[0074] In some embodiments of the disclosed methods, the viral capsid preparation comprises about 6.7 x 10 per ml of AEX medium. 14 ~3.1x10 15 In some embodiments of the disclosed methods, the viral capsid preparation contains about 6.7 x 10 vp per ml of AEX medium, and about 1% to 60% intact capsid particles. 14 ~3.1x10 15 In some embodiments of the disclosed methods, the viral capsid preparation contains about 6.7 x 10 vp per ml of AEX medium, and about 4% to 11% intact capsid particles. 14 ~3.1x10 15 In some embodiments of the disclosed methods, the viral capsid preparation contains about 6.7x10 vp per ml of AEX medium, and about 4% intact capsid particles. 14 ~3.1x10 15 of vp, and approximately 11% intact capsid particles.

[0075] In some embodiments of the disclosed methods, the volume of the viral capsid preparation is 6.5x10 per ml of AEX medium. 14 ~5.5x10 15 In some embodiments of the disclosed methods, the volume of the viral capsid preparation is 6.5 x 10 per ml of AEX medium. 14 ~5.5x10 15 In some embodiments of the disclosed methods, the volume of the viral capsid preparation is 6.5x10 vp per ml of AEX medium, and about 4% intact capsid particles. 14 ~5.5x10 15 vp between 100 and 1100, and about 11% of intact capsid particles.

[0076] In some embodiments of the disclosed methods, the volume of the viral capsid preparation is 6.5x10 per ml of AEX medium. 14 ~5.5x10 15 In some embodiments of the disclosed methods, the volume of the viral capsid preparation is 6.5 x 10 per ml of AEX medium. 14 ~5.5x10 15 In some embodiments of the disclosed methods, the volume of the viral capsid preparation is 6.5 x 10 per ml of AEX medium. 14 ~5.5x10 15 vp between 100 and 150 μg / ml, and approximately 4% of intact capsid particles.

[0077] In some embodiments of the disclosed methods, the volume of the viral capsid preparation is 5.5x10 per ml of AEX medium. 14 ~3.5x10 15 In some embodiments of the disclosed methods, the volume of the viral capsid preparation is 5.5x10 per ml of AEX medium. 14 ~3.5x10 15 In some embodiments of the disclosed methods, the volume of the viral capsid preparation is 5.5 x 10 per ml of AEX medium. 14 ~3.5x10 15 In some embodiments of the disclosed methods, the volume of the viral capsid preparation is 5.5x10 vp per ml of AEX medium, and about 4% intact capsid particles. 14 ~3.5x10 15 vp between 100 and 1100, and about 11% of intact capsid particles.

[0078] In some embodiments of the disclosed methods, the volume of the viral capsid preparation is 7x10 per ml of AEX medium. 14 ~3x10 15In some embodiments of the disclosed methods, the volume of the viral capsid preparation is 7x10 per ml of AEX medium. 14 ~3x10 15 In some embodiments of the disclosed methods, the volume of the viral capsid preparation is 7x10 per ml of AEX medium. 14 ~3x10 15 In some embodiments of the disclosed methods, the volume of the viral capsid preparation is 7x10 vp per ml of AEX medium, and about 4% intact capsid particles. 14 ~3x10 15 vp between 100 and 1100, and about 11% of intact capsid particles.

[0079] In some embodiments of the disclosed methods, the volume of viral capsid preparation applied to or contacted with the AEX medium is between 100 and 1000 ml (e.g., 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000, 200-900, 300-800, 400-700, 450-650, 500-600, and all integers between 100 and 1000, inclusive) per ml of AEX medium. In some embodiments of the disclosed methods, the volume of viral capsid preparation applied to or contacted with the AEX medium is between 200 and 800 ml (e.g., 200-300, 300-400, 400-500, 500-600, 600-700, or 700-800, 300-700, 400-600, 450-550, and all integers between 200 and 800, inclusive) ml per ml of AEX medium.

[0080] In some embodiments of the disclosed methods, the flow-through produced by passing a viral capsid preparation through at least one AEX medium comprises at least 50% (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) of the empty capsids present in the volume of the viral capsid preparation. In some embodiments of the disclosed methods, the flow-through produced by passing a viral capsid preparation through at least one AEX medium comprises at least 90% (e.g., at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the empty capsids present in the volume of the viral capsid preparation. In some embodiments of the disclosed methods, the flow-through produced by passing a viral capsid preparation through at least one AEX medium comprises 99% of the empty capsids present in the volume of the viral capsid preparation.

[0081] In some embodiments of the disclosed methods, the flow-through produced by passing a viral capsid preparation through at least one AEX medium comprises 5% or less (e.g., 5%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or 0%) of the complete capsid particles present in the volume of the viral capsid preparation. In some embodiments of the disclosed methods, the flow-through produced by passing a viral capsid preparation through at least one AEX medium comprises less than 5% (e.g., less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or 0%) of the complete capsid particles present in the volume of the viral capsid preparation. In some embodiments of the disclosed methods, the flow-through produced by passing a viral capsid preparation through at least one AEX medium comprises 5% of the complete capsid particles present in the volume of the viral capsid preparation.

[0082] In some embodiments of the disclosed methods, the flow-through comprises 40% to 100% of the empty capsids present in the viral capsid preparation (e.g., 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100%, 45% to 95%, 50% to 90%, 55% to 85%, 60% to 80%, 65% to 75%, and all percentage amounts between and including 40% to 100%). In some embodiments of the disclosed methods, the flow-through comprises at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, 97%, at least, at least 99%, or 100%) of the empty capsids present in the viral capsid preparation. In some embodiments of the disclosed methods, the flow-through comprises 100% of the empty capsids present in the viral capsid preparation.

[0083] In some embodiments of the disclosed methods, the flow-through produced by passing a viral capsid preparation through at least one AEX medium comprises 5% or less of the complete capsid particles present in the viral capsid preparation (e.g., 5%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or 0%, and all percentage amounts between 5% and 0%, inclusive). In some embodiments of the disclosed methods, the flow-through produced by passing a viral capsid preparation through at least one AEX medium comprises less than 5% of the complete capsid particles present in the viral capsid preparation (e.g., less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or 0%). In some embodiments of the disclosed methods, the flow-through produced by passing a viral capsid preparation through at least one AEX medium comprises 5% of the complete capsid particles present in the viral capsid preparation.

[0084] The disclosed methods typically include an elution step using an elution solution containing a salt, wherein the concentration of the salt in the elution solution remains constant (i.e., isocratically maintained) or varies throughout the wash step. In some embodiments, the salt in the elution solution may include a quaternary ammonium salt. In some embodiments, the concentrations of all salts in the elution solution containing a quaternary ammonium salt are isocratically maintained throughout the elution step. In some embodiments, the concentrations of all components in the elution solution containing a quaternary ammonium salt are isocratically maintained throughout the elution step.

[0085] Isocratic Separation As stated in many embodiments, (i) the concentration of the quaternary salt in the elution solution remains constant throughout the steps in which the at least one elution solution is passed through or applied to at least one anion exchange medium, and / or (ii) the salt composition of the elution solution remains constant throughout the steps in which the elution solution is applied to or contacted with at least one anion exchange medium. In some embodiments, the concentration of additional components of the at least one elution solution also remains constant throughout the steps. In some embodiments, the concentration of additional components of the at least one elution solution also remains constant throughout the steps.

[0086] In some embodiments, (1) one or both of the steps of applying or contacting the second or subsequent elution solution with at least one anion exchange medium comprises an isocratic separation, hi some embodiments, the salt composition of the second or subsequent elution solution and / or the salt composition of the second or subsequent elution solution remains constant throughout the steps in which the solution is passed through or applied to the anion exchange medium.

[0087] In some embodiments, the salt composition of the first elution solution, the salt composition of the second elution solution, and the salt composition of any subsequent elution solutions remains constant throughout each individual wash or elution step.

[0088] Gradient Separation In certain embodiments, one or more of the elution steps in the methods disclosed herein involve using a solution whose composition changes during that or those steps. For example, in a gradient separation step, the concentration of salt in the elution solution can be gradually and continuously (e.g., linearly or stepwise) increased with time throughout the step.

[0089] In some embodiments of the disclosed methods, the concentration of salt in the elution solution increases continuously with time throughout each individual elution step. In some embodiments of the disclosed methods, the concentration of salt in the elution solution increases linearly with time throughout each individual elution step. In some embodiments of the disclosed methods, the concentration of salt in the elution solution increases stepwise throughout each individual elution step.

[0090] equilibration solution Any of a variety of equilibration solutions may be suitable for use with the disclosed methods. In some embodiments, the pH and ionic strength of the buffered equilibration solution are selected based on the characteristics of the viral capsid preparation and / or the type of anion exchange medium used.

[0091] In some embodiments, the equilibration solution contains NaCl. For example, in some embodiments, the elution solution contains NaCl at a concentration of about 2 mM to about 200 mM (e.g., about 2 mM to about 200 mM, about 2 mM to about 175 mM, about 2 mM to about 150 mM, about 2 mM to about 125 mM, about 2 mM to about 100 mM, about 2 mM to about 90 mM, about 2 mM to about 80 mM, about 5 mM to about 70 mM, about 5 mM to about 60 mM, about 5 mM to about 50 mM, about 10 mM to about 50 mM, about 15 mM to about 50 mM, about 15 mM to about 40 mM, about 20 mM to about 40 mM, or about 20 mM to about 30 mM). For example, in some embodiments, the equilibration solution comprises NaCl at a concentration of about 20 mM, about 25 mM, or about 30 mM. In some embodiments, the equilibration solution comprises NaCl at a concentration of about 25 mM.

[0092] In some embodiments, the equilibration solution contains a divalent salt (e.g., MgCl). For example, in some embodiments, the equilibration solution contains MgCl at a concentration of about 1 mM to about 10 mM (e.g., about 1 mM to about 10 mM, about 1 mM to about 9 mM, about 1 mM to about 8 mM, about 1 mM to about 7 mM, about 1 mM to about 6 mM, about 1 mM to about 5 mM, 1 mM to about 4 mM, or about 1 mM to about 3 mM). In some embodiments, the equilibration solution contains MgCl at a concentration of about 1 mM, about 2 mM, or about 3 mM. In some embodiments, the equilibration solution contains MgCl at a concentration of about 2 mM.

[0093] In some embodiments, the equilibration solution further comprises bis-tris-propane (BTP). For example, in some embodiments, the equilibration solution comprises BTP at a concentration of between about 1 mM and 100 mM (e.g., about 2 mM to about 200 mM, about 2 mM to about 175 mM, about 2 mM to about 150 mM, about 2 mM to about 125 mM, about 2 mM to about 100 mM, about 2 mM to about 90 mM, about 2 mM to about 80 mM, about 5 mM to about 70 mM, about 5 mM to about 60 mM, about 5 mM to about 50 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, or 10 mM to about 30 mM). For example, in some embodiments, the equilibration solution comprises BTP at a concentration of about 10 mM, 15 mM, about 20 mM, or about 25 mM. In some embodiments, the equilibration solution comprises BTP at a concentration of about 20 mM.

[0094] In some embodiments, the equilibration solution contains both NaCl and a divalent salt (e.g., MgCl2).

[0095] In some embodiments, the equilibration solution comprises NaCl, a divalent salt (e.g., MgCl2), and bis-tris-propane.

[0096] In some embodiments, the equilibration solution has a pH near or within a range of values, such as about 6.0 to about 10.0, e.g., about 7.0 to about 9.5. In some embodiments, the equilibration solution has a pH of 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 equilibration solution has a pH of about 9.0.

[0097] As a non-limiting example, in some embodiments, the equilibration solution is 20 mM BTP, 25 mM NaCl, 2 mM MgCl2, pH 9.0.

[0098] Elution solutions and fractions The disclosed methods generally include passing or applying an elution solution to an anion exchange medium, for example, to elute intact capsid particles in an elution fraction. In some embodiments, the elution solution includes a quaternary ammonium salt. In some embodiments, the elution solution does not include a quaternary ammonium salt.

[0099] In some embodiments of the disclosed methods, the eluate produced by passing the elution solution through at least one AEX medium comprises at least 50% (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) of the intact capsid particles present in the volume of the virus capsid preparation. In some embodiments of the disclosed methods, the eluate produced by passing the elution solution through at least one AEX medium comprises at least 90% (e.g., at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the intact capsids present in the volume of the virus preparation. In some embodiments of the disclosed methods, the eluate produced by passing the elution solution through at least one AEX medium comprises at least 99% of the intact capsid particles present in the volume of the virus preparation.

[0100] In some embodiments of the disclosed methods, the concentration of salt in the elution solution increases continuously with time throughout each individual elution step. In some embodiments of the disclosed methods, the concentration of salt in the elution solution increases linearly with time throughout each individual elution step. In some embodiments of the disclosed methods, the concentration of salt in the elution solution increases stepwise throughout each individual elution step.

[0101] In some embodiments, the disclosed methods include passing a total of 1 to 200 (e.g., 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 10 to 90, 20 to 80, 30 to 70, 40 to 60, or 45 to 55, and all integers therebetween, inclusive) column volumes of elution solution through at least one AEX medium. In some embodiments, the disclosed methods comprise passing a total of 50-150 (e.g., 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 60-140, 70-130, 80-120, 90-110, and all integers between and including 50-150) column volumes of elution solution through at least one AEX medium. In some embodiments, the disclosed methods comprise passing a total of 90 column volumes of elution solution through at least one AEX medium.

[0102] In some embodiments, the flow-through comprises no more than 5% of the intact capsid particles present in the viral capsid preparation.

[0103] In some embodiments, the methods of the disclosure provide that the eluate from at least one AEX medium comprises at least 20% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the intact capsid particles present in the volume of the viral capsid preparation. In some embodiments, the disclosed methods comprise eluate from at least one AEX medium comprising 20% ​​to 50% (e.g., 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 20% to 30%, 20% to 45%, 25% to 45%, 30% to 45%, 35% to 45%, and all percentages between and including 20% ​​to 50%) of intact capsid particles present in the volume of the viral capsid preparation. In some embodiments, the disclosed methods comprise eluate from at least one AEX medium comprising 50% to 100% (e.g., 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100%, 60% to 90%, 50% to 70%, 60% to 80%, 70% to 90, and all percentage amounts between and including 50% to 100%) of intact capsid particles present in the volume of the viral capsid preparation.

[0104] In some embodiments of the disclosed methods, the eluate from at least one AEX medium comprises 10% or less of the empty capsid particles present in the volume of the viral capsid preparation (e.g., 10% to 9%, 9% to 8%, 8% to 7%, 7% to 6%, 6% to 5%, 5% to 4%, 4% to 3%, 3% to 2%, 2% to 1%, 1% to 0%, 9% to 1%, 8% to 2%, 7% to 3%, 8% to 4%, 9% to 6%, or 6% to 5%, and all percentage amounts between and including 10% to 0%). In some embodiments of the disclosed methods, the eluate from at least one AEX medium contains 5% or less (e.g., 5% to 4%, 4% to 3%, 3% to 2%, 2% to 1%, 1% to 0%, 4% to 1%, or 3% to 2%, and all percentage amounts therebetween, including 5% to 0%) of empty capsid particles present in the volume of the viral capsid preparation. In some embodiments of the disclosed methods, the eluate from at least one AEX medium contains 1% or less of empty capsid particles present in the volume of the viral capsid preparation (e.g., 1% to 0.9%, 0.9% to 0.8%, 0.8% to 0.7%, 0.7% to 0.6%, 0.6% to 0.5%, 0.5% to 0.4%, 0.4% to 0.3%, 0.3% to 0.2%, 0.2% to 0.1%, 0.1% to 0%, 0.9% to 0.1%, 0.8% to 0.2%, 0.7% to 0.3%, 0.6% to 0.4%, or 0.55% to 0.45%, and all percentage amounts therebetween, inclusive, between 1% and 0%). In some embodiments of the disclosed methods, the eluate from at least one AEX medium contains no empty capsid particles.

[0105] In some embodiments, the disclosed method further comprises combining all of the eluates generated from each of the at least one AEX medium to form a combined eluate. In some embodiments, the combined eluate comprises 1 to 20 (e.g., 1 to 2, 2 to 4, 4 to 6, 6 to 8, 8 to 10, 10 to 12, 12 to 14, 14 to 16, 16 to 18, 18 to 20, 2 to 18, 4 to 16, 6 to 16, 8 to 14, 14 to 15, or 15 to 16, and all integers therebetween, inclusive) column volumes for each of the at least one AEX medium. In some embodiments, the disclosed method further comprises combining all of the eluates generated from each of the at least one AEX medium to form a combined eluate. In some embodiments, the combined eluate comprises 1 to 10 (e.g., 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 2 to 9, 3 to 8, 4 to 7, 4 to 6, or 5 to 7, and all integers therebetween, inclusive) column volumes of at least one AEX medium.

[0106] Elution Solution Components For example, in some embodiments, the elution solution may be about 25 mM to about 375 mM (e.g., about 25 mM to about 50 mM, about 50 mM to about 75 mM, about 75 mM to about 100 mM, about 100 mM to about 125 mM, about 125 mM to about 150 mM, about 150 mM to about 175 mM, about 175 mM to about 200 mM, about 200 mM to about 225 mM, about 225 mM to about 250 mM, about 250 mM to about 275 mM, about 2 The solution contains NaCl at a concentration of 75 mM to about 300 mM, about 300 mM to about 325 mM, about 325 mM to about 375 mM, about 50 mM to 325 mM, about 50 mM to about 325 mM, about 75 mM to about 300 mM, about 100 mM to about 275 mM, about 125 mM to about 250 mM, about 150 mM to about 225 mM, about 175 mM to about 200 mM, and all integers therebetween, inclusive. For example, in some embodiments, the elution solution contains between about 70 mM and about 140 mM NaCl (e.g., about 70 mM to about 80 mM, about 80 mM to about 90 mM, about 90 mM to about 100 mM, about 100 mM to about 110 mM, about 110 mM to about 120 mM, about 120 mM to about 130 mM, about 130 mM to about 140 mM, about 80 mM to about 130 mM, about 90 mM to about 120 mM, and all integers between and including 70 mM and 140 mM).

[0107] In some embodiments of the disclosed methods, one or more of the first elution solution, the second elution solution, and any subsequent elution solutions comprise a pH near or within a range of values, about 6 to about 10 (e.g., about 6 to about 7, about 7 to about 8, about 8 to about 9, about 9 to about 10, about 6 to about 8, about 7 to about 9, or about 8 to about 10, and all integers therebetween, inclusive). In some embodiments, one or more of the first elution solution, the second elution solution, and any subsequent elution solutions comprise a pH of 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, one or more of the first elution solution, the second elution solution, and any subsequent elution solutions comprise a pH of about pH 9.0.

[0108] In some embodiments, one or more of the first elution solution, the second elution solution, and any subsequent elution solutions comprise a buffer system to maintain a pH of about 6 to about 10. Non-limiting examples of suitable buffer systems include bis-tris propane-based buffers, such as a 20 mM bis-tris propane system.

[0109] In some embodiments, one or more of the first elution solution, the second elution solution, and any subsequent elution solutions include a stabilizer 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 stabilizer 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 stabilizer or surfactant is present in the solution at a concentration of about 0.001% or less.

[0110] cleaning solution In some embodiments, the disclosed method further comprises applying at least a first wash solution comprising salt over at least one AEX medium between each of one or more repeats. In some embodiments, the disclosed method further comprises applying a first and a second wash solution comprising salt over at least one AEX medium between each of one or more repeats. In some embodiments, the disclosed method further comprises applying a first, second, and at least a third wash solution comprising salt over at least one AEX medium between each of one or more repeats. In some embodiments, the disclosed method further comprises applying a first and a second wash solution comprising salt over at least one AEX medium between each of one or more repeats. In some embodiments, the disclosed method further comprises applying a first, a second, a third, and at least a fourth wash solution comprising salt over at least one AEX medium between each of one or more repeats.

[0111] Wash solutions suitable for use in the methods of the present disclosure generally contain one or more salts and, optionally, a buffer, such as those described herein.

[0112] In some embodiments, the first wash solution does not contain a quaternary ammonium salt. In some embodiments, the second wash solution does not contain a quaternary ammonium salt. In some embodiments, one or more of the first wash solution and the second or any subsequent wash solutions does not contain a quaternary ammonium salt.

[0113] In some embodiments, the first wash solution comprises a quaternary ammonium salt. In some embodiments, the second wash solution comprises a quaternary ammonium salt. In some embodiments, one or more of the first wash solution and the second or any subsequent wash solution comprises a quaternary ammonium salt.

[0114] In some embodiments, the concentration of the quaternary ammonium salt in the first, second, and / or any subsequent wash solutions is about 30 mM to about 200 mM (e.g., about 30 mM to about 50 mM, about 50 mM to about 75 mM, about 75 mM to about 100 mM, about 100 mM to about 125 mM, about 125 mM to about 150 mM, about 150 mM to about 175 mM, about 175 mM to about 200 mM, about 50 mM to 175 mM, about 75 mM to 150 mM, or about 100 mM to about 125 mM, and all integers therebetween, inclusive, between 30 mM and 200 mM). In some embodiments, the concentration of the quaternary ammonium salt in the first, second, and / or any subsequent wash solutions is at least 30 mM, at least 50 mM, at least 70 mM, at least 90 mM, at least 95 mM, at least 98 mM, at least 99 mM, or at least 100 mM. In some embodiments, the concentration of the quaternary ammonium salt in the first, second, and / or any subsequent wash solutions is no more than 200 mM, no more than 180 mM, no more than 160 mM, no more than 150 mM, no more than 140 mM, no more than 130 mM, or no more than 120 mM.

[0115] 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).

[0116] 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. In some embodiments, the quaternary ammonium salt is choline chloride.

[0117] In some embodiments, the first wash solution contains a divalent salt, such as MgCl2, in an amount of about 1 mM to about 10 mM (e.g., about 1 mM to about 2 mM, about 2 mM to about 3 mM, about 3 mM to about 4 mM, about 4 mM to about 5 mM, about 5 mM to about 6 mM, about 6 mM to about 7 mM, about 7 mM to about 8 mM, about 9 mM to about 10 mM, about 2 mM to about 9 mM, about 3 mM to about 8 mM, about 4 mM to about 7 mM, about 5 mM to about 6 mM, and all integers between about 1 mM and about 10 mM).

[0118] In some embodiments, the first wash solution comprises NaCl, Na2SO4, MgSO4, or any combination thereof. For example, in some embodiments, the first wash solution contains about 25 mM to about 375 mM (e.g., about 25 mM to about 50 mM, about 50 mM to about 75 mM, about 75 mM to about 100 mM, about 100 mM to about 125 mM, about 125 mM to about 150 mM, about 150 mM to about 175 mM, about 175 mM to about 200 mM, about 200 mM to about 225 mM, about 225 mM to about 250 mM, about 250 mM to about 275 mM, about The solution contains NaCl at a concentration of 275 mM to about 300 mM, about 300 mM to about 325 mM, about 325 mM to about 375 mM, about 50 mM to 325 mM, about 50 mM to about 325 mM, about 75 mM to about 300 mM, about 100 mM to about 275 mM, about 125 mM to about 250 mM, about 150 mM to about 225 mM, about 175 mM to about 200 mM, and all integers therebetween, inclusive. For example, in some embodiments, the first wash solution contains about 70 mM to about 140 mM NaCl (e.g., about 70 mM to about 80 mM, about 80 mM to about 90 mM, about 90 mM to about 100 mM, about 100 mM to about 110 mM, about 110 mM to about 120 mM, about 120 mM to about 130 mM, about 130 mM to about 140 mM, about 80 mM to about 130 mM, about 90 mM to about 120 mM, or about 100 mM to about 120 mM, and all integers between and including 70 mM and 140 mM).

[0119] In some embodiments, the first wash fraction comprises more than 50%, more than 60%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, 98% or less, 99% or less, or 100% of the salts present in the elution solution that has passed through one or more AEX media.

[0120] In some embodiments, the first wash fraction comprises less than about 5%, less than about 3%, less than about 2%, or less than about 1% or 0% of the intact capsid particles present in the virus capsid preparation.

[0121] Second and / or subsequent wash solutions The disclosed methods of applying at least a first wash solution containing salt through at least one AEX medium during each of one or more repetitions generally include passing the second or subsequent wash solution through or applying the second or subsequent wash solution to an anion exchange medium to obtain second and / or subsequent wash fractions, e.g., wash fractions containing salt that was present in the first wash solution. In many embodiments, the second or subsequent wash solutions do not contain salt.

[0122] In some embodiments, the second or subsequent wash solution comprises a divalent salt, such as MgCl. For example, in some embodiments, the second or subsequent wash solution comprises about 2 mM to about 10 mM MgCl (e.g., 1 mM to about 2 mM, about 2 mM to about 3 mM, about 3 mM to about 4 mM, about 4 mM to about 5 mM, about 5 mM to about 6 mM, about 6 mM to about 7 mM, about 7 mM to about 8 mM, about 9 mM to about 10 mM, about 2 mM to about 9 mM, about 3 mM to about 8 mM, about 4 mM to about 7 mM, about 5.5 mM to about 7.5 mM, and all integers between and including about 1 mM to about 10 mM).

[0123] In some embodiments, the second and / or any subsequent wash solution comprises NaCl, NaSO, MgSO, or any combination thereof. For example, in some embodiments, the second wash solution contains about 25 mM to about 375 mM (e.g., about 25 mM to about 50 mM, about 50 mM to about 75 mM, about 75 mM to about 100 mM, about 100 mM to about 125 mM, about 125 mM to about 150 mM, about 150 mM to about 175 mM, about 175 mM to about 200 mM, about 200 mM to about 225 mM, about 225 mM to about 250 mM, about 250 mM to about 275 mM, about The solution contains NaCl at a concentration of 275 mM to about 300 mM, about 300 mM to about 325 mM, about 325 mM to about 375 mM, about 50 mM to 325 mM, about 50 mM to about 325 mM, about 75 mM to about 300 mM, about 100 mM to about 275 mM, about 125 mM to about 250 mM, about 150 mM to about 225 mM, about 175 mM to about 200 mM, and all integers therebetween, inclusive. For example, in some embodiments, the second and / or subsequent wash solutions contain about 70 mM to about 140 mM NaCl (e.g., about 70 mM to about 80 mM, about 80 mM to about 90 mM, about 90 mM to about 100 mM, about 100 mM to about 110 mM, about 110 mM to about 120 mM, about 120 mM to about 130 mM, about 130 mM to about 140 mM, about 80 mM to about 130 mM, about 90 mM to about 120 mM, or about 100 mM to about 120 mM, and all integers therebetween, inclusive, between 70 mM and 140 mM).

[0124] In some embodiments, the second and / or subsequent wash solutions elute at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% of the salts present in the wash solutions before they are passed through the one or more AEX media.

[0125] In some embodiments, the second and / or subsequent wash fractions contain less than about 5%, less than about 3%, less than about 2%, less than about 1%, or 0% of the intact capsid particles present in the virus capsid preparation.

[0126] In some embodiments of the disclosed methods, the salt composition of at least the first wash solution and / or the second and subsequent wash solutions remains constant throughout each individual wash. In some embodiments of the disclosed methods, the salt composition of at least the first wash solution and / or the second or any subsequent wash solutions continues to change throughout each individual wash.

[0127] In some embodiments of the disclosed method, the salt composition in at least the first wash solution increases continuously through each individual wash. In some embodiments of the disclosed method, the salt composition in at least the first wash solution increases linearly through each individual wash. In some embodiments of the disclosed method, the salt composition in at least the first wash solution increases stepwise through each individual wash. In some embodiments of the disclosed method, the salt composition in the second or any subsequent wash solution increases continuously through each individual wash. In some embodiments of the disclosed method, the salt composition in the second or any subsequent wash solution increases linearly through each individual wash. In some embodiments of the disclosed method, the salt composition in the second or any subsequent wash solution increases stepwise through each individual wash.

[0128] In some embodiments, one or more of the first wash solution, the second wash solution, and any subsequent wash solutions include a stabilizer 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 stabilizer 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 stabilizer or surfactant is present in the solution at a concentration of about 0.001% or less.

[0129] Evaluation of viral capsid preparations and / or fractions In some embodiments, the viral capsid preparation and / or elution fraction, or a sample thereof, is evaluated. The first, second, or any subsequent wash fraction may or may not be collected. In some embodiments, at least one sample of one or more wash fractions is collected and evaluated, for example, for quality control purposes.

[0130] Full and empty capsid particles In some embodiments, the presence and / or quantity of intact capsid particles and / or empty capsid particles in a virus capsid preparation and / or one or more eluates (or elution fractions) are evaluated.A variety of methods for determining or quantifying the presence of intact or empty particles are known in the art, and many of these methods can also be used to determine the quantity, for example, the relative amount of intact 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, for example, for quality control purposes, measuring capsids and genome copies by ELISA and qPCR.

[0131] 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 high rotational speeds that apply centrifugal force. The sedimentation rate, measured by the sedimentation coefficient s, is related to the buoyant mass, density, specific volume, and frictional force of the molecule in the formulation matrix. When normalized to standard solution conditions (standard temperature and pressure) in water at 20°C, the bs value is s 20,wThe sedimentation coefficient distribution is known as the σ value, which is a fundamental molecular parameter that defines the mass, shape, and 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 molecules at that sedimentation coefficient. The degree of accuracy of the peak area in representing the true population depends in part on the suitability of the detection system and the number of data points collected.

[0132] SV-AUC can be applied, for example, to separate different types of capsid particles, such as separating complete capsid particles from empty capsid particles. The mass of virus particles with the same virus type and serotype can differ depending on the presence of a complete vector genome (as in the case of complete capsid particles), or the presence of only a portion of the vector genome, or the complete absence of the vector genome (as in the case of empty capsid particles). For example, empty capsid particles, which contain less DNA than complete capsids, are lighter than complete capsids and sediment more slowly than complete capsids. Therefore, the s value in the SV-AUC method reflects the size of the DNA packaged within the virus capsid particle.

[0133] In addition, some impurities in a sample may absorb light at a wavelength of 230 nm, and these contaminants typically outnumber those absorbing at 280 nm. Therefore, the A260 / A230 ratio may provide some indication of the purity of a sample. In some embodiments, the A230 and / or A260 / A230 values ​​are evaluated.

[0134] 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 A254 / A280 or A260 / A280 ratio of one fraction (from which empty capsid particles are preferentially released) is smaller than the A254 / A280 or A260 / A280 ratio of a subsequent fraction (from which full capsid particles are preferentially released) (e.g., as shown in Figure 1). In some embodiments, the A254 / A280 or A260 / A280 ratio of one or more fractions is directly proportional to the amount of full capsid particles in the flow-through or eluate.

[0135] The ordering of preferential release may be related to one or more of a variety of aspects, such as (but not limited to) a) characteristics of the nucleic acid payload, b) serotype of the capsid, c) preparation conditions of the viral capsid (e.g., rAVV), and d) characteristics of the AEX medium.

[0136] Other evaluations In some embodiments, the fraction or a sample thereof is evaluated to determine the presence or amount of an analyte, such as, for example, a component of a washing solution. For example, in some embodiments, a sample of the second washing fraction is evaluated to determine the presence or amount of quaternary ammonium salts in the sample. Methods for detecting or quantifying quaternary ammonium salts are known in the art, and include, for example, liquid chromatography-mass spectrometry (LC-MS) and reverse-phase high-performance liquid chromatography (RP-HPLC).

[0137] Anion Exchange Medium 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.

[0138] In some embodiments, the anion exchange (AEX) medium is in the form of a packed bed. In some embodiments, the anion exchange medium is a chromatographic monolithic column. By way of example only, a CIMmultus® monolithic column (e.g., a CIMmultus® monolithic QA column) can be used. In some embodiments, the monolithic column is any of a CIMmultus® QA column, a CIMac® QA column, a NuviaQ® column, a POROS® HQ column, an Eshmuno® Q column, a POROS® XQ column, a FractoGel® TMAE column, and a CaptoQ® column. In some embodiments, the monolithic column is a CIMmultus® QA column or a CIMac QA® column.

[0139] Viral capsid preparation The disclosed methods 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 recombinant viral particles containing heterologous nucleic acid. Typically, the full and empty capsid particles in a given viral capsid preparation are capsid particles of the same virus and the same serotype. In some embodiments, the capsid is derived from an AAV capsid of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, rh10, or hu37, or a mutant thereof. In some embodiments, the capsid is an AAV capsid of serotype 8 or 9, or a mutant thereof. In some embodiments, the capsid is an AAV capsid of serotype 8 or a mutant thereof. In some embodiments, the capsid is derived from an AAV capsid of serotype 9, or a mutant thereof. In some embodiments, the capsid is derived from an AAV capsid of serotype rh10 or hu37, or a variant thereof. In some embodiments, the capsid is derived from an AAV capsid of serotype rh10, or a variant thereof. In some embodiments, the capsid is derived from an AAV capsid of serotype hu37, or a variant thereof. [Example]

[0140] material and method AAV material containing empty and intact recombinant AAV (rAAV) capsids was obtained from a pilot-scale 250 L bioreactor run as an exemplary AAV loading material. The clarified harvest was affinity captured, and an affinity eluate sample was retained for a lab-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 load conductivity to approximately 4 mS / cm. Then, 10 mL of the diluted affinity eluate was loaded into AEX medium (in this case, 0.1 mL of CIMac-QA) as a scaled-down model of CIMac-QA (1 mL) pre-equilibrated with a pH-matched equilibration buffer (20 mM BTP, 25 mM NaCl, 2 mM MgCl, pH 9.0). For elution with NaCl, a linear gradient from approximately 25 mM to approximately 182.5 mM NaCl was applied over 90 column volumes (CV). The eluate was collected in fractions, and fractions corresponding to the "empty" and "full" peaks were pooled accordingly.

[0141] Example 1: AEX scale-down model of dynamic binding capacity Objective: The CIMmultus-QA column was found to exhibit superior separation of empty and intact rAAV compared to other AEX stationary phases evaluated, and therefore can be used in AEX-WPC operations. However, given the CIMmultus-QA's minimum 1 mL column volume (CV), column overpacking requires prohibitively large amounts of rAAV material. To circumvent this obstacle, the study described herein determined the feasibility of using a small AEX column, termed CIMac-QA, with a column volume of 0.1 mL to determine the overpacking range of rAAV viral particles for integrating weak partitioning chromatography into an AEX process (AEX-WPC).

[0142] Results: Both CIMac-QA and CIMmultus-QA are manufactured by Sartorius AG and have a monolith-based stationary phase and quaternized amine ligands. To demonstrate the feasibility of CIMac-QA (0.1 ml) as a scale-down model of CIMmultus-QA (1 ml), we performed a normal batch-loading run (column underloading conditions) on both columns using rAAV AEX packing material containing 4% intact rAAV and empty and intact capsid particles. The results are shown in Figure 1A and Figure 1B. Figure 1A and Figure 1B show that the AEX elution chromatograms were qualitatively comparable between the CIMac-QA and CIMmultus-QA runs, with the empty rAAV peak (denoted "E"), intact rAAV peak (denoted "F"), and a third peak (denoted "T" and known to contain damaged viral particles as assessed by transmission electron microscopy) sequentially resolving with increasing salt concentration. Table 1 shows that the peak conductance and A254 / A280 area ratios of empty and full rAAV were similar between CIMac-QA and CIMmultus-QA runs.

[0143] Conclusion: Based on the results of the studies described herein, the 0.1 ml CIMac-QA column was demonstrated as a suitable scale-down model of the 1 ml CIMmultus-QA column for AEX-WPC development.

[0144] [Table 1]

[0145] Example 2: AEX Breakthrough Curve Analysis The study described herein determined the overloading range for AEX-WPC operation by measuring the dynamic binding capacity (DBC) of rAAV AEX packing material on a 1 ml CIMmultus-QA column using a second lot of AEX packing material containing 11% intact rAAV. The fraction under the UV breakthrough curve was quantified for both viral particle concentration (vp / ml) and viral genome concentration (vg / ml). The results are shown in Figures 2A-2C. Figure 2A shows the breakthrough of viral particle concentration (shown as open bars) prior to viral genome concentration (shown as diagonally shaded bars) during the packing phase, suggesting that empty rAAV breaks through earlier than intact rAAV on the AEX column, demonstrating WPC behavior. Consequently, during the elution phase, the empty rAAV peak (shown as "E") (Figure 2B) was significantly reduced compared to that of the normal batch packing run (Figure 2C), confirming that empty rAAV was indeed reduced during AEX column overloading. Based on the results of the studies described herein, it was concluded that to ensure minimal flow-through of intact rAAV during overfilling, the upper loading limit for the AEX-WPC operation should not exceed the breakthrough position of viral genome concentration in the DBC run.

[0146] Example 3: Multi-column chromatography implementation All studies described here integrated AEX-WPC with multiple column chromatography (MCC), which allows for parallel column processing capabilities, to generate the AEX-WPC-MCC method in order to reduce redundant AEX processing times due to column overpacking.

[0147] Study Design: The study described herein used a commercially available instrument, the AKTA PCC75, which allows parallel multicolumn processing, to perform AEX-WPC-MCC runs. One three-column MCC loop (comprising four AEX runs) is illustrated by six schematic diagrams in Figure 3, A–F, each showing a representative phase of the MCC loop. For a single column, the process begins with an equilibration phase (denoted "Eq"), followed by a loading phase (denoted "L"), with shaded columns representing saturation with intact rAAV. After column loading, intact rAAV was eluted from the AEX column by a linear salt gradient elution phase (denoted "G"), and fractions below the intact rAAV peak were pooled. The eluted column then entered a wash phase (denoted "C") before being cycled through the next equilibration phase. The AEX-WPC-MCC method developed in this study did not include a column interconnection phase, which is the phase when a volume of rAAV-loaded material passes between any two columns in a multiple AEX column system. For the column interconnection phase to occur, each column of multiple AEX columns, as depicted in Figures 3A-3F, must be connected to the next AEX column so that the same volume of rAAV-loaded material passes through all of the multiple AEX columns. In the study described herein, because there was no complete rAAV breakthrough detected at the end of each column load, there was no need to interconnect the columns, and therefore the system did not need to have a column interconnection phase.

[0148] Example 4: Effect of % of complete AAV in AEX loading on AEX-WPC-MCC Objective: The percentage of complete rAAV in the AEX packing material can alter the relative breakthrough position between viral particle and viral genome concentrations, thus affecting the available packing range in the operation of AEX-WPC-MCC. In the study described herein, robust packing ranges (lower and upper packing limits of viral capsid preparation / packing material) were identified by DBC runs on a 0.1 ml CIMac-QA column (a scaled-down model used to conserve AEX packing material) using both 4% and 11% complete rAAV AEX packing material.

[0149] Results: The results are presented in Figures 4A-4B and Table 2. In Figures 4A and 4B, both the viral particle breakthrough curves and the viral genome breakthrough curves were plotted as a function of viral particle loading per column volume (vg / ml-column) for both 11% and 4% complete rAAV material, respectively. Due to material limitations, a maximum of approximately 1.4 x 10 viral particles was achieved for the 4% complete rAAV material. 16 Compared to vp / ml column loading, the maximum DBC loading achieved for 11% intact rAAV material was approximately 3.5x10 15 vp / ml column. A detailed comparison of the loading ranges is shown in Table 2, where 4% intact rAAV material was 6.7x10 14 vp / ml-column and 5.3x10 15 vp / ml-column column loading was achieved at 5% viral particle concentration breakthrough and 5% viral genome concentration breakthrough, respectively. In contrast, 11% intact rAAV material was 5.8x10 14 vp / ml column and 3.1x10 15 Column loading of 1000 vp / ml column was achieved at 5% viral particle concentration breakthrough and 5% viral genome concentration breakthrough, respectively. This resulted in 4% complete rAAV material (4.6 x 10 15 The differential viral particle loading range in vp / ml-column was 11% complete AAV material (2.6x10 15The differential viral particle loading range (vp / ml-column) was shown to be 1.8 times that of the 6.7x10 bounded by 4% total rAAV loaded material and 11% total rAAV loaded material, provided that the % of total rAAV in the exemplary AEX loaded material ranged from 4 to 11%. 14 ~3.1x10 15 It was concluded that the overlapping range of vp / ml-columns served as a robust packing space for the operation of AEX-WPC-MCC.

[0150] [Table 2]

[0151] In addition, Figures 4A-4B show the ng / 1x10 observed during the loading phase. 12 Figure 4C shows that the host cell DNA (HCDNA) flow-through concentration (shown as open triangles) decreased with increasing viral particle loading for both 11% and 4% full rAAV loaded materials. Based on the viral particle and viral genome breakthrough plotted against the viral particle loading of the loaded material, we defined the operational space of the AEX-WPC-MCC method for isolating / enriching full rAAV from capsid preparations / loaded materials (Figure 4C).

[0152] Conclusion: With a well-defined loading range, it was concluded that the AEX-WPC-MCC method can also reduce HCDNA impurities from the product stream. Given that the HCDNA detectable at this stage of purification (post-nuclease digestion) is primarily encapsidated and appears as intermediate rAAV in analytical ultracentrifugation (AUC) analysis, it was concluded that intermediate rAAV can also be reduced to some extent in the AEX-WPC-MCC procedure.

[0153] Example 5: Evaluation of the loading range of AEX-WPC-MCC Objective: The study described herein aims to determine the proposed filling range (6.7x10 14 ~3.1x1015 The process performance and product quality of the AEX-WPC-MCC operation in vp / ml-column was evaluated using a lower limit load run (7.0x10) using AAV serotype 8 AEX packing material containing 11% intact rAAV. 14 vp / ml-column, indicated by the circled "L" in Figure 4B) and the upper-load run (2.8x10 15 vp / ml-column, indicated by a circled "H" in Figure 4B).

[0154] Results: Figures 5A-5E and Table 3 show the results of the study described herein. In the lower-load AEX-WPC-MCC run, there was no detectable UV280 breakthrough during loading (Figure 5A), but the gradient elution profile (Figure 5B) showed a significant enhancement of the intact rAAV peak compared to that of the regular batch-loaded run in Figure 2C. In contrast, the upper-load AEX-WPC-MCC run resulted in UV280 breakthrough in the loading profile (Figure 5C) and a reduction of the empty peak in the gradient elution profile (Figure 5D), similar to that of the DBC run in Figure 2B. In Figure 5E, the overlay of the gradient elution profiles of both the lower-load and upper-load runs (run 4 from each group) showed that the intact rAAV peak was further enhanced in the upper-load run.

[0155] Additionally, fractions below the peak of intact rAAV (8 CV adjacent to the apex) from both the lower- and upper-load runs were collected as AEX pools, and their viral genome recoveries and pool intact rAAV% were determined. The results, listed in Table 3, showed that the viral genome recoveries were 34%, 38%, and 55%, and the pool intact rAAV% were 14%, 22%, and 36%, for the normal batch loading run, the lower-load AEX-WPC-MCC run, and the upper-load AEX-WPC-MCC run, respectively. These data were further plotted in Figure 5F to visualize the trade-off between viral genome recovery and pool intact rAAV%. The AEX regular batch load run achieved only 1% of the DBC cutoff (viral particle load at 5% breakthrough of viral particle concentration using 11% full rAAV AEX load material, as shown in Table 3), compared to 22% and 90% loads of the DBC cutoff for the low-load and high-load AEX-WPC-MCC runs, respectively. Based on the results described herein, it was determined that the higher pool full rAAV% for the AEX-WPC-MCC run was due to empty rAAV flow-through during overloading, while the higher viral genome recovery for the AEX-WPC-MCC run was due to reduced product loss due to column overloading. Furthermore, the results described in Table 3 indicate that the viral genome concentration (vg / ml) of the AEX pool was also significantly lower than that of the AEX regular batch run (1.2x10 11 vg / ml) to the lower limit filled AEX-WPC-MCC run (5.2x10 12 vg / ml), and the upper-filled AEX-WPC-MCC run (3.0x10 13 The results showed a significant increase in serum creatinine (mg / ml).

[0156] Conclusion: Based on the results described herein, it was determined that the AEX-WPC-MCC procedure within the loading range described herein is effective and qualitatively efficient in separating empty and intact AAV capsids in the AAV capsid loading material. Furthermore, the high viral genome concentration after the AEX step is beneficial for downstream operations, including the final ultrafiltration and diafiltration (UFDF) step, in which recombinant AAV (rAAV) is concentrated and buffer exchanged into the drug substance. The rAAV drug substance viral genome concentration was 10 13 vg / ml, 3.0 × 10 13 A viral genome concentration of 0.1 vg / ml can enable the UFDF step to skip the concentration phase and enter directly into the buffer exchange phase, alleviating the current workload imposed by the high concentration factor (typically around 100x) in the UFDF step.

[0157] [Table 3]

[0158] For two different rAAV preparation loading material / capsid preparations (rAAV preparation 1 and rAAV preparation 2), 6.7x10 14 ~3.1x10 15 Overpacked runs of AEX-WPC-MCC were performed over the vp / ml column loading range. Results from these overpacking experiments demonstrated a higher degree of purification of intact viral particles (as indicated by % viral genome recovery) compared to runs with regular AEX, as well as comparable separation of empty, intermediate, and intact viral particles (Figures 6A-6B). The method of overpacking AEX-WPC-MCC using the loading range described herein resulted in an approximately 33-fold reduction in column volume, an approximately 65-fold reduction in buffer volume, and an approximately 34-fold increase in productivity (complete rAAV / ml AEX column / hour) compared to methods using AEX regular loading (Figure 7).

[0159] Example 6: Evaluation of host cell protein (HCP) levels in fractions isolated from overloaded runs of AEX-WPC-MCC Objective: In addition to separating empty and intact rAAV, AEX chromatography is also responsible for removing certain process-related impurities, such as host cell proteins (HCPs). In this example, the effect of AEX overloading chromatography of an AAV preparation on HCP levels in the isolated fraction was determined.

[0160] Results: In rAAV AEX salt linear gradient elution, HCPs typically appeared in the tailing fractions of the full AAV capsid particle peak ("full peak," denoted "F" in Figure 8), suggesting that these HCPs have a higher net negative charge than full rAAV and are retained on the AEX column until column wash. These characteristics of HCPs allow for full peak tail cutting as a robust method to exclude HCPs from entering the product stream in the AEX chromatography step. AAV material was loaded onto the AEX chromatography column for both batch loading runs (without loading breakthrough) and DBC loading runs (with loading breakthrough).

[0161] Table 4 below shows the genome recovery, % intact AAV, and HCP levels in the AAV fractions separated using AEX overpacking chromatography. Both genome recovery and % intact rAAV were higher for the AAV material from the AEX DBC-packing run (50.5% and 20.8%, respectively) compared to the AEX batch-packing run (39.0% and 17.9%, respectively). Separating empty and complete AAV capsids from the AAV material using AEX weak partitioning chromatography demonstrated that even though HCP levels were detectable in the AEX-packing material (12 ng / mL), the HCP levels were below the detection limit (<4 ng / mL) in the AEX eluate from both the AEX batch-packing and AEX DBC-packing runs. In addition, the high genome titer (10^1^3 GC / mL) of the AEX eluate from the AEX DBC-packing run, which is very close to the drug substance concentration, indicated that similar (<4 ng / mL) HCP levels were also observed in the drug substance.

[0162] [Table 4]

[0163] Conclusion: This example demonstrated that despite overloading the AEX column, HCPs are sufficiently removed throughout the AEX step, thereby mitigating concerns about HCP removal in AEX weak partitioning chromatography methods. AAV overloading and separation using AEX chromatography has been shown to maintain robustness in HCP removal (see Table 4). Furthermore, even when HCP removal is a concern, the HCP reduction potential from other equipment operations prior to the AEX step, such as harvest clarification, harvest tangential flow filtration, and affinity chromatography, can be leveraged to reduce the burden of the AEX step.

[0164] Based on the results of the studies described herein, the disclosed method, which incorporates weak partitioning chromatography (WPC) and multiple column chromatography (MCC) into the AEX method, achieves improved separation of empty rAAV and complete rAAV, with the empty rAAV flowing through the column during column overload. In summary, the methods disclosed herein result in enhanced viral genome recovery and the percentage of complete rAAV in the AEX pool, as well as reduced AEX column volume and buffer usage, contributing to cost savings in downstream manufacturing processes. Previous reports have shown that HCPs can be removed by synthetic depth filters and affinity chromatography, and these examples can be utilized and adapted to rAAV manufacturing processes to achieve optimal HCP reduction through AEX chromatography during column overload.

Claims

1. A method for separating empty capsid particles from a viral capsid preparation containing empty and complete capsid particles, a) A step of applying an equilibration solution to at least one anion exchange (AEX) chromatography column, b) A step of passing the volume of the viral capsid preparation through the at least one AEX chromatography column to generate a flow-through containing empty capsid particles. Includes, The volume of the virus capsid preparation is 1 x 10⁶ per milliliter of the AEX chromatography column. １４ Virus particles (vp / ml) ~ 5.5 x 10 １５ A method including the range of vp / ml.

2. A method for separating complete capsid particles and empty capsid particles in a viral capsid preparation containing empty capsid particles and complete capsid particles, a) A step of applying an equilibration solution to at least one anion exchange (AEX) chromatography column, b) A step of passing the volume of the viral capsid preparation through the at least one AEX chromatography column to generate a flow-through containing empty capsid particles, c) A step of passing the salt-containing elution solution through at least one AEX chromatography column to produce an eluate containing complete capsid particles. Methods that include...

3. A method for increasing the proportion of complete capsid particles in a viral capsid preparation containing empty capsid particles and complete capsid particles, a) A step of applying an equilibration solution to at least one anion exchange (AEX) chromatography column, b) A step of passing the volume of the viral capsid preparation through at least one AEX chromatography column to generate a flow-through, c) A step of passing the salt-containing elution solution through at least one AEX chromatography column to produce an eluate, d) A step of collecting the eluate from step c) and Includes, The volume of the eluate in step d) contains complete capsid particles in a higher proportion than the proportion of complete capsid particles in an equivalent volume of the virus capsid preparation. The volume of the virus capsid preparation is 1 x 10⁶ per milliliter of the AEX chromatography column. １４ Virus particles (vp / ml) ~ 5.5 x 10 １５ A method including the range of vp / ml.

4. The method according to claim 1, further comprising step b) collecting at least a portion of the flow-through and analyzing the content of empty capsids and complete capsids in the collected portion of the flow-through, wherein passing the volume of the viral capsid preparation through the at least one AEX chromatography column in step b) is stopped if complete capsid particles are detected in the collected portion of the flow-through.

5. At least two, at least three, or at least four AEX chromatography columns are used, and / or The aforementioned at least two, at least three, or at least four AEX chromatography columns are used in sequential use, and / or Each of the above steps is repeated at least once, at least twice, or at least three times, each time using a different AEX chromatography column. The method according to claim 1.

6. The volume of the viral capsid preparation applied in step b) is constant among each of the at least two, at least three, or at least four AEX chromatography columns, and the sequential use is (i) complete each of the steps using one AEX chromatography column, and then repeat each of the steps starting from step a) using a subsequent AEX chromatography column, or (ii) completing at least one of the steps using one AEX chromatography column, and then performing each of the steps starting from step a) using a subsequent AEX chromatography column. The method according to claim 5, including the method described in claim 5.

7. The method according to claim 5, comprising at least one, at least two, or at least three repetitions of the step, each time using a different AEX chromatography column, wherein the volume of the viral capsid preparation applied to the at least one AEX chromatography column remains constant between each repetition.

8. The method according to claim 7, further comprising passing at least one first washing solution through the at least one AEX chromatography column between each iteration.

9. A method of elution, a) A step of passing a volume of a viral capsid preparation containing complete capsid particles and empty capsid particles through at least one anion exchange (AEX) chromatography column to generate a flow-through containing empty capsid particles, b) passing the salt-containing elution solution through at least one AEX chromatography column to produce an eluate containing complete capsid particles, A method wherein the volume of the viral capsid preparation contains between 1 x 10¹⁴ viral particles (vp / ml) and 5.5 x 10¹⁵ vp / ml per milliliter of the AEX chromatography column.

10. The method according to claim 9, wherein step a) further comprises collecting at least a portion of the flow-through and analyzing the content of empty capsids and complete capsids in the collected portion of the flow-through, and passing the volume of the viral capsid preparation through the at least one AEX chromatography column in step a) is stopped if complete capsid particles are detected in the collected portion of the flow-through.

11. i) Steps a) to b) are carried out using at least two, at least three, or at least four AEX chromatography columns, ii) Each of the steps described above is repeated at least once, at least twice, or at least three times, each time using a different AEX chromatography column, and / or iii) The method according to claim 9, wherein at least two, at least three, or at least four AEX chromatography columns are used in sequential use.

12. A sequential use of at least two, at least three, or at least four AEX chromatography columns, The sequential use includes completing each of the steps using one AEX chromatography column, and then repeating each of the steps starting from step a) using a subsequent AEX chromatography column, or The sequential use includes completing at least one of the steps using one AEX chromatography column, and then performing each of the steps, starting from step a), using subsequent AEX chromatography columns. The method according to claim 11.

13. The method according to claim 11, comprising at least one, at least two, or at least three repetitions of the step, each using a different AEX chromatography column, wherein the volume of the viral capsid preparation applied to the at least one AEX chromatography column remains constant between each repetition.

14. The volume of the viral capsid preparation applied to or in contact with the AEX chromatography column is (i) Between 6.7 x 10¹⁴ virus particles (vp / ml) and 3.1 x 10¹⁵ vp / ml per milliliter of the AEX chromatography column, (ii) Between 6.5 x 10¹⁴ virus particles (vp / ml) and 5.5 x 10¹⁵ vp / ml per milliliter of the AEX chromatography column, (iii) Between 5.5 x 10¹⁴ virus particles (vp / ml) and 3.5 x 10¹⁵ vp / ml per milliliter of the AEX chromatography column, or (iv) Between 7 x 10¹⁴ virus particles (vp / ml) and 3 x 10¹⁵ vp / ml per milliliter of the AEX chromatography column. including and / or The volume of the viral capsid preparation applied to or in contact with the AEX chromatography column is between 100 and 1000 ml per ml of the AEX chromatography column, and / or The aforementioned viral capsid preparation contains approximately 1% to approximately 60% complete capsid particles. The method according to claim 1.

15. (i) The flow-through comprises at least 50%, at least 90%, or at least 99% of the empty capsid present in the volume of the viral capsid preparation, and / or (ii) The flow-through comprises 5% or less of the complete capsid particles present in the volume of the viral capsid preparation, The method according to claim 1.

16. The method according to claim 2, comprising the step of passing a total column volume of 1 to 200 of the elution solution through at least one AEX chromatography column.

17. i) (1) The eluate comprises at least 20%, at least 50%, at least 90%, or at least 99% of the complete capsid particles present in the volume of the virus capsid preparation, (2) The eluate contains 20% to 50% or 50% to 100% of the complete capsid particles present in the virus capsid preparation. (3) The eluate contains 10% or less, 5% or less, or 1% or less of the empty capsid particles present in the virus capsid preparation, and / or (4) The eluate does not contain any empty capsid particles. ii) The elution solution contains MgCl₂ or NaCl, iii) The elution solution contains approximately 25 mM to approximately 375 mM NaCl, iv) The elution solution has a pH of about 9, and / or v) The concentration of the salt in the elution solution increases continuously, linearly, or stepwise over time through each individual elution step. The method according to claim 2.

18. The method according to claim 2, further comprising the step of combining all of the eluates generated from each of the at least one AEX chromatography columns to form a combined eluate.

19. The method according to claim 18, wherein the combined eluate comprises 1 to 20 column volumes for each of the at least one AEX chromatography columns.

20. c) Between each iteration, applying at least one first washing solution containing a salt across the at least one AEX chromatography column, and / or d) Between each iteration, apply at least a second washing solution containing salt across the at least one AEX chromatography column. The method according to claim 13, further comprising:

21. (i) The first washing solution does not contain a quaternary ammonium salt, (ii) The second cleaning solution or subsequent cleaning solution does not contain a quaternary ammonium salt. (iii) The composition of the salts in at least the first washing solution and / or the second washing solution or any subsequent washing solution remains constant throughout each individual washing. (iv) The composition of the salt in at least the first washing solution increases continuously, linearly, or stepwise through each individual washing, and / or (v) The composition of the salt in the second washing solution or any subsequent washing solution increases continuously, linearly, or stepwise through each individual washing. The method according to claim 20.

22. The method according to claim 1, wherein the AEX chromatography column is a monolithic column.

23. The method according to claim 22, wherein the monolithic column is selected from the group consisting of CIMmultius® QA column, CIMac® QA column, NuviaQ® column, POROS® HQ column, Eshmuno® Q column, POROS® XQ column, FractoGel® TMAE column, or CaptoQ® column.

24. The method according to claim 1, wherein the capsid is derived from an AAV capsid of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, rh10, or hu37, or a variant thereof.

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