Methods for purifying intact recombinant AAV particles

Abstract

Provided herein is a method for purifying recombinant adeno-associated virus (rAAV) particles on a large scale using chromatography. Also provided are rAAV particles and pharmaceutical compositions prepared by the method. The present disclosure relates to a method for purifying rAAV particles. The method enables the purification and concentration of intact rAAV particles suitable for clinical application. In some embodiments, the rAAV particles comprise the capsid protein of AAVhu68.

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 378,997, filed October 10, 2022, the disclosure of which is incorporated herein by reference in its entirety. [Background technology]

[0002] (2.Background) Adeno-associated virus (AAV) is a non-enveloped virus belonging to the Parvoviridae family. AAV has a linear, single-stranded DNA (ssDNA) genome of approximately 4.7 kilobases (kb) and contains three genes: Rep (replication), Cap (capsid), and aap (assembly). The Rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40) required for viral genome replication and packaging, while the Cap gene encodes viral capsid proteins (VP; VP1, VP2, and VP3) required for encapsidation, target cell binding, and internalization.

[0003] Due to their low genetic complexity, AAVs have attracted significant interest in the field of gene therapy. This low genetic complexity facilitates the cloning, packaging, and delivery of therapeutic gene expression cassettes into target cells. rAAVs, which lack viral genes and contain the gene expression cassette along with a gene of interest, have been shown to be safe and effective gene therapy vehicles capable of delivering the gene of interest to targets in vivo. rAAVs have become the dominant form of gene therapy, and rAAV-based therapeutics have received regulatory approval in Europe and the United States.

[0004] rAAV particles can be produced in packaging host cell cultures by co-expressing the AAV Rep gene and AAV Cap gene, which are helper viruses for replication and packaging. Typically, the host cells are lysed to release the rAAV particles and maximize the yield of recovered rAAV. However, the cell lysate contains various cellular components (e.g., host cell DNA, host cell proteins), media components, and in some cases, helper virus or helper virus plasmid DNA. Furthermore, not all rAAV particles produced and released from host cells contain the genomic DNA along with the gene of interest (complete rAAV particles). A significant portion of the rAAV particles contain no DNA (empty rAAV particles) or only a partial genome (partially filled rAAV particles). Empty and partially filled rAAV particles are considered impurities because they increase the total AAV dose administered for efficient transduction. Therefore, rAAV particles collected from the medium and / or cell lysates should be further purified to be suitable for therapeutic use.

[0005] Currently available methods for rAAV purification are not scalable and / or do not comply with good manufacturing practices. rAAV particles purified using cesium chloride gradient ultracentrifugation as a purification step have been used in some clinical trials, but these purification methods are not easily scalable. Other methods, such as heparin-based affinity column chromatography and ion exchange chromatography, have been used to purify rAAV. However, unlike density gradient centrifugation, chromatographic methods do not generally achieve separation of empty capsids from intact capsids. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, a scalable purification method that allows enrichment of rAAV full capsids from empty and partial capsids is needed to meet the burgeoning demand for rAAV, particularly for rAAV-based therapeutics. [Means for solving the problem]

[0007] (3. Summary) The present disclosure relates to methods for purifying rAAV particles, which allow for the purification and concentration of intact rAAV particles suitable for clinical application. rAAV particles and pharmaceutical compositions produced by the methods are also provided.

[0008] In one aspect, the present disclosure provides a method for purifying recombinant adeno-associated virus (rAAV) particles, the method comprising: a) providing a feed composition comprising the rAAV particles, wherein the rAAV particles in the feed composition include empty rAAV particles and complete rAAV particles; b) contacting the feed composition with a chromatography medium under conditions that allow binding of the rAAV particles to the chromatography medium for a medium contact time, wherein the medium contact time is at least 0.5 hours; c) eluting the rAAV particles from the chromatography medium; and d) recovering the purified rAAV particles, thereby enriching for intact rAAV particles Includes:

[0009] In some embodiments, the medium contact time is greater than 2 hours. In some embodiments, the medium contact time is between 2 and 24 hours. In some embodiments, the medium contact time is greater than 3 hours, greater than 4 hours, greater than 5 hours, greater than 6 hours, greater than 7 hours, greater than 8 hours, greater than 9 hours, greater than 10 hours, greater than 11 hours, or greater than 12 hours.

[0010] In some embodiments, the contacting step comprises retaining the rAAV particles bound to the chromatography medium in a retention buffer for a retention period of 0.5 hours or longer. In some embodiments, the retention period is 0.5 hours to 24 hours. In some embodiments, the retention period is 3 hours or less. In some embodiments, the retention period is 0.5 hours to 3 hours. In some embodiments, the retention period is about 3 hours.

[0011] In some embodiments, the holding buffer has a pH of 9.0 to 11. In some embodiments, the holding buffer has a pH of about pH 10.2.

[0012] In some embodiments, the contacting step comprises loading the rAAV particles onto the chromatography medium for a loading period. In some embodiments, the loading period is greater than 0.5 hours, greater than 1 hour, greater than 2 hours, greater than 3 hours, greater than 4 hours, greater than 5 hours, greater than 6 hours, greater than 12 hours, or greater than 18 hours. In some embodiments, the loading period is between 0.5 hours and 24 hours.

[0013] In some embodiments, the loading period and the holding period total between 0.5 hours and 24 hours, hi some embodiments, the loading period and the holding period total at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, or at least 23 hours.

[0014] In some embodiments, the contacting step comprises washing the rAAV particles bound to the chromatography medium with a wash buffer having a pH of 9.0 to 11 after loading the feed composition onto the chromatography medium but before retaining the rAAV particles bound to the chromatography medium. In some embodiments, the wash buffer has a pH between pH 9.5 and pH 10.5. In some embodiments, the wash buffer has a pH of about pH 10.2. In some embodiments, the wash buffer comprises bis-tris propane (BTP) or glycine.

[0015] In some embodiments, the feed composition has a pH between 8.0 and 8.9.

[0016] In some embodiments, the chromatography medium is an anion exchange chromatography medium. In some embodiments, the chromatography medium is an affinity chromatography medium.

[0017] In some embodiments, the eluting step is performed using a linear salt gradient. In some embodiments, the linear salt gradient comprises between about 0.001 mM NaCl and about 1000 mM NaCl. In some embodiments, the linear salt gradient comprises between about 0.001 mM NaCl and about 100 mM NaCl. In some embodiments, the linear salt gradient comprises between about 0.001 mM NaCl and about 100 mM NaCl. In some embodiments, the eluting step is performed using a step salt gradient. In some embodiments, the step salt gradient comprises between about 7 mM NaCl and about 500 mM NaCl. In some embodiments, the step salt gradient comprises between about 70 mM NaCl and about 100 mM NaCl.

[0018] In some embodiments, the loading step is carried out by flowing the feed composition through the chromatographic medium at a flow rate between 0.1 CV / min and 5 CV / min.

[0019] In some embodiments, the chromatography medium comprises one or more amine functional groups. In some embodiments, the one or more amine functional groups are selected from primary amine, secondary amine, tertiary amine, quaternary amine functional groups, or combinations thereof. In some embodiments, the one or more amine functional groups comprise quaternary amine functional groups. In some embodiments, the one or more amine functional groups are bonded to a resin chromatography medium, a membrane chromatography medium, and / or a nanofiber chromatography medium. In some embodiments, the chromatography medium comprises a monolith.

[0020] In some embodiments, the eluting step is carried out in a buffer having a pH between 9.5 and 10.5.

[0021] In some embodiments, the rAAV particles are selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, AAV-16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.rh79, AAV.RHM4-1, AAV.hu37, AAVhu68, AAV.Anc80, AAV.Anc80L65, AAV.7 m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and / or AAV.HSC16.

[0022] In some embodiments, the rAAV particles comprise capsid proteins of AAVhu68. In some embodiments, the rAAV particles comprise capsid proteins of AAV1. In some embodiments, the rAAV particles comprise capsid proteins of AAV9.

[0023] In some embodiments, the method further comprises determining the yield of the purified rAAV particles, hi some embodiments, the yield of the purified rAAV particles is between 65% and 99%.

[0024] In some embodiments, the method further comprises determining the enrichment of intact rAAV particles in the purified rAAV particles. In some embodiments, at least 80% of the purified rAAV particles are intact rAAV particles. In some embodiments, at least 85% of the purified rAAV particles are intact rAAV particles. In some embodiments, between 1% and 40% of the rAAV particles in the feed composition are intact rAAV particles.

[0025] In some embodiments, the rAAV particles in the feed composition further comprise partially filled rAAV particles.

[0026] In some embodiments, the chromatography medium is a pre-packed monolithic chromatography column medium, hi some embodiments, the chromatography medium is a rigid, high-flow agarose matrix modified with a dextran surface extender and a quaternary ammonium (Q) strong anion exchanger.

[0027] In some embodiments, the purified rAAV particles have at least 95% of the titer of the rAAV particles in the feed composition.

[0028] In some embodiments, the delivery composition comprises poloxamer 188.

[0029] In some embodiments, the method further comprises the preceding step of contacting a sample containing rAAV particles with an affinity chromatography medium, thereby providing the feed composition. In some embodiments, the method further comprises the preceding step of preparing the sample containing the rAAV particles by depth filtration, concentration, or diafiltration. In some embodiments, the method further comprises the preceding step of preparing the sample containing the rAAV particles by depth filtration, concentration, and diafiltration.

[0030] In another aspect, the present disclosure provides a population of rAAV particles prepared by the methods disclosed herein. In some embodiments, the population of rAAV particles includes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, AAV-16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.rh79, AAV.RHM4-1, AAV.hu37, AAVhu68, AAV.Anc80, AAV.Anc80L65, AAV. In some embodiments, the population of rAAV particles comprises capsid proteins of an AAV selected from AAV1, AAV9, and AAVhu68.

[0031] In some embodiments, at least 70% of the rAAV particles in the population are complete rAAV particles. In some embodiments, at least 80% of the rAAV particles in the population are complete rAAV particles. In some embodiments, at least 85% or at least 90% of the rAAV particles in the population are complete rAAV particles.

[0032] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising a population of rAAV disclosed herein and a pharmaceutically acceptable excipient. [Brief explanation of the drawings]

[0033] (4. Brief description of some figures in the drawing) These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description and accompanying drawings.

[0034] [Figure 1A] Figures 1A-1B present UV absorbance (mAU) profiles at 280 nM and 260 nM of rAAV particles containing AAVhu68 capsids purified by AEX under low loading (1 column volume (1 CV)) conditions (Figure 1A) or high loading (4 column volumes (4 CV)) conditions (Figure 1B). [Figure 1B] Figures 1A-1B present UV absorbance (mAU) profiles at 280 nM and 260 nM of rAAV particles containing AAVhu68 capsids purified by AEX under low loading (1 column volume (1 CV)) conditions (Figure 1A) or high loading (4 column volumes (4 CV)) conditions (Figure 1B).

[0035] [Figure 2]Figure 2 presents UV absorbance (mAU) profiles at 280 nM and 260 nM of rAAV particles collected from anion exchange chromatography (AEX) purification performed under high loading (3.65E+14 GC / mL resin) and low loading (2.44E+14 GC / mL resin) conditions without a hold period on the column.

[0036] [Figure 3] Figure 3 presents the UV absorbance (mAU) profiles at 280 nM and 260 nM of rAAV particles purified from a feed composition containing 20 mM BTP buffer at pH 8.8 and eluted at pH 8.8.

[0037] [Figure 4] Figure 4 presents the UV absorbance (mAU) profiles at 280 nM and 260 nM of rAAV particles containing AAVhu68 capsids purified from sample dilutions without poloxamer. Omission of poloxamer reduced the yield and percentage of intact AAVhu68 particles.

[0038] [Figure 5A] Figures 5A-5C show UV absorbance (mAU) profiles at 280 nM and 260 nM of rAAV particles purified from conditions involving a short loading period (0.85 h) (Figure 5A) or a long loading period (14.57 h for Figure 5B and 20.54 h for Figure 5C). The short loading period was achieved by decreasing the loading amount, and the long loading period was achieved by increasing the loading amount. [Figure 5B]Figures 5A-5C show UV absorbance (mAU) profiles at 280 nM and 260 nM of rAAV particles purified from conditions involving a short loading period (0.85 h) (Figure 5A) or a long loading period (14.57 h for Figure 5B and 20.54 h for Figure 5C). The short loading period was achieved by decreasing the loading amount, and the long loading period was achieved by increasing the loading amount. [Figure 5C] Figures 5A-5C show UV absorbance (mAU) profiles at 280 nM and 260 nM of rAAV particles purified from conditions involving a short loading period (0.85 h) (Figure 5A) or a long loading period (14.57 h for Figure 5B and 20.54 h for Figure 5C). The short loading period was achieved by decreasing the loading amount, and the long loading period was achieved by increasing the loading amount.

[0039] [Figure 6] Figure 6 presents UV absorbance (mAU) profiles at 280 nM and 260 nM of rAAV particles purified by a method comprising holding a feed composition in solution for 24 hours prior to loading into AEX medium. The feed composition comprises rAAV particles suspended in 20 mM BTP buffer at high pH (pH 10.2).

[0040] [Figure 7] Figure 7 presents UV absorbance (mAU) profiles at 280 nM and 260 nM of rAAV particles purified by a method comprising loading a feed composition at low pH (pH 8.8) onto a pre-packed chromatography monolith column medium, followed by holding the composition on the column (in the chromatography medium) for 3 hours at high pH (pH 10.2). The feed composition comprises rAAV particles suspended in 20 mM BTP buffer at low pH (pH 8.8).

[0041] [Figure 8] Figure 8 presents UV absorbance (mAU) profiles at 280 nM and 260 nM of rAAV particles purified by a method comprising holding the feed composition in solution at high pH (pH 10.2) for 24 hours ("24 hour hold") prior to loading into AEX medium, or holding the feed composition on a column (in AEX medium) at high pH (pH 10.2) for 24 hours ("column hold") after loading. The feed composition comprises rAAV particles suspended in 20 mM BTP buffer at high pH (pH 10.2).

[0042] [Figure 9A-B] Figures 9A and 9B present the percent intact rAAV particles (y-axis) measured by the SEC-MALS detector plotted as a function of media contact time (x-axis) at pH 10.2. While not every molecule remains in contact with the media for this period, it is an approximation of the initial molecules in contact with the media until the onset of elution. An exception is circled in Figure 9B, where loading was performed at a lower pH and a 3-hour hold was added. Figure 9B is an expanded view of a subset of the data from Figure 9A (0-400 on the x-axis).

[0043] [Figure 10] Figure 10 shows the UV absorbance (mAU) profiles at 280 nM and 260 nM of rAAV particles purified by AEX after loading the feed composition and retaining it on the column for 3 hours. The results are from low loading ratio conditions (3.83E+13).

[0044] [Figure 11] Figure 11 presents the UV absorbance (mAU) profiles at 280 nM and 260 nM of rAAV particles purified by AEX without a holding step after loading a feed composition at a high loading pH (pH 10.2). The feed composition included rAAV particles suspended in 20 mM BTP buffer at a high pH (pH 10.2).

[0045] [Figure 12] Figure 12 presents the UV absorbance (mAU) profiles at 280 nM and 260 nM of rAAV particles purified by AEX after loading the feed composition in glycine buffer at a low loading pH (pH 8.8) followed by retention on the column (in the chromatography medium) at a high pH (pH 10.2) for a period of 3 hours. The feed composition comprises rAAV particles suspended in 20 mM glycine buffer at a high pH (pH 8.8).

[0046] [Figure 13A] Figures 13A and 13B present UV absorbance (mAU) profiles at 280 nM and 260 nM of rAAV particles purified by AEX after loading the feed composition at a low loading pH (pH 8.8) followed by retention on the column (in the chromatography medium) at a high pH (pH 10.2) for a 24 hour period. The feed compositions in Figure 13A (fresh) and Figure 13B (cleaned in place through storage (CIP)) contain rAAV particles suspended in 20 mM glycine buffer at a low pH (pH 8.8). [Figure 13B] Figures 13A and 13B present UV absorbance (mAU) profiles at 280 nM and 260 nM of rAAV particles purified by AEX after loading the feed composition at a low loading pH (pH 8.8) followed by retention on the column (in the chromatography medium) at a high pH (pH 10.2) for a 24 hour period. The feed compositions in Figure 13A (fresh) and Figure 13B (cleaned in place through storage (CIP)) contain rAAV particles suspended in 20 mM glycine buffer at a low pH (pH 8.8).

[0047] [Figure 14]Figure 14 presents the UV absorbance (mAU) profiles at 280 nM and 260 nM of rAAV particles purified by AEX after loading the feed composition at pH 8.9 followed by a 3-hour retention period on the column (in the chromatography medium) at high pH (pH 10.2). The loading ratio was 12.8E+13. The feed composition comprises rAAV particles suspended in a 20 mM glycine buffer at low pH (pH 8.9).

[0048] [Figure 15] Figure 15 presents the UV absorbance (mAU) profiles at 280 nM and 260 nM of rAAV particles purified by AEX after loading the feed composition at a low loading pH (pH 8.8) followed by a 3 hour column hold at a high pH (pH 10.2). The feed composition comprises rAAV particles suspended in 20 mM BTP buffer at low pH (pH 8.8). DETAILED DESCRIPTION OF THE INVENTION

[0049] (5. Detailed Description) (5.1 Definition) As used herein, the term "recombinant adeno-associated virus particle" or "rAAV particle" refers to a nuclease-resistant particle (NRP) containing an AAV capsid. The AAV capsid can package a heterologous nucleic acid molecule containing an AAV 5' inverted terminal repeat sequence and / or a 3' inverted terminal repeat sequence. In some cases, the AAV capsid does not package a heterologous nucleic acid molecule, forming an empty rAAV capsid. The heterologous nucleic acid molecule can include an expression cassette containing a coding sequence operably linked to an expression control sequence. The coding sequence can encode a therapeutic protein. Alternatively, the expression cassette can include sequences for gene editing, shRNA, miRNA, or other therapeutic functions.

[0050] In many cases, rAAV particles are called DNase-resistant particles (DRP).However, in addition to this endonuclease (DNase), exonucleases can also be used to remove contaminating nucleic acids in the purification process described herein.Such nucleases can be selected to degrade single-stranded DNA and / or double-stranded DNA, as well as RNA.Such processes can include a single nuclease, a mixture of nucleases for various targets, and can be endonucleases or exonucleases.

[0051] The term "nuclease-resistant" refers to a fully assembled AAV capsid. In the case of a complete rAAV particle, the AAV capsid surrounds an expression cassette designed to deliver a transgene into a host cell, and the AAV capsid protects these packaged genomic sequences from degradation (digestion) during a nuclease incubation step designed to remove possible contaminating nucleic acids from the production process.

[0052] As used herein, the term "complete rAAV particle" refers to an rAAV particle containing an AAV capsid enclosing a heterologous nucleic acid molecule containing an AAV 5' inverted terminal repeat and / or 3' inverted terminal repeat. The heterologous nucleic acid molecule is also referred to as a "vector genome." As used herein, the term "empty rAAV particle" refers to an rAAV particle lacking such a heterologous nucleic acid molecule. As used herein, the term "partially filled rAAV particle" refers to an rAAV viral particle containing only a partially packaged nucleic acid molecule that is insufficient to achieve expression of its gene product. These empty or partially filled rAAV particles are not functional for transferring the heterologous nucleic acid molecule (e.g., a gene of interest, a minigene) into a host cell.

[0053] Compositions containing rAAV particles can be analyzed by ultraviolet absorbance at about 260 nm and about 280 nm. Because the nucleic acid content of the capsid has a significant effect on the absorbance data at A260 and A280, the A260 / A280 ratio obtained in a plot of the absorbance data can be used to help identify complete AAV particles and empty or partially filled AAV particles. Typically, complete rAAV particles containing nucleic acid content within the capsid have UV260 detection higher than 280 nm, while empty and partially filled particles have UV280 detection higher than UV260 nm. The A260 / A280 ratio can be obtained by integrating the peak areas of individual species in a plot of UV absorbance (mAU) at both wavelengths and dividing the A260 measurement by the A280 measurement.

[0054] Compositions containing rAAV particles can be analyzed and characterized using analytical ultracentrifugation (AUC) or multi-angle static light scattering (SEC-MALS) as described in Brunham et al., Analytical Ultracentrifugation as an Approach to Characterize Recombinant Adeno-Associated Viral Vectors, Hum Gene Ther Methods, 2015 Dec;26(6):228-42, and McIntosh et al., Comprehensive characterization and quantification of adeno-associated vectors by size exclusion chromatography and multi-angle light scattering, Sci Rep., 2021 Feb;4;11(1):3012, which are incorporated by reference herein in their entireties.

[0055] As used herein, the term "rAAV particle titer" or "titer" refers to the ability of the rAAV particle to transfer its vector genome into target cells. The titer can be measured by detecting gene expression from the viral genome transferred into target cells. In some cases, the titer is measured by quantifying the vector genome transferred into target cells. The titer can be measured in vitro or in vivo.

[0056] As used herein, the term "loading period" or "feed composition loading period" refers to the time it takes to load a chromatography medium with a feed composition. The feed composition loading period can be measured as the time between the initial contact of the feed composition with the chromatography medium and the final application of the feed composition to the chromatography medium. The loading period can vary depending on the volume of the feed composition, the capacity of the chromatography medium, and the loading rate.

[0057] As used herein, the term "holding period" or "hold period" refers to a period of holding during which a composition is maintained at a steady state. The holding is often performed under static conditions. However, holding can be performed under dynamic conditions, provided that any changes applied to the composition are consistent and constant. For example, "holding duration on column" can refer to the time a feed composition is maintained in contact with a column without any new sample or buffer being applied to the column, or with continuous, steady application of the same sample or buffer to the column.

[0058] "Retention period in solution" refers to the time that the feed composition is maintained in solution (e.g., holding buffer) in the absence of the chromatography medium, typically prior to loading the feed composition onto the chromatography medium.

[0059] As used herein, the term "column volume" or "CV" refers to the volume inside a packed column that is not occupied by the media. The volume can include both the interstitial volume (the volume outside the particles) and the internal voids (pore volume) of the media itself. The column volume can be used as a unit. For example, 50 CV or 50 column volumes refers to 50 times the column volume.

[0060] As used herein, the term "time-on-media" refers to the time between initial contact of a rAAV particle with a chromatography medium and the elution of the rAAV particle from the chromatography medium. Although each rAAV particle is not in contact with the chromatography medium for this period, it is determined by the time from initial loading of the feed composition onto the chromatography medium to the start of elution.

[0061] As used herein, the term "time-on-column" refers to the time between initial contact of the rAAV particles with the chromatography column and the elution of the rAAV particles from the chromatography column.

[0062] 5.2 Methods for Purifying rAAV Particles The present disclosure provides a method for purifying recombinant adeno-associated virus (rAAV) particles. The method effectively separates rAAV particles containing heterologous nucleic acid molecules (complete rAAV particles) that contain AAV 5' inverted terminal repeats and / or 3' inverted terminal repeats adjacent to the DNA sequence from rAAV particles that lack the heterologous nucleic acid molecule (empty rAAV particles) or contain partial heterologous nucleic acid molecule DNA (partially filled rAAV particles). In a preferred embodiment, the rAAV composition purified and recovered by the method disclosed herein contains significantly enriched complete rAAV particles compared to the source composition.

[0063] The method for purifying the rAAV particles includes subjecting a feed composition containing rAAV particles to a chromatography medium under conditions that allow binding of the rAAV particles to the chromatography medium. The rAAV particles bound to the chromatography medium are washed and eluted. To improve performance, the rAAV particles bound to the chromatography medium can be incubated in a retention buffer for at least 0.5 hours before being eluted.

[0064] Thus, in some embodiments, the method comprises: a) providing a feed composition comprising the rAAV particles, wherein the rAAV particles in the feed composition include empty rAAV particles, partially filled rAAV particles, and complete rAAV particles; b) loading the feed composition onto a chromatography medium for a loading period under conditions that allow binding of the rAAV particles to the chromatography medium; c) optionally, holding the rAAV particles bound to the chromatography medium in a holding buffer for a holding period, wherein the holding period is 0.5 hours or longer; d) eluting the rAAV particles from the chromatography medium; and e) recovering the purified rAAV particles, thereby enriching for intact rAAV particles Includes:

[0065] In some embodiments, the method includes retaining the rAAV particles bound to the chromatography medium for a retention period of at least 0.5 hours.

[0066] In some other embodiments, the method comprises: a) providing a feed composition comprising the rAAV particles, wherein the rAAV particles in the feed composition include empty rAAV particles, partially filled rAAV particles, and complete rAAV particles; b) contacting the feed composition with a chromatography medium under conditions that allow binding of the rAAV particles to the chromatography medium for a medium contact time, wherein the medium contact time is at least 0.5 hours; c) eluting the rAAV particles from the chromatography medium; and d) recovering the purified rAAV particles, thereby enriching for intact rAAV particles Includes:

[0067] In some embodiments, the chromatographic purification methods are used in combination with rAAV production and purification methods known in the art. Additional steps for the production of rAAV particles may be referred to as upstream or downstream processes, depending on the order of the steps relative to the chromatographic purification methods disclosed herein. Various modifications to these upstream and downstream processes may be made.

[0068] 5.2.1 Providing Feed Composition The feed composition is a composition containing rAAV particles to be purified. The rAAV particles in the feed composition include empty, partially filled, and complete rAAV particles. The feed composition is loaded onto a chromatography medium.

[0069] In some embodiments, the feed composition comprises a loading buffer that is compatible with a chromatography medium. In some embodiments, the feed composition comprises a loading buffer that is compatible with an anion exchange chromatography medium. In some embodiments, the feed composition comprises a loading buffer that is compatible with an affinity chromatography medium.

[0070] In some embodiments, the loading buffer comprises bis-tris propane (BTP). In some embodiments, the loading buffer comprises 10 mM to 30 mM BTP. In some embodiments, the loading buffer comprises 10 mM BTP, 15 mM BTP, 20 mM BTP, 25 mM BTP, or 30 mM BTP. In some embodiments, the loading buffer comprises glycine. In some embodiments, the loading buffer comprises 10 mM to 30 mM glycine. In some embodiments, the loading buffer comprises 10 mM glycine, 15 mM glycine, 20 mM glycine, 25 mM glycine, or 30 mM glycine.

[0071] In some embodiments, the loading buffer further comprises poloxamer 188. In some embodiments, the loading buffer lacks poloxamer 188.

[0072] In some embodiments, the loading buffer further comprises NaCl. In some embodiments, the loading buffer further comprises 100 mM to 400 mM NaCl. In some embodiments, the loading buffer further comprises 10 mM to 200 mM NaCl. In some embodiments, the loading buffer further comprises 10 mM to 100 mM NaCl. In some embodiments, the loading buffer further comprises 10 mM to 50 mM NaCl.

[0073] In some embodiments, the loading buffer comprises 20 mM BTP and 10 mM NaCl at pH 10.2. In some embodiments, the loading buffer comprises 20 mM glycine and 10 mM NaCl at pH 10.2.

[0074] In some embodiments, the loading buffer, when applied to the chromatography medium, provides conditions that allow binding of rAAV particles to the chromatography medium. In some embodiments, the feed composition has a pH between 9.5 and 10.5. In some embodiments, the feed composition has a pH between 10 and 10.5. In some embodiments, the feed composition has a pH of about 10.2. In some embodiments, the feed composition has a pH between 7.0 and 9.5. In some embodiments, the feed composition has a pH between 7.5 and 9.5. In some embodiments, the feed composition has a pH between 8.0 and 9.5. In some embodiments, the feed composition has a pH between 8.0 and 8.9. In some embodiments, the feed composition has a pH between 8.2 and 8.8. In some embodiments, the feed composition has a pH of 8.3, 8.4, 8.5, 8.6, 8.7, or 8.8.

[0075] In some embodiments, less than 50% of the rAAV particles in the feed composition are whole. In some embodiments, less than 40% of the rAAV particles in the feed composition are whole. In some embodiments, less than 30% of the rAAV particles in the feed composition are whole. In some embodiments, less than 20% of the rAAV particles in the feed composition are whole. In some embodiments, less than 10% of the rAAV particles in the feed composition are whole.

[0076] In some embodiments, at least 45% of the rAAV particles in the feed composition are whole. In some embodiments, at least 40% of the rAAV particles in the feed composition are whole. In some embodiments, at least 30% of the rAAV particles in the feed composition are whole. In some embodiments, at least 20% of the rAAV particles in the feed composition are whole. In some embodiments, at least 10% of the rAAV particles in the feed composition are whole. In some embodiments, at least 5% of the rAAV particles in the feed composition are whole. In some embodiments, at least 1% of the rAAV particles in the feed composition are whole.

[0077] In some embodiments, between 1% and 40% of the rAAV particles in the feed composition are whole. In some embodiments, between 1% and 35% of the rAAV particles in the feed composition are whole. In some embodiments, between 1% and 30% of the rAAV particles in the feed composition are whole. In some embodiments, between 1% and 25% of the rAAV particles in the feed composition are whole. In some embodiments, between 1% and 20% of the rAAV particles in the feed composition are whole.

[0078] 5.2.1.1 Preparation of Feed Composition The feed composition can be prepared using upstream processes known in the art, for example, the feed composition can be made by an rAAV production process followed by a concentration and / or purification process.

[0079] In some embodiments, the feed composition is prepared by a process comprising (i) rAAV production, (ii) harvest processing and lysis, and (iii) filtration. In some embodiments, the feed composition is prepared by a process comprising (i) rAAV production, (ii) harvest processing and lysis, and (iii) depth filtration and filtration. In some embodiments, the feed composition is prepared by a process comprising (i) rAAV production, (ii) harvest processing and lysis, (iii) depth filtration and filtration, and (vi) TFF1 concentration and buffer exchange. In some embodiments, the process further comprises affinity chromatography purification. In some embodiments, the feed composition is prepared by a process comprising (i) cell bank thawing, (ii) inoculum growth, (iii) rAAV production in a production bioreactor, (iv) harvest processing and lysis, (v) depth filtration and filtration, (vi) TFF1 concentration and buffer exchange, and (vii) affinity chromatography. These steps may be used in various orders. Some exemplary processes are presented in FIG.

[0080] 5.2.1.1.1 rAAV Production Many methods are known in the art for the production of rAAV vectors, including, but not limited to, production from cell culture using transient transfection, stable cell line production, and infectious hybrid virus production systems (including adenovirus-AAV hybrids, herpesvirus-AAV hybrids, and baculovirus-AAV hybrids), as described in U.S. Pat. No. 11,098,286, which is incorporated herein by reference in its entirety. rAAV production cultures for the production of rAAV viral particles generally require: 1) suitable host cells, such as a human-derived cell line (e.g., HeLa, A549, or 293 cells) or, in the case of baculovirus production systems, an insect-derived cell line (e.g., SF-9); 2) appropriate helper virus functions provided by wild-type or mutant adenovirus (e.g., temperature-sensitive adenovirus), herpesvirus, baculovirus, or nucleic acid constructs providing helper functions in trans or cis; 3) functional AAV rep gene, functional cap gene, and gene products; 4) a transgene (e.g., a therapeutic transgene) flanked by AAV ITR sequences; and 5) media and media components suitable for supporting rAAV production.

[0081] A variety of suitable cells and cell lines have been described for use in AAV production. The cells can be selected from any organism, including prokaryotic (e.g., bacterial) cells and eukaryotic cells (including insect cells, yeast cells, and mammalian cells). Particularly desirable host cells are selected from any mammalian species, including, but not limited to, A549, WEHI, 3T3, 10T1 / 2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, HEK 293 cells (which express functional adenovirus E1), Saos, C2C12, L cells, HT1080, HepG2, and primary fibroblasts, hepatocytes, and myoblasts from mammals (including humans, monkeys, mice, rats, rabbits, and hamsters). In certain embodiments, the cells are suspension-culture compatible cells. In some embodiments, the cell line used for the rAAV particle purification methods described herein is the HEK 293 cell line.

[0082] The host cell may be a cell that has been stably transformed with the rep-encoding sequence and the cap-encoding sequence and transfected with the DNA of adenovirus E1, E2a, and E4 ORF6 and a construct carrying the expression cassette described above. Other stable cell lines that express rep and / or cap (e.g., B-50, described in WO 99 / 15685, or the cell line described in U.S. Pat. No. 5,658,785, which are incorporated herein by reference in their entirety) may be used. Another desirable host cell contains the minimal adenovirus DNA sufficient to express E4 ORF6.

[0083] The components required for AAV production (e.g., adenoviral E1a, E1b, E2a, and / or E4ORF6 gene products, rep or fragments thereof, cap, the expression cassettes described above, and any other desired helper functions) can be delivered to a packaging host cell, separately or in combination, in the form of any genetic elements that transfer the sequences they carry. Alternatively, one or more of the components required to be cultured in a host cell for packaging an expression cassette into an AAV capsid can be provided in trans to the host cell using the appropriate genetic elements.

[0084] Suitable media known in the art can be used for the production of rAAV vectors, including, but not limited to, media manufactured by Hyclone Laboratories and JRH, including modified Eagle's medium (MEM), Dulbecco's modified Eagle's medium (DMEM), custom formulations (e.g., the custom formulations described in U.S. Patent No. 6,566,118, and Sf-900 II SFM medium described in U.S. Patent No. 6,723,551, each of which is incorporated herein by reference in its entirety, particularly with respect to custom media formulations for use in the production of recombinant AAV vectors).

[0085] rAAV production culture medium can be supplemented with serum or serum-derived recombinant proteins at levels of 0.5% (v / v or w / v) to 20% (v / v or w / v). Alternatively, as is known in the art, rAAV vectors can be produced in serum-free conditions (which may also be referred to as animal-derived product-free media). Those skilled in the art will understand that commercially available or specialized media designed to support rAAV vector production can also be supplemented with one or more cell culture components known in the art (including, but not limited to, glucose, vitamins, amino acids, and / or growth factors) to increase the titer of rAAV in the production culture.

[0086] rAAV production cultures can be grown under a variety of conditions (such as over a wide temperature range and for various lengths of time) appropriate for the particular host cells utilized. As is known in the art, rAAV production cultures include attachment-dependent cultures that can be cultured in suitable attachment-dependent vessels (e.g., roller bottles, hollow fiber filters, microcarriers, packed-bed or fluidized-bed bioreactors, etc.). rAAV vector production cultures can also include suspension-adapted host cells (e.g., HeLa, 293, and SF-9 cells) that can be cultured in a variety of formats (e.g., shake flasks, spinner flasks, stirred-tank bioreactors, and disposable systems (e.g., Wave bioreactor systems)). In some embodiments, the host cells are cultured in a bioreactor (e.g., the iCellis® bioreactor system (Pall)). In some embodiments, the host cells are cultured in a shake flask or wave bioreactor for inoculum propagation prior to large-scale cultivation. In some embodiments, the host cells are cultured in a production bioreactor (eg, an iCellis® bioreactor system (Pall)) after growth of the inoculum.

[0087] (5.2.1.2 Collection and Lysis) In some embodiments, the rAAV production culture is subjected to harvesting and subsequent lysis of the producer cell culture.The target rAAV vector particles can be collected from the rAAV production culture by lysing the host cells of the production culture or by collecting the medium from the production culture when the cells are cultured under conditions known in the art to cause the release of rAAV particles from intact cells into the medium, as more fully described in U.S. Patent No. 6,566,118 (which is incorporated herein in its entirety).Suitable methods for lysing cells are also known in the art, and include, for example, multiple freeze / thaw cycles, sonication, microfluidization, and treatment with chemicals (for example, detergents and / or proteases).

[0088] At harvest, rAAV production cultures may contain one or more of the following in addition to rAAV particles: (1) host cell proteins; (2) host cell DNA; (3) plasmid DNA; (4) helper virus; (5) helper virus proteins; (6) helper virus DNA; and (7) media components (including, for example, serum proteins, amino acids, transferrin, and other low molecular weight proteins).

[0089] In some embodiments, the harvest is treated with a detergent (e.g., 0.16% lauryldimethylamine oxide (LDAO), 2 mM MgCl, 20 mM Tris, 400 mM NaCl, pH 8.0) at a slightly alkaline pH. In some embodiments, the harvest is treated with a nuclease or combination of nucleases to digest any high molecular weight contaminating nucleic acids present in the production culture. Such nucleases may target single-stranded DNA, double-stranded DNA, or RNA. Although the examples show the use of a deoxyribonuclease (DNase) (e.g., Benzonase or Turbonuclease), other suitable nucleases are known, many of which are commercially available. The nuclease can be a DNase (e.g., Benzonase®) digestion performed under standard conditions known in the art. An appropriate nuclease or combination of nucleases can be selected accordingly. Furthermore, the nuclease selected for this step can be the same as or different from the nuclease used during upstream processing or in a step immediately following harvest of the cell culture. In some embodiments, the harvest is treated with a high salt-tolerant nuclease (Salt Active Nuclease High Quality (SanHQ) (25 U / mL)).

[0090] 5.2.1.3 Clarification and Concentration of rAAV Particles In some embodiments, the process for preparing the feed composition further comprises tangential flow filtration (TFF) to concentrate the rAAV particles, heat inactivation of helper virus, rAAV capture by hydrophobic interaction chromatography, buffer exchange by size exclusion chromatography (SEC), and / or filtration. These steps can be used alone, in various combinations, or in various orders.

[0091] In some embodiments, the rAAV production culture harvest is clarified to remove host cell debris by filtration through a series of depth filters (e.g., including a grade DOHC Millipore Millistak+HC Pod Filter, a grade A1HC Millipore Millistak+HC Pod Filter, a 0.2 μm Filter Opticap XL10 Millipore Express SHC Hydrophilic Membrane filter, and a bilayer media grade PDK11 (2-20 μM retention) and Pall Supracap 50 depth filter capsule with a Luer-lock connection).

[0092] Clarification can also be achieved by a variety of other standard techniques known in the art, such as centrifugation or filtration through any cellulose acetate filter known in the art with a pore size of 0.2 μm or larger. Still other suitable depth filters (e.g., in the range of about 0.045 μm to about 0.2 μm) or other filtration techniques can be used. In some embodiments, clarification is carried out by 0.2 μm filtration using a Pall EKV 0.2 μm filter. In some embodiments, the clarification step does not include centrifugation. In some embodiments, the clarification is carried out by ultrafiltration or diafiltration.

[0093] 5.2.1.4 Tangential Flow Filtration (TFF) and Buffer Exchange In some embodiments, the rAAV composition is concentrated by tangential flow filtration ("TFF"). Large-scale virus concentration using TFF ultrafiltration was described by R. Paul et al., HUMAN GENE THERAPY, 4:609-615 (1993). TFF concentration of the feed composition allows for technically manageable volumes of the feed composition to be subjected to the chromatography steps of the present invention, allowing for more reasonable column sizing without the need for long recirculation times. In some embodiments, the rAAV feed composition is concentrated between at least 2-fold and at least 10-fold. In some embodiments, the feed composition is concentrated between at least 10-fold and at least 20-fold. In some embodiments, the feed composition is concentrated between at least 20-fold and at least 50-fold. In some embodiments, the TFF step is performed using a membrane cassette (Pall Omega 100 kDa filter) to remove salts and proteins. TFF can also be used at any step in the purification process, where it may be desirable to exchange the buffer before performing the next step in the purification process. Those skilled in the art will also recognize that. In some embodiments, tangential flow filtration (TFF) is performed more than once (eg, both before and after chromatographic purification).

[0094] 5.2.2 Contacting Feed Composition with Chromatography Media The methods provided herein include contacting a feed composition containing rAAV particles with a chromatography medium under conditions that allow binding of the rAAV particles to the chromatography medium. The duration of the contacting step is referred to as the "time-on-media." The media contact time can be greater than 2 hours. In some embodiments, the media contact time is between 2 and 24 hours. In some embodiments, the media contact time is greater than 3 hours, greater than 4 hours, greater than 5 hours, greater than 6 hours, greater than 7 hours, greater than 8 hours, greater than 9 hours, greater than 10 hours, greater than 11 hours, or greater than 12 hours. In some embodiments, the media contact time is less than 24 hours, less than 22 hours, less than 20 hours, less than 18 hours, less than 16 hours, less than 14 hours, or less than 12 hours. In some embodiments, the media contact time is less than 48 hours, less than 36 hours, or less than 24 hours.

[0095] In some embodiments, the contacting step comprises loading the rAAV particles onto the chromatography medium for a loading period. The loading period can be greater than 0.5 hours, greater than 1 hour, greater than 2 hours, greater than 3 hours, greater than 4 hours, greater than 5 hours, greater than 6 hours, greater than 12 hours, or greater than 18 hours. In some embodiments, the loading period is between 0.5 hours and 24 hours. The loading period can be adjusted by changing the loading volume or loading ratio.

[0096] In some embodiments, the contacting step comprises holding the rAAV particles bound to the chromatography medium in a holding buffer for a holding period. In preferred embodiments, the holding period is 0.5 hours or longer. In some embodiments, the holding buffer is the same as the loading buffer.

[0097] In some embodiments, the loading period and holding period combined is between 0.5 hours and 24 hours, hi some embodiments, the loading period and holding period combined is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, or at least 23 hours.

[0098] In some embodiments, the contacting step comprises washing the rAAV particles bound to the chromatography medium with a wash buffer having a pH of 9.0-11. The wash can be performed after loading the feed composition onto the chromatography medium but before retaining the rAAV particles bound to the chromatography medium. In some embodiments, the wash can be performed after retaining the rAAV particles bound to the chromatography medium. In some embodiments, the wash buffer can be the same as the loading buffer and / or the retaining buffer.

[0099] 5.2.3 Loading of Feed Composition onto Chromatography Media The feed composition can be loaded onto a chromatography medium for rAAV particle purification and enrichment of intact rAAV particles. After loading, the feed composition can be contacted with the chromatography medium under conditions that allow binding of the rAAV particles to the chromatography medium.

[0100] In some embodiments, the feed composition is buffer exchanged with a column equilibration / loading buffer. In some embodiments, the feed composition is purified on an affinity chromatography medium. In some embodiments, the feed composition is purified by anion exchange chromatography (AEX). In some embodiments, the feed composition is purified by affinity chromatography followed by anion exchange chromatography. In some embodiments, the feed composition is purified by anion exchange chromatography followed by affinity chromatography.

[0101] In some embodiments, the process of preparing the feed composition for the anion exchange chromatography comprises a step of affinity chromatography. In some embodiments, the process of preparing the feed composition for the affinity chromatography comprises a step of anion exchange chromatography.

[0102] The chromatography media can have a variety of load capacities. In some embodiments, the load capacity is between 5E+10 vg / mL and 5E+15 vg / mL. In some embodiments, the load capacity is between 5E+11 vg / mL and 5E+15 vg / mL. In some embodiments, the load capacity is between 5E+11 vg / mL and 5E+14 vg / mL. In some embodiments, the load capacity is between 5E+12 vg / mL and 5E+14 vg / mL. In some embodiments, the load capacity is between 5E+13 vg / mL and 10E+13 vg / mL.

[0103] In some embodiments, the loading takes at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, or at least 24 hours.

[0104] In some embodiments, the loading takes less than 30 hours, less than 24 hours, less than 23 hours, less than 22 hours, less than 21 hours, less than 20 hours, less than 19 hours, less than 18 hours, less than 17 hours, less than 16 hours, less than 15 hours, less than 14 hours, less than 13 hours, less than 12 hours, less than 6 hours, or less than 3 hours.

[0105] In some embodiments, the loading takes between 3 hours and 24 hours, between 6 hours and 24 hours, between 12 hours and 24 hours, between 18 hours and 24 hours, or longer than 24 hours.

[0106] The loading time can be increased by increasing the loading volume or decreasing the flow rate. The loading time can be decreased by decreasing the loading volume or increasing the flow rate. In some embodiments, loading is performed as a continuous flow. In some embodiments, loading is performed as a discontinuous flow.

[0107] 5.2.3.1 Affinity Chromatography In some embodiments, the feed composition is loaded onto an affinity chromatography medium having binding specificity for rAAV. In some embodiments, the affinity chromatography is performed using an antibody-capture affinity chromatography medium. In some embodiments, the affinity chromatography medium comprises an rAAV-specific antibody (e.g., an AAV1-specific antibody, an AAV9-specific antibody, or an AAVhu68-specific antibody) or other immunoglobulin construct specific for some rAAV serotype.

[0108] In one embodiment, the chromatographic medium comprises a solid support, which is cross-linked poly(styrene-divinylbenzene) having an average particle size of about 50 μm and bearing rAAV-specific antibodies. An example of such a commercially available affinity resin is the POROS™ high-performance affinity resin (POROS CaptureSelect AAVX Affinity Resin) available from Thermo Fisher Scientific. The resin comprises a ligand, generated by a technique based on a camelid-derived single-domain antibody fragment, linked to the resin via carbonyldiimidazole (CDI). The ligand may comprise a single-domain fragment containing three CDRs that form the antigen-binding domain. In some embodiments, the solid support comprises a polymer matrix material (e.g., agarose, Sepharose, Sephadex, among others).

[0109] In some embodiments, the loading amount is about 2×10 12 GC / mL medium ~ approx. 5 x 10 14 In some embodiments, the loading amount is in the range of about 2×10 12 GC / mL medium ~ approx. 5 x 10 13 In some embodiments, the loading amount is in the range of about 2×10 12GC / mL medium ~ approx. 5 x 10 12 Within the range of GC / mL media.

[0110] In some embodiments, the maximum flow rate is between about 100 cm / hr and about 600 cm / hr (e.g., 350 cm / hr). In some embodiments, the maximum flow rate is between 100 cm / hr and 400 cm / hr. In some embodiments, the maximum flow rate is between 100 cm / hr and 300 cm / hr. In some embodiments, the maximum flow rate is between 100 cm / hr and 200 cm / hr. In some embodiments, the maximum flow rate is between 200 cm / hr and 400 cm / hr. In some embodiments, the maximum flow rate is between 300 cm / hr and 400 cm / hr. In some embodiments, the maximum flow rate is about 150 cm / hr. In some embodiments, the maximum flow rate is about 300 cm / hr. In some embodiments, the maximum flow rate is less than 150 cm / hr. In some embodiments, the maximum flow rate is less than 300 cm / hr.

[0111] In one embodiment, a feed composition containing the rAAV particles (including empty, partially filled, and full particles) is loaded onto the chromatography medium in a buffer having a high salt concentration (e.g., about 400 nM NaCl to about 650 mM NaCl, or other salts with comparable ionic strength).

[0112] 5.2.3.2 Anion Exchange Chromatography (AEX) In some embodiments, the methods provided herein include purification using anion exchange chromatography (AEX). Anion exchange chromatography (AEX) is a form of ion exchange chromatography that separates samples based on the net surface charge of the sample. Anion exchange chromatography (AEX) specifically uses positively charged ligands that have affinity for targets with a negative net surface charge.

[0113] The present invention relates to methods for purifying rAAV particles using various anion exchange chromatography (AEX) media. The interaction between the AEX media and rAAV particles is affected by several factors (e.g., anion exchanger, flow rate, resin particle size, binding capacity, etc.). The present invention further relates to specific conditions under which rAAV particles can be effectively isolated, purified, or partially fractionated using the AEX media. For example, specific buffer conditions for purifying rAAV particles using AEX are disclosed.

[0114] In some embodiments, the AEX medium comprises one or more amine functional groups. In some embodiments, the one or more amine functional groups are selected from primary amine, secondary amine, tertiary amine, quaternary amine functional groups, or combinations thereof. In some embodiments, the one or more amine functional groups comprise quaternary amine functional groups. In some embodiments, the one or more amine functional groups are bonded to a resin chromatography medium, a membrane chromatography medium, and / or a nanofiber chromatography medium.

[0115] In some embodiments, the AEX medium comprises a monolith. In some embodiments, the AEX medium is a monolith anion exchange medium. In some embodiments, the AEX medium is a monolith column. In some embodiments, the AEX medium is a pre-packed monolith column medium for chromatography. In some embodiments, the AEX medium is a CIMQA Monolith column (Sartorius). In some embodiments, the AEX medium comprises a rigid high-flow agarose matrix modified with a dextran surface extender and a quaternary ammonium (Q) strong anion exchanger.

[0116] Prior to loading, the AEX medium may be equilibrated. In some embodiments, the AEX medium is equilibrated with a loading buffer. In some embodiments, the AEX medium is equilibrated in multiple steps with various buffers.

[0117] In some embodiments, the feed composition has a high salt concentration when it is loaded onto the AEX column. In one embodiment, the feed composition has a salt concentration of about 400 mM NaCl to about 650 mM NaCl or equivalent before being applied to the AEX medium. In one embodiment, the feed composition has a salt concentration of about 10 mM to about 300 mM NaCl. In one embodiment, the feed composition has a salt concentration of about 10 mM to about 200 mM NaCl. In one embodiment, the feed composition has a salt concentration of about 10 mM to about 100 mM NaCl. In one embodiment, the feed composition has a salt concentration of about 10 mM.

[0118] In some embodiments, the feeding composition is present in a loading buffer (Buffer A). In some embodiments, the buffer comprises 20 mM bis-tris-propane (BTP). In some embodiments, the buffer comprises glycine. In some embodiments, the buffer comprises 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, or 50 mM bis-tris-propane (BTP). In some embodiments, the buffer comprises 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, or 50 mM glycine. In some embodiments, the loading buffer comprises 20 mM BTP and 10 mM NaCl. In some embodiments, the loading buffer comprises 20 mM glycine and 10 mM NaCl.

[0119] In some embodiments, the feed composition has a pH between 9.5 and 10.5 before being applied to the AEX medium. In some embodiments, the feed composition has a pH between 10 and 11 before being applied to the AEX medium. In some feed embodiments, the composition has a pH of about 10.2 before being applied to the AEX medium. In some embodiments, the feed composition has a pH between 8 and 9 before being applied to the AEX medium. In some embodiments, the feed composition has a pH between 8.5 and 9.5 before being applied to the AEX medium. In some embodiments, the feed composition has a pH between 8.5 and 9 before being applied to the AEX medium. In some embodiments, the feed composition has a pH of about 8.8 before being applied to the AEX medium.

[0120] In some embodiments, the loading amount is about 2×10 12 GC / mL medium ~ approx. 5 x 10 14 In some embodiments, the loading amount is in the range of about 2×10 12 GC / mL medium ~ approx. 5 x 10 13 In some embodiments, the loading amount is in the range of about 5×10 12 GC / mL medium ~ approx. 10×10 13 In some embodiments, the loading amount is in the range of about 1×10 13 GC / mL medium ~ approx. 10×10 13 In some embodiments, the loading amount is in the range of about 1×10 13 GC / mL medium ~ approx. 5 x 10 13 In some embodiments, the loading amount is in the range of about 3.0 x 10 13 GC / mL ~ approx. 3.5×10 13In some embodiments, the loading amount is in the range of about 2.0×10 GC / mL. 14 GC / mL ~ approx. 3.5×10 14 It is in the range of GC / mL.

[0121] In some embodiments, the loading is performed at a loading rate of 0.1 to 5.0 column volumes (CV) mL / min of the AEX medium. In some embodiments, the loading is performed at a loading rate of 0.5 to 3.0 CV mL / min of the AEX medium. In some embodiments, the loading is performed at a loading rate of 0.5 to 2.0 CV mL / min of the AEX medium. In some embodiments, the loading is performed at a loading rate of 0.5 to 1.5 CV mL / min of the AEX medium. In some embodiments, the loading is performed at a loading rate of 0.5 to 1.0 CV mL / min of the AEX medium. In some embodiments, the loading is performed at a loading rate of about 0.5 CV, about 0.6 CV, about 0.7 CV, or about 0.8 CV mL / min of the AEX medium. In some embodiments, the loading is performed at a loading rate of about 1.0 CV mL / min of the AEX medium. In some embodiments, the loading time is adjusted by changing the loading speed or by changing the loading volume.

[0122] 5.2.4 Washing of rAAV particles bound to chromatography media In some embodiments, loading is followed by a washing step using a wash buffer. This washing step can improve purity or further aid in concentrating, removing, or isolating the rAAV particles. The wash buffer can be a solution having a specific pH range, salt, organic solvent, small molecule, surfactant, zwitterion, amino acid, polymer, or any combination of the above. In some embodiments, the washing step is omitted. In this case, the rAAV particles bound to the chromatography medium are retained in the retention buffer without being washed with a wash buffer. In some embodiments, the loading buffer is used for washing.

[0123] The wash steps for the affinity chromatography may be performed using a wash buffer having a higher salt concentration than the loading buffer (eg, in the range of about 750 mM to about 1 M NaCl or equivalent).

[0124] The wash step for the anion exchange chromatography can be performed using a wash buffer having a high pH. The wash buffer can have a pH between 9.0 and 11. In some embodiments, the wash buffer has a pH between 9.5 and 10.5. In some embodiments, the wash buffer has a pH of about 10.2.

[0125] In some embodiments, the washing is performed at a washing rate of 0.5 to 5 column volumes / minute of the chromatography medium. In some embodiments, the washing is performed at a washing rate of about 1 column volume / minute of the chromatography medium. In some embodiments, the washing is performed at a washing rate of 1.0 to 10.0 column volumes / minute of the chromatography medium. In some embodiments, the washing is performed at a washing rate of 1.0 to 5.0 column volumes / minute of the chromatography medium. In some embodiments, the washing is performed at a washing rate of 2.0 to 4.0 column volumes / minute of the chromatography medium. In some embodiments, the washing is performed at a washing rate of 3.0 to 4.0 column volumes / minute of the chromatography medium. In some embodiments, the washing is performed at a loading rate of about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, or about 4.5 column volumes / minute of the chromatography medium.

[0126] In some embodiments, washing is performed with 0.5 to 50 column volumes of the wash buffer. In some embodiments, washing is performed with 1 to 40 column volumes of the wash buffer. In some embodiments, washing is performed with 1 to 25 column volumes of the wash buffer. In some embodiments, washing is performed with 5 to 25 column volumes of the wash buffer. In some embodiments, washing is performed with 10 to 20 column volumes of the wash buffer.

[0127] In some embodiments, the washing step is performed using a wash buffer. In some embodiments, the wash buffer comprises 20 mM bis-tris-propane (BTP). In some embodiments, the buffer comprises 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, or 50 mM bis-tris-propane (BTP). In some embodiments, the wash buffer comprises 20 mM bis-tris-propane (BTP) and NaCl. In some embodiments, the wash buffer comprises glycine. In some embodiments, the wash buffer comprises 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, or 50 mM glycine. In some embodiments, the wash buffer comprises 20 mM glycine and NaCl. In some embodiments, the wash buffer comprises 20 mM BTP and 10 mM NaCl at pH 10.2. In some embodiments, the wash buffer comprises 20 mM glycine and 10 mM NaCl at pH 10.2.

[0128] 5.2.5 Retention of rAAV Particles Bound to Chromatography Media In some embodiments, the method of purifying rAAV particles includes retaining the rAAV particles bound to the chromatography medium in a retention buffer for a retention period of at least 0.5 hours, which may improve purity or further aid in concentrating, removing, or isolating intact rAAV particles.

[0129] In some embodiments, the holding period is 0.5 hours or longer. In some embodiments, the holding period is at least 1 hour. In some embodiments, the holding period is at least 2 hours. In some embodiments, the holding period is at least 3 hours. In some embodiments, the holding period is at least 6 hours. In some embodiments, the holding period is at least 12 hours. In some embodiments, the holding period is at least 15 hours.

[0130] In some embodiments, the holding period is less than 24 hours. In some embodiments, the holding period is less than 20 hours. In some embodiments, the holding period is less than 18 hours. In some embodiments, the holding period is less than 15 hours. In some embodiments, the holding period is less than 12 hours. In some embodiments, the holding period is less than 6 hours. In some embodiments, the holding period is less than 3 hours. In some embodiments, the holding period is 3 hours or less.

[0131] In some embodiments, the holding period is 0.5 hours to 24 hours. In some embodiments, the holding period is 1 hour to 24 hours. In some embodiments, the holding period is 2 hours to 24 hours. In some embodiments, the holding period is 3 hours to 24 hours. In some embodiments, the holding period is 3 hours to 12 hours. In some embodiments, the holding period is 3 hours to 6 hours. In some embodiments, the holding period is 0.5 hours to 3 hours. In some embodiments, the holding period is about 3 hours to about 12 hours. In some embodiments, the holding period is about 3 hours. In some embodiments, the holding period is about 12 hours. In some embodiments, the holding period is about 24 hours. In some embodiments, the holding period is less than 24 hours.

[0132] In some embodiments, the holding buffer has a pH of 9.0 to 12. In some embodiments, the holding buffer has a pH of 9.0 to 11. In some embodiments, the holding buffer has a pH of 9.0 to 10.5. In some embodiments, the holding buffer has a pH of 9.5 to 10.5. In some embodiments, the holding buffer has a pH of about 10.2. In some embodiments, the holding buffer has the same pH as the wash buffer. In some embodiments, the holding buffer is the same as the wash buffer. In some embodiments, the holding buffer is the same as the loading buffer. In some embodiments, the holding buffer comprises 20 mM BTP. In some embodiments, the holding buffer comprises 20 mM glycine.

[0133] In some embodiments, the retention is performed under static conditions without application of sample to the chromatographic medium, hi some embodiments, the retention is performed under dynamic conditions with continuous application of sample or buffer to the chromatographic medium.

[0134] In some embodiments, the holding step is omitted if the loading period of the feeding composition is longer than 1 hour. In some embodiments, the holding step is omitted if the loading period of the feeding composition is longer than 2 hours, longer than 3 hours, longer than 4 hours, longer than 5 hours, longer than 6 hours, longer than 7 hours, longer than 8 hours, longer than 9 hours, longer than 10 hours, longer than 11 hours, or longer than 12 hours. In some embodiments, the total loading and holding period is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, or at least 23 hours. In some embodiments, the medium contact time of the feed composition is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, or at least 23 hours. In some embodiments, the medium contact time of the feed composition is less than 24 hours, less than 23 hours, less than 22 hours, less than 21 hours, less than 20 hours, less than 19 hours, less than 18 hours, less than 17 hours, less than 16 hours, less than 15 hours, less than 14 hours, less than 13 hours, or less than 12 hours. In some embodiments, the medium contact time of the feed composition is between 1 hour and 24 hours. In some embodiments, the medium contact time of the feed composition is between 2 hours and 24 hours. In some embodiments, the media contact time of the feed composition is between 3 hours and 24 hours.In some embodiments, the media contact time of the feed composition is between 6 hours and 24 hours, hi some embodiments, the media contact time of the feed composition is between 6 hours and 12 hours.

[0135] 5.2.6 Elution of rAAV Particles Selective elution can be achieved by altering the salt, phosphate, or calcium concentration; altering the pH; changing the temperature; adding an organic modifier, an organic solvent, a small molecule, a surfactant, a zwitterion, an amino acid, a polymer, a polyol (sucrose, glucose, trehalose, mannose, sorbitol, mannitol, glycerol, etc.), an antioxidant (e.g., methionine), EDTA, EGTA, polysorbate 20, polysorbate 80, ethylene glycol, propylene glycol, polyethylene glycol, polypropylene glycol, or urea; adding an excipient that alters the surface tension of the solvent; adding an excipient that alters the polarity of the solvent; changing the residence time to take advantage of differential desorption rates between intact and empty rAAV particles; or any combination of the above.

[0136] Elution can be achieved using multiple elution buffers with different properties (e.g., pH, salt, organic solvent, small molecule, detergent, zwitterion, amino acid, polymer, temperature, and any combination of the above). Multiple eluted fractions can be collected, with the rAAV particles collected in each fraction having different properties. For example, the rAAV particles collected in one fraction have higher purity, smaller or larger average size, more preferred composition, etc. than the rAAV particles in other fractions. Such elution buffers with different properties can be applied as a continuous stream, while multiple eluted fractions are collected.

[0137] In some embodiments, the eluting step is performed using a linear salt gradient. In some embodiments, the linear salt gradient comprises between about 0.001 mM NaCl and about 1000 mM NaCl. In some embodiments, the linear salt gradient comprises between about 0.001 mM NaCl and about 100 mM NaCl. In some embodiments, the linear salt gradient comprises between about 0.01 mM NaCl and about 100 mM NaCl. In some embodiments, the linear salt gradient comprises between about 1 mM NaCl and about 100 mM NaCl.

[0138] In some embodiments, the eluting step is performed using a step salt gradient. In some embodiments, the step salt gradient comprises between about 7 mM NaCl and about 500 mM NaCl. In some embodiments, the step salt gradient comprises between about 70 mM NaCl and about 500 mM NaCl. In some embodiments, the step salt gradient comprises between about 70 mM NaCl and about 100 mM NaCl.

[0139] 5.2.7 Recovery of purified rAAV particles The method of purifying the rAAV particles includes recovering or collecting the rAAV particles from the elution. The purified rAAV particles can be present in an eluate from the elution. The eluate can be analyzed to determine the purity of the purified rAAV particles (e.g., the percentage [%] of intact rAAV particles).

[0140] Thus, the method may further comprise analyzing the purified rAAV particles. In some embodiments, the method comprises determining the yield of the purified rAAV particles. In some embodiments, the method comprises determining the yield of the intact rAAV particles. The yield is determined as the ratio between total purified rAAV particles in the eluate and total rAAV particles in the feed composition.

[0141] In some embodiments, the yield is at least 50%. In some embodiments, the yield is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In some embodiments, the yield is between 65% and 99%. In some embodiments, the yield is between 70% and 99%. In some embodiments, the yield is between 75% and 99%. In some embodiments, the yield is between 80% and 99%. In some embodiments, the yield is between 80% and 95%.

[0142] In some embodiments, the method comprises determining the enrichment of intact rAAV particles in the purified or recovered rAAV particles. In some embodiments, the method comprises determining the enrichment of intact rAAV particles in the purified rAAV particles relative to intact rAAV particles in the feed composition. In some embodiments, the method comprises determining the percentage (%) of intact rAAV particles in the purified rAAV particles.

[0143] In some embodiments, at least 60% of the purified rAAV particles are complete rAAV particles. In some embodiments, at least 65% of the purified rAAV particles are complete rAAV particles. In some embodiments, at least 70% of the purified rAAV particles are complete rAAV particles. In some embodiments, at least 75% of the purified rAAV particles are complete rAAV particles. In some embodiments, at least 80% of the purified rAAV particles are complete rAAV particles. In some embodiments, at least 85% of the purified rAAV particles are complete rAAV particles. In some embodiments, at least 90% of the purified rAAV particles are complete rAAV particles. In some embodiments, at least 95% of the purified rAAV particles are complete rAAV particles.

[0144] In some embodiments, the purified rAAV particles have at least 99% of the titer of the rAAV particles in the feed composition. In some embodiments, the purified rAAV particles have at least 98% of the titer of the rAAV particles in the feed composition. In some embodiments, the purified rAAV particles have at least 95% of the titer of the rAAV particles in the feed composition. In some embodiments, the purified rAAV particles have at least 90% of the titer of the rAAV particles in the feed composition. In some embodiments, the purified rAAV particles have at least 85% of the titer of the rAAV particles in the feed composition. In some embodiments, the purified rAAV particles have at least 80% of the titer of the rAAV particles in the feed composition. In some embodiments, the purified rAAV particles have at least 75% of the titer of the rAAV particles in the feed composition.

[0145] 5.3 Purified rAAV particle populations In another aspect, the disclosure provides a population of rAAV particles purified using the methods disclosed herein. In some embodiments, the population comprises enriched, intact rAAV particles.

[0146] In some embodiments, at least 95% of the population are complete rAAV particles. In some embodiments, at least 90% of the population are complete rAAV particles. In some embodiments, at least 85% of the population are complete rAAV particles. In some embodiments, at least 80% of the population are complete rAAV particles. In some embodiments, at least 75% of the population are complete rAAV particles. In some embodiments, at least 70% of the population are complete rAAV particles. In some embodiments, at least 99%, at least 98%, at least 97%, at least 98%, at least 96%, or at least 95% of the population are complete rAAV particles.

[0147] In some embodiments, less than 5% of the population are empty rAAV particles. In some embodiments, less than 10% of the population are empty rAAV particles. In some embodiments, less than 15% of the population are empty rAAV particles. In some embodiments, less than 20% of the population are empty rAAV particles. In some embodiments, less than 25% of the population are empty rAAV particles. In some embodiments, less than 30% of the population are empty rAAV particles. In some embodiments, less than 1%, less than 2%, less than 3%, less than 4%, or less than 5% of the population are empty rAAV particles.

[0148] In some embodiments, less than 5% of the population are partially filled rAAV particles. In some embodiments, less than 10% of the population are partially filled rAAV particles. In some embodiments, less than 15% of the population are partially filled rAAV particles. In some embodiments, less than 20% of the population are partially filled rAAV particles. In some embodiments, less than 25% of the population are partially filled rAAV particles. In some embodiments, less than 30% of the population are partially filled rAAV particles. In some embodiments, less than 1%, less than 2%, less than 3%, less than 4%, or less than 5% of the population are partially filled rAAV particles.

[0149] In some embodiments, the rAAV particles in the population are selected from the group consisting of AAVhu68, AAV9, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, AAV-16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.rh79, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV In some embodiments, the rAAV particles in the population comprise capsid proteins of an AAV selected from AAV9, AAV1, and AAVhu68.

[0150] 5.4 Pharmaceutical Compositions In yet another aspect, the present disclosure provides a pharmaceutical composition comprising the rAAV particles prepared by the methods disclosed herein. The pharmaceutical composition may comprise a population of purified rAAV particles and a pharmaceutically acceptable excipient.

[0151] In some embodiments, the rAAV particle comprises a heterologous nucleic acid molecule together with a coding sequence for a therapeutic protein. In some embodiments, the heterologous nucleic acid molecule comprises a sequence for gene editing, shRNA, miRNA, or other therapeutic function. In some embodiments, the heterologous nucleic acid molecule further comprises a regulatory sequence operably linked to the therapeutic gene.

[0152] The pharmaceutical composition may be formulated for intravenous, intramuscular, subcutaneous, intrathecal, intracranial, intraventricular, intradermal, rectal, oral, intravaginal, intranasal, or inhalation administration, hi some embodiments, the pharmaceutical composition is formulated for injection or infusion.

[0153] The pharmaceutical composition can be used to deliver the rAAV to a mammalian subject (e.g., a human subject) in need thereof.

[0154] The pharmaceutical compositions may be formulated using one or more carriers, excipients, stabilizers, and adjuvants to, for example: (1) increase stability; (2) increase cell transfection or transduction; (3) enable sustained or delayed release; (4) alter biodistribution (e.g., to target rAAV particles to specific tissues or cell types); (5) increase translation of the encoded protein in vivo; and / or (6) alter the release profile of the encoded protein in vivo.

[0155] Formulations of the pharmaceutical compositions provided herein can include, but are not limited to, saline, which can be formulated using various buffer solutions (e.g., phosphate buffered saline), lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, water, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, nanoparticle mimetics, and combinations thereof.

[0156] Formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. Generally, such preparative methods include bringing into association the active ingredient (i.e., rAAV particles) with the carrier and / or one or more other accessory ingredients (e.g., excipients, stabilizers, and adjuvants).

[0157] Pharmaceutical compositions according to the present disclosure may be prepared, packaged, and / or sold in bulk, as a unit dose, and / or as a plurality of unit doses. As used herein, a unit dose refers to a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient, which amount is generally equal to the dose of the active ingredient that would be administered to a subject and / or a convenient fraction of such a dose (e.g., one-half or one-third of such a dose).

[0158] The relative amounts of the active ingredient (i.e., rAAV particles), pharmaceutically acceptable carrier, and / or any additional components in a pharmaceutical composition according to the present disclosure may vary depending on the identity, size, and / or condition of the subject being treated, as well as the route by which the composition is administered.

[0159] Various carriers, excipients, stabilizers and adjuvants for formulating pharmaceutical compositions and techniques for preparing the compositions are known in the art (see Remington: The Science and Practice of Pharmacy, Revised 22nd Edition, Pharmaceutical Press, 2012, which is incorporated herein by reference in its entirety). The use of suitable conventional carriers, excipients, stabilizers and adjuvants is contemplated within the scope of the present disclosure. [Example]

[0160] 6. Working Example 6.1. Example 1: AEX Purification of rAAV Particles rAAV particles were purified by a process involving anion exchange chromatography (AEX). Various purification conditions, such as buffer composition, were tested, and preferred conditions were selected. Table 1 presents two exemplary conditions (Process A and Process B) tested for the purification of rAAV particles. [Table 1]

[0161] As summarized in Table 1, in Process A, a feed composition containing a mixture of empty, partial, and full capsids partially purified by upstream processing (e.g., depth filtration / diafiltration, TFF concentration, and affinity chromatography) was diluted 50-fold in a loading buffer containing bis-tris-propane (BTP) buffer (20 mM BTP, 10 mM NaCl, pH 10.2). The same buffer can be used to equilibrate an anion exchange column. The process involved a two-step load adjustment to pH 10.2. The feed composition was diluted in BTP buffer (20 mM) at pH 10.2 containing 0.01% poloxamer 188, and then diluted in 0.2 M BTP at pH 10.2. In Process A, there was no maximum range setting for the loading capacity (dynamic binding capacity, DBC). The feed composition was applied to an anion exchange column (e.g., a CIMmultus-QA™ column, a CaptoQ column) equilibrated at pH 10.2. The rAAV particles bound to the chromatography medium were washed with BTP wash buffer (20 mM BTP, 10 mM NaCl, pH 10.2) at 1 CV / min for 10 CV. The bound rAAV particles were eluted with BTP buffer B (20% to 80% buffer B over 60 CV) using a linear salt gradient. UV absorbance was monitored at 260 nm and 280 nm. Fractions where the A260 / A280 ratio reached an inflection point (≥1) were collected and then stripped using multiple buffers, which included eight steps.

[0162] Process B shows alternative purification conditions for anion exchange chromatography to purify rAAV particles. This is a simplified process with fewer steps and reduced buffer usage. This process included a one-step load adjustment (instead of two) to pH 8.8 by adding BTP buffer (20 mM) at pH 8.9 containing 0.01% poloxamer 188. The load capacity (dynamic binding capacity, DBC) was limited to approximately 8.5E+13 VG / mL monolith. Similar to process A, rAAV particles bound to the chromatography medium in an anion exchange column (e.g., a CIMmultus-QA™ column) were washed with glycine wash buffer (20 mM glycine, 10 mM NaCl, pH 10.2) at 1 CV / min for 20 CV, followed by a 3-hour hold on the column at pH 10.2. The bound rAAV particles were eluted in a linear salt gradient (20% to 50% Buffer B over 30 CV) using glycine buffer B. UV absorbance was monitored at 260 nm and 280 nm. Fractions where the A260 / A280 ratio reached an inflection point (≥1) were collected and then stripped with reducing buffer, which involved two steps.

[0163] Process B offers several advantageous features over conventional methods. For example, Process B significantly reduces the number of steps, reduces processing time, and reduces the amount of buffer and the total number of buffers prepared during the clean-in-place (CIP) and equilibration (CIP / EQ) steps. It was noted that the CIP / EQ steps do not impact processing.

[0164] Process B replaced the BTP buffer with a glycine buffer, an adjustment that enhanced the buffering capacity of glycine for the pH control step of the gradient (glycine pKa=9.7 vs. BTP pKa=9.0).

[0165] Process A required a two-step load adjustment at a high pH (pH 10.2). In contrast, Process B used a one-step load adjustment to a low pH (pH 8.8). Process B reduced the pH to 8.8 or 8.9 to limit the amount of time the product remained at a high pH (e.g., pH 10.2).

[0166] Process A had no maximum range for loading capacity (dynamic binding capacity (DBC)), while Process B limited its loading capacity to approximately 8.5E+13 vg / mL monolith. Typically, DBC loading was evaluated at a pH of approximately 8.8. At 8.5E+13 vg / mL, 10% breakthrough was observed. Production-scale loading was 35% (3 x 200 ml) of DBC. 2 -400mL CIMQA) and 59% (1 x 200m 2 -80mL CIMQA).

[0167] During the wash step, Process B replaced BTP with 20 mM glycine, 10 mM NaCl, pH 10.2 at 1 CV / min for 20 CV, followed by a 3-hour hold on the column. This adjustment increased the amount of media contact time of intact capsids at pH 10.2, which increased the yield of collected intact capsids.

[0168] Process B reduces the buffer gradient range from 20% to 80% buffer B over 60 CV to 20% to 50% buffer B over 30 CV, with an additional starting criteria: ≧ The adjustments included a conductivity (mS / cm) of 10, milliabsorbance units at 280 nm (mAU) of ≥ 5, and an A260 / A280 (inflection point) of ≥ 1.0. The adjustments reduced elution time and the time before neutralization adjustment (time at pH 10.2). A final harvest with 50% Buffer B provided simplification for manufacturing. Additional starting criteria (e.g., A260 / A280 ratio) could be used for potential elimination and simplification for manufacturing.

[0169] During the stripping step, Process A included 8 steps using multiple buffers, while Process B included 2 steps using reduced buffers. These adjustments reduced processing time, the amount of buffer, and the total number of buffers prepared.

[0170] 6.2. Example 2: Conditions for Purification of rAAV Particles by Anion Exchange Chromatography The Convective Interactive Media (CIM) QA Monolith (CIMmultus-QA™, Sartorius) is a strong anion-exchange monolith chromatography column containing a cross-linked porous polymethacrylate material with a defined channel size distribution with a diameter greater than 1,000 nm. This monolith chromatography column was used to separate rAAV particles. The rAAV particles processed through upstream processes (e.g., depth filtration / diafiltration, TFF concentration, and capture chromatography) were diluted 50-fold into bis-tris-propane (BTP) buffer A (20 mM BTP, 10 mM NaCl, pH 10.2) (also referred to as "loading buffer") to form a feed composition. The pH of the feed composition was adjusted to 10.2.

[0171] To determine the effect of loading volume on the percentage of intact rAAV capsids collected, a control experiment was performed using a feed composition containing AAVhu68, whose UV absorbance (mAU) profiles at 280 nM and 260 nM are shown in Figures 1A and 1B.

[0172] Approximately 1 mL (low volume) of a feed composition containing AAVhu68 (including empty capsids, partially filled capsids, and complete capsids) was loaded onto a CIMmultus-QA™. The loading ratio was 3.67E+13. The loading rate was 0.67 CV / min, and the processing rate was 3.13 CV / min. The loading period was approximately 2.23 hours at pH 10.2. As shown in Figure 1A, these conditions resulted in an AAVhu68 particle yield of approximately 78.2% and approximately 70.7% complete AAVhu68 capsids.

[0173] The process was repeated using a total of 4 mL (high volume) of AAVhu68 particles. The loading ratio was 3.7E+13. The loading rate was 1 CV / min, and the processing rate was 1 CV / min. The loading period was approximately 1.25 hours at pH 10.2. These conditions provided a comparable loading ratio but resulted in a shorter amount of time on the column compared to 1 mL (low volume) loaded at a slower rate (0.67 CV / min). Figure 1B shows that the AAVhu68 particle yield was approximately 79% and that approximately 68.5% intact AAVhu68 capsids were present.

[0174] The data show that increasing the medium contact time slightly increases the percentage of particle yield or the percentage of complete capsids (as distinguished from empty and partially filled capsids) collected.

[0175] To determine the effect of high loading ratios on purification efficiency, a feed composition containing AAVhu68 particles (9E+13 vg / mL (vector genomes / milliliter)) was loaded onto a 1 mL CIMmultus-QA™ column at a loading ratio (GC / mL resin) of 2.44E+14 or a loading ratio of 3.65E+14. Where necessary, the column was pre-equilibrated with loading buffer. The feed composition was run through the column at a loading rate of 0.67 mL / min. The column was washed with wash buffer (20 mM BTP, 20 mM NaCl, pH 10.2) and then eluted with a salt gradient (20% to 50% gradient) in elution buffer (20 mM BTP, 1 M NaCl, pH 10.2). The volumetric flow rate was 1.54 CV / min. UV absorbance was measured at 260 and 280 nm. Fractions were collected when the A260 / A280 ratio reached the inflection point (≧1) and then stripped with buffer. The UV absorbance (mAU) profiles at 280 nM and 260 nM are shown in FIG. 2.

[0176] Referring to Figure 2, a low loading ratio (2.44E+14) resulted in an approximately 81.8% yield and approximately 84.1% intact rAAV capsids, with an elution conductivity of approximately 10.9 mS / cm. The A260 / A280 ratio was greater than 1 (1.26). In comparison, a high loading ratio (3.65E+14) resulted in an approximately 66.33% yield and approximately 83.1% intact rAAV capsids, with an elution conductivity of approximately 11.71 mS / cm. The A260 / A280 ratio was also greater than 1 (1.37).

[0177] The data show that although a high loading ratio at high pH (pH 10.2) reduces rAAV particle yield, it results in a significant percentage of collected intact rAAV capsids. The slow flow rate in the low loading ratio experiment likely provided more time for the rAAV particles to flow through the column because the feed composition was dynamically loaded.

[0178] To determine the effect of loading pH on yield and purity, the supernatant containing rAAV particles was diluted 50-fold in loading buffer (20 mM BTP, 10 mM NaCl, pH 10) as described above, except that magnesium chloride was omitted from the buffer, to form a feed composition. The feed composition was adjusted to have a pH of 8.8. Approximately 4 mL of the feed composition having a pH of 8.8 was loaded onto a CIMmultus-QA™ column at a normal loading ratio of 3.05E+13. The loading rate was approximately 1 CV / min, and the processing rate was approximately 1 CV / min. The percentage of intact rAAV capsids separated from empty and partially filled rAAV capsids was approximately 57.5%, as shown in Figure 3.

[0179] The data show that loading at low pH (e.g., pH 8.8) results in a lower percentage of intact rAAV capsids, likely due to overlap of full and empty peaks (band broadening effect).

[0180] Poloxamer 188 (P188) is a nonionic linear copolymer with an average molecular weight of 8400 daltons and is also known as PLURONIC® F68, FLOCOR, and RheothRx. Poloxamer 188 is commonly used as a surfactant to stabilize rAAV particles. To determine whether the presence of poloxamer 188 affects the purification of intact rAAV capsids, a feed composition containing rAAV particles (e.g., AAVhu68) was prepared in loading buffer (20 mM BTP, 10 mM NaCl, pH 10.2). No poloxamer 188 was added. The mixture was diluted 50-fold for loading. Approximately 1 mL of the feed composition, having a pH of 10.2, was loaded onto a CIMmultus-QA™ column. The loading ratio was 3.03E+13, the loading rate was approximately 0.67 CV / min, and the throughput rate was approximately 3.13 CV / min. Purification in the absence of poloxamer 188 resulted in a low yield of AAVhu68 particles (approximately 55.28%) and a low percentage of intact rAAV capsids (approximately 61%) separated from empty and partially filled AAVhu68 capsids, as shown in Figure 4. The data suggest that poloxamer 188 helps increase the yield and purity of rAAV particles.

[0181] To determine the effect of "time-on-media" on rAAV particle purification, a feed composition was prepared in loading buffer (20 mM BTP, 10 mM NaCl, pH 10.2). The mixture was diluted 20-fold before loading. Approximately 1 mL (loading volume) of the feed composition was loaded onto a CIMmultus-QA™ column. The loading ratio was 3.05E+13, the loading rate was approximately 0.67 CV / min, and the processing rate was approximately 3.13 CV / min. The loading period was approximately 0.85 hours at pH 10.2. Under these conditions, as shown in Figure 5A, an rAAV particle yield of approximately 82.7% was observed, while the percentage of intact rAAV capsids, separated from empty and partially filled rAAV capsids, was approximately 51.8%.

[0182] In another experiment, 1 mL of a 50-fold diluted feed composition in loading buffer (20 mM BTP, 10 mM NaCl, pH 10.2) was loaded onto a CIMmultus-QA™ column. The loading ratio was 2.44E+14, the loading rate was approximately 0.67 CV / min, and the processing rate was approximately 3.13 CV / min. Compared with the 20-fold diluted (higher concentration) described above, the loading period for this 50-fold diluted loading composition was approximately 14.57 hours, which was increased by more than 12 hours. Notably, these conditions increased both the yield of rAAV particles (approximately 81.8%) and the purity of intact rAAV capsids (approximately 84.1%). The UV absorption (mAU) profiles at 280 nM and 260 nM are shown in Figure 5B.

[0183] In another experiment, the loading ratio was increased to 3.65E+14, the loading volume (1 mL), loading rate (approximately 0.67 CV / min), and processing rate (approximately 3.13 CV / min) remained the same, and the loading period was approximately 20.54 hours. The data show that a higher loading ratio increases the loading period by approximately 18 hours when compared to the loading period for a smaller load as shown in Figure 5A. Figure 5C shows that under such conditions, the rAAV yield was approximately 66.33%, and the percentage of intact capsids was approximately 83.1%.

[0184] In this experiment, the medium contact time could be decreased by loading a smaller amount, and increased by loading a larger amount (e.g., increased column time beyond 12 hours as shown in Figure 5B, or increased column time beyond 18 hours as shown in Figure 5C). Overall, the data from this experiment indicate that medium contact time may be a determining factor for improving intact rAAV capsid isolation. When the medium contact time was increased, the yield and purity of intact rAAV particles also increased.

[0185] 6.3. Example 3: Retention on Media Improves Purification of rAAV Particles by Anion Exchange Chromatography rAAV particles were purified by AEX under conditions in which the medium contact time was increased by holding the loaded sample on the medium for a holding period.

[0186] In the first experiment, a feed composition was prepared in loading buffer (20 mM BTP, 10 mM NaCl, pH 10.2) and diluted 50-fold. Prior to loading onto a CIMmultus-QA™ column, the feed composition was held in solution at pH 10.2 for a period of 24 hours. After the holding period, 1 mL of the feed composition was loaded onto the column. The loading ratio was approximately 5.66E+13, the loading rate was approximately 1.54 CV / min, and the throughput rate was approximately 1.54 CV / min. The loading period was approximately 1.49 hours. The column was washed with wash buffer (20 mM BTP, 20 mM NaCl, pH 10.2) at 1 CV / min for 10 CV and eluted with a salt gradient (20% to 50% gradient) in elution buffer (20% to 80% Buffer B, 20 mM BTP, 310 mM NaCl, pH 10.2 over 60 CV). UV absorbance was monitored at 260 nm and 280 nm. Fractions where the A260 / A280 ratio reached an inflection point (≥1) were collected and then stripped with buffer. The UV absorbance (mAU) profiles at 280 nM and 260 nM in Figure 6 indicate a yield of approximately 60.06% and approximately 70.9% intact rAAV capsids. The data indicate that maintaining the solution at pH 10.2 does not increase the purity of intact capsids.

[0187] A second experiment was performed under identical conditions, except for the retention step. The loading ratio was approximately 3.0E+13 VG / mL, and the loading rate was approximately 0.67 CV / min. After washing, the rAAV particles were retained (retained on the column in contact with the medium) at pH 10.2 for a period of approximately 3 hours (e.g., medium contact time). As shown in Figure 7, there was a yield of approximately 53.4% ​​and approximately 87% intact rAAV capsids. Compared to retention in solution (approximately 70.9%, Figure 6), retention in medium (approximately 87%) significantly improved the percentage of intact rAAV capsids by approximately 16.1%, as shown in the side-by-side comparison in Figure 8. Furthermore, loading at pH 8.8 and elution at pH 10.2 (approximately 87%) dramatically increases the percentage of intact rAAV capsids by approximately 29.5%, compared to loading and elution at pH 8.8 (approximately 57.5%) shown in Figure 4.

[0188] 6.4. Example 4: Increased Medium Contact Time Positively Correlates with Percentage of Intact rAAV Capsids To determine the effect of media contact time on purification efficiency, the experimental runs were plotted with the percentage of size-exclusion chromatography (Y-axis) measured by multi-angle static light scattering (SEC-MALS) versus media contact time (X-axis), as shown in Figures 9A and 9B. Experiments with all variables using comparable conditions were included in the statistical analysis. "Time-on-media" refers to the loading period at pH 10.2. While not every molecule resides on the column in contact with the media for this period, it is an approximation of the first molecule to reside on the column in contact with the media until the onset of elution. An exception is circled in Figure 9B, where loading was performed at a lower pH and a 3-hour hold was added. Figure 9B is a subset of Figure 9A, showing the time range between 0 and 400 minutes. Figure 9B excludes high loading with a long time on the column in contact with the media because the effect is not linear at this point (circled in Figure 9A). An experiment was included where the column was loaded at pH 8.8 and held in contact with the medium at pH 10.2 (18 min) (circle in Figure 9B).

[0189] The data show that decreasing the flow rate increases the media contact time at pH 10.2. Similarly, increasing the loading dose increases the media contact time at pH 10.2. The effect is statistically significant (Prob ≥ |t| = 0.024, which is less than 0.05) when including or excluding the long loading period at pH 10.2 (increased media contact time at 10.2). The data show that media contact time at pH 10.2 was positively correlated with the percentage of purified and collected intact rAAV capsids.

[0190] 6.5. Example 5: Effect of Buffer Type on Purification of rAAV Particles by Anion Exchange Chromatography To determine the effect of buffer type on the percentage of intact rAAV capsids collected, feed compositions were prepared in a loading buffer containing BTP (20 mM BTP, 10 mM NaCl, pH 10.2) or a loading buffer containing glycine (20 mM glycine, 10 mM NaCl, pH 10.2). Each of these mixtures was diluted 50-fold before loading onto a 1 mL CIMmultus-QA™ column. The loading pH was adjusted to pH 8.8.

[0191] The feed composition in BTP loading buffer was loaded onto the column at a loading pH of 8.8, washed, and eluted in BTP buffer at pH 10.2. The loading ratio was 3.05E+13. After washing, the rAAV particles were retained on the column in contact with the medium at pH 10.2 for a period of 3 hours (column retention). The results are shown in Figure 7.

[0192] The feed composition in glycine buffer was loaded onto the column at pH 8.8. The loading ratio was 3.83E+13. The column was washed with glycine wash buffer (20 mM glycine, 10 mM NaCl, pH 10.2) at 1 CV / min for 20 CV, followed by a 3-hour column retention period in contact with media at pH 10.2. The bound rAAV particles were eluted with a linear salt gradient (20% to 50% Buffer B over 30 CV) using glycine buffer B. UV absorbance was monitored at 260 nm and 280 nm. Fractions where the A260 / A280 ratio reached the inflection point (≥1) were collected and then stripped with buffer. The results are shown in Figure 10.

[0193] The UV absorbance (mAU) profiles at 280 nM and 260 nM shown in Figures 7 and 10 indicate that loading, washing, retention on the column in contact with the medium, and elution in BTP buffer yielded a yield of about 53.4% ​​and about 87% intact rAAV capsids. In comparison, loading, washing, retention on the column in contact with the medium, and elution in glycine buffer yielded a yield of about 79.2% and about 85.3% intact rAAV capsids. The data suggest that the percentage of intact capsids is not affected by buffer composition but is affected by retention on the column in contact with the medium.

[0194] 6.6. Example 6: Modification of an Anion Exchange Chromatography Process for Purification of rAAV Particles This example demonstrates modifications to the purification process to improve the efficiency of rAAV particle purification by anion exchange chromatography.

[0195] The feed composition in glycine buffer was diluted 50-fold and loaded onto the column at pH 8.8. The loading ratio was approximately 3.83E+13 VG / mL, and the loading rate was approximately 0.67 CV / min. The column was washed with glycine wash buffer (20 mM glycine, 10 mM NaCl, pH 10.2) at 1 CV / min for 20 CV to wash away unbound components. The bound components (e.g., rAAV particles) were retained on the column for a period of 3 hours at pH 10.2. The bound rAAV particles were eluted in a linear salt gradient (20% to 50% buffer B over 30 CV) using glycine buffer B at pH 10.2. As shown in Figure 10, this method resulted in a yield of approximately 79.2% and approximately 85.3% intact rAAV capsids. The data show that reducing the loading pH of the sample to 8.8, washing with glycine buffer, and retention on the column for 3 hours dramatically increases the amount of intact rAAV capsid.

[0196] Compared to the BTP buffer experiment described above (Figure 7), which yielded approximately 87% intact rAAV capsids, the use of glycine buffer (approximately 85.3%, Figure 10) yielded a slightly lower percentage of intact capsids, although the difference was small. The data suggest that the improvement in intact rAAV capsid production is independent of the BTP buffer. Furthermore, glycine buffer has a higher pKa than BTP, providing increased pH control.

[0197] Overall, the data suggest that at pH 8.8 there is a significantly lower dynamic binding capacity, as determined by the breakthrough at the top of the graph.

[0198] 6.7. Example 7: Improved purification of rAAV particles by anion exchange chromatography is not AAV serotype dependent The rAAV particle purification procedure described in Example 6 was tested using another AAV serotype, AAV1. A feed composition containing AAV1 was loaded onto a CIMmultus-QA™ column at pH 10.2 with a loading ratio of 2.0E+13. The column was washed and eluted with BTP buffer at pH 10.2. No retention was performed. Figure 11 shows that this method resulted in a 54.9% yield and 62.1% intact AAV1 capsids.

[0199] The feed composition containing AAV1 was loaded onto a CIMmultus-QA™ column at pH 8.8 with a loading ratio of 4.0E+13. The column was washed and eluted with glycine buffer at pH 10.2, and the column was allowed to remain in contact with the medium at pH 10.2 for approximately 3 hours. Figure 12 shows that this method resulted in a yield of 88.7%. The A260 / A280 ratio was higher than the control, indicating an increased percentage of intact AAV1 capsids.

[0200] A third experiment was performed in which the retention time on the column in contact with the medium was extended to 24 hours, and the FPLC analysis results are shown in Figures 13A and 13B. The A260 / A280 ratio was higher than the control, indicating an increased percentage of intact AAV1 capsids.

[0201] The data demonstrate that the modified rAAV particle purification process described in Example 6 is applicable to multiple AAV serotypes.

[0202] 6.8. Example 8: Scalable Anion Exchange Chromatography Process for Purification of rAAV Particles This example demonstrates that the performance of the rAAV particle purification process described in Examples 6 and 7 is scalable for large-scale manufacturing and production of high-quality intact rAAV capsids. The following experiment was performed to isolate intact capsids of AAV1.

[0203] This experiment was performed with a slightly adjusted loading pH (pH 8.9 vs. pH 8.8). A feed composition containing AAVhu68 was loaded onto a CIMmultus-QA™ column at pH 8.9 with a loading ratio of 1.28E+13. The column was washed and eluted with glycine buffer at pH 10.2. rAAV particles bound to the chromatography medium were retained on the column for approximately 3 hours at pH 10.2. The UV absorbance (mAU) profiles at 280 nM and 260 nM are shown in Figure 14. This modified process achieved a yield of approximately 76.9% and approximately 88.3% (by AUC) of intact AAVhu68 capsids. Notably, while trace amounts of partial particles were typically detected in intact capsid pools collected using conventional methods, there was no detection of partial particles in intact capsid pools using this modified process.

[0204] 6.9. Example 9: Application of the Improved Process in an Alternative Anion Exchange Chromatography System for Purification of rAAV Particles This experiment demonstrates the application of a modified process described in Examples 6 and 7 using an alternative anion exchange chromatography system for the purification of rAAV particles.

[0205] Capto Q resin (Cytiva) is a strong anion-exchange resin chromatography column containing a robust, high-flow agarose matrix modified with a dextran surface extender and a quaternary ammonium (Q). It was used to purify rAAV particles obtained from upstream purification processes. This experiment was performed with an adjustment to the loading pH. The feed composition was loaded at a low pH (pH 8.8) with a loading ratio of 9.7E+12, washed with BTP buffer at pH 10.2, and retained on the column at pH 10.2 for approximately 3 hours. The UV absorbance (mAU) profiles at 280 nM and 260 nM in Figure 15 indicate that AAV1 particles likely bound more tightly to the chromatography medium, broadening the peak and potentially reducing yield. This method achieved a yield of approximately 56% and approximately 76% intact capsids. The data indicate that additional horizontal gradient runs (eg, extended gradients) can improve this production.

[0206] 7. EQUIVALENTS AND INCORPORATION BY REFERENCE While the present invention has been particularly shown and described with reference to preferred and various alternative embodiments, it will be understood by those skilled in the art that various changes in form and detail can be made therein without departing from the spirit and scope of the invention.

[0207] All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.

Claims

1. 1. A method for purifying recombinant adeno-associated virus (rAAV) particles, comprising: e) providing a feed composition comprising the rAAV particles, wherein the rAAV particles in the feed composition include empty rAAV particles and complete rAAV particles; f) contacting the feed composition with a chromatography medium under conditions that allow binding of the rAAV particles to the chromatography medium for a medium contact time, wherein the medium contact time is at least 0.5 hours; g) eluting the rAAV particles from the chromatography medium; and h) recovering the purified rAAV particles, thereby enriching for intact rAAV particles A method comprising:

2. The method of claim 1 , wherein the medium contact time is greater than 2 hours.

3. 3. The method of claim 2, wherein the medium contact time is from 2 hours to 24 hours.

4. 3. The method of claim 2, wherein the medium contact time is greater than 3 hours, greater than 4 hours, greater than 5 hours, greater than 6 hours, greater than 7 hours, greater than 8 hours, greater than 9 hours, greater than 10 hours, greater than 11 hours, or greater than 12 hours.

5. 5. The method of any one of claims 1 to 4, wherein the contacting step comprises retaining the rAAV particles bound to the chromatography medium in a retention buffer for a retention period, wherein the retention period is 0.5 hours or longer.

6. 6. The method of claim 5, wherein the holding period is from 0.5 hours to 24 hours.

7. 7. The method of claim 6, wherein the holding period is 3 hours or less.

8. 8. The method of claim 7, wherein the holding period is from 0.5 hours to 3 hours.

9. 9. The method of claim 8, wherein the holding period is about 3 hours.

10. The method of any one of claims 5 to 9, wherein the storage buffer has a pH of 9.0 to 11.

11. 11. The method of claim 10, wherein the holding buffer has a pH of about pH 10.

2.

12. 12. The method of any one of claims 1 to 11, wherein the contacting step comprises loading the rAAV particles onto the chromatography medium for a loading period.

13. 13. The method of claim 12, wherein the loading period is greater than 0.5 hours, greater than 1 hour, greater than 2 hours, greater than 3 hours, greater than 4 hours, greater than 5 hours, greater than 6 hours, greater than 12 hours, or greater than 18 hours.

14. 14. The method of claim 13, wherein the loading period is from 0.5 hours to 24 hours.

15. 15. The method of any one of claims 12 to 14, wherein the loading period and the holding period together are from 0.5 hours to 24 hours.

16. 16. The method of any one of claims 12 to 15, wherein the loading period and the holding period together are at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, or at least 23 hours.

17. 17. The method of any one of claims 12-16, wherein the contacting step comprises washing the rAAV particles bound to the chromatography medium with a wash buffer having a pH of 9.0 to 11 after loading the feed composition onto the chromatography medium but before retaining the rAAV particles bound to the chromatography medium.

18. 18. The method of claim 17, wherein the wash buffer has a pH between pH 9.5 and pH 10.

5.

19. 20. The method of claim 18, wherein the wash buffer has a pH of about pH 10.

2.

20. The method of any one of claims 17 to 19, wherein the wash buffer comprises bis-tris propane (BTP) or glycine.

21. The method of any one of claims 1 to 20, wherein the feed composition has a pH between 8.0 and 8.

9.

22. The method of any one of claims 1 to 21, wherein the chromatography medium is an anion exchange chromatography medium.

23. The method of any one of claims 1 to 22, wherein the chromatography medium is an affinity chromatography medium.

24. 24. The method of any one of claims 1 to 23, wherein the eluting step is carried out using a linear salt gradient.

25. 25. The method of claim 24, wherein the linear salt gradient comprises between about 0.001 mM NaCl and about 1000 mM NaCl.

26. 25. The method of claim 24, wherein the linear salt gradient comprises between about 0.001 mM NaCl and about 100 mM NaCl.

27. 24. The method of any one of claims 1 to 23, wherein the eluting step is carried out using a step salt gradient.

28. 28. The method of claim 27, wherein the step salt gradient comprises between about 7 mM NaCl and about 500 mM NaCl.

29. 28. The method of claim 27, wherein the step salt gradient comprises between about 70 mM NaCl and about 100 mM NaCl.

30. 29. The method of any one of claims 12 to 28, wherein the loading step is carried out by flowing a feed composition through the chromatography medium at a flow rate between 0.1 CV / min and 5 CV / min.

31. The method of any one of claims 1 to 30, wherein the chromatographic medium comprises one or more amine functional groups.

32. 32. The method of claim 31 , wherein the one or more amine functional groups are selected from primary amine, secondary amine, tertiary amine, quaternary amine functional groups, or combinations thereof.

33. 32. The method of claim 31 , wherein the one or more amine functional groups comprise a quaternary amine functional group.

34. 34. The method of claims 31-33, wherein the one or more amine functional groups are attached to a resin chromatography medium, a membrane chromatography medium, and / or a nanofiber chromatography medium.

35. The method of any one of claims 1 to 34, wherein the chromatography medium comprises a monolith.

36. 36. The method of any one of claims 1 to 35, wherein the eluting step is carried out in a buffer having a pH between 9.5 and 10.

5.

37. The rAAV particles may be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, AAV-16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. rh79, AAV. RHM4-1, AAV. hu37, AAVhu68, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP.

37. The method of any one of claims 1 to 36, wherein the capsid protein comprises a capsid protein of an AAV selected from AAV.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and / or AAV.HSC16.

38. 38. The method of claim 37, wherein the rAAV particles comprise capsid proteins of AAVhu68.

39. 38. The method of claim 37, wherein the rAAV particles comprise capsid proteins of AAV1.

40. 38. The method of claim 37, wherein the rAAV particles comprise capsid proteins of AAV9.

41. determining the yield of the purified rAAV particles The method of any one of claims 1 to 40, further comprising:

42. 42. The method of any one of claims 1 to 41, wherein the yield of purified rAAV particles is between 65% and 99%.

43. determining the enrichment of intact rAAV particles in said purified rAAV particles.

43. The method of any one of claims 1 to 42, further comprising:

44. 44. The method of any one of claims 1 to 43, wherein at least 80% of the purified rAAV particles are complete rAAV particles.

45. 45. The method of claim 44, wherein at least 85% of the purified rAAV particles are complete rAAV particles.

46. 46. ​​The method of any one of claims 1-45, wherein between 1% and 40% of the rAAV particles in the feed composition are complete rAAV particles.

47. 47. The method of any one of claims 1-46, wherein the rAAV particles in the feed composition further comprise partially loaded rAAV particles.

48. 48. The method of any one of claims 1 to 47, wherein the chromatography medium is a pre-packed monolithic chromatography column medium.

49. 48. The method of any one of claims 1 to 47, wherein the chromatography medium is a rigid, high-flow agarose matrix modified with a dextran surface extender and a quaternary ammonium (Q) strong anion exchanger.

50. 50. The method of any one of claims 1-49, wherein the purified rAAV particles have a titer of at least 95% of the rAAV particles in the feed composition.

51. 51. The method of any one of claims 1 to 50, wherein the feed composition comprises poloxamer 188.

52. a prior step of contacting a sample containing rAAV particles with an affinity chromatography medium, thereby providing said feed composition.

52. The method of any one of claims 1 to 51, further comprising:

53. a preceding step of preparing a sample containing said rAAV particles by depth filtration, concentration, or diafiltration.

53. The method of any one of claims 1 to 52, further comprising:

54. a preceding step of preparing a sample containing said rAAV particles by depth filtration, concentration, and diafiltration; 54. The method of claim 53, further comprising:

55. A population of rAAV particles prepared by the method of any one of claims 1 to 54.

56. AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, AAV-16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. rh79, AAV. RHM4-1, AAV. hu37, AAVhu68, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP.

56. The population of rAAV particles of claim 55, comprising capsid proteins of an AAV selected from AAV.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and / or AAV.HSC16.

57. 57. The population of rAAV particles of claim 56, comprising capsid proteins of an AAV selected from AAV1, AAV9, and AAVhu68.

58. 58. A population of rAAV particles according to any one of claims 55 to 57, wherein at least 70% of the rAAV particles in the population are complete rAAV particles.

59. 59. A population of rAAV particles described in claim 58, wherein at least 80% of the rAAV particles in the population are complete rAAV particles.

60. 60. The population of rAAV particles of claim 59, wherein at least 85% or at least 90% of the rAAV particles in the population are complete rAAV particles.

61. 61. A pharmaceutical composition comprising a population of rAAVs according to any one of claims 55 to 60 and a pharmaceutically acceptable excipient.

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