Formulations for AAV gene therapy

Abstract

A composition for preserving and maintaining viral viability, comprising a balance of ionic strength and osmolality, that results in reproducible and stable product quality at room temperature, 4°C, and has the ability to withstand 10 freeze / thaw events.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of the earlier filing date of U.S. Provisional Patent Application No. 63 / 379,117, filed October 11, 2022, which provisional application is incorporated by reference in its entirety.

[0002] The present disclosure relates to formulations for preserving adeno-associated virus (AAV) and maintaining its viability or infectivity after a lyophilization cycle. [Background technology]

[0003] AAV gene therapies in liquid formulations are rarely considered stable under refrigeration and are typically stabilized by freezing at -80°C. However, the freezing and thawing process can compromise product quality, and shipping and storage of frozen materials is often complicated and expensive. Furthermore, freezing at -80°C reduces the number of available drug delivery device options, such as prefilled syringes, which can hinder product accessibility. Therefore, novel formulations for preserving AAV gene therapy products and maintaining viability / infectivity are urgently needed. Summary of the Invention

[0004] Embodiments of the present disclosure relate to formulations that maintain the quality of AAV products at a variety of storage temperatures, including from about ambient temperature down to -80°C, and / or upon exposure to multiple freezing / drying conditions over time.

[0005] Thus, in certain aspects, a formulation for AAV particles is provided, the formulation comprising a sugar, a buffer, a surfactant, a salt, or a combination thereof. In certain embodiments, the sugar comprises one or more sugars. In certain embodiments, the one or more sugars comprise trehalose, cyclodextrin, sucrose, or a combination thereof. In certain embodiments, the salt comprises a sodium salt, a magnesium salt, a calcium salt, a potassium salt, a phosphate salt, a sulfate salt, triethylamine, guanidine, an N-substituted guanidine salt, acetamidine, an N-substituted acetamidine, pyridine, picoline, ethanolamine, triethanolamine, dicyclohexylamine, or N,N'-dibutylethylenediamine salt, or a combination thereof. In certain embodiments, the sodium salt comprises sodium chloride, sodium phosphate, or a combination thereof. In certain embodiments, the magnesium salt is magnesium sulfate. In certain embodiments, the salt comprises sodium chloride, sodium phosphate, magnesium sulfate, or a combination thereof. In certain embodiments, the formulation further comprises a buffer comprising phosphate-buffered saline (PBS), sodium phosphate, citric acid, acetic acid, tromethamine, aspartic acid, glutamic acid, HEPES, Tris, bicine, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, lysine, arginine, succinate, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS (3-N-morpholino)propanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid), triethanolamine buffer, and combinations thereof. In certain embodiments, the surfactant is a non-ionic surfactant. In certain embodiments, the non-ionic surfactant comprises a polysorbate. In certain embodiments, the polysorbate is polysorbate 80. In certain embodiments, the formulation comprises an ionic strength of about 100 mM to about 700 mM. In certain embodiments, the formulation comprises an osmolality of about 100 mOsm / kg to about 800 mOsm / kg, hi certain embodiments, the formulation comprises a pH of about 7.0 to about 8.0.In certain embodiments, the formulation comprises a pH of about 7.5. In certain embodiments, the formulation is utilized to preserve or maintain the viability of adeno-associated virus (AAV) particles over a period of time at temperatures of about 20°C to about -80°C. In certain embodiments, the AAV particles are stable over multiple lyophilization cycles. In certain embodiments, the AAV particles are derived from an AAV having an AAV serotype selected from serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, DJ, or DJ / 8. In certain embodiments, the AAV particles comprise a genome derived from AAV serotype 2. In certain embodiments, the AAV particles comprise a capsid derived from AAV serotype 2.

[0006] In another aspect, a pharmaceutical composition for preserving, maintaining infectivity, and / or maintaining viability of AAV particles comprises a cryoprotectant, a buffer, a surfactant, and a salt. In certain embodiments, the cryoprotectant comprises one or more sugars. In certain embodiments, the sugar comprises trehalose, cyclodextrin, sucrose, or a combination thereof. In certain embodiments, the salt comprises a sodium salt, a magnesium salt, a calcium salt, a potassium salt, a phosphate salt, a sulfate salt, or a combination thereof. In certain embodiments, the sodium salt comprises sodium chloride, sodium phosphate, or a combination thereof. In certain embodiments, the magnesium salt is magnesium sulfate. In certain embodiments, the surfactant is a non-ionic surfactant. In certain embodiments, the non-ionic surfactant comprises a polysorbate. In certain embodiments, the polysorbate is polysorbate 80. In certain embodiments, the pharmaceutical composition comprises an ionic strength of about 100 mM to about 700 mM. In certain embodiments, the pharmaceutical composition comprises an osmolality of about 100 mOsm / kg to about 800 mOsm / kg. In certain embodiments, the pharmaceutical composition comprises a pH of about 7.0 to about 8.0. In certain embodiments, the pharmaceutical composition comprises a pH of about 7.5. In certain embodiments, the pharmaceutical composition is utilized to store or maintain viability of AAV particles over a period of time at temperatures of about 20°C to about -80°C. In certain embodiments, the AAV particles remain stable over multiple freeze / dry cycles.

[0007] In another aspect, a cryoprotective formulation for preserving, maintaining infectivity, and / or maintaining viability of AAV particles comprises sodium phosphate, sodium chloride, cyclodextrin, and polysorbate. In certain embodiments, the cryoprotective formulation comprises an ionic strength of about 100 mM to about 700 mM. In certain embodiments, the cryoprotective formulation comprises an ionic strength of about 100 mM to about 400 mM. In certain embodiments, the cryoprotective formulation comprises an osmolality of about 100 mOsm / kg to about 800 mOsm / kg. In certain embodiments, the cryoprotective formulation comprises an osmolality of about 200 mOsm / kg to about 700 mOsm / kg. In certain embodiments, the sodium phosphate concentration is about 1 mM to about 20 mM. In certain embodiments, the sodium chloride concentration is about 100 mM to about 400 mM. In certain embodiments, the cyclodextrin comprises from about 0.1% weight / volume (w / v) up to about 20% (w / v). In certain embodiments, the polysorbate comprises from about 0.01% weight / volume (w / v) up to about 5% (w / v). In certain embodiments, a cryoprotective formulation is utilized to store or maintain viability of AAV particles for a period of time at temperatures between about 20°C and about -80°C. In certain embodiments, the cryoprotective formulation comprises a pH of about 7.0 to about 8.0. In certain embodiments, the AAV particles remain stable over multiple freeze / dry cycles.

[0008] In another aspect, a cryoprotective formulation for preserving, maintaining infectivity, and / or maintaining viability of AAV particles comprises a cryoprotective sugar, a buffer, a salt, and a non-ionic surfactant. In certain embodiments, the cryoprotective sugar comprises cyclodextrin, trehalose, or a combination thereof. In certain embodiments, the cryoprotective sugar is trehalose. In certain embodiments, the trehalose is at a concentration of about 1% weight / volume (w / v) to about 20% w / v. In certain embodiments, the non-ionic surfactant is a polysorbate. In certain embodiments, the polysorbate is polysorbate 80. In certain embodiments, the polysorbate 80 is at a concentration of 0.001% w / v to about 1% w / v. In certain embodiments, the buffer is Tris buffer. In certain embodiments, the Tris buffer is at a concentration of about 1 mM to about 20 mM. In certain embodiments, the salt is a magnesium salt. In certain embodiments, the magnesium salt is magnesium sulfate. In certain embodiments, the magnesium sulfate is at a concentration of about 10 mM to about 250 mM. In certain embodiments, the cryoprotective formulation comprises an ionic strength of about 100 mM to about 700 mM. In certain embodiments, the cryoprotective formulation comprises an ionic strength of about 200 mM to about 500 mM. In certain embodiments, the cryoprotective formulation comprises an osmolality of about 100 mOsm / kg to about 800 mOsm / kg. In certain embodiments, the cryoprotective formulation comprises an osmolality of about 200 mOsm / kg to about 600 mOsm / kg. In certain embodiments, the cryoprotective formulation comprises a pH of about 7.0 to about 8.0. In certain embodiments, the cryoprotective formulation further comprises one or more pharmaceutical agents, cell culture media, proteins, lipids, or combinations thereof. In certain embodiments, formulations are utilized to store or maintain the viability of AAV particles for periods of time at temperatures between about 20° C. and about −80° C. In certain embodiments, the AAV particles remain stable over multiple freeze / dry cycles.

[0009] In another aspect, a composition for preserving, maintaining infectivity, and / or maintaining viability of AAV particles comprises about 1 mM to about 20 mM sodium phosphate, about 100 mM to about 400 mM sodium chloride, about 0.1% weight / volume (w / v) to up to about 30% (w / v) cyclodextrin, and about 0.01% weight / volume (w / v) to about 5% (w / v) polysorbate. In certain embodiments, the composition is utilized to preserve or maintain viability of AAV particles for a period of time at temperatures between about 20°C and about -80°C. In certain embodiments, the AAV particles remain stable over multiple freeze / dry cycles.

[0010] In another aspect, a composition for preserving, maintaining infectivity, and / or maintaining viability of AAV particles comprises about 1% weight / volume (w / v) to about 20% w / v trehalose, 0.001% w / v to about 1% w / v polysorbate, about 1 mM to about 20 mM Tris buffer, about 10 mM to about 250 mM magnesium sulfate, about 0.1% weight / volume (w / v) to up to about 30% w / v cyclodextrin, and about 0.01% weight / volume (w / v) to about 5% w / v polysorbate. In certain embodiments, the composition is utilized to preserve or maintain viability of adeno-associated virus (AAV), recombinant AAV (rAAV) particles, adenovirus particles, or other virus particles over a period of time at temperatures between about 20°C and about -80°C. In certain embodiments, the virus particles remain stable over multiple freeze / dry cycles.

[0011] In another aspect, a formulation for storing or maintaining viability of AAV, rAAV particles, adenovirus particles, or other virus particles at ambient temperature, or at temperatures of about 20°C to about -80°C, comprises about 1 mM to about 20 mM sodium phosphate, about 80 mM to about 30 mM sodium chloride (NaCl), about 0.1% to about 10% cyclodextrin, and about 0.001% to about 5% polysorbate 80, wherein the formulation has an ionic strength of about 100 mM to about 500 mM, and an osmolality of about 150 mOsm / kg to about 600 mOsm / kg.

[0012] In certain embodiments, formulations are utilized to store or maintain the viability of AAV particles for a period of time at temperatures between about 20°C and about -80°C. In certain embodiments, the AAV particles are stable at about 4°C. In certain embodiments, the AAV particles are stable at 4°C for at least 6 months to at least 1 year. In certain embodiments, the AAV particles are stable over multiple lyophilization cycles. In certain embodiments, the AAV particles are stable over at least 5 lyophilization cycles.

[0013] In certain embodiments, the formulation comprises a buffer composition shown in Tables 1 and 2. In certain embodiments, the formulation comprises a cryoprotectant formulation shown in Table 1 or 2. In certain embodiments, the pharmaceutical composition comprises a buffer composition shown in Table 1 or 2.

[0014] In one aspect, a cryoprotective formulation for preserving, maintaining infectivity, and / or maintaining viability of adeno-associated virus (AAV) particles is provided, the formulation comprising a sugar, a salt, a buffer, a surfactant, or a combination thereof.

[0015] In one aspect, a pharmaceutical composition for preserving, maintaining infectivity, and / or maintaining viability of AAV particles is provided, comprising a sugar, a salt, a buffer, a surfactant, or a combination thereof.

[0016] In embodiments, the sugar comprises one or more sugars. In embodiments, the one or more sugars comprise trehalose, cyclodextrin, sucrose, or a combination thereof. In embodiments, the sugar is cyclodextrin. In embodiments, the cyclodextrin is at a concentration of about 0.1% weight / volume (w / v) up to about 20% (w / v). In embodiments, the cyclodextrin is at a concentration of about 0.1% (w / v) up to about 1% (w / v). In embodiments, the cyclodextrin is at a concentration of about 0.4% (w / v).

[0017] In an embodiment, the sugar is trehalose. In an embodiment, the trehalose is at a concentration of about 0.5% (w / v) to about 20% (w / v). In an embodiment, the trehalose is at a concentration of about 0.5% (w / v) to about 5% (w / v). In an embodiment, the trehalose is at a concentration of about 1.1% (w / v).

[0018] In embodiments, the salt includes a sodium salt, a magnesium salt, a calcium salt, a potassium salt, a phosphate salt, a sulfate salt, a triethylamine, a guanidine, an N-substituted guanidine salt, an acetamidine, an N-substituted acetamidine, a pyridine, a picoline, an ethanolamine, a triethanolamine, a dicyclohexylamine, or an N,N'-dibutylethylenediamine salt, or a combination thereof. In embodiments, the salt is a sodium salt. In embodiments, the sodium salt is sodium chloride, sodium phosphate, or both. In embodiments, the sodium chloride is at a concentration of about 100 mM to about 400 mM. In embodiments, the sodium chloride is at a concentration of about 200 mM to about 350 mM. In embodiments, the sodium chloride is at a concentration of about 280 mM. In embodiments, the salt is a magnesium salt. In embodiments, the magnesium salt is magnesium sulfate. In an embodiment, the magnesium sulfate is at a concentration of about 10 mM to about 250 mM. In an embodiment, the magnesium sulfate is at a concentration of about 50 mM to about 200 mM. In an embodiment, the magnesium sulfate is at a concentration of about 125 mM.

[0019] In embodiments, the buffer comprises phosphate buffered saline (PBS), sodium phosphate, citric acid, acetic acid, tromethamine, aspartic acid, glutamic acid, HEPES, Tris, bicine, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, lysine, arginine, succinate, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS (3-N-morpholino)propanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid), triethanolamine buffer, or a combination thereof. In embodiments, the buffer comprises sodium phosphate. In embodiments, the sodium phosphate is at a concentration of about 1 mM to about 20 mM. In embodiments, the sodium phosphate is at a concentration of about 10 mM. In embodiments, the buffer is Tris. In one embodiment, Tris is at a concentration of about 1 mM to about 20 mM, In one embodiment, Tris is at a concentration of about 10 mM.

[0020] In embodiments, the surfactant is a non-ionic surfactant. In embodiments, the non-ionic surfactant comprises a polysorbate. In embodiments, the polysorbate is polysorbate 80. In embodiments, the polysorbate is at a concentration of about 0.01% (w / v) to about 5% (w / v). In embodiments, the polysorbate is at a concentration of about 0.001% (w / v) to about 1% (w / v). In embodiments, the polysorbate is at a concentration of 0.02% (w / v). In embodiments, the cryoprotected formulation or pharmaceutical composition has an ionic strength of about 100 mM to about 700 mM. In embodiments, the cryoprotected formulation or pharmaceutical composition has an ionic strength of about 200 mM to about 600 mM. In embodiments, the cryoprotected formulation or pharmaceutical composition has an ionic strength of about 300 mM to about 500 mM. In embodiments, the cryoprotected formulation or pharmaceutical composition has an ionic strength of about 300 mM. In embodiments, the cryoprotectant formulation or pharmaceutical composition has an ionic strength of about 500 mM.

[0021] In embodiments, the cryoprotected formulation or pharmaceutical composition has an osmolality of about 100 mOsm / kg to about 800 mOsm / kg. In embodiments, the cryoprotected formulation or pharmaceutical composition has an osmolality of about 200 mOsm / kg to about 600 mOsm / kg. In embodiments, the cryoprotected formulation or pharmaceutical composition has an osmolality of less than about 400 mOsm / kg. In embodiments, the cryoprotected formulation or pharmaceutical composition has an osmolality of about 200 mOsm / kg. In embodiments, the cryoprotected formulation or pharmaceutical composition has an osmolality of about 350 mOsm / kg.

[0022] In embodiments, the cryoprotected formulation or pharmaceutical composition has a pH of about 7.0 to about 8.0, In embodiments, the cryoprotected formulation or pharmaceutical composition has a pH of about 7.5.

[0023] In embodiments, the cryoprotective formulation or pharmaceutical composition comprises (1) about 0.1% (w / v) to about 20% (w / v) cyclodextrin, (2) about 100 mM to about 400 mM sodium chloride, (3) about 1 mM to about 20 mM sodium phosphate, and (4) 0.001% (w / v) to about 1% (w / v) polysorbate. In embodiments, the cryoprotective formulation or pharmaceutical composition comprises (1) about 0.4% (w / v) cyclodextrin, (2) about 280 mM sodium chloride, (3) about 10 mM sodium phosphate, and (4) about 0.02% (w / v) polysorbate 80. In embodiments, the cryoprotective formulation or pharmaceutical composition comprises (1) about 1% (w / v) to about 20% (w / v) trehalose, (2) about 10 mM to about 250 mM magnesium sulfate, (3) about 1 mM to about 20 mM Tris, and (4) 0.001% (w / v) to about 1% (w / v) polysorbate. In embodiments, the cryoprotective formulation or pharmaceutical composition comprises (1) about 1.1% (w / v) trehalose, (2) about 125 mM magnesium sulfate, (3) about 10 mM Tris, and (4) about 0.02% (w / v) polysorbate 80.

[0024] In embodiments, the cryoprotective formulation or pharmaceutical composition further comprises one or more of a pharmaceutical agent, a vehicle, a protein, or a combination thereof.

[0025] In embodiments, the AAV particles are derived from an AAV serotype selected from serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, DJ, or DJ / 8. In embodiments, the AAV particles comprise a genome derived from AAV serotype 2. In embodiments, the AAV particles comprise a capsid derived from AAV serotype 2.

[0026] In one aspect, a method for preserving or maintaining the viability and / or infectivity of AAV particles at various temperatures is provided, the method comprising attaching AAV particles to a cryoprotective formulation disclosed herein or to a pharmaceutical composition disclosed herein. In embodiments, the AAV particles are derived from an AAV serotype selected from serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, DJ, or DJ / 8. In embodiments, the AAV particles comprise a genome derived from AAV serotype 2. In embodiments, the AAV particles comprise a capsid derived from AAV serotype 2. In embodiments, the AAV particles are stable over multiple lyophilization cycles. In embodiments, the AAV particles are stable over at least five lyophilization cycles. In embodiments, the AAV particles are stable at temperatures from about +20°C to about -80°C. In embodiments, the AAV particles are stable at temperatures of about +4°C. In embodiments, the AAV particles are stable at +4°C for at least 6 months. [Brief explanation of the drawings]

[0027] [Figure 1] Figure 1 shows the change in osmolality with increasing cyclodextrin concentration. (A) Represents a buffer with an ionic strength of 200 mM. (B) Represents a buffer with an ionic strength of 300 mM. A line was fitted through the three data points to generate an equation. Osmolality was measured with an osmometer, n=1. [Figure 2] 1 is a series of graphs showing the change in osmolality with increasing concentrations of trehalose. (A) Represents a buffer with an ionic strength of 200 mM. (B) Represents a buffer with an ionic strength of 350 mM. (C) Represents a buffer with an ionic strength of 500 mM. A line was fitted through the three data points to generate an equation. Osmolality was measured with an osmometer, n=1. [Figure 3] This is a series of graphs showing the stability of AAV2 over time in various buffers and storage conditions from Formulation A. Changes in VP (viral particle) titer, VG (vector genome) titer, monomeric region, and high molecular weight species (HMW) were monitored. Samples were thawed simultaneously at room temperature for at least 1 hour. Sample analysis consisted of the following techniques or instruments: Gyrolab for VP, qPCR for VG titer, and HPLC-SEC (high-performance liquid chromatography-size exclusion chromatography) for both monomeric region and HMW content. Replicate-to-replicate variation for VP titer was less than 5%, while replicate-to-replicate variation for VG titer was less than 20%. The starting AAV2 VG titer was 1.2 x 10 VG / mL for all buffers. Graphs include confidence regions for fitted lines showing the expected trend of the data over time. See Table 1 for buffer compositions belonging to Formulation A. [Figure 4] A series of graphs showing the relative standard deviation (RSD) of total VP and HMW data for two formulations are shown. (A) VP and HMW data for Buffer 3 for Formulation A (see Table 1). (B) VP and HMW data for Buffer 7 for Formulation B (see Table 2). RSD was calculated as a percentage using all storage conditions. [Figure 5]Figure 1 shows plots of AAV2 stability in two selected buffers with 10-fold lower product concentrations. (A) Data for Buffer 3 is shown for Formulation 3. (B) Data for Buffer 7 is shown for Formulation B (see Tables 1 and 2). Changes in VP titer, monomeric range, and high molecular weight species (HMW) for the two formulations were monitored. Samples were thawed simultaneously at room temperature for at least 1 hour. Analyses performed consisted of the following techniques or instruments: Gyrolab for VP titer and HPLC-SEC for both monomeric range and HMW content. Replicate variation for VP titer was below 5%. The starting AAV2 VG titer was 1.0 x 10 VG / mL for all buffers. The graph includes confidence regions for fitted lines showing the expected trend of the data over time. At -80°C, the resulting monomeric range for Formulation A-Buffer 3 at the sixth freeze / thaw cycle is 1802 mAU. [Figure 6] Figure 1 shows a summary of early stability data using relative standard deviation. RSD was calculated as a percentage using all storage conditions. Formulation A-3 is Buffer 3 for Formulation A, and Formulation B-7 is Buffer 7 for Formulation B. [Figure 7] Figure 1 shows the ratio of infectious particles to vector genomes before and after exposure to various storage conditions. The control was a sample frozen at -80°C on the day of production and thawed only once. Error bars represent ±1SD, generated by broadening the error of the VG titer assay and the infectious titer assay. DETAILED DESCRIPTION OF THE INVENTION

[0028] It is shown herein that the appropriate selection of excipients and balance of ionic strength and osmolality results in reproducible and stable product quality of viruses, such as AAV particles, at, for example, room temperature or 4° C., and / or confers the ability to withstand at least 10 freeze / thaw events. The embodiments provided herein reduce reliance on the −80° C. cold chain, thus enabling alternative product storage options that can be implemented to improve manufacturability and distribution, as well as increase product accessibility.

[0029] Adeno-associated viruses (AAVs) are small, single-stranded DNA viruses that require a helper virus to promote efficient replication. The 4.7-kb genome of AAV is characterized by two inverted terminal repeats (ITRs) and two open reading frames encoding the Rep and Cap proteins, respectively. The rep reading frame encodes four proteins with molecular weights of 78 kD, 68 kD, 52 kD, and 40 kD. These proteins primarily function in AAV replication and rescue, as well as in regulating AAV integration into host cell chromosomes. The cap reading frame encodes three structural proteins with molecular weights of 85 kD (VP1), 72 kD (VP2), and 61 kD (VP3), which form the virion capsid. VP3 accounts for more than 80% of the total protein in AAV virions. Adjacent to the 5' and 3' ends of the rep and cap open reading frames are inverted terminal repeats (ITRs) approximately 145 bp long. The two ITRs are the only cis elements essential for AAV replication, rescue, packaging, and integration of the AAV genome. The entire rep and cap domains can be excised and replaced with therapeutic or reporter transgenes.

[0030] The AAV particles described herein are not limited to a particular serotype, and any AAV serotype, as well as AAV variants, are suitable for the pharmaceutical compositions described herein.

[0031] In some embodiments, the AAV is selected from the group consisting of AAV1 (i.e., an AAV containing AAV1 ITRs and AAV1 capsid proteins), AAV2 (i.e., an AAV containing AAV2 ITRs and AAV2 capsid proteins), AAV3 (i.e., an AAV containing AAV3 ITRs and AAV3 capsid proteins), AAV4 (i.e., an AAV containing AAV4 ITRs and AAV4 capsid proteins), AAV5 (i.e., an AAV containing AAV5 ITRs and AAV5 capsid proteins), AAV6 (i.e., an AAV containing AAV6 ITRs and AAV6 capsid proteins), AAV7 (i.e., an AAV containing AAV7 ITRs and AAV7 capsid proteins), AAV8 (i.e., an AAV containing AAV8 ITRs and AAV8 capsid proteins), AAV9 (i.e., an AAV containing AAV9 ITRs and AAV9 capsid proteins), AAVrh74 (i.e., an AAVrh74 ITRs and AAVrh74 capsid protein), AAVrh.8 (i.e., an AAV containing AAVrh.8 ITRs and AAVrh.8 capsid protein), or AAVrh.10 (i.e., an AAV containing AAVrh.10 ITRs and AAVrh.10 capsid protein).

[0032] In some embodiments, the AAV is pseudotyped, comprising ITRs from one AAV serotype and capsid proteins from a different AAV serotype. In some embodiments, the pseudotyped AAV is AAV2 / 9 (i.e., an AAV containing AAV2 ITRs and AAV9 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2 / 10 (i.e., an AAV containing AAV2 ITRs and AAV10 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2 / 7m8 (i.e., an AAV containing AAV2 ITRs and AAV 7m8 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2 / 8 (i.e., an AAV containing AAV2 ITRs and AAV capsid proteins). In some embodiments, the pseudotyped AAV is AAV2 / 1 (i.e., an AAV containing AAV2 ITRs and AAV1 capsid proteins).

[0033] In some embodiments, the AAV contains a recombinant capsid protein, such as a capsid protein containing a chimera of one or more capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh74, AAVrh.8, or AAVrh.10. In embodiments, the capsid is a variant AAV capsid, such as the AAV2 variant rAAV2-retro (SEQ ID NO: 44 from WO2017 / 218842, incorporated herein by reference).

[0034] In some embodiments, the AAV contains two or more capsid proteins selected from different serotypes, hi some embodiments, the AAV contains rAAV2 retro and AAVrh.10 capsid proteins.

[0035] In embodiments, the AAV genome and / or AAV capsid is selected from serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, DJ, or DJ / 8.

[0036] In certain embodiments, the AAV particles comprise a genome derived from AAV serotype 2. In certain embodiments, the AAV particles comprise a capsid derived from AAV serotype 2.

[0037] The described AAV particles can be used in any downstream application compatible with AAV particles. For example, in some embodiments, the AAV particles are suitable for use in gene therapy. In some embodiments, the purified AAV particles contain a therapeutic gene. However, it is understood that AAV particles may also be useful as vaccines. Furthermore, downstream applications can extend beyond therapeutic uses. For example, the AAV particles described herein may also be useful for imaging applications or other non-therapeutic uses. In some embodiments, the AAV particles contain encapsidated recombinant vector sequences. In some embodiments, it may be desirable to have AAV particles comprising empty capsids; AAV particles comprising a mixture of complete and empty capsids; AAV particles comprising a mixture of complete, partially complete, and empty capsids; or AAV particles comprising a mixture of partially complete and empty capsids. In some embodiments, the formulations and pharmaceutical compositions disclosed herein comprise AAV particles having empty capsids. In some embodiments, the formulations and pharmaceutical compositions disclosed herein comprise AAV particles having complete capsids, partially complete full capsids, or a mixture of complete and empty capsids. In some embodiments, the formulations and pharmaceutical compositions disclosed herein comprise AAV particles having a mixture of full capsids, partially full capsids, and empty capsids. In some embodiments, the AAV particles have a mixture of partially complete and empty capsids.

[0038] Recombinant AAV—"rAAV"—includes any AAV derived from any adeno-associated virus serotype. rAAV can have one or more of the AAV wild-type genes, preferably the rep and / or cap genes, deleted in whole or in part, but retain functional flanking ITR sequences.

[0039] As used herein, a "vector" is a vehicle containing a polynucleotide that can be delivered to a host cell either in vitro or in vivo. The term includes, by way of example, plasmids, expression vectors, viral vectors, and viruses. In embodiments, a vector refers to a virion containing a recombinant viral genome (e.g., rAAV), where the viral genome includes one or more ITRs and a transgene.

[0040] As used herein, the terms "AAV particle," "rAAV particle," "virus particle," and "AAV virion," unless otherwise specified, are intended to mean genome-containing (also known as "full capsid" or "partially full capsid") as well as empty capsids, or any combination of full, partially full, and empty capsids.

[0041] "Empty capsid" and "empty particle" refer to an AAV particle (e.g., an rAAV particle) that has an AAV capsid but lacks all or part of the AAV genome (e.g., a recombinant AAV genome containing a transgene sequence and one or two ITRs). Such empty capsids are incapable of transferring a transgene into one or more target cells.

[0042] As used herein, the term "titer" is intended to mean the number of viruses in a given volume. Viral titer can include "physical titer" or "functional titer." Physical titer is a measure of how much virus is present and is generally expressed as the number of virus particles per mL (VP / mL) or the number of vector genomes per mL (VG / mL), which is interchangeable with genome copies per mL (GC / mL). Functional titer, or infectious titer, is a measure of how much virus actually infects target cells and is generally expressed in the form of transducing units per mL (TU / mL) or, for adenovirus, plaque-forming units per mL (pfu / mL) or infectious units per mL (ifu / mL). Functional titers are generally understood to be about 10 to about 100 times smaller than physical titers.

[0043] In various embodiments, the virus is a chimeric virus, a synthetic virus, a recombinant virus, a mosaic virus, or a pseudotyped virus.

[0044] As used herein, the terms "freeze-thawing cycles," "freeze / drying cycles," or "freeze-thaw cycles" refer to when the temperature changes from above freezing to below freezing and then back above freezing.

[0045] Ranges: Throughout this disclosure, various aspects of the present disclosure can be expressed in range format. The description of numerical values ​​in range format should be understood merely for convenience and simplicity and should not be construed as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be construed as including all the specifically disclosed possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be construed as including the specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. The recitation of numerical ranges by endpoints includes all fractions and subfractions contained within that range (e.g., a recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, etc.), and includes all numbers, e.g., whole integers, as well as any range within that range.

[0046] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on the method for measuring or determining the value, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more standard deviations, as is customary in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value or range. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and preferably within 2-fold, of a value. Unless otherwise stated, when a particular value is recited in this application and claims, the term "about" is deemed to mean within an acceptable error range for the particular value. All numerical values ​​are intended to be modified herein by the term "about," whether explicitly stated or not. The recitation of numerical ranges by endpoints includes all numbers within that range (e.g., 1 to 5 includes 1.5, 2, 2.75, 3, 3.80, 4, and 5).

[0047] Any formulation, pharmaceutical composition or method provided herein can be combined with one or more of any other formulations, pharmaceutical compositions and methods provided herein.

[0048] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limit of that range, to one-tenth of the unit of the lower limit, and any other stated or intervening value in that stated range, is encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed herein, subject to any specific excluded limits in the stated range. Where a stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the invention.

[0049] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. Furthermore, to the extent that "including," "includes," "having," "has," "involving," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising." The transitional phrase "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, the term "comprising" can be replaced with "containing" or "including," or, in some cases, with the term "having," as used herein. In contrast, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in a claim. The transitional phrase "consisting essentially of" limits the scope of a claim to the materials or steps specified and that do not materially affect the basic and novel characteristic(s) of the claimed disclosure.

[0050] As used in this specification and the appended claims, the term "or" is generally used in its sense including "and / or" unless the content clearly dictates otherwise.

[0051] As used herein, the term "and / or" means any one of the items, any combination of the items, or all of the items with which this term is associated.

[0052] Any and all examples provided within this specification, or the use of exemplary language (e.g., "etc."), are intended merely to better clarify the invention and do not impose limitations on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0053] cryoprotectants In certain embodiments, the formulations and pharmaceutical compositions disclosed herein comprise one or more cryoprotectants. As used herein, the terms "cryoprotectant" or "cryoprotection" refer to a chemical or chemical solution that facilitates the process of cryoprotection by reducing damage to the stored components (e.g., viral particles, viruses, cells, and / or tissues) during freezing and drying, or that reduces damage to the stored components at room temperature of about +20°C or about +4°C. A cryoprotective formulation or composition protects the stored components (i.e., AAV particles) from damage associated with storage and / or freezing at sub-zero temperatures, e.g., damage to the viral capsid or cell membrane due to ice crystal formation, or damage at room temperature of about +20°C or about +4°C. In some embodiments, multiple different cryoprotectants can be combined.

[0054] In some embodiments, the cryoprotectant is a sugar. In some embodiments, the cryoprotectant is a sugar.

[0055] In certain embodiments, the sugar comprises trehalose, cyclodextrin, sucrose, or a combination thereof. In certain embodiments, the sugar is trehalose. In certain embodiments, the sugar is cyclodextrin.

[0056] As used herein, the term "sugar" refers to any carbohydrate, including monosaccharides (e.g., glucose, ribose, fructose, galactose, etc.), disaccharides (e.g., sucrose, lactose, maltose, cellobiose, trehalose, dextrans such as dextran-40, melibiose, etc.), oligosaccharides (e.g., raffinose, stachyose, amylose, etc.), and polysaccharides (e.g., starch, glycogen, cellulose, chitin, xylan, arabinoxylan, mannan, fucoidan, galactomannan, callose, laminarin, chrysolaminarin, amylopectin, dextran, dextrin, maltodextrin, inulin, oligofructose, polydextrose, etc.). The term encompasses complex carbohydrates in addition to simple carbohydrates. Indeed, it is not intended that the present disclosure be limited to any particular sugar, as a variety of sugars and forms of sugars find use in the present disclosure.

[0057] In certain embodiments, the cryoprotective formulation for preserving, maintaining infectivity, and / or maintaining viability of AAV particles comprises sodium phosphate, sodium chloride, cyclodextrin, and polysorbate.

[0058] In certain aspects, the cryoprotective formulation for preserving, maintaining infectivity, and / or maintaining viability of AAV particles comprises a cryoprotective sugar, a buffer, a salt, and a non-ionic surfactant.

[0059] In certain embodiments, the cryoprotective formulation comprises a buffer composition shown in Table 1 or 2.

[0060] In certain embodiments, a formulation or pharmaceutical composition embodied herein comprises between about 0.001% w / v and about 30% w / v, between about 0.005% w / v and about 25% w / v, between about 0.01% w / v and about 24% w / v, between about 0.05% w / v and about 23% w / v, between about 0.1% w / v and about 22% w / v, between about 0.1% w / v and about 21% w / v, between 0.1% w / v and about 20% w / v, between about 0.5% w / v and about 19% w / v, between about 1% w / v and about 18% w / v, between about 1% w / v and about 17% w / v, or between about 1% w / v and about 16% w / v of sugar.

[0061] In certain embodiments, formulations or pharmaceutical compositions embodied herein contain about 0.001% w / v to about 30% w / v, about 0.005% w / v to about 25% w / v, about 0.01% w / v to about 24% w / v, about 0.05% w / v to about 23% w / v, or about 0.1% w / v to about 22% w / v of cyclodextrin. In certain embodiments, formulations or pharmaceutical compositions embodied herein contain about 0.375% w / v of cyclodextrin.

[0062] In certain embodiments, formulations or pharmaceutical compositions embodied herein contain between about 0.1% w / v and about 20% w / v, between about 0.5% w / v and about 19% w / v, between about 0.1% w / v and about 18% w / v, between about 1% w / v and about 17% w / v, or between about 1% w / v and about 16% w / v of trehalose. In certain embodiments, formulations or pharmaceutical compositions embodied herein contain about 1.1% w / v of trehalose.

[0063] Sugars and other cryoprotectants In some embodiments, a formulation or pharmaceutical composition embodied herein comprises one or more cryoprotectants, wherein the cryoprotectant is a solvent (e.g., an organic solvent), a polyol, a polymer, a sugar, or a combination thereof. In some embodiments, the cryoprotectant is DMSO (dimethyl sulfoxide), ethylene glycol, glycerol, propylene glycol, 2-methyl-2,4-pentanediol (MP), glycerol-3-phosphate, diethyl glycol, triethylene glycol, polyvinyl alcohol, PEG, hydroxyethyl starch, sorbitol, mannitol, lactose, sucrose, trehalose, or a combination thereof. In some embodiments, the cryoprotectant is an organic solvent. In some embodiments, the cryoprotectant is a polyol. In some embodiments, the cryoprotectant is a polymer. In some embodiments, the cryoprotectant is DMSO (dimethyl sulfoxide). In some embodiments, the cryoprotectant is ethylene glycol. In some embodiments, the cryoprotectant is glycerol. In some embodiments, the cryoprotectant is propylene glycol. In some embodiments, the cryoprotectant is (MPD)2-methyl-2,4-pentanediol. In some embodiments, the cryoprotectant is glycerol-3-phosphate. In some embodiments, the cryoprotectant is diethyl glycol. In some embodiments, the cryoprotectant is triethylene glycol. In some embodiments, the cryoprotectant is polyvinyl alcohol. In some embodiments, the cryoprotectant is PEG. In some embodiments, the cryoprotectant is hydroxyethyl starch. In some embodiments, the cryoprotectant is sorbitol. In some embodiments, the cryoprotectant is mannitol. In some embodiments, the cryoprotectant is lactose.

[0064] In embodiments, multiple (eg, 2, 3, 4, 5, etc.) cryoprotectants may be combined.

[0065] solvent In certain aspects, as described above, the formulation or pharmaceutical composition can include one or more solvents. Any solvent suitable for stably maintaining AAV particles can be incorporated into the composition according to the present disclosure. Some non-limiting examples include Dulbecco's Modified Eagle Medium (DMEM), Eagle's Minimum Essential Medium (EMEM), X-VIVO, water, saline, dextrose, and combinations thereof. In certain embodiments, the composition includes DMEM. In certain embodiments, the composition includes EMEM.

[0066] pharmaceutically acceptable salts The formulations and pharmaceutical compositions disclosed herein contain one or more pharmaceutically acceptable salts. As used herein, "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic and organic acids and bases. Suitable pharmaceutically acceptable base addition salts of the pharmaceutically acceptable compositions disclosed herein include, but are not limited to, metal salts prepared from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc, or organic salts prepared from lysine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methyl-glucamine), and procaine. Suitable non-toxic acids include, but are not limited to, inorganic and organic acids, such as acetic acid, alginic acid, anthranilic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethenesulfonic acid, formic acid, fumaric acid, furoic acid, galacturonic acid, gluconic acid, glucuronic acid, glutamic acid, glycolic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, propionic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, sulfuric acid, tartaric acid, and p-toluenesulfonic acid. Specific non-toxic acids include hydrochloric acid, hydrobromic acid, maleic acid, phosphoric acid, sulfuric acid, and methanesulfonic acid. Specific examples of salts include hydrochloride and mesylate.

[0067] In certain embodiments, pharmaceutically acceptable salts are metal salts and salts of ammonia or organic amines that are safe for administration to subjects (e.g., humans) in drug formulations. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium, potassium, magnesium, calcium, cesium, ammonium, triethylamine, guanidine and N-substituted guanidine salts, acetamidine and N-substituted acetamidine, pyridine, picoline, ethanolamine, triethanolamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine salts. Pharmaceutically acceptable salts (of the basic nitrogen center) include, but are not limited to, inorganic acid salts such as hydrochloride, hydrobromide, sulfate, and phosphate; organic acid salts such as trifluoroacetic acid and maleate; sulfonates such as methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, and naphthalenesulfonate; amino acid salts such as arginate, alanine, aspartate, and glutamate; and carbohydrate salts such as gluconate and galacturonate. Other salts that can be used herein are well known in the art, see, for example, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton Pa. (1990) or Remington: The Science and Practice of Pharmacy, 19th ed., Mack Publishing, Easton Pa. (1995), which are incorporated herein by reference in their entirety for all intents and purposes. In some embodiments, the pharmaceutically acceptable salt is a sodium salt, a magnesium salt, a calcium salt, a potassium salt, a phosphate salt, or a sulfate salt. In some embodiments, the pharmaceutically acceptable salt is a metal salt. In some embodiments, the pharmaceutically acceptable salt is a sodium salt. In some embodiments, the pharmaceutically acceptable salt is a magnesium salt. In some embodiments, the pharmaceutically acceptable salt is a calcium salt. In some embodiments, the pharmaceutically acceptable salt is a potassium salt.In some embodiments, the pharmaceutically acceptable salt is a phosphate salt. In some embodiments, the pharmaceutically acceptable salt is a sulfate salt. In certain embodiments, the sodium salt comprises sodium chloride, sodium phosphate, or a combination thereof. In some embodiments, the pharmaceutically acceptable salt is sodium chloride. In some embodiments, the pharmaceutically acceptable salt is sodium phosphate. In some embodiments, the magnesium salt is magnesium sulfate.

[0068] In certain embodiments, the salt comprises a sodium salt, a magnesium salt, a calcium salt, a potassium salt, a phosphate salt, a sulfate salt, a triethylamine salt, a guanidine salt, an N-substituted guanidine salt, an acetamidine salt, an N-substituted acetamidine salt, a pyridine salt, a picoline salt, an ethanolamine salt, a triethanolamine salt, a dicyclohexylamine salt, or an N,N'-dibutylethylenediamine salt, or a combination thereof. In certain embodiments, the sodium salt comprises sodium chloride, sodium phosphate, or a combination thereof. In certain embodiments, the magnesium salt is magnesium sulfate. In certain embodiments, the salt comprises sodium chloride, sodium phosphate, magnesium sulfate, or a combination thereof.

[0069] In some embodiments, the pharmaceutical composition comprises about 1 mM to about 300 mM of a pharmaceutically acceptable salt. In some embodiments, the pharmaceutically acceptable salt is about 2 mM to about 295 mM. In some embodiments, the pharmaceutically acceptable salt is about 3 mM to about 290 mM. In some embodiments, the pharmaceutically acceptable salt is about 4 mM to about 285 mM. In some embodiments, the pharmaceutically acceptable salt is about 6 mM to about 284 mM. In some embodiments, the pharmaceutically acceptable salt is about 7 mM to about 283 mM. In some embodiments, the pharmaceutically acceptable salt is about 8 mM to about 282 mM. In some embodiments, the pharmaceutically acceptable salt is about 9 mM to about 281 mM. In some embodiments, the pharmaceutically acceptable salt is about 10 mM to about 280 mM.

[0070] In some embodiments, the pharmaceutically acceptable salt is sodium chloride. In certain embodiments, the concentration of sodium chloride is about 280 mM.

[0071] In some embodiments, the pharmaceutically acceptable salt is sodium phosphate. In some embodiments, the concentration of sodium phosphate is about 10 mM.

[0072] In certain embodiments, the formulation or pharmaceutical composition comprises sodium phosphate at a concentration of about 10 mM and sodium chloride at a concentration of about 280 mM.

[0073] In certain embodiments, the pharmaceutically acceptable salt is magnesium sulfate. In certain embodiments, the magnesium sulfate is at a concentration of about 30 mM to about 200 mM, about 35 mM to about 190 mM, about 40 mM to about 180 mM, about 40 mM to about 170 mM, about 45 mM to about 160 mM, about 46 mM to about 150 mM, about 47 mM to about 140 mM, about 48 mM to about 130 mM, about 49 mM to about 128 mM, or about 50 mM to about 125 mM. In certain embodiments, the magnesium sulfate is at a concentration of about 50 mM.

[0074] In some aspects of the disclosure, the formulations and pharmaceutical compositions comprise pharmaceutically acceptable salts, buffers, cryoprotectants, and non-ionic surfactants.

[0075] In certain embodiments, formulations or pharmaceutical compositions may further comprise one or more pharmaceutically acceptable excipients, or be diluted in pharmaceutically acceptable excipients to obtain the desired ratio of agents in compositions or formulations.As used herein, pharmaceutically acceptable excipients include any pharmaceutically acceptable excipients that may be or include solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, etc., as appropriate for the specific formulation desired.Remington's The Science and Practice of Pharmacy, 21st Edition, AR Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006, incorporated herein by reference) discloses various excipients used in the formulation of pharmaceutical compositions, and these excipients are useful in the preparation of this composition. The use of any conventional excipient is contemplated within the scope of the present disclosure, except insofar as it is incompatible with the substance or its derivatives, such as by producing any undesirable biological effects or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition. In certain embodiments, a pharmaceutically acceptable excipient is at least 95%, 96%, 97%, 98%, 99%, or 100% pure. In some embodiments, the excipient is approved for use in human and veterinary medicine. In some embodiments, the excipient is approved for human use by the U.S. Food and Drug Administration (FDA). In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0076] In certain embodiments, formulations or pharmaceutical compositions may further comprise one or more pharmaceutically acceptable excipients, or be diluted in pharmaceutically acceptable excipients to obtain the desired ratio of agents in compositions or formulations.As used herein, pharmaceutically acceptable excipients include any pharmaceutically acceptable excipients that may be or include solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, etc., as appropriate for the specific formulation desired.Remington's The Science and Practice of Pharmacy, 21st Edition, AR Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006, incorporated herein by reference) discloses various excipients used in the formulation of pharmaceutical compositions, and these excipients are useful in the preparation of this composition. The use of any conventional excipient is contemplated within the scope of the present disclosure, except insofar as it is incompatible with the substance or its derivatives, such as by producing any undesirable biological effects or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition. In certain embodiments, a pharmaceutically acceptable excipient is at least 95%, 96%, 97%, 98%, 99%, or 100% pure. In some embodiments, the excipient is approved for use in human and veterinary medicine. In some embodiments, the excipient is approved for human use by the U.S. Food and Drug Administration (FDA). In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0077] Nonionic surfactants In certain embodiments, the formulations or pharmaceutical compositions disclosed herein contain one or more nonionic surfactants. As used herein, the term "nonionic surfactant" refers to a surfactant composed of an uncharged polar head group. In some embodiments, the nonionic surfactant is a copolymer. In some embodiments, the nonionic surfactant is a poloxamer. Poloxamers are nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene poly(propylene oxide) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). Poloxamers are also known under the trade name Pluronic®. Because the length of the polymer blocks can be customized, many different poloxamers exist, each with slightly different properties. With respect to the general term "poloxamer," these copolymers are generally named with the letter "P" (for poloxamer) followed by three digits, where the first two digits x 100 indicate the approximate molecular weight of the polyoxypropylene core and the last digit x 10 indicates the percentage polyoxyethylene content (e.g., P407 is a poloxamer with a polyoxypropylene molecular weight of 4,000 g / mol and a polyoxyethylene content of 70%). In some embodiments, the poloxamer is P188, P237, P338, or P407. In some embodiments, the poloxamer is P188. In some embodiments, the poloxamer is P237. In some embodiments, the poloxamer is P338. In some embodiments, the poloxamer is P407.In some embodiments, the nonionic surfactant is a polyoxyethylene sorbitan ester surfactant (commonly known as Tween), such as PS-20 and PS-80; a copolymer of ethylene oxide (EO), a phospholipid, such as phosphatidylcholine (lecithin); a polyoxyethylene fatty acid ether derived from lauryl, cetyl, stearyl, and oleyl alcohol (known as Brij surfactants), such as triethylene glycol monolauryl ether (Brij 30) or polyoxyethylene (23) lauryl ether (Brij™ 35); and a sorbitan ester (commonly known as SPAN), such as sorbitan trioleate (Span™ 85) and sorbitan monolaurate. In some embodiments, the nonionic surfactant is polysorbate 20 (PS-20), polysorbate 80 (PS-80), or a Brij surfactant, or a combination thereof. In some embodiments, the nonionic surfactant is a polysorbate. In some embodiments, the polysorbate is PS-20. In some embodiments, the polysorbate is PS-40. In some embodiments, the polysorbate is PS-60. In some embodiments, the polysorbate is PS-80. In some embodiments, the nonionic surfactant is a Brij surfactant. In some embodiments, the nonionic surfactant is an EO copolymer. In some embodiments, the nonionic surfactant is a phospholipid. In some embodiments, the nonionic surfactant is a phosphatidylcholine (lecithin). In some embodiments, the nonionic surfactant is a polyoxyethylene fatty acid ether derived from lauryl alcohol. In some embodiments, the nonionic surfactant is a polyoxyethylene fatty acid ether derived from cetyl alcohol. In some embodiments, the nonionic surfactant is a polyoxyethylene fatty acid ether derived from stearyl alcohol. In some embodiments, the nonionic surfactant is a polyoxyethylene fatty acid ether derived from oleyl alcohol.In some embodiments, the nonionic surfactant is triethylene glycol monolauryl ether (Brij™ 30). In some embodiments, the nonionic surfactant is polyoxyethylene (23) lauryl ether (Brij™ 35). In some embodiments, the nonionic surfactant is a sorbitan ester. In some embodiments, the nonionic surfactant is sorbitan trioleate (Span™ 85). In some embodiments, the nonionic surfactant is sorbitan monolaurate.

[0078] In some embodiments, the formulations or pharmaceutical compositions disclosed herein may be at a concentration of from about 0.0001% (w / v) to about 0.1% (w / v), from about 0.0005% (w / v) to about 0.005% (w / v), from about 0.00075% (w / v) to about 0.0025% (w / v), from about 0.0050% (w / v) to about 0.0075% (w / v), or from about 0. In some embodiments, the formulation or pharmaceutical composition disclosed herein comprises about 0.01% (w / v) to about 0.015% (w / v), about 0.0175% (w / v) to about 0.018% (w / v), about 0.019% (w / v) to about 0.02% (w / v), about 0.025% (w / v) to about 0.03% (w / v), or about 0.04% (w / v) to about 0.5% (w / v) of non-ionic surfactant(s). In some embodiments, the formulation or pharmaceutical composition disclosed herein comprises about 0.02% (w / v) of a non-ionic surfactant.

[0079] Ionic strength As provided herein, formulations or pharmaceutical compositions of the present disclosure can have an ionic strength of about 50 mM to about 800 mM. In certain embodiments, the ionic strength is about 75 mM to about 750 mM, about 100 mM to about 700 mM, about 150 mM to about 675 mM, about 175 mM to about 650 mM, about 180 mM to about 600 mM, about 190 mM to about 550 mM, or about 200 mM to about 500 mM. In certain embodiments, formulations or pharmaceutical compositions described herein have an ionic strength of about 300 mM or about 500 mM.

[0080] osmolality As provided herein, formulations or pharmaceutical compositions of the present disclosure can have an osmolality of about 100 mOsm / kg to about 800 mOsm / kg. In certain embodiments, the osmolality is about 150 mOsm / kg to about 750 mOsm / kg, about 175 mOsm / kg to about 700 mOsm / kg, about 180 mOsm / kg to about 675 mOsm / kg, about 185 mOsm / kg to about 650 mOsm / kg, or about 190 mOsm / kg to about 628 mOsm / kg. In certain embodiments, the osmolality is about 200 mOsm / kg, about 350 mOsm / kg, about 500 mOsm / kg, or about 600 mOsm / kg. In certain embodiments, the formulation or pharmaceutical composition of the present disclosure has an osmolality of about 200 mOsm / kg or about 350 mOsm / kg. In certain embodiments, the formulation or pharmaceutical composition of the present disclosure has an osmolality of about 200 mOsm / kg or about 600 mOsm / kg. In certain embodiments, the formulation or pharmaceutical composition of the present disclosure has an osmolality of less than about 400 mOsm / kg.

[0081] pH conditions As provided herein, the formulations or pharmaceutical compositions of the present disclosure can have a pH that is about 4.0 to about 9.0. In some embodiments, the pH of the formulation or pharmaceutical composition is about 7.0 to about 8.0. In certain embodiments, the pH of the formulation or pharmaceutical composition is about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 6.10, about 6.11, about 6.12, about 6.13, about 6.14, about 6.15, about 6.16, about 6.17, about 6.18, about 6.19 ...9, about 6.19, about 6.19, about 6.19, about 6.19, about 6.19, about 6.19, about 6.19, about 6.19, about 6.19, about 6.19, about 6.19, about 6.19, about 6.19, about 6.19, about In some embodiments, the pH of a formulation or pharmaceutical composition of the present disclosure is about 7.5.

[0082] Formulations and Pharmaceutical Compositions The formulation or pharmaceutical composition provided herein can be either a liquid composition or a frozen composition.Therefore, in some embodiments, the formulation or pharmaceutical composition is in a liquid state.In other embodiments, the formulation or pharmaceutical composition is in a solid or semi-solid state.

[0083] In certain embodiments, formulations for preserving, maintaining infectivity, and / or maintaining viability of AAV particles comprise a sugar, a buffer, a detergent, a salt, or a combination thereof, hi certain embodiments, pharmaceutical compositions for preserving, maintaining infectivity, and / or maintaining viability of AAV particles comprise a cryoprotectant, a buffer, a detergent, and a salt.

[0084] In certain embodiments, a composition for preserving, maintaining infectivity, and / or maintaining viability of AAV particles comprises about 1 mM to about 20 mM sodium phosphate, about 100 mM to about 400 mM sodium chloride, about 0.1% weight / volume (w / v) up to about 20% (w / v) cyclodextrin, and about 0.01% weight / volume (w / v) to about 5% (w / v) polysorbate.

[0085] In certain embodiments, a composition for preserving AAV particles, maintaining infectivity, and / or maintaining viability comprises about 1% weight / volume (w / v) to about 20% w / v trehalose, 0.001% w / v to about 1% w / v polysorbate, about 1 mM to about 20 mM Tris buffer, about 10 mM to about 250 mM magnesium sulfate, about 0.1% weight / volume (w / v) to up to about 20% (w / v) cyclodextrin, and about 0.01% weight / volume (w / v) to about 5% (w / v) polysorbate.

[0086] In certain embodiments, the pharmaceutical composition comprises a buffer composition shown in Table 1 or 2.

[0087] In certain embodiments, the formulation comprises a buffer composition as shown in Table 1 or 2.

[0088] In certain embodiments, the formulation comprises about 5 mM to about 15 mM sodium phosphate, about 150 mM to about 200 mM NaCl, about 1% to about 2% cyclodextrin, and about 0.01% to about 0.5% polysorbate 80, and the formulation comprises an ionic strength of about 180 mM to about 250 mM and an osmolality of about 300 mOsm / kg to about 400 mOsm / kg.

[0089] In certain embodiments, the formulation comprises about 10 mM sodium phosphate, about 180 mM NaCl, about 1.5% cyclodextrin, and about 0.1% polysorbate 80, wherein the formulation has an ionic strength of about 206 mM and an osmolality of about 367 mOsm / kg.

[0090] In certain embodiments, the formulation comprises about 10 mM sodium phosphate, about 180 mM NaCl, about 0.375% cyclodextrin, and about 0.02% polysorbate 80, wherein the formulation has an ionic strength of about 200 mM and an osmolality of about 350 mOsm / kg.

[0091] In certain embodiments, the formulation comprises about 10 mM sodium phosphate, about 180 mM NaCl, about 21.0% cyclodextrin, and about 0.02% polysorbate 80, wherein the formulation has an ionic strength of about 200 mM and an osmolality of about 600 mOsm / kg.

[0092] In certain embodiments, the formulation comprises about 10 mM sodium phosphate, about 280 mM NaCl, about 0.375% cyclodextrin, and about 0.02% polysorbate 80, wherein the formulation has an ionic strength of about 300 mM and an osmolality of about 350 mOsm / kg.

[0093] In certain embodiments, the formulation comprises about 10 mM sodium phosphate, about 280 mM NaCl, about 11.5% cyclodextrin, and about 0.02% polysorbate 80, wherein the formulation has an ionic strength of about 300 mM and an osmolality of about 600 mOsm / kg.

[0094] In certain embodiments, the formulation comprises about 5 mM to about 20 mM Tris, about 30 mM to about 80 mM MgSO, about 0.5% to about 3% trehalose, and about 0.01% to about 0.5% polysorbate 80, and the formulation comprises an ionic strength of about 180 mM to about 250 mM and an osmolality of about 100 mOsm / kg to about 200 mOsm / kg.

[0095] In certain embodiments, the formulation comprises about 5 mM to about 15 mM Tris, about 40 mM to about 70 mM MgSO, about 1% to about 2% trehalose, and about 0.05% to about 0.5% polysorbate 80, and the formulation comprises an ionic strength of about 200 mM to about 240 mM and an osmolality of about 110 mOsm / kg to about 140 mOsm / kg.

[0096] In certain embodiments, the formula comprises about 10 mM Tris, about 125 MgSO4, about 1.1% trehalose, and about 0.02% polysorbate 80, and the formulation comprises an ionic strength of about 500 mM and an osmolality of about 200 mOsm / kg.

[0097] In certain embodiments, the formulation comprises about 10 mM Tris, about 55 mM MgSO4, about 1.5% trehalose, and about 0.1% polysorbate 80, wherein the formulation has an ionic strength of about 220 mM and an osmolality of about 128 mOsm / kg.

[0098] Storage conditions In certain embodiments, a formulation or pharmaceutical composition disclosed herein is stored at ambient or room temperature, e.g., about 25°C. In some embodiments, a disclosed composition is stored below about 25°C. In some embodiments, a disclosed composition is stored between about 0°C and about 25°C. In some embodiments, a composition is stored between about 0°C and about 10°C. In some embodiments, a composition is stored between about 2°C and about 8°C. In some embodiments, a composition is stored at about 4°C. In some embodiments, a composition is stored below 0°C. In some embodiments, a composition is stored between about -20°C and about -80°C. In some embodiments, a composition is stored at about -20°C. In some embodiments, a composition is stored at about -70°C. In some embodiments, a composition is stored at about -80°C. In certain embodiments, a composition is stored below 0°C, and stability is maintained or improved after one or more freeze / thaw cycles. In certain embodiments, the composition is stored at about -20°C and stability is maintained or improved after one or more freeze / thaw cycles. In certain embodiments, the composition is stored at about -20°C and stability is maintained or improved after one or more freeze / thaw cycles. In certain embodiments, the composition is stored at about -70°C and stability is maintained or improved after one or more freeze / thaw cycles. In certain embodiments, the composition is stored at about -80°C and stability is maintained or improved after one or more freeze / thaw cycles. In some embodiments, stability is maintained after one freeze / thaw cycle. In some embodiments, stability is maintained after two or more freeze / thaw cycles. In some embodiments, stability is improved. In some embodiments, stability is improved after one freeze / thaw cycle. In some embodiments, stability is improved after two or more freeze / thaw cycles.

[0099] The formulations or pharmaceutical compositions disclosed herein can maintain or improve stability, or reduce or prevent aggregation, of AAV particles after one or more freeze / thaw cycles. In some embodiments, the disclosed compositions maintain AAV particle stability. In some embodiments, the disclosed compositions reduce aggregation after one freeze / thaw cycle. In some embodiments, the disclosed compositions reduce aggregation after two or more freeze / thaw cycles. In some embodiments, the disclosed compositions prevent aggregation after one freeze / thaw cycle. In some embodiments, the disclosed compositions prevent aggregation after two or more freeze / thaw cycles.

[0100] In some embodiments, stability is maintained or improved after two or more freeze / thaw cycles. In some embodiments, stability is maintained or improved after three or more freeze / thaw cycles. In some embodiments, stability is maintained or improved after four or more freeze / thaw cycles. In some embodiments, stability is maintained or improved after four or more freeze / thaw cycles. In some embodiments, stability is maintained or improved after five or more freeze / thaw cycles. In some embodiments, stability is maintained or improved after six or more freeze / thaw cycles. In some embodiments, stability is maintained or improved after seven or more freeze / thaw cycles. In some embodiments, stability is maintained or improved after eight or more freeze / thaw cycles. In some embodiments, stability is maintained or improved after nine or more freeze / thaw cycles. In some embodiments, stability is maintained or improved after ten or more freeze / thaw cycles.

[0101] In another aspect, embodied herein is a method for preserving or maintaining the viability of AAV particles, the method comprising attaching the AAV particles to a formulation or pharmaceutical composition. In certain embodiments, a formulation is utilized to preserve or maintain the viability of AAV particles for a period of time at temperatures between about 20°C and about -80°C. In certain embodiments, the AAV particles remain stable over multiple freeze / dry cycles. In certain embodiments, the AAV particles are stable over multiple lyophilization cycles. In certain embodiments, the AAV particles are stable over at least five multiple lyophilization cycles. In certain embodiments, the AAV particles are stable at temperatures between about 20°C and about -80°C. In certain embodiments, the AAV particles are stable at about 4°C. In certain embodiments, the AAV particles are stable for at least six months to at least one year.

[0102] In another aspect, embodied herein is a method for cryoprotecting a virus, the method comprising attaching AAV particles to a cryoprotective formulation or pharmaceutical composition. In certain embodiments, the formulation is utilized to preserve or maintain the viability of AAV particles for a period of time at temperatures between about 20°C and about -80°C. In certain embodiments, the AAV particles remain stable over multiple freeze / dry cycles. In certain embodiments, the AAV particles are stable over multiple lyophilization cycles. In certain embodiments, the AAV particles are stable over at least five multiple lyophilization cycles. In certain embodiments, the AAV particles are stable at temperatures between about 20°C and about -80°C. In certain embodiments, the AAV particles are stable at about 4°C.

[0103] Stability assay As provided herein, formulations or pharmaceutical compositions of the present disclosure can also prevent aggregation and / or improve stability of AAV particles. As used herein, a composition or formulation comprising AAV particles is stable if it exhibits less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% reduction in viral particle infectivity upon exposure to freeze / thaw cycles. As used herein, a composition or formulation comprising AAV particles is stable if it exhibits less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% reduction in viral particle infectivity when stored at a target storage temperature, e.g., for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months.

[0104] Various techniques for measuring physical properties (e.g., AAV particle size), viral titer, and / or purity of AAV particles are well known in the art. Exemplary assays are provided below, and in some cases, the assays can measure multiple properties. However, it should be understood that the application is not limited to the assays described below. As provided herein, the formulations and pharmaceutical compositions can prevent aggregation and / or improve the stability of AAV particles. Various techniques for measuring physical properties (e.g., AAV particle size), viral titer, and / or purity of AAV particles are well known in the art. Exemplary assays are provided below, and in some cases, the assays can measure multiple properties. However, it should be understood that the application is not limited to the assays described below.

[0105] In certain embodiments, AAV particle stability is measured by various assays, such as, for example, dynamic light scattering (DLS), analytical ultracentrifugation (AUC), light microscopy, size exclusion chromatography (SEC), transmission electron microscopy (TEM), field-flow fractionation with multi-angle static light scattering (FFF-MALS), infectivity, immunocytochemistry, and image analysis.

[0106] Various assays for assessing the identity of AAV particle preparations are well known in the art. In some embodiments, the identity of the AAV particles is derived from an AAV serotype selected from serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, DJ, or DJ / 8. In some embodiments, the identity of the AAV particles is AAV2. For example, assays analyzing viral protein expression can be useful in assessing the identity of AAV particles. Such assays include, but are not limited to, SDS-PAGE, mass spectrometry, immunoblotting, and ELISA. The appropriate number, molecular weight, and stoichiometry of viral proteins can be used to positively identify the presence of vectors as well as impurities. In addition, PCR or high-throughput NGS (next-generation genome sequencing) can be used to assess the vector genome and ensure positive identity.

[0107] AAV particles can also be characterized by their viral titer. Viral titer can include physical titer as well as functional titer. Physical titer is calculated by calculating the total number of live and dead viral particles present and expressed as the number of viral particles per mL (VP / mL) or, for AAV, as genome copies per mL (GC / mL). Various methods can be used to measure the physical titer of a virus based on quantifying the concentration of viral genomes or viral proteins. Suitable techniques include DNA hybridization, real-time PCR (including, but not limited to, quantitative PCR (qPCR) and digital drop PCR (dPCR)), optical density (A 260 / 280 ), Nanosight, and high performance liquid chromatography (HPLC).

[0108] For example, optical density (A 260 / 280 The HPLC assay measures the concentration of viral DNA and proteins. It is a physical assay that measures the concentration of viral particles (VP). HPLC is also a rapid method for quantifying total viral particles by separating intact viral particles from other cellular contaminants or viral particle fragments.

[0109] Functional titers measure how many viruses enter target cells and can be determined by examining the number of colony-forming units after antibiotic selection if the vector contains an antibiotic resistance gene, or by flow cytometry or immunofluorescence analysis of target cells if the vector contains a fluorescent protein. Alternatively, if the vector does not express a fluorescent protein, qPCR can be used to measure the number of integrated proviral DNA copies per cell, providing a fast and easy way to assess functional titers. [Example]

[0110] Example 1: Design and screening of formulations to improve the manufacturability and distribution of AAV gene therapy

[0111] Materials and Methods The AAV-AQP1 (Aquaporin-1) clarified lysate was separated by affinity chromatography and divided into two pots, which were then processed separately by ultrafiltration and diafiltration (UF / DF). The first aliquot was buffer-exchanged into Formulation A: 10 mM sodium phosphate, 180 mM NaCl, 1.5% cyclodextrin, 0.02% polysorbate 80 (pH 7.5), while the second aliquot was buffer-exchanged into Formulation B: 10 mM Tris, 50 mM MgSO, 1.5% trehalose, 0.02% polysorbate 80 (pH 7.5). 2The UF / DF step was performed using an AKTA™ Flux system (Cytiva, Marlborough, MA, USA) equipped with a 100 kDa mPES hollow fiber membrane (Repligen Corporation, Rancho Dominguez, CA, USA). The buffer-exchanged and concentrated material was then filtered using a 0.2 μm syringe filter. Five mL volumes of the processed material were used to generate different buffer combinations belonging to one of Formulation A (final buffer compositions were created based on equations generated from known buffer components). Target ionic strength and osmolality concentrations, as well as exact buffer components, can be found in Tables 1 and 2.

[0112] Formulation A (Table 1) or Formulation B (Table 2) made up a final volume of 20 mL. Stock solutions were prepared to accommodate a variety of buffer compositions. Target product concentrations ranged from 1 x 10 12 The samples were aliquoted and kept at various storage conditions, including room temperature at +20°C, a refrigerator at +4°C, and freezers at -20°C and -80°C over time (Table 3).

[0113] Buffer solution design Buffer ionic strength (I) was calculated as a function of the ions present in solution, i.e., the molar concentration (c) of each ion multiplied by the square of its valency (z) and divided by 2 (Equation 1).

number

[0114] Buffers with different osmolality were designed based on ionic strength and sugar content. To investigate the changes in osmolality, buffers containing a set ionic strength and various concentrations of cyclodextrin or trehalose were prepared (Figures 1A-1B and 2A-2C). Using three sugar concentrations, a line could be fitted to generate an equation that can be used to predict the amount of sugar needed to reach a desired osmolality.

[0115] The final buffer composition was made based on an equation generated from known buffer components. Target ionic strength and osmolality concentrations, as well as exact buffer components, can be found in Tables 1 and 2. [Table 1] [Table 2]

[0116] Retention Research Plan Samples were held at various temperatures for a series of time periods (Table 3) and thawed at room temperature for 1 hour before analysis. New virus was thawed each time as all analyses were completed on separate days. Upon completion of the sample's holding time at +20°C and +4°C, the samples were immediately placed in -80°C storage for analysis. Another series of samples was stored at -20°C and -80°C and thawed at room temperature for 1 hour to generate different freeze / thaw cycles. [Table 3]

[0117] Analytical assays Vector genome concentrations were measured by qPCR.

[0118] Viral particle concentrations were measured using the Gyrolab® AAVX Titer Kit (Gyros Protein Technologies).

[0119] Monomeric regions and HMW species were determined by HPLC-SEC (Agilent Technologies) using an Xbridge BEH450 SEC 3.5 μm 7.8×300 mm (Waters™) column.

[0120] After transduction of COS-7 cells with the AQP1 product, infectivity assays were performed by cell lysis, single-stranded DNA degradation, and end-point qPCR in a validated AQP1 VG titer assay.

[0121] All statistical analyses and model development were performed using JMP® (SAS Institute).

[0122] result Stability—Formulation A: AAV2 stability was assessed by measuring VP titer, VG titer, monomeric region, and the presence of high molecular weight species (HMW) before and after exposure to various storage conditions. Storage in a refrigerator for up to 4 weeks, rather than at room temperature for up to 1 week (168 hours), was generally found to have less of an effect on the changes in the parameters studied (Figure 3). Storage of the product in both -20°C and -80°C freezers was found to have a comparable effect on AAV2 stability. Buffer 3 resulted in the least amount of variability, with the smallest changes of 7% for VP titer, 15% for VG titer, 5% for monomeric region, and 22% for HMW among all conditions studied (Figure 3). Changes in HMW were greatest during storage at -80°C, while the remaining conditions resulted in less than 5% differences when using Buffer 3.

[0123] Data Summary Using Relative Standard Deviation (RSD): Data were summarized using RSD to ensure that the selected formulation provided maximum product stability in terms of VP titer and HMW content across all storage conditions. Increasing osmolality resulted in increased VP titer variability for both formulations, while increasing ionic strength had no effect (Figures 4A-4B). Specifically, osmolality below 400 mOsm / kg and 250 mOsm / kg was found to result in less than 7% variability in VP titer for Formulation A and Formulation B, respectively. In contrast, ionic strength was the key determinant of the shift to HMW species, with increasing ionic strength resulting in decreased HMW variability for Formulation B (Figure 4B). Surprisingly, the same trend was not observed for Formulation A, where increasing osmolality with ionic strength decreased the RSD for HMW and showed no significant effect. Overall, the maximum RSD% was found to be 20% for the VP titer and 10% for the HMW species for both formulations.

[0124] Example 2: Buffer Formulation

[0125] method The AAV-AQP1 clarified lysate processed in the affinity chromatography step was divided into two pots and processed separately during the ultrafiltration and diafiltration (UF / DF) step. The first aliquot was buffer-exchanged into Formulation A-Buffer 3: 10 mM sodium phosphate, 280 mM NaCl, 0.4% cyclodextrin, 0.02% polysorbate 80 (pH 7.5), while the second aliquot was buffer-exchanged into Formulation B-Buffer 7: 10 mM Tris, 125 mM MgSO, 1.1% trehalose, 0.02% polysorbate 80 (pH 7.5). 2The UF / DF step was performed using an Ambr® Crossflow system (both from Sartorius Stedim UK Limited, Surrey, UK) equipped with a 100 kDa Ambr® CF PESU filter. The buffer-exchanged and concentrated material was then filtered using a 0.2 μm syringe filter. The target product concentration was 1×10 11 The concentration was VG / mL. Samples were aliquoted and kept under various storage conditions (Table 4).

[0126] Retention Research Plan The retention study design was carried out similarly to that described in Example 1 with a sustained retention time at a temperature of +20°C (Table 4). [Table 4]

[0127] result Stability: Based on early results (Figures 4A-4B), buffer 3 from formulation A and buffer 7 from formulation B were selected for subsequent studies using a 10-fold lower product concentration. AAV2 stability was assessed by measuring VP titer, monomeric regions, and the presence of high molecular weight species before and after exposure to various storage conditions. Formulation A - buffer 3 generally has lower variability in the measured data than formulation B - buffer 7 (Figures 5A-5B).

[0128] Data Summary Using Relative Standard Deviation (RSD): Relative standard deviations were calculated for VP titer, monomer area, and HMW content at all storage conditions. Formulation B-Buffer 7 had a 4-fold higher RSD than Formulation A-Buffer 3 in all assays tested (Figure 6). Note in Figure 6 that the monomer area result for Formulation A-Buffer 3 at the sixth freeze / thaw cycle at -80°C of 1802 mAU did not follow any trend and was above the control value, making it an outlier and therefore excluded from the RSD calculation. The overall maximum RSD values ​​were less than 25% for Formulation B and less than 10% for Formulation A.

[0129] Infectivity: AAV2 infectivity was assessed for two formulations stored at room temperature of +20°C for 2 weeks and after 10 freeze-thaw (F / T) cycles in a -80°C freezer. The ratio of infectious VP to total vector genome (VG) was comparable across the formulation buffers and storage conditions studied (Figure 7).

[0130] Sodium phosphate and sodium chloride-based formulation buffers generally provided a more stable environment for AAV. A buffer ionic strength of 300–500 mM and a water osmolality of less than 400 mOsm / kg were found to be most beneficial for AAV2 stability, resulting in minimal variation in VP titer, VG titer, monomer region, and HMW. Using relative standard deviations was helpful in deconvolving complex data sets. Different virus concentrations may result in different stability.

[0131] From the foregoing description it will be apparent that variations and modifications can be made to the invention described herein to adapt it to various usages and conditions, and such embodiments also fall within the scope of the following claims.

[0132] All citations to sequences, patents, and publications in this specification are incorporated herein by reference to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference. The publications disclosed herein are provided solely for their invention prior to the filing date of this application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

Claims

1. A cryoprotective formulation for preserving, maintaining infectivity, and / or maintaining viability of adeno-associated virus (AAV) particles, the formulation comprising a sugar, a salt, a buffer, a surfactant, or a combination thereof.

2. A pharmaceutical composition for preserving, maintaining infectivity, and / or maintaining viability of AAV particles, comprising a sugar, a salt, a buffer, a surfactant, or a combination thereof.

3. 3. The cryoprotective formulation of claim 1, or the pharmaceutical composition of claim 2, wherein the sugar comprises one or more sugars.

4. 4. The cryoprotective formulation of claim 1 or 3, or the pharmaceutical composition of claim 2 or 3, wherein the one or more sugars comprise trehalose, cyclodextrin, sucrose, or a combination thereof.

5. 4. The cryoprotective formulation or pharmaceutical composition of claim 3, wherein the sugar is a cyclodextrin.

6. 6. The cryoprotective formulation or pharmaceutical composition of claim 5, wherein the cyclodextrin is at a concentration of about 0.1% weight / volume (w / v) up to about 20% (w / v).

7. 7. The cryoprotective formulation or pharmaceutical composition of claim 6, wherein the cyclodextrin is at a concentration of about 0.1% (w / v) up to about 1% (w / v).

8. 8. The cryoprotective formulation or pharmaceutical composition of claim 7, wherein the cyclodextrin is at a concentration of about 0.4% (w / v).

9. 4. The cryoprotective formulation or pharmaceutical composition of claim 3, wherein the sugar is trehalose.

10. 10. The cryoprotective formulation or pharmaceutical composition of claim 9, wherein the trehalose is at a concentration of about 0.5% (w / v) to about 20% (w / v).

11. 11. The cryoprotective formulation or pharmaceutical composition of claim 10, wherein the trehalose is at a concentration of about 0.5% (w / v) to about 5% (w / v).

12. 12. The cryoprotective formulation or pharmaceutical composition of claim 11, wherein the trehalose is at a concentration of about 1.1% (w / v).

13. 13. The cryoprotective formulation of any one of claims 1 or 3-12, or the pharmaceutical composition of claims 2-12, wherein the salt comprises a sodium salt, a magnesium salt, a calcium salt, a potassium salt, a phosphate salt, a sulfate salt, triethylamine, guanidine, an N-substituted guanidine salt, acetamidine, an N-substituted acetamidine, pyridine, picoline, ethanolamine, triethanolamine, dicyclohexylamine, or N,N'-dibutylethylenediamine salt, or a combination thereof.

14. 14. The cryoprotective formulation or pharmaceutical composition of claim 13, wherein the salt is a sodium salt, a magnesium salt, a calcium salt, a potassium salt, a phosphate salt, a sulfate salt, or a combination thereof.

15. 15. The cryoprotective formulation or pharmaceutical composition of claim 14, wherein the salt is a sodium salt.

16. 16. The cryoprotective formulation or pharmaceutical composition of claim 15, wherein the sodium salt is sodium chloride, sodium phosphate, or both.

17. 17. The cryoprotective formulation or pharmaceutical composition of claim 16, wherein the sodium chloride is at a concentration of about 100 mM to about 400 mM.

18. 18. The cryoprotective formulation or pharmaceutical composition of claim 17, wherein the sodium chloride is at a concentration of about 200 mM to about 350 mM.

19. 20. The cryoprotective formulation or pharmaceutical composition of claim 18, wherein the sodium chloride is at a concentration of about 280 mM.

20. 15. The cryoprotective formulation or pharmaceutical composition of claim 14, wherein the salt is a magnesium salt.

21. 21. The cryoprotective formulation or pharmaceutical composition of claim 20, wherein the magnesium salt is magnesium sulfate.

22. 22. The cryoprotective formulation or pharmaceutical composition of claim 21, wherein the magnesium sulfate is at a concentration of about 10 mM to about 250 mM.

23. 23. The cryoprotective formulation or pharmaceutical composition of claim 22, wherein the magnesium sulfate is at a concentration of about 50 mM to about 200 mM.

24. 24. The cryoprotective formulation or pharmaceutical composition of claim 23, wherein the magnesium sulfate is at a concentration of about 125 mM.

25. 25. The cryoprotective formulation of any one of claims 1 or 3-24, or the pharmaceutical composition of claims 2-24, wherein the buffer comprises phosphate buffered saline (PBS), sodium phosphate, citric acid, acetic acid, tromethamine, aspartic acid, glutamic acid, HEPES, Tris, bicine, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, lysine, arginine, succinate, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid), triethanolamine buffer, or a combination thereof.

26. 26. The cryoprotective formulation or pharmaceutical composition of claim 25, wherein the buffer comprises sodium phosphate.

27. 27. The cryoprotective formulation or pharmaceutical composition of claim 26, wherein the sodium phosphate is at a concentration of about 1 mM to about 20 mM.

28. 28. The cryoprotective formulation or pharmaceutical composition of claim 27, wherein the sodium phosphate is at a concentration of about 10 mM.

29. 26. The cryoprotective formulation or pharmaceutical composition of claim 25, wherein the buffer comprises Tris.

30. 30. The cryoprotective formulation or pharmaceutical composition of claim 29, wherein the Tris is at a concentration of about 1 mM to about 20 mM.

31. 31. The cryoprotective formulation or pharmaceutical composition of claim 30, wherein the Tris is at a concentration of about 10 mM.

32. 32. The cryoprotective formulation of claim 1, or any one of claims 3 to 31, or the pharmaceutical composition of claims 2 to 31, wherein the surfactant is a non-ionic surfactant.

33. 33. The cryoprotective formulation or pharmaceutical composition of claim 32, wherein the non-ionic surfactant comprises a polysorbate.

34. 34. The cryoprotective formulation or pharmaceutical composition of claim 33, wherein the polysorbate is polysorbate 80.

35. 35. The cryoprotective formulation or pharmaceutical composition of any one of claims 32 to 34, wherein the polysorbate is at a concentration of about 0.01% (w / v) to about 5% (w / v).

36. 36. The cryoprotective formulation or pharmaceutical composition of claim 35, wherein the polysorbate is at a concentration of 0.001% (w / v) to about 1% (w / v).

37. 37. The cryoprotective formulation or pharmaceutical composition of claim 36, wherein the polysorbate is at a concentration of 0.02% (w / v).

38. 38. The cryoprotected formulation of any one of claims 1 or 3 to 37, or the pharmaceutical composition of claims 2 to 37, wherein the cryoprotected formulation or pharmaceutical composition has an ionic strength of about 100 mM to about 700 mM.

39. 39. The cryoprotective formulation or pharmaceutical composition of claim 38, wherein the cryoprotective formulation or pharmaceutical composition has an ionic strength of about 200 mM to about 600 mM.

40. 40. The cryoprotective formulation or pharmaceutical composition of claim 39, wherein the cryoprotective formulation or pharmaceutical composition has an ionic strength of about 300 mM to about 500 mM.

41. 41. The cryoprotective formulation or pharmaceutical composition of claim 40, wherein the cryoprotective formulation or pharmaceutical composition has an ionic strength of about 300 mM.

42. 41. The cryoprotective formulation or pharmaceutical composition of claim 40, wherein the cryoprotective formulation or pharmaceutical composition has an ionic strength of about 500 mM.

43. 43. The cryoprotected formulation of any one of claims 1 or 3-42, or the pharmaceutical composition of claims 2-42, wherein the cryoprotected formulation or pharmaceutical composition has an osmolality of about 100 mOsm / kg to about 800 mOsm / kg.

44. 44. The cryoprotective formulation or pharmaceutical composition of claim 43, wherein the cryoprotective formulation or pharmaceutical composition has an osmolality of about 200 mOsm / kg to about 600 mOsm / kg.

45. 44. The cryoprotective formulation or pharmaceutical composition of claim 43, wherein the cryoprotective formulation or pharmaceutical composition has an osmolality of less than about 400 mOsm / kg.

46. 44. The cryoprotective formulation or pharmaceutical composition of claim 43, wherein the cryoprotective formulation or pharmaceutical composition has an osmolality of about 200 mOsm / kg.

47. 44. The cryoprotective formulation or pharmaceutical composition of claim 43, wherein the cryoprotective formulation or pharmaceutical composition has an osmolality of about 350 mOsm / kg.

48. 48. The cryoprotected formulation of any one of claims 1 or 3 to 47, or the pharmaceutical composition of claims 2 to 47, wherein the cryoprotected formulation or pharmaceutical composition has a pH of about 7.0 to about 8.

0.

49. 49. The cryoprotective formulation or pharmaceutical composition of claim 48, wherein the cryoprotective formulation or pharmaceutical composition has a pH of about 7.

5.

50. 50. The cryoprotective formulation of claim 1, or any one of claims 38-49, or the pharmaceutical composition of claim 2, or any one of claims 38-49, wherein the cryoprotective formulation or pharmaceutical composition comprises: (1) about 0.1% (w / v) to up to about 20% (w / v) cyclodextrin; (2) about 100 mM to about 400 mM sodium chloride; (3) about 1 mM to about 20 mM sodium phosphate; and (4) 0.001% (w / v) to about 1% (w / v) polysorbate.

51. 51. The cryoprotective formulation or pharmaceutical composition of claim 50, wherein the cryoprotective formulation or pharmaceutical composition comprises: (1) about 0.4% (w / v) cyclodextrin; (2) about 280 mM sodium chloride; (3) about 10 mM sodium phosphate; and (4) about 0.02% (w / v) polysorbate 80.

52. 50. The cryoprotective formulation of claim 1, or any one of claims 38-49, or the pharmaceutical composition of claim 2, or any one of claims 38-49, wherein the cryoprotective formulation or pharmaceutical composition comprises: (1) about 1% (w / v) to about 20% (w / v) trehalose, (2) about 10 mM to about 250 mM magnesium sulfate, (3) about 1 mM to about 20 mM Tris, and (4) 0.001% (w / v) to about 1% (w / v) polysorbate.

53. 53. The cryoprotective formulation or pharmaceutical composition of claim 52, wherein the cryoprotective formulation or pharmaceutical composition comprises: (1) about 1.1% (w / v) trehalose, (2) about 125 mM magnesium sulfate, (3) about 10 mM Tris, and (4) about 0.02% (w / v) polysorbate 80.

54. 54. The cryoprotective formulation of any one of claims 1 or 3-53, or the pharmaceutical composition of claims 2-53, wherein the cryoprotective formulation or pharmaceutical composition further comprises one or more of a pharmaceutical agent, a vehicle, a protein, or a combination thereof.

55. 55. The cryoprotected formulation of any one of claims 1 or 3-54, or the pharmaceutical composition of claims 2-54, wherein the AAV particles are derived from an AAV serotype selected from serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, DJ, or DJ / 8.

56. 55. The cryoprotected formulation of claim 1, or any one of claims 3 to 54, or the pharmaceutical composition of claims 2 to 54, wherein the AAV particles comprise a genome derived from AAV serotype 2.

57. 57. The cryoprotected formulation of any one of claims 1, 3-54, or 56, or the pharmaceutical composition of claims 2-54, or 56, wherein the AAV particles comprise capsids derived from AAV serotype 2.

58. 54. A method for preserving or maintaining the viability and / or infectivity of AAV particles at various temperatures, the method comprising attaching the AAV particles to a cryoprotected formulation of any one of claims 1 or 3 to 54, or to a pharmaceutical composition of claims 2 to 54.

59. 59. The method of claim 58, wherein the AAV particles are derived from an AAV serotype selected from serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, DJ, or DJ / 8.

60. 59. The method of claim 58, wherein the AAV particles comprise a genome derived from AAV serotype 2.

61. 61. The method of claim 58 or 60, wherein the AAV particles comprise capsids derived from AAV serotype 2.

62. 62. The method of any one of claims 58-61, wherein the AAV particles are stable over multiple lyophilization cycles.

63. 62. The method of any one of claims 58-61, wherein the AAV particles are stable for at least five lyophilization cycles.

64. 64. The method of any one of claims 58 to 63, wherein the AAV particles are stable at temperatures from about +20°C to about -80°C.

65. 64. The method of any one of claims 58 to 63, wherein the AAV particles are stable at a temperature of about +4°C.

66. 66. The method of claim 65, wherein the AAV particles are stable at +4°C for at least 6 months.

67. 66. The method of claim 65, wherein the AAV particles are stable at +4°C for at least one year.

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