Methods for separating or depleting empty AAV capsids from full AAV capsids

Abstract

A method for separating or depleting empty AAV capsids from full AAV capsids in an aqueous mixture containing empty AAV capsids and full AAV capsids, comprising contacting the mixture with a solid surface bearing primary amino groups in a first alkaline environment, thereby (i) binding the full AAV capsids to the solid surface while not binding at least a portion of the empty AAV capsids to the solid surface, or (ii) binding both the full AAV capsids and the empty AAV capsids to the solid surface, and subsequently eluting at least a portion of the empty AAV capsids with a second alkaline environment having a pH value higher than the pH value of the first alkaline environment, provided that the second alkaline environment does not elute the full AAV capsids from the solid surface.

Description

[Technical Field]

[0001] The present invention relates to a method for separating or depleting empty AAV capsids from full AAV capsids using solid phase extraction. [Background technology]

[0002] Recombinant adeno-associated virus (AAV) preparations generally consist of two capsid subpopulations. The desired capsids are properly filled with the desired therapeutic DNA payload. These are called full capsids. Impure preparations also contain capsids that are not filled with the desired DNA load. These are called empty capsids. Some preparations contain more than 90% empty capsids, while others contain less than 50%. Current clinical guidelines recommend reducing the empty AAV capsid content to less than 10%. This can be achieved by ultracentrifugation, but the equipment and process are difficult to scale up and prone to failure due to minor operator errors. Chromatography is considered by many to have advantages over centrifugation.

[0003] Chromatographic methods for separating empty from full AAV capsids are known [1-3]. Using strong anion exchangers with a salt gradient at alkaline pH has been shown to adequately reduce empty AAV capsids for many AAV serotypes. However, results can be unsatisfactory in one or more respects. First, resolution tends to be limited. While baseline resolution is achieved in some cases, i.e., the peaks corresponding to empty and full AAV capsids are completely separated, this is rare. Generally, the elution peaks overlap to some extent, and in some cases almost completely. This requires the sacrifice of full AAV capsids that overlap with empty capsids in order to fully remove empty capsids. Therefore, full capsid recovery is compromised.

[0004] Related difficulties also relate to the limited separation achieved by strong anion exchangers, making the technique unsuitable for industrial-scale use. Many users prefer to employ step gradient elution in large-scale chromatography techniques because it is simpler and saves costs associated with equipment, buffer preparation, fraction collection, and analysis. Unfortunately, step gradients present challenges in reproducibility because every single process variable must remain constant between processing lots. Among other variables, buffers must be of exactly the same composition with respect to both pH and conductivity, chromatography media must be of exactly the same composition, and processing temperatures must be identical. Since virtually none of these parameters are constant, results will inevitably vary with respect to the extent of recovery of full AAV capsids and / or removal of empty AAV capsids.

[0005] Linear gradient elution improves reproducibility. Although the elution profile may vary slightly with changes in materials or conditions, the relative relationship between the eluting full capsid peak and the empty capsid peak remains consistent regardless of changes in materials or conditions, maintaining separation. However, scale equipment for linear gradient elution is more expensive to build than step gradients and requires more training, more buffers, analysis of more fractions, and more time.

[0006] A third limitation relates to the extreme chemical conditions required for empty-full separation on strong anion exchangers. The required pH is often above pH 9.0, sometimes up to pH 10.0 or even higher [1-3]. In many cases, the higher the pH, the better the separation. Unfortunately, however, the chemical stress caused by pH values ​​above pH 9.0 is known to reduce the stability of many biologics, and the higher the pH, the greater the level of chemical stress. Chromatograms of well-purified AAV capsids often show additional peaks in addition to empty and full AAV capsids, which are believed to represent partially dissociated capsids and free capsid proteins, respectively. DNA released by damage to intact full AAV capsids may also be evident.

[0007] The term strong anion exchanger is understood to refer to chromatographic media carrying quaternary amine ligands. The adjective "strong" specifically refers to the ability to maintain a full electrostatic charge over a wide pH range, such as from about pH 2.0 to pH 12.0. Commercial product names generally include Q, QA, and QAE, which refer to quaternary, quaternary amine, and quaternary aminoethyl, respectively. Other names for strong anion exchangers include TEAE (triethylaminoethyl) or TMAE (trimethylaminoethyl), which correspond to the same degree of amine derivatization but have different naming conventions. TEAE and TMAE also refer to quaternary amine ligands.

[0008] Examples of less derivatized amino chromatography ligands are commonly known by names such as DEAE (diethylaminoethyl). DEAE stands for tertiary amino ligand. Tertiary amino ligands maintain their full charge over a relatively limited pH range and are therefore examples of so-called weak anion exchangers. DEAE ligands begin to lose charge at pH values ​​as low as 7.5. DEAE ligands lose most of their charge at pH 9.0 and essentially all of their charge at pH 12. The low charge at pH 9.0 results in lower capacity for AAV and reduced productivity. It has also been suggested that weaker anion exchange ligands, such as secondary or primary amino ligands, are more likely to produce unfavorable results than quaternary amino ligands. In this regard, it is noteworthy that weak exchangers such as DEAE have rarely been investigated for the separation of empty and full AAV capsids.

[0009] Anion exchangers are typically eluted at a fixed pH using a salt gradient, e.g., by increasing the concentration of sodium chloride. Anion exchangers may also be eluted with a decreasing pH gradient. While the charge of strong exchangers remains constant within a range of approximately 2–12, the charge of proteins changes. Protein electronegativity decreases with decreasing pH, while protein electropositivity increases. A decrease in electronegativity indicates a decrease in attraction to a positively charged anion exchange surface. An increase in electropositivity indicates an increase in repulsion from a positively charged anion exchange surface. These two phenomena work in concert to achieve elution with decreasing pH. Exposing a strong anion exchanger to an increasing pH gradient causes bound components to bind more tightly.

[0010] Cation exchangers are widely used for AAV purification, most often using a salt gradient elution, which elutes empty and full AAV capsids together in a single peak. They can also be eluted with a pH gradient, but gradient elution with a cation exchanger requires increasing pH because anion exchangers are positively charged and cation exchangers are negatively charged. Elution with a cation exchanger in an increasing pH gradient allows some separation of empty and full AAV capsids, but the resolution is inferior to that achieved by salt elution with a strong anion exchanger. Summary of the Invention

[0011] The present invention is based on the surprising discovery that separation of full AAV capsids from empty AAV capsids can be achieved using a solid phase modified with primary amino groups. The method of the present invention overcomes several practical limitations associated with known methods of removing empty AAV capsids using strong (quaternary amine) anion exchangers eluted with an increasing salt gradient at a constant alkaline pH [1-3]. A key feature enabling the method of the present invention is the surprising discovery that a large proportion of empty AAV capsids can be selectively removed by increasing the pH in the alkaline range, even in the absence of excess salt. This is contrary to the behavior of empty AAV capsids in known anion exchange methods, where capsid binding is strong at alkaline pH in the absence of excess salt [1-3] and becomes even stronger at higher pH values. As used herein, the term "excess salt" refers to salt in excess of the buffer components used to control the pH of the operating solutions, e.g., sodium chloride (NaCl), or other salts that may be added to Tris buffers or Bis-Tris-propane buffers or other buffers to achieve elution of bound components.

[0012] In one general aspect, the present invention relates to a method for separating or depleting full AAV capsids from empty AAV capsids in an aqueous mixture comprising full and empty AAV capsids using solid phase extraction, wherein the aqueous mixture comprising full and empty AAV capsids is contacted with a primary amino group bearing solid phase.

[0013] In another aspect, the present invention provides a method for separating or depleting empty AAV capsids from full AAV capsids in an aqueous mixture comprising empty and full AAV capsids, by contacting the mixture with a solid surface bearing primary amino groups in a first alkaline environment: (i) binding full AAV capsids to a solid surface while at least a portion of empty AAV capsids are not bound to the solid surface; or (ii) binding both full and empty AAV capsids to a solid surface, and subsequently eluting at least a portion of the empty AAV capsids with a second alkaline environment having a pH value higher than the pH value of the first alkaline environment, provided that the second alkaline environment does not elute the full AAV capsids from the solid surface; Regarding the method.

[0014] In one embodiment of the present invention, the pH value of the first alkaline environment may be greater than pH 7, in particular greater than pH 7 and up to pH 8.

[0015] In another embodiment of the invention, the second alkaline environment can have a pH value that is higher than the pH value of the first alkaline environment. In yet another embodiment of the invention, after eluting at least a portion of the empty AAV capsids, the solid surface can be contacted with a third alkaline environment having a pH value that is higher than the pH value of the second alkaline environment.

[0016] In yet another embodiment of the invention, after eluting at least a portion of the empty AAV capsids, the solid surface may be contacted with a fourth alkaline environment having a pH value lower than the pH value of the third alkaline environment or the pH value of the second alkaline environment and having a salt concentration higher than the salt concentration of the first alkaline environment, the second alkaline environment, or the third alkaline environment.

[0017] According to the invention, the pH value of the second alkaline environment may in particular be in the range pH 8.0 to pH 9.0, in the range pH 8.1 to pH 8.9, in the range pH 8.2 to pH 8.8, in the range pH 8.3 to pH 8.7 or in the range pH 8.4 to pH 8.6.

[0018] According to the invention, the pH value of the third alkaline environment may in particular be in the range of pH 8.5 to pH 10.5, or in the range of pH 8.5 to pH 10.0, or in the range of pH 8.5 to pH 9.5.

[0019] The overlap of the pH ranges of the first pH value, the second pH value, and the third pH value is virtual. If the pH value of the first alkaline environment is selected to be pH 8 (a range greater than pH 7 and less than or equal to pH 8) and the pH values ​​of the second alkaline environment are selected to be pH 8.0 to pH 9.0, this means that the pH value of the second alkaline environment must not be at the lowest value of the pH range, i.e., pH 8, but must be a higher value, such as 8.1 or 8.2, as can be easily understood by those skilled in the art. Similar considerations apply to the substantial overlap of the pH ranges of the second and third alkaline environments.

[0020] In another embodiment of the method of the present invention, the solution contains a salt concentration equivalent to a concentration of 1 M or less of an alkali metal salt such as NaCl, in particular a concentration of 1 mM to 1,000 mM, or a concentration of 2.5 mM to 250 mM, for example a concentration of 2.5 mM, or a concentration of 5 mM, or a concentration of 12.5 mM, or a concentration of 25 mM, or a concentration of 50 mM, or a concentration of 100 mM, or a concentration of 150 mM, or a concentration of 200 mM, or a concentration of 250 mM.

[0021] In particular embodiments of the invention, the alkaline environment may contain magnesium salts at a magnesium ion concentration in the range of 1.0 mM to 5.0 mM, in particular in the range of 1.5 mM to 3.0 mM or in the range of 2.0 mM to 2.5 mM.

[0022] An advantageous aspect of the present invention is that the full and empty AAV capsids may belong to any serotype, in particular may be selected from the group consisting of natural or recombinant serotypes, chimeras (mixed), and combinations thereof.

[0023] The method of the present invention can be applied to any device conventional in the art, for example, a solid phase surface bearing primary amino groups can be placed in a chromatography device. Typically, the solid phase surface bearing primary amino groups can be a monolith, a column of packed particles, a column of packed nanofibers, a membrane adsorber, or a hydrogel.

[0024] The subject of the present invention is also the use of a solid-phase extraction material having a solid-phase surface carrying primary amino groups for separating or depleting empty AAV capsids in an aqueous mixture containing empty and full AAV capsids.

[0025] The method of the present invention may be used after, before, or after additional processing steps to reduce empty capsid content to a greater extent than would be achievable with either of these additional processing steps alone. Additional processing steps currently known to be useful for reducing the amount of empty capsids include ion exchange chromatography using a quaternary amine ion exchanger and density gradient ultracentrifugation. In one embodiment, combining the method of the present invention with ion exchange chromatography using a quaternary anion exchanger may produce a full capsid fraction with a sufficiently low concentration of empty capsids that no additional processing is required to remove empty capsids. The two steps may be performed in any order. In another embodiment, the method of the present invention may be followed by density gradient ultracentrifugation. In another embodiment, combining the method of the present invention with ion exchange chromatography using a quaternary amine anion exchanger may be followed by density gradient ultracentrifugation.

[0026] Although no theory has been developed regarding the specific mechanism or mechanisms by which the present invention functions; the present invention is fundamentally different from known cation exchange or anion exchange chromatography, and the present invention achieves superior separation of empty from full AAV capsids compared to salt elution with a strong anion exchanger. Superior separation refers to any combination of the following three properties: separation can be performed under milder chemical conditions that favor maintaining full capsid stability, and / or the degree of separation between empty and full AAV capsids is improved, and / or the degree of separation is sufficient to make step gradient elution a viable approach.

[0027] The method can be used to perform analytical or preparative applications. The method is applicable to all AAV serotypes. Specific chromatographic conditions and the degree of resolution between empty and full AAV capsids may vary between serotypes or between different recombinant constructs within a serotype. This is a well-known feature of empty-full separation performed by known methods of ion-exchange chromatography and simply reflects the inherent variation in capsid surface chemistry between different AAV serotypes. [Brief explanation of the drawings]

[0028] [Figure 1] FIG. 1 shows the results of a comparison of the separation of empty AAV capsids from full AAV capsids by salt elution using quaternary and primary amino solid phases. [Figure 2] FIG. 2 shows the results of a comparison of the separation of empty AAV capsids from full AAV capsids by salt elution on a quaternary amino solid phase and a primary amino solid phase in an ascending pH gradient. [Figure 3] Figure 3 shows the results of equilibrating and loading a primary amine column under conditions that bind both empty and full AAV capsids, removing the empty AAV capsids in a second wash step, and eluting the full AAV capsids with an ascending pH gradient. [Figure 4] Figure 4 shows the results of equilibration and sample loading of a primary amine column under conditions in which empty AAV capsids do not bind, followed by step gradient elution of full AAV capsids with decreasing pH and increasing salt. [Figure 5] Figures 5-10 show the results of a series of experiments. Samples containing empty and full AAV8 capsids were applied to a solid phase bearing primary amino groups at pH 8.0. In each experiment, the loaded column was washed with buffers of higher pH and eluted with an ascending pH gradient, ending at pH 9.5. [Figure 6] Figures 5-10 show the results of a series of experiments. Samples containing empty and full AAV8 capsids were applied to a solid phase bearing primary amino groups at pH 8.0. In each experiment, the loaded column was washed with buffers of higher pH and eluted with an ascending pH gradient, ending at pH 9.5. [Figure 7] Figures 5-10 show the results of a series of experiments. Samples containing empty and full AAV8 capsids were applied to a solid phase bearing primary amino groups at pH 8.0. In each experiment, the loaded column was washed with buffers of higher pH and eluted with an ascending pH gradient, ending at pH 9.5. [Figure 8] Figures 5-10 show the results of a series of experiments. Samples containing empty and full AAV8 capsids were applied to a solid phase bearing primary amino groups at pH 8.0. In each experiment, the loaded column was washed with buffers of higher pH and eluted with an ascending pH gradient, ending at pH 9.5. [Figure 9] Figures 5-10 show the results of a series of experiments. Samples containing empty and full AAV8 capsids were applied to a solid phase bearing primary amino groups at pH 8.0. In each experiment, the loaded column was washed with buffers of higher pH and eluted with an ascending pH gradient, ending at pH 9.5. [Figure 10]Figures 5-10 show the results of a series of experiments. Samples containing empty and full AAV8 capsids were applied to a solid phase bearing primary amino groups at pH 8.0. In each experiment, the loaded column was washed with buffers of higher pH and eluted with an ascending pH gradient, ending at pH 9.5. [Figure 11] FIG. 11 graphically depicts the relative recovery of empty and full AAV capsids by gradient. [Figure 12] FIG. 12 shows comparative chromatograms showing the effect of magnesium ions and the absence of magnesium ions on the elution of empty and full AAV capsids. DETAILED DESCRIPTION OF THE INVENTION

[0029] The term "primary amine-bearing solid phase" refers to a solid phase that primarily or exclusively bears primary amine ligands on its surface, where the term primary amino ligand refers to a nitrogen atom bound to two hydrogen atoms by a single covalent bond and also to a carbon atom by a single covalent bond. Secondary amines will be absent or present in minority on the solid phase surface. Tertiary and quaternary amines will be absent or present in minority. Negatively charged residues will be absent. Uncharged hydrophobic or hydrogen-bonding residues may be present. The primary amino ligand may be directly covalently bound to the solid phase through the carbon atom of the ligand. Alternatively, the primary amino ligand may be indirectly bound to the solid phase by covalent bonding of a carbon atom of the ligand to a so-called spacer arm that is covalently bound to the solid phase. The primary amino ligand may also be part of a polymer structure covalently bound to the solid phase. The solid phase may be a chromatographic solid phase in the form of one or more porous membranes, one or more fibers, one or more porous particles, or one or more non-porous particles, or a monolithic solid phase including a monolith synthesized from a single polymer mixture or a monolith first synthesized as a macroscaffold and having a secondary ligand-bearing polymer phase synthesized thereon. Any of the above solid phase materials may be provided within a housing to facilitate performing chromatography. A chromatographic solid phase within a housing is commonly referred to as a chromatographic device, often referred to as a column.

[0030] Chromatographic solid phases carrying primary amines are known and commercially available. One example is sold under the name Toyopearl NH2-750F (Tosoh Bioscience), where "NH2" refers to the primary amine [https: / / www.separations.eu.tosohbioscience.com / solutions / process-media-products / by-mode / ion-exchange / anion-exchange / toyopearl-nh2-750f]. Sales materials indicate that the primary amine ligand is in the form of a polyamine, meaning that it is a polymer with repeating primary amine subunits covalently anchored to the solid phase. Another example is sold under the name Sartobind STIC PA (Sartorius), where "PA" refers to the primary amine.

[0031]

number

[0032] Sales literature indicates that the primary amine ligands are in the form of polyamines, specifically polyallylamine, with repeating primary amine subunits covalently anchored to the solid phase. All major manufacturers of chromatography solid phases produce products with amine derivatives on their surfaces, providing the necessary knowledge and resources in the field for routine or experimental preparation of primary amine-bearing solid phases.

[0033] Considering that chromatographic solid phases bearing primary amines may not be named in a manner that reveals their composition, it would be useful to have an analytical method for determining whether a product has the appropriate properties for practicing the present invention. One convenient way to make this determination is to equilibrate the chromatographic solid phase with a buffer such as 10 mM Tris, 10 mM Bis-Tris-propane, 15 mM NaCl, pH 7.2, apply a sample consisting of a protein standard for isoelectric focusing, wash the solid phase with the equilibration buffer, and then apply a 50 CV linear gradient beginning with the equilibration buffer and ending at 10 mM Tris, 10 mM Bis-Tris-propane, 15 mM NaCl, pH 9.5, with a 10 column volume (CV) hold at the end buffer. Elution of the protein over the linear gradient would indicate that the solid phase bears a primary amine. A control experiment consisting of a solid phase bearing a quaternary amine may be run in parallel; no protein will elute. Protein standards for isoelectric focusing are commercially available from a variety of suppliers, including ThermoFisher, Bio-Rad, and Serva.

[0034] In some cases, polyethyleneimine (PEI)-bearing solid phases may allow elution of proteins with alkaline isoelectric points according to the above suitability test, allowing some separation of empty and full capsids. Linear PEI is a polymer in which only the terminal primary amines are present, and each repeating subunit of the polymer has secondary amines. Branched PEI polymers also contain tertiary amines at the branch points. PEI solid phases are known to allow for descending pH gradient separation of some proteins, but, like quaternary amine solid phases, only ascending pH gradient elution is known.

[0035] The terms "equilibrated" or "equilibration" refer to a chemical conditioning step performed on a solid phase and / or a sample to create a specific chemical environment. The solid phase is typically conditioned by exposure to a buffer embodying a desired pH, salt composition, and salt concentration. The sample is typically conditioned by pH titration, or optionally dilution to lower the salt concentration, or optionally buffer exchange techniques including chromatography, dialysis, or diafiltration using tangential flow filtration membranes. In this case, equilibration conditions can be established such that the solid phase binds both empty and full AAV capsids. Alternatively, the subset of empty capsids, which may include most or all of the empty capsids, can be established such that they do not bind to the solid phase during sample loading. All of these techniques and the criteria for selecting between them have been well known in the art for decades.

[0036] The full and empty AAV capsids in any sample may belong to any serotype, including natural or recombinant serotypes, including chimeric (mixed) and novel serotypes. It will be understood that capsids of different serotypes have different charge characteristics, including different levels of charge characteristics for empty and full AAV capsids, and that such differences may also be observed between different recombinant constructs within a serotype. This is important because the behavior of any preparation may vary on a strong anion exchanger and therefore on a primary amino solid phase. This also means that, as with known methods using salt gradients on strong anion exchangers to separate empty and full AAV capsids, some optimization is likely required when applying the method to untried AAV samples. The sample may be in the form of a partially purified preparation such as a cell culture harvest, a cell lysate, an elution fraction from an affinity chromatography column, a partially purified fraction eluted from an ion exchange chromatography column, a partially purified fraction eluted from a hydrophobic interaction chromatography column, or a capsid mixture from any other purification method or combination thereof used to process AAV.

[0037] The term "loaded" refers to the process of contacting an equilibrated sample with an equilibrated primary amino solid phase, typically accomplished using a chromatographic device by forcing the sample through the device by gravity or an external force such as pumping.

[0038] The term "adsorption" refers to the process of binding a biological product to a chemically complementary surface. Complementarity in this case is understood to involve electrostatic charge. The negative electrostatic charge on the surface of the AAV capsid mediates adsorption to a positively charged solid surface by covalent immobilization of primary amine residues. Adsorption of a biological product to a solid phase used in chromatography is often referred to as "binding."

[0039] The term "selective adsorption" refers to the application of conditions that allow adsorption of at least one species while preventing adsorption of one or more other species. In the present case, the operating pH can be adjusted to the alkaline range to allow adsorption of desired full AAV capsids while preventing adsorption of undesired empty AAV capsids.

[0040] The term "desorption" refers to the process of releasing a biological product from a chemically complementary surface to which it is adsorbed. In the present application, such desorption can be achieved by a variety of means, including, but not limited to, decreasing the electronegativity of the biological product (AAV), for example, by lowering the pH; or by decreasing the electropositivity of the solid phase (e.g., by increasing the pH); or by disrupting electrostatic interactions by introducing a competing substance such as a salt; or by any combination of these techniques. Desorption of a biological product from a chromatographic medium is often referred to as elution.

[0041] The term "selective desorption" refers to a situation in which one adsorbed species is released from a solid surface by a change in conditions that leaves one or more additional species adsorbed, followed by a different set of conditions that release different species. The change in conditions may be stepwise or continuous, with weakly bound species desorbing early in the continuum and strongly bound species eluting later in the continuum, ideally well separated from each other.

[0042] The term "washed" refers to the process of exposing a loaded column to a clean buffer with the purpose of displacing unbound species from the solid phase. In the context of this specification, the term "rinsed" has the same meaning. In the most basic case, the wash buffer is the same formulation as the equilibration buffer. In more complex configurations, the wash buffer may have the additional role of displacing a subset of weakly bound impurities from the solid phase so that they can be removed before the desired product is eluted. Alternatively, there may be multiple wash steps, one using the same conditions as the equilibration buffer and a second using conditions that displace a subset of weakly bound impurities from the solid phase so that they can be removed before elution.

[0043] The term "elution" or "eluted" refers to the process of changing the chemical environment in which the solid resides to disrupt interactions between the primary amino solid phase and species that remain bound after the loading and washing steps.

[0044] Once the empty AAV capsids have been removed from the solid phase, the full AAV capsids can be eluted by any of a variety of techniques. As described above, the full AAV capsids may be eluted by further increasing the pH in the absence of salt or in the presence of excess salt. Alternatively, once the empty AAV capsids have been removed, the full AAV capsids may be eluted by increasing the pH in the presence of salt. Alternatively, once the empty AAV capsids have been removed, the full AAV capsids may be eluted by introducing excess salt while maintaining a constant pH. Alternatively, once the empty AAV capsids have been removed, the full AAV capsids may be eluted by introducing excess salt while decreasing the pH.

[0045] The following general, non-limiting descriptions of a series of basic method options illustrate variations in how each method may be performed and provide a basis for a detailed discussion of operational variables. The buffer conditions mentioned in each of these scenarios are intended to provide a general idea of ​​how to perform each method, and it will be understood that optimization of buffer formulations may be necessary to accommodate capsid mixtures from different serotypes or different recombinant constructs within a serotype.

[0046] In one embodiment utilizing only ascending pH gradient elution, a primary amino solid phase in the form of a chromatographic device, such as a monolith, is equilibrated to a near-neutral pH value, such as 20 mM Tris, 20 mM Bis-Tris-propane, pH 7.5±0.2. A sample containing a mixture of empty and full AAV capsids is equilibrated to 20 mM Tris, 20 mM Bis-Tris-propane, pH 7.5±0.2 by buffer exchange. The sample is loaded onto the chromatographic device. The chromatographic device is then eluted with a linear pH gradient from the equilibration buffer to an endpoint buffer of 20 mM Tris, 20 mM Bis-Tris-propane, pH 9.5±0.2, over 20 device volumes, or over 50 device volumes, or over 100 device volumes. The number of device volumes is used as a means to control the rate at which pH changes during the gradient, i.e., the gradient slope. This technique may be particularly useful for analytical purposes because the entire empty and full capsid populations elute within the gradient. It may also be valuable as a starting point for developing preparative conditions. While it can be used for preparative purposes, it should be noted that this technique may expose full AAV capsids to various pH values, which may adversely affect capsid stability.

[0047] In an embodiment utilizing only ascending salt gradient elution, a primary amino solid phase in the form of a chromatographic device, such as a monolith, is equilibrated to a slightly alkaline value, such as 20 mM Hepes, pH 7.5±0.2. A sample containing a mixture of empty and full AAV capsids is equilibrated to the same conditions by buffer exchange. The sample is loaded onto the chromatographic device. The chromatographic device is then eluted with a linear sodium chloride gradient from the equilibration buffer, 20 mM Hepes, pH 7.5±0.2, to an end-point buffer, 20 mM Hepes, 200 mM NaCl, pH 7.5±0.2, over 20 device volumes, 50 device volumes, or 100 device volumes. The number of device volumes is used as a means to control the rate at which the salt concentration changes during the gradient, i.e., the gradient slope. This approach may also be suitable for analytical purposes, since, as described above, all empty and full capsids bind to the primary amino solid phase during sample load. The resolution of empty and full AAV capsids is generally poorer than with elution with an ascending pH gradient, but is superior to fractionation on a strong anion exchanger eluted with a salt gradient at a fixed pH.

[0048] In a closely related embodiment utilizing only ascending salt gradient elution, a primary amino solid phase in the form of a chromatographic device, such as a monolith, is equilibrated to a mildly alkaline pH, such as 10 mM Tris, 10 mM Bis-Tris-propane, pH 8.7±0.2. A sample containing a mixture of empty and full AAV capsids is equilibrated to the same conditions by buffer exchange and applied to the solid surface. The chromatographic device is then eluted with a linear sodium chloride gradient from the equilibration buffer, 10 mM Tris, 10 mM Bis-Tris-propane, 100 mM NaCl, pH 8.7±0.2, over 20, 50, or 100 device volumes. The number of device volumes is used as a means to control the rate at which the salt concentration changes during the gradient, i.e., the gradient slope. This approach may also be suitable for analytical purposes, since, as noted above, all empty and full capsids bind to the primary amino solid phase during sample loading. The resolution of empty and full AAV capsids is better than with salt gradients at lower fixed pH values ​​and generally superior to fractionation on strong anion exchangers eluted with salt gradients at fixed pH, but generally inferior to elution with an increasing pH gradient on primary amino solid phases.

[0049] In an embodiment utilizing a mixed pH-salt elution, a primary amino solid phase in the form of a chromatographic device, such as a monolith, is equilibrated to a near-neutral pH, such as 20 mM Hepes, pH 7.0±0.2. A sample containing a mixture of empty and full AAV capsids is equilibrated to 20 mM Hepes, pH 7.0±0.2 by buffer exchange. The sample is loaded onto the chromatographic device. Under these conditions, both empty and full particles bind to the solid phase. The device is washed with equilibration buffer to remove unbound species. Empty AAV capsids are washed / eluted from the device by changing the chemical environment by exposing them to a buffer with a higher pH, such as 20 mM Tris, pH 8.5±0.2. This buffer is run through the chromatographic device until the UV absorbance of the column effluent reaches baseline. Once the empty AAV capsids have been removed from the device, the chemical environment is again changed by exposure to a buffer containing excess salt at a lower pH, such as 20 mM Hepes, 50 mM NaCl, pH 7.0. This approach can be advantageous for preparative separations due to the milder conditions for elution of full capsids. This is also an example of a step gradient elution format, which some users may find advantageous.

[0050] In another embodiment utilizing a combination of pH and salt elution, a primary amino solid phase in the form of a chromatography device, such as a monolith, is equilibrated to an alkaline pH, such as 20 mM Tris, pH 8.5±0.2. A sample containing a mixture of empty and full AAV capsids is equilibrated to 20 mM Tris, pH 8.5±0.2 by buffer exchange. The sample is then loaded onto the chromatography device. Under these conditions, full particles bind to the solid phase, but empty particles do not. Once the empty AAV capsids are removed from the device, the chemical environment is again changed by exposure to a buffer containing a lower pH and a small amount of salt, such as 20 mM Hepes, 50 mM NaCl, pH 7.0. This approach is attractive for preparative purposes because it is procedurally simpler than the previously described approach. However, the equilibration conditions are close to the threshold that prevents full capsid binding, potentially reducing the binding capacity of full AAV capsids.

[0051] In another method embodiment utilizing pH and salt in the elution of full AAV capsids, a primary amino solid phase in the form of a chromatographic device, such as a monolith, is equilibrated to a near-neutral pH, such as 20 mM Bis-Tris-propane, pH 8.5±0.2. A sample containing a mixture of empty and full AAV capsids is equilibrated to 20 mM Bis-Tris-propane, pH 8.5 by buffer exchange. The sample is loaded onto the chromatographic device. The chromatographic device is then eluted with a linear salt gradient from the equilibration buffer (pH 7.5) to an endpoint buffer of 20 mM Bis-Tris-propane, 100 mM NaCl, pH 8.5, over 20 device volumes, or over 50 device volumes, or over 100 device volumes. The number of device volumes is used as a means to control the rate at which the salt concentration is changed during the gradient, i.e., the gradient slope. Preliminary data indicate that salt gradients provide similar separation of empty and full AAV capsids to that achieved by strong anion exchangers under milder conditions than strong anion exchangers, but are inferior to pH gradients. In many cases, the higher the pH of the salt gradient, the better the resolution of empty and full capsids.

[0052] It will be apparent to those skilled in the art that one or more elements of any one of the above basic configurations may be applied to other configurations. For example, an elution gradient can be applied that simultaneously changes both pH and salt concentration, increasing both parameters, or simultaneously changes both pH and salt concentration, increasing one parameter while decreasing the other; a single-step elution can be converted to a multiple-step elution; a linear gradient elution can be converted to a step elution; a linear gradient elution can be applied after a step that significantly reduces but does not completely eliminate the remaining bound fraction; or a linear gradient elution can be applied after loading under equilibration conditions that prevent most empty AAV capsids from binding. These and many other variations can be applied without departing from the characteristic elements and essence of the method.

[0053] In one embodiment, the primary amino solid phase and sample are equilibrated to an operating pH ranging from pH 5.5±0.2 to pH 8.5±0.2, or from pH 6.0±0.2 to pH 8.5±0.2, or from pH 6.5±0.2 to pH 8.5±0.2, or from pH 7.0±0.2 to pH 8.5±0.2, or from pH 7.5±0.2 to pH 8.5±0.2. It is understood that the exact conditions may vary depending on the AAV serotype and may also vary for different recombinant constructs within a serotype. For samples known to tolerate higher pH ranges, the pH range may be higher. Generally, the higher the pH within the above range, the fewer empty AAV capsids will bind. Simple experimentation with untested AAVs may be required to identify a pH value at which essentially all full AAV capsids bind but empty AAV capsids do not bind. Within the above range, the lower the pH, the stronger the binding of full AAV capsids and the higher the binding capacity of full capsids. However, it will be clear to those skilled in the art that empty AAV capsids may compete with full AAV capsids for binding space, and such competition may limit the binding capacity of full capsids. These are routine process optimization issues that are considered during the development of any industrial purification method.

[0054] In some embodiments, where empty capsids are not suspended at pH 8.5±0.2 and higher pH values ​​that may compromise capsid stability are particularly desirable, salt may be added to the equilibration buffer, for example, at 2 mM NaCl, 5 mM NaCl, 10 mM NaCl, or 20 mM NaCl, or intermediate or higher concentrations. The lowest salt concentration that achieves suspension of bound empty capsids at moderate pH is generally most advantageous. Care can be taken to evaluate the use of higher concentrations, such as 30 mM, 40 mM, 50 mM, 60 mM, or 70 mM, or intermediate or higher concentrations, to displace empty AAV capsids, particularly to avoid inadvertent displacement and loss of full capsids. This approach can be considered as needed to reduce empty capsid binding, particularly at acidic, neutral, or very alkaline pH values.

[0055] Many methods for equilibrating a sample to the conditions for loading the sample onto a chromatography column are known to those skilled in the art. Any of these methods can be used without changing the essence of the method. These methods include laboratory-scale dialysis, diafiltration using a tangential flow filtration membrane, and buffer exchange chromatography. In some cases, sufficient sample equilibration can be achieved by titrating the sample to the desired pH and diluting it with water or a buffer containing a small amount of salt or no salt, as needed.

[0056] Although equilibration prevents binding by empty AAV capsids, the conditions for doing so may weaken binding of full AAV capsids and reduce the capacity of the solid phase for full capsids. Because the goal of preparative separation is to provide the maximum practical load of the desired full capsid, washing offers a potentially less disruptive approach to minimizing the content of empty capsids before elution. Once the most effective washing conditions are used, equilibration conditions can be selected that favor high-capacity binding of the desired full capsid, generally at neutral or lower pH with minimal or no additional salt.

[0057] In one embodiment, the primary amine solid phase loaded with a mixture of empty and full AAV capsids is washed with a buffer of the same composition as the buffer used to equilibrate the solid phase.

[0058] In another embodiment, a primary amine solid phase loaded with a mixture of empty and full AAV capsids is washed with a buffer having a higher pH than the equilibration buffer. For example, a primary amine solid phase equilibrated and loaded at pH 7.5 may be first washed with a buffer of the same composition as the buffer used to equilibrate the solid phase, followed by a second wash with a buffer of a higher pH, such as pH 8.0±0.2, or pH 8.1±0.2, or pH 8.2±0.2, or pH 8.3±0.2, or pH 8.4±0.2, or pH 8.5±0.2, or pH 8.6±0.2, or pH 8.7±0.2. It is understood that the exact conditions may vary depending on the AAV serotype and may vary between different recombinant constructs within a serotype. If increasing the pH does not completely remove empty AAV capsids, salt may be added. For example, to a buffer solution of pH 8.5±0.2, NaCl may be added in an amount equivalent to 2 mM, 5 mM, 10 mM, or 20 mM, or an intermediate concentration thereof, or a higher concentration.

[0059] In a related embodiment, washing at different pH conditions than those used for sample equilibration may be performed in a different manner, simplifying the overall method. The sample and column can be equilibrated at different conditions. Specifically, the sample can be equilibrated to a pH and / or salt concentration that promotes maximum capacity binding of full capsids, while the column can be equilibrated to conditions that significantly remove empty AAV capsids prior to the full capsid elution step. For example, the sample can be equilibrated to 50 mM Hepes, pH 7.5±0.2, and the column can be equilibrated to 50 mM Tris, pH 8.5±0.2. Loading the sample onto the column will have the effect of at least partially re-equilibrating the column to the sample conditions. At the end of sample loading, the higher pH condition of the equilibration / wash buffer will immediately begin to displace empty AAV capsids that bound during sample application. A second wash step would not be necessary, simplifying buffer preparation and the overall method.

[0060] In one embodiment, only full AAV capsids are eluted by raising the pH, which may consist of a single elution step, multiple step gradient elution, or linear gradient elution to pH 8.6±0.2, or 8.7±0.2, or 8.8±0.2, or 8.9±0.2, or 9.0±0.2, or 9.1±0.2, or 9.2±0.2, or 9.3±0.2, or 9.4±0.2, or 9.5±0.2, or lower, intermediate, or higher, depending on the AAV capsid serotype.

[0061] If it is recognized that pH values ​​approaching and exceeding 9.0±0.2 pose an increased risk of destabilizing full AAV capsids, the elution pH may be moderated by the addition of salt. For example, instead of a step or gradient elution to pH 9.5±0.2, a step or gradient elution to pH 8.5±0.2 may be performed in the presence of 2 mM NaCl, or 5 mM NaCl, or 10 mM NaCl, or 15 mM NaCl, or 25 mM NaCl, or lower values, or intermediate values, or higher values. Alternatively, a step or pH gradient may be performed at pH 8.0±0.2, or pH 7.5±0.2, or pH 7.0±0.2, or pH 6.5±0.2, or pH 6.0±0.2, or pH 5.5±0.2, or lower values, intermediate values, or higher values ​​enhanced by the addition of salt. It will be apparent to one skilled in the art that the more salt added, the lower the pH can be. If necessary, the amount of salt added at any pH value can be increased to any value within the range of 2.5 mM to 250 mM, for example, 2.5 mM, or 5 mM, or 12.5 mM, or 25 mM, or 50 mM, or 100 mM, or 150 mM, or 200 mM, or 250 mM, or any value lower, intermediate, or higher than these.

[0062] Any of the above embodiments may be performed in the presence of a divalent metal cation, such as magnesium and / or calcium, at a concentration in the range of 0.1 mM to 10 mM, or 0.5 mM to 5 mM, or 1.0 mM to 2.75 mM, or 1.5 mM to 2.5 mM, or 1.75 mM to 2.25 mM, or 2.0 mM ± 0.1 mM, or higher (e.g., up to 5 mM).

[0063] In one embodiment, the inclusion of divalent metal cations, such as magnesium and / or calcium, can be analytically advantageous because they help maintain the integrity of empty AAV capsids and tend to cause the empty capsids to elute as a single peak, making it easier to determine the total amount of empty AAV capsids and the relative amounts of empty and full AAV capsids in a given sample.

[0064] In some embodiments, the presence of divalent metal cations, such as magnesium and / or calcium, often improves the separation of full capsids from empty capsids and may also be advantageous in preparative separations, as it stabilizes the full capsids.

[0065] The presence of magnesium is generally expected to result in more desirable results in terms of AAV purity and / or recovery. However, in other embodiments, divalent metal ions such as magnesium and / or calcium may be deficient or absent. This is particularly true when full AAV capsids exhibit sufficient stability under the selected separation conditions; and when impurities are distributed throughout the fraction such that fractionation in the absence of divalent metal cations allows for isolation of a full capsid fraction with less contamination by empty AAV capsids than separation performed in the presence of divalent metal cations.

[0066] Any of the foregoing embodiments may be performed in the presence of arginine or histidine to stabilize the full capsids, and / or to improve the solubility of the capsids, and / or to reduce non-specific interactions between the capsids and the solid phase that may reduce the efficiency of the separation or reduce the recovery of the full capsids, and / or simply to reduce the conductivity during the separation.

[0067] In some embodiments, arginine or histidine may be used directly in place of NaCl or other salts to perform gradient separations. The aqueous solubility of arginine is limited to about 600 mM, and the aqueous solubility of histidine is limited to about 200 mM. While arginine is more convenient to use due to its high solubility, histidine, which has more limited solubility, can often be used effectively.

[0068] In embodiments where a sufficient arginine or histidine concentration cannot be achieved to elute capsids, either may be used in combination with a salt, such as arginine and sodium chloride, or histidine and sodium chloride. In some such embodiments, a baseline level of arginine may be added to the equilibration buffer, wash buffer, and / or elution buffer, or to the wash buffer and elution buffer only, or to the elution buffer only. Such a baseline level may be 25 mM or 50 mM, or intermediate or higher concentrations within their solubility ranges.

[0069] In another embodiment, arginine or histidine may be used at baseline levels during the ascending pH gradient, for example at a concentration of 5 mM, or 10 mM, or intermediate or higher concentrations within their solubility ranges.

[0070] Any of the above embodiments may be practiced in the presence of low molecular weight zwitterions, such as glycine or alanine or betaine. Such additives may have the effect of improving capsid solubility and stability without increasing conductivity. Such zwitterions increase polarity but do not contribute to conductivity. They may also be preferentially excluded from the protein surface, thereby imparting a stabilizing effect. Such additives may be particularly useful in conjunction with a pH gradient to overcome the desolubilizing effect of low conductivity, where the additive may have the effect of improving peak sharpness and / or capsid stability.

[0071] In some embodiments, betaine may be used at pH 5.0 to pH 10.0, while alanine and glycine are limited to the range of 5.0 to about 8.5. Above pH 8.5, the nitrogen atom of glycine or alanine begins to lose its positive charge, resulting in an overall negative charge of the molecule. This affects the conductivity of the sample and temporarily negates the other beneficial effects of their zwitterionic forms. The nitrogen atom of betaine is a quaternary amine that retains its charge below about pH 12, remaining in the zwitterionic form throughout that range.

[0072] In one embodiment where the operating pH is 8.5 or less, glycine or alanine may be present at a concentration of up to 2M or more, or 0.5M to 2.5M, or 1.0M to 2.0M, or 1.25M to 1.75M, or 1.4M to 1.6M, or about 1.5M, or any other range.

[0073] In one embodiment where the pH is less than 10, betaine may be present at a concentration of up to 2M or more, or 0.5M to 2.5M, or 1.0M to 2.0M, or 1.25M to 1.75M, or 1.4M to 1.6M, or about 1.5M, or any other range.

[0074] Any of the above embodiments may be practiced in the presence of sugars, such as, but not limited to, sucrose, sorbitol, xylose, mannitol, trehalose, or other non-ionic sugars, at concentrations ranging from 0.5% to 25% or more, for example, to stabilize the full capsid.

[0075] Any of the above embodiments may be carried out in the presence of glycerol, for example at a concentration of 0.5% to 25% or more, for example to stabilize the full capsid.

[0076] Any of the above embodiments may be performed in the presence of organic additives to minimize nonspecific interactions between the capsid and the primary amino solid surface. Such additives include surfactants, including nonionic surfactants, at concentrations ranging from 0.01% to 1.0%. Such additives also include agents such as ethylene glycol or propylene glycol at concentrations ranging from 1% to 10%.

[0077] Those skilled in the art will recognize that the methods of the present invention can be performed on ligands other than primary amino groups that have no or only a small proportion of more highly derivatized amines, such as secondary, tertiary, or quaternary amines. Similarly, it will be recognized that the inclusion of hydrophobic residues or residues that enhance hydrogen bonding between the capsid and the solid phase can improve results.

[0078] All references cited herein are incorporated by reference in their entirety, unless such incorporation would contradict an explicit teaching herein.

[0079] The present invention is further illustrated by the following non-limiting examples. [Example]

[0080] Example 1 Comparison of separation of empty AAV capsids from full AAV capsids by salt elution on quaternary and primary amino solid phases.

[0081] A 100-microliter monolith bearing a primary amine surface was equilibrated in 20 mM Bis-Tris-propane, 2 mM magnesium chloride, pH 8.8. A sample of cation-exchange-purified AAV8 was applied to the column and washed with the equilibration buffer. The monolith was then eluted with a 50-bed-volume linear gradient ending with 20 mM Bis-Tris-propane, 2 mM magnesium chloride, 100 mM NaCl, pH 8.8. A similar basic procedure was performed with a quaternary amine (strong anion-exchange monolith, CIMac QA), except that the NaCl concentration in the buffer at the gradient end point was 200 mM. While complete separation of empty and full AAV capsids was not achieved with either column, the primary amine solid phase achieved better results (Figure 1).

[0082] Example 2 Comparison of empty / full capsid separation on a strong (quaternary amino) anion exchanger using a salt gradient and a primary amino solid phase eluted by increasing pH.

[0083] A 100-microliter monolith bearing a primary amino surface was equilibrated in 10 mM Bis-Tris-propane, 10 mM Tris, 2 mM magnesium chloride, pH 8.0. A sample of cation-exchange-purified AAV8 was applied to the column and washed with equilibration buffer. The column was then eluted with a 100-bed-volume linear gradient ending with 10 mM Bis-Tris-propane, 10 mM Tris, 2 mM magnesium chloride, pH 9.5. Separation of empty and full AAV capsids was compared with that of a strong anion-exchange (quaternary amine) monolith eluted with a salt gradient. The strong anion-exchange monolith was equilibrated in 20 mM Bis-Tris-propane, 2 mM magnesium chloride, pH 9.0. A sample of cation-exchange-purified AAV8 was applied to the column and washed with equilibration buffer (the same sample used for the primary amine column experiments). The column was then eluted with a 50-column-volume linear gradient ending in 20 mM Bis-Tris-propane, 2 mM magnesium chloride, and 200 mM sodium chloride, pH 9.0. The comparative results are shown in Figure 2. As shown, elution of the primary amine column using an ascending pH gradient without excess salt dramatically outperformed the separation achieved with the strong anion exchanger using a salt gradient. In addition to demonstrating excellent separation of empty and full capsids, the elution order of other impurities demonstrates the fundamentally different selectivity of the primary amine column compared to the strong anion exchanger. Impurities that eluted before empty AAV capsids on the strong anion exchanger eluted after full AAV capsids on the primary amine column and were better separated from the full AAV capsids.

[0084] Example 3 Removal of bound empty AAV capsids by a washing step prior to pH gradient elution of full capsids.

[0085] A primary amine monolith was equilibrated in 10 mM Bis-Tris-propane, 10 mM Tris, 2 mM magnesium chloride, pH 8.0. A sample of cation-exchange-purified AAV8 was applied to the column and washed with equilibration buffer (the same sample used in Example 2). Empty AAV capsids were subsequently removed in a wash step with 10 mM Bis-Tris-propane, 10 mM Tris, 2 mM magnesium chloride, pH 8.7. The column was then returned to its original equilibration state and eluted with a 40-bed-volume linear gradient ending with 10 mM Bis-Tris-propane, 10 mM Tris, 2 mM magnesium chloride, pH 9.5. The results are shown in Figure 3. This experiment demonstrates that the primary amine column has the ability to bind both empty and full AAV capsids at a slightly alkaline pH (8.0) and supports the selective removal of empty AAV capsids in an ascending pH gradient (up to pH 9.5) in the absence of excess salt.

[0086] Example 4 Elution of full AAV capsids by a single step of simultaneously increasing salt concentration and decreasing pH after equilibration under conditions in which empty AAV capsids do not bind.

[0087] A primary amine monolith was equilibrated in 10 mM Bis-Tris-propane, 10 mM Tris, 2 mM magnesium chloride, pH 8.5. A sample of cation-exchange-purified AAV8 was applied to the column and washed with equilibration buffer (the same sample used in Examples 2 and 3). Empty AAV capsids hardly bound under these conditions and were almost completely removed during a lengthy wash process. Full AAV capsids were then eluted from the column in steps of 10 mM Bis-Tris-propane, 10 mM Tris, 2 mM magnesium chloride, 50 mM NaCl, pH 7.5. The results are shown in Figure 4. This experiment specifically demonstrates the ability of a primary amine column to separate empty from full AAV capsids using a highly simplified step gradient approach that is readily available on even the simplest chromatography equipment.

[0088] Example 5 The following examples illustrate the methodology of the methods of the invention, starting at pH 8, employing various methods for isolating empty AAV capsids by increasing the pH.

[0089] It is understood that capsids of different serotypes respond differently to the same conditions, and conditions must be tailored individually for each product. A series of diagrams demonstrates how easily capsids from any serotype can be screened for the most effective conditions.

[0090] In a series of experiments, samples containing empty and full AAV8 capsids were applied to a solid phase bearing primary amino groups at pH 8.0 (20 mM Tris, 20 mM Bis-Tris-propane). In each experiment, the loaded column was washed with a higher pH buffer (20 mM Tris, 20 mM Bis-Tris-propane) and subsequently eluted with an ascending pH gradient ending at pH 9.5 (20 mM Tris, 20 mM Bis-Tris-propane). The original chromatograms are shown in Figures 5-10. The relative recoveries of empty and full AAV capsids by gradient are graphically depicted in Figure 11.

[0091] Figure 5. Empty and full AAV capsids are both bound at pH 8.0. Both remain bound during washes at pH 8.3. Full capsids begin to elute immediately upon application of the pH elution gradient, as indicated by a UV absorbance ratio of 0.68. They are clearly partially separated from the later-eluting full capsids, which exhibit a distinct wavelength ratio of 1.32.

[0092] Figure 6. Empty and full AAV capsids are both bound at pH 8.0. A portion of the empty AAV capsids is removed by washing at pH 8.4. The remaining empty AAV capsids begin to elute immediately upon application of a pH elution gradient. Full AAV capsids remain bound and are eluted with the pH gradient.

[0093] Figure 7: Empty and full AAV capsids are both bound at pH 8.0. Most of the empty AAV capsids are removed by washing at pH 8.5. The remaining empty AAV capsids begin to elute immediately upon application of a pH elution gradient. Full AAV capsids remain bound and are eluted with the pH gradient.

[0094] Figure 8: Both empty and full AAV capsids bind at pH 8.0. All empty AAV capsids are removed by washing at pH 8.6. Full AAV capsids remain bound and are eluted with a pH gradient.

[0095] Figure 9: Both empty and full AAV capsids are bound at pH 8.0. All empty AAV capsids are removed by washing at pH 8.7, but some full AAV capsids also elute during the wash. The remaining full AAV capsids remain bound and are eluted with the pH gradient.

[0096] Figure 10: Both empty and full AAV capsids bind at pH 8.0. All empty AAV capsids are removed by washing at pH 8.8, but most of the full AAV capsids are also removed. The remaining full AAV capsids are eluted with a pH gradient.

[0097] Figure 11: The graph shows the relative amounts of full and empty AAV capsids gradient eluted after a wash step at different pH values. The shaded vertical bars indicate the wash pH range that most effectively removes empty AAV capsids and maximizes recovery of full capsids.

[0098] Example 6 Changes in selectivity with or without magnesium ions.

[0099] Figure 12 compares the elution profiles of two chromatographic runs on a primary amine-bearing solid phase. One profile was generated using 2 mM magnesium ions in both gradient buffers, and the other profile was generated in the absence of magnesium ions. The buffers used in both runs were the same. The equilibration buffer was 10 mM Tris, 10 mM Bis-Tris-propane, 15 mM NaCl, pH 7.0 (with or without 2 mM magnesium chloride). The gradient endpoint was 10 mM Tris, 10 mM Bis-Tris-propane, 15 mM NaCl, pH 9.5 (with or without 2 mM magnesium chloride). In the presence of magnesium, full capsids eluted at a pH of approximately 8.77 (peak center). In the absence of magnesium, full capsids eluted at a pH of approximately 9.23 (peak center), approximately a half pH unit higher than when magnesium was present. The separation of empty and full capsids was also clearer in the presence of magnesium, with the recovery of full capsids being approximately 50% higher.

[0100] References All references cited herein are incorporated by reference in their entirety, unless such incorporation would contradict an explicit teaching herein. [1] M. Lock, M. Alvira, J. Wilson, Analysis of particle content of recombinant adeno-associated virus serotype 8 vectors by ion exchange chromatography, Human Genetherapy Methods: Part B 23(2012)56-64. [2] M. Lock, M. Alvira, Scalable Purification Method for AAV9, U.S. Patent Application Publication No. 20190002842(A1), priority to U.S. Provisional Patent Application No. 201562266357(P), International Publication No. 2017160360(A9). [3] X. Fu, W. Chen, C. Argento, P. Clarner, V. Bhatt, R. Dickerson, G. Bou-Assaf, M. Bahshaveshi, X. Lu, S. Bergelson, J. Pieracci, Analytical strategies for quantification of adeno-associated virus empty AAV capsids to support process development, Hum. Gene Ther. Met. 30 (2019) 144-152. Aspects of the present disclosure include the following. <Section 1> 1. A method for separating or depleting empty adeno-associated virus (AAV) capsids from full AAV capsids in an aqueous mixture comprising empty and full AAV capsids, the method comprising contacting the mixture with a solid phase surface bearing primary amino groups in a first alkaline environment, (i) binding full AAV capsids to the solid surface while at least a portion of empty AAV capsids are not bound to the solid surface, and eluting at least a portion of the bound empty AAV capsids by a second alkaline environment having a pH value higher than the pH value of the first alkaline environment, wherein the second alkaline environment does not elute the full AAV capsids from the solid surface; or (ii) binding both full and empty AAV capsids to the solid surface, and subsequently eluting at least a portion of the empty AAV capsids with a second alkaline environment having a pH value higher than the pH value of the first alkaline environment, wherein the second alkaline environment does not elute the full AAV capsids from the solid surface; method. <Section 2> Item 1. The method according to Item 1, wherein the pH value of the first alkaline environment is greater than pH 7 and less than or equal to pH 8. <Section 3> Item 1 or Item 2. The method of claim 1 or 2, wherein after at least a portion of the empty AAV capsids are eluted by the second environment, the solid surface is contacted with a third alkaline environment having a pH value higher than the pH value of the second environment. <Section 4> Item 4. The method of Item 3, wherein the solid surface is contacted with a fourth alkaline environment having a pH value lower than the pH value of the third alkaline environment or the pH value of the second alkaline environment and a salt concentration higher than the salt concentration of the first alkaline environment, the salt concentration of the second alkaline environment, or the salt concentration of the third alkaline environment. <Section 5> Item 5. The method according to any one of Items 1 to 4, wherein the pH value of the second alkaline environment is in the range of pH 8.0 to pH 9.0, pH 8.1 to pH 8.9, pH 8.2 to pH 8.8, pH 8.3 to pH 8.7, or pH 8.4 to pH 8.6. <Section 6> Item 6. The method according to any one of Items 1 to 5, wherein the pH value of the third alkaline environment is in the range of pH 8.5 to pH 10.5, or in the range of pH 8.5 to 10.0, or in the range of pH 8.5 to 9.5. <Section 7> Item 7. The method according to any one of Items 1 to 6, wherein the first alkaline environment, the second alkaline environment, the third alkaline environment, and / or the fourth alkaline environment contains a salt concentration equivalent to a concentration of an alkali metal salt such as NaCl of 1 M or less, particularly a concentration of 1 mM to 1,000 mM or a concentration of 2.5 mM to 250 mM. <Section 8> Item 8. The method according to any one of Items 1 to 7, wherein the alkaline environment contains magnesium ions at a concentration in the range of 1.0 mM to 5.0 mM, particularly in the range of 1.5 mM to 3.0 mM or in the range of 2.0 mM to 2.5 mM. <Section 9> Item 9. The method of any one of items 1 to 8, wherein the full AAV capsid and empty AAV capsid belong to any serotype, particularly selected from the group consisting of natural or recombinant serotypes, chimeric, mixed, and combinations thereof. <Section 10> Item 10. The method according to any one of Items 1 to 9, wherein the solid phase surface carrying the primary amino groups is placed in a chromatography device. <Section 11> Item 11. The method according to any one of Items 1 to 10, wherein the solid surface carrying primary amino groups is a monolith, a column of packed particles, a column of packed nanofibers, a membrane adsorbent, or a hydrogel. <Section 12> Use of a solid phase extraction material having a solid phase surface bearing primary amino groups to separate or deplete empty AAV capsids in an aqueous mixture containing empty and full AAV capsids.

Claims

1. 1. A method for separating or depleting empty adeno-associated virus (AAV) capsids from full AAV capsids in an aqueous mixture comprising empty AAV capsids and full AAV capsids, comprising contacting the aqueous mixture with a solid phase surface bearing primary amino groups in a first alkaline environment, (i) binding full AAV capsids to the solid surface while at least a portion of empty AAV capsids are not bound to the solid surface, and eluting at least a portion of the bound, empty AAV capsids by a second alkaline environment in the range of pH 8.0 to pH 9.0 and having a pH value higher than the pH value of the first alkaline environment, wherein the second alkaline environment does not elute full AAV capsids from the solid surface; or (ii) binding both full and empty AAV capsids to the solid surface, and subsequently eluting at least a portion of the empty AAV capsids with a second alkaline environment in the range of pH 8.0 to pH 9.0 and having a pH value higher than the pH value of the first alkaline environment, wherein the second alkaline environment does not elute the full AAV capsids from the solid surface; 1. A method comprising: The method, wherein empty AAV capsids are selectively removed by increasing the pH in the alkaline range.

2. 2. The method of claim 1, wherein the pH value of the first alkaline environment is greater than pH 7 and less than or equal to pH 8.

3. 3. The method of claim 1 or claim 2, wherein after at least a portion of the empty AAV capsids are eluted by the second alkaline environment, the solid surface is contacted with a third alkaline environment having a pH value higher than the pH value of the second alkaline environment.

4. The solid phase surface is treated with a pH value of the third alkaline environment or a pH value of the second alkaline environment.

4. The method of claim 3, wherein the first alkaline environment is contacted with a fourth alkaline environment having a pH value lower than the first alkaline environment and a salt concentration higher than the salt concentration of the first alkaline environment, the salt concentration of the second alkaline environment, or the salt concentration of the third alkaline environment.

5. The method according to any one of claims 1 to 4, wherein the pH value of the second alkaline environment is in the range of pH 8.1 to pH 8.

9.

6. The method according to any one of claims 3 to 5, wherein the pH value of the third alkaline environment is in the range of pH 8.5 to pH 10.

5.

7. 7. The method of any one of claims 1 to 6, wherein the first alkaline environment and / or the second alkaline environment comprises a salt concentration corresponding to a concentration of an alkali metal salt (e.g. NaCl) of 1 M or less.

8. The third alkaline environment and / or the fourth alkaline environment may be an alkali metal salt (e.g., 8. The method according to claim 4, further comprising a salt concentration equivalent to a concentration of 1 M or less of NaCl.

9. 9. The method of any one of claims 1 to 8, wherein the first alkaline environment and / or the second alkaline environment comprises magnesium ions at a concentration in the range of 1.0 mM to 5.0 mM.

10. 10. The method of any one of claims 4 to 9, wherein the third alkaline environment and / or the fourth alkaline environment comprises magnesium ions at a concentration in the range of 1.0 mM to 5.0 mM.

11. 11. The method of any one of claims 1 to 10, wherein the full AAV capsid and empty AAV capsid belong to any serotype selected from the group consisting of natural or recombinant serotypes, chimeric, mixed, and combinations thereof.

12. The method according to any one of claims 1 to 11, wherein the solid surface carrying primary amino groups is placed in a chromatographic device.

13. 13. The method of any one of claims 1 to 12, wherein the solid surface carrying primary amino groups is a monolith, a column of packed particles, a column of packed nanofibers, a membrane adsorber, or a hydrogel.

14. 1. Use of a solid phase extraction material having a solid phase surface bearing primary amino groups for separating or depleting empty AAV capsids in an aqueous mixture containing empty and full AAV capsids, comprising: The above use, wherein empty AAV capsids are selectively removed by increasing the pH in the alkaline range.

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