Formulations for AAV gene therapy

Abstract

A composition for storing and maintaining virus viability comprising a balance of ionic strength and osmolality moles, which results in reproducible and stable product quality at room temperature, at 4 DEG C, and has the ability to tolerate 10 freeze-thaw events.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of the earlier filing date of U.S. Provisional Patent Application No. 63 / 379,117, filed on October 11, 2022, under 35 U.S.C.§119(e), and the entire disclosure of the provisional application is hereby incorporated by reference in its entirety. Technical field

[0003] The present disclosure relates to formulations for storing adeno - associated virus (AAV) and maintaining the viability or infectivity of adeno - associated virus (AAV) after freeze - thaw cycles. Background art

[0004] AAV gene therapies in liquid formulations are rarely considered stable under refrigerated conditions and are typically stabilized by freezing at - 80°C. However, the freezing and thawing process can damage product quality, while the transportation and storage of frozen materials are generally complex and expensive. In addition, the use of freezing at - 80°C reduces the number of available drug delivery device options (e.g., pre - filled syringes), which may hinder product accessibility. Thus, there is an urgent need for new formulations for storing AAV gene therapy products and maintaining the viability / infectivity of AAV gene therapy products. Summary of the invention

[0005] Embodiments of the present disclosure relate to formulations that maintain AAV product quality over time at various storage temperatures (ranging from approximately ambient temperature up to and including - 80°C) and / or when exposed to multiple freeze - thaw conditions.

[0006] Accordingly, in some aspects, there is provided a formulation for AAV particles, wherein the formulation comprises a sugar, a buffer, a surfactant, a salt, or a combination thereof. In some embodiments, the sugar comprises one or more sugars. In some embodiments, the one or more sugars comprise trehalose, cyclodextrin, sucrose, or a combination thereof. In some embodiments, the salt comprises a sodium salt, a magnesium salt, a calcium salt, a potassium salt, a phosphate, a sulfate, triethylamine, guanidine, an N-substituted guanidine salt, acetamidine, an N-substituted acetamidine, pyridine, picoline, ethanolamine, triethanolamine, dicyclohexylamine, or an N,N'-dibenzylethylenediamine salt, or a combination thereof. In some embodiments, the sodium salt comprises sodium chloride, sodium phosphate, or a combination thereof. In some embodiments, the magnesium salt is magnesium sulfate. In some embodiments, the salt comprises sodium chloride, sodium phosphate, magnesium sulfate, or a combination thereof. In some embodiments, the formulation further comprises a buffer, which 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, and combinations thereof. In some embodiments, the surfactant is a non-ionic surfactant. In some embodiments, the non-ionic surfactant comprises polysorbate. In some embodiments, the polysorbate is polysorbate 80. In some embodiments, the formulation comprises an ionic strength of about 100 mM to about 700 mM. In some embodiments, the formulation comprises a weight osmolarity (mOsm / kg) of about 100 mOsm / kg to about 800 mOsm / kg. In some embodiments, the formulation comprises a pH of about 7.0 to about 8.0. In some embodiments, the formulation comprises a pH of about 7.5. In some embodiments, the formulation is for storing adeno-associated virus (AAV) particles or maintaining the viability of adeno-associated virus (AAV) particles over a period of time at a temperature of about 20°C to about -80°C. In some embodiments, the AAV particles are stable during multiple freeze-thaw cycles. In some embodiments, the AAV particles are derived from an AAV having an AAV serotype selected from serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, DJ, or DJ / 8. In some embodiments, the AAV particles comprise a genome derived from AAV serotype 2. In some embodiments, the AAV particles comprise a capsid derived from AAV serotype 2.

[0007] On the other hand, a pharmaceutical composition for storing AAV particles, maintaining the infectivity of AAV particles and / or maintaining the 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, a sulfate 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 nonionic surfactant. In certain embodiments, the nonionic surfactant comprises polysorbate. In certain embodiments, the polysorbate is polysorbate 80. In certain embodiments, the pharmaceutical composition has an ionic strength of about 100 mM to about 700 mM. In certain embodiments, the pharmaceutical composition has a weight osmolarity (mOsm / kg) of about 100 mOsm / kg to about 800 mOsm / kg. In certain embodiments, the pharmaceutical composition has a pH of about 7.0 to about 8.0. In certain embodiments, the pharmaceutical composition has a pH of about 7.5. In certain embodiments, the pharmaceutical composition is used to store AAV particles or maintain the viability of AAV particles at a temperature of about 20°C to about -80°C over a period of time. In certain embodiments, the AAV particles remain stable during multiple freeze-thaw cycles.

[0008] On the other hand, a cryoprotective formulation for storing AAV particles, maintaining the infectivity of AAV particles and / or maintaining the viability of AAV particles comprises sodium phosphate, sodium chloride, cyclodextrin and polysorbate. In certain embodiments, the cryoprotective formulation has an ionic strength of from about 100 mM to about 700 mM. In certain embodiments, the cryoprotective formulation has an ionic strength of from about 100 mM to about 400 mM. In certain embodiments, the cryoprotective formulation has a weight osmolarity (mOsm / kg) of from about 100 mOsm / kg to about 800 mOsm / kg. In certain embodiments, the cryoprotective formulation has a weight osmolarity (mOsm / kg) of from about 200 mOsm / kg to about 700 mOsm / kg. In certain embodiments, the concentration of the sodium phosphate is from about 1 mM to about 20 mM. In certain embodiments, the concentration of the sodium chloride is from about 100 mM to about 400 mM. In certain embodiments, the cyclodextrin comprises from about 0.1% weight / volume (w / v) to up to about 20% (w / v). In certain embodiments, the polysorbate comprises from about 0.01% weight / volume (w / v) to up to about 5% (w / v). In certain embodiments, the cryoprotective formulation is used to store AAV particles or maintain the viability of AAV particles at a temperature of from about 20 °C to about -80 °C over a period of time. In certain embodiments, the cryoprotective formulation has a pH of from about 7.0 to about 8.0. In certain embodiments, the AAV particles remain stable during multiple freeze-thaw cycles.

[0009] On the other hand, a cryoprotective formulation for storing AAV particles, maintaining the infectivity of AAV particles and / or maintaining the viability of AAV particles comprises a cryoprotective sugar, a buffer, a salt and a nonionic 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 concentration of the trehalose is from about 1% weight / volume (w / v) to about 20% w / v. In certain embodiments, the nonionic surfactant is polysorbate. In certain embodiments, the polysorbate is polysorbate 80. In certain embodiments, the concentration of the polysorbate 80 is from 0.001% w / v to about 1% w / v. In certain embodiments, the buffer is a tris buffer. In certain embodiments, the concentration of the tris buffer is from 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 concentration of the magnesium sulfate is from about 10 mM to about 250 mM. In certain embodiments, the cryoprotective formulation comprises an ionic strength of from about 100 mM to about 700 mM. In certain embodiments, the cryoprotective formulation comprises an ionic strength of from about 200 mM to about 500 mM. In certain embodiments, the cryoprotective formulation comprises a weight osmolarity (mOsm / kg) of from about 100 mOsm / kg to about 800 mOsm / kg. In certain embodiments, the cryoprotective formulation comprises a weight osmolarity (mOsm / kg) of from about 200 mOsm / kg to about 600 mOsm / kg. In certain embodiments, the cryoprotective formulation comprises a pH of from about 7.0 to about 8.0. In certain embodiments, the cryoprotective formulation further comprises one or more medicaments, cell culture medium, proteins, lipids or a combination thereof. In certain embodiments, the formulation is for storing AAV particles or maintaining the viability of AAV particles at a temperature of from about 20°C to about -80°C over a period of time. In certain embodiments, the AAV particles remain stable during multiple freeze-thaw cycles.

[0010] On the other hand, a composition for storing AAV particles, maintaining the infectivity of AAV particles and / or maintaining the viability of AAV particles comprises from about 1 mM to about 20 mM of sodium phosphate, from about 100 mM to about 400 mM of sodium chloride, from about 0.1% weight / volume (w / v) to up to about 30% (w / v) of cyclodextrin, and from about 0.01% weight / volume (w / v) to up to about 5% (w / v) of polysorbate. In certain embodiments, the composition is for storing AAV particles or maintaining the viability of AAV particles at a temperature of from about 20°C to about -80°C over a period of time. In certain embodiments, the AAV particles remain stable during multiple freeze-thaw cycles.

[0011] In another aspect, a composition for storing AAV particles, maintaining the infectivity of AAV particles and / or maintaining the viability of AAV particles comprises trehalose at about 1% weight / volume (w / v) to about 20% w / v, polysorbate at 0.001% w / v to about 1% w / v, tris buffer at about 1 mM to about 20 mM, magnesium sulfate at about 10 mM to about 250 mM, cyclodextrin at about 0.1% weight / volume (w / v) to up to about 30% (w / v), and polysorbate at about 0.01% weight / volume (w / v) to up to about 5% (w / v). In certain embodiments, the composition is used to store adeno-associated virus (AAV), recombinant AAV (rAAV) particles, adenovirus particles or other virus particles or maintain the viability of adeno-associated virus (AAV), recombinant AAV (rAAV) particles, adenovirus particles or other virus particles over a period of time at a temperature of about 20°C to about -80°C. In certain embodiments, the virus particles remain stable during multiple freeze-thaw cycles.

[0012] In another aspect, a formulation for storing AAV, rAAV particles, adenovirus particles or other virus particles or maintaining the viability of AAV, rAAV particles, adenovirus particles or other virus particles at ambient temperature or at a temperature of about 20°C to about -80°C comprises about 1 mM to about 20 mM sodium phosphate, about 80 mM to about 300 mM sodium chloride (NaCl), about 0.1% to about 10% cyclodextrin, 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.

[0013] In certain embodiments, the formulation is used to store AAV particles or maintain the viability of AAV particles over a period of time at a temperature of about 20°C to 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 six months to at least one year. In certain embodiments, the AAV particles are stable during multiple freeze-thaw cycles. In certain embodiments, the AAV particles are stable during at least 5 freeze-thaw cycles.

[0014] In certain embodiments, the formulation comprises the buffer compositions provided in Tables 1 and 2. In certain embodiments, the cryoprotective formulation comprises the buffer compositions provided in Table 1 or Table 2. In certain embodiments, the pharmaceutical composition comprises the buffer compositions provided in Table 1 or Table 2.

[0015] In one aspect, there is provided a cryoprotective formulation for storing adeno-associated virus (AAV) particles, maintaining the infectivity of adeno-associated virus (AAV) particles and / or maintaining the viability of adeno-associated virus (AAV) particles, the formulation comprising a sugar, a salt, a buffer, a surfactant or a combination thereof.

[0016] In one aspect, there is provided a pharmaceutical composition for storing AAV particles, maintaining the infectivity of AAV particles and / or maintaining the viability of AAV particles, the pharmaceutical composition comprising a sugar, a salt, a buffer, a surfactant or a combination thereof.

[0017] 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 concentration of the cyclodextrin is from about 0.1% weight / volume (w / v) up to about 20% (w / v). In embodiments, the concentration of the cyclodextrin is from about 0.1% (w / v) up to about 1% (w / v). In embodiments, the concentration of the cyclodextrin is about 0.4% (w / v).

[0018] In embodiments, the sugar is trehalose. In embodiments, the concentration of the trehalose is from about 0.5% (w / v) to about 20% (w / v). In embodiments, the concentration of the trehalose is from about 0.5% (w / v) to about 5% (w / v). In embodiments, the concentration of the trehalose is about 1.1% (w / v).

[0019] In embodiments, the salt comprises a sodium salt, a magnesium salt, a calcium salt, a potassium salt, a phosphate, a sulfate, triethylamine, guanidine, an N-substituted guanidine salt, acetamidine, an N-substituted acetamidine, pyridine, picoline, ethanolamine, triethanolamine, dicyclohexylamine or an N,N'-dibenzylethylenediamine salt or a combination thereof. In embodiments, the salt is a sodium salt, a magnesium salt, a calcium salt, a potassium salt, a phosphate, a sulfate 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 concentration of the sodium chloride is from about 100 mM to about 400 mM. In embodiments, the concentration of the sodium chloride is from about 200 mM to about 350 mM. In embodiments, the concentration of the sodium chloride is about 280 mM. In embodiments, the salt is a magnesium salt. In embodiments, the magnesium salt is magnesium sulfate. In embodiments, the concentration of the magnesium sulfate is from about 10 mM to about 250 mM. In embodiments, the concentration of the magnesium sulfate is from about 50 mM to about 200 mM. In embodiments, the concentration of the magnesium sulfate is about 125 mM.

[0020] In an embodiment, 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 an embodiment, the buffer comprises sodium phosphate. In an embodiment, the concentration of the sodium phosphate is from about 1 mM to about 20 mM. In an embodiment, the concentration of the sodium phosphate is about 10 mM. In an embodiment, the buffer comprises Tris. In an embodiment, the concentration of the Tris is from about 1 mM to about 20 mM. In an embodiment, the concentration of the Tris is about 10 mM.

[0021] In an embodiment, the surfactant is a nonionic surfactant. In an embodiment, the nonionic surfactant comprises polysorbate. In an embodiment, the polysorbate is polysorbate 80. In an embodiment, the concentration of the polysorbate is from about 0.01% (w / v) to about 5% (w / v). In an embodiment, the concentration of the polysorbate is from 0.001% (w / v) to about 1% (w / v). In an embodiment, the concentration of the polysorbate is 0.02% (w / v). In an embodiment, the cryoprotective formulation or pharmaceutical composition has an ionic strength of from about 100 mM to about 700 mM. In an embodiment, the cryoprotective formulation or pharmaceutical composition has an ionic strength of from about 200 mM to about 600 mM. In an embodiment, the cryoprotective formulation or pharmaceutical composition has an ionic strength of from about 300 mM to about 500 mM. In an embodiment, the cryoprotective formulation or pharmaceutical composition has an ionic strength of about 300 mM. In an embodiment, the cryoprotective formulation or pharmaceutical composition has an ionic strength of about 500 mM.

[0022] In embodiments, the cryoprotective formulation or pharmaceutical composition has a weight osmolarity of from about 100 mOsm / kg to about 800 mOsm / kg. In embodiments, the cryoprotective formulation or pharmaceutical composition has a weight osmolarity of from about 200 mOsm / kg to about 600 mOsm / kg. In embodiments, the cryoprotective formulation or pharmaceutical composition has a weight osmolarity of less than about 400 mOsm / kg. In embodiments, the cryoprotective formulation or pharmaceutical composition has a weight osmolarity of about 200 mOsm / kg. In embodiments, the cryoprotective formulation or pharmaceutical composition has a weight osmolarity of about 350 mOsm / kg.

[0023] In embodiments, the cryoprotective formulation or pharmaceutical composition has a pH of from about 7.0 to about 8.0. In embodiments, the cryoprotective formulation or pharmaceutical composition has a pH of about 7.5.

[0024] In embodiments, the cryoprotective formulation or pharmaceutical composition comprises (1) from about 0.1% (w / v) to up to about 20% (w / v) cyclodextrin, (2) from about 100 mM to about 400 mM sodium chloride, (3) from about 1 mM to about 20 mM sodium phosphate, and (4) from 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) from about 1% (w / v) to about 20% (w / v) trehalose, (2) from about 10 mM to about 250 mM magnesium sulfate, (3) from about 1 mM to about 20 mM Tris, and (4) from 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.

[0025] In embodiments, the cryoprotective formulation or the pharmaceutical composition further comprises one or more of the following: medicaments, culture media, proteins, or combinations thereof.

[0026] 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.

[0027] In one aspect, a method of storing AAV particles or maintaining the viability and / or infectivity of AAV particles at different temperatures is provided, the method comprising depositing the AAV particles into a cryoprotective formulation disclosed herein or into 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 in multiple freeze-thaw cycles. In embodiments, the AAV particles are stable in at least 5 freeze-thaw 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 a temperature of about +4°C. In embodiments, the AAV particles are stable at +4°C for at least six months. In embodiments, the AAV particles are stable at +4°C for at least one year. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figures 1A to 1B is a graph showing the change in weight osmolarity with increasing cyclodextrin concentration. Figure 1A represents a buffer with an ionic strength of 200 mM, Figure 1B represents a buffer with an ionic strength of 300 mM. A line is fitted through three data points and an equation is generated. Weight osmolarity is measured with an osmometer, n = 1.

[0029] Figures 2A to 2C is a series of graphs showing the change in weight osmolarity with increasing trehalose concentration. Figure 2A represents a buffer with an ionic strength of 200 mM. Figure 2B represents a buffer with an ionic strength of 350 mM. Figure 2C shows a buffer with an ionic strength of 500 mM. A line is fitted through three data points and an equation is generated. Weight osmolarity is measured with an osmometer, n = 1.

[0030] Figure 3is a series of graphs that show the stability of AAV2 over time in different buffers and storage conditions from Formulation A. Changes in VP (viral particle) titer, VG (vector genome) titer, monomer area, and high molecular weight material (HMW) are monitored. Samples are thawed simultaneously at room temperature for at least 1 hour. Sample analysis consists of the following techniques or instruments: Gyrolab for VP titer, qPCR for VG titer, and HPLC-SEC (high performance liquid chromatography - size exclusion chromatography) for both monomer area and HMW content. The variation between replicates for VP titer is less than 5%, while the variation between replicates for VG titer is less than 20%. The starting AAV2 VG titer for all buffers is 1.2×10 12 VG / mL. The graphs contain confidence bands of the fitted lines that illustrate the predicted trend of the data over time. The buffer compositions belonging to Formulation A are shown in Table 1.

[0031] Figures 4A to 4B is a series of graphs that show the relative standard deviation (RSD) of the total VP and HMW data for two formulations. Figure 4A Shows the VP and HMW data for Buffer 3 of Formulation A (see Table 1). Figure 4B Depicts the VP and HMW data for Buffer 7 of Formulation B (see Table 2). RSD values are calculated as a percentage using all storage conditions.

[0032] Figure 5 A to Figure 5 B are graphs that show the stability of AAV2 in two selected buffers at 10-fold lower product concentrations. Figure 5 A shows the data for Buffer 3 of Formulation A, and Figure 5 B shows the data for Buffer 7 of Formulation B (see Tables 1 and 2). Changes in VP titer, monomer area, and high molecular weight material (HMW) are monitored for these two formulations. Samples are thawed simultaneously at room temperature for at least 1 hour. The analysis performed consists of the following techniques or instruments: Gyrolab for VP titer and HPLC-SEC for both monomer area and HMW content. The variation between replicates for VP titer is less than 5%. The starting AAV2 VG titer for all buffers is 1.0×10 11 VG / mL. The graphs contain confidence bands of the fitted lines that illustrate the predicted trend of the data over time. The monomer area result for Formulation A - Buffer 3 in the 6th freeze-thaw cycle at -80°C is 1802 mAU.

[0033] Figure 6It is a chart showing the summary of the aforementioned stability data using relative standard deviation. The RSD values are calculated in percentage using all storage conditions. Formulation A-3 is Buffer 3 of Formulation A, and Formulation B-7 is Buffer 7 of Formulation B.

[0034] Figure 7 It is a chart showing the ratio of infectious particles to vector genome before and after exposure to different storage conditions. The control is a sample frozen at -80 °C on the day of generation and thawed only once. Error bars represent ±1SD generated by the error of the propagated VG titer assay and the infectious titer assay. Detailed Description

[0035] It is demonstrated herein that the proper selection of excipients and the balance of ionic strength and weight osmolarity result in the product quality of viruses such as AAV particles being reproducible and stable, for example, at room temperature or at 4 °C, and / or provide the ability to tolerate at least 10 freeze-thaw cycles. The embodiments provided herein reduce the dependence on the -80 °C cold chain, thus allowing for improved manufacturability and distribution, as well as alternative product containment options that can be implemented to improve product accessibility.

[0036] Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires a helper virus to facilitate efficient replication. The 4.7 kb genome of AAV is characterized by two inverted terminal repeats (ITRs) and two open reading frames that encode the Rep protein and the Cap protein, respectively. The rep reading frame encodes four proteins with molecular weights of 78 kD, 68 kD, 52 kD, and 40 kD. The functions of these proteins are mainly to regulate AAV replication, rescue AAV, and integrate AAV into the host cell chromosome. 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 viral particle capsid. More than 80% of the total protein in the AAV viral particle contains VP3. The rep and cap open reading frames are flanked on both the 5' and 3' ends by inverted terminal repeats (ITRs) approximately 145 bp in length. These two ITRs are the only cis-elements required for AAV replication, rescue, packaging, and integration of the AAV genome. The entire rep and cap domains can be excised and replaced with a therapeutic transgene or a reporter transgene.

[0037] The AAV particles described herein are not limited to a specific serotype, and any AAV serotype as well as AAV variants are suitable for the pharmaceutical compositions described herein.

[0038] In some embodiments, the AAV is AAV1 (i.e., AAV containing AAV1 ITR and AAV1 capsid protein), AAV2 (i.e., AAV containing AAV2 ITR and AAV2 capsid protein), AAV3 (i.e., AAV containing AAV3 ITR and AAV3 capsid protein), AAV4 (i.e., AAV containing AAV4 ITR and AAV4 capsid protein), AAV5 (i.e., AAV containing AAV5 ITR and AAV5 capsid protein), AAV6 (i.e., AAV containing AAV6 ITR and AAV6 capsid protein), AAV7 (i.e., AAV containing AAV7 ITR and AAV7 capsid protein), AAV8 (i.e., AAV containing AAV8 ITR and AAV8 capsid protein), AAV9 (i.e., AAV containing AAV9 ITR and AAV9 capsid protein), AAVrh74 (i.e., AAV containing AAVrh74 ITR and AAVrh74 capsid protein), AAVrh.8 (i.e., AAV containing AAVrh.8 ITR and AAVrh.8 capsid protein), or AAVrh.10 (i.e., AAV containing AAVrh.10 ITR and AAVrh.10 capsid protein).

[0039] In some embodiments, the AAV is a pseudotyped AAV that contains ITRs from one AAV serotype and capsid proteins from a different AAV serotype. In some embodiments, the pseudotyped AAV is AAV2 / 9 (i.e., AAV containing AAV2 ITR and AAV9 capsid protein). In some embodiments, the pseudotyped AAV is AAV2 / 10 (i.e., AAV containing AAV2 ITR and AAV10 capsid protein). In some embodiments, the pseudotyped AAV is AAV2 / 7m8 (i.e., AAV containing AAV2 ITR and AAV7m8 capsid protein). In some embodiments, the pseudotyped AAV is AAV2 / 8 (i.e., AAV containing AAV2 ITR and AAV capsid protein). In some embodiments, the pseudotyped AAV is AAV2 / 1 (i.e., AAV containing AAV2 ITR and AAV1 capsid protein).

[0040] In some embodiments, the AAV contains a recombinant capsid protein, such as a capsid protein of a chimeric body containing one or more of the capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh74, AAVrh.8, or AAVrh.10. In an embodiment, the capsid is a variant AAV capsid, such as AAV2 variant rAAV2-retro (SEQ ID NO:44 from WO 2017 / 218842, which is incorporated herein by reference).

[0041] In some embodiments, the AAV contains two or more capsid proteins selected from different serotypes. In some embodiments, the AAV contains rAAV2-retro and AAVrh.10 capsid proteins.

[0042] In embodiments, the AAV genome and / or AAV capsid are selected from serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, DJ, or DJ / 8.

[0043] In certain embodiments, the AAV particle contains a genome derived from AAV serotype 2. In certain embodiments, the AAV particle contains a capsid derived from AAV serotype 2.

[0044] The AAV particles described herein can be used in any downstream application compatible with the 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 should also be understood that the AAV particles may also be used as vaccines. Additionally, the downstream applications can extend beyond therapeutic uses. For example, the AAV particles described herein may also be useful in imaging applications or other non-therapeutic uses. In some embodiments, the AAV particles contain a capsidated recombinant vector sequence. In some embodiments, it may be desirable to have AAV particles that include: AAV particles that are empty capsids; AAV particles that include a mixture of full capsids and empty capsids; AAV particles that include a mixture of full capsids, partially full capsids, and empty capsids; or AAV particles that include a mixture of partially full capsids and empty capsids. In some embodiments, the formulations and pharmaceutical compositions disclosed herein contain AAV particles that are empty capsids. In some embodiments, the formulations and pharmaceutical compositions disclosed herein contain AAV particles that are full capsids, partially full capsids, or a mixture of full capsids and empty capsids. In some embodiments, the formulations and pharmaceutical compositions disclosed herein contain AAV particles that are a mixture of full capsids, partially full capsids, and empty capsids. In some embodiments, the AAV particles have a mixture of partially full capsids and empty capsids.

[0045] Recombinant AAV (i.e., "rAAV") includes any AAV derived from any adeno-associated virus serotype. The rAAV may have one or more (preferably the rep and / or cap genes) of the AAV wild-type genes that are fully or partially deleted, but retains functional flanking ITR sequences.

[0046] As used herein, a "vector" is a vehicle that contains a polynucleotide to be delivered to a host cell, either in vitro or in vivo. The term includes, for example, plasmids, expression vectors, viral vectors, and viruses. In an embodiment, a vector refers to a viral particle that contains a recombinant viral genome (e.g., rAAV), wherein the viral genome contains one or more ITRs and a transgene.

[0047] As used herein, the terms "AAV particle", "rAAV particle", "viral particle", and "AAV virion" are intended to mean a capsid containing a genome (also referred to as a "full capsid" or "partially full capsid") and empty capsids, or any combination of full capsids, partially full capsids, and empty capsids, unless otherwise specified.

[0048] "Empty capsid" and "empty particle" refer to an AAV particle (e.g., an rAAV particle) that has an AAV capsid but is missing 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 do not function to transfer a transgene to one or more target cells.

[0049] As used herein, the term "titer" is intended to mean the amount of virus in a given volume. Virus titer can include "physical titer" or "functional titer". Physical titer is a measure of how much virus is present and is typically expressed as the number of virus particles per mL (VP / mL), or vector genomes per mL (VG / mL), which can be interchanged 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 typically expressed as transduction units per mL (TU / mL), or for adenovirus as plaque-forming units per mL (pfu / mL) or infectious units per mL (ifu / mL). It should be understood that functional titer will generally be lower than physical titer, typically by a factor of about 10-fold to about 100-fold.

[0050] In various embodiments, the virus is a chimeric virus, a synthetic virus, a recombinant virus, a mosaic virus, or a pseudotyped virus.

[0051] As used herein, the term "freeze-thawing cycle", "freeze / thaw cycle", or "freeze-thaw cycle" refers to the process of changing the temperature from above freezing to below freezing and then back to above freezing.

[0052] Scope: Throughout this disclosure, various aspects of the disclosure may be presented in a range format. It should be understood that describing numerical values in a range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the disclosure. Accordingly, a description of a range should be considered to have specifically disclosed all the possible sub-ranges within that range as well as the individual numerical values. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed sub-ranges within that range, 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, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of how broad the range is. A numerical range expressed by endpoints includes all the numbers within that range, e.g., integers, including fractions of integers (e.g., the expression 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, such as 1.5, 2.25, 3.75, 4.1, etc.) and any range within that range.

[0053] The term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, in accordance with the practice in the art, "about" can mean within 1 standard deviation or greater than 1 standard deviation. 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 one order of magnitude of the value, preferably within 5-fold, and still more preferably within 2-fold. In cases where a particular value is described in this application and the claims, unless otherwise stated, it should be assumed that the term "about" means within the acceptable error range of the particular value. It is assumed herein that all numerical values are modified by the term "about" whether or not explicitly indicated. A numerical range expressed by endpoints includes all the numbers within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5).

[0054] Any formulation, pharmaceutical composition, or method provided herein can be combined with one or more of any other formulation, pharmaceutical composition, and method provided herein.

[0055] The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the invention. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit (unless the context clearly dictates otherwise), between the upper and lower limits of that range, as well as any other stated value or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0056] As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly dictates otherwise. Additionally, for the use of the terms “including,” “includes,” “having,” “has,” “with” or variants thereof in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term “comprising.” The transitional term “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. When used herein, the term “comprising” may be replaced with the term “including” or “containing,” or sometimes may be replaced with the term “having” when used herein. In contrast, the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of” limits the scope of the claim to the specified materials or steps, “and materials or steps that do not materially affect the basic and novel characteristics of the claimed disclosure.”

[0057] As used in this specification and the appended claims, the term “or” is generally used in its inclusive sense of “and / or” unless the context clearly dictates otherwise.

[0058] As used herein, the term “and / or” means any one of the items associated with that term, any combination of the said items, or all of the said items.

[0059] The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illustrate the invention and does not impose a limitation on the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0060] Cryoprotectant

[0061] In certain embodiments, the formulations and pharmaceutical compositions disclosed herein comprise one or more cryoprotectants. As used herein, the term "cryoprotectant" or "cryoprotected" refers to a chemical or chemical solution that facilitates the cryoprotection process by reducing damage to stored components (e.g., viral particles, viruses, cells, and / or tissues) during freezing and thawing, or reduces damage to the stored components at room temperature of about +20 °C or at about +4 °C. The cryoprotective formulation or cryoprotective composition protects the stored components (i.e., AAV particles) from damage associated with sub-zero temperatures and / or cryogenic storage (e.g., viral capsid or cell membrane damage due to ice crystal formation), or damage associated with storage at room temperature of about +20 °C or at about +4 °C. In some embodiments, multiple different cryoprotectants can be combined.

[0062] In some embodiments, the cryoprotectant is a sugar. In some embodiments, the cryoprotectant is a sugar.

[0063] In certain embodiments, the sugar includes trehalose, cyclodextrin, sucrose, or a combination thereof. In certain embodiments, the sugar is trehalose. In certain embodiments, the sugar is cyclodextrin.

[0064] 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, dextran (e.g., 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, fructooligosaccharide, polydextrose, etc.). The term includes simple carbohydrates and complex carbohydrates. Indeed, the present disclosure is not intended to be limited to any particular sugar, as various sugars and sugar forms can be used in the present disclosure.

[0065] In certain aspects, a cryoprotective formulation for storing AAV particles, maintaining the infectivity of AAV particles, and / or maintaining the viability of AAV particles comprises sodium phosphate, sodium chloride, cyclodextrin, and polysorbate.

[0066] In certain aspects, a cryoprotective formulation for storing AAV particles, maintaining the infectivity of AAV particles, and / or maintaining the viability of AAV particles comprises a cryoprotective sugar, a buffer, a salt, and a nonionic surfactant.

[0067] In certain aspects, the cryoprotective formulation comprises the buffer composition provided in Table 1 or Table 2.

[0068] In certain embodiments, the formulations or pharmaceutical compositions embodied herein comprise sugars in the following amounts: from about 0.001% w / v to about 30% w / v, from about 0.005% w / v to about 25% w / v, from about 0.01% w / v to about 24% w / v, from about 0.05% w / v to about 23%, from about 0.1% to about 22% w / v, 0.1% w / v to about 21% w / v, 0.1% w / v to about 20% w / v, from about 0.5% w / v to about 19% w / v, from about 1% w / v to about 18% w / v, from about 1% w / v to about 17% or from about 1% to about 16% w / v.

[0069] In certain embodiments, the formulations or pharmaceutical compositions embodied herein comprise cyclodextrins in the following amounts: from about 0.001% w / v to about 30% w / v, from about 0.005% w / v to about 25% w / v, from about 0.01% w / v to about 24% w / v, from about 0.05% w / v to about 23% or from about 0.1% to about 22% w / v. In certain embodiments, the formulations or pharmaceutical compositions embodied herein comprise about 0.375% w / v cyclodextrin.

[0070] In certain embodiments, the formulations or pharmaceutical compositions embodied herein comprise trehalose in the following amounts: from about 0.1% w / v to about 20% w / v, from about 0.5% w / v to about 19% w / v, from about 1% w / v to about 18% w / v, from about 1% w / v to about 17% or from about 1% to about 16% w / v. In certain embodiments, the formulations or pharmaceutical compositions embodied herein comprise about 1.1% w / v trehalose.

[0071] Sugars and other cryoprotectants

[0072] In some embodiments, the formulations or pharmaceutical compositions embodied herein comprise one or more cryoprotectants, where the cryoprotectants are solvents (e.g., organic solvents), polyols, polymers, sugars, or combinations thereof. In some embodiments, the cryoprotectants are DMSO (dimethyl sulfoxide), ethylene glycol, glycerol, propylene glycol, 2-methyl-2,4-pentanediol (MP), glycerol-3-phosphate, diethylene glycol, triethylene glycol, polyvinyl alcohol, PEG, hydroxyethyl starch, sorbitol, mannitol, lactose, sucrose, trehalose, or combinations thereof. In some embodiments, the cryoprotectants are organic solvents. In some embodiments, the cryoprotectants are polyols. In some embodiments, the cryoprotectants are polymers. In some embodiments, the cryoprotectants are DMSO (dimethyl sulfoxide). In some embodiments, the cryoprotectants are ethylene glycol. In some embodiments, the cryoprotectants are glycerol. In some embodiments, the cryoprotectants are propylene glycol. In some embodiments, the cryoprotectants are (MPD) 2-methyl-2,4-pentanediol. In some embodiments, the cryoprotectants are glycerol-3-phosphate. In some embodiments, the cryoprotectants are diethylene glycol. In some embodiments, the cryoprotectants are triethylene glycol. In some embodiments, the cryoprotectants are polyvinyl alcohol. In some embodiments, the cryoprotectants are PEG. In some embodiments, the cryoprotectants are hydroxyethyl starch. In some embodiments, the cryoprotectants are sorbitol. In some embodiments, the cryoprotectants are mannitol. In some embodiments, the cryoprotectants are lactose.

[0073] In embodiments, multiple (e.g., 2, 3, 4, 5, etc.) cryoprotectants can be combined.

[0074] Solvent

[0075] In certain aspects, as described above, the formulations or pharmaceutical compositions can comprise one or more solvents. Any solvent suitable for the stable maintenance of AAV particles can be incorporated into the compositions 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 comprises DMEM. In certain embodiments, the composition comprises EMEM.

[0076] Pharmaceutically acceptable salts

[0077] In embodiments, the formulations and pharmaceutical compositions disclosed herein include one or more pharmaceutically acceptable salts. As used herein, the term "pharmaceutically acceptable salt" 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 made from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc, or organic salts made from N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, glucosamine (N-methyl-glucosamine), 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, ethanesulfonic 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. Thus, examples of specific salts include hydrochloride and mesylate salts.

[0078] In certain embodiments, a pharmaceutically acceptable salt is a salt of a metal and ammonia or an organic amine that is safe for administration to a subject (e.g., a human) in a pharmaceutical formulation. 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, methylpyridine, ethanolamine, triethanolamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine salts. Pharmaceutically acceptable salts (of basic nitrogen centers) include, but are not limited to, inorganic acid salts such as hydrochloride, hydrobromide, sulfate, phosphate; organic acid salts such as trifluoroacetate and maleate; sulfonate salts such as mesylate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, and naphthalenesulfonate; amino acid salts such as arginine salt, alanine salt, aspartate salt, and glutamate salt; and carbohydrate salts such as gluconate and galacturonate. Other salts that may 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 hereby incorporated 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 includes 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.

[0079] In certain embodiments, the salt includes 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, methylpyridine, ethanolamine, triethanolamine, dicyclohexylamine, or an N,N'-dibenzylethylenediamine salt, or a combination thereof. In certain embodiments, the sodium salt includes sodium chloride, sodium phosphate, or a combination thereof. In certain embodiments, the magnesium salt is magnesium sulfate. In certain embodiments, the salt includes sodium chloride, sodium phosphate, magnesium sulfate, or a combination thereof.

[0080] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable salt concentration of from about 1 mM to about 300 mM. In some embodiments, the pharmaceutically acceptable salt concentration is from about 2 mM to about 295 mM. In some embodiments, the pharmaceutically acceptable salt concentration is from about 3 mM to about 290 mM. In some embodiments, the pharmaceutically acceptable salt concentration is from about 4 mM to about 285 mM. In some embodiments, the pharmaceutically acceptable salt concentration is from about 6 mM to about 284 mM. In some embodiments, the pharmaceutically acceptable salt concentration is from about 7 mM to about 283 mM. In some embodiments, the pharmaceutically acceptable salt concentration is from about 8 mM to about 282 mM. In some embodiments, the pharmaceutically acceptable salt concentration is from about 9 mM to about 281 mM. In some embodiments, the pharmaceutically acceptable salt concentration is from about 10 mM to about 280 mM.

[0081] In some embodiments, the pharmaceutically acceptable salt is sodium chloride. In certain embodiments, the sodium chloride concentration is about 280 mM.

[0082] In some embodiments, the pharmaceutically acceptable salt is sodium phosphate. In some embodiments, the sodium phosphate concentration is about 10 mM.

[0083] 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.

[0084] In certain embodiments, the pharmaceutically acceptable salt is magnesium sulfate. In certain embodiments, the concentration of magnesium sulfate is from about 30 mM to about 200 mM, from about 35 mM to about 190 mM, from about 40 mM to about 180 mM, from about 40 mM to about 170 mM, from about 45 mM to about 160 mM, from about 46 mM to about 150 mM, from about 47 mM to about 140 mM, from about 48 mM to about 130 mM, from about 49 mM to about 128 mM, from about 50 mM to about 125 mM. In certain embodiments, the concentration of magnesium sulfate is about 50.

[0085] In some aspects of the present disclosure, the formulation and pharmaceutical composition comprise a pharmaceutically acceptable salt, a buffer, a cryoprotectant, and a nonionic surfactant.

[0086] In some aspects, the formulation or pharmaceutical composition may also include one or more pharmaceutically acceptable excipients or be diluted in a pharmaceutically acceptable excipient to obtain a desired dosage ratio in the composition or formulation. As used herein, pharmaceutically acceptable excipients include any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, etc. suitable for the particular formulation desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro, (Lippincott, Williams & Wilkins, Baltimore, Maryland, 2006; incorporated herein by reference) discloses various excipients for formulating pharmaceutical compositions, and such excipients can be used to prepare the compositions of the present invention. The use of any conventional excipient is contemplated within the scope of the present disclosure, except where the conventional excipient is incompatible with the substance or its derivatives, such as due to the production of any undesirable biological effects or otherwise interacting in a detrimental manner with any one or more other components of the pharmaceutical composition. In certain embodiments, the pharmaceutically acceptable excipient has a purity of at least 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient is approved by the U.S. Food and Drug Administration (FDA) for human use. In some embodiments, the excipient is of pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopeia (USP), European Pharmacopeia (EP), British Pharmacopeia, and / or International Pharmacopeia.

[0087] In some aspects, the formulation or pharmaceutical composition may also include one or more pharmaceutically acceptable excipients or be diluted in a pharmaceutically acceptable excipient to obtain a desired dosage ratio in the composition or formulation. As used herein, pharmaceutically acceptable excipients include any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, etc. suitable for the particular formulation desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro, (Lippincott, Williams & Wilkins, Baltimore, Maryland, 2006; incorporated herein by reference) discloses various excipients for formulating pharmaceutical compositions, and such excipients can be used to prepare the compositions of the present invention. The use of any conventional excipient is contemplated within the scope of this disclosure, except if the conventional excipient is incompatible with the substance or its derivatives, such as due to producing any undesirable biological effects or otherwise interacting in a detrimental manner with any one or more other components of the pharmaceutical composition. In certain embodiments, the pharmaceutically acceptable excipient has a purity of at least 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient is approved by the U.S. Food and Drug Administration (FDA) for human use. In some embodiments, the excipient is of pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopeia (USP), European Pharmacopeia (EP), British Pharmacopeia, and / or International Pharmacopeia.

[0088] Nonionic surfactant

[0089] In certain embodiments, the formulations or pharmaceutical compositions disclosed herein comprise one or more nonionic surfactants. As used herein, the term "nonionic surfactant" refers to a surfactant composed of a polar head group lacking a charge. In some embodiments, the nonionic surfactant is a copolymer. In some embodiments, the nonionic surfactant is a poloxamer. A poloxamer is a nonionic triblock copolymer composed of a central hydrophobic polyoxypropylene (polypropylene oxide) chain flanked by two hydrophilic polyoxyethylene (polyethylene oxide) chains. Poloxamers are also sold under the trade name and is known as such. Since the lengths of the polymer blocks can be customized, there are many different poloxamers, which have slightly different properties. For the general term "poloxamer", these copolymers are generally named with the letter "P" (indicating poloxamer) followed by three digits. The first two digits multiplied by 100 give the approximate molecular weight of the polyoxypropylene core, and the last digit multiplied by 10 gives the percentage of polyoxyethylene content (e.g., P407 = poloxamer with a polyoxypropylene molecular weight of 4,000 g / mol and a 70% polyoxyethylene content). 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; an ethylene oxide (EO) copolymer; a phospholipid, such as phosphatidylcholine (lecithin); a polyoxyethylene fatty ether derived from lauryl alcohol, cetyl alcohol, stearyl alcohol, and oleyl alcohol (called Brij surfactant), such as triethylene glycol monolauryl ether (Brij 30) or polyoxyethylene(23) lauryl ether (Brij TM 35); and sorbitan esters (commonly known as SPAN), such as sorbitan trioleate (Span TM 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 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 a copolymer of EO. In some embodiments, the nonionic surfactant is a phospholipid. In some embodiments, the nonionic surfactant is phosphatidylcholine (lecithin). In some embodiments, the nonionic surfactant is a polyoxyethylene fatty ether derived from lauryl alcohol. In some embodiments, the nonionic surfactant is a polyoxyethylene fatty ether derived from cetyl alcohol. In some embodiments, the nonionic surfactant is a polyoxyethylene fatty ether derived from stearyl alcohol. In some embodiments, the nonionic surfactant is a polyoxyethylene fatty ether derived from oleyl alcohol. In some embodiments, the nonionic surfactant is triethylene glycol monolauryl ether (Brij TM30). In some embodiments, the nonionic surfactant is polyoxyethylene (23) lauryl ether (Brij TM 35). In some embodiments, the nonionic surfactant is a sorbitan ester. In some embodiments, the nonionic surfactant is sorbitan trioleate (Span TM 85). In some embodiments, the nonionic surfactant is sorbitan monolaurate.

[0090] In some embodiments, the formulations or pharmaceutical compositions disclosed herein comprise one or more nonionic surfactants in the following amounts: about 0.0001% (w / v) to about 0.1% (w / v), about 0.0005% (w / v) to about 0.005% (w / v), about 0.00075% (w / v) to about 0.0025% (w / v), about 0.0050% (w / v) to about 0.0075% (w / v), 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 0.4% (w / v) to about 0.5% (w / v). In some embodiments, the formulations or pharmaceutical compositions disclosed herein comprise about 0.02% (w / v) nonionic surfactant.

[0091] Ionic strength

[0092] As provided herein, the 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, the formulations or pharmaceutical compositions described herein have an ionic strength of about 300 mM or about 500 mM.

[0093] Osmolality

[0094] As provided herein, the formulations or pharmaceutical compositions of the present disclosure can have a weight osmolarity of from about 100 mOsm / kg to about 800 mOsm / kg. In certain embodiments, the weight osmolarity is from about 150 mOsm / kg to about 750 mOsm / kg, from about 175 mOsm / kg to about 700 mOsm / kg, from about 180 mOsm / kg to about 675 mOsm / kg, from about 185 mOsm / kg to about 650 mOsm / kg, or from about 190 mOsm / kg to about 628 mOsm / kg. In certain embodiments, the weight osmolarity is about 200 mOsm / kg, or about 350 mOsm / kg, or about 500 mOsm / kg, or about 600 mOsm / kg. In certain embodiments, the formulations or pharmaceutical compositions of the present disclosure have a weight osmolarity of about 200 mOsm / kg or about 350 mOsm / kg. In certain embodiments, the formulations or pharmaceutical compositions of the present disclosure have a weight osmolarity of about 200 mOsm / kg or about 600 mOsm / kg. In certain embodiments, the formulations or pharmaceutical compositions of the present disclosure have a weight osmolarity of less than about 400 mOsm / kg.

[0095] pH conditions

[0096] As provided herein, the formulations or pharmaceutical compositions of the present disclosure can have a pH of from about 4.0 to about 9.0. In some embodiments, the pH of the formulation or pharmaceutical composition is from 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 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0. In some embodiments, the pH of the formulations or pharmaceutical compositions of the present disclosure is about 7.5.

[0097] Formulations and pharmaceutical compositions

[0098] The formulations or pharmaceutical compositions provided herein can be liquid compositions or frozen compositions. Thus, 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.

[0099] In certain aspects, formulations for storing AAV particles, maintaining the infectivity of AAV particles and / or maintaining the viability of AAV particles comprise sugars, buffers, surfactants, salts or combinations thereof. In certain aspects, pharmaceutical compositions for storing AAV particles, maintaining the infectivity of AAV particles and / or maintaining the viability of AAV particles comprise cryoprotectants, buffers, surfactants and salts.

[0100] In certain aspects, the composition for storing AAV particles, maintaining the infectivity of AAV particles and / or maintaining the viability of AAV particles comprises from about 1 mM to about 20 mM sodium phosphate, from about 100 mM to about 400 mM sodium chloride, from about 0.1% weight / volume (w / v) to up to about 20% (w / v) cyclodextrin and from about 0.01% weight / volume (w / v) to up to about 5% (w / v) polysorbate.

[0101] In certain aspects, the composition for storing AAV particles, maintaining the infectivity of AAV particles and / or maintaining the viability of AAV particles comprises from about 1% weight / volume (w / v) to about 20% w / v trehalose, from 0.001% w / v to about 1% w / v polysorbate, from about 1 mM to about 20 mM tris buffer, from about 10 mM to about 250 mM magnesium sulfate, from about 0.1% weight / volume (w / v) to up to about 20% (w / v) cyclodextrin and from about 0.01% weight / volume (w / v) to up to about 5% (w / v) polysorbate.

[0102] In certain aspects, the pharmaceutical composition comprises the buffer composition provided in Table 1 or Table 2.

[0103] In certain aspects, the formulation comprises the buffer composition provided in Table 1 or Table 2.

[0104] In certain aspects, the formulation comprises from about 5 mM to about 15 mM sodium phosphate, from about 150 mM to about 200 mM NaCl, from about 1% to about 2% cyclodextrin, from about 0.01% to about 0.5% polysorbate 80, wherein the formulation comprises from about 180 mM to about 250 mM ionic strength and from about 300 mOsm / kg to about 400 mOsm / kg weight osmolarity.

[0105] In some aspects, the formulation comprises about 10 mM sodium phosphate, about 180 mM NaCl, about 1.5% cyclodextrin, about 0.1% polysorbate 80, wherein the formulation has an ionic strength of about 206 mM and a weight osmolarity of about 367 mOsm / kg.

[0106] In some aspects, the formulation comprises about 10 mM sodium phosphate, about 180 mM NaCl, about 0.375% cyclodextrin, about 0.02% polysorbate 80, wherein the formulation has an ionic strength of about 200 mM and a weight osmolarity of about 350 mOsm / kg.

[0107] In some aspects, the formulation comprises about 10 mM sodium phosphate, about 180 mM NaCl, about 21.0% cyclodextrin, about 0.02% polysorbate 80, wherein the formulation has an ionic strength of about 200 mM and a weight osmolarity of about 600 mOsm / kg.

[0108] In some aspects, the formulation comprises about 10 mM sodium phosphate, about 280 mM NaCl, about 0.375% cyclodextrin, about 0.02% polysorbate 80, wherein the formulation has an ionic strength of about 300 mM and a weight osmolarity of about 350 mOsm / kg.

[0109] In some aspects, the formulation comprises about 10 mM sodium phosphate, about 280 mM NaCl, about 11.5% cyclodextrin, about 0.02% polysorbate 80, wherein the formulation has an ionic strength of about 300 mM and a weight osmolarity of about 600 mOsm / kg.

[0110] In some aspects, the formulation comprises about 5 mM to about 20 mM tris, about 30 mM to about 80 mM MgSO4, about 0.5% to about 3% trehalose, about 0.01% to about 0.5% polysorbate 80, wherein the formulation has an ionic strength of about 180 mM to about 250 mM and a weight osmolarity of about 100 mOsm / kg to about 200 mOsm / kg.

[0111] In some aspects, the formulation comprises about 5 mM to about 15 mM tris, about 40 mM to about 70 mM MgSO4, about 1% to about 2% trehalose, about 0.05% to about 0.5% polysorbate 80, wherein the formulation has an ionic strength of about 200 mM to about 240 mM and a weight osmolarity of about 110 mOsm / kg to about 140 mOsm / kg.

[0112] In some aspects, the formulation comprises about 10 mM tris, about 125 MgSO4, about 1.1% trehalose, about 0.02% polysorbate 80, wherein the formulation has an ionic strength of about 500 mM and a weight osmolarity of about 200 mOsm / kg.

[0113] In some aspects, the formulation comprises about 10 mM tris, about 55 mM MgSO4, about 1.5% trehalose, about 0.1% polysorbate 80, wherein the formulation comprises an ionic strength of about 220 mM and a weight osmolarity of about 128 mOsm / kg.

[0114] Storage conditions

[0115] In certain embodiments, the formulations or pharmaceutical compositions disclosed herein are stored at ambient temperature or room temperature, such as about 25°C. In some embodiments, the compositions disclosed herein are stored at below about 25°C. In some embodiments, the compositions disclosed herein are stored between about 0°C and about 25°C. In some embodiments, the compositions are stored between about 0°C and about 10°C. In some embodiments, the compositions are stored between about 2°C and about 8°C. In some embodiments, the compositions are stored at about 4°C. In some embodiments, the compositions are stored below 0°C. In some embodiments, the compositions are stored between about -20°C and about -80°C. In some embodiments, the compositions are stored at about -20°C. In some embodiments, the compositions are stored at about -70°C. In some embodiments, the compositions are stored at about -80°C. In certain embodiments, the compositions are stored at 0°C or below 0°C and stability is maintained or enhanced after one or more freeze-thaw cycles. In certain embodiments, the compositions are stored at about -20°C and stability is maintained or enhanced after one or more freeze-thaw cycles. In certain embodiments, the compositions are stored at about -20°C and stability is maintained or enhanced after one or more freeze-thaw cycles. In certain embodiments, the compositions are stored at about -70°C and stability is maintained or enhanced after one or more freeze-thaw cycles. In certain embodiments, the compositions are stored at about -80°C and stability is maintained or enhanced 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 more than one freeze-thaw cycle. In some embodiments, stability is enhanced. In some embodiments, stability is enhanced after one freeze-thaw cycle. In some embodiments, stability is enhanced after more than one freeze-thaw cycle.

[0116] The formulations or pharmaceutical compositions disclosed herein can maintain or enhance the stability of AAV particles and / or reduce or prevent aggregation after one or more freeze-thaw cycles. In some embodiments, the compositions disclosed herein maintain the stability of AAV particles. In some embodiments, the compositions disclosed herein reduce aggregation after one freeze-thaw cycle. In some embodiments, the compositions disclosed herein reduce aggregation after more than one freeze-thaw cycle. In some embodiments, the compositions disclosed herein prevent aggregation after one freeze-thaw cycle. In some embodiments, the compositions disclosed herein prevent aggregation after more than one freeze-thaw cycle.

[0117] In some embodiments, the stability is maintained or enhanced after two or more freeze-thaw cycles. In some embodiments, the stability is maintained or enhanced after three or more freeze-thaw cycles. In some embodiments, the stability is maintained or enhanced after four or more freeze-thaw cycles. In some embodiments, the stability is maintained or enhanced after four or more freeze-thaw cycles. In some embodiments, the stability is maintained or enhanced after five or more freeze-thaw cycles. In some embodiments, the stability is maintained or enhanced after six or more freeze-thaw cycles. In some embodiments, the stability is maintained or enhanced after seven or more freeze-thaw cycles. In some embodiments, the stability is maintained or enhanced after eight or more freeze-thaw cycles. In some embodiments, the stability is maintained or enhanced after nine or more freeze-thaw cycles. In some embodiments, the stability is maintained or enhanced after ten or more freeze-thaw cycles.

[0118] In another aspect, a method of storing AAV particles or maintaining the viability of AAV particles, the method comprising storing the AAV particles in a formulation or pharmaceutical composition embodied herein. In certain embodiments, the formulation is used to store AAV particles or maintain the viability of AAV particles over a period of time at a temperature of from about 20°C to about -80°C. In certain embodiments, the AAV particles remain stable during multiple freeze-thaw cycles. In certain embodiments, the AAV particles are stable during multiple freeze-thaw cycles. In certain embodiments, the AAV particles are stable during at least 5 multiple freeze-thaw cycles. In certain embodiments, the AAV particles are stable at a temperature of from about 20°C to 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.

[0119] In another aspect, a method of cryopreserving a virus, the method comprising storing AAV particles in a cryoprotective formulation or pharmaceutical composition as embodied herein. In certain embodiments, the formulation is used to store AAV particles or maintain the viability of AAV particles over a period of time at a temperature of about 20°C to about -80°C. In certain embodiments, the AAV particles remain stable during multiple freeze-thaw cycles. In certain embodiments, the AAV particles are stable during multiple freeze-thaw cycles. In certain embodiments, the AAV particles are stable during at least 5 multiple freeze-thaw cycles. In certain embodiments, the AAV particles are stable at a temperature of about 20°C to about -80°C. In certain embodiments, the AAV particles are stable at about 4°C.

[0120] Stability determination

[0121] As provided herein, the formulations or pharmaceutical compositions of the present disclosure are capable of preventing AAV particle aggregation and / or enhancing the stability of AAV particles. As used herein, a composition or formulation comprising AAV particles is stable if it exhibits a less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% decrease in viral particle infectivity after exposure to freeze-thaw cycles. As used herein, a composition or formulation comprising AAV particles is stable if it exhibits a less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% decrease in viral particle infectivity when stored at the intended storage temperature for, for example, 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.

[0122] A variety of techniques for measuring the physical properties of AAV particles (e.g., AAV particle size), viral titer, and / or purity are 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 this application is not limited to the assays described below. As provided herein, the formulations and pharmaceutical compositions are capable of preventing AAV particle aggregation and / or enhancing the stability of AAV particles. A variety of techniques for measuring the physical properties of AAV particles (e.g., AAV particle size), viral titer, and / or purity are 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 this application is not limited to the assays described below.

[0123] In certain embodiments, the stability of AAV particles is measured by a variety of assays such as dynamic light scattering (DLS), analytical ultracentrifugation (AUC), optical microscopy, size exclusion chromatography (SEC), transmission electron microscopy (TEM), field flow fractionation - multi - angle static light scattering (FFF - MALS), infectivity, immunocytochemistry, image analysis, and the like.

[0124] A variety of assays for evaluating the identity of AAV particle preparations are 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 that analyze viral protein expression may be used to evaluate the identity of AAV particles. Such assays include, but are not limited to, SDS - PAGE, mass spectrometry, immunoblotting, and ELISA. The appropriate quantity, molecular weight, and stoichiometry of viral proteins can be used to definitively identify the vector and the presence of impurities. In addition, PCR or high - throughput NGS (next - generation genome sequencing) can be used to evaluate the vector genome to ensure a clear identity.

[0125] AAV particles can also be characterized by their viral titer. The viral titer can include a physical titer as well as a functional titer. The physical titer calculates the total number of live and dead virus particles present and is expressed as the number of virus particles per mL (VP / mL), or for AAV as the number of genome copies per mL (GC / mL). Various methods can be used to determine the physical titer of the virus based on quantifying the concentration of the viral genome or viral proteins. Suitable techniques include, but are not limited to, DNA hybridization, real - time PCR (including, but not limited to, quantitative PCR (qPCR) and digital droplet PCR (dPCR)), absorbance (A 260 / 280 ), NanoSight, and high - performance liquid chromatography (HPLC).

[0126] For example, the absorbance (A 260 / 280 ) assay measures the concentration of viral DNA and proteins. It is a physical assay for measuring the concentration of virus particles (VP). HPLC is also a rapid method for quantifying total virus particles, which is performed by separating intact virus particles from other cellular contaminants or virus particle fragments.

[0127] Functional titer measures how much virus enters target cells and can involve assessing the number of colony-forming units after antibiotic selection if the vector contains an antibiotic resistance gene, or performing flow cytometry or immunofluorescence analysis on the target cells if the vector contains a fluorescent protein. Alternatively, if the vector does not express a fluorescent protein, determining the number of proviral DNA copies integrated per cell by qPCR provides a rapid and easy method for assessing functional titer.

[0128] Example

[0129] Example 1: Design and Screen Formulations to Improve the Manufacturability and Distribution of AAV Gene Therapy

[0130] Materials and Methods

[0131] The AAV-AQP1 (aquaporin-1) clarified lysate was separated into two batches by affinity chromatography and 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 Mg2SO4, 1.5% trehalose, 0.02% polysorbate 80, pH 7.5. The UF / DF steps were performed using an AKTA 2 Flux system (Cytiva, Marlborough, Massachusetts, USA) with an 115 cm TM 100 kDa mPES hollow fiber membrane (Repligen Corporation, Rancho Dominguez, California, USA). The buffer-exchanged and concentrated materials were then filtered using a 0.2 μm syringe filter. Five milliliters of the processed material was used to generate different buffer combinations belonging to Formulation A (the final buffer composition was created based on equations generated from known buffer components). The target ionic strength and weight osmolarity as well as the exact buffer components are shown in Tables 1 and 2.

[0132] Formulation A (Table 1) or Formulation B (Table 2) was made up to a final volume of 20 mL. Stock solutions were prepared to accommodate multiple buffer compositions. The target product concentration was 1×10 12 VG / mL. The samples were aliquoted and held at different storage conditions over time, including room temperature at +20 °C, a refrigerator at +4 °C, and freezers at -20 °C and -80 °C (Table 3).

[0133] Buffer Design

[0134] The ionic strength (I) of the buffer is calculated based on the ions present in the solution, i.e., the sum of the molar concentrations (c) of each ion multiplied by the square of the valence (z), and divided by 2 (Equation 1).

[0135]

[0136] Buffers are designed based on ionic strength and sugar content, and the weight osmolarity of the buffers varies. Buffers containing a fixed ionic strength and different concentrations of cyclodextrin or trehalose are generated to evaluate the change in weight osmolarity ( Figures 1A to 1B and Figures 2A to 2C ). Using three sugar concentrations, a linear line can be fitted and an equation can be generated, which can be used to predict the amount of sugar required to achieve the desired weight osmolarity.

[0137] The final buffer composition is generated based on the equation derived from the known buffer components. The target ionic strength and weight osmolarity, as well as the exact buffer components, are shown in Tables 1 and 2.

[0138] Table 1. Final buffer composition belonging to formulation A.

[0139]

[0140] Table 2. Final buffer composition belonging to formulation B.

[0141]

[0142] Maintain the research plan

[0143] Before analysis, the samples are maintained at different temperatures for a fixed period of time (Table 3) and thawed at room temperature for 1 hour. All analyses are done on separate dates, so each time a new vial is thawed. Once the holding time of the samples at +20 °C and +4 °C is completed, the samples are immediately placed in -80 °C storage for analysis. Separate sample groups are stored at -20 °C and -80 °C and thawed at room temperature for 1 hour to generate different freeze-thaw cycles.

[0144] Table 3. First holding research plan for all buffers used in formulations A and B.

[0145]

[0146] Analytical assays

[0147] Determine the vector genome concentration by qPCR.

[0148] Use the AAVX titer kit (Gyros Protein Technologies) to determine the virus particle concentration.

[0149] The monomer area and HMW substances were determined by HPLC-SEC (Agilent Technologies) using an Xbridge BEH450 SEC 3.5 μm 7.8 x 300 mm (Waters TM ) column.

[0150] The infectivity assay was performed as follows: COS-7 cells were transduced with the AQP1 product and then lysed, single-stranded DNA was degraded, and endpoint qPCR was performed in a validated AQP1 VG titer assay.

[0151] All statistical analyses and model development were performed using (SAS Institute).

[0152] Results

[0153] Stability – Formulation A: The stability of AAV2 was evaluated by measuring the VP titer, VG titer, monomer area, and the presence of high molecular weight substances (HMW) before and after exposure to different storage conditions. Overall, it was found that storage in the refrigerator for up to 4 weeks had less impact on the changes in the parameters studied than storage at room temperature for up to one week (168 hours) ( Figure 3 ). Storage of the product in both -20 °C and -80 °C freezers was found to have comparable effects on AAV2 stability. Buffer 3 provided the least amount of variability, with a maximum change in VP titer of 7%, a maximum change in VG titer of 15%, a maximum change in monomer area of 5%, and a maximum change in HMW of 22% under all conditions studied ( Figure 3 ). The change in HMW was greatest under -80 °C storage, while the differences caused by the remaining conditions were less than 5% when using Buffer 3.

[0154] Data summarization was performed using the relative standard deviation (RSD): To summarize the data and ensure that the selected formulations provide the best product stability in terms of VP titer and HMW content under all storage conditions, the RSD was utilized. An increase in weight osmolality led to an increase in the change in VP titer for both formulations, while an increase in ionic strength had no effect ( Figures 4A to 4B ). Specifically, it was found that for Formulation A and Formulation B, weight osmolalities below 400 mOsm / kg and 250 mOsm / kg provided VP titer changes of less than 7%, respectively. In contrast, ionic strength was a key determinant of the change in HMW substances, whereby an increase in ionic strength led to a decrease in the HMW variability of Formulation B ( Figure 4B)。Surprisingly, the same trend was not observed for Formulation A, where the RSD of HMW decreased with increasing weight osmolarity and the ionic strength showed no significant effect. In summary, it was found that for both formulations, the maximum RSD percentage for VP titer was 20% and for HMW substances was 10%.

[0155] Example 2: Buffer Formulations

[0156] Method

[0157] The AAV-AQP1 clarified lysate processed by the affinity chromatography step was divided into two tanks 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 Mg2SO4, 1.1% trehalose, 0.02% polysorbate 80, pH 7.5. A crossflow system with a 10 cm 2 、100 kDa CF PESU filter (both from Sartorius Stedim UK Limited, Surrey, UK) was used for the UF / DF step. The buffer-exchanged and concentrated material was then filtered through a 0.2 μm syringe filter. The target product concentration was 1×10 11 VG / mL. The samples were aliquoted and kept under different storage conditions (Table 4).

[0158] Holding Study Protocol

[0159] The holding study protocol was carried out in the same manner as described in Example 1, but with an extended holding time at +20 °C (Table 4).

[0160] Table 4. Second holding study protocol for Formulation A - Buffer 3 and Formulation B - Buffer 7.

[0161]

[0162]

[0163] Results

[0164] Stability: Based on the foregoing results( Figures 4A to 4B), Buffer 3 of Formulation A and Buffer 7 of Formulation B were selected for the next study, which used a product concentration 10 times lower. The stability of AAV2 was evaluated by measuring the VP titer, monomer area, and presence of high molecular weight substances before and after exposure to different storage conditions. The changes in the measurement data of Formulation A - Buffer 3 were generally lower than those of Formulation B - Buffer 7( Figure 5 A to Figure 5 B).

[0165] Data summary was performed using the relative standard deviation (RSD): The relative standard deviations of the VP titer, monomer area, and HMW content were calculated for all storage conditions. In all the assays tested, the RSD of Formulation B - Buffer 7 was 4 times that of Formulation A - Buffer 3( Figure 6 ). Note that in Figure 6 , the monomer area result of Formulation A - Buffer 3 at the 6th freeze - thaw cycle at - 80°C was 1802 mAU, which was considered an outlier as it did not follow any trend and was higher than the control value, and thus was excluded from the RSD calculation. The overall maximum RSD value of Formulation B was less than 25%, and the overall maximum RSD value of Formulation A was less than 10%.

[0166] Infectivity: The infectivity of AAV2 of the two formulations was evaluated. The two formulations were stored at +20°C room temperature for 2 weeks and then stored in a - 80°C freezer, followed by 10 freeze - thaw (F / T) cycles. The ratio of infectious VP to total vector genome (VG) was comparable among the formulation buffers and storage conditions studied( Figure 7 ).

[0167] Phosphate - based and sodium chloride - based formulation buffers generally provide a more stable environment for AAV. It was found that a buffer ionic strength of 300 - 500 mM and a weight osmolarity of less than 400 mOsm / kg water were most beneficial for AAV2 stability, providing the smallest variation in VP titer, VG titer, monomer area, and HMW. The use of relative standard deviation helps to deconvolute complex data sets. Differences in virus concentration may lead to changes in stability.

[0168] It is clear from the foregoing description that the invention described herein can be varied and modified to be suitable for various uses and conditions. Such embodiments are also within the scope of the following claims.

[0169] All sequences, patents, and publications cited in this specification are hereby incorporated by reference as if each individual patent and publication was specifically and individually indicated to be incorporated by reference. The publications disclosed herein are provided only for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the provided publication dates may differ from the actual publication dates, which may need to be independently confirmed.

Claims

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

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

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

5. The cryoprotective formulation or pharmaceutical composition according to claim 3, wherein the sugar is cyclodextrin.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

25. The cryoprotective formulation according to any one of claims 1 or 3 - 24 or the pharmaceutical composition according to 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. The cryoprotective formulation or pharmaceutical composition according to claim 25, wherein the buffer comprises sodium phosphate.

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

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

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

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

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

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

33. The cryoprotective formulation or pharmaceutical composition according to claim 32, wherein the nonionic surfactant comprises a polysorbate.

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

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

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

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

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

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

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

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

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

43. The cryoprotective formulation according to claim 1 or any one of claims 3 - 42 or the pharmaceutical composition according to claims 2 - 42, wherein the cryoprotective formulation or pharmaceutical composition has a weight osmolarity of from about 100 mOsm / kg to about 800 mOsm / kg.

44. The cryoprotective formulation or pharmaceutical composition according to claim 43, wherein the cryoprotective formulation or pharmaceutical composition has a weight osmolarity of from about 200 mOsm / kg to about 600 mOsm / kg.

45. The cryoprotective formulation or pharmaceutical composition according to claim 43, wherein the cryoprotective formulation or pharmaceutical composition has a weight osmolarity of less than about 400 mOsm / kg.

46. The cryoprotective formulation or pharmaceutical composition according to claim 43, wherein the cryoprotective formulation or pharmaceutical composition has a weight osmolarity of about 200 mOsm / kg.

47. The cryoprotective formulation or pharmaceutical composition according to claim 43, wherein the cryoprotective formulation or pharmaceutical composition has a weight osmolarity of about 350 mOsm / kg.

48. A cryoprotective formulation as claimed in any one of claims 1 or 3 - 47 or a pharmaceutical composition as claimed in claims 2 - 47, wherein the cryoprotective formulation or pharmaceutical composition has a pH of from about 7.0 to about 8.

0.

49. A cryoprotective formulation or pharmaceutical composition as claimed in claim 48, wherein the cryoprotective formulation or pharmaceutical composition has a pH of about 7.

5.

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

51. A cryoprotective formulation or pharmaceutical composition as claimed in 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. A cryoprotective formulation as claimed in any one of claims 1 or 38 - 49 or a pharmaceutical composition as claimed in any one of claims 2 or 38 - 49, wherein the cryoprotective formulation or pharmaceutical composition comprises (1) from about 1% (w / v) to about 20% (w / v) trehalose, (2) from about 10 mM to about 250 mM magnesium sulfate, (3) from about 1 mM to about 20 mM Tris, and (4) from 0.001% (w / v) to about 1% (w / v) polysorbate.

53. A cryoprotective formulation or pharmaceutical composition as claimed in 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. A cryoprotective formulation as claimed in any one of claims 1 or 3 - 53 or a pharmaceutical composition as claimed in claims 2 - 53, the cryoprotective formulation or pharmaceutical composition further comprising one or more of the following: medicaments, culture media, proteins, or combinations thereof.

55. A cryoprotective formulation as claimed in any one of claims 1 or 3 - 54 or a pharmaceutical composition as claimed in 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. A cryoprotective formulation as claimed in any one of claims 1 or 3 - 54 or a pharmaceutical composition as claimed in claims 2 - 54, wherein the AAV particles comprise a genome derived from AAV serotype 2.

57. A cryoprotective formulation as claimed in any one of claims 1, 3 - 54 or 56 or a pharmaceutical composition as claimed in claims 2 - 54 or 56, wherein the AAV particles comprise a capsid derived from AAV serotype 2.

58. A method of storing AAV particles or maintaining the viability and / or infectivity of AAV particles at different temperatures, the method comprising storing the AAV particles in a cryoprotective formulation as claimed in any one of claims 1 or 3 - 54 or a pharmaceutical composition as claimed in claims 2 - 54.

59. The method as claimed in 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. The method as claimed in claim 58, wherein the AAV particles comprise a genome derived from AAV serotype 2.

61. The method as claimed in claim 58 or 60, wherein the AAV particles comprise a capsid derived from AAV serotype 2.

62. The method as claimed in any one of claims 58 - 61, wherein the AAV particles are stable during multiple freeze - thaw cycles.

63. The method as claimed in any one of claims 58 - 61, wherein the AAV particles are stable during at least 5 freeze - thaw cycles.

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

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

66. The method as claimed in claim 65, wherein the AAV particles are stable at +4°C for at least six months.

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

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