Method for enhancing separation of complete adeno-associated virus (AAV) capsid

Abstract

A method of enriching, by chromatography, intact adeno-associated virus (AAV) capsids from a mixture comprising intact AAV capsids, partially filled and / or empty AAV capsids, comprising the steps of:-contacting the mixture with a strong or weak anion exchange material; eluting the load mixture with a neutral to alkaline buffer comprising an organic modulator having a relative measurement of solvent polarity of 0.4 to 0.8; and-collecting a fraction rich in intact AAV capsids.

Description

[0001] The present invention relates to a method for enriching intact adeno-associated virus (AAV) capsids from a mixture comprising intact AAV capsids, partially filled and / or empty AAV capsids. The method of the present invention is particularly suitable for obtaining an enriched intact AAV capsid fraction, wherein contaminants such as empty capsids, partially filled capsids, heavy capsids, damaged capsids, capsid aggregates, etc. have been removed. BACKGROUND

[0002] Gene therapy is a promising field of medicine, which is based on the principle of modifying cells by genes to produce a therapeutic effect, or to treat a disease by repairing or reconstructing defective genetic material. Genetic material is administered to patients suffering from a disease caused by a defective gene. One way of administering a therapeutic genetic material is by using the capsid of a virus or viroid particle as a carrier, in particular the capsid of an adeno-associated virus (AAV).

[0003] AAV is a widely used vector in gene therapy, which is mainly advantageous due to its good safety profile and efficient transduction into a variety of target tissues. The production process of AAV viral vectors is complex and requires innovative methods to meet the stringent safety and efficacy requirements, while complying with the demanding clinical and market needs.

[0004] Despite its widespread use in clinical applications, there is a need to improve the purification process to remove product- and process-related impurities, ensuring the quality and potency of the AAV product throughout the production process.

[0005] Both empty capsids and capsids that are partially filled with the target vector genome are product-related impurities, which pose a technical challenge for their separation from the target vector during downstream purification. Removing empty capsids from AAV preparations and maximizing the ratio of intact capsids to empty capsids is one of the goals of the purification process, which is directly related to the safety and regulatory requirements for AAV-based gene therapy [1-3].

[0006] Depending on the specific packaging sequence, partially filled capsids can still participate in transduction of target cells [4]. However, AAV capsids containing host cell and / or helper DNA and product-related impurities can pose an immunological risk to patients [5].

[0007] This has prompted the development and evaluation of a variety of different materials and methods for purifying AAV. For example, metal affinity chromatography has been used to purify AAV capsids [6]. However, this method does not distinguish between empty capsids and intact capsids and is serotype-dependent. After contacting the crude sample (containing the target AAV capsids and impurities) with the metal affinity material, the AAV capsids are theoretically bound to the affinity material, while the impurities are not. The unbound impurities are removed by elution, and finally the AAV capsids are recovered by chemically disrupting the interaction between the affinity material and the AAV capsids.

[0008] Downstream processing, including purification, remains one of the main bottlenecks for the production of Adeno-Associated Vector. BIA Separations, a Sartorius company, offers an Adeno-Associated Vaccine purification platform, using market leading monolith chromatography columns and an analytical toolbox for monitoring the Adeno-Associated Vaccine production process.

[0009] The simplified purification process of AAV capsids comprises typical downstream steps including complex lysis, clarification, tangential flow filtration (TFF), and chromatographic capture on a pre-packed monolithic sulfonate (SO3) chromatography column, followed by enrichment of intact AAV capsids by a pre-packed monolithic quaternary ammonium (QA) chromatography column.

[0010] Although this process allows the preparation of pharmaceutical grade Adeno- Associated Viruses, there is still a need to further improve the purity of AAVs for gene therapy, in particular to separate empty capsids and partially filled AAV capsids, as well as impurities carrying DNA or other contaminants, from the intact AAV capsids required for therapy. The latter should carry genetic material (such as plasmids) and be free of contaminants.

[0011] Object of the invention

[0012] It is an object of the present invention to provide a method for isolating intact AAV capsids from empty and partially filled Adeno-Associated Virus (AAV) capsids.

[0013] It is another object of the present invention to provide a method for enriching intact AAV capsids from a mixture comprising intact AAV capsids, empty capsids, partially filled capsids, heavy capsids, damaged capsids, capsid aggregates, etc.

[0014] It is another object to provide a method that allows obtaining intact AAV capsids as free as possible from contaminants, such as DNA (more specifically, hcDNA and pDNA).

[0015] It is a further object to provide a method that can be used both for analytical detection and for preparative scale. SUMMARY

[0016] The subject of the present invention is a method for enriching intact Adeno- Associated Virus (AAV) capsids from a mixture comprising intact AAV capsids, partially filled and / or empty AAV capsids, by chromatography, comprising the following steps:

[0017] - contacting the mixture with a strong or weak anion exchange material;

[0018] - eluting the loaded mixture using a neutral to basic buffer containing an organic modifier having a relative measure of solvent polarity of 0.4 to 0.8; and

[0019] - Collect components rich in intact AAV capsids.

[0020] The relative polarity of a solvent, RPM, is defined by the following equation:

[0021] RPM = E T (n-hexane)–E T / E T (n-Hexane)

[0022] (Proposed by Dukiie et al. [8])

[0023] E T – Transition energies [kJ / mol]

[0024] The RPM values ​​in Table 1 are negative, but the absolute RPM values ​​can be used when performing correlation analysis [7].

[0025] The method of the present invention can be advantageously used for analytical purposes, and can also be used for the preparative production of complete AAV capsids.

[0026] According to one embodiment of the present invention, the organic regulator may be selected from the group consisting of: methanol, 1,3-propanediol, 1,2-propanediol, N-methylformamide, diethylene glycol, triethylene glycol, 1,3-butanediol, 2-propynyl-1-ol (propynyl alcohol), 2-methoxyethanol, 2-propenyl-1-ol (allyl alcohol), N-methylacetamide, ethanol, 2-aminoethanol, acetic acid, benzyl alcohol, 1-propanol, 1-butanol, 2-hydroxymethylfuran (furfuryl alcohol), 2-phenylethanol, 1-pentanol, 2-methyl-1-propanol (isobutanol), 1-hexanol, 2-propanol, 3-phenyl-1-propanol, 1-heptanol, 1 -Octanol, cyclopentanol, 1-decanol, 2,6-dimethylphenol (2,6-dimethylphenol), 2-butanol, 3-methyl-1-butanol (isoamyl alcohol), cyclohexanol, 1-dodecyl alcohol, 1-phenylethanol, acrylonitrile, 4-methyl-1,3-dioxolane-2-one (propylene carbonate), 2-pentanol, nitromethane, acetonitrile, dimethyl sulfoxide, methyl acrylate, aniline, tetra-N-hexylbenzoate, tetrahydrothiophene-1,1-dioxide (sulfolane), 2-methyl-2-propanol (tert-butanol), acetic anhydride, N,N-dimethylformamide, N,N-dimethylacetamide, propionitrile, and nitroethane.

[0027] Preferred organic modifiers include acetonitrile, propylene carbonate, 2-propanol, 1-butanol, 1-propanol, tert-butanol, ethanol, methanol, and mixtures thereof. One embodiment of the invention is a method for enriching intact adeno-associated virus (AAV) capsids from a mixture containing intact AAV capsids, partially filled and / or empty AAV capsids by chromatography, comprising the following steps:

[0028] - contacting the mixture with a strong or weak anion exchanger;

[0029] - eluting the loaded mixture using a neutral to basic buffer comprising an organic modifier selected from the group consisting of methanol, 1,3-propanediol, 1,2-propanediol, N-methylformamide, diethylene glycol, triethylene glycol, 1,3-butanediol, 2-propyn-1-ol (propargyl alcohol), 2-methoxyethanol, 2-propen-1-ol (allyl alcohol), N-methylacetamide, ethanol, 2-aminoethanol, acetic acid, benzyl alcohol, 1-propanol, 1-butanol, 2-hydroxymethylfuran (furfuryl alcohol), 2-phenylethanol, 1-pentanol, 2-methyl-1-propanol (isobutanol), 1-hexanol, 2-propanol, 3-phenyl-1-propanol, 1-heptanol, 1-octanol, cyclopentanol, 1-decanol, 2,6-dimethylphenol (2,6-xylenol), 2-butanol, 3-methyl-1-butanol (isopentanol), cyclohexanol, 1-dodecanol, 1-phenylethanol, acrylonitrile, 4-methyl-1,3-dioxolan-2-one (propiolactone), 2-pentanol, nitromethane, acetonitrile, dimethyl sulfoxide, methyl acrylate, aniline, tetra-N-hexylbenzoate, tetrahydrothiophene-1,1-dioxide (sulfolane), 2-methyl-2-propanol (tert-butanol), acetic anhydride, N,N-dimethylformamide, N,N-dimethylacetamide, propionitrile and nitroethane; and

[0030] - collecting the fraction enriched in intact AAV capsids.

[0031] In another embodiment of the present application, the buffer can comprise alkaline earth metal salts, in particular magnesium or calcium salts and mixtures thereof.

[0032] In yet another embodiment of the present application, the alkaline earth metal salt can be magnesium acetate or formate, calcium acetate or formate and mixtures thereof, and / or kosmotropic alternatives thereof. Preferred kosmotropic alternatives are magnesium and calcium salts of inorganic acids, organic acids or organic hydroxy acids, amino acids or polycarboxylic acids containing not more than 10 carbon atoms (e.g. oxalate or citrate).

[0033] In another embodiment of the present application, the pH of the buffer can be about 7.0 to 10.5, in particular about 7.5 to 9.5.

[0034] In a further embodiment of the present application, the buffer can comprise an isotonicity agent selected from the group consisting of sucrose, sorbitol, mannitol and xylitol.

[0035] According to another embodiment of the present application, the strong or weak anion exchange material can be a strong or weak anion exchange material with hydrogen bonding properties and complexed with a positively charged metal affinity ligand to form a multimodal material, a monolithic anion exchanger or multimodal material, a particulate anion exchanger or multimodal material, and / or an anion exchanger or multimodal material disposed in a membrane, and / or a particulate packed anion exchanger or multimodal column, and / or a fibrous chromatography anion exchanger or fibrous column.

[0036] Multimodal chromatography (also known as mixed mode chromatography, MMC) refers to a chromatographic method that utilizes multiple interaction forms between the stationary phase and the analyte for separation [16, 17, 18]. MMC can be classified into physical MMC and chemical MMC. The former is constructed by combining two or more packings to form the stationary phase, while the latter uses a single packing containing two or more functional groups. One implementation is to connect two commercial chromatography columns in series, which is called "tandem column". Another method is "dual-phase chromatography column", i.e. two stationary phases are filled into the two ends of the same chromatography column. Another method is to uniformly mix two or more different types of stationary phases in a single chromatography column, which is called "mixed chromatography column" or "mixed bed chromatography column".

[0037] According to another embodiment of the present application, the AAV can be selected from the following serotype groups: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.10, AAV11, AAV12, and different serotypes (such as hybrid serotypes). The AAV serotype analyzed by the method of the present application can be a recombinant hybrid serotype (such as AAV2 / 8), other hybrid serotypes, chimeras, surface-modified AAVs, and any synthetic derivative AAV-like particles.

[0038] The chimeras or chimeric viruses refer to viruses containing genetic material derived from two or more different viruses.

[0039] Surface-modified viruses are known, for example described in the document

[20] .

[0040] Synthetic derivative AAV-like particles are known, for example described in the document

[21] .

[0041] The subject of the present application also includes an aqueous solution having a neutral to basic pH value, comprising a buffer substance and an organic modifier having a relative polarity measure (RPM) of 0.4 to 0.8 solvent equivalents.

[0042] According to another embodiment of the aqueous solution of the present application, the organic modifier can be selected from the group consisting of methanol, 1,3-propanediol, 1,2-propanediol, N-methylformamide, diethylene glycol, triethylene glycol, 1,3-butanediol, 2-propyn-1-ol (propargyl alcohol), 2-methoxyethanol, 2-propen-1-ol (allyl alcohol), N-methylacetamide, ethanol, 2-aminoethanol, acetic acid, benzyl alcohol, 1-propanol, 1-butanol, 2-hydroxymethylfuran (furfuryl alcohol), 2-phenylethanol, 1-pentanol, 2-methyl-1-propanol (isobutyl alcohol), 1-hexanol, 2-propanol, 3-phenyl-1-propanol, 1-heptanol, 1-octanol, cyclopentanol, 1-decanol, 2,6-dimethylphenol (2,6-xylenol), 2-butanol, 3-methyl-1-butanol (isopentanol), cyclohexanol, 1-dodecanol, 1-phenylethanol, acrylonitrile, 4-methyl-1,3-dioxolan-2-one (propiolactone), 2-pentanol, nitromethane, acetonitrile, dimethylsulfoxide, methyl acrylate, aniline, tetra-N-hexylbenzenesulfonate, tetrahydrothiophene-1,1-dioxide (sulfolane), 2-methyl-2-propanol (tert-butyl alcohol), acetic anhydride, N,N-dimethylformamide, N,N-dimethylacetamide, propionitrile and nitroethane.

[0043] Preferred organic modifiers are acetonitrile, n-butanol, tert-butanol, propylene carbonate, isopropyl alcohol, ethanol, methanol and propanol.

[0044] According to another embodiment of the aqueous solution of the present application, the solution can comprise an alkaline earth metal salt.

[0045] According to yet another embodiment of the aqueous solution of the present application, the alkaline earth metal salt can be a magnesium or calcium salt and mixtures thereof, in particular magnesium acetate or magnesium formate, and / or calcium acetate or calcium formate, and / or a more lyotropic substitute thereof. More preferred lyotropic substitutes are magnesium and calcium salts of inorganic acids, organic acids, organic hydroxy acids, amino acids or polycarboxylic acids containing up to 10 carbon atoms (e.g. oxalic acid or citric acid).

[0046] According to a further embodiment of the aqueous solution of the present application, the buffering substance is used for buffering the aqueous solution to a pH in the range of pH 6 to pH 12, in particular, the buffering substance can be selected from the group consisting of 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol (Bis-Tris), 2,2',2"-nitrilotriacetic acid (ADA), 2-[(2-amino-2-oxoethyl)amino]ethane-1 -sulfonic acid (ACES), 2,2'-(piperazin-1,4-diyl)bis(ethane-1 -sulfonic acid) (PIPES), 2-hydroxy-3-(morpholin-4-yl)propane-1 -sulfonic acid (MOPSO), 2,2'-[propane-1,3-diylbis(azanediyl)]bis[2-(hydroxymethyl)propane-1,3-diol] (BTP), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-(morpholin-4-yl)propane-1 -sulfonic acid (MOPS), 2-{[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}ethane-1 -sulfonic acid (TES), 2-[4-(2-hydroxyethyl)piperazin-1 -yl]ethane-1 -sulfonic acid (HEPES), 3-[N,N-bis(2-hydroxyethylamino)-2-hydroxy-1 -propane sulfonic acid (DIPSO), 4-(4-morpholinyl)butane sulfonic acid (MOBS), 2-hydroxy-3-[tris(hydroxymethyl)methylamino]-1 -propane sulfonic acid (TAPSO), 2-amino-2-(hydroxymethyl)-1,3-propanediol (Trizma), 4-(2-hydroxyethyl)piperazine-1 -(2-hydroxypropane-3-sulfonic acid) (HEPPSO), Piperazine-N,N'-bis(2-hydroxypropane sulfonic acid) (POPSO), triethylamine (TEA), 4-(2-hydroxyethyl)-1 -piperazine-propane sulfonic acid (EPPS), N-tris(hydroxymethyl)methylglycine (Tricine), N,N-bis(2-hydroxyethyl)glycine (Bicine), N-(2-hydroxyethyl)piperazine-N'-(4-butane sulfonic acid) (HEPBS), N-tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid (TAPS), 2-amino-2-methyl-1,3-propanediol (AMPD), N-tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid (TABS), N-(1,1 -dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropane sulfonic acid (AMPSO), 2-(N-cyclohexylamino)ethanesulfonic acid (CHES), 3-cyclohexylamino-2-hydroxypropane sulfonic acid sodium salt (CAPSO), N-(1,1 -dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropane sulfonic acid (AMPS), 3-(cyclohexylamino)-1 -propane sulfonic acid (CAPS) and 4-[cyclohexylamino]-1 -butane sulfonic acid (CABS).

[0047] According to another embodiment of the aqueous solution of the present application, the buffer can comprise an isotonicity additive, in particular, the isotonicity additive is selected from the group consisting of sucrose, sorbitol, mannitol and xylitol.

[0048] The buffer can further comprise a non-ionic surfactant, such surfactants help to suppress adverse effects, e.g. to suppress interactions between the components of the mixture comprising the AAV capsids. In particular, so-called poloxamers (e.g. poloxamer 188) can be used.

[0049] The subject matter of the present application also comprises the use of the aqueous solution of the present application for separating intact AAV capsids from empty AAV capsids in a method according to the present application.

[0050] Table 1 lists some organic solvents and their corresponding RPM values. The table data is derived from reference [7]. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 The influence of different organic modulators on the intact / empty AAV capsid separation is depicted.

[0052] Figure 2A and 2B The influence of organic modulators on the intact / empty AAV capsid separation is depicted. Figure 2C The influence of organic modulators on the intact / empty AAV capsid separation in the presence of magnesium chloride (MgCl2), magnesium acetate (MgAc2) and magnesium formate (MgFor2) is depicted.

[0053] Figure 3 The influence of preferred organic modulators on the intact / empty AAV capsid separation compared to poloxamer 188 is depicted.

[0054] Figure 4 The influence of the percentage of organic modulators on the empty / intact AAV capsid separation is depicted.

[0055] Figure 5 The influence of the Mg 2+ ion concentration in the elution buffer on the separation is depicted.

[0056] Figure 6 The pH range of the elution buffer for separating empty / intact AAV8 capsids is depicted.

[0057] Figure 7 The influence of the pH value on the AAV8 and AAV9 serotype capsid separation is depicted.

[0058] Figure 8A The sample preparation (purification) run with different AAV capsid populations is depicted. The collected fractions were analyzed and the multi-detector results are presented, e.g.Figure 8B , 8C , 8D and 8E.

[0059] Figure 9 Depicts the effect of preferred organic modifiers on the resolution of full / empty AAV capsids on a weak anion exchanger monolith.

[0060] Figure 10 Depicts the effect of preferred organic modifiers on the resolution of full / empty AAV capsids on a multimodal exchanger monolith or membrane.

[0061] Figure 11A and 11B Depicts the effect of higher concentrations of organic modifiers on AAV capsid resolution, especially in terms of baseline separation of empty capsids from partial capsids from full capsids.

[0062] Figure 12 depicts the elution fractions in a preparative run analyzed by orthogonal density gradient ultracentrifugation combined with PATfix TM (Sartorius BIA Separations) multi-detector system.

[0063] Figure 13 Depicts comparative results of AAV capsid separation using a QA chromatography column versus an anion exchange membrane adsorber.

[0064] Figure 14 Depicts the resolution of full / empty AAV capsids under different loading and elution strategies.

[0065] Figure 15 Depicts chromatograms presenting the tryptophan fluorescence and light scattering resolution of the same batch of harvested AAV8 and full / empty AAV8 capsids by a two-dimensional chromatography system. DETAILED DESCRIPTION

[0066] The term "resolution" is well known to those skilled in the art. It is calculated by dividing the difference in peak retention times between different chromatographic peaks by the peak width at half height of each peak, as follows [9].

[0067] R = 1.18 (t R2 - tr R1 ) / (W h1 +W h2 ), where t R2 > t R1

[0068] t R1 , t R2 - retention time of the peak

[0069] W h1 , Wh2 - peak width at half height

[0070] The term "full AAV capsids" refers to capsids that carry a sufficient amount of vector genome to exert a therapeutic effect.

[0071] The term "empty AAV capsids" refers to capsids that lack a sufficient amount of vector genome to exert a therapeutic effect.

[0072] When referring to temperatures or when not specified, room temperature (23°C) is meant.

[0073] When referring to volumes or when not specified, room temperature is meant.

[0074] According to the method of the present application, full adeno-associated virus (AAV) capsids are separated from empty AAV capsids by a chromatographic method using a strong anion exchange material in contact with the mixture to be separated. After contacting the mixture, the mixture is eluted from the strong anion exchange material using a neutral to basic buffer containing an organic modifier having a solvent relative polarity measurement of 0.4 to 0.8 under suitable conditions known to those skilled in the art. During the elution step, the empty AAV capsids are particularly separated from the full AAV capsids and can be collected in a fraction that is separated from other components of the mixture, in particular empty AAV capsids.

[0075] Table 1 lists the RPM values for various organic compounds. Table 1 lists exemplary organic modifiers that can be used in the method of the present application, including acetonitrile, 1-butanol, t-butanol, propylene carbonate, isopropanol, ethanol, methanol or propanol, or combinations thereof.

[0076] Figure 1 Results obtained using exemplary organic modifiers are summarized. A series of experiments demonstrating the results shown in Figure 2 and Figure 3 In a series of experiments demonstrating the results shown in Figure 2 and Table 2, samples containing mixtures of empty AAV and full AAV capsids (and other impurities) were subjected to a monolithic QA anion exchanger in buffers containing different organic modifiers. These buffers used TRIS as the buffering agent, sorbitol to stabilize the capsids, magnesium acetate as the elution salt, and different organic modifiers were added. In the standard buffer, the organic modifier was replaced by the non-ionic surfactant Poloxamer 188.

[0077] The separation of empty AAV capsids from full AAV capsids was significantly better when using the organic modifiers described in the present application than when using elution buffers without the organic modifier. Poloxamer 188, although an organic compound, is outside the RPM range of 0.4 to 0.8, and as shown in Figure 2 and Table 2, the resolution was only 1.89; whereas the resolution obtained using the organic modifiers of the present application was between 2.00 and 2.40. Figure 3

[0078] ​Figure 3 The experimental results shown demonstrate that the presence of at least one organic modifier in the present application can significantly improve the resolution of full / empty AAV capsids. A resolution of 2.40 was achieved when only acetonitrile was used as the organic modifier in the elution buffer with the addition of magnesium acetate. In contrast, a resolution of only 1.89 was achieved with the buffer containing the organic component Poloxamer 188, which is significantly reduced.

[0079] Advantageously, the elution buffer contains not only an organic modifier but also an alkaline earth metal salt (in particular, such as magnesium or calcium salts and mixtures thereof). Typically, the alkaline earth metal salt is magnesium acetate and / or calcium acetate and / or magnesium formate or calcium formate. As shown in the experimental results Figure 2A and Figure 3 The experimental results shown demonstrate that the addition of an alkaline earth metal salt (e.g. magnesium acetate at a regular concentration) to the elution buffer can improve the resolution of full / empty AAV capsids compared to the case without the alkaline earth metal salt. Figure 2A Potassium acetate was used in the experiments to replace the magnesium acetate in Figure 3 . Figure 2A The resolution was 1.57 in Figure 3 , and 2.40 in Figure 2C When the inorganic anion magnesium chloride was replaced by the organic magnesium formate in the experiments, the resolution was improved from 1.75 to 2.06. Organic anions containing two carbon atoms (or more carbon atoms but water-soluble) can enhance the resolution. In the case of the addition of acetonitrile as an organic modifier, the resolution of magnesium formate Figure 2C ) was higher than that of magnesium chloride Figure 2C .

[0080] In another embodiment, Figure 2B shows the effect of using only acetonitrile as an organic modifier. The resolution of full and empty AAV capsids was improved from 1.51 in the presence of Poloxamer 188 to 1.75 in the presence of acetonitrile.

[0081] Figure 2A , 2B and 2C show that when acetonitrile is used as an organic modifier in the elution buffer, the resolution of empty / full AAV capsids can be significantly improved regardless of the alkaline earth metal salt (different cations or anions present in the elution buffer), such as potassium acetate, magnesium acetate or magnesium chloride. The addition of acetonitrile as an organic modifier in the elution buffer used in the present application has a significant advantage in the resolution of empty / full AAV capsids.

[0082] The concentration of the organic modifier in the elution buffer should be as high as necessary and as low as possible. The upper limit for the organic modifier depends on factors such as its miscibility with water and its compatibility with other components in the elution buffer. The skilled person can easily estimate the range of concentrations for the organic modifier. Typically, the concentration of the organic modifier ranges from 1 % (v / v) to 5 % (v / v). When the concentration exceeds 5 %, as shown in Figure 2, the separation of empty capsids from full capsids decreases as the percentage of the organic modifier increases. Although it does not appear to be critical to use a high concentration of the organic modifier, the skilled person should avoid using unnecessarily high concentrations of the organic modifier. Figure 4

[0083] In principle, the skilled person can easily adjust the appropriate concentration of the alkaline earth metal salt in the elution buffer used for the preparation of AAV capsids. Typically, the concentration ranges from about 0.5 mM (weight / volume) to 10 mM (weight / volume).

[0084] Figure 5 The results obtained by varying the concentration of magnesium acetate in the elution buffer are shown in Figure 3. Although it does not appear to be critical to use a high concentration of magnesium acetate, the skilled person should avoid using unnecessarily high concentrations of magnesium acetate. The different concentrations of magnesium acetate have a very small effect on the separation, but a concentration of 5 mM of magnesium acetate appears to be more desirable. As shown in Figure 3, the pH of the elution buffer is typically between about 7.5 and about 9.25. The optimal pH of the buffer depends on the serotype of the corresponding AAV capsid, and its optimal pH can be easily evaluated by simple experiments. Figure 6

[0085] The AAV capsids can be selected, for example, from the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.10, AAV1 1, AAV12, and mixtures of different serotypes, such as hybrid serotypes, chimeras, surface-modified AAVs, and any synthetically derived AAV-like particles. The AAV serotype analyzed by the method of the present application can be a recombinant hybrid serotype (such as AAV2 / 8) or other hybrid serotypes.

[0086] For example, the separation of full and empty AAV8 serotype capsids is typically performed at a pH of about 8.5, while the separation of full and empty AAV9 capsids requires a higher pH for optimal separation, as shown in Figure 4. Figure 7

[0087] ​​​Buffering substances for adjusting the appropriate pH in the separation process, which can provide a buffering capacity for aqueous solutions in the range from pH 7 to pH 12. Typically, they are selected from the group of 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol (Bis-Tris), 2,2',2"-nitrilotriacetic acid (ADA), 2-[(2-amino-2-oxoethyl)amino]ethane-1 -sulfonic acid (ACES), 2,2'-(piperazin-1,4-diyl)bis(ethane-1 -sulfonic acid) (PIPES), 2-hydroxy-3-(morpholin-4-yl)propane-1 -sulfonic acid (MOPSO), 2,2'-[propane-1,3-diylbis(azanediyl)]bis[2-(hydroxymethyl)propane-1,3-diol] (BTP), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-(morpholin-4-yl)propane-1 -sulfonic acid (MOPS), 2-{[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}ethane-1 -sulfonic acid (TES), 2-[4-(2-hydroxyethyl)piperazin-1 -yl]ethane-1 -sulfonic acid (HEPES), 3-[N,N-bis(2-hydroxyethylamino)-2-hydroxy-1 -propane sulfonic acid (DIPSO, 4-(4-morpholinyl)butane sulfonic acid (MOBS), 2-hydroxy-3-[tris(hydroxymethyl)methylamino]-1 -propane sulfonic acid (TAPSO), 2-amino-2-(hydroxymethyl)-1,3-propanediol (Trizma), 4-(2-hydroxyethyl)piperazine-1 -(2-hydroxypropane-3-sulfonic acid) (HEPPSO), Piperazine-N,N'-bis(2-hydroxypropane sulfonic acid) (POPSO), triethylamine (TEA), 4-(2-hydroxyethyl)-1 -piperazine propane sulfonic acid (EPPS), N-tris(hydroxymethyl)methylglycine (Tricine), N,N-bis(2-hydroxyethyl)glycine (Bicine), N-(2-hydroxyethyl)piperazine-N'-(4-butane sulfonic acid) (HEPBS), N-tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid (TAPS), 2-amino-2-methyl-1,3-propanediol (AMPD), N-tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid (TABS), N-(1,1 -dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropane sulfonic acid (AMPSO), 2-(N-cyclohexylamino)ethanesulfonic acid (CHES), 3-cyclohexylamino-2-hydroxypropane sulfonic acid sodium salt (CAPSO), N-(1,1 -dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropane sulfonic acid (AMPS), 3-(cyclohexylamino)-1 -propane sulfonic acid (CAPS) and 4-[cyclohexylamino]-1 -butane sulfonic acid (CABS).

[0088] In the elution buffer an isotonicity agent can be present. Typically selected from the group of sucrose, sorbitol, mannitol, xylitol.

[0089] The buffer can also comprise a non-ionic surfactant. In particular, so-called poloxamers (e.g. Poloxamer 188) can be used. Poloxamers are non-ionic copolymers with a tri-block structure consisting of a central hydrophobic polyoxypropylene (polypropylene oxide) chain flanked by two hydrophilic polyoxyethylene (polyethylene oxide) chains [US 3,740,421 A]. Poloxamers are also known under the trade names Pluronic®, Lutrol®, and Kolliphor®. are known.

[0090] Strong anion exchanger materials comprise quaternary ammonium salt ligands, which are commercially known as Q, QA, QAE, QAM, TEAE, TMAM or TMAE, which can maintain a stable charge in the pH range of about 2 to 13. Quaternary ammonium salt anion exchanger materials have been used in the literature

[10] to separate empty capsids from full capsids.

[0091] Weak anion exchanger DEAE (diethylaminoethyl) materials employ tertiary amine ligands with a pKa of about 11.5, which can achieve the elution of empty capsids from full capsids at moderate pH conditions, as shown in Figure 9

[11] .

[0092] The material can be a monolithic anion exchanger material, a particulate anion exchanger material, and / or an anion exchanger material arranged in a membrane, and / or a particulate packed anion exchanger column, and / or a fibrous chromatographic anion exchanger or a multimodal fibrous column.

[0093] The material can be a multimodal metal affinity exchanger material. It combines the properties of positively charged metal affinity ligands with the properties of weak anion exchangers with hydrogen bonding properties [6, 12]. The material can achieve the separation of an empty capsid subpopulation before full capsids in a linear magnesium chloride gradient, followed by the elution of most empty capsids in a high salt step, as shown in Figure 10 .

[0094] Figure 11A Figures A and B show the effect of higher concentrations of organic modifiers on AAV capsid separation, in particular the effect of using a strong anion exchanger to separate empty capsids from partially filled capsids.

[0095] The method of the present application also tests various loading and elution combination schemes, for example: loading with different salts as elution salts (e.g. using more / less kosmotropic salts), or loading with different organic modifiers as elution organic modifiers. In both cases, the elution gradient uses a mixed salt or organic modifier gradient Figure 14 .

[0096] Furthermore, different sample pre-treatment methods (pre-treatment of the sample in different salt solutions or final use of different organic modifiers) as well as decreasing organic modifier gradients (from 2.5% to 0.0% or from 20.0% to 0.0%) were tested. Figure 14

[0097] The method of the present application is equally applicable for the analysis of different AAV serotype harvest or lysate samples by one-dimensional or more preferably two-dimensional chromatography. The use of a two-dimensional chromatography system PATfix TM The AAV Switcher (Sartorius BIA Separations, Ajdovscina, Slovenia) enables the analysis of complex crude samples in the upstream of process development. The first chromatographic column uses strong cation exchange (CEX) technology for sample pre-purification and the second chromatographic column uses anion exchange (AEX) technology, enabling the separation of empty capsids, partially filled capsids, full capsids or other types of capsids, as shown in Figure 15

[0098] Example

[0099] Example 1

[0100] AAV capsid preparation

[0101] rAAV2 / 8 was generated by triple plasmid transfection of a suspension HEK293 cell line in a chemically defined medium. Rep2-Cap8 was used in combination with a helper plasmid and combined with a c / s construct containing a GFP expression cassette flanked by inverted terminal repeat sequences (ITRs) derived from AAV2. The plasmids were mixed in a molar ratio of 1 : 1 : 1 and transfected into the cells using PEI MAX transfection reagent (Polysciences). The transfection was performed in a 5 L stirred tank Biostat B-DCU bioreactor (Sartorius) in a fed-batch mode. Cell lysis was performed 72 hours post transfection by direct addition of Tween 20 (Sigma-Aldrich) detergent to the bioreactor. The harvested material was stored frozen at -80°C for later use. The lysed AAV 2 / 8 serotype harvest was treated by a pre-capture step using TFF in combination with DNase treatment after clarification. After capture, the sample was subjected to additional purification using a cation exchange chromatography column CIMmultus TM SO3 (Sartorius BIA Separations). After concentration of the capture step eluate, anion exchange CIMmultus TM ​​The QA chromatography column enriched for full AAV capsids. The final fractions containing empty and full capsids were collected separately and buffer exchanged into the formulation buffer using Vivaspin Turbo 100 kDa PES concentrators. All analytical runs used samples prepared by mixing empty and full capsid fractions at a 1 :2 ratio, while preparative runs used samples prepared by mixing empty and full capsid fractions at a 2: 1 ratio.

[0102] Example 2

[0103] 2a - Effect of different organic modifiers on the separation of empty / full AAV capsids

[0104] A 100 μL strong anion exchanger CIMac TM AAV full / empty column for analytical separation of empty and full capsid samples. The column was equilibrated with 2 mM magnesium acetate, 2.5% organic modifier, 20 mM TRIS, and 1% sorbitol (pH 8.5) followed by a linear salt gradient to 80 mM magnesium acetate, 2.5% organic modifier, 20 mM TRIS, and 1% sorbitol (pH 8.5). The volume flow rate was 1 mL / min. 2000 mM potassium acetate, 2.5% organic modifier, 20 mM TRIS, and 1% sorbitol (pH 8.5) was used as the elution buffer.

[0105] Figure 1 The screening results for some readily available organic modifiers are shown, where isopropyl alcohol, acetonitrile (followed by ethanol, t-butanol, and n-butanol) provided the highest separation of about 2.28 for empty and full AAV capsids (as shown in Figure 1 Figure 1). Other tested organic solvents (such as propylene carbonate and methanol) provided lower separation and are less recommended for use.

[0106] In addition to achieving empty and full AAV capsid separation, all tested organic modifiers were able to achieve subpopulation separation.

[0107] 2b - Effect of replacing the conventionally used Poloxamer 188 as an organic modifier, and the effect of different loading and elution salts on the separation of empty / full AAV capsids

[0108] A 100 μL strong anion exchanger CIMac TM AAV full / empty column for analytical separation of empty and full capsid samples. The column was equilibrated and eluted using the following conditions:

[0109] · Figure 2A: Equilibrated with 2 mM magnesium acetate, 2.5% acetonitrile, or 0.1% Poloxamer 188, 20 mM TRIS buffer, and 1% sorbitol (pH 8.5) and eluted with a linear salt gradient to 50 mM magnesium acetate, 2.5% acetonitrile, or 0.1% Poloxamer 188, 20 mM TRIS buffer, and 1% sorbitol (pH 8.5). The volumetric flow rate was 1 mL / min. Elution buffer was 2000 mM potassium acetate, 2.5% acetonitrile, or 0.1% Poloxamer 188, 20 mM TRIS solution (pH 8.5).

[0110] Figure 2B : Equilibrated with 2 mM magnesium acetate, 2.5% acetonitrile, or 0.1% Poloxamer 188, 20 mM TRIS buffer, and 1% sorbitol (pH 8.5) and eluted with a linear salt gradient to 50 mM magnesium acetate, 2.5% acetonitrile, or 0.1% Poloxamer 188, 20 mM TRIS buffer, and 1% sorbitol (pH 8.5). The volumetric flow rate was 1 mL / min. Elution buffer was 2000 mM potassium acetate, 2.5% acetonitrile, or 0.1% Poloxamer 188, 20 mM TRIS solution (pH 8.5).

[0111] Figure 2A 2B The comparison using Poloxamer 188 with acetonitrile as the organic modifier is shown. In both sets of experiments, the resolution improved by about 20-40% just by replacing Poloxamer 188 with acetonitrile, regardless of the salt used. Furthermore, in the high salt elution, the AAV capsid percentage was lower in the acetonitrile treated group than the Poloxamer 188 treated group in both cases (Fig. 2B and Fig. 2C). Figure 2A 2B

[0112] Figure 2C : Equilibrated with 2 mM magnesium acetate, 2.5% acetonitrile, or 0.1% Poloxamer 188, 20 mM TRIS buffer, and 1% sorbitol (pH 8.5) and eluted with a linear salt gradient to 50 mM magnesium acetate, 2.5% acetonitrile, or 0.1% Poloxamer 188, 20 mM TRIS buffer, and 1% sorbitol (pH 8.5). The volumetric flow rate was 1 mL / min. Elution buffer was 2000 mM potassium acetate, 2.5% acetonitrile, or 0.1% Poloxamer 188, 20 mM TRIS solution (pH 8.5).

[0113] It is clear from Fig. 3A and Fig. 3B that magnesium acetate has the best resolution compared to formate salts, and especially compared to the inorganic chloride salts tested. Figure 2C

[0114] ​​​​​​2c - Effect of organic modifier as the only component on the separation of empty / full AAV capsid compared to Poloxamer 188

[0115] 100 μL strong anion exchanger CIMac TM AAV full / empty capsid column for analytical separation of empty and full AAV capsid samples. The column was equilibrated with 2 mM magnesium acetate, 2.5% acetonitrile, 20 mM TRIS and 1% sorbitol (pH 8.5) and eluted with a linear salt gradient to 80 mM magnesium acetate, 2.5% acetonitrile, 20 mM TRIS and 1% sorbitol (pH 8.5). The volumetric flow rate was 1 mL / min. 2000 mM potassium acetate, 2.5% acetonitrile, 20 mM TRIS and 1% sorbitol (pH 8.5) was used as elution buffer.

[0116] As shown in Figure 3 The corresponding buffer combination enabled a high degree of separation of empty and full AAV capsid 2.40, while using Poloxamer 188 resulted in a separation degree of only 1.89. The combination of Poloxamer 188 with an organic modifier (e.g. acetonitrile) was less effective than acetonitrile alone. Elution was performed with a linear salt gradient of 2 mM to 80 mM magnesium acetate with an elution volume of 160 column volumes (CV). In addition to enabling separation of empty and full AAV capsid, this buffer combination also enabled separation of subpopulations.

[0117] 2d - Effect of different percentages of organic modifier on the separation degree of empty / full AAV capsid

[0118] 100 μL strong anion exchanger CIMac TM AAV full / empty capsid column for analytical separation of empty and full AAV capsid samples. The column was equilibrated with 2 mM magnesium acetate, X% acetonitrile, 20 mM TRIS and 1% sorbitol (pH 8.5) and eluted with a linear salt gradient to 50 mM magnesium acetate, X% acetonitrile, 20 mM TRIS and 1% sorbitol (pH 8.5). The volumetric flow rate was 1 mL / min. 2000 mM potassium acetate, X% acetonitrile, 20 mM TRIS and 1% sorbitol was used as elution buffer (pH 8.5). In the control group, 1% Poloxamer 188 was used instead of acetonitrile.

[0119] X: 1%, 2.5%, 5%, 10%, 20% acetonitrile

[0120] As shown in Figure 4As shown, the best results occurred at 5% and 2.5% acetonitrile concentrations (highest resolution of empty vs. full AAV8 and lowest percentage of AAV in the high salt elution). However, for possible process scale-up to manufacturing scale and safety considerations, it is preferable to use the lowest possible concentration of organic modifier (e.g., acetonitrile). Too high or too low acetonitrile concentrations can result in decreased resolution. When 1% Poloxamer 188 was used, the resolution was the lowest and the highest percentage of AAV in the high salt elution (see Figure 4 ).

[0121] 2e - Effect of different magnesium acetate or calcium acetate loading concentrations on empty / full AAV capsid resolution

[0122] 100 μL strong anion exchanger, CIMac TM AAV full / empty chromatography column, empty and full AAV capsid samples were analyzed. The column was equilibrated with X mM magnesium acetate or calcium acetate, 2.5% acetonitrile, 20 mM TRIS, and 1% sorbitol (pH 8.5) and eluted with a linear salt gradient to Y mM magnesium acetate or calcium acetate, 2.5% acetonitrile, 20 mM TRIS, and 1% sorbitol (pH 8.5). The volume flow rate was 1 mL / min. 2000 mM potassium acetate, 2.5% acetonitrile, 20 mM TRIS, and 1% sorbitol (pH 8.5) was used as the elution buffer.

[0123] X: 0, 0.5, 5, 10, 20 mM magnesium acetate or calcium acetate

[0124] Y: The concentration of magnesium acetate or calcium acetate in the eluent was adjusted to achieve the same gradient rate as the standard 2-80 mM magnesium acetate method.

[0125] Overall, calcium acetate was not as ideal as magnesium acetate Figure 5 , which provided significantly better resolution. In addition, the preferred concentration for loading with magnesium acetate was about 5 mM. Magnesium acetate concentrations of 0.5 mM and 10 mM resulted in slightly decreased results.

[0126] 2f - Effect of different pH values on empty / full AAV capsid resolution

[0127] 100 μL strong anion exchanger, CIMac TMAn AAV full / empty column was used to perform analytical separations of empty and full AAV capsid samples. The column was equilibrated with 2 mM magnesium acetate, 2.5% ethanol, 20 mM TRIS, and 1% sorbitol (pH 7.50-9.25) and eluted with a linear salt gradient to 50 mM magnesium acetate, 2.5% ethanol, 20 mM TRIS, and 1% sorbitol (pH 7.50-9.25). The volumetric flow rate was 1 mL / min. 2000 mM potassium acetate, 2.5% ethanol, 20 mM TRIS, and 1% sorbitol (pH 7.50-9.25) was used as the elution buffer.

[0128] As shown in Figure 1, a buffer pH of 8.50 achieved the highest degree of separation of empty and full AAV8 capsids while minimizing the percentage of high salt elution. Other pH values, particularly values below 8.00 and above 8.75, resulted in poorer results. Figure 6

[0129] 2g - The method was extended to other AAV serotypes

[0130] A 100 μL strong anion exchanger CIMac TM An AAV full / empty column was used to perform analytical separations of empty and full AAV capsid samples. The column was equilibrated with 2 mM magnesium acetate, 2.5% acetonitrile, 20 mM TRIS, and 1% sorbitol (pH 8.50) and eluted with a linear salt gradient to 80 mM magnesium acetate, 2.5% acetonitrile, 20 mM TRIS, and 1% sorbitol (pH 8.50). The volumetric flow rate was 1 mL / min. 2000 mM potassium acetate, 2.5% acetonitrile, 20 mM TRIS, and 1% sorbitol was used as the elution buffer (pH 8.50).

[0131] • 2 mM magnesium acetate, 2.5% acetonitrile, 20 mM TRIS buffer, and 1% sorbitol (pH 8.50) eluted with a linear salt gradient to 80 mM magnesium acetate, 2.5% acetonitrile, 20 mM TRIS buffer, and 1% sorbitol (pH 8.50). The volumetric flow rate was 1 mL / min. 2000 mM potassium acetate, 2.5% acetonitrile, 20 mM TRIS, and 1% sorbitol was used as the elution buffer (pH 8.50).

[0132] The method of the present invention was also tested on AAV2 and AAV9 serotypes Figure 7 ). However, it is recommended that the method be adjusted for each serotype to achieve better degrees of separation.

[0133] Example 3

[0134] Preparative separations were performed using QA

[0135] CIMmultus was used TM A QA-1 mL (2 μm) column was used to perform preparative separations of empty and full AAV capsids. The buffer and elution conditions were identical to those described in Example 2 except that ethanol was substituted for acetonitrile as the organic modifier. As shown in Figure 2, a buffer pH of 8.50 achieved the highest degree of separation of empty and full AAV8 capsids while minimizing the percentage of high salt elution. Other pH values, particularly values below 8.00 and above 8.75, resulted in poorer results. Figure 8A ​The preparative separation fractions E1 to E4 were collected as shown. Each fraction was analyzed separately under analytical conditions using a multi-detector system, with detection parameters including: UV 260 and 280 nm wavelengths, light scattering, intrinsic protein fluorescence induced mainly by tryptophan, and extrinsic fluorescence determined using an intercalating dye. Tryptophan is abundant in AAV capsid proteins, representing 2.2% of the total capsid [13, 14], and its fluorescence was determined at an absorbance wavelength of 280 nm and an emission wavelength of 348 nm. Extrinsic fluorescence was determined using the intercalating dye PicoGreen at an absorbance wavelength of 485 nm and an emission wavelength of 520 nm to enhance the sensitivity of detection of nucleic acid impurities

[15] . Figure 8B UV spectral results are shown, where the E1 fraction represents empty capsids with a 260 / 280 wavelength ratio of 0.64. The E2 fraction was collected between the empty capsid peak and the full capsid peak (valley fraction), and exhibited two populations with 260 / 280 wavelength ratios of 0.82 and 0.73, respectively. This fraction is most likely representative of partially filled capsids or DNA attached to the surface of empty capsids (due to extrinsic PicoGreen fluorescence). The E3 fraction is rich in full capsids with a 260 / 280 wavelength ratio of 1.37. The E4 fraction (tail fraction) is most likely representative of damaged AAV capsids and / or aggregates. Figure 8C Elution fractions monitored by light scattering detector are shown. Elution fractions monitored by tryptophan fluorescence are shown in Figure 8D , and elution fractions monitored by PicoGreen fluorescence are shown in Figure 8E . The E2 fraction ( Figure 8E ) exhibited a high PicoGreen fluorescence signal, indicating a significantly higher content of DNA-related impurities than the other elution fractions.

[0136] Notes: Figure 8B The UV 260 / 280 ratio is abbreviated as Rat.

[0137] Example 4

[0138] Separation of empty and full AAV capsids using a weak anion exchanger

[0139] A 100 μL CIMac TM DEAE column was used to separate empty AAV capsids from full AAV capsids.

[0140] The column was equilibrated with 20 mM TRIS buffer, 0.5% acetonitrile or 1% Poloxamer 188, 1% sorbitol (pH 9.0). A linear salt gradient was used to elute to 50 mM magnesium acetate, 20 mM TRIS buffer, 0.5% acetonitrile or 1% Poloxamer 188, and 1% sorbitol (pH 9.0). The volume flow rate was 1 mL / min. No salt was added to the equilibration buffer due to weak binding of the sample.Figure 9 Results show a slight increase in resolution when organic modifiers are introduced into the buffer. 2000 mM potassium acetate, 0.5% acetonitrile, or 0.1% Poloxamer 188, 20 mM TRIS (pH 9.0) were used as elution buffers.

[0141] Example 5

[0142] Separation of empty and full AAV capsids using multi-modal material

[0143] Through 100 μL CIMac TM The PrimaT column achieved separation of empty and full AAV capsids. The column employed multi-modal metal affinity ligands, combining the properties of hydrophobic and anion exchange chromatography.

[0144] Samples were bound to the column through dominant hydrogen bonding interactions at neutral pH using 25 mM HEPES, 1% sucrose, 0.1% Poloxamer 188, or 2.5% acetonitrile as the buffer; pH shift to 50 mM Tris, 13.6 mM borate, 1% sucrose, 0.1% Poloxamer 188, or 2.5% acetonitrile (pH 9.0) to replace hydrogen bonding with electrostatic interactions. Most full and partial empty AAV capsids were eluted with a linear salt gradient to 50 mM magnesium chloride, 50 mM Tris, 9.6 mM borate, 1% sucrose, 0.1% Poloxamer 188, or 2.5% acetonitrile (pH 9.0), followed by a high salt linear gradient to 2 M NaCl, 50 mM Tris, 12 mM borate, 1% sucrose, 0.1% Poloxamer 188, or 2.5% acetonitrile (pH 9.0) to elute the remaining empty AAV capsids. The volumetric flow rate was 1 mL / min.

[0145] Higher resolution was achieved when acetonitrile was used in place of Poloxamer 188, and less empty AAV capsids were observed in the high salt linear gradient Figure 10 ].

[0146] Example 6

[0147] Effect of organic modifier concentration

[0148] Through CIMac TMA QA column was used to investigate the effect of higher percentage organic modifier with salt gradient on baseline separation of AAV capsids. The column was equilibrated with 10 mM magnesium acetate, 50 mM TRIS, 2% acetonitrile, 1% sorbitol (pH 8.5, buffer A). The first elution used a linear gradient of acetonitrile to 30% with 10 mM magnesium acetate, 50 mM TRIS, 1% sorbitol (pH 8.5). After the acetonitrile gradient elution was complete, the elution was stopped with buffer A to reduce the acetonitrile concentration and a salt gradient was used to elute to 50 mM magnesium acetate, 50 mM TRIS, 1% sorbitol (pH 8.5). The volume flow rate was 1 mL / min.

[0149] Baseline separation of empty AAV capsids from full AAV capsids and other components was achieved using higher concentrations of organic modifier for elution followed by salt gradient techniques. Figure 11A The UV chromatograms at 260 and 280 nm shown demonstrate the separation of AAV capsids achieved by the following combination: reverse phase chromatography conditions (empty capsids eluted with a 260 / 280 wavelength ratio of 0.64, partially filled capsids with a ratio of 1.08), followed by elution using anion exchange conditions, full capsids with a 260 / 280 ratio of 1.32 as the main peak, followed by a tailing peak with a 260 / 280 ratio of 1.25. Baseline separation of empty capsids and partially filled AAV capsids from other subpopulations of full capsids (in particular, full capsids, heavy full capsids, or aggregates) was achieved by increasing the temperature to 40 °C in combination with reverse phase and anion exchange conditions. Figure 11B The tryptophan fluorescence chromatogram shows the same trend.

[0150] Example 7

[0151] Using density gradient ultracentrifugation in combination with PATfix TM A multi-detector array was used to perform orthogonal analysis of the preparative fractions. During density gradient ultracentrifugation, different capsid populations were separated based on density differences: full capsids sedimented at the bottom of the cesium chloride gradient, and empty capsids floated at the top. After ultracentrifugation, the fractions were pumped for analysis of the UV 260 and 280 nm signals Figure 12A ), and tryptophan endogenous fluorescence Figure 12B ). The 260 and 280 nm UV signal results for the E1 fraction Figure 12A ) eluted at 6.85 minutes corresponded to empty AAV capsids. Partially filled AAV capsids were observed for the E2 fraction at 6.78 minutes. The E2 fraction showed a pronounced fronting phenomenon between about 5.5 and 6.5 minutes, indicating the presence of slightly heavier capsids than empty capsids. The E3 fraction (5.17 minutes) showed primarily full capsids. The E4 fraction eluted earlier than the E3 fraction, with a fronting peak between 3 and 4 minutes, indicating the presence of heavier AAV capsids or aggregates.

[0152] Density ultracentrifugation results supported both the analytical and preparative results.

[0153] Example 8

[0154] Comparative study of AAV capsid separation using QA chromatography column versus anion exchange membrane adsorber

[0155] Preparative runs were performed using the sample material described in Example 1, loaded onto both the monolithic anion exchange column and the membrane adsorber Q - 3 mL column (Sartorius). The buffers and elution conditions described in Example 3 were used. As shown, both the QA column and the membrane adsorber gave similar separation profiles. Figure 13

[0156] Example 9

[0157] AAV capsid preparation

[0158] rAAV2 / 8 was generated by triple plasmid transfection of a suspension HEK293 cell line in a chemically defined medium. The vector was composed of a Rep2-Cap8 plasmid, a helper plasmid and a cis construct containing a GFP expression cassette flanked by inverted terminal repeat sequences (ITRs) derived from AAV2. The plasmids were mixed at a 1 : 1 : 1 molar ratio and cells were transfected using PEI MAX transfection reagent (Polysciences). Transfection was performed in a 5 L stirred tank Biostat B-DCU bioreactor (Sartorius) in a fed-batch mode. Cell lysis was performed 72 hours post-transfection by direct addition of Tween 20 (Sigma-Aldrich) detergent to the bioreactor. The harvested material was stored frozen at -80°C for later use. The lysed harvest of AAV 2 / 8 serotype (used for the experiment of Example 2) was clarified and treated by a TFF pre-capture step combined with DNase treatment. The sample was captured and further purified on a SO3 (Sartorius BIA Separations) cation exchange chromatography column. The capture step eluate was concentrated on a Vivaspin Turbo 100 kDa PES concentrator and exchanged into formulation buffer for later experiments. This sample was used only for the experiments of Example 10 and Example 11. TM

[0159] Example 10

[0160] Effect of different loading and elution variables on the separation of empty / full AAV capsids

[0161] A 100 μL strong anion exchanger CIMac TM ​​AAV full / empty chromatography column, analytical separation of empty capsids and full AAV capsid samples. The chromatography column was equilibrated with an application buffer (buffer A) containing different magnesium salt concentrations, different organic solvents or different percentages of organic solvents as organic modifier. In two cases the percentage of organic solvent was 0.0% (organic modifier step down gradient).

[0162] Buffer A (application buffer): X mM magnesium salt, Y% organic modifier, 20 mM TRIS and 1% sorbitol, pH 8.5.

[0163] Buffer B (elution buffer): 50 mM magnesium acetate, Y% organic modifier, 20 mM TRIS and 1% sorbitol, pH 8.5. The samples were applied in buffer A and eluted with a linear salt gradient to buffer B. The volume flow rate was 1 mL / min.

[0164] Buffer C was used to elute more negatively charged compounds that remained after elution with buffer B.

[0165] Buffer C (high salt eluent): 2000 mM potassium acetate, 2.5% ethanol, 20 mM TRIS, pH 8.5.

[0166] 1. Experiment:

[0167] Buffer A: 5 mM magnesium acetate, 2.5% ethanol, 20 mM TRIS and 1% sorbitol, pH 8.5

[0168] Buffer B: 50 mM magnesium acetate, 2.5% ethanol, 20 mM TRIS and 1% sorbitol, pH 8.5

[0169] 2. Experiment:

[0170] Buffer A: 5 mM magnesium lactate, 2.5% ethanol, 20 mM TRIS and 1% sorbitol, pH 8.5 Buffer B: 50 mM magnesium acetate, 2.5% ethanol, 20 mM TRIS and 1% sorbitol, pH 8.5

[0171] 3. Experiment:

[0172] Buffer A: 5 mM magnesium formate, 2.5% ethanol, 20 mM TRIS and 1% sorbitol, pH 8.5 Buffer B: 50 mM magnesium acetate, 2.5% ethanol, 20 mM TRIS and 1% sorbitol, pH 8.5

[0173] 4. Experiment:

[0174] Buffer A: 5 mM magnesium chloride, 2.5% ethanol, 20 mM TRIS and 1% sorbitol, pH 8.5

[0175] Buffer B: 50 mM Magnesium Acetate, 2.5% Ethanol, 20 mM TRIS, and 1% Sorbitol, pH 8.5

[0176] 5. Experiment:

[0177] Buffer A: 5 mM Magnesium Acetate, 2.5% Ethanol, 20 mM TRIS, and 1% Sorbitol, pH 8.5

[0178] Buffer B: 50 mM Magnesium Acetate, 2.5% Ethanol, 20 mM TRIS, and 1% Sorbitol, pH 8.5

[0179] Samples were prepared in 5 mM Magnesium Chloride, 2.5% Ethanol, 20 mM TRIS, and 1% Sorbitol at a pH of 8.5.

[0180] 6. Experiment:

[0181] Buffer A: 5 mM Magnesium Acetate, 2.5% Acetonitrile, 20 mM TRIS, and 1% Sorbitol, pH 8.5

[0182] Buffer B: 50 mM Magnesium Acetate, 2.5% Ethanol, 20 mM TRIS, and 1% Sorbitol, pH 8.5

[0183] 7. Experiment:

[0184] Buffer A: 5 mM Magnesium Acetate, 2.5% Ethanol, 20 mM TRIS, and 1% Sorbitol, pH 8.5

[0185] Buffer B (no organic modifier): 50 mM Magnesium Acetate, 20 mM TRIS, and 1% Sorbitol, pH 8.5

[0186] 8. Experiment:

[0187] Buffer A: 5 mM Magnesium Acetate, 20.0% Ethanol, 20 mM TRIS, and 1% Sorbitol, pH 8.5

[0188] Buffer B (no organic modifier): 50 mM Magnesium Acetate, 20 mM TRIS, and 1% Sorbitol, pH 8.5

[0189] The results above show that all protocols yielded comparable results with no significant differences between experiments in the resolution of empty AAV capsids from intact AAV capsids and the estimated percentage of intact AAV8. As shown in Sample 8, the lowest resolution and lowest estimated percentage of intact AAV8 was obtained when loading at high concentration of ethanol and eluting at zero concentration of ethanol. In addition, using a less lyotropic salt (magnesium chloride) for loading, Figure 14 Sample 8. Furthermore, using a less lyotropic salt (magnesium chloride) for loading, Figure 14Sample 4 in Figure 6 shows a clear decrease in resolution. On the other hand, as expected, the resolution is slightly improved when using a more lyotropic salt (magnesium lactate) for loading compared to the magnesium acetate loading condition (Sample 1). Figure 14 In Figure 6, the resolution is slightly improved when using a more lyotropic salt (magnesium lactate) for loading compared to the magnesium acetate loading condition (Sample 1).

[0190] When combining multiple potential different elution strategies, it is helpful to have robust binding and loading conditions to obtain reproducible results.

[0191] Example 11

[0192] The PATfix AAV Switcher (Sartorius BIA Separations, Ajdovscina, Slovenia) was used for the lysis sample

[0193] The following buffers were used for the AEX separation:

[0194] Buffer A: 20 Tris + 5 mM magnesium acetate + 1% sorbitol + 2.5% ethanol; pH 8.50.

[0195] Buffer B: 20 Tris + 65 mM magnesium acetate + 1% sorbitol + 2.5% ethanol; pH 8.50.

[0196] Buffer C: 500 mM sodium acetate; pH 5.50.

[0197] Buffer D: 100 mM sodium hydroxide + 2000 mM sodium chloride.

[0198] By using similar conditions as described above, separation of empty, possibly partially filled, full and / or other capsids in complex lysis samples was achieved on the CIMac TM QA HR chromatography column. The lysis sample was first eluted on a cation exchange column with a pH gradient (pH 4.50 to pH 9.50) for purification. Subsequently, the pH gradient eluate was transferred in proportion to a second AEX column in series to achieve separation of the subpopulations. In addition to empty and full capsids, partially filled capsids and other impurities were observed Figure 15). The pre-peak appearing at about 8 to about 10 minutes indicates the presence of certain protein impurities in the harvest sample (visible by tryptophan fluorescence detection, but not by light scattering detection). The peak at about 13.7 minutes corresponds to other more likely macromolecular protein related impurities in the harvest sample (visible by both fluorescence and light scattering detection). The remaining contaminants elute in the AEX column cleaning step (starting at about 18 minutes). While one-dimensional analysis can yield similar results, it is more likely to co-elute more impurities from the harvest sample with the AAV capsid. Since these highly negatively charged impurities (which can be removed by pre-purification on a tandem cation exchange column) will continue to adsorb to the AEX column, this will result in a shortened column lifetime.

[0199]

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Claims

1. A method for enriching intact adeno-associated virus (AAV) capsids from a mixture comprising intact AAV capsids, partially filled and / or empty AAV capsids by chromatography, comprising the following steps: -Contact the mixture with a strong or weak anion exchange material; - The loaded mixture was eluted using a neutral to alkaline buffer containing an organic modifier with a solvent polarity relative of 0.4 to 0.8; and - Collect fractions rich in intact AAV capsids.

2. The method as described in claim 1, characterized in that, The organic regulator is selected from the group consisting of acetonitrile, 1-butanol, tert-butanol, propylene carbonate, isopropanol, ethanol, methanol, propanol, or combinations thereof.

3. The method as described in claim 1 or 2, characterized in that, The buffer solution contains an alkaline earth metal salt, particularly magnesium acetate, magnesium formate, calcium acetate or calcium formate, or combinations thereof, and / or more hydrophilic alternatives thereof.

4. The method according to any one of claims 1 to 3, characterized in that, The pH value of the buffer solution is from pH 7.0 to pH 10.

50.

5. The method according to any one of claims 1 to 4, characterized in that, The buffer solution contains: -Isotonic substances selected from sucrose, sorbitol, mannitol, xylitol, and mixtures thereof; and / or - Nonionic surfactants, such as Poloxamer 188.

6. The method according to any one of claims 1 to 5, characterized in that, The strong or weak anion exchange material is: (i) Strong or weak anion exchange materials with or without hydrogen bonding properties, which can be combined or not combined with positively charged metal affinity ligands to form multimodal materials. (ii) Monolithic anion exchanger; (iii) Monolithic multimodal materials; (iv) Particulate anion exchangers; (v) Particulate multimodal materials; (vi) Anion exchangers or multimodal materials disposed in the membrane; and / or (vii) Packed anion exchange resins or multimodal chromatographic columns; and / or (viii) Fiber chromatography using anion exchangers or multimodal fiber chromatography columns.

7. The method according to any one of claims 1 to 6, characterized in that, The AAVs are selected from serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.10, AAV11, AAV12, and other serotypes, such as hybrid serotypes, especially recombinant hybrid serotypes such as AAV2 / 8, chimeras, surface-modified AAVs, and synthetically derived AAV-like particles.

8. An aqueous solution with a neutral to alkaline pH, comprising a buffer substance and an organic regulator, wherein the solvent polarity relative to the measured value of the regulator is 0.4 to 0.

8.

9. The aqueous solution as described in claim 8, characterized in that, The organic regulator is selected from the group consisting of acetonitrile, 1-butanol, tert-butanol, propylene carbonate, isopropanol, ethanol, methanol, propanol, or combinations thereof.

10. The aqueous solution as described in claim 8 or 9, characterized in that, The aqueous solution contains alkaline earth metal salts.

11. The aqueous solution as described in claim 10, characterized in that, The alkaline earth metal salt is a magnesium or calcium salt, or a combination thereof, wherein the alkaline earth metal salt is in particular magnesium acetate, magnesium formate, calcium acetate or calcium formate, or a combination thereof, and / or a more hydrophilic alternative thereof.

12. The aqueous solution according to any one of claims 8 to 11, characterized in that, The buffering substances buffer the pH of the aqueous solution within the range of pH 7 to pH 12, particularly buffering substances selected from the group consisting of: 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol (Bis-Tris), 2,2',2”-aminotriacetic acid (ADA), 2-[(2-amino-2-oxoethyl)amino]ethane-1-sulfonic acid (ACES), 2,2'-(piperazin-1,4-diyl)bis(ethane-1-sulfonic acid) (PIPES), 2-hydroxy-3-(morpholin-4-yl)propane-1-sulfonic acid (MOPSO), 2,2'-[propane-1,3-diylbis(azadiyl)]bis[2-(hydroxymethyl)propane-1,3-diol] (B TP), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-(morpholin-4-yl)propane-1-sulfonic acid (MOPS), 2-{[1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl]amino}ethane-1-sulfonic acid (TES), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid (HEPES), 3-[N,N-bis(2-hydroxyethylamino)-2-hydroxy-1-propanesulfonic acid (DIPSO), 4-(4-morpholinyl)butanesulfonic acid (MOBS), 2-hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid (TAPSO), 2 -Amino-2-(hydroxymethyl)-1,3-propanediol (Trizma), 4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropane-3-sulfonic acid) (HEPPSO), piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) (POPSO), triethylamine (TEA), 4-(2-hydroxyethyl)-1-piperazine propanesulfonic acid (EPPS), N-tris(hydroxymethyl)methylglycine (Tricine), N,N-bis(2-hydroxyethyl)glycine (Bicine), N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid) (HEPBS), N-tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid (T APS), 2-amino-2-methyl-1,3-propanediol (AMPD), N-tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid (TABS), N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO), 2-(N-cyclohexylamino)ethanesulfonic acid (CHES), sodium 3-cyclohexylamino-2-hydroxypropanesulfonate (CAPSO), N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPS), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), and 4-[cyclohexylamino]-1-butanesulfonic acid (CABS).

13. The aqueous solution according to any one of claims 8 to 12, characterized in that, The buffer solution contains: -Isotonic additives, particularly those selected from the group consisting of sucrose, sorbitol, mannitol, xylitol, and mixtures thereof; and / or - Nonionic surfactants, such as Poloxamer 188.

14. Use of the aqueous solution as described in any one of claims 8 to 13 for separating an intact adeno-associated virus (AAV) capsid from an empty AAV capsid, particularly by means of the method described in any one of claims 1 to 7.

15. A method for enriching intact adeno-associated virus (AAV) capsids from a mixture comprising intact AAV capsids, partially filled and / or empty AAV capsids by chromatography, comprising the following steps: -Contact the mixture with a strong or weak anion exchange material; - Elute the loaded mixture using a neutral to alkaline buffer containing an organic modifier selected from the group consisting of: methanol, 1,3-propanediol, 1,2-propanediol, N-methylformamide, diethylene glycol, triethylene glycol, 1,3-butanediol, 2-propynyl-1-ol (propynyl alcohol), 2-methoxyethanol, 2-propenyl-1-ol (allyl alcohol), N-methylacetamide, ethanol, 2-aminoethanol, acetic acid, benzyl alcohol, 1-propanol, 1-butanol, 2-hydroxymethylfuran (furfuryl alcohol), 2-phenylethanol, 1-pentanol, 2-methyl-1-propanol (isobutanol), 1-hexanol, 2-propanol, 3-phenyl-1-propanol, 1 -Heptanol, 1-Octanol, Cyclopentanol, 1-Decanol, 2,6-Dimethylphenol (2,6-dimethylphenol), 2-Butanol, 3-Methyl-1-butanol (isoamyl alcohol), Cyclohexanol, 1-Dodecanol, 1-Phenyleneethanol, Acrylonitrile, 4-Methyl-1,3-dioxolane-2-one (propylene carbonate), 2-Pentanol, Nitromethane, Acrylonitrile, Dimethyl sulfoxide, Methyl acrylate, Aniline, Tetra-N-hexylbenzoate, Tetrahydrothiophene-1,1-dioxide (sulfolane), 2-Methyl-2-propanol (tert-butanol), Acetic anhydride, N,N-dimethylformamide, N,N-dimethylacetamide, Propanolitrile, and Nitroethane; and - Collect fractions rich in intact AAV capsids.

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