Adeno-associated viral vector formulations and methods

Abstract

To provide improved formulations comprising recombinant AAV particles.SOLUTION: A stable formulation comprises recombinant adeno-associated virus (rAAV) particles and a) Tris, b) a sugar, and c) an amorphous salt, wherein the formulation is suitable for lyophilization, and the rAAV particles include AAV9 capsid protein.SELECTED DRAWING: None

Description

[Technical field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application is a joint venture with U.S. Provisional Patent Application No. 62 / 836,115, filed April 19, 2019. No. 6,399,433, filed on May 13, 2003, which is incorporated herein by reference in its entirety.

[0002] References to Electronically Submitted Sequence Listings This application is incorporated by reference in its sequence listing, which is submitted herewith as a text file. The text file will have the name "12656-131-228_Seque nce_Listing.txt", creation date April 16, 2020, and file size It is 70,092 bytes.

[0003] 2. Introduction Stable formulations of recombinant adeno-associated virus (rAAV) products and methods for their formulation are described. The formulations and methods provide for the delivery of therapeutic rAAV products to humans to treat a variety of diseases and disorders. Suitable for delivery to elephants. [Background technology]

[0004] 3. Background of the invention Recombinant adeno-associated virus (AAV) vectors are being developed as gene therapy products The rAAV vector system is currently the most widely used. This is due, in part, to the lack of disease associated with wild-type virus and the lack of differentiation of dividing cells. The ability of AAV to transduce not only mitotic but also non-dividing cells, and the long-term robust transduction observed in clinical trials The observed results in gene expression suggest that this may be a valuable delivery strategy in gene therapy applications. In addition, natural and recombinant rAAV vectors have been shown to be serotype specific. Differentiated, they can specifically target different tissues, organs, and cells and provide resistance to vectors. This will help the virus evade any existing immune response to AAV, making AAV-based gene therapy a promising option. Therapeutic uses are expanding.

[0005] AAV products must be manufactured, transported, stored, and administered without fail, as well as demonstrated to be safe and effective. It must remain stable and powerful throughout the life of the drug. If commercialized, the transport or storage requirements may be ambient temperature or under refrigerated conditions. We have identified formulations that provide long-term stability, whether in storage or under frozen conditions. In the case of rAAV production, the formulation can be induced by various conditions. The product must be able to withstand the stresses that are introduced into it and still maintain its quality. Thus, improved formulations containing recombinant AAV particles are needed. . Summary of the Invention

[0006] The present disclosure relates to recombinant adeno-associated virus (rAAV) particles, as well as buffers, cryoprotectants, and and a lyoprotectant excipient, and an amorphous salt. Provided herein are recombinant adeno-associated virus (rAAV) particles, as well as A stable formulation comprising a buffer, a sugar, and an amorphous salt. In some embodiments, the formulation is , suitable for freeze-drying.

[0007] In some embodiments, the buffer comprises Tris, sucrose, and less than about 100 mM quercetin. In some embodiments, the formulation further comprises a non-ionic surfactant, For example, poloxamer 188. In some embodiments, the formulation further comprises a plasticizer or In some embodiments, the rAAV comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16, rAAV.rh8, rAAV.rh10, rAAV.rh20, rA AV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, r AAV.Anc80, rAAV.Anc80L65, rAAV.7m8, rAAV.PH PB, rAAV2.5, rAAV2tYF, rAAV3B, rAAV.LK03, AA V.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HS C5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, A AV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, A AV.HSC14, AAV.HSC15, or AAV.HSC16 capsid protein In some embodiments, the rAAV comprises a capsule of the AAV-8 or AAV-9 serotype. Contains cytoprotein.

[0008] The present disclosure relates to recombinant adeno-associated virus (rAAV) particles, as well as buffers, sugars, and and a salt comprising sodium citrate. In some embodiments, the buffering agent is , Tris, sucrose, and less than about 100 mM sodium citrate. In some cases, the formulation further comprises a non-ionic surfactant, e.g., poloxamer 188. In some embodiments, the formulation further comprises a plasticizer or stabilizer, such as glycerol. In some embodiments, the rAAV comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AA V-12, AAV-13, AAV-14, AAV-15 and AAV-16, rAAV.r h8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh 74, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV. Anc80L65, rAAV.7m8, rAAV.PHP.B, rAAV2.5, rAA V2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2 , AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV .HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HS C11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HS In some embodiments, the capsid protein comprises a capsid protein of AAV.HSC15, or AAV.HSC16. The rAAV contains capsid proteins of the AAV-8 or AAV-9 serotypes.

[0009] In some embodiments, the formulation is suitable for lyophilization. The composition is a freeze-dried version of the liquid composition disclosed herein. In some cases, the pharmaceutical composition is a reconstituted lyophilized formulation.

[0010] In some embodiments, the formulation is a liquid formulation. In some embodiments, the formulation is a frozen formulation. In some embodiments, the formulation is a lyophilized formulation of the liquid formulation disclosed herein. In some embodiments, the formulation is a reconstituted lyophilized formulation.

[0011] In some embodiments, the formulation is a lyophilized formulation having a residual moisture content of about 1% to about 7%. .

[0012] The present disclosure also provides methods for producing stable formulations containing recombinant adeno-associated virus (rAAV) particles. do.

[0013] The present disclosure also provides methods for reducing rAAV genome release from rAAV particles.

[0014] The present disclosure also provides the use of sugars to reduce rAAV genome release from rAAV particles. do.

[0015] The present disclosure also relates to the use of plasticizers to reduce rAAV genome release from rAAV particles. provide.

[0016] The present disclosure relates to a method for treating a disease or disorder in a subject in need thereof. Also provided is a method for administering a formulation comprising the recombinant AAV particles disclosed herein. Includes:

[0017] In some embodiments, the present disclosure provides: [1] Recombinant adeno-associated virus (rAAV) particles, and a) a buffering agent, b) sugar, and c) Salts containing sodium citrate A stable formulation comprising:

[0018] [2] The formulation according to [1], wherein the buffer comprises about 1 mM to about 50 mM Tris.

[0019] [3] About 1mM to about 30mM, about 1mM to about 20mM, about 5mM to about 30mM, about 5m M to about 20 mM, about 10 mM to about 30 mM, about 10 mM to about 20 mM, or about 20 mM The formulation described in [2], comprising about 50 mM Tris.

[0020] [4] About 1mM, about 2mM, about 3mM, about 5mM, about 10mM, about 15mM, about 20m [2] The formulation of [2], comprising about 25 mM, about 30 mM, or about 40 mM Tris. .

[0021] [5] The formulation of [2], comprising about 5 mM Tris.

[0022] [6] The formulation according to any one of [1] to [4], wherein the pH is about 6.5 to 8.0. .

[0023] [7] The formulation described in [6], having a pH of about 7.2 to 7.8.

[0024] [8] The pH is approximately 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8. The formulation described in [6].

[0025] [9] The formulation described in [6], having a pH of about 7.5.

[0026]

[10] A method according to any one of [1] to [9], comprising: The formulation described above.

[0027]

[11] About 50mM to about 350mM, about 50mM to about 300mM, about 50mM to about 25 The formulation according to

[0010] , comprising 0 mM, about 50 mM to about 200 mM, or about 50 mM to about 150 mM sugar.

[0028]

[12] About 100 mM to about 400 mM, about 150 mM to about 400 mM, about 200 mM to About 400 mM, about 250 mM to about 400 mM, or about 300 mM to about 400 mM sugar The formulation described in

[10] , which comprises

[0029]

[13] Approx. 100mM~Approx. 300mM, Approx. 150mM~Approx. 250mM, Approx. 200mM~ The formulation according to

[10] , comprising about 300 mM, or about 250 mM to about 350 mM sugar.

[0030]

[14] Approx. 50mM, approx. 100mM, approx. 150mM, approx. 160mM, approx. 170mM, approx. 180mM, approx. 190mM, approx. 200mM, approx. 210mM, approx. 220mM, approx. 230mM , about 240mM, about 250mM, about 260mM, about 270mM, about 280mM, about 290 The formulation of

[10] , comprising about 300 mM, about 300 mM, or about 350 mM sugar.

[0031]

[15] About 190 mM to about 230 mM, about 170 mM to about 250 mM, or about 150 mM The formulation according to

[10] , comprising from 2 mM to about 270 mM sugar.

[0032]

[16] The formulation of

[10] , comprising about 210 mM sugar.

[0033]

[17] The formulation according to any one of [1] to

[16] , wherein the sugar is a non-reducing sugar. .

[0034]

[18] The formulation described in

[0017] , wherein the non-reducing sugar is sucrose, trehalose, or raffinose.

[0035]

[19] The formulation described in

[17] , wherein the non-reducing sugar is sucrose.

[0036]

[20] The formulation according to any one of [1] to

[16] , wherein the sugar is a reducing sugar.

[0037]

[21] The reducing sugar is glucose, fructose, mannose, galactose, or

[20] The formulation described in

[20] , wherein

[0038]

[22] The formulation described in

[20] , wherein the reducing sugar is glucose.

[0039]

[23] Any of [1] to

[22] containing less than about 100 mM sodium citrate. or the formulation described in one of the above.

[0040]

[24] The composition according to

[23] , comprising about 10 mM to about 100 mM sodium citrate. Agent.

[0041]

[25] About 10 mM to about 100 mM, about 20 mM to about 100 mM, about 30 mM to about 10 0 mM, about 40 mM to about 100 mM, about 50 mM to about 100 mM, about 10 mM to about 80 mM M, about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, The formulation according to

[23] , comprising about 10 mM to about 30 mM sodium citrate.

[0042]

[26] About 10mM to about 50mM, about 20mM to about 60mM, about 30mM to about 70mM or about 10 mM to about 30 mM sodium citrate.

[0043]

[27] About 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, or about 6 0 mM sodium citrate.

[0044]

[28] The formulation of

[23] , comprising about 20 mM sodium citrate.

[0045]

[29] Further, about 0.0005% to about 0.01% of a nonionic surfactant is contained, The formulation according to any one of claims 1 to 28.

[0046]

[30] The formulation of

[29] , comprising about 0.002% nonionic surfactant.

[0047]

[31] The nonionic surfactant is poloxamer 188, poloxamer 407, poloxamer Resorbate 80, Polysorbate 20, Pluronic F-68, or BRIJ The formulation described in

[28] or

[29] , including 35.

[0048]

[32] The method according to

[31] , wherein the nonionic surfactant comprises poloxamer 188. formulation.

[0049]

[33] Recombinant adeno-associated virus (rAAV) particles, and a) about 1 mM to about 25 mM Tris, b) about 50 mM to about 400 mM sugar; c) about 10 mM to about 100 mM sodium citrate, and d) about 0.0005% to about 0.01% of a nonionic surfactant; A stable formulation comprising: The formulation has a pH of about 7.2 to about 7.8.

[0050]

[34] Recombinant adeno-associated virus (rAAV) particles, and a) about 2 mM to about 10 mM Tris, b) about 150 mM to about 250 mM sugar; c) about 10 mM to about 20 mM sodium citrate, and d) about 0.001% to about 0.005% of a nonionic surfactant; A stable formulation comprising: The formulation has a pH of about 7.2 to about 7.8.

[0051]

[35] Recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sugar; c) about 20 mM sodium citrate, and d) about 0.002% of a nonionic surfactant; A stable formulation comprising: The formulation has a pH of about 7.5.

[0052]

[36] The method according to any one of

[33] to

[35] , wherein the sugar is a non-reducing sugar. Agent.

[0053]

[37] The formulation described in

[0036] , wherein the non-reducing sugar is sucrose, trehalose, or raffinose.

[0054]

[38] The formulation described in

[17] , wherein the non-reducing sugar is sucrose.

[0055]

[39] Approximately 1.0E+11 genome copies / mL (GC / mL) and approximately 1.0E+15G A formulation described in any one of [1] to

[38] , comprising C / mL of rAAV particles.

[0056]

[40] Approx. 1.0E+11GC / mL, approx. 1.0E+12GC / mL, approx. 1.0E+1 rA of 3GC / mL, about 1.0E+14GC / mL, or about 1.0E+15GC / mL The formulation described in

[39] , comprising AV particles.

[0057]

[41] The rAAV particles may be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16, rAAV.rh8, rA AV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rA AV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80 L65, rAAV.7m8, rAAV.PHP.B, rAAV2.5, rAAV2tYF , rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV. HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7 , AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, A AV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 capsid protein, any one of [1] to

[40] The formulation described in one.

[0058]

[42] The rAAV particles comprise a capsid protein comprising the AAV-8 or AAV-9 serotype. The formulation described in

[41] , which contains a protein.

[0059]

[43] In addition, glycerol, xylitol, sorbitol, or mannitol were used. The formulation according to any one of [1] to

[42] , further comprising a plasticizer selected from the group consisting of: .

[0060]

[44] The process according to any one of [1] to

[42] , further comprising glycerol. Agent.

[0061]

[45] The formulation of

[44] , comprising about 0.1% to about 5% glycerol.

[0062]

[46] The formulation of

[44] , comprising about 0.1% to about 2% glycerol.

[0063]

[47] The formulation of

[44] , comprising about 0.25% to about 2% glycerol.

[0064]

[48] ​​The formulation described in any one of [1] to

[47] , which is a liquid formulation.

[0065]

[49] The formulation according to any one of [1] to

[47] , which is a frozen formulation.

[0066]

[50] A lyophilized preparation or a reconstituted lyophilized preparation, from [1] to

[47] The formulation according to any one of the preceding claims.

[0067]

[51] The formulation according to

[50] , wherein the residual moisture content is about 1% to about 7%.

[0068]

[52] The residual moisture content is about 1% to about 7%, about 2% to about 7%, about 3% to about 7%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to about 10%, about 10% to about 12%, about 10% to about 14%, about 10% to about 15%, about 1 % to about 7%, about 5% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, and The formulation described in

[51] , wherein the content is about 1% to about 3%.

[0069]

[53] The residual moisture content is about 3% to about 7%, about 3% to about 6%, or about 3% to about 5%. The formulation according to

[51] ,

[0070]

[54] The residual moisture content is about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5 The formulation described in

[51] , wherein the concentration is about 0.5%, or about 6%.

[0071]

[55] The relative potency of the rAAV particles was slightly improved after storage of the formulation at room temperature for 3 months. at least about 60%, at least about 70%, or at least about 80%, but rAAV particles are cultured in Dulbecco's phosphate buffered saline containing 0.001% poloxamer 188 buffer. Stored in saline (DPBS) at -70°C, in one of [1] to

[54] The formulation described.

[0072]

[56] The relative potency of the rAAV particles was slightly improved after storage of the formulation at room temperature for 6 months. at least about 60%, at least about 70%, or at least about 80%, but rAAV particles were incubated at -70°C in DPBS containing 0.001% poloxamer 188 buffer. A formulation described in any one of [1] to

[54] , which is stored.

[0073]

[57] The relative potency of the rAAV particles was measured after storing the formulation at 35° C. for 1 week. at least about 30%, at least about 40%, at least about 50%, or at least 60% where the reference rAAV particles are DP The formulation according to any one of [1] to

[54] , stored in BS at -70°C.

[0074]

[58] The relative potency of the rAAV particles was measured after storage of the formulation at 35°C for 2 weeks. at least about 30%, at least about 40%, at least about 50%, or at least 60% where the reference rAAV particles are DP The formulation according to any one of [1] to

[54] , stored in BS at -70°C.

[0075]

[59] The relative potency of the rAAV particles was measured after storage of the formulation at 35° C. for 4 weeks. at least about 30%, at least about 40%, at least about 50%, or at least 60% where the reference rAAV particles are DP The formulation according to any one of [1] to

[54] , stored in BS at -70°C.

[0076]

[60] A method for producing a stable formulation containing recombinant adeno-associated virus (rAAV) particles, , the rAAV particles are combined with a buffer, sugar, salt, or the like of the formulation described in any one of [1] to

[47] . Optionally, the rAAV is mixed with a plasticizer and, optionally, a non-ionic surfactant. and producing the formulation comprising:

[0077]

[61] A method for reducing rAAV genome release from rAAV particles, comprising: preparing a formulation comprising a buffer, a sugar, a salt, and optionally a non-ionic surfactant; Release of the rAAV genome from the rAAV particles after three freeze-thaw cycles was The method of claim 1, wherein rAAV genome release is reduced compared to that in a preparation not containing the rAAV.

[0078]

[62] A method for reducing rAAV genome release from rAAV particles, comprising: preparing a formulation comprising a buffer, a sugar, a salt, and optionally a non-ionic surfactant; Release of the rAAV genome from the rAAV particles after lyophilization and reconstitution was achieved without the sugar. The method further comprises:

[0079]

[63] Furthermore, the preparation is freeze-dried to reduce the residual water content to about 1% to about 5%. The formulation according to

[61] or

[62] ,

[0080]

[64] A method for reducing rAAV genome release from rAAV particles, comprising: Producing a formulation containing a buffer, a sugar, a salt, a plasticizer, and optionally a non-ionic surfactant. and the rAAV genome release from the rAAV particles after three freeze-thaw cycles is The method, wherein rAAV genome release is reduced compared to that in a sugar-free formulation.

[0081]

[65] A method for reducing rAAV genome release from rAAV particles, comprising: Producing a formulation containing a buffer, a sugar, a salt, a plasticizer, and optionally a non-ionic surfactant. and release of the rAAV genome from the rAAV particles after lyophilization and reconstitution is controlled by the presence of the glycosylation agent. The method of claim 1, wherein the rAAV genome release is reduced compared to a formulation not containing the rAAV genome release.

[0082]

[0023]

[0024] The use of sugars to reduce rAAV genome release from rAAV particles. A formulation is prepared comprising rAAV particles, a buffer, a sugar, a salt, and optionally a non-ionic surfactant. and producing an rAAV genome from the rAAV particles after three freeze-thaw cycles. The release is reduced compared to the release of the rAAV genome in a formulation that does not contain the sugar. use.

[0083]

[67] Use of sugars to reduce rAAV genome release from rAAV particles. A formulation is prepared comprising rAAV particles, a buffer, a sugar, a salt, and optionally a non-ionic surfactant. and releasing the rAAV genome from the rAAV particles after lyophilization and reconstitution. is reduced compared to rAAV genome release in a formulation that does not contain the sugar. .

[0084]

[68] Furthermore, the preparation is freeze-dried to reduce the residual water content to about 1% to about 7%. The use according to

[66] or

[67] , comprising:

[0085]

[69] The use of plasticizers to reduce rAAV genome release from rAAV particles. The rAAV particles, buffer, sugar, salt, plasticizer, and optionally a non-ionic surfactant are then mixed together. and producing a formulation comprising: AAV genome release was reduced compared to rAAV genome release in the sugar-free formulation. The method according to claim 1,

[0086]

[70] The use of plasticizers to reduce rAAV genome release from rAAV particles. The rAAV particles, buffer, sugar, salt, plasticizer, and optionally a non-ionic surfactant are then mixed together. and producing a formulation comprising rAA V from the rAAV particles after lyophilization and reconstitution. V genome release was reduced compared to rAAV genome release in the sugar-free formulation. The above-mentioned use.

[0087]

[71] rAAV genome release is measured by measuring relative fluorescence in the presence of a DNA-specific fluorescent stain. The method according to any one of

[61] to

[65] or the use according to any one of

[0066] to

[70] , characterized by the

[0088]

[72] Freezing-induced rAAV genome release was at least approximately 10%, 20%, or A decrease of 50%, 80%, or 90% in any of

[61] to

[65] and

[71] The method according to any one of claims 1 to 7 or the use according to any one of claims 66 to 71.

[0089]

[73] The method according to any one of

[0061] to

[65] and

[71] , or

[66] to

[71] , in which freezing-induced rAAV genome release is substantially eliminated. 2. The use according to any one of the preceding claims.

[0090]

[74] The sugar is a non-reducing sugar,

[61] to

[65] and

[71] to

[73] . or the use according to any one of

[66] to

[73] .

[0091]

[75] The method according to

[0074] or the use according to

[74] , wherein the non-reducing sugar is sucrose, trehalose, or raffinose.

[0092]

[76] The method according to

[74] or the method according to

[74] , wherein the non-reducing sugar is sucrose. Use as described.

[0093]

[77] The sugar according to any one of

[61] to

[65] and

[71] to

[73] , wherein the sugar is a reducing sugar. 2. The method according to any one of claims 1 to 11 or the use according to any one of claims 1 to 11, wherein the method is selected from the group consisting of:

[0094]

[78] The reducing sugar may be glucose, fructose, mannose, galactose, or The method according to

[77] or the use according to

[77] , wherein

[0095]

[79] The method according to

[77] or the method according to

[77] , wherein the reducing sugar is glucose. Use of.

[0096]

[80] The plasticizer comprises glycerol.

[79] or any one of the methods described in

[66] to

[79] Use of.

[0097]

[81] The preparation is any one of [1] to

[47] , any one of

[0061] to

[65] and

[71] to

[80] , or any one of

[66] to

[67] .

[80] .

[0098] In some embodiments, the methods disclosed herein involve the use of recombinant adeno-associated viruses (rAAVs). The present invention relates to a method for producing a stable formulation comprising rAAV particles, the rAAV particles being isolated from a supply containing impurities. by isolating rAAV particles from a feed (e.g., an rAAV-producing culture); The method for isolating rAAV particles includes one or more processing steps. For purposes of this specification, processing may include harvesting the cell culture, clarifying the harvested cell culture (e.g., by centrifugation or deep distillation), layer filtration), tangential flow filtration, affinity chromatography, Anion exchange chromatography, cation exchange chromatography, size exclusion chromatography at least one of the following: chromatography, hydrophobic interaction chromatography, and sterile filtration. In a further embodiment, the processing comprises harvesting the cell culture, clarifying the harvested cell culture, purification (e.g., by centrifugation or depth filtration), tangential flow filtration, affinity -chromatography, anion exchange chromatography, cation exchange chromatography size exclusion chromatography, hydrophobic interaction chromatography, and sterile filtration. At least two, at least three, at least four, at least five, or at least In some embodiments, the processing does not include centrifugation of the harvested cell culture. stomach.

[0099] The present disclosure provides a method for producing a stable formulation comprising isolated recombinant adeno-associated virus (rAAV) particles. The present invention provides a method for the preparation of a cellulose membrane comprising the steps of: (a) centrifugation, depth filtration, tangential flow filtration, ultrafiltration, filtration, affinity chromatography, size exclusion chromatography, ion exchange chromatography and hydrophobic interaction chromatography. and isolating rAAV particles from an impure feed, as well as isolating the isolated rAAV particles. and formulating the compound to produce a stable formulation.

[0100] The present disclosure provides a pharmaceutical unit of a stable formulation comprising isolated recombinant adeno-associated virus (rAAV) particles. A method for producing a dosage form is provided, the method comprising: (a) filtering the dosage form by centrifugation, depth filtration, tangential flow filtration, or the like; - Filtration, ultrafiltration, affinity chromatography, size exclusion chromatography , ion exchange chromatography, and hydrophobic interaction chromatography. and isolating the rAAV particles from an impure feed by one or more of the following: and formulating the rAAV particles.

[0101] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate, and d) about 0.002% (w / v) poloxamer 188; It is a stable formulation comprising:

[0102] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate; d) about 0.002% (w / v) poloxamer 188, and e) about 0.25% (w / v) glycerol; It is a stable formulation comprising:

[0103] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate; d) about 0.002% (w / v) poloxamer 188, and e) about 0.5% (w / v) sorbitol; It is a stable formulation comprising:

[0104] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) about 5 mM Tris, b) about 30 mM sodium sulfate; c) about 263 mM sucrose, and d) about 0.005% (w / v) poloxamer 188; It is a stable formulation comprising:

[0105] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) Tris, b) sucrose, c) sodium citrate, and d) Poloxamer 188 and A stable formulation comprising: However, the formulation has an ionic strength of 60 mM to 150 mM.

[0106] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) Tris, b) sucrose, c) sodium citrate, d) Poloxamer 188, and e) glycerol and A stable formulation comprising: However, the formulation has an ionic strength of 60 mM to 150 mM.

[0107] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) Tris, b) sucrose, c) sodium citrate, d) Poloxamer 188, and e) Sorbitol A stable formulation comprising: However, the formulation has an ionic strength of 60 mM to 150 mM.

[0108] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) Tris, b) sodium sulfate, c) sucrose, and d) Poloxamer 188 and A stable formulation comprising: However, the formulation has an ionic strength of 60 mM to 150 mM.

[0109] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate, and d) about 0.002% (w / v) poloxamer 188; It is a stable formulation comprising:

[0110] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate; d) about 0.002% (w / v) poloxamer 188, and e) about 0.25% (w / v) glycerol; It is a stable formulation comprising:

[0111] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate; d) about 0.002% (w / v) poloxamer 188, and e) about 0.5% (w / v) sorbitol; It is a stable formulation comprising:

[0112] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) about 5 mM Tris, b) about 30 mM sodium sulfate; c) about 263 mM sucrose, and d) about 0.005% (w / v) poloxamer 188; It is a stable formulation comprising:

[0113] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) Tris, b) sucrose, c) sodium citrate, and d) Poloxamer 188 and A stable formulation comprising: However, the formulation has an ionic strength of 60 mM to 150 mM.

[0114] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) Tris, b) sucrose, c) sodium citrate, d) Poloxamer 188, and e) glycerol and A stable formulation comprising: However, the formulation has an ionic strength of 60 mM to 150 mM.

[0115] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) Tris, b) sucrose, c) sodium citrate, d) Poloxamer 188, and e) Sorbitol A stable formulation comprising: However, the formulation has an ionic strength of 60 mM to 150 mM.

[0116] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) Tris, b) sodium sulfate, c) sucrose, and d) Poloxamer 188 and A stable formulation comprising: However, the formulation has an ionic strength of 60 mM to 150 mM.

[0117] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate, and d) about 0.002% (w / v) poloxamer 188; It is a stable formulation comprising:

[0118] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate; d) about 0.002% (w / v) poloxamer 188, and e) about 0.25% (w / v) glycerol; It is a stable formulation comprising:

[0119] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate; d) about 0.002% (w / v) poloxamer 188, and e) about 0.5% (w / v) sorbitol; It is a stable formulation comprising:

[0120] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) about 5 mM Tris, e) about 30 mM sodium sulfate; f) about 263 mM sucrose, and g) about 0.005% (w / v) poloxamer 188; It is a stable formulation comprising:

[0121] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) Tris, b) sucrose, c) sodium citrate, and d) Poloxamer 188 and A stable formulation comprising: However, the formulation has an ionic strength of 60 mM to 150 mM.

[0122] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) Tris, b) sucrose, c) sodium citrate, d) Poloxamer 188, and e) glycerol and A stable formulation comprising: However, the formulation has an ionic strength of 60 mM to 150 mM.

[0123] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) Tris, b) sucrose, c) sodium citrate, d) Poloxamer 188, and e) Sorbitol A stable formulation comprising: However, the formulation has an ionic strength of 60 mM to 150 mM.

[0124] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) Tris, b) sodium sulfate, c) sucrose, and d) Poloxamer 188 and A stable formulation comprising: However, the formulation has an ionic strength of 60 mM to 150 mM.

[0125] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) Tris, b) sucrose, c) sodium citrate, and d) Poloxamer 188 and A stable formulation comprising: However, the formulation has an ionic strength of less than 200 mM.

[0126] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) Tris, b) sucrose, c) sodium citrate, d) Poloxamer 188, and e) glycerol and A stable formulation comprising: However, the formulation has an ionic strength of less than 200 mM.

[0127] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) Tris, b) sucrose, c) sodium citrate, d) Poloxamer 188, and e) Sorbitol A stable formulation comprising: However, the formulation has an ionic strength of less than 200 mM.

[0128] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) Tris, b) sodium sulfate, c) sucrose, and d) Poloxamer 188 and A stable formulation comprising: However, the formulation has an ionic strength of less than 200 mM.

[0129] In certain embodiments, the stable formulation does not include mannitol.

[0130] In certain embodiments, the stable formulation comprises about 150 mM, about 140 mM, about 130 mM M, approx. 120mM, approx. 110mM, approx. 100mM, approx. 90mM, approx. 80mM, approx. 70mM , about 60 mM, about 50 mM, about 40 mM, about 30 mM, about 20 mM, or less than about 10 mM Contains 100% mannitol.

[0131] In certain embodiments, the stable formulation comprises sucrose at a concentration of about 210 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 263 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 409 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 14.6 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 45 mM. In one embodiment, the stable formulation comprises sucrose at a concentration of about 0 mM to about 20 mM. In certain embodiments, the stable formulation contains sucrose at a concentration of about 20 mM to about 50 mM. In certain embodiments, the stable formulation comprises about 50 mM to about 100 mM sucrose. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 100 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 200 mM. In certain embodiments, the stable formulation comprises: In certain embodiments, the sucrose is present at a concentration of about 300 mM to about 400 mM. The stable formulation contains sucrose at a concentration of about 400 mM to about 500 mM. In one embodiment, the stable formulation contains sucrose at a concentration of about 500 mM to about 600 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 600 mM to about 700 mM. Including.

[0132] In certain embodiments, the stable formulation comprises sorbitol at a concentration of about 0.5%. In certain embodiments, the stable formulation comprises sorbitol at a concentration of about 0.25%. In certain embodiments, the stable formulation comprises sorbitol at a concentration of about 0.10%.

[0133] In certain embodiments, the stable formulation contains sorbitol at a concentration of about 0% to about 0.10%. In certain embodiments, the stable formulation contains from about 0.10% to about sorbitol. In certain embodiments, the stable formulation contains sorbitol at a concentration of about 0.20%. In certain embodiments, the stable formulation comprises a concentration of about 0.20% to about 0.30%. In certain embodiments, the composition further comprises sorbitol at a concentration of about 0.30% to about 0.40%. The stable formulation contains sorbitol at a concentration of about 0.40% to about 0.50%. In one embodiment, the stable formulation contains sorbitol at a concentration of about 0.50% to about 0.60%. In certain embodiments, the stable formulation contains about 0.60% to about 0.70% sorbitol. In certain embodiments, the stable formulation contains sorbitol at a concentration of about 0.70%. In certain embodiments, the stable formulation comprises sorbitol at a concentration of about 0.80%. In certain embodiments, the stable formulation comprises , containing sorbitol at a concentration of about 0.90% to about 1.00%.

[0134] In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.5%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.25%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.10%.

[0135] In certain embodiments, the stable formulation contains glycerol at a concentration of about 0% to about 0.10%. In certain embodiments, the stable formulation contains glycerol at about 0.10% to about In certain embodiments, the stable formulation contains glycerol at a concentration of about 0.20%. In certain embodiments, the stable formulation comprises 0.20% to about 0.30%. Glycerol at a concentration of about 0.30% to about 0.40%. The stable formulation contains glycerol at a concentration of about 0.40% to about 0.50%. In one embodiment, the stable formulation contains glycerol at a concentration of about 0.50% to about 0.60%. In certain embodiments, the stable formulation contains about 0.60% to about 0.70% glycerol. In certain embodiments, the stable formulation contains glycerol at a concentration of about 0.70%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.80%. In certain embodiments, the stable formulation comprises , containing glycerol at a concentration of about 0.90% to about 1.00%.

[0136] In certain embodiments, the stable formulation contains about 0.001% (by weight) poloxamer 188. In certain embodiments, the stable formulation comprises: It contains poloxamer 188 at a concentration of approximately 0.002% (weight / volume, 0.02 g / L). In certain embodiments, the stable formulation contains poloxamer 188 at about 0.005% (w / w) Containing a concentration of 0.05 g / L (by volume).

[0137] In certain embodiments, the stable formulation contains poloxamer 188 at about 0.0005% ( At concentrations of about 0.005 g / L (weight / volume) to about 0.05% (weight / volume, 0.5 g / L) In certain embodiments, the stable formulation comprises about 0.0001% poloxamer 188. % (weight / volume, 0.001 g / L) to approximately 0.01% (weight / volume, 0.1 g / L) concentration In certain embodiments, the stable formulation contains poloxamer 188 at about 0.00 0.05% (weight / volume, 0.005 g / L) to approximately 0.001% (weight / volume, 0.01 g / In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.001% (weight / volume, 0.01 g / L) to approximately 0.05% (weight / volume, 0.5 g / L) In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about In certain embodiments, the concentration is 0.0005% (weight / volume, 0.005 g / L). In this study, the stable formulation contained approximately 0.0006% (weight / volume, 0.006 In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of 100 g / L. at a concentration of about 0.0007% (weight / volume, 0.007 g / L). In this embodiment, the stable formulation contains poloxamer 188 at approximately 0.0008% (weight / volume, 0.0 In certain embodiments, the stable formulation contains poloxamer 1.08 g / L. 88 at a concentration of approximately 0.0009% (weight / volume, 0.009 g / L). In an embodiment, the stable formulation contains poloxamer 188 at about 0.001% (weight / volume, 0. In certain embodiments, the stable formulation contains poloxamer 1.01 g / L. 88 at a concentration of about 0.002% (weight / volume, 0.02 g / L). In this embodiment, the stable formulation contains poloxamer 188 at approximately 0.003% (weight / volume, 0.03 In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of 100 g / L. In certain embodiments, the composition comprises a concentration of about 0.004% (weight / volume, 0.04 g / L). In this study, the stable formulation contained approximately 0.005% (weight / volume, 0.05 g / mL) poloxamer 188. In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about In certain embodiments, the concentration is 0.01% (weight / volume, 0.1 g / L). The stable formulation contains poloxamer 188 at a concentration of approximately 0.05% (weight / volume, 0.5 g / L). include.

[0138] In some embodiments, the present disclosure provides a method for the preparation of a recombinant adeno-associated virus (AAV), a salt excipient, a sugar In some embodiments, the stable formulation also includes a surfactant. In some embodiments, the stable formulation also includes poloxamer 188. The agent is suitable for lyophilization.

[0139] In some embodiments, provided herein are recombinant adeno-associated viruses (rAA viruses). V) Particles, as well as buffers, sugars, and amorphous materials having an ionic strength of 60 mM to 150 mM. and a salt thereof, the formulation being suitable for lyophilization. In some embodiments, the stable formulation also includes a surfactant. Also includes 8. In some embodiments, the formulation is a pre-lyophilized formulation.

[0140] In some embodiments, provided herein are recombinant adeno-associated viruses (rAA viruses). V) Particles, as well as buffers, sugars, and amorphous materials having an ionic strength of 30 mM to 100 mM. and a salt thereof, the formulation being suitable for lyophilization. In some embodiments, the stable formulation also includes a surfactant. Also includes 8. In some embodiments, the formulation is a pre-lyophilized formulation.

[0141] In some embodiments, provided herein are recombinant adeno-associated viruses (rAA viruses). V) particles, as well as a buffer, a sugar, and an amorphous salt having an ionic strength of greater than 200 mM. In some embodiments, the stable formulation is suitable for lyophilization. The agent also includes a surfactant. In some embodiments, the stable formulation also includes poloxamer 188. In some embodiments, the formulation is a pre-lyophilized formulation.

[0142] In some embodiments, provided herein are recombinant adeno-associated viruses (rAA viruses). V) particles, as well as a buffer, a sugar, and an amorphous salt having an ionic strength of 60 mM to 150 mM; and a surfactant, wherein the formulation is suitable for lyophilization. In some embodiments, the stable formulation also includes a surfactant. It also contains xamer 188. In some embodiments, the formulation is a pre-lyophilized formulation.

[0143] In some embodiments, provided herein are recombinant adeno-associated viruses (rAA viruses). V) particles, as well as a buffer, a sugar, and an amorphous salt having an ionic strength of 30 mM to 100 mM; and a surfactant, wherein the formulation is suitable for lyophilization. In some embodiments, the stable formulation also includes a surfactant. It also contains xamer 188. In some embodiments, the formulation is a pre-lyophilized formulation.

[0144] In some embodiments, provided herein are recombinant adeno-associated viruses (rAA viruses). V) particles, as well as a buffer, a sugar, and an amorphous salt having an ionic strength greater than 200 mM; and and a surfactant, the formulation being suitable for lyophilization. Thus, the stable formulation also includes a surfactant. In some embodiments, the stable formulation includes poloxamer. It also contains Mer 188. In some embodiments, the formulation is a pre-lyophilized formulation.

[0145] In some embodiments, the formulations disclosed herein contain from about 0.0001% to about 0.5% In some embodiments, the formulations disclosed herein contain about 0.5% to about 10% by weight of a non-ionic surfactant. In some embodiments, the composition includes from 0.0005% to about 0.1% of a non-ionic surfactant. The formulations disclosed herein may contain about 0.001%, about 0.002%, about 0.003%, about 0.00 4%, about 0.005%, about 0.007%, or about 0.01% of a nonionic surfactant include.

[0146] In some embodiments, the formulations disclosed herein contain from about 0.0001% to about 0.5% Poloxamer 188. In some embodiments, the formulations disclosed herein contain about 0. In some embodiments, the composition described herein comprises from about 0.05% to about 0.1% poloxamer 188. The disclosed formulations include about 0.001%, about 0.002%, about 0.003%, about 0.004% , about 0.005%, about 0.007%, or about 0.01% poloxamer 188.

[0147] In some embodiments, the formulations disclosed herein contain about 0.0005% poloxamer. 188. In some embodiments, the formulations disclosed herein contain about 0.001% poly In some embodiments, the formulations disclosed herein contain about 0.0 0.02% Poloxamer 188. In some embodiments, the formulations disclosed herein include and about 0.003% poloxamer 188. In some embodiments, the compositions disclosed herein include The formulation contains about 0.004% poloxamer 188. In some embodiments, the formulation The formulation disclosed herein contains about 0.005% poloxamer 188. The formulations disclosed herein contain about 0.008% poloxamer 188.

[0148] In some embodiments, the salt is a pharma- ceutically acceptable salt. It is an amorphous salt that does not crystallize. In some embodiments, the salt is a sodium salt. In some embodiments, the salt is sodium citrate, sodium acetate, or sodium chloride. In some embodiments, the salt is sodium citrate, sodium sulfate, ammonium sulfate. In some embodiments, the salt is a multivalent salt, and the salt is a multiply charged ion with a higher ionic strength (compared to monovalent sodium chloride) The compound consists of 100% excipients per molecule, which prevents AAV aggregation while minimizing crystallization. can harm.

[0149] In some embodiments, salt excipients are added or adjusted to achieve the desired ionic strength. In some embodiments, the amorphous salt has an ionic strength of about 150 mM, about 145 mM, or about 150 mM. , about 140mM, about 135mM, about 130mM, about 125mM, about 120mM, about 115 100 mM, about 110 mM, about 110 mM, about 105 mM, or about 100 mM or less. In certain embodiments, the stable formulation has an ionic strength of about 150 mM, about 145 mM, or about 150 mM. mM, approx. 140mM, approx. 135mM, approx. 130mM, approx. 125mM, approx. 120mM, approx. 1 The concentration is about 15 mM, about 110 mM, about 105 mM, or about 100 mM or less. That is, the rAAV particles in the formulation are rAAV8 particles or rAAV9 particles.

[0150] In some embodiments, the stable formulation has an ionic strength of 150 mM, 145 mM, 140 mM , 135mM, 130mM, 125mM, 120mM, 115mM, or 110mM or more In certain embodiments, the stable formulation has a buffer ionic strength of 150 mM , 145mM, 140mM, 135mM, 130mM, 125mM, 120mM, 115 In some embodiments, the rAAV particles in the formulation have an A Contains the AV8 capsid.

[0151] In certain embodiments, the stable formulation has an ionic strength of greater than 60 mM. In one embodiment, the stable formulation has an ionic strength of about 60 mM to 150 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 115 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 100 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM. In certain embodiments, the stable formulation has an ionic strength of about 65 mM. The formulation has an ionic strength of about 70 mM. In certain embodiments, the stable formulation has an ionic strength of about 80 mM. In certain embodiments, the stable formulation has an ionic strength of about 85 mM. In embodiments, the stable formulation has an ionic strength of about 90 mM. The rAAV particles in the formulation have AAV8 capsids.

[0152] In certain embodiments, the stable formulation has an ionic strength of greater than 30 mM. In one embodiment, the stable formulation has an ionic strength of about 30 mM to 100 mM. In certain embodiments, the stable formulation has an ionic strength of about 30 mM. In certain embodiments, the stable formulation has an ionic strength of about 35 mM. In certain embodiments, the stable formulation has an ionic strength of about 40 mM. In certain embodiments, the stable formulation has an ionic strength of about 50 mM. In certain embodiments, the stable formulation has an ionic strength of about 55 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM. The stable formulation has an ionic strength of about 65 mM. In certain embodiments, the stable formulation has an ionic strength of about 70 mM. In certain embodiments, the stable formulation has an ionic strength of about 80 mM. In certain embodiments, the stable formulation has an ionic strength of about 85 mM. In certain embodiments, the stable formulation has an ionic strength of about 90 mM. The formulation has an ionic strength of about 95 mM. In certain embodiments, the stable formulation has an ionic strength of about 95 mM. In some embodiments, the rAAV particles in the formulation contain AAV9 cascade. It has a psid.

[0153] In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 150 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 115 mM. In certain embodiments, the stable formulation has an ionic strength of about 65 mM to 95 mM. In certain embodiments, the stable formulation has an ionic strength of about 70 mM to 90 mM. In certain embodiments, the stable formulation has an ionic strength of about 75 mM to 85 mM. In some embodiments, the rAAV particles in the formulation have AAV8 capsids.

[0154] In certain embodiments, the stable formulation has an ionic strength of about 30 mM to 100 mM. In certain embodiments, the stable formulation has an ionic strength of about 35 mM to 95 mM. In certain embodiments, the stable formulation has an ionic strength of about 40 mM to 90 mM. In certain embodiments, the stable formulation has an ionic strength of about 45 mM to 85 mM. In certain embodiments, the stable formulation has an ionic strength of about 50 mM to 80 mM. In certain embodiments, the stable formulation has an ionic strength of about 55 mM to 75 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 70 mM. In some embodiments, the rAAV particles in the formulation do not have AAV8 capsids. In some configurations, the rAAV particles in the formulation have AAV9 capsids.

[0155] In some embodiments, the stable formulation has an ionic strength of greater than about 200 mM. In other words, the rAAV particles in the formulation have AAV2 capsids. The rAAV particles in the agent do not have rAAV8 or rAAV9 capsids.

[0156] In some embodiments, the stable formulation has an ionic strength of 200 mM, 210 mM, 220 mM , 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, 290 In some embodiments, the rAAV particles in the formulation are In some embodiments, the rAAV particles in the formulation have an rAAVV2 capsid. It does not contain the rAAV9 capsid or the rAAV9 capsid.

[0157] In certain embodiments, the stable formulation has an ionic strength of about 200 mM to about 210 mM In certain embodiments, the stable formulation has an ionic strength of about 210 mM to about 22 mM. In certain embodiments, the stable formulation has an ionic strength of about 220 mM to about 250 mM. In certain embodiments, the stable formulation has an ionic strength of about 230 mM. In certain embodiments, the stable formulation has an ionic strength of about In certain embodiments, the stable formulation has an ionic strength of about 240 mM to about 250 mM. In certain embodiments, the stable formulation has a concentration of about 250 mM to about 260 mM. In certain embodiments, the stable formulation has an on strength of about 260 mM to about 270 mM. In a particular embodiment, the ionic strength is about 270 mM to about 280 mM. The fixed formulation has an ionic strength of about 280 mM to about 290 mM. In some embodiments, the stable formulation has an ionic strength of about 290 mM to about 300 mM. , the rAAV particles in the formulation do not have AAV8 or AAV9 capsids.

[0158] In some embodiments, the formulations disclosed herein contain from about 0.0001% to about 0.5% In some embodiments, the formulations disclosed herein contain about 0.5% to about 10% by weight of a non-ionic surfactant. In some embodiments, the composition includes from 0.0005% to about 0.1% of a non-ionic surfactant. The formulations disclosed herein may contain about 0.001%, about 0.002%, about 0.003%, about 0.00 4%, about 0.005%, about 0.007%, or about 0.01% of a nonionic surfactant include.

[0159] In some embodiments, the formulations disclosed herein contain from about 0.0001% to about 0.5% Poloxamer 188. In some embodiments, the formulations disclosed herein contain about 0. In some embodiments, the composition described herein comprises from about 0.05% to about 0.1% poloxamer 188. The disclosed formulations include about 0.001%, about 0.002%, about 0.003%, about 0.004% , about 0.005%, about 0.007%, or about 0.01% poloxamer 188.

[0160] In some embodiments, the formulations disclosed herein contain about 0.0005% poloxamer. 188. In some embodiments, the formulations disclosed herein contain about 0.001% poly In some embodiments, the formulations disclosed herein contain about 0.0 0.02% Poloxamer 188. In some embodiments, the formulations disclosed herein include and about 0.003% poloxamer 188. In some embodiments, the compositions disclosed herein include The formulation contains about 0.004% poloxamer 188. In some embodiments, the formulation The formulation disclosed herein contains about 0.005% poloxamer 188. The formulations disclosed herein contain about 0.008% poloxamer 188.

[0161] In some embodiments, the salt is a pharma- ceutically acceptable salt. It is an amorphous salt that does not crystallize. In some embodiments, the salt is a sodium salt. In some embodiments, the salt is sodium citrate, sodium acetate, or sodium chloride. In some embodiments, the salt is sodium citrate, sodium sulfate, ammonium sulfate. In some embodiments, the salt is a multivalent salt, and the salt has a higher ionic strength (per molecule of excipient added compared to monovalent sodium chloride) ), which prevents AAV aggregation while minimizing crystallization. can harm.

[0162] In some embodiments, the stable formulation has an ionic strength of about 200 mM, about 195 mM, about 180 mM, or about 200 mM. mM, 175mM, 170mM, 165mM, 160mM, 155mM, 150mM, 1 45mM, 140mM, 135mM, 130mM, 125mM, 120mM, 115mM , or 110 mM or less.

[0163] In some embodiments, salt excipients are added or adjusted to achieve the desired ionic strength. In some embodiments, the stable formulation has an ionic strength of about 200 mM, about 190 mM , about 180mM, about 170mM, about 160mM, about 155mM, about 150mM, about 145 mM, approx. 140mM, approx. 135mM, approx. 130mM, approx. 125mM, approx. 120mM, approx. 1 15 mM, or about 110 mM or less. In certain embodiments, the stable formulation comprises: The ionic strength of the buffer is about 150 mM, about 145 mM, about 140 mM, about 135 mM, about 1 30 mM, about 125 mM, about 120 mM, about 115 mM, or about 110 mM or less. In some embodiments, the rAAV particles in the formulation are rAAV8 particles. Thus, the rAAV particles in the formulation have AAV9 capsids.

[0164] In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 150 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 115 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 100 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM. In certain embodiments, the stable formulation has an ionic strength of about 65 mM. In certain embodiments, the stable formulation has an ionic strength of about 70 mM. In certain embodiments, the stable formulation has an ionic strength of about 75 mM. In certain embodiments, the stable formulation has an ionic strength of about 85 mM. In certain embodiments, the stable formulation has an ionic strength of about 90 mM. Thus, the rAAV particles in the formulation have AAV8 capsids.

[0165] In certain embodiments, the stable formulation has an ionic strength of about 30 mM to 100 mM. In certain embodiments, the stable formulation has an ionic strength of about 30 mM. In certain embodiments, the stable formulation has an ionic strength of about 35 mM. The stable formulation has an ionic strength of about 40 mM. In certain embodiments, the stable formulation has an ionic strength of about 5. 0 mM. In certain embodiments, the stable formulation has an ionic strength of about 55 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM. In certain embodiments, the stable formulation has an ionic strength of about 65 mM. The formulation has an ionic strength of about 70 mM. In certain embodiments, the stable formulation has an ionic strength of about In certain embodiments, the stable formulation has an ionic strength of about 80 mM. In certain embodiments, the stable formulation has an ionic strength of about 85 mM. In certain embodiments, the stable formulation has an ionic strength of about 90 mM. In certain embodiments, the stable formulation has an ionic strength of about 95 mM. In some embodiments, the rAAV particles in the formulation have an ionic strength of about 100 mM. Contains the AAV9 capsid.

[0166] In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 150 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 140 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 130 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 120 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 115 mM. In certain embodiments, the stable formulation has an ionic strength of about 65 mM to 95 mM. In certain embodiments, the stable formulation has an ionic strength of about 70 mM to 90 mM. In certain embodiments, the stable formulation has an ionic strength of about 75 mM to about 85 mM. In some embodiments, the rAAV particles in the formulation have an AAV8 capsid. .

[0167] In certain embodiments, the stable formulation has an ionic strength of about 30 mM to 100 mM. In certain embodiments, the stable formulation has an ionic strength of about 35 mM to 95 mM. In certain embodiments, the stable formulation has an ionic strength of about 40 mM to 90 mM. In certain embodiments, the stable formulation has an ionic strength of about 45 mM to 85 mM. In certain embodiments, the stable formulation has an ionic strength of about 50 mM to 80 mM. In certain embodiments, the stable formulation has an ionic strength of about 55 mM to 75 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 70 mM. In some embodiments, the rAAV particles in the formulation have AAV9 capsids.

[0168] In certain embodiments, the pH of the stable formulation is about 7.4.

[0169] In certain embodiments, the pH of the stable formulation is about 6.0 to 8.8. In certain embodiments, the pH of the stable formulation is about 6.0 to 9.0. In certain embodiments, the pH of the stable formulation is about 6.0. The pH is about 6.1. In certain embodiments, the pH of the stable formulation is about 6.2. In certain embodiments, the pH of the stable formulation is about 6.3. In certain embodiments, the pH of the stable formulation is about 6.4. The pH of the stable formulation is about 6.5. In certain embodiments, the pH of the stable formulation is about In certain embodiments, the pH of the stable formulation is about 6.7. In certain embodiments, the pH of the stable formulation is about 6.8. In certain embodiments, the pH of the stable formulation is about 6.9. H is about 7.0. In certain embodiments, the pH of the stable formulation is about 7.1. In certain embodiments, the pH of the stable formulation is about 7.2. In certain embodiments, the pH of the stable formulation is about 7.3. The pH of the formulation is about 7.4. In certain embodiments, the pH of the stable formulation is about 7. In certain embodiments, the pH of the stable formulation is about 7.6. In certain embodiments, the pH of the stable formulation is about 7.7. In certain embodiments, the pH of the stable formulation is about 7.8. is about 7.9. In certain embodiments, the pH of the stable formulation is about 8.0. In certain embodiments, the pH of the stable formulation is about 8.1. In certain embodiments, the pH of the stable formulation is about 8.2. The pH of the formulation is about 8.3. In certain embodiments, the pH of the stable formulation is about 8. In certain embodiments, the pH of the stable formulation is about 8.5. In certain embodiments, the pH of the stable formulation is about 8.6. In certain embodiments, the pH of the stable formulation is about 8.7. , about 8.8. In certain embodiments, the pH of the stable formulation is about 8.9. In certain embodiments, the pH of the stable formulation is about 9.0.

[0170] In certain embodiments, the vector genome concentration (VGC) of the stable formulation is about 3×1 0 9 GC / mL, 4×109 GC / mL, 5×10 9 GC / mL, 6×10 9 GC / mL ,7×10 9 GC / mL, 8×10 9 GC / mL, 9×10 9 GC / mL, about 1×10 1 0 GC / mL, about 2×10 10 GC / mL, about 3×10 10 GC / mL, about 4×10 10 GC / mL, about 5×10 10 GC / mL, about 6×10 10 GC / mL, about 7×10 10 G C / mL, about 8×10 10 GC / mL, about 9×10 10 GC / mL, about 1×10 11 GC / mL, about 2×10 11 GC / mL, about 3×10 11 GC / mL, about 4×10 11 GC / mL, about 5×10 11 GC / mL, about 6×10 11 GC / mL, about 7×10 11 GC / m L, about 8×10 11 GC / mL, about 9×10 11 GC / mL, about 1×10 12 GC / mL , about 2×10 12 GC / mL, about 3×10 12 GC / mL, about 4×10 12 GC / mL, About 5×10 12 GC / mL, about 6×10 12 GC / mL, about 7×10 12 GC / mL, approx. 8×10 12 GC / mL, about 9×10 12 GC / mL, about 1×10 13 GC / mL, about 1 ×10 13 GC / mL, about 2×10 13GC / mL, or approximately 3 x 10 13 In GC / mL be.

[0171] In certain embodiments, recombinant adeno-associated virus (rAAV) particles in a stable formulation The results showed that the rAAV particles in the lyophilization or reconstitution process were significantly higher than those in the reference formulation. , stability is at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 1 7%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 1 00%, approximately 2x, approximately 3x, approximately 5x, approximately 10x, approximately 100x, or approximately 1000 or higher In certain embodiments, the stability of rAAV particles can be determined using the avidin-based methods disclosed in Section 6.8. The test is identified by a test sequence (single or multiple).

[0172] In certain embodiments, the rAAV particles in the stable formulation contain the same rAAAV as in the reference formulation. Compared to V particles, the infectivity is at least about 2%, 5%, 7%, 10%, 12%, About 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, Approximately 50%, approximately 100%, approximately 2 times, approximately 3 times, approximately 5 times, approximately 10 times, approximately 100 times, or approximately 10 In certain embodiments, the viral infectivity of the rAAV particles is higher than 100. Identified by the assay(s) disclosed in Section 6.8. In certain embodiments, infectivity is measured before or after lyophilization. Infectivity is measured before or after reconstitution of the lyophilized preparation.

[0173] In certain embodiments, the rAAV particles in the stable formulation contain the same rAAAV as in the reference formulation. Compared to V particles, aggregation is at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, approximately 100%, approximately 2x, approximately 3x, approximately 5x, approximately 10x, approximately 100x, or approximately 100 In certain embodiments, rAAV particle aggregation is reduced by 100% as disclosed in Section 6.8. In certain embodiments, the agglutination is identified by an assay or assays. is measured before or after lyophilization. In certain embodiments, aggregation is measured after lyophilization. Measured before or after reconstitution of the dry formulation.

[0174] In certain embodiments, the stable formulation is lyophilized prior to storage.

[0175] In certain embodiments, the stable formulation is reconstituted after storage.

[0176] In certain embodiments, the formulation is maintained at -80°C, -70°C, -20°C, 4°C, 20°C. , stored at 25°C, 30°C, 35°C, 37°C, or 40°C.

[0177] In certain embodiments, the formulation may be administered for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, approx. 10 months, approx. 11 months, 12 months, approx. 15 months, approx. 18 months, approx. 24 months, approx. It is stored for 2 years, about 3 years, or about 4 years.

[0178] In certain embodiments, recombinant adeno-associated virus (rAAV) particles in a stable formulation The results showed that the rAAV particles in the lyophilization or reconstitution process were significantly higher than those in the reference formulation. , stability is at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 1 7%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 1 00%, approximately 2 times, approximately 3 times, approximately 5 times, approximately 10 times, approximately 100 times, or approximately 1000 times higher. In certain embodiments, the stability of rAAV particles is determined using the assays disclosed in Section 6.8. Identified by (singular or plural).

[0179] In certain embodiments, the rAAV particles in the stable formulation contain the same rAAAV as in the reference formulation. Compared to V particles, the infectivity is at least about 2%, 5%, 7%, 10%, 12%, About 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, Approximately 50%, approximately 100%, approximately 2 times, approximately 3 times, approximately 5 times, approximately 10 times, approximately 100 times, or approximately 10 In certain embodiments, the viral infectivity of the rAAV particles is 6.80 times higher. In certain embodiments, the method is characterized by the assay(s) disclosed in the section. In certain embodiments, infectivity is measured before or after lyophilization. Infecting activity is measured before and after reconstitution of the lyophilized formulation.

[0180] In certain embodiments, the rAAV particles in the stable formulation contain the same rAAAV as in the reference formulation. Compared to V particles, aggregation is at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, approximately 100%, approximately 2x, approximately 3x, approximately 5x, approximately 10x, approximately 100x, or approximately 100 In certain embodiments, rAAV particle aggregation is reduced by 100% as disclosed in Section 6.8. In certain embodiments, the agglutination is identified by an assay or assays. is measured before or after lyophilization. In certain embodiments, aggregation is measured after lyophilization. Measured before or after reconstitution of the dry formulation.

[0181] In certain embodiments, the stable formulation is lyophilized prior to storage.

[0182] In certain embodiments, the stable formulation is reconstituted after storage.

[0183] In certain embodiments, the formulation is maintained at -80°C, -70°C, -20°C, 4°C, 20°C. , stored at 25°C, 30°C, 35°C, 37°C, or 40°C.

[0184] In certain embodiments, the formulation may be administered for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, approx. 10 months, approx. 11 months, 12 months, approx. 15 months, approx. 18 months, approx. 24 months, approx. In certain embodiments, the composition is stored for about 2 years, about 3 years, about 4 years, or about 5 years. The rAAV particles in the stable formulation can be used to inactivate the formulation over a period of time, e.g., about one week, about two weeks, , about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months , about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about After storage for 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years, The stability is at least about 2% greater than that of the same rAAV particles in a reference formulation stored under the same conditions. , about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, approx. 35%, approx. 40%, approx. 45%, approx. 50%, approx. 100%, approx. 2x, approx. 3x, approx. 5x fold, about 10 times, about 100 times, or more than about 1000 times higher. The stability of rAAV particles over a period of time can be assessed using the assays disclosed in Section 6.8. The term "personal information" is sometimes referred to as "personal information" or "personal information."

[0185] In certain embodiments, the rAAV particles in a stable formulation can be stable over a period of time. , for example, at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, About 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months , about 11 months, 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, Stability compared to the same rAAV particles in a reference formulation after storage for approximately 4 or 5 years However, at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 100%, About 2 times, about 3 times, about 5 times, about 10 times, about 100 times, or more than about 1000 times higher. In certain embodiments, the stability of the rAAV particles over a period of time can be determined using methods described in Section 6.8. The antibody is identified by the assay or assays performed.

[0186] In certain embodiments, the rAAV particles in the stable formulation contain the same rAAAV as in the reference formulation. Compared to V particles, the in vitro relative potency (IVRP) is at least about 2% and at least about 5% , about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, Approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 100%, approximately 2 times, approximately 3 times, approximately 5 times, approximately 1 0 fold, about 100 fold, or about 1000 fold higher. The in vitro relative potency (IVRP) of the compound was determined using the assays disclosed in Chapter 6.8 (singular In certain embodiments, the IVRP is identified by a lyophilized In certain embodiments, the IVRP is measured before or after reconstitution of a lyophilized formulation. Measured before or after construction.

[0187] In certain embodiments, the rAAV particles in the stable formulation contain the same rAAAV as in the reference formulation. Free DNA is at least about 2%, about 5%, about 7%, about 10%, about 12%, or about 16% relative to V particles. %, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45 %, about 50%, about 100%, about 2x, about 3x, about 5x, about 10x, about 100x, or about In certain embodiments, the free DNA of the rAAV particles is 6. In certain embodiments, the method is characterized by the assay(s) disclosed in Section 8. In certain embodiments, the free DNA is measured before or after lyophilization. In this study, free DNA was measured before or after reconstitution of the lyophilized formulation.

[0188] In certain embodiments, the rAAV particles in the stable formulation contain the same rAAAV as in the reference formulation. rAAV genome release is at least about 2%, about 5%, about 7%, about 10% compared to V particles. , about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40% , about 45%, about 50%, about 100%, about 2 times, about 3 times, about 5 times, about 10 times, about 100 times, or about 1000-fold less. In certain embodiments, rAAV genome release is A is identified by measuring the relative fluorescence in the presence of a specific fluorescent stain. In one embodiment, rAAV genome release can be measured using the assay (s) disclosed in Section 6.8. In certain embodiments, the rAAV genome release is determined by lyophilization. In certain embodiments, rAAV genome release is measured before or after freezing. Measured before or after reconstitution of the dry formulation.

[0189] In certain embodiments, the rAAV particles in a stable formulation can be stable over a period of time. For example, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, About 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months Months, approx. 12 months, approx. 15 months, approx. 18 months, approx. 24 months, approx. 2 years, approx. 3 years, approx. 4 years or after about 5 years of storage, the dimensional change is at most about 20%, about 15%, about 10%, about 8%, %, about 5%, about 4%, about 3%, about 2%, or about 1%. Therefore, rAAV particle size can be characterized by the assay(s) disclosed in Section 6.8. In certain embodiments, the dimensions are measured before or after a freeze / thaw cycle. It is determined.

[0190] In certain embodiments, the rAAV particles in a stable formulation can be stable over a period of time. , for example, at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, About 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months , about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years After storage for about 4 years or about 5 years, the dimensional changes were at most 20%, 15%, 10%, 8%, 5%, 4%, 3%, 2%, or about 1%. In certain embodiments, r AAV particle size is determined by the assay(s) disclosed in Section 6.8. In certain embodiments, the dimensions are measured before or after lyophilization. In certain embodiments, the dimensions are measured before or after reconstitution of the lyophilized formulation.

[0191] In certain embodiments, the rAAV particles in the stable formulation can be stored at -80° C., -70° C., - When stored at 20℃, 4℃, 20℃, 25℃, 30℃, 35℃, 37℃, or 40℃ , stability is at least about 2%, about 5%, about 7%, or more than the same rAAV particles in a reference formulation. %, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35 %, approx. 40%, approx. 45%, approx. 50%, approx. 100%, approx. 2x, approx. 3x, approx. 5x, approx. 10x, In certain embodiments, the stability of the rAAV particles is about 100-fold or about 1000-fold higher. The assay is determined by the assay(s) disclosed in Section 6.8.

[0192] In certain embodiments, the rAAV particles in the stable formulation can be stored at -80° C., -70° C., - 20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time. For example, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months , about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or approximately 5 years of storage, the infectivity was reduced compared to the same rAAV particles in the reference formulation. At most 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2x, 3x, 5x, 10x, 100x, In certain embodiments, the viral infectivity of the rAAV particles is Identified by the assay(s) disclosed in Section 6.8.

[0193] In certain embodiments, the rAAV particles in the stable formulation can be stored at -80° C., -70° C., - 20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time. For example, at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, About 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, When stored for approximately 4 or 5 years, the infectivity increased 100% compared to the same rAAV particles in a reference formulation. Power is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25% , 30%, 35%, 40%, 45%, 50%, 100%, 2x, 3x, 5x, 10x, 1 In certain embodiments, the viral load of the rAAV particle is 100, 1000, or 1000 times higher. Infectivity is determined by the assay(s) disclosed in Section 6.8.

[0194] In certain embodiments, the rAAV particles in the stable formulation can be stored at -80° C., -70° C., - 20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time. For example, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months , about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or approximately 5 years of storage, showed less aggregation compared to the same rAAV particles in a reference formulation. 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 3 5%, 40%, 45%, 50%, 100%, 2x, 3x, 5x, 10x, 100x, and In certain embodiments, aggregation of rAAV particles is reduced by 1000 times. The antibodies are identified by the assay(s) disclosed in.

[0195] In certain embodiments, the rAAV particles in the stable formulation can be stored at -80° C., -70° C., - 20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time. For example, at least about 11 weeks, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, About 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months , about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years , 4 years, or 5 years of storage, compared to the same rAAV particles in a reference formulation. The collection is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25% , 30%, 35%, 40%, 45%, 50%, 100%, 2x, 3x, 5x, 10x, 1 In certain embodiments, rAAV particle aggregation is , identified by the assay(s) disclosed in Section 6.8.

[0196] In certain embodiments, the rAAV particles in the stable formulation can be stored at -80° C., -70° C., - 20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time. For example, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months , about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or approximately 5 years of storage, the stability was less than that of the same rAAV particles in a reference formulation. At most 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2x, 3x, 5x, 10x, 100x, In certain embodiments, the stability of the rAAV particles over a period of time is increased by 1000 times. The assay is determined by the assay(s) disclosed in Section 6.8.

[0197] In certain embodiments, the rAAV particles in the stable formulation can be stored at -80° C., -70° C., - 20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time. For example, at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, About 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, Stable compared to the same rAAV particles in a reference formulation when stored for approximately 4 or 5 years Sexuality is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, and 25% , 30%, 35%, 40%, 45%, 50%, 100%, 2x, 3x, 5x, 10x, 1 In certain embodiments, the rAAV particles may be expressed for a period of time that is greater than or equal to 1000 times the rAAV particle expression level. The stability over a period of time is determined by the assay(s) disclosed in Section 6.8. do.

[0198] In certain embodiments, the rAAV particles in the stable formulation can be stored at -80° C., -70° C., - 20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time. For example, 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, About 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, rAAV particles in a reference formulation when stored for 5 years or more in vitro The relative potency (IVRP) is at least 2%, 5%, 7%, 10%, 12%, 15%, 17% %, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2x, 3x , 5 times, 10 times, 100 times, or 1000 times higher. The in vitro relative potency (IVRP) of AAV particles was determined using the assay disclosed in Section 6.8. It is identified by (singular or plural).

[0199] In certain embodiments, the rAAV particles in the stable formulation can be stored at -80° C., -70° C., - 20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time. For example, at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, About 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, When stored for approximately 4 or 5 years, in comparison to the same rAAV particles in a reference formulation In vitro relative potency (IVRP) of at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100% , 2x, 3x, 5x, 10x, 100x, or 1000x higher. The in vitro relative potency (IVRP) of rAAV particles is disclosed in Section 6.8. The antibody is identified by the assay or assays performed.

[0200] In certain embodiments, the rAAV particles in the stable formulation contain the same rAAAV as in the reference formulation. Compared to V particles, -80℃, -70℃, -20℃, 4℃, 20℃, 25℃, 30℃, 35℃ ℃, 37 ℃, or 40 ℃ for a period of time, for example, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, Weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 2 The same efficacy was observed in the reference formulation when stored for 4 months, about 2 years, about 3 years, about 4 years, or about 5 years. The in vitro relative potency (IVRP) is at least 2% compared to the rAAV particles 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40 %, 45%, 50%, 100%, 2x, 3x, 5x, 10x, 100x, or 1000 In certain embodiments, the in vitro relative potency of the rAAV particles ( The IVRPs are identified by the assay(s) disclosed in Section 6.8.

[0201] In certain embodiments, the rAAV particles in the stable formulation can be stored at -80° C., -70° C., - 20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time. For example, at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, About 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, When stored for approximately 4 or 5 years, in comparison to the same rAAV particles in a reference formulation In vitro relative potency (IVRP) of at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100% , 2x, 3x, 5x, 10x, 100x, or 1000x higher. The in vitro relative potency (IVRP) of rAAV particles is disclosed in Section 6.8. The antibody is identified by the assay or assays performed.

[0202] In certain embodiments, the rAAV particles in the stable formulation can be stored at -80° C., -70° C., - 20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time. For example, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months , about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or approximately 5 years of storage, compared to the same rAAV particles in a reference formulation, At least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30 %, 35%, 40%, 45%, 50%, 100%, 2x, 3x, 5x, 10x, 100x , or 1000 times less. In certain embodiments, the free DNA of the rAAV particles is , identified by the assay(s) disclosed in Section 6.8.

[0203] In certain embodiments, the rAAV particles in the stable formulation can be stored at -80° C., -70° C., - 20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time. For example, at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, About 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, When stored for approximately 4 or 5 years, the free DNA is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 2 5%, 30%, 35%, 40%, 45%, 50%, 100%, 2x, 3x, 5x, 10x , 100-fold, or 1000-fold less. The isolated DNA is identified by the assay(s) disclosed in Section 6.8.

[0204] In certain embodiments, the rAAV particles in the stable formulation can be stored at -80° C., -70° C., - 20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time. For example, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months , about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or when stored for about 5 years, the particle size changes by up to 20%, 15%, 10%, and 8% , 5%, 4%, 3%, 2%, or 1%. Particle size is determined by the assay(s) disclosed in Section 6.8.

[0205] In certain embodiments, the rAAV particles in the stable formulation can be stored at -80° C., -70° C., - 20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time. For example, at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, About 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, When stored for approximately 4 or 5 years, compared to the same rAAV particles in a reference formulation ,The change in particle size is up to 20%, 15%, 10%, 8%, 5%, 4%, 3%, 2%, or 1%. In certain embodiments, the rAAV particle size is Identified by the assay(s) indicated.

[0206] In certain embodiments, rAAV particle size is determined using the assays disclosed in Section 6.8. Identified by (singular or plural).

[0207] In certain embodiments, the reference formulation is 0.001% poloxamer 188 buffer The DPBS includes:

[0208] In certain embodiments, the reference formulation is a sugar-free formulation. In the embodiment, the reference formulation is a formulation without plasticizer.

[0209] In certain embodiments, the formulation is lyophilized at a temperature of about -20°C during the lyophilization process. In certain embodiments, the frozen formulation, when frozen at -20°C, In certain embodiments, the frozen formulation is When frozen at 20°C, the pH value is within ±1 unit of the pre-freezing pH value. Maintain.

[0210] In certain embodiments, the glass transition temperature (Tg) of the lyophilized cake of the formulation is 3 Higher than 5℃.

[0211] In certain embodiments, the maximally freezing solution of the formulation The glass transition temperature (Tg') of the eze-concentrated solution is , higher than -40℃.

[0212] In certain embodiments, the moisture content is about 0.5% to about 1%. In certain embodiments, the water content is about 1% to about 2%. In a particular embodiment, the water content is about 3% to about 4%. In a particular embodiment, the water content is about 4% to about 5%.

[0213] In certain embodiments, the moisture content is about 0.1%, about 0.2%, about 0.3%, about 0.5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 70%, about 80%, about 90%, about 100%, about 15 ... .4%, about 0.5%, about 0.6%, about 0.8%, about 1.0%, about 1.2%, about 1.2%, Approximately 1.3%, approximately 1.4%, approximately 1.5%, approximately 1.6%, approximately 1.7%, approximately 1.8%, approximately 1.9 %, about 2.0%, about 3.0%, about 4.0%, or about 5.0%.

[0214] In some embodiments, the formulations disclosed herein include a plasticizer or stabilizer. Acceptable stabilizers or plasticizers include glycerol, xylitol, and sorbitol. In some embodiments, the methods disclosed herein include, but are not limited to, The formulation includes glycerol, xylitol, sorbitol, or a combination thereof. In some embodiments, the formulations disclosed herein include glycerol.

[0215] In some embodiments, the formulations disclosed herein contain from about 0.1% to about 5% plasticizer or In some embodiments, the formulations disclosed herein contain from about 0.1% to about 2 % of a plasticizer or stabilizer. In some embodiments, the formulations disclosed herein comprise about Contains 0.25% to about 2% plasticizer.

[0216] In some embodiments, the formulations disclosed herein contain from about 0.1% to about 5% glycerol. In some embodiments, the formulations disclosed herein contain from about 0.1% to about 2% gravidol. In some embodiments, the formulations disclosed herein contain about 0.25% to about glycerol. Contains approximately 2% glycerol.

[0217] In some embodiments, the term "about" refers to a range, plus or minus, a given value or range. In some embodiments, the term "about" refers to the indicated number or In certain embodiments, the range is about 10-20% greater or less than the range. The term "about" is inclusive of the exact number recited.

[0218] Further features and advantages of the compositions and methods described herein may be seen in conjunction with the accompanying drawings. This will become clearer as you read the detailed explanation below.

[0219] 4.1 Illustrative Embodiments 4.1.1 Set 1 1. Recombinant adeno-associated virus (rAAV) particles, and a) a buffering agent, b) sugar, and c) Sodium citrate and A stable formulation comprising:

[0220] 2. The formulation of clause 1, wherein said buffer comprises about 1 mM to about 50 mM Tris.

[0221] 3. About 1mM to about 30mM, about 1mM to about 20mM, about 5mM to about 30mM, about 5mM ~ about 20 mM, ~ about 10 mM to about 30 mM, ~ about 10 mM to about 20 mM, or ~ about 20 mM The formulation of clause 2 comprising about 50 mM Tris.

[0222] 4. About 1mM, about 2mM, about 3mM, about 5mM, about 10mM, about 15mM, about 20mM 3. The formulation of clause 2, comprising about 25 mM, about 30 mM, or about 40 mM Tris.

[0223] 5. The formulation of clause 2 comprising about 5 mM Tris.

[0224] 6. The formulation according to any one of clauses 1 to 4, wherein the pH is about 6.5 to 8.0.

[0225] 7. The formulation according to clause 6, wherein the pH is about 7.2 to 7.8.

[0226] 8. The pH is about 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8 7. The formulation according to clause 6.

[0227] 9. The formulation of clause 6, wherein the pH is about 7.5.

[0228] 10. The method according to any one of clauses 1 to 9, comprising about 50 mM to about 400 mM sugar. Agent.

[0229] 11. Approx. 50mM ~ Approx. 350mM, Approx. 50mM ~ Approx. 300mM, Approx. 50mM ~ Approx. 250 1, comprising about 50 mM to about 200 mM, or about 50 mM to about 150 mM of sugar. 0. The formulation described in

[0230] 12. About 100 mM to about 400 mM, about 150 mM to about 400 mM, about 200 mM to about 400 mM, about 250 mM to about 400 mM, or about 300 mM to about 400 mM sugar. 11. The formulation according to claim 10.

[0231] 13. Approx. 100mM~Approx. 300mM, Approx. 150mM~Approx. 250mM, Approx. 200mM~Approx. 11. The formulation of clause 10, comprising 300 mM, or about 250 mM to about 350 mM sugar.

[0232] 14. About 50mM, about 100mM, about 150mM, about 160mM, about 170mM, about 1 80mM, about 190mM, about 200mM, about 210mM, about 220mM, about 230mM, Approx. 240mM, Approx. 250mM, Approx. 260mM, Approx. 270mM, Approx. 280mM, Approx. 290m 11. The formulation of clause 10, comprising about 300 mM, about 300 mM, or about 350 mM sugar.

[0233] 15. About 190 mM to about 230 mM, about 170 mM to about 250 mM, or about 150 mM 11. The formulation of clause 10, comprising M to about 270 mM sugar.

[0234] 16. The formulation of clause 10, comprising about 210 mM sugar.

[0235] 17. The formulation according to any one of clauses 1 to 16, wherein the sugar is a non-reducing sugar.

[0236] 18. The method according to claim 1, wherein the non-reducing sugar is sucrose, trehalose, or raffinose. 17. The formulation according to claim 17.

[0237] 19. The formulation according to clause 17, wherein the non-reducing sugar is sucrose.

[0238] 20. The formulation according to any one of clauses 1 to 16, wherein the sugar is a reducing sugar.

[0239] 21. The reducing sugar is glucose, fructose, mannose, galactose, or 21. The formulation according to clause 20, which is lactose.

[0240] 22. The formulation according to clause 20, wherein the reducing sugar is glucose.

[0241] 23. Any one of clauses 1 to 22, comprising less than about 100 mM sodium citrate. The formulation described in

[0242] 24. The formulation of clause 23, comprising about 10 mM to about 100 mM sodium citrate. .

[0243] 25. About 10 mM to about 100 mM, about 20 mM to about 100 mM, about 30 mM to about 100 mM, about 40mM to about 100mM, about 50mM to about 100mM, about 10mM to about 80mM , about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, or 24. The formulation of clause 23, comprising about 10 mM to about 30 mM sodium citrate.

[0244] 26. About 10mM to about 50mM, about 20mM to about 60mM, about 30mM to about 70mM, or about 10 mM to about 30 mM sodium citrate.

[0245] 27. About 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, or about 60 mM 24. The formulation of clause 23, comprising 1 mM sodium citrate.

[0246] 28. The formulation of clause 23, comprising about 20 mM sodium citrate.

[0247] 29. The method according to claim 1, further comprising: 29. The formulation according to any one of 1 to 28.

[0248] 30. The formulation of clause 29, comprising about 0.002% of a non-ionic surfactant.

[0249] 31. The nonionic surfactant is poloxamer 188, poloxamer 407, poly Sorbate 80, Polysorbate 20, Pluronic F-68, or BRIJ 35. A formulation according to clause 28 or clause 29, including

[0250] 32. The method according to claim 31, wherein the nonionic surfactant comprises poloxamer 188. Agent.

[0251] 33. Recombinant adeno-associated virus (rAAV) particles, and a) about 1 mM to about 25 mM Tris, b) about 50 mM to about 400 mM sugar; c) about 10 mM to about 100 mM sodium citrate, and d) about 0.0005% to about 0.01% of a nonionic surfactant; A stable formulation comprising: The formulation has a pH of about 7.2 to about 7.8.

[0252] 34. Recombinant adeno-associated virus (rAAV) particles, and a) about 2 mM to about 10 mM Tris, b) about 150 mM to about 250 mM sugar; c) about 10 mM to about 20 mM sodium citrate, and d) about 0.001% to about 0.005% of a nonionic surfactant; A stable formulation comprising: The formulation has a pH of about 7.2 to about 7.8.

[0253] 35. Recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sugar; c) about 20 mM sodium citrate, and d) about 0.002% of a nonionic surfactant; A stable formulation comprising: The formulation has a pH of about 7.5.

[0254] 36. The formulation according to any one of clauses 33 to 35, wherein the sugar is a non-reducing sugar.

[0255] 37. The method according to claim 3, wherein the non-reducing sugar is sucrose, trehalose, or raffinose. 36. The formulation according to claim 36.

[0256] 38. The formulation according to clause 17, wherein the non-reducing sugar is sucrose.

[0257] 39. Approximately 1.0E+11 genome copies / mL (GC / mL) to approximately 1.0E+15 GC / mL 40. The formulation of any one of clauses 1 to 38, comprising 1 mL of rAAV particles.

[0258] 40. Approx. 1.0E+11GC / mL, approx. 1.0E+12GC / mL, approx. 1.0E+13 GC / mL, approximately 1.0E+14GC / mL, or approximately 1.0E+15GC / mL of rAA 40. The formulation of clause 39, comprising V particles.

[0259] 41. The rAAV particles are selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV17, AAV18, AAV19, AAV20, AAV21, AAV22, AAV23, AAV24 AV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, A AV-13, AAV-14, AAV-15 and AAV-16, rAAV.rh8, rAA V.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAA V.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L 65, rAAV.7m8, rAAV.PHP.B, rAAV2.5, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.H SC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AA V.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and The capsid protein of AAV.HSC16 is contained in any one of clauses 1 to 40. The formulation described above.

[0260] 42. The rAAV particles contain capsid proteins of AAV-8 or AAV-9 serotypes. 42. The formulation according to clause 41, comprising:

[0261] 43. In addition, from glycerol, xylitol, sorbitol, or mannitol 43. The formulation according to any one of the preceding clauses, comprising a plasticizer selected from the group consisting of:

[0262] 44. The formulation according to any one of clauses 1 to 42, further comprising glycerol.

[0263] 45. The formulation of clause 44, comprising about 0.1% to about 5% glycerol.

[0264] 46. ​​The formulation of clause 44, comprising about 0.1% to about 2% glycerol.

[0265] 47. The formulation of clause 44, comprising about 0.25% to about 2% glycerol.

[0266] 48. A formulation according to any one of clauses 1 to 47, which is a liquid formulation.

[0267] 49. The formulation according to any one of clauses 1 to 47, which is a frozen formulation.

[0268] 50. Any of clauses 1 to 47 which is a lyophilized preparation or a reconstituted lyophilized preparation. or the formulation described in one of the above.

[0269] 51. The formulation according to clause 50, having a residual moisture content of about 1% to about 7%.

[0270] 52. The residual moisture content is about 1% to about 7%, about 2% to about 7%, about 3% to about 7%, about 4% ~ about 7%, about 5% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, or 52. The formulation of clause 51, wherein the concentration is from about 1% to about 3%.

[0271] 53. The residual moisture content is about 3% to about 7%, about 3% to about 6%, or about 3% to about 5%. 52. The formulation according to clause 51,

[0272] 54. The residual moisture content is about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5. 52. The formulation of clause 51, wherein the concentration is 5%, or about 6%.

[0273] 55. The relative potency of the rAAV particles was at least 100% after storage of the formulation at room temperature for 3 months. at least about 60%, at least about 70%, or at least about 80%, but AAV particles were stored at -70°C in DPBS containing 0.001% poloxamer 188 buffer. 55. The formulation according to any one of clauses 1 to 54, wherein the formulation is stored.

[0274] 56. The relative potency of the rAAV particles was at least 100% after storage of the formulation at room temperature for 6 months. at least about 60%, at least about 70%, or at least about 80%, but AAV particles are prepared in Dulbecco's Phosphate Buffered Saline with 0.001% Poloxamer 188 buffer. 55. The method according to any one of clauses 1 to 54, stored in water (DPBS) at -70°C. formulation.

[0275] 57. The relative potency of the rAAV particles was at least 100% after storing the formulation at 35° C. for 1 week. at least about 30%, at least about 40%, at least about 50%, or at least 60% Yes, except that the reference rAAV particles were prepared in DPB containing 0.001% poloxamer 188 buffer. 55. The formulation of any one of clauses 1 to 54, stored in S at -70°C.

[0276] 58. The relative potency of the rAAV particles was at least 100% after storage of the formulation at 35° C. for 2 weeks. at least about 30%, at least about 40%, at least about 50%, or at least 60% Yes, except that the reference rAAV particles were prepared in DPB containing 0.001% poloxamer 188 buffer. 55. The formulation of any one of clauses 1 to 54, stored in S at -70°C.

[0277] 59. The relative potency of the rAAV particles was at least 100% after storage of the formulation at 35° C. for 4 weeks. at least about 30%, at least about 40%, at least about 50%, or at least 60% Yes, except that the reference rAAV particles were prepared in DPB containing 0.001% poloxamer 188 buffer. 55. The formulation of any one of clauses 1 to 54, stored in S at -70°C.

[0278] 60. A method for producing a stable formulation containing recombinant adeno-associated virus (rAAV) particles, comprising: The rAAV particles are mixed with a buffer, sugar, salt, optional, of the formulation described in any one of clauses 1 to 47. and optionally a plasticizer, and optionally a non-ionic surfactant, thereby forming a polymeric dispersion containing rAAV. manufacturing said formulation.

[0279] 61. A method for reducing rAAV genome release from rAAV particles, comprising: 3. The method of claim 1, further comprising: The release of the rAAV genome from the rAAV particles after three freeze-thaw cycles was The method further comprises:

[0280] 62. A method for reducing rAAV genome release from rAAV particles, comprising: The method includes preparing a formulation comprising a freeze-dried glycerol, a sugar, a salt, and optionally a non-ionic surfactant. Release of the rAAV genome from the rAAV particles after drying and reconstitution is consistent with the sugar-free The method, wherein the rAAV genome release is reduced compared to that in the formulation.

[0281] 63. Furthermore, the preparation is freeze-dried to reduce the residual water content to about 1% to about 5%. 63. The method according to clause 61 or 62,

[0282] 64. A method for reducing rAAV genome release from rAAV particles, comprising: and preparing a formulation comprising a glycerol, a sugar, a salt, a plasticizer, and optionally a non-ionic surfactant. and rAAV genome release from the rAAV particles after three freeze-thaw cycles is The method, wherein rAAV genome release is reduced compared to that in a sugar-free formulation.

[0283] 65. A method for reducing rAAV genome release from rAAV particles, comprising: and preparing a formulation comprising a glycerol, a sugar, a salt, a plasticizer, and optionally a non-ionic surfactant. and release of the rAAV genome from the rAAV particles after lyophilization and reconstitution is The method, wherein rAAV genome release is reduced compared to a formulation not containing the rAAV genome release.

[0284] 66. Use of sugars to reduce rAAV genome release from rAAV particles, comprising: Produce a formulation containing rAAV particles, buffer, sugar, salt, and optionally a non-ionic surfactant and releasing the rAAV genome from the rAAV particles after three freeze-thaw cycles. The release of the rAAV genome is reduced compared to that in a formulation that does not contain the sugar. For.

[0285] 67. Use of sugars to reduce rAAV genome release from rAAV particles, comprising: Produce a formulation containing rAAV particles, buffer, sugar, salt, and optionally a non-ionic surfactant and releasing the rAAV genome from the rAAV particles after lyophilization and reconstitution. The use, wherein the rAAV genome release is reduced compared to a formulation that does not contain the sugar.

[0286] 68. The preparation is further freeze-dried to reduce the residual water content to about 1% to about 7%. 66 or 67, including the use described in clause 66 or 67.

[0287] 69. The use of plasticizers to reduce rAAV genome release from rAAV particles. The composition comprises rAAV particles, a buffer, a sugar, a salt, a plasticizer, and optionally a non-ionic surfactant. and producing a formulation containing rAAV from the rAAV particles after three freeze-thaw cycles. rAAV genome release was reduced compared to rAAV genome release in the sugar-free formulation. The above-mentioned use.

[0288] 70. The use of plasticizers to reduce rAAV genome release from rAAV particles. The composition comprises rAAV particles, a buffer, a sugar, a salt, a plasticizer, and optionally a non-ionic surfactant. and producing a formulation comprising an rAAV from the rAAV particles after lyophilization and reconstitution. Genome release is reduced compared to rAAV genome release in the sugar-free formulation , said use.

[0289] 71. rAAV genome release can be measured by measuring relative fluorescence in the presence of a DNA-specific fluorescent stain. The method according to any one of clauses 61 to 65 or clauses 66 to 68, 70. The use according to any one of claims 1 to 70.

[0290] 72. Freezing-induced rAAV genome release is at least about 10%, 20%, or 5%. 0%, 80% or 90% reduction in any one of Articles 61 to 65 and 71 2. The method or use according to any one of clauses 66 to 71.

[0291] 73. Freezing-induced rAAV genome release is substantially eliminated, clause 6 The method according to any one of clauses 1 to 65 and 71 or any one of clauses 66 to 71 The use described in.

[0292] 74. Any one of clauses 61 to 65 and 71 to 73, wherein the sugar is a non-reducing sugar. or the use according to any one of clauses 66 to 73.

[0293] 75. The method according to claim 1, wherein the non-reducing sugar is sucrose, trehalose, or raffinose. 74 or the use according to clause 74.

[0294] 76. The method according to claim 74 or the method according to claim 74, wherein the non-reducing sugar is sucrose. Use of the above.

[0295] 77. The method according to any one of clauses 61 to 65 and 71 to 73, wherein the sugar is a reducing sugar. 74. The method or use according to any one of clauses 66 to 73.

[0296] 78. The reducing sugar is glucose, fructose, mannose, galactose, or 78. The method according to clause 77 or the use according to clause 77, wherein the sugar is lactose.

[0297] 79. The method according to claim 77 or the method according to claim 77, wherein the reducing sugar is glucose. use.

[0298] 80. Any of clauses 61 to 65 and 71 to 79, wherein the plasticizer comprises glycerol. or the use according to any one of clauses 66 to 79.

[0299] 81. The formulation according to any one of clauses 61 to 6, wherein the formulation is according to any one of clauses 1 to 47. 5 and 71 to 80 or any one of clauses 66 to 80. The use described in.

[0300] 4.1.2 Set 2 1. Recombinant adeno-associated virus (rAAV) particles, and a) a buffering agent, b) sugar, and c) Amorphous salts and A stable formulation comprising: The formulation is suitable for lyophilization.

[0301] 2. The formulation of clause 1, wherein said buffer comprises about 1 mM to about 50 mM Tris.

[0302] 3. About 1mM to about 30mM, about 1mM to about 20mM, about 5mM to about 30mM, about 5mM ~ about 20 mM, ~ about 10 mM to about 30 mM, ~ about 10 mM to about 20 mM, or ~ about 20 mM The formulation of clause 2 comprising about 50 mM Tris.

[0303] 4. About 1mM, about 2mM, about 3mM, about 5mM, about 10mM, about 15mM, about 20mM 3. The formulation of clause 2, comprising about 25 mM, about 30 mM, or about 40 mM Tris.

[0304] 5. The formulation of clause 2 comprising about 5 mM Tris.

[0305] 6. Recombinant adeno-associated virus (rAAV) particles, and a) a buffering agent, b) sugar, and c) Amorphous salts with an ionic strength greater than 60 mM A stable formulation comprising: The formulation is suitable for lyophilization.

[0306] 7. The formulation of clause 6, wherein the rAAV particles have AAV8 capsids.

[0307] 8. Recombinant adeno-associated virus (rAAV) particles, and a) a buffering agent, b) sugar, and c) Amorphous salts having an ionic strength of 60 mM to 150 mM; A stable formulation comprising: The formulation is suitable for lyophilization.

[0308] 9. The formulation of clause 8, wherein the rAAV particles have AAV8 capsids.

[0309] 10. Recombinant adeno-associated virus (rAAV) particles, and a) a buffering agent, b) sugar, and c) Amorphous salts having an ionic strength of 30 to 100 mM; A stable formulation comprising: The formulation is suitable for lyophilization.

[0310] 11. The formulation of clause 10, wherein the rAAV particles have rAAV9 capsids.

[0311] 12. Recombinant adeno-associated virus (rAAV) particles, and a) a buffering agent, b) sugar, and c) Amorphous salts with an ionic strength greater than 200 mM A stable formulation comprising: The formulation is suitable for lyophilization.

[0312] 13. The rAAV particles do not have rAAV8 or rAAV9 capsids. Item 13. The formulation according to item 12.

[0313] 14. Any one of clauses 1 to 13, wherein the amorphous salt is sodium citrate. The formulation described.

[0314] 15. The method according to any one of clauses 1 to 13, wherein the amorphous salt is sodium sulfate. Formulation.

[0315] 16. The method according to any one of clauses 1 to 13, wherein the amorphous salt is ammonium sulfate. The formulation described above.

[0316] 17. The method according to any one of clauses 1 to 13, wherein the amorphous salt is magnesium sulfate. The formulation described above.

[0317] 18. The amorphous salt is sodium citrate, sodium sulfate, ammonium sulfate, sulfate 1. The compound according to any one of clauses 1 to 13, The formulation described above.

[0318] 19. A formulation according to any one of clauses 1 to 13, comprising sodium citrate.

[0319] 20. A formulation according to any one of clauses 1 to 13, comprising sodium sulfate.

[0320] 21. A formulation according to any one of clauses 1 to 13, comprising ammonium sulfate.

[0321] 22. A formulation according to any one of clauses 1 to 13, comprising magnesium sulfate.

[0322] 23. Sodium citrate, sodium sulfate, ammonium sulfate, magnesium sulfate, or a combination thereof.

[0323] 24. The formulation may contain about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, approx. 60mM, approx. 70mM, approx. 80mM, approx. 90mM, approx. 100mM, approx. 120mM, approx. 1 40 mM, about 150 mM, or about 200 mM sodium sulfate. The formulation according to any one of the preceding claims.

[0324] 25. A formulation according to any one of clauses 1 to 24, having a pH of about 6.5 to 8.0.

[0325] 26. The formulation according to clause 25, having a pH of about 7.2 to 7.8.

[0326] 27. If the pH is approximately 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8, 26. The formulation according to clause 25,

[0327] 28. The formulation according to clause 25, wherein the pH is about 7.5.

[0328] 29. The method according to any one of clauses 1 to 28, comprising about 50 mM to about 400 mM sugar. formulation.

[0329] 30. Approx. 50mM ~ Approx. 350mM, Approx. 50mM ~ Approx. 300mM, Approx. 50mM ~ Approx. 250 Clause 2, comprising about 50 mM to about 200 mM, or about 50 mM to about 150 mM sugar. 9. The formulation according to claim 9.

[0330] 31. About 100 mM to about 400 mM, about 150 mM to about 400 mM, about 200 mM to about 400 mM, about 250 mM to about 400 mM, or about 300 mM to about 400 mM sugar. 29. The preparation according to claim 29.

[0331] 32. Approximately 100mM to approximately 300mM, approximately 150mM to approximately 250mM, approximately 200mM to approximately 30. The formulation of clause 29, comprising 300 mM, or about 250 mM to about 350 mM sugar.

[0332] 33. About 50mM, about 100mM, about 150mM, about 160mM, about 170mM, about 1 80mM, about 190mM, about 200mM, about 210mM, about 220mM, about 230mM, Approx. 240mM, Approx. 250mM, Approx. 260mM, Approx. 270mM, Approx. 280mM, Approx. 290m 30. The formulation of clause 29, comprising about 300 mM, or about 350 mM sugar.

[0333] 34. About 190 mM to about 230 mM, about 170 mM to about 250 mM, or about 150 mM 30. The formulation of clause 29, comprising from M to about 270 mM sugar.

[0334] 35. The formulation of clause 29, comprising about 210 mM sugar.

[0335] 36. The formulation according to any one of clauses 1 to 35, wherein the sugar is a non-reducing sugar.

[0336] 37. The method according to claim 3, wherein the non-reducing sugar is sucrose, trehalose, or raffinose. 36. The formulation according to claim 36.

[0337] 38. The formulation according to clause 36, wherein the non-reducing sugar is sucrose.

[0338] 39. The formulation according to any one of clauses 1 to 35, wherein the sugar is a reducing sugar.

[0339] 40. The reducing sugar is glucose, fructose, mannose, galactose, or 40. The formulation according to clause 39, which is lactose.

[0340] 41. Any one of clauses 1 to 40, comprising less than about 100 mM sodium citrate. The formulation described in

[0341] 42. The formulation according to clause 41, comprising about 10 mM to about 100 mM sodium citrate. .

[0342] 43. About 10 mM to about 100 mM, about 20 mM to about 100 mM, about 30 mM to about 100 mM, about 40mM to about 100mM, about 50mM to about 100mM, about 10mM to about 80mM , about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, or 42. The formulation of clause 41, comprising about 10 mM to about 30 mM sodium citrate.

[0343] 44. About 10mM to about 50mM, about 20mM to about 60mM, about 30mM to about 70mM, or about 10 mM to about 30 mM sodium citrate.

[0344] 45. About 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, or about 60 mM 42. The formulation of clause 41, comprising mM sodium citrate.

[0345] 46. ​​The formulation of clause 41, comprising about 20 mM sodium citrate.

[0346] 47. The method according to claim 1, further comprising: 47. The formulation according to any one of 1 to 46.

[0347] 48. The formulation according to clause 47, comprising about 0.002% of a non-ionic surfactant.

[0348] 49. The nonionic surfactant is poloxamer 188, poloxamer 407, poly Sorbate 80, Polysorbate 20, Pluronic F-68, or BRIJ 35. A formulation as defined in clause 46 or clause 47, including

[0349] 50. The method according to claim 49, wherein the nonionic surfactant comprises poloxamer 188. Agent.

[0350] 51. Recombinant adeno-associated virus (rAAV) particles, and a) about 1 mM to about 25 mM Tris, b) about 50 mM to about 400 mM sugar; c) about 10 mM to about 100 mM sodium citrate, and d) about 0.0005% to about 0.01% of a nonionic surfactant; A stable formulation comprising: The formulation has a pH of about 7.2 to about 7.8.

[0351] 52. Recombinant adeno-associated virus (rAAV) particles, and a) about 2 mM to about 10 mM Tris, b) about 150 mM to about 250 mM sugar; c) about 10 mM to about 20 mM sodium citrate, and d) about 0.001% to about 0.005% of a nonionic surfactant; A stable formulation comprising: The formulation has a pH of about 7.2 to about 7.8.

[0352] 53. Recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sugar; c) about 20 mM sodium citrate, and d) about 0.002% of a nonionic surfactant; A stable formulation comprising: The formulation has a pH of about 7.5.

[0353] 54. The formulation according to any one of clauses 51 to 53, wherein the sugar is a non-reducing sugar.

[0354] 55. The method according to claim 1, wherein the non-reducing sugar is sucrose, trehalose, or raffinose. 54. The formulation according to claim 54.

[0355] 56. The formulation according to clause 55, wherein the non-reducing sugar is sucrose.

[0356] 57. Recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate, and d) about 0.002% (w / v) poloxamer 188; A stable formulation comprising:

[0357] 58. Recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate; d) about 0.002% (w / v) poloxamer 188, and e) about 0.25% (w / v) glycerol; A stable formulation comprising:

[0358] 59. Recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate; d) about 0.002% (w / v) poloxamer 188, and e) about 0.5% (w / v) sorbitol; A stable formulation comprising:

[0359] 60. Recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 30 mM sodium sulfate; c) about 263 mM sucrose, and d) about 0.005% (w / v) poloxamer 188; A stable formulation comprising:

[0360] 61. A formulation according to any one of clauses 1 to 60, suitable for lyophilization.

[0361] 62. The formulation according to any one of clauses 1 to 61, wherein the pH is about 7.5.

[0362] 63. The pH is approximately 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8. 62. The formulation according to any one of clauses 1 to 61,

[0363] 64. The formulation according to any one of clauses 1 to 61, wherein the pH is about 7.1.

[0364] 65. A formulation according to any one of clauses 1 to 61, having a pH of about 6.5 to 8.0.

[0365] 66. The method according to any one of claims 1 to 61, wherein the pH is about 7.2 to about 7.8. Agent.

[0366] 67. Approximately 1.0E+11 genome copies / mL (GC / mL) to approximately 1.0E+15 GC / mL 67. The formulation of any one of clauses 1 to 66, comprising 1 mL of rAAV particles.

[0367] 68. Approx. 1.0E+11GC / mL, approx. 1.0E+12GC / mL, approx. 1.0E+13 GC / mL, approximately 1.0E+14GC / mL, or approximately 1.0E+15GC / mL of rAA 68. The formulation according to clause 67, comprising V particles.

[0368] 69. The rAAV particles are selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV17, AAV18, AAV19, AAV20, AAV21, AAV22, AAV23, AAV24 AV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, A AV-13, AAV-14, AAV-15 and AAV-16, rAAV.rh8, rAA V.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAA V.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L 65, rAAV.7m8, rAAV.PHP.B, rAAV2.5, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.H SC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AA V.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and 68. The method according to claim 1, further comprising administering to said patient a capsid protein comprising said first and second capsid proteins of said first and second capsid proteins of said second ... The formulation described above.

[0369] 70. The rAAV particles contain capsid proteins of AAV-8 or AAV-9 serotypes. 70. The formulation according to clause 69, comprising:

[0370] 71. The composition further comprises a stabilizer selected from the group consisting of glycerol or sorbitol. 71. A formulation according to any one of clauses 1 to 70.

[0371] 72. The formulation according to any one of clauses 1 to 70, further comprising glycerol.

[0372] 73. The method according to any one of clauses 1 to 70, comprising about 0.1% to about 5% glycerol. The formulation described above.

[0373] 74. The method according to any one of clauses 1 to 70, comprising about 0.1% to about 2% glycerol. The formulation described above.

[0374] 75. Any one of clauses 1 to 70, comprising about 0.25% to about 2% glycerol. The formulation described.

[0375] 76. A formulation according to any one of clauses 1 to 70, which does not contain mannitol.

[0376] 77.150 mM, 100 mM, 50 mM, 20 mM, or less than 10 mM mannitol 71. The formulation according to any one of clauses 1 to 70, comprising

[0377] 78. The formulation according to any one of clauses 1 to 77, which is a pre-lyophilized formulation.

[0378] 79. A formulation as described in clause 1, having a residual moisture content of about 1% to about 7%.

[0379] 80. The residual moisture content is about 1% to about 7%, about 2% to about 7%, about 3% to about 7%, about 4% ~ about 7%, about 5% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, or 80. The formulation of clause 79, wherein the concentration is from about 1% to about 3%.

[0380] 81. The residual moisture content is about 3% to about 7%, about 3% to about 6%, or about 3% to about 5%. 79. The formulation according to claim 79,

[0381] 82. The residual moisture content is about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5. 5%, or about 6%.

[0382] 83. The formulation according to clause 79, wherein the water content is from about 1% to about 2%.

[0383] 84. The moisture content is about 1.5%, about 1.4%, about 1.3%, about 1.2%, or about 1. 79. The formulation according to claim 79, wherein the concentration is 0.1%.

[0384] 85. The formulation according to clause 79, wherein the water content is about 1%.

[0385] 86. The glass transition temperature (Tg) of the lyophilized cake of the formulation is greater than 35° C.; 79. A formulation according to any one of clauses 1 to 78.

[0386] 87. The glass transition temperature (Tg') of the maximum freeze-concentrated solution of said formulation is greater than -40°C. 79. A formulation according to any one of clauses 1 to 78.

[0387] 88. The relative potency of the rAAV particles was at least 100% after storage of the formulation at room temperature for 3 months. at least about 60%, at least about 70%, or at least about 80%, but AAV particles were stored at -70°C in DPBS containing 0.001% poloxamer 188 buffer. 79. A formulation according to any one of clauses 1 to 78, wherein the formulation is in storage.

[0388] 89. The relative potency of the rAAV particles was at least 100% after storage of the formulation at room temperature for 6 months. at least about 60%, at least about 70%, or at least about 80%, but AAV particles are prepared in Dulbecco's Phosphate Buffered Saline with 0.001% Poloxamer 188 buffer. 79. The method according to any one of clauses 1 to 78, stored in water (DPBS) at -70°C. formulation.

[0389] 90. The relative potency of the rAAV particles was at least 100% after storing the formulation at 35° C. for 1 week. at least about 30%, at least about 40%, at least about 50%, or at least 60% Yes, except that the reference rAAV particles were prepared in DPB containing 0.001% poloxamer 188 buffer. 79. The formulation of any one of clauses 1 to 78, stored in S at -70°C.

[0390] 91. The relative potency of the rAAV particles was at least 100% after storage of the formulation at 35° C. for 2 weeks. at least about 30%, at least about 40%, at least about 50%, or at least 60% Yes, except that the reference rAAV particles were prepared in DPB containing 0.001% poloxamer 188 buffer. 79. The formulation of any one of clauses 1 to 78, stored in S at -70°C.

[0391] 92. The relative potency of the rAAV particles was at least 100% after storage of the formulation at 35° C. for 4 weeks. at least about 30%, at least about 40%, at least about 50%, or at least 60% Yes, except that the reference rAAV particles were prepared in DPB containing 0.001% poloxamer 188 buffer. 79. The formulation of any one of clauses 1 to 78, stored in S at -70°C.

[0392] 93. The method according to any one of clauses 87 to 92, wherein the formulation is freeze-dried before storage. Formulation.

[0393] 94. The formulation according to clause 94, wherein the lyophilized formulation is reconstituted after storage.

[0394] 95. The relative potency of the rAAV particles is at least about 25% immediately after lyophilization, At least about 30%, at least about 40%, at least about 50%, at least about 60%, at least at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 79. The formulation of any one of clauses 1 to 78, wherein the affinity is at least about 99%.

[0395] 96. The level of rAAV particle aggregation in the formulation is compared to that in a reference formulation. When compared to the level, it is about 10%, about 20%, about 30%, about 40%, about 50%, about 60% %, about 70%, about 80%, about 90%, or about 100% lower, The formulation according to any one of the preceding claims.

[0396] 97. The stability of the formulation is evaluated by vector genome content or viral titer assay. The formulations were evaluated for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 Weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 After storage for months, 9 months, 10 months, 11 months, 1 year, 2 years, or 3 years under the same conditions When compared to the genomic content of a stored reference preparation, the genomic content is at least About 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, approximately 90%, approximately 100%, or approximately 200% more of any one of clauses 1 to 78 The formulation described in

[0397] 98. The stability of the formulation is evaluated by measuring the relative potency of the formulation. The formulations are available in 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, After storage for 10 months, 11 months, 1 year, 2 years, or 3 years, the reference samples were stored under the same conditions. The relative potency, when compared to the genomic content of the formulation, is at least about 5%, about 10%, About 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, 79. The formulation of any one of clauses 1 to 78, which is about 100%, or about 200% higher.

[0398] 99. The stability of the formulation was evaluated by the reduction in infectivity, and the formulation was stable for 1 day, 2 days, 1 hour, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months month, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, After storage for 1, 2, or 3 years, the decrease in infectivity of the reference preparation stored under identical conditions was In comparison, the decrease in infectivity is at least about 5%, about 10%, about 20%, about 30%, or more. %, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or 79. The formulation of any one of clauses 1 to 78, wherein the

[0399] 100. The stability of the formulation is evaluated by rAAV genome release, and the rAAV genome DNA release was identified by measuring relative fluorescence in the presence of a DNA-specific fluorescent stain. The above formulations are administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, and 3 weeks. , 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months After storage for 10, 11, 1, 2, or 3 years, the samples stored under the same conditions were the relative fluorescence level is at least about 5% when compared to the relative fluorescence level of a control formulation; About 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, About 90%, about 100%, or about 200% lower, as described in any one of clauses 1 to 78. formulation.

[0400] 101. The method according to any one of clauses 97 to 100, wherein the formulation is freeze-dried before storage. The formulation described.

[0401] 102. The formulation according to clause 101, wherein the lyophilized formulation is reconstituted after storage.

[0402] 103. The formulation is stored at -80°C, -70°C, -20°C, 4°C, 20°C, 25°C, and 30°C. 103. The formulation according to any one of clauses 97 to 102, stored at 35°C or 40°C. .

[0403] 104. The reference formulation is DPBS containing 0.001% poloxamer 188 buffer. 104. The formulation according to any one of clauses 96 to 103,

[0404] 105. Any one of clauses 96 to 104, wherein the reference formulation is a sugar-free formulation. The formulation described in one.

[0405] 106. Any of clauses 96 to 105, wherein the reference formulation is a formulation that does not contain a plasticizer. or the formulation described in one of the above.

[0406] 107. The formulation is frozen to a temperature of about -20°C during the freeze-drying process. 107. A formulation according to any one of clauses 1 to 106.

[0407] 108. The frozen preparation has a pH of about pH 6 to about pH 9 when frozen at −20° C. 108. The formulation according to any one of clauses 1 to 107,

[0408] 109. When the frozen preparation is frozen at −20° C., the pH value of the frozen preparation is changed from the pH value before freezing to The product according to any one of clauses 1 to 107, maintained within a range of plus or minus 1 unit. Agent.

[0409] 110. Any one of clauses 1 to 109, which is a stabilized aqueous rAAV formulation for lyophilization. The formulation described in one.

[0410] 111. A method for producing a stable formulation containing recombinant adeno-associated virus (rAAV) particles, 108. The rAAV particles are then mixed with a buffer, sugar, salt, or any other suitable agent of the formulation of any one of clauses 1 to 108. Optionally, the rAAV is mixed with a plasticizer and, optionally, a non-ionic surfactant, thereby and producing the formulation comprising:

[0411] 112. A method for reducing rAAV genome release from rAAV particles, comprising: preparing a formulation comprising a buffer, a sugar, a salt, and optionally a non-ionic surfactant; Release of the rAAV genome from the rAAV particles after three freeze-thaw cycles was The method of claim 1, wherein rAAV genome release is reduced compared to that in a preparation not containing the rAAV.

[0412] 113. A method for reducing rAAV genome release from rAAV particles, comprising: preparing a formulation comprising a buffer, a sugar, a salt, and optionally a non-ionic surfactant; Release of the rAAV genome from the rAAV particles after lyophilization and reconstitution was achieved without the sugar. The method further comprises:

[0413] 114. Furthermore, the preparation is freeze-dried to reduce the residual water content to about 1% to about 5%. 114. The method according to claim 112 or 113, comprising:

[0414] 115. A method for reducing rAAV genome release from rAAV particles, comprising: Producing a formulation containing a buffer, a sugar, a salt, a plasticizer, and optionally a non-ionic surfactant. and the rAAV genome release from the rAAV particles after three freeze-thaw cycles is The method, wherein rAAV genome release is reduced compared to that in a sugar-free formulation.

[0415] 116. A method for reducing rAAV genome release from rAAV particles, comprising: Producing a formulation containing a buffer, a sugar, a salt, a plasticizer, and optionally a non-ionic surfactant. and release of the rAAV genome from the rAAV particles after lyophilization and reconstitution is controlled by the presence of the glycosylation agent. The method of claim 1, wherein the rAAV genome release is reduced compared to a formulation not containing the rAAV genome release.

[0416] 117. Use of sugars to reduce rAAV genome release from rAAV particles. A formulation is prepared comprising rAAV particles, a buffer, a sugar, a salt, and optionally a non-ionic surfactant. and producing an rAAV genome from the rAAV particles after three freeze-thaw cycles. The release is reduced compared to the release of the rAAV genome in a formulation that does not contain the sugar. method.

[0417] 118. Use of sugars to reduce rAAV genome release from rAAV particles. A formulation is prepared comprising rAAV particles, a buffer, a sugar, a salt, and optionally a non-ionic surfactant. and releasing the rAAV genome from the rAAV particles after lyophilization and reconstitution. is reduced compared to rAAV genome release in a formulation that does not contain the sugar. .

[0418] 119. Furthermore, the preparation is freeze-dried to reduce the residual water content to about 1% to about 7%. 19. The method according to claim 117 or 118, comprising:

[0419] 120. Use of plasticizers to reduce rAAV genome release from rAAV particles. The rAAV particles, buffer, sugar, salt, plasticizer, and optionally a non-ionic surfactant are then mixed together. and producing a formulation comprising: AAV genome release was reduced compared to rAAV genome release in the sugar-free formulation. The method according to claim 1,

[0420] 121. Use of plasticizers to reduce rAAV genome release from rAAV particles. The rAAV particles, buffer, sugar, salt, plasticizer, and optionally a non-ionic surfactant are then mixed together. and producing a formulation comprising rAA V from the rAAV particles after lyophilization and reconstitution. V genome release was reduced compared to rAAV genome release in the sugar-free formulation. The method.

[0421] 122. rAAV genome release is measured by measuring relative fluorescence in the presence of a DNA-specific fluorescent stain The method according to any one of clauses 112 to 116 or clause 1 17 to 121. The use according to any one of claims 17 to 121.

[0422] 123. Freezing-induced rAAV genome release is at least about 10%, 20%, 50%, 80% or 90% reduction in any of Articles 112 to 116 and 122 or the use according to any one of clauses 117 to 121.

[0423] 124. Freezing-induced rAAV genome release is substantially eliminated. 112 to 116 and 122 or the method according to any one of clauses 117 to 121 2. Use according to any one of the preceding claims.

[0424] 125. The method according to any one of clauses 112 to 116 and 122 to 124, wherein the sugar is a non-reducing sugar. 125. The method according to any one of the preceding clauses or the use according to any one of the clauses 117 to 124.

[0425] 126. The non-reducing sugar is sucrose, trehalose, or raffinose. 126. The method according to clause 125 or the use according to clause 125.

[0426] 127. The method according to claim 125 or claim 12, wherein the non-reducing sugar is sucrose. 5. Use as described in 5.

[0427] 128. Any of clauses 112 to 116 and 122 to 124, wherein the sugar is a reducing sugar. 125. The method according to any one of the preceding clauses or the use according to any one of the clauses 117 to 124.

[0428] 129. The reducing sugar is glucose, fructose, mannose, galactose, or 123. The method according to clause 122 or the use according to clause 122, wherein is lactose.

[0429] 130. The method according to claim 128 or the method according to claim 128, wherein the reducing sugar is glucose. Use as described.

[0430] 131. The plasticizer comprises glycerol, 30 or the use according to any one of clauses 82 to 105. .

[0431] 132. The formulation is according to any one of claims 1 to 110. The method according to any one of paragraphs 111 to 117 and 122 to 131 or the method according to paragraph 82 106. The use according to any one of claims 1 to 106.

[0432] 133. A method for producing a stable lyophilized formulation of a rAAV product, comprising: and subjecting the pre-lyophilized formulation to lyophilization, the pre-lyophilized formulation being The method according to any one of the above.

[0433] 134. A method for treating or preventing a disease, comprising administering to a subject in need of such treatment or prevention administering a therapeutically effective amount of a rAAV formulation that has been reconstituted from a stable lyophilized formulation; The pre-lyophilized formulation of the stable lyophilized formulation comprises 10. The method according to any one of claims 1 to 9. [Brief description of the drawings]

[0434] 5. Brief description of the drawings [Figure 1] Viral particle aggregation is affected by ionic strength. (A) Viral particle aggregation observed upon dilution into buffers of different ionic strength. Salt 1 is sodium chloride and salt 2 is sodium citrate. (B) Viral particle aggregation increases over time. [Diagram 2] Minimum ionic strength to prevent aggregation. Effective diameter of AAV8 particles at 1.8E+13GC / mL prepared with different NaCl concentrations. Based on the particle diameter, it appears that an ionic strength of 90 mM or higher is required to prevent particle aggregation. [Diagram 3] The minimum ionic strength is serotype dependent. Effective diameter of AAV8 (open squares) and AAV9 (open triangles) adjusted with different NaCl concentrations. The vector concentration was 6.0E+11 GC / mL. [Figure 4] Prevention of aggregation is necessary but not sufficient to ensure a stable formulation. Size distribution of virus particles after storage at -80°C for 6 months (6 months, -80°C) or at 25°C for 6 months (6 months, 25°C) is shown. [Diagram 5] Detection of AAV genome release in the presence of SYBR gold, a dye that exhibits a significant increase in fluorescence intensity when bound to DNA. Solutions contain 9.7E+11 GC / mL AAV8 particles in Tris buffer containing poloxamer 188 in the presence of either (A) NaCl or (B) sodium citrate. The solid line is a non-frozen control, and the dashed line is after three freeze-thaw cycles. [Figure 6] DNA release is increased in the presence of salts that crystallize during freezing. Salt 1 is sodium citrate and salt 2 is sodium chloride. DNA release in control samples and after three freeze-thaw cycles (3xF / T) is shown. Buffer 1 is Tris, buffer 2 is sodium phosphate, and buffer 3 is histidine. [Figure 7] Detection of AAV genome release in the presence of SYBR gold. Solutions contain 1E+12GC / mL AAV8 in either phosphate buffered saline (PBS) (A) or PBS with 4% sucrose (B). The solid line is the non-frozen control, and the dashed line is after 10 freeze-thaw cycles. [Figure 8] The addition of amorphous sugar inhibits cosolute crystallization and results in less DNA release. Shown are DNA release after control samples and three freeze-thaw cycles (3×F / T) in buffers containing different ratios of crystalline mannitol and amorphous sucrose components. [Figure 9] The reduction in GC leads to a concomitant decrease in relative potency after lyophilization. The relative potency and viral DNA titer of the formulations after lyophilization are shown. The sample labeled "C" contained crystalline bulking agent. [Figure 10] The pH of the freezing solution affects the stability of the lyophilized solid. The pH of the liquid and freezing solution is indicated by color. The relative potency of the lyophilized formulations after 6 months storage at 25° C. is shown. [Figure 11]Percent relative potency of AAV8 formulations after non-lyophilized control ("Liquid"), after lyophilization ("Post Lyo"), and after 3 and 6 months of storage at room temperature. Formulations are either Tris buffer (solid black) or phosphate buffer (solid grey). [Figure 12] Percent relative potency of lyophilized AAV8 formulations during storage at 35° C. Residual moisture content is 5.8% (circles), 4.7% (triangles), 3.6% (diamonds), and 2.1% (squares). [Figure 13] Relative percent potency of lyophilized AAV8 formulations during storage at 5°C (triangles) or room temperature (squares). [Figure 14] Detection of AAV genome release in the presence of SYBR gold. Solutions contained formulation buffer only (thick solid line) or 2.4E+11 GC / mL AAV8 either before lyophilization (solid line) or after lyophilization to different residual moisture levels: 4.7% (dotted line), 3.6% (dashed line), and 2.1% (dash-dotted line), followed by reconstitution with water to the original volume. As the residual moisture decreases, more AAV genomes are released. [Figure 15] Comparison of AAV stability of formulations 1A, 6A, and 7A after lyophilization. A shows free DNA release due to AAV capsid damage. Higher fluorescence intensity indicates more free DNA. B shows logarithm of viral titer reduction. C shows in vitro relative potency. [Figure 16] Thermal transitions of formulation 4A characterized by low-temperature DSC. [Figure 17] Comparison of formulations 1A, 2A, 3A, 4A, and 5A after 2 weeks at 35° C. A shows free DNA release due to AAV capsid damage. B shows virus titer. C shows in vitro relative potency. [Figure 18] Particle size of AAV after dilution in NaCl or Na2SO4 solutions of different ionic strengths: A, diameter at TO, B, diameter after 8 hours, C, diameter after 24 hours at room temperature in the presence of increasing sucrose concentrations in the presence of 30 mM Na2SO4. [Figure 19]1 shows the pH shift of dPBS, Formulation 4A, and Formulation 5A when frozen to -20°C. [Figure 19] 1 shows the pH shift of dPBS, Formulation 4A, and Formulation 5A when frozen to -20°C. [Figure 20] Clustal multiple sequence alignment of AAV capsids 1-9 (SEQ ID NOs: 41-51). Amino acid substitutions (indicated in bold in the last line) can be introduced into AAV9 and AAV8 capsids by "recruiting" amino acid residues from corresponding positions in other aligned AAV capsids. Sequence regions designated "HVR" = hypervariable regions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0435] 6. Detailed Description Provided herein are formulations comprising rAAV particles. In some embodiments, the The formulations disclosed herein may contain a buffer, one or more stabilizers (e.g., sugars, polyols, etc. , amino acids), salts, and optionally a non-ionic surfactant. The formulations disclosed herein are frozen or lyophilized formulations. In some embodiments, the formulations disclosed herein remain amorphous during freezing. The formulations comprising the rAAV particles disclosed herein are stable when stored at room temperature for extended periods of time. and does not significantly reduce infectivity.

[0436] In some embodiments, provided herein are stable formulations, treatments involving stable formulations, The present invention relates to a method for producing a pharmaceutical composition and a kit including a stable formulation.

[0437] In some embodiments, the formulations described in Section 6.2 may be prepared by mixing one or more of the formulations described in Section 6.2. In some embodiments, the compositions are formulated to have one or more functional properties. The stable preparations described in Chapter 2 have one or more of the properties described in Chapter 6.2. In certain embodiments, the formulations provided herein are formulated to have , various advantages, such as improved stability after lyophilization and long-term stability under various conditions. Also provided herein are improvements that may be used in Section 6.8. The present inventors have surprisingly found that the minimum ionic strength required to prevent aggregation is We found that the degree of AAV aggregation was AAV serotype-dependent. AAV9 requires a lower ionic strength than AAV8. The inventors further surprisingly found that crystallization of the formulation components during freezing reduced the size of the rAAV particles. To the best of our knowledge, this species The genome reduction of the virus and methods to prevent genome reduction through drug development are also publicly available. The present inventors have demonstrated that genome reduction is achieved by one of the following two formulation approaches: We found that this could be mitigated by either: (a) replacing salt that crystallizes during freezing; (b) The formulation is formulated using a salt that does not crystallize, and (b) the formulation contains a component that inhibits crystallization (e.g. In some embodiments, the compositions disclosed herein further include a unique The combination of sodium citrate and sucrose is one of the most effective ways to improve the taste compared to other salts. The freeze-drying process provides unique advantages. In some embodiments, the methods disclosed herein The formulations used use buffers that prevent pH shifts during freezing.

[0438] The inventors further surprisingly found that, in some embodiments, lyophilized rAAV formulations can be produced in a lyophilized form. We found that drying as much as possible did not provide the best stability in the dry state. As a result, in some embodiments, the stable rAAV formulations disclosed herein have a yield of about Residual moisture content of 1% to 7%. In addition, depending on the embodiment, there is no intention to adhere to a specific theory. Although not guaranteed, the stable rAAV formulations disclosed herein can be made in a dry state as a substitute for water. The plasticizers that may be used include, for example, glycerol.

[0439] 6.1 Definition Unless otherwise defined, all technical and scientific terms used herein are within the meaning of the present disclosure. The terms have the same meaning as commonly understood by one of ordinary skill in the relevant art. For ease of understanding, a number of terms and phrases are defined below.

[0440] "About" can refer to, for example, the amount of a component in a composition ... Concentrations of components in the medium, flow rates, rAAV particle yields, feed rates, salt concentrations, and similar values, as well as Although the term modifies the scope of these, it is not intended to limit the scope of the invention, for example, to the use of the term to make a concentrate or to use a solution. Through typical measurement and handling procedures that can be performed, and through unintentional errors in such procedures As such, differences in manufacture, source, or purity of ingredients used in making the composition or carrying out the method may be The term "about" is intended to indicate certain variations in the quantities that may occur through variations in the quantities specified, and similar considerations. It also encompasses amounts that vary due to aging of a composition having a given initial concentration or mixture state. The term also encompasses amounts that vary due to mixing or processing of compositions having a particular initial concentration or mixture state. Whether or not modified by the term "about," the claims are to be interpreted as including the In some embodiments, the term "about" refers to the amount by which the number is expressed. or a range that is about 10-20% greater or less than the range. In this case, "about" means plus or minus 10% of the stated figure or range. For example, "about "10%" indicates a range of 9% to 11%. In certain embodiments, the use of "about" The words are inclusive of the exact numbers recited.

[0441] "AAV" is an abbreviation for adeno-associated virus, and can refer to the virus itself or to any of its modifications. It can be used to indicate a variant, derivative, or pseudotype. The term applies to all subtypes. "Includes both natural and recombinant types, unless otherwise required." The abbreviation "rAAV" refers to recombinant adeno-associated virus. The term "AAV" refers to AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), A AV4 type (AAV-4), AAV5 type (AAV-5), AAV6 type (AAV-6), AA V7 type (AAV-7), AAV8 type (AAV-8), AAV9 type (AAV-9), avian A AV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. "Primate AAV" includes diAAV, as well as modifications, derivatives, or pseudotypes thereof. "Non-primate AAV" refers to AAV that infects non-primate mammals. V, "bovine AAV" refers to an AAV that infects bovine mammals, and so on.

[0442] "Recombinant," as applied to an AAV particle, refers to an AAV particle that is different from a naturally occurring AAV particle. is the product of one or more techniques that result in distinct AAV particle constructs. Taste.

[0443] A recombinant adeno-associated virus particle, or "rAAV particle," is a virus that contains at least one AAV capsid. AAV protein and a heterologous polynucleotide encapsidated therein (i.e., wild-type AAV Polynucleotides containing non-genomic polynucleotides, e.g., transgenes, that are delivered to mammalian cells. 1 shows a viral particle composed of a ribonucleic acid nucleotide and a rAAV vector. Any AAV serotype may be used, and such particles may include any modified, derivative, or pseudotypes (e.g., AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, AAV-16, AAV-17, AAV-18, AAV-19, AAV-20, AAV-21, AV-5, AAV-6, AAV-7, AAV-8, AAV-9, or AAV-10, Such AAV serotypes and derivatives / modifications / pseudotypes thereof. Pseudotypes, as well as methods for generating such serotypes / derivatives / modifications / pseudotypes, are known in the art. It is known (e.g., Asokan et al., Mol. Ther. 20(4 ):699-708 (2012).

[0444] The rAAV particles of the present disclosure can be of any serotype, or any combination of serotypes (e.g., A population of rAAV particles containing two or more serotypes, e.g., rAAV2 particles, rAAV8 particles. and rAAV9 particles). For the rAAV particles, rAAV1, rAAV2, rAAV3, rAAV4, and rAAV5 are , rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, or other rAA In some embodiments, the rAAV V particle is a rAAV V particle, or a combination of two or more thereof. The particles are rAAV8 particles or rAAV9 particles.

[0445] In some embodiments, the rAAV particles include AAV-1, AAV-2, AAV-3, AAV -4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, and AAV AAV capsid proteins of serotypes selected from the group consisting of AAV-16, AAV-20, AAV-30, AAV-40, AAV-50, AAV-60, AAV-70, AAV-80, AAV-90, AAV-110, AAV-120, AAV-130, AAV-140, AAV-150, AAV-160, AAV-161, AAV-162, AAV-163, AAV-164, AAV-165, AAV-166, AAV-167, AAV-168, AAV-169 ...9, AAV-161, A In some embodiments, the rAAV particles include AAV- 8. AAV capsid protein of the AAV-9 serotype, or a derivative or modification thereof; Or has a pseudotype.

[0446] "Purifying", "purification", "fractionation" "separate", "separating", "separation" "isolation", "isolate", "isolation" The terms "isolation" and "isolation" are used herein. Increasing the purity of rAAV particles from samples containing the desired product and one or more impurities Typically, the purity of the target product is determined by removing at least one impurity from the sample. In some embodiments, the increase is due to the removal (completely or partially) of The purity of rAAV can be determined by extracting one or more species from a sample using the methods described herein. is increased by removing (completely or partially) several impurities.

[0447] As used in this disclosure and claims, the singular forms "a," "an," and "th" are used interchangeably. "e" includes the plural unless the context clearly indicates otherwise.

[0448] It will be understood that embodiments are described herein using the term "comprising." Whenever stated herein, "consisting of" and / or is written as "consisting essentially of" Other similar embodiments are provided, as described below. The term "consisting essentially of" Whenever described herein using the term "consisting of" or "consisting of Further similar embodiments are also provided, as described under "f)".

[0449] The term "and / or" is used herein in phrases such as "A and / or B." When used in a sentence, it includes both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" is intended to include "A, B, and / or C." " and the like, is intended to encompass each of the following embodiments: : A, B, and C; A, B, or C; A or C; A or B; B or C; A and C ;A and B;B and C;A (alone);B (alone);and C (alone).

[0450]

[0023] Embodiments of the present disclosure may be described in Markush groups or other groupings of options. If a compound is included in the recited group, the method disclosed includes not only the entire recited group in its entirety, but also Each individual member of the group and all possible subgroups of the main group, as well as the group The disclosure also includes the main group from which one or more of the group members are absent. The law also envisages the explicit exclusion of any one or more of the group members of the disclosed method. is doing.

[0451] 6.2 Formulations containing rAAV particles In some embodiments, the present disclosure provides recombinant adeno-associated virus (rAAV) particles, and and a buffer, a sugar, and a salt. In some embodiments, the salt is pharma- ceutical In some embodiments, the salt is an amorphous salt that does not crystallize. In some embodiments, the salt is a sodium salt. In some embodiments, the salt is quenched. The preferred salts are sodium phosphate, sodium sulfate, ammonium sulfate, or magnesium sulfate. Multivalent salts contain multiple charged ions (compared to monovalent sodium chloride) that have a higher ionic strength. (per molecule of excipient added) and this salt is This allows the AAV aggregation to be inhibited, thus maintaining the amorphous matrix of the composition. Therefore, rAAV formulations containing salts other than monovalent sodium chloride may be useful. It's hidden.

[0452] 6.2.1 Salt In some embodiments, salt excipients are added or adjusted to achieve the desired ionic strength. It will be adjusted.

[0453] In some embodiments, provided herein are recombinant adeno-associated viruses (rAA viruses). V) particles, as well as a buffer, sugar, and amorphous salt, and having an ionic strength of 60 mM to 15 mM. 0 mM, and the formulation is suitable for lyophilization. In some embodiments, the stable formulation also includes a surfactant. Including 8.

[0454] In some embodiments, provided herein are recombinant adeno-associated viruses (rAA viruses). V) particles, as well as a buffer, sugar, and amorphous salt, having an ionic strength of greater than 200 mM. In some embodiments, the stable formulation is suitable for lyophilization. The agent also includes a surfactant. In some embodiments, the stable formulation also includes poloxamer 188. .

[0455] In some embodiments, provided herein are recombinant adeno-associated viruses (rAA viruses). V) particles, as well as a buffer, sugar, and amorphous salt, having an ionic strength of 200 mM, 2 10mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM , 280 mM, 290 mM, or greater than 300 mM, and the formulation is stable under freezing. In some embodiments, the stable formulation also includes a surfactant. Thus, the stable formulation also contains poloxamer 188.

[0456] In some embodiments, the salt is a pharma- ceutically acceptable salt. It is an amorphous salt that does not crystallize. In some embodiments, the salt is a sodium salt. In some embodiments, the salt is sodium citrate, sodium acetate, or sodium chloride. In some embodiments, the salt is sodium citrate, sodium sulfate, ammonium sulfate. In some embodiments, the salt is a multivalent salt, and the salt is a multiply charged ion with a higher ionic strength (compared to monovalent sodium chloride) The compound consists of 100% excipients per molecule, which prevents AAV aggregation while minimizing crystallization. can harm.

[0457] In some embodiments, the present disclosure provides a method for the preparation of a recombinant adeno-associated virus (AAV), a salt excipient, a sugar In some embodiments, the stable formulation also includes a surfactant. In some embodiments, the stable formulation also includes poloxamer 188. The agent is suitable for lyophilization.

[0458] In some embodiments, provided herein are recombinant adeno-associated viruses (rAA viruses). V) Particles, as well as buffers, sugars, and amorphous materials having an ionic strength of 60 mM to 150 mM. and a salt thereof, the formulation being suitable for lyophilization. In some embodiments, the stable formulation also includes a surfactant. Also includes 8. In some embodiments, the formulation is a pre-lyophilized formulation.

[0459] In some embodiments, provided herein are recombinant adeno-associated viruses (rAA viruses). V) Particles, as well as buffers, sugars, and amorphous materials having an ionic strength of 30 mM to 100 mM. and a salt thereof, the formulation being suitable for lyophilization. In some embodiments, the stable formulation also includes a surfactant. Also includes 8. In some embodiments, the formulation is a pre-lyophilized formulation.

[0460] In some embodiments, provided herein are recombinant adeno-associated viruses (rAA viruses). V) particles, as well as a buffer, a sugar, and an amorphous salt having an ionic strength greater than 200 mM. In some embodiments, the stable formulation is suitable for lyophilization. The agent also includes a surfactant. In some embodiments, the stable formulation also includes poloxamer 188. In some embodiments, the formulation is a pre-lyophilized formulation.

[0461] In some embodiments, provided herein are recombinant adeno-associated viruses (rAA viruses). V) particles, as well as a buffer, a sugar, and an amorphous salt having an ionic strength of 60 mM to 150 mM; and a surfactant, wherein the formulation is suitable for lyophilization. In some embodiments, the stable formulation also includes a surfactant. It also contains xamer 188. In some embodiments, the formulation is a pre-lyophilized formulation.

[0462] In some embodiments, provided herein are recombinant adeno-associated viruses (rAA viruses). V) particles, as well as a buffer, a sugar, and an amorphous salt having an ionic strength of 30 mM to 100 mM; and a surfactant, wherein the formulation is suitable for lyophilization. In some embodiments, the stable formulation also includes a surfactant. It also contains xamer 188. In some embodiments, the formulation is a pre-lyophilized formulation.

[0463] In some embodiments, provided herein are recombinant adeno-associated viruses (rAA viruses). V) particles, as well as buffers, sugars, and amorphous salts having an ionic strength greater than 200 mM; and and a surfactant, the formulation being suitable for lyophilization. Thus, the stable formulation also includes a surfactant. In some embodiments, the stable formulation includes poloxamer. It also contains Mer 188. In some embodiments, the formulation is a pre-lyophilized formulation.

[0464] In some embodiments, the formulations disclosed herein contain from about 0.0001% to about 0.5% In some embodiments, the formulations disclosed herein contain about 0.5% to about 10% by weight of a non-ionic surfactant. In some embodiments, the composition includes from 0.0005% to about 0.1% of a non-ionic surfactant. The formulations disclosed herein may contain about 0.001%, about 0.002%, about 0.003%, about 0.00 4%, about 0.005%, about 0.007%, or about 0.01% of a nonionic surfactant include.

[0465] In some embodiments, the formulations disclosed herein contain from about 0.0001% to about 0.5% Poloxamer 188. In some embodiments, the formulations disclosed herein contain about 0. In some embodiments, the composition described herein comprises from about 0.05% to about 0.1% poloxamer 188. The disclosed formulations include about 0.001%, about 0.002%, about 0.003%, about 0.004% , about 0.005%, about 0.007%, or about 0.01% poloxamer 188.

[0466] In some embodiments, the formulations disclosed herein contain about 0.0005% poloxamer. 188. In some embodiments, the formulations disclosed herein contain about 0.001% poly In some embodiments, the formulations disclosed herein contain about 0.0 0.02% Poloxamer 188. In some embodiments, the formulations disclosed herein include and about 0.003% poloxamer 188. In some embodiments, the compositions disclosed herein include The formulation contains about 0.004% poloxamer 188. In some embodiments, the formulation The formulation disclosed herein contains about 0.005% poloxamer 188. The formulations disclosed herein contain about 0.008% poloxamer 188.

[0467] In some embodiments, the salt is a pharma- ceutically acceptable salt. It is an amorphous salt that does not crystallize. In some embodiments, the salt is a sodium salt. In some embodiments, the salt is sodium citrate, sodium acetate, or sodium chloride. In some embodiments, the salt is sodium citrate, sodium sulfate, ammonium sulfate. In some embodiments, the salt is a multivalent salt, and the salt is a multiply charged ion with a higher ionic strength (compared to monovalent sodium chloride) The compound consists of 100% excipients per molecule, which prevents AAV aggregation while minimizing crystallization. can harm.

[0468] In some embodiments, the salt excipient is added until the desired ionic strength is achieved. In some embodiments, the amorphous salt has an ionic strength of about 150 mM, about 140 mM, or about 200 mM. 5mM, about 140mM, about 135mM, about 130mM, about 125mM, about 120mM, about 115 mM, about 110 mM, about 110 mM, about 105 mM, or about 100 mM or less In certain embodiments, the stable formulation has a buffer ionic strength of about 150 mM, about 1 45mM, about 140mM, about 135mM, about 130mM, about 125mM, about 120mM, About 115 mM, about 110 mM, about 105 mM, or about 100 mM or less. In some cases, the rAAV particles in the formulation are rAAV8 particles or rAAV9 particles.

[0469] In some embodiments, the stable formulation has an ionic strength of 150 mM, 145 mM, 140 mM , 135mM, 130mM, 125mM, 120mM, 115mM, or 110mM or more In certain embodiments, the stable formulation has a buffer ionic strength of 150 mM, 145mM, 140mM, 135mM, 130mM, 125mM, 120mM, 115m In some embodiments, the rAAV particles in the formulation have an AA It has a V8 capsid.

[0470] In certain embodiments, the stable formulation has an ionic strength of greater than 60 mM. In one embodiment, the stable formulation has an ionic strength of about 60 mM to 150 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 115 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 100 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM. In certain embodiments, the stable formulation has an ionic strength of about 65 mM. The formulation has an ionic strength of about 70 mM. In certain embodiments, the stable formulation has an ionic strength of about 80 mM. In certain embodiments, the stable formulation has an ionic strength of about 85 mM. In an embodiment, the stable formulation has an ionic strength of about 90 mM. The rAAV particles in the formulation have AAV8 capsids.

[0471] In certain embodiments, the stable formulation has an ionic strength of greater than about 30 mM. In certain embodiments, the stable formulation has an ionic strength of about 30 mM to 100 mM. In certain embodiments, the stable formulation has an ionic strength of about 30 mM. In certain embodiments, the stable formulation has an ionic strength of about 35 mM. In certain embodiments, the stable formulation has an ionic strength of about 40 mM. In certain embodiments, the stable formulation has an ionic strength of about 50 mM. In certain embodiments, the stable formulation has an ionic strength of about 55 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM. In certain embodiments, the stable formulation has an ionic strength of about 65 mM. In certain embodiments, the stable formulation has an ionic strength of about 70 mM. In certain embodiments, the stable formulation has an ionic strength of about 80 mM. In certain embodiments, the stable formulation has an ionic strength of about 85 mM. In certain embodiments, the stable formulation has an ionic strength of about 90 mM. The stable formulation has an ionic strength of about 95 mM. In some embodiments, the rAAV particles in the formulation have an affinity for AAV9. It has a capsid.

[0472] In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 115 mM. In certain embodiments, the stable formulation has an ionic strength of about 65 mM to 95 mM. In certain embodiments, the stable formulation has an ionic strength of about 70 mM to 90 mM. In certain embodiments, the stable formulation has an ionic strength of about 75 mM to 85 mM. In some embodiments, the rAAV particles in the formulation have AAV8 capsids.

[0473] In certain embodiments, the stable formulation has an ionic strength of about 30 mM to 100 mM. In certain embodiments, the stable formulation has an ionic strength of about 35 mM to 95 mM. In certain embodiments, the stable formulation has an ionic strength of about 40 mM to 90 mM. In certain embodiments, the stable formulation has an ionic strength of about 45 mM to 85 mM. In certain embodiments, the stable formulation has an ionic strength of about 50 mM to 80 mM. In certain embodiments, the stable formulation has an ionic strength of about 55 mM to 75 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 70 mM. In some embodiments, the rAAV particles in the formulation do not have AAV8 capsids. In some configurations, the rAAV particles in the formulation have AAV9 capsids.

[0474] In some embodiments, the stable formulation has an ionic strength of greater than about 200 mM. In other words, the rAAV particles in the formulation have AAV2 capsids. The rAAV particles in the agent do not have AAV8 or AAV9 capsids.

[0475] In some embodiments, the stable formulation has an ionic strength of 200 mM, 210 mM, 220 mM , 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, 290 In some embodiments, the rAAV particles in the formulation are It has a V2 capsid.

[0476] In certain embodiments, the stable formulation has an ionic strength of about 200 mM to about 210 mM In certain embodiments, the stable formulation has an ionic strength of about 210 mM to about 22 mM. In certain embodiments, the stable formulation has an ionic strength of about 220 mM to about 250 mM. In certain embodiments, the stable formulation has an ionic strength of about 230 mM. In certain embodiments, the stable formulation has an ionic strength of about In certain embodiments, the stable formulation has an ionic strength of about 240 mM to about 250 mM. In certain embodiments, the stable formulation has a concentration of about 250 mM to about 260 mM. In certain embodiments, the stable formulation has an on strength of about 260 mM to about 270 mM. In a particular embodiment, the ionic strength is about 270 mM to about 280 mM. The fixed formulation has an ionic strength of about 280 mM to about 290 mM. In some embodiments, the stable formulation has an ionic strength of about 290 mM to about 300 mM. , the rAAV particles in the formulation do not have AAV8 or AAV9 capsids.

[0477] In some embodiments, the formulations disclosed herein contain less than about 100 mM salt. In some embodiments, the formulations disclosed herein contain about 10 mM to about 100 mM salt. .

[0478] In some embodiments, the formulations disclosed herein contain from about 10 mM to about 100 mM, about 2 0 mM to about 100 mM, about 30 mM to about 100 mM, about 40 mM to about 100 mM, about 50 mM ~ approx. 100mM, approx. 10mM ~ approx. 80mM, approx. 10mM ~ approx. 60mM, approx. 10mM ~ Contains about 50 mM, about 10 mM to about 40 mM, or about 10 mM to about 30 mM salt.

[0479] In some embodiments, the formulations disclosed herein contain from about 10 mM to about 50 mM, about 20 mM 10 mM to about 60 mM, about 30 mM to about 70 mM, or about 10 mM to about 30 mM salt .

[0480] In some embodiments, the formulations disclosed herein contain about 10 mM, about 20 mM, about 30 mM, or about 40 mM of glycerol. 50 mM, about 40 mM, about 50 mM, or about 60 mM salt.

[0481] In some embodiments, the formulations disclosed herein comprise about 20 mM salt.

[0482] In some embodiments, the formulations disclosed herein contain less than about 100 mM sodium citrate. In some embodiments, the formulations disclosed herein contain about 10 mM to about 10 mM sodium. Contains 0 mM sodium citrate.

[0483] In some embodiments, the formulations disclosed herein contain from about 10 mM to about 100 mM quencher. In some embodiments, the formulations disclosed herein contain about 20 mM sodium phosphate. In some embodiments, the compositions disclosed herein comprise about 100 mM sodium citrate. In some embodiments, the formulation contains about 30 mM to about 100 mM sodium citrate. The formulations disclosed herein contain about 40 mM to about 100 mM sodium citrate. In some embodiments, the formulations disclosed herein contain about 50 mM to about 100 mM quercetin. In some embodiments, the formulations disclosed herein contain about 10 mM sodium phosphate. In some embodiments, the compositions disclosed herein include a sodium citrate solution having about 80 mM sodium citrate. The formulation comprises about 10 mM to about 60 mM sodium citrate. The formulations disclosed herein contain about 10 mM to about 50 mM sodium citrate. In some embodiments, the formulations disclosed herein contain from about 10 mM to about 40 mM sodium citrate. In some embodiments, the formulations disclosed herein contain from about 10 mM to about 30 mM sodium. Contains mM sodium citrate.

[0484] In some embodiments, the formulations disclosed herein contain about 10 mM to about 50 mM citric acid. In some embodiments, the formulations disclosed herein contain about 20 mM to about 20 mM sodium phosphate. In some embodiments, the compositions disclosed herein contain about 60 mM sodium citrate. In some embodiments, the present invention comprises about 30 mM to about 70 mM sodium citrate. The formulations disclosed herein contain from about 10 mM to about 30 mM sodium citrate.

[0485] In some embodiments, the formulations disclosed herein contain about 10 mM sodium citrate. In some embodiments, the formulations disclosed herein contain about 20 mM sodium citrate. In some embodiments, the formulations disclosed herein contain about 30 mM citrate. In some embodiments, the formulations disclosed herein contain about 40 mM sodium phosphate. In some embodiments, the formulations disclosed herein contain about 50 In some embodiments, the formulations disclosed herein comprise: Contains approximately 60 mM sodium citrate.

[0486] In some embodiments, the formulations disclosed herein contain about 20 mM sodium citrate. Includes.

[0487] 6.2.2 Buffers Buffers are well known in the art, and examples of buffers include, but are not limited to, phosphate buffer, histidine buffer, and the like. Thyidine, Sodium Citrate, HEPES, Tris, Bicine, Glycine, N-Glu Lysylglycine, sodium acetate, sodium carbonate, glycylglycine, lysine, arginine In certain embodiments, the present invention includes phosphate buffer, sodium phosphate, and mixtures thereof. In some embodiments, the buffering agent is histidine (e.g., L-histidine). The buffer is a pharma- ceutically acceptable buffer. In some embodiments, the buffer contains Tris. nothing.

[0488] In some embodiments, the formulations disclosed herein contain from about 1 mM to about 50 mM of a buffering agent. In some embodiments, the formulations disclosed herein contain from about 1 mM to about 30 mM, about 1 mM~about 20mM, about 5mM~about 30mM, about 5mM~about 20mM, about 10mM~about 30 50 mM, about 10 mM to about 20 mM, or about 20 mM to about 50 mM of a buffering agent. In some embodiments, the formulations disclosed herein contain about 1 mM, about 2 mM, about 3 mM, about 5 mM , about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, or about 40 mM In some embodiments, the formulations disclosed herein contain about 5 mM of a buffering agent. include.

[0489] In some embodiments, the formulations disclosed herein contain about 1 mM to about 50 mM Tris In some embodiments, the formulations disclosed herein contain from about 1 mM to about 30 mM, about 1mM to about 20mM, about 5mM to about 30mM, about 5mM to about 20mM, about 10mM to about 3 0 mM, about 10 mM to about 20 mM, or about 20 mM to about 50 mM Tris. In some embodiments, the formulations disclosed herein may contain about 1 mM, about 2 mM, about 3 mM, about 5 mM, or about 6 mM of glycerol. mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, or about 40 mM In some embodiments, the formulations disclosed herein contain about 5 mM Tris. Contains ris.

[0490] In some embodiments, the formulations disclosed herein include liquid formulations. and a buffer capable of providing substantially the same pH in the frozen state.

[0491] In some embodiments, the formulations disclosed herein have a pH of about 6.5 to 8.0. In some embodiments, the formulations disclosed herein have a pH of about 7.0 to 8.0. In some embodiments, the formulations disclosed herein have a pH of about 7.2 to 7.8.

[0492] In some embodiments, the formulations disclosed herein have a pH of about 6.6. In some embodiments, the formulations disclosed herein have a pH of about 6.8. In some embodiments, the formulations disclosed herein have a pH of about 7.0. The formulations disclosed herein have a pH of about 7.2. The formulation has a pH of about 7.3. In some embodiments, the formulations disclosed herein include The pH is about 7.4. In some embodiments, the formulations disclosed herein have a pH of about 7. In some embodiments, the formulations disclosed herein have a pH of about 7.6. In some embodiments, the formulations disclosed herein have a pH of about 7.7. In some cases, the formulations disclosed herein have a pH of about 7.8.

[0493] In some embodiments, the formulations disclosed herein have a pH of about 7.5.

[0494] In some embodiments, the formulations disclosed herein have a substantially constant pH in liquid and frozen states. is the same as.

[0495] In some embodiments, the formulations disclosed herein have a pH of about 6.0 in liquid and frozen states. In some embodiments, the formulations disclosed herein are available in liquid and frozen form. In some embodiments, the formulations disclosed herein have a pH of about 7.0 to 8.0. In the liquid and frozen state, the pH is about 7.2 to 7.8. The disclosed formulations have a pH of about 6.6, 6.8, 7.0, or 7.2 in liquid and frozen states. In some embodiments, the formulations disclosed herein have a pH in liquid and frozen states. In some embodiments, the β-amino acid is about 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8. Thus, the formulations disclosed herein have a pH of about 7.5 in liquid and frozen states.

[0496] 6.2.3 sugar In some embodiments, the formulations disclosed herein include a non-reducing sugar. For purposes of this specification, the non-reducing sugars are sucrose, trehalose, raffinose, or combinations thereof. In some embodiments, the formulations disclosed herein include sucrose.

[0497] In some embodiments, the formulations disclosed herein include a reducing sugar. Reducing sugars are glucose, fructose, mannose, galactose, lactose, or or a combination thereof. In some embodiments, the formulations disclosed herein include Contains glucose.

[0498] In some embodiments, the formulations disclosed herein contain from about 50 mM to about 400 mM sugar. In some embodiments, the formulations disclosed herein contain from about 50 mM to about 350 mM, About 50 mM to about 300 mM, about 50 mM to about 250 mM, about 50 mM to about 200 mM, or about 50 mM to about 150 mM sugar.

[0499] In some embodiments, the formulations disclosed herein contain from about 100 mM to about 400 mM, about 150mM to about 400mM, about 200mM to about 400mM, about 250mM to about 400mM or about 300 mM to about 400 mM sugar.

[0500] In some embodiments, the formulations disclosed herein contain from about 100 mM to about 300 mM, about 150 mM to about 250 mM, about 200 mM to about 300 mM, or about 250 mM to about 35 Contains 0 mM sugar.

[0501] In some embodiments, the formulations disclosed herein contain about 50 mM, about 100 mM, about 1 50mM, about 160mM, about 170mM, about 180mM, about 190mM, about 200mM, Approx. 210mM, Approx. 220mM, Approx. 230mM, Approx. 240mM, Approx. 250mM, Approx. 260m M, about 270 mM, about 280 mM, about 290 mM, about 300 mM, or about 350 mM Contains sugar.

[0502] In some embodiments, the formulations disclosed herein contain from about 190 mM to about 230 mM, about Contains 170 mM to about 250 mM, or about 150 mM to about 270 mM sugar.

[0503] In some embodiments, the formulations disclosed herein comprise about 210 mM sugar.

[0504] In some embodiments, the formulations disclosed herein contain from about 50 mM to about 400 mM sucrose. In some embodiments, the formulations disclosed herein contain from about 50 mM to about 35 mM In some embodiments, the formulations disclosed herein contain about 500 mM sucrose. In some embodiments, the compositions disclosed herein comprise from about 20 mM to about 300 mM sucrose. In some embodiments, the composition comprises about 50 mM to about 250 mM sucrose. The disclosed formulations include about 50 mM to about 200 mM sucrose. Thus, the formulations disclosed herein contain from about 50 mM to about 150 mM sucrose.

[0505] In some embodiments, the formulations disclosed herein contain from about 100 mM to about 400 mM S. In some embodiments, the formulations disclosed herein contain from about 150 mM to about 400 mM sucrose. In some embodiments, the formulations disclosed herein contain about In some embodiments, the present invention comprises a method for preparing a sucrose-based food comprising the steps of: The formulation comprises about 250 mM to about 400 mM sucrose. The formulations disclosed herein contain about 300 mM to about 400 mM sucrose.

[0506] In some embodiments, the formulations disclosed herein contain from about 100 mM to about 300 mM S. In some embodiments, the formulations disclosed herein contain from about 150 mM to about 250 mM sucrose. In some embodiments, the formulations disclosed herein contain about In some embodiments, the compositions disclosed herein include a sucrose extract. The formulation contains about 250 mM to about 350 mM sucrose.

[0507] In some embodiments, the formulations disclosed herein contain about 50 mM sucrose. In some embodiments, the formulations disclosed herein contain about 100 mM sucrose. In some embodiments, the formulations disclosed herein contain about 150 mM sucrose. In some embodiments, the formulations disclosed herein contain about 160 mM sucrose. In some embodiments, the formulations disclosed herein contain about 170 mM sucrose. In some embodiments, the formulations disclosed herein contain about 180 mM sucrose. In some embodiments, the formulations disclosed herein contain about 190 mM sucrose. In some embodiments, the formulations disclosed herein contain about 200 mM sucrose. In some embodiments, the formulations disclosed herein contain about 210 mM sucrose. In some embodiments, the formulations disclosed herein contain about 220 mM sucrose. In some embodiments, the formulations disclosed herein contain about 230 mM sucrose. In some embodiments, the formulations disclosed herein contain about 240 mM sucrose. In some embodiments, the formulations disclosed herein contain about 250 mM sucrose. In some embodiments, the formulations disclosed herein contain about 260 mM sucrose. In some embodiments, the formulations disclosed herein contain about 270 mM sucrose. In some embodiments, the formulations disclosed herein contain about 280 mM sucrose. In some embodiments, the formulations disclosed herein contain about 290 mM sucrose. In some embodiments, the formulations disclosed herein contain about 300 mM sucrose. In some embodiments, the formulations disclosed herein contain about 350 mM sucrose.

[0508] In some embodiments, the formulations disclosed herein contain from about 190 mM to about 230 mM S. In some embodiments, the formulations disclosed herein contain from about 170 mM to about 250 mM sucrose. In some embodiments, the formulations disclosed herein contain about Contains 150 mM to about 270 mM sucrose.

[0509] In some embodiments, the formulations disclosed herein contain about 210 mM sucrose. .

[0510] In some embodiments, the formulations disclosed herein include sugars and salts in specific ratios. In some embodiments, the sugar to salt ratio is a molar ratio. In some embodiments, the sugar to salt ratio is , about 1, about 2, about 3, about 5, about 10, about 20, about 40, or about 60. Thus, the molar ratio of sugar to salt is from about 5 to about 20. In some embodiments, the molar ratio of sugar to salt is from about 5 to about 20. The ratio is about 10. In some embodiments, the sugar comprises sucrose and the salt comprises sodium citrate. Includes Umu.

[0511] In some embodiments, the formulations disclosed herein include salts and sugars in specific ratios. In some embodiments, the ratio of salt to sugar is a weight to weight (w:w) ratio. The w:w ratio of salt to sugar is about 3:1, about 1:1, or about 1:3. In some embodiments, the w:w ratio of salt to sugar is about 3:1 to about 1:3. The w ratio is about 1:1. In some embodiments, the sugar comprises sucrose and the salt comprises sodium citrate. Contains thorium.

[0512] 6.2.4 Surfactants In some embodiments, the formulations disclosed herein further comprise a non-ionic surfactant. Pharmaceutically acceptable nonionic surfactants include, but are not limited to, poloxamer 18, 8, Poloxamer 407, Polysorbate 80, Polysorbate 20, Pluronic F-68, or BRIJ 35. In some embodiments, the compositions disclosed herein may be The formulations used are poloxamer 188, poloxamer 407, polysorbate 80, and polysorbate 100. Rubet 20, Pluronic F-68, BRIJ 35, or a combination thereof In some embodiments, the formulations disclosed herein include poloxamer 188. nothing.

[0513] In some embodiments, the formulations disclosed herein further comprise from about 0.0001% to about 0 In some embodiments, the formulations disclosed herein contain 0.5% non-ionic surfactant. The composition further comprises about 0.0005% to about 0.1% of a non-ionic surfactant. Thus, the formulations disclosed herein may further comprise about 0.001%, about 0.002%, about 0.003%, approximately 0.004%, approximately 0.005%, approximately 0.007%, or approximately 0.01% The nonionic surfactants include

[0514] In some embodiments, the formulations disclosed herein further comprise from about 0.0001% to about 0 0.5% Poloxamer 188. In some embodiments, the formulations disclosed herein include and further comprising about 0.0005% to about 0.1% poloxamer 188. For example, the formulations disclosed herein may further comprise about 0.001%, about 0.002%, about 0. 0.003%, approximately 0.004%, approximately 0.005%, approximately 0.007%, or approximately 0.01% Contains Loxamer 188.

[0515] In some embodiments, the formulations disclosed herein further comprise about 0.0005% porphyrin. In some embodiments, the formulations disclosed herein further comprise about 0.001% Poloxamer 188. In some embodiments, the compositions disclosed herein include The formulation further comprises about 0.002% poloxamer 188. In some embodiments, the The formulations disclosed herein further include about 0.003% poloxamer 188. In some embodiments, the formulations disclosed herein further comprise about 0.004% poloxamer. 188. In some embodiments, the formulations disclosed herein further comprise about 0.00 5% Poloxamer 188. In some embodiments, the formulations disclosed herein include: It further contains about 0.008% poloxamer 188.

[0516] 6.2.5 Stabilizers or plasticizers In some embodiments, the formulations disclosed herein further comprise a plasticizer or stabilizer. Pharmaceutically acceptable stabilizers or plasticizers include glycerol, xylitol, and sorbitol. In some embodiments, the present invention includes, but is not limited to, ribulitol. The formulations shown may further comprise glycerol, xylitol, sorbitol, or their equivalents. In some embodiments, the formulations disclosed herein further comprise glycerol. Includes rolls.

[0517] In some embodiments, the formulations disclosed herein further comprise about 0.1% to about 5% of a soluble formaldehyde. In some embodiments, the formulations disclosed herein further comprise: In some embodiments, the composition disclosed herein comprises about 0.1% to about 2% of a plasticizer or stabilizer. The formulation further comprises about 0.25% to about 2% of a plasticizer.

[0518] In some embodiments, the formulations disclosed herein further comprise about 0.1% to about 5% of glycerol. Glycerol. In some embodiments, the formulations disclosed herein further comprise about 0. In some embodiments, the formulations disclosed herein contain from 1% to about 2% glycerol. , and further contains about 0.25% to about 2% glycerol.

[0519] 6.2.6 Formulation Characteristics In some embodiments, the formulations disclosed herein have glass transition temperatures greater than or equal to those of the corresponding pure In some embodiments, the glass transition temperature of the present invention is higher than that of pure sugar solutions and pure salt solutions. The formulation disclosed herein contains sucrose and sodium citrate and has a glass transition temperature of The glass transition temperatures are higher than those of the corresponding pure sucrose and pure sodium citrate. In some embodiments, the formulations disclosed herein contain from about 50 mM to about 400 mM sucrose. and about 10 mM to 100 mM sodium citrate.

[0520] In some embodiments, the formulations disclosed herein have a concentration of from about 1.0E+11 GC / mL to about In some embodiments, the rAAV particles comprise 1.0E+14GC / mL. The formulation is a rAAV particle size of about 1.0E+11GC / mL to about 1.0E+15GC / mL. In some embodiments, the formulations disclosed herein comprise a GC / mL, approx. 1.0E+12GC / mL, approx. 1.0E+13GC / mL, approx. 1.0E+14G C / mL, or about 1.0E+15GC / mL of rAAV particles. The rAAV particles are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-1 3, AAV-14, AAV-15 and AAV-16, rAAV.rh8, rAAV.rh 10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RH M4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, r AAV.7m8, rAAV.PHP.B, rAAV2.5, rAAV2tYF, rAAV 3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV. HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HS C12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AA V. HSC16, or a combination thereof. In particular, the rAAV particles may be of the AAV-8 serotype, the AAV-9 serotype, or a combination thereof. It contains a combination of capsid proteins.

[0521] In some embodiments, the formulations disclosed herein are liquid formulations.

[0522] In some embodiments, the formulations disclosed herein are frozen formulations.

[0523] In some embodiments, the formulations disclosed herein can be prepared by freezing the liquid formulations disclosed herein. It is a freeze-dried formulation.

[0524] In some embodiments, the formulations disclosed herein are reconstituted lyophilized formulations. .

[0525] In some embodiments, the formulations disclosed herein are pre-lyophilized formulations.

[0526] In some embodiments, the formulations disclosed herein have a residual moisture content of about 1% to about 7%. In some embodiments, the residual water content is determined by Karl Fischer titration. In some embodiments, the residual moisture content is between about 1% and about 7%. In some embodiments, the residual moisture content is about 2% to about 7%. In some embodiments, the residual moisture content is about 4% to about 7%. In some embodiments, the residual moisture content is about 5% to about 7%. The residual moisture content is about 1% to about 6%. In some embodiments, the residual moisture content is about 1% to about 5%. In some embodiments, the residual moisture content is about 1% to about 4%. Therefore, the residual moisture content is about 1% to about 3%.

[0527] In some embodiments, the formulations disclosed herein have a residual moisture content of about 1% to about 7%. In some embodiments, the residual water content is about 3% to about 7%. In some embodiments, the residual moisture content is about 3% to about 6%. The amount is about 3% to about 5%.

[0528] In some embodiments, the formulations disclosed herein have a residual moisture content of about 1% to about 7%. In some embodiments, the residual water content is about 3%. Thus, the residual moisture content is about 3.5%. In some embodiments, the residual moisture content is about 4. %. In some embodiments, the residual moisture content is about 4.5%. In some embodiments, the remaining moisture content is about 5%. In some embodiments, the remaining moisture content is about 5.5%. In some embodiments, the residual moisture content is about 6%.

[0529] In some embodiments of the formulations disclosed herein, the relative potency of the rAAV particles is At least about 60%, at least about 70%, or less after storage at room temperature for 3 months. In some embodiments of the formulations disclosed herein, the rAAV particle The percent relative efficacy is at least about 60% after the formulation is stored at room temperature for 3 months. In some embodiments of the disclosed formulations, the relative potency of the rAAV particles is measured after incubation of the formulation at room temperature for 3 h. After storage for 1 month, the retention time is at least about 70%. Thus, the relative potency of the rAAV particles was at least 10% after storage of the formulation at room temperature for 6 months. At least about 60%, at least about 70%, or at least about 80%. In some embodiments of the formulation, the relative potency of the rAAV particles is measured after storage of the formulation at room temperature for 6 months. In some embodiments of the formulations disclosed herein, The relative potency of the rAAV particles was at least approximately 70% after storage of the formulation at room temperature for 6 months. In some embodiments of the formulations disclosed herein, the relative potency % of the rAAV particles is: After storage of the formulation at 35° C. for 1 week, the composition is at least about 30%, at least about 40%, The percentage of glycerol in the formulations disclosed herein is about 50%, or at least 60%. Thus, the relative potency of the rAAV particles was at least about 100% after storage of the formulation at 35° C. for 1 week. In some embodiments of the formulations disclosed herein, the relative efficacy of rAAV particles is The potency is at least about 50% after the formulation is stored at 35° C. for 1 week. Depending on the embodiment of the formulation used, the relative potency of the rAAV particles can be measured by incubation of the formulation at 35° C. for 2 weeks. After storage, at least about 30%, at least about 40%, at least about 50%, or less In some embodiments of the formulations disclosed herein, the rAAV particles The relative efficacy of the formulation is at least about 30% after storage of the formulation at 35° C. for 2 weeks. In some embodiments of the formulations disclosed herein, the relative potency of the rAAV particles is determined by measuring the relative potency of the formulation at 35° C. At least about 50% after 2 weeks of storage at RT. In some cases, the relative potency of the rAAV particles was at least 10% after storage of the formulation at 35°C for 4 weeks. at least about 30%, at least about 40%, at least about 50%, or at least 60%. In some embodiments of the formulations disclosed herein, the relative potency of the rAAV particles is determined by the is at least about 30% after storage for 4 weeks at 35° C. In some embodiments, the relative potency of the rAAV particles is measured after storage of the formulation at 35° C. for 4 weeks. At least about 50%. In some embodiments, the reference rAAV particles are at least about 0.001% poly It is stored at -70°C in DPBS with Loxamer 188 buffer. , Relative potency % is PCT International Application No. PCT / US19 / 56042, October 2019 Filed on the 14th, titled "METHODS FOR MEASURING THE INFECTION TIVITY OF REPLICATION DEFECTIVE VIRAL VE The present application is filed on the same date herewith as a part hereof, and is hereby incorporated by reference in its entirety as if fully set forth herein. The entire contents of which are incorporated herein by reference. In some embodiments, the formulation is a frozen formulation. In some embodiments, the formulation is a lyophilized formulation. is a pre-lyophilized formulation. In some embodiments, the formulation is a liquid formulation. Depending on the medium, the rAAV particles may be mixed with about 1 mM to about 25 mM Tris, about 50 mM M to about 400 mM sucrose, about 10 mM to about 100 mM sodium citrate, and and about 0.0005% to about 0.01% of a nonionic surfactant, and the formulation has a pH It is about 7.2 to about 7.8.

[0530] In some embodiments, the formulations disclosed herein comprise rAAV particles and about 1 ml 25 mM to about 25 mM buffer, 50 mM to about 400 mM sugar, 10 mM to about 100 mM Sodium citrate, and about 0.0005% to about 0.01% of a nonionic surfactant. In some embodiments, the buffer is T In some embodiments, the buffer comprises Tris, and the sugar comprises sucrose. The sugar comprises sucrose and the non-ionic surfactant comprises poloxamer 188. Therefore, the buffer contains Tris, the sugar contains sucrose, and the non-ionic surfactant contains polyoxyethylene. The formulation further contains a plasticizer (e.g., about 0.1% to about 2% glycerol). In some embodiments, the formulation is a frozen formulation. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the formulation is a liquid formulation. In one embodiment, the rAAV particles include rAAV-8, rAAV-9, or a combination thereof. In some embodiments, the formulation contains from about 1.0E+11 GC / mL to about 1.0E+14 GC / mL. mL of rAAV particles. In some embodiments, the formulation contains about 1.0E+11 GC / mL ~Contains approximately 1.0E+15GC / mL of rAAV particles.

[0531] In some embodiments, the formulations disclosed herein comprise rAAV particles and about 2m 10 mM to about 10 mM buffer, about 150 mM to about 250 mM sugar, about 10 mM to about 20 mM Sodium citrate, and about 0.001% to about 0.005% of a nonionic surfactant. and the formulation has a pH of about 7.2 to about 7.8. In some embodiments, the buffer is T In some embodiments, the buffer comprises Tris, and the sugar comprises sucrose. The sugar comprises sucrose and the non-ionic surfactant comprises poloxamer 188. Therefore, the buffer contains Tris, the sugar contains sucrose, and the non-ionic surfactant contains polyoxyethylene. The formulation further contains a plasticizer (e.g., about 0.1% to about 2% glycerol). In some embodiments, the formulation is a frozen formulation. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the formulation is a liquid formulation. In one embodiment, the rAAV particles include rAAV-8, rAAV-9, or a combination thereof. In some embodiments, the formulation contains about 1.0E+11 GC / mL to about 1.0E+14 GC / mL In some embodiments, the formulation comprises about 1.0E+11 GC / mL to about Contains 1.0E+15GC / mL of rAAV particles.

[0532] In some embodiments, the formulations disclosed herein comprise rAAV particles and about 5 μM of M buffer, about 210 mM sugar, about 20 mM sodium citrate, and about 0.002% and a non-ionic surfactant, and the formulation has a pH of about 7.5. In some embodiments, the buffer comprises Tris and the sugar comprises sucrose. The sugar contains sucrose, and the non-ionic surfactant is poloxamer 188. In some embodiments, the buffer comprises Tris, the sugar comprises sucrose, and the non-ionic The reactive surfactant comprises poloxamer 188, and the formulation further comprises a plasticizer (e.g., about 0.1% to About 2% glycerol). In some embodiments, the formulation is a frozen formulation. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the formulation is a liquid formulation. In some embodiments, the rAAV particles are rAAV-8, rAAV-9, or derivatives thereof. In some embodiments, the formulation comprises from about 1.0E+11 GC / mL to about 1. 0E+14GC / mL of rAAV particles. In some embodiments, the formulation contains about 1.0E Contains rAAV particles at +11GC / mL to approximately 1.0E+15GC / mL.

[0533] The formulations disclosed herein can be used to prepare capsid proteins from any AAV capsid serotype. In some embodiments, the rAAV particles can include an AAV particle comprising 1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV 9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV- 15 and AAV-16, rAAV.rh8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37 , rAAV.Anc80, rAAV.Anc80L65, rAAV.7m8, rAAV. PHP.B, rAAV2.5, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV. HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9 , AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13 , AAV.HSC14, AAV.HSC15, or AAV.HSC16-derived capsids In some embodiments, the rAAV particles comprise AAV1, AAV2, AAV 3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AA V-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16 , rAAV.rh8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, rAAV.Anc8 0, rAAV.Anc80L65, rAAV.7m8, rAAV.PHP.B, rAAV 2.5, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, A AV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.H SC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14 Derivatives and modifications of AAV.HSC15 or AAV.HSC16 capsid proteins The capsid protein may be a pseudotype or a pseudotyped form.

[0534] In some embodiments, the rAAV particles comprise an AAAV molecule selected from AAV-8 and AAV-9. In some embodiments, the rAAV comprises a capsid protein derived from a V capsid serotype. The particles have an AAV capsid serotype of AAV-8. The offspring have an AAV capsid serotype of AAV-9.

[0535] In some embodiments, the rAAV particles contain AAV-8 or AAV-9 capsid proteins. In some embodiments, the capsid protein may be a derivative, modification, or pseudotype of the capsid protein. The rAAV particles contain AAV-8 capsid proteins VP1, VP2, and / or VP 3 sequences that are at least 80% identical to each other, e.g., 85%, 85%, 87%, 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 8%, 99%, 99.5%, etc., i.e., up to 100% identical AAV-8 capsids It contains a capsid protein having a protein structure.

[0536] In some embodiments, the rAAV particle composition comprises a derivative of the AAV-9 capsid protein. In some embodiments, the feed composition comprises a capsid protein that is a variant, modified, or pseudotyped. The rAAV particles in the preparation contain the AAV-9 capsid proteins VP1, VP2, and / or At least 80% identical to the VP3 sequence, e.g., 85%, 85%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99%, 99.5%, etc., i.e., up to 100% identical AAV-9 capsules. It comprises a capsid protein having a ribozyme protein.

[0537] In further embodiments, the rAAV particles comprise a mosaic capsid. In some embodiments, the rAAV particles include pseudotyped rAAV particles. The rAAV particles are capsids that contain chimeric capsid proteins from two or more AAV capsid serotypes. Including psids.

[0538] In some embodiments, the formulations disclosed herein include a pharma- ceutically acceptable carrier. It is a stable formulation.

[0539] As used herein, the term "pharmaceutical acceptable" means biologically acceptable. The formulation, gas, liquid, or solid, or mixture thereof, which is administered in vivo "Pharmaceutical acceptable" means one that is suitable for one or more routes of delivery or contact. A "tolerable" composition is one that is free of any biological or other undesirable properties. For example, such materials may be capable of being administered without causing substantial undesirable biological effects. That is, such a stable formulation can be administered to a subject, for example, according to the method of the present disclosure. The rAAV isolated according to the method can be administered to a subject. Incorporating compounds (e.g., preservatives, antibacterial, antiviral, and antifungal agents) into the composition The pharmaceutical compositions can be prepared as described herein or as known to those skilled in the art. , can be formulated to be compatible with a particular route of administration or delivery. The pharmaceutical compositions may contain carriers, diluents, or excipients suitable for administration by various routes. The rAAV particles and methods of the invention, as well as pharmaceutical compositions and delivery methods suitable for use therein, are well known in the art. (e.g., Remington: The Science and Pr actice of Pharmacy (2003) 20th ed., Mack Publishing Co., Easton, Pa.;Remington's Pharmaceutical Sciences (1990) 18th ed. , Mack Publishing Co., Easton, Pa.;The M erck Index (1996) 12th ed., Merck Publicis hing Group, Whitehouse, NJ; Pharmaceuti cal Principles of Solid Dosage Forms (19 93), Technonic Publishing Co., Inc., Lan caster, Pa.;Ansel and Stoklosa, Pharmace utical Calculations (2001) 11th ed., Lip pincott Williams & Wilkins, Baltimore, M d.; and Poznansky et al., Drug Delivery Sys. tems (1980), RL Juliano, ed., Oxford, (See NY, pp. 253-315).

[0540] In some embodiments, the formulations disclosed herein are prepared in their entirety for administration by known methods. A pharma- ceutically acceptable composition suitable for administration to a patient in need of such a formulation. Known methods include, for example, intravenous administration (e.g., as a bolus or over a period of time). by continuous infusion over a period of 12 hours, or by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, or Intrathecal, intrathecal, oral, topical, or by inhalation route. In particular, the formulations disclosed herein are suitable for systemic or local administration. The drug may be administered orally, subdermally, intraperitoneally, subcutaneously, nasally, sublingually, or rectally, without particular limitation. Examples of local administration include, without particular limitation, topical, subcutaneous, intramuscular, subretinal, and subarachnoid administration. Intravenous, intravenous, and intraperitoneal routes of administration are included.

[0541] In some embodiments, the formulations disclosed herein are pharmaceutical unit doses. " denotes physically discrete units suitable for unitary administration to a subject to be treated, each unit being A predetermined amount of the compound is added to the mixture, optionally with a pharmaceutical carrier (such as an excipient, diluent, vehicle, or or filler), which amount may be any amount when administered in one or more doses. The unit dose is calculated to produce a desired effect (e.g., a prophylactic or therapeutic effect). The form may be, for example, in ampoules and vials, which are , liquid compositions, or freeze-dried or lyophilized The composition may be in a philized state, e.g., a sterile liquid carrier may be added to the or prior to in vivo delivery. The individual unit dosage forms may be included in a multi-dose kit. Recombinant vector (e.g., AAV) sequences, plasmids, The vector genomes and recombinant viral particles, as well as pharmaceutical compositions thereof, are Packaged in single or multiple unit dosage forms for convenience and uniformity of dosage In some embodiments, the composition may comprise AAV1, AAV2, AAV3, AAV 4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16, rAAV .rh8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV. Rh74, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAA V.Anc80L65, rAAV.7m8, rAAV.PHP.B, rAAV2.5, r AAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HS C2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, A AV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV. HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV. rAAV containing AAV capsid proteins derived from HSC15 or AAV.HSC16 In some embodiments, the AAV capsid serotype is AAV-8. In some embodiments, the AAV capsid serotype is AAV-9.

[0542] In some embodiments, the present disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising the recombinant adeno-associated virus (rAAV) vector disclosed herein. The present invention provides a method for producing a formulation comprising rAAV particles, the method comprising the steps of: The buffer, sugar, salt, optional plasticizer, and optional non-ionic surfactant of the formulation to be and mixing the components of the formulation with each other, thereby producing said formulation containing rAAV. Methods for combining and preparing formulations are known to those skilled in the art.

[0543] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate, and d) about 0.002% (w / v) poloxamer 188; It is a stable formulation comprising:

[0544] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate; d) about 0.002% (w / v) poloxamer 188, and e) about 0.25% (w / v) glycerol; It is a stable formulation comprising:

[0545] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate; d) about 0.002% (w / v) poloxamer 188, and e) about 0.5% (w / v) sorbitol; It is a stable formulation comprising:

[0546] In certain embodiments, provided herein are recombinant adeno-associated viruses. (rAAV) particles, and a) about 5 mM Tris, b) about 30 mM sodium sulfate; c) about 263 mM sucrose, and d) about 0.005% (w / v) poloxamer 188; It is a stable formulation comprising:

[0547] In certain embodiments, the stable formulation does not include mannitol.

[0548] In certain embodiments, the stable formulations include 150 mM, 140 mM, 130 mM, 1 20mM, 110mM, 100mM, 90mM, 80mM, 70mM, 60mM, 50m M, 40 mM, 30 mM, 20 mM, or less than 10 mM mannitol.

[0549] In certain embodiments, the stable formulation comprises sucrose at a concentration of about 210 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 263 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 409 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 14.6 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 45 mM. In one embodiment, the stable formulation comprises sucrose at a concentration of about 0 mM to about 20 mM. In certain embodiments, the stable formulation contains sucrose at a concentration of about 20 mM to about 50 mM. In certain embodiments, the stable formulation comprises about 50 mM to about 100 mM sucrose. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 100 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 200 mM. In certain embodiments, the stable formulation comprises: In certain embodiments, the sucrose is present at a concentration of about 300 mM to about 400 mM. The stable formulation contains sucrose at a concentration of about 400 mM to about 500 mM. In one embodiment, the stable formulation contains sucrose at a concentration of about 500 mM to about 600 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 600 mM to about 700 mM. Including.

[0550] In certain embodiments, the stable formulation comprises sorbitol at a concentration of about 0.5%. In certain embodiments, the stable formulation comprises sorbitol at a concentration of about 0.25%. In certain embodiments, the stable formulation comprises sorbitol at a concentration of about 0.10%.

[0551] In certain embodiments, the stable formulation contains sorbitol at a concentration of about 0% to about 0.10%. In certain embodiments, the stable formulation contains from about 0.10% to about sorbitol. In certain embodiments, the stable formulation contains sorbitol at a concentration of about 0.20%. In certain embodiments, the stable formulation comprises a concentration of about 0.20% to about 0.30%. In certain embodiments, the composition further comprises sorbitol at a concentration of about 0.30% to about 0.40%. The stable formulation contains sorbitol at a concentration of about 0.40% to about 0.50%. In one embodiment, the stable formulation contains sorbitol at a concentration of about 0.50% to about 0.60%. In certain embodiments, the stable formulation contains about 0.60% to about 0.70% sorbitol. In certain embodiments, the stable formulation contains sorbitol at a concentration of about 0.70%. In certain embodiments, the stable formulation comprises sorbitol at a concentration of about 0.80%. In certain embodiments, the stable formulation comprises , containing sorbitol at a concentration of about 0.90% to about 1.00%.

[0552] In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.5%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.25%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.10%.

[0553] In certain embodiments, the stable formulation contains glycerol at a concentration of about 0% to about 0.10%. In certain embodiments, the stable formulation contains glycerol at about 0.10% to about In certain embodiments, the stable formulation contains glycerol at a concentration of about 0.20%. In certain embodiments, the stable formulation comprises a concentration of about 0.20% to about 0.30%. Glycerol at a concentration of about 0.30% to about 0.40%. The stable formulation contains glycerol at a concentration of about 0.40% to about 0.50%. In one embodiment, the stable formulation contains glycerol at a concentration of about 0.50% to about 0.60%. In certain embodiments, the stable formulation contains about 0.60% to about 0.70% glycerol. In certain embodiments, the stable formulation contains glycerol at a concentration of about 0.70%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.80%. In certain embodiments, the stable formulation comprises , containing glycerol at a concentration of about 0.90% to about 1.00%.

[0554] In certain embodiments, the stable formulation contains about 0.001% (by weight) poloxamer 188. In certain embodiments, the stable formulation comprises: It contains poloxamer 188 at a concentration of approximately 0.002% (weight / volume, 0.02 g / L). In certain embodiments, the stable formulation contains poloxamer 188 at about 0.005% (w / w) Containing a concentration of 0.05 g / L (by volume).

[0555] In certain embodiments, the stable formulation contains poloxamer 188 at about 0.0005% ( At concentrations of about 0.005 g / L (weight / volume) to about 0.05% (weight / volume, 0.5 g / L) In certain embodiments, the stable formulation comprises about 0.0001% poloxamer 188. % (weight / volume, 0.001 g / L) to approximately 0.01% (weight / volume, 0.1 g / L) concentration In certain embodiments, the stable formulation contains poloxamer 188 at about 0.00 0.05% (weight / volume, 0.005 g / L) to approximately 0.001% (weight / volume, 0.01 g / In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.001% (weight / volume, 0.01 g / L) to approximately 0.05% (weight / volume, 0.5 g / L) In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about In certain embodiments, the concentration is 0.0005% (weight / volume, 0.005 g / L). In this study, the stable formulation contained approximately 0.0006% (weight / volume, 0.006 In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of 100 g / L. at a concentration of about 0.0007% (weight / volume, 0.007 g / L). In this embodiment, the stable formulation contains poloxamer 188 at approximately 0.0008% (weight / volume, 0.0 In certain embodiments, the stable formulation contains poloxamer 1.08 g / L. 88 at a concentration of approximately 0.0009% (weight / volume, 0.009 g / L). In an embodiment, the stable formulation contains poloxamer 188 at about 0.001% (weight / volume, 0. In certain embodiments, the stable formulation contains poloxamer 1.01 g / L. 88 at a concentration of about 0.002% (weight / volume, 0.02 g / L). In this embodiment, the stable formulation contains poloxamer 188 at approximately 0.003% (weight / volume, 0.03 In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of 100 g / L. In certain embodiments, the composition comprises a concentration of about 0.004% (weight / volume, 0.04 g / L). In this study, the stable formulation contained approximately 0.005% (weight / volume, 0.05 g / mL) poloxamer 188. In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about In certain embodiments, the concentration is 0.01% (weight / volume, 0.1 g / L). The stable formulation contains poloxamer 188 at a concentration of approximately 0.05% (weight / volume, 0.5 g / L). include.

[0556] In certain embodiments, the pH of the pharmaceutical composition is about 7.4.

[0557] In certain embodiments, the pH of the pharmaceutical composition is about 6.0 to 8.8. In certain embodiments, the pH of the pharmaceutical composition is about 6.0 to 9.0. In certain embodiments, the pH of the pharmaceutical composition is about 6.0. The pH of the pharmaceutical composition is about 6.1. In certain embodiments, the pH of the pharmaceutical composition is In certain embodiments, the pH of the pharmaceutical composition is about 6.3. In certain embodiments, the pH of the pharmaceutical composition is about 6.4. In certain embodiments, the pH of the pharmaceutical composition is about 6.5. The pH of the pharmaceutical composition is about 6.6. In certain embodiments, the pH of the pharmaceutical composition is In certain embodiments, the pH of the pharmaceutical composition is about 6.8. In certain embodiments, the pH of the pharmaceutical composition is about 6.9. In certain embodiments, the pH of the pharmaceutical composition is about 7.0. The pH of the pharmaceutical composition is about 7.1. In certain embodiments, the pH of the pharmaceutical composition is In certain embodiments, the pH of the pharmaceutical composition is about 7.3. In certain embodiments, the pH of the pharmaceutical composition is about 7.4. In certain embodiments, the pH of the pharmaceutical composition is about 7.5. The pH of the pharmaceutical composition is about 7.6. In certain embodiments, the pH of the pharmaceutical composition is In certain embodiments, the pH of the pharmaceutical composition is about 7.8. In certain embodiments, the pH of the pharmaceutical composition is about 7.9. In certain embodiments, the pH of the pharmaceutical composition is about 8.0. The pH of the pharmaceutical composition is about 8.1. In certain embodiments, the pH of the pharmaceutical composition is In certain embodiments, the pH of the pharmaceutical composition is about 8.3. In certain embodiments, the pH of the pharmaceutical composition is about 8.4. In certain embodiments, the pH of the pharmaceutical composition is about 8.5. The pH of the pharmaceutical composition is about 8.6. In certain embodiments, the pH of the pharmaceutical composition is In certain embodiments, the pH of the pharmaceutical composition is about 8.8. In certain embodiments, the pH of the pharmaceutical composition is about 8.9. In this embodiment, the pH of the pharmaceutical composition is about 9.0.

[0558] In certain embodiments, the vector genome concentration (VGC) of the stable formulation is about 3×1 0 9 GC / mL, 4×10 9 GC / mL, 5×10 9 GC / mL, 6×10 9 GC / mL , 7×10 9 GC / mL, 8×10 9 GC / mL, 9×10 9 GC / mL, approximately 1×10 1 0 GC / mL, about 2×10 10 GC / mL, about 3×10 10 GC / mL, about 4×10 10 GC / mL, about 5×10 10 GC / mL, about 6×10 10 GC / mL, about 7×10 10 G C / mL, about 8×10 10 GC / mL, about 9×10 10 GC / mL, about 1×10 11 GC / mL, about 2×10 11 GC / mL, about 3×10 11 GC / mL, about 4×10 11 GC / mL, about 5×10 11 GC / mL, about 6×10 11 GC / mL, about 7×10 11 GC / m L, about 8×10 11 GC / mL, about 9×10 11 GC / mL, about 1×10 12 GC / mL , about 2×10 12 GC / mL, about 3×10 12 GC / mL, about 4×10 12 GC / mL, About 5×10 12 GC / mL, about 6×10 12 GC / mL, about 7×10 12 GC / mL, approx. 8×10 12 GC / mL, about 9×10 12 GC / mL, about 1×10 13 GC / mL, about 1 ×10 13 GC / mL, about 2×10 13 GC / mL, about 3×10 13 GC / mL ある.

[0559] 6.3 How to reduce the release and reduction of rAAV particles In some embodiments, the present disclosure reduces rAAV genome release from rAAV particles. The present invention provides a method for the preparation of a rAAV particle, a buffer, a sugar, a salt, and optionally a non-ionic boundary agent. The method includes preparing a formulation disclosed herein that includes a surfactant, the method comprising: Release of the rAAV genome from rAAV particles after freeze-thaw cycling was significantly greater in sugar-free formulations. In some embodiments, the formulation is a frozen formulation. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the salt is In some embodiments, the salt comprises sodium citrate and the sugar comprises In some embodiments, the rAAV genome release inhibitors disclosed herein include A further method for reducing the residual moisture content is to freeze-dry the formulation to a residual moisture content of about 1% to about 5%. Includes.

[0560] In some embodiments, the present disclosure reduces rAAV genome release from rAAV particles. The present invention provides a method for the preparation of a rAAV particle, a buffer, a sugar, a salt, and optionally a non-ionic boundary agent. The method includes preparing a formulation disclosed herein that includes a surfactant, the method comprising: freeze-drying and rAAV genome release from rAAV particles after reconstitution was compared with that in sugar-free formulations. The release of the AAV genome is reduced compared to that of the frozen formulation. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the salt comprises sodium citrate and the sugar comprises sucrose. In some embodiments, the method for reducing rAAV genome release disclosed herein comprises The method further comprises freeze-drying the formulation to reduce the residual moisture content to about 1% to about 5%. include.

[0561] In some embodiments, the present disclosure provides methods for reducing rAAV genome release from rAAV particles. The present invention provides a method for producing rAAV particles, a buffer, a sugar, a salt, a plasticizer, and optionally a non-isopropyl alcohol. The method includes preparing a formulation disclosed herein that includes an ionic surfactant, the method comprising: Release of the rAAV genome from rAAV particles after three freeze-thaw cycles was In some embodiments, the formulation is cryopreserved and has reduced rAAV genome release compared to that in cryopreserved formulations. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the salt comprises sodium citrate. In some embodiments, the salt comprises sodium citrate and the sugar comprises sucrose. In some embodiments, the plasticizer comprises glycerol. The method for reducing rAAV genome release further includes lyophilizing the formulation. nothing.

[0562] In some embodiments, the present disclosure provides methods for reducing rAAV genome release from rAAV particles. The present invention provides a method for producing rAAV particles, a buffer, a sugar, a salt, a plasticizer, and optionally a non-isopropyl alcohol. The method includes preparing a formulation disclosed herein that includes an ionic surfactant, the method comprising: rAAV genome release from rAAV particles after lyophilization and reconstitution in sugar-free formulations In some embodiments, the formulation is a frozen preparation. In some embodiments, the formulation is a lyophilized formulation. comprises sodium citrate. In some embodiments, the salt comprises sodium citrate; The sugar comprises sucrose. In some embodiments, the salt comprises sodium citrate and the sugar comprises sucrose. In some embodiments, the plasticizer comprises glycerol. The method for reducing rAAV genome release further includes lyophilizing the formulation.

[0563] In some embodiments, the present disclosure reduces rAAV genome release from rAAV particles. The present invention provides a method for the preparation of an rAAV particle, a buffer, a sugar, a salt, and optionally a non- The use includes preparing a formulation disclosed herein that includes an ionic surfactant. , rAAV genome release from rAAV particles after three freeze-thaw cycles does not include sugar In some embodiments, the formulation comprises: In some embodiments, the formulation is a freeze-dried formulation. In some embodiments, the salt comprises sodium citrate. In some embodiments, the rAAV genome disclosed herein comprises The use of sugar to reduce glycerol release can also be achieved by freeze-drying the formulation to reduce the residual water content to approximately This includes increasing the ratio from 1% to about 7%.

[0564] In some embodiments, the present disclosure reduces rAAV genome release from rAAV particles. The present invention provides a method for the preparation of an rAAV particle, a buffer, a sugar, a salt, and optionally a non- The use includes preparing a formulation disclosed herein that includes an ionic surfactant. rAAV genome release from rAAV particles after lyophilization and reconstitution in sugar-free formulations In some embodiments, the formulation is frozen, and the rAAV genome release is reduced compared to that in frozen In some embodiments, the formulation is a lyophilized formulation. The salt comprises sodium citrate. In some embodiments, the salt comprises sodium citrate. In some embodiments, the sugar comprises sucrose. The use of sugar to reduce exudation further allows the formulation to be freeze-dried to reduce the residual moisture content to approximately 1%. ~About 7%.

[0565] In some embodiments, the present disclosure reduces rAAV genome release from rAAV particles. The present invention provides a method for producing an rAAV particle, comprising the steps of: Manufacture of a formulation as disclosed herein, optionally including a non-ionic surfactant. In this study, the release of the rAAV genome from rAAV particles after three freeze-thaw cycles was The release of the rAAV genome is reduced compared to that in the sugar-free formulation. In some embodiments, the formulation is a frozen formulation. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the salt comprises sodium citrate. In some embodiments, the salt comprises sodium citrate and the sugar comprises sucrose. In some embodiments, the plasticizer comprises glycerol, the sugar comprises sucrose, and the plasticizer comprises glycerol. The use of sugars to reduce rAAV genome release as disclosed herein further comprises the steps of: The method includes freeze-drying the

[0566] In some embodiments, the present disclosure reduces rAAV genome release from rAAV particles. The present invention provides a method for producing an rAAV particle, comprising the steps of: Manufacture of a formulation as disclosed herein, optionally including a non-ionic surfactant. In this application, rAAV genome release from rAAV particles after lyophilization and reconstitution is achieved by the addition of sugars. In some embodiments, the release of the rAAV genome is reduced compared to that in formulations that do not contain the rAAV. The formulation is a frozen formulation. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the salt comprises sodium citrate. In some embodiments, the salt comprises sodium citrate and the sugar comprises sucrose. In some embodiments, the sugar comprises sucrose and the plasticizer comprises glycerol. The use of sugars to reduce rAAV genome release as disclosed herein further reduces the risk of developing rAAV genomes being released by freezing the formulation. This includes freezing and drying.

[0567] Methods for reducing rAAV genome release or rAAV genome release disclosed herein In some embodiments of the use of the compound to reduce rAAV genome release, the release of the rAAV genome is The identification is performed by measuring the relative fluorescence in the presence of a specific fluorescent stain. The DNA specific fluorescent stain is SYBR Gold.

[0568] Methods for reducing rAAV genome release or rAAV genome release disclosed herein In some embodiments of the use of the compound to reduce freezing-induced rAAV Genome release is reduced by at least about 10%, 20%, 50%, 80%, or 90%. In some embodiments, freezing-induced rAAV genome release is at least about 2 In some embodiments, freezing-induced rAAV genome release is In some embodiments, the freezing-induced rAAV Genome release is reduced by at least about 80%. In some embodiments, induced by freezing In vivo, rAAV genome release is virtually eliminated.

[0569] Methods for reducing rAAV genome release or rAAV genome release disclosed herein In some embodiments of the use of the compound to reduce In some cases, the non-reducing sugar may be sucrose, trehalose, raffinose, or any of their derivatives. In some embodiments, the non-reducing sugar is sucrose.

[0570] Methods for reducing rAAV genome release or rAAV genome release disclosed herein In some embodiments of the use of the compound to reduce Depending on the type, reducing sugars can be glucose, fructose, mannose, galactose, or lactose. In some embodiments, the reducing sugar is glucose. do.

[0571] Methods for reducing rAAV genome release or rAAV genome release disclosed herein In some embodiments of the use of the compound to reduce In some embodiments, the glycerol may be glycerol, sorbitol, mannitol, or a combination thereof. In this example, the plasticizer is glycerol.

[0572] 6.4 rAAV particles The formulations provided are suitable for containing any isolated recombinant AAV particle and may be any In a subject in need of treatment for a disease or disorder, the treatment comprising administering an isolated recombinant AAV particle of The methods provided are suitable for use in treating any disease or disorder. The present invention is suitable for formulating isolated recombinant AAV particles of any isolated recombinant AAV. It is suitable for reducing rAAV genome release from particles. Any serotype, modification or derivative thereof known in the art, or a variant thereof known in the art. Any combination thereof known in the art (e.g., a rAAV particle population comprising two or more serotypes) For example, two or more of rAAV2 particles, rAAV8 particles, and rAAV9 particles. In some embodiments, the rAAV particles can be AAV1, AAV2, , AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV1 0, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AA V-16, rAAV.rh8, rAAV.rh10, rAAV.rh20, rAAV.r h39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, rAAV. Anc80, rAAV.Anc80L65, rAAV.7m8, rAAV.PHP.B, rAAV2.5, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HS C1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, A AV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.H SC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.H SC14, AAV.HSC15, or AAV.HSC16, or other rAAV particles , or a combination of two or more of them.

[0573] In some embodiments, the rAAV particles include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV17, AAV18, AAV19, AAV20, AAV21, AAV22, AAV23, AAV24, AAV25, AV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV -12, AAV-13, AAV-14, AAV-15 and AAV-16, rAAV.rh 8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh7 4, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.A nc80L65, rAAV.7m8, rAAV.PHP.B, rAAV2.5, rAAV 2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV. HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC 11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC a capsid protein from an AAV serotype selected from AAV.HSC16, AAV.HSC17, or AAV.HSC18 or a derivative, modification, or pseudotype thereof. V particles include, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV17, AAV18, AAV19, AAV20, AAV21, AAV22, AAV23, AAV24, AAV25, AAV26 AV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13 , AAV-14, AAV-15 and AAV-16, rAAV.rh8, rAAV.rh1 0, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM 4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, rA AV.7m8, rAAV.PHP.B, rAAV2.5, rAAV2tYF, rAAV3 B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, A AV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.H SC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC 12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV. VP1, VP2, and / or VP3 of an AAV capsid serotype selected from HSC16 At least 80% identical to the sequence, e.g., 85%, 85%, 87%, 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 Capsid proteins that are 8%, 99%, 99.5%, etc., i.e. up to 100% identical include.

[0574] In some embodiments, the rAAV particles include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV17, AAV18, AAV19, AAV20, AAV21, AAV22, AAV23, AAV24, AAV25, AV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV -12, AAV-13, AAV-14, AAV-15 and AAV-16, rAAV.rh 8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh7 4, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.A nc80L65, rAAV.7m8, rAAV.PHP.B, rAAV2.5, rAAV 2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV. HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC 11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC A capsid derived from an AAV capsid serotype selected from AAV.HSC16, AAV.HSC20, or AAV.HSC30. In some embodiments, the present invention includes a polypeptide or a derivative, modification, or pseudotype thereof. The rAAV particles can be, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV 6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV -13, AAV-14, AAV-15 and AAV-16, rAAV.rh8, rAAV. rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV. RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65 , rAAV.7m8, rAAV.PHP.B, rAAV2.5, rAAV2tYF, rA AV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC 3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AA V.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV. HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or A VP1, VP2, and / or VP.HSC16 of an AAV capsid serotype selected from At least 80% identical to the VP3 sequence, e.g., 85%, 85%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99%, 99.5%, etc., i.e., capsid proteins that are up to 100% identical Includes quality.

[0575] In certain embodiments, the rAAV particles are , Cell Rep. 12(6): 1056-1068. c80 or Anc80L65, which is incorporated by reference in its entirety. In certain embodiments, the rAAV particles are prepared as described in U.S. Pat. Nos. 9,458,517 and 9,587,282, and U.S. Patent Application Publication No. 2003 / 0133666. LGETTRP or LALGETTRP as described in No. 016 / 0376323 Each of these references is incorporated herein in its entirety. In certain embodiments, the rAAV particles can be prepared as described in U.S. Pat. Nos. 193,956, 9,458,517, and 9,587,282, and including the AAV.7m8 as described in U.S. Patent Application Publication No. 2016 / 0376323. Each of these documents is incorporated herein by reference in its entirety. In one embodiment, the rAAV particles are those disclosed in U.S. Pat. No. 9,585,971. In certain embodiments, the rAAV includes any AAV, such as AAV-PHP.B. AV particles are disclosed in U.S. Pat. No. 9,840,719 and WO 2015 / 013313. Any AAV known in the art, including AAV.Rh74 and RHM4-1, Each of the above is incorporated herein by reference in its entirety. In the present invention, the rAAV particle can be any AAV disclosed in WO 2014 / 172669, See, for example, AAV rh.74, which is incorporated herein by reference in its entirety. In certain embodiments, the rAAV particles are prepared using the method described in Georgiadis et al. t al., 2016, Gene Therapy 23: 857-862 and G eorgiadis et al., 2018, Gene Therapy 25: 450, each of which is incorporated herein by reference in its entirety. In certain embodiments, the rAAV particles are Any of the AAVs disclosed in US Pat. No. 17 / 070491, e.g., AAV2tYF, No. 6,399,433, which is incorporated herein by reference in its entirety. In certain embodiments, The rAAV particles were AAVLK03 or AA as described in l. Med. 29(9): 418 V3B, which is incorporated by reference in its entirety. In the present invention, the rAAV particles are prepared as described in U.S. Pat. Nos. 8,628,966 and 8,927,514. Nos. 9,923,120, 9,923,120, and WO 2016 / 049230. AAV, e.g., HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10, HSC11, HSC12, HSC13, H SC14, HSC15, or HSC16, each of which is incorporated herein in its entirety. is incorporated by reference.

[0576] In certain embodiments, the rAAV particles are described in any of the following patents and patent applications: Each of these references is incorporated herein by reference in its entirety, including the AAVs disclosed therein. Incorporated by reference: U.S. Patent Nos. 7,282,199, 7,906,111, 8,5 No. 24,446, No. 8,999,678, No. 8,628,966, No. 8,92 No. 7,514, No. 8,734,809, No. 9,284,357, No. 9,409 , No. 953, No. 9,169,299, No. 9,193,956, No. 945851 Nos. 7, 9,587,282, and U.S. Patent Application Publication No. 2015 / 0374803 , same No. 2015 / 0126588, same No. 2017 / 0067908, same No. 2013 / No. 0224836, No. 2016 / 0215024, No. 2017 / 0051257 , and International Patent Application Nos. PCT / US2015 / 034799 and PCT / EP2 In some embodiments, the rAAV particles are disclosed in the following patents and publications: The VP1, VP2, and / or VP3 sequences of the AAV capsid disclosed in any of the applications herein are At least 80% identical to, e.g., 85%, 85%, 87%, 88%, 8 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9 9%, 99.5%, etc., i.e., up to 100% identical capsid proteins, Each of these documents is incorporated by reference in its entirety herein: U.S. Pat. No. 282,199, No. 7,906,111, No. 8,524,446, No. 8,9 No. 99,678, No. 8,628,966, No. 8,927,514, No. 8,73 No. 4,809, No. 9,284,357, No. 9,409,953, No. 9,169 ,299, ... 282, U.S. Patent Application Publication Nos. 2015 / 0374803 and 2015 / 01265 No. 88, No. 2017 / 0067908, No. 2013 / 0224836, No. 20 16 / 0215024, 2017 / 0051257, and International Patent Application No. P CT / US2015 / 034799, PCT / EP2015 / 053335.

[0577] In some embodiments, the rAAV particles are prepared using the methods described in International Application Publication WO 2003 / 052051 ( See, e.g., SEQ ID NO:2), WO 2005 / 033321 (see, e.g., SEQ ID NO:123 and 88), WO 03 / 042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97), WO 2006 / 068888 (see, e.g., SEQ ID NOs: 1 and 3-6) , WO 2006 / 110689, (see, for example, SEQ ID NOs: 5-38), WO 200 9 / 104964 (see, for example, SEQ ID NOs: 1-5, 7, 9, 20, 22, 24, and 31) WO 2010 / 127097 (see, e.g., SEQ ID NOs: 5-38), and WO 2015 / 191508 (see, e.g., SEQ ID NOs: 80-294), and U.S. Patent Application Publication No. The compounds disclosed in Patent Publication No. 20150023924 (see, for example, SEQ ID NOs: 1, 5 to 10) The contents of each of these references are incorporated herein by reference in their entirety. In some embodiments, the rAAV particles are prepared according to the method described in International Publication WO 2003 / 052051 (see, e.g., SEQ ID NO:2), WO 2005 / 033321 (see, e.g., , see SEQ ID NOs: 123 and 88), WO 03 / 042397 (e.g., SEQ ID NOs: 2, 81, 85, and 97), WO 2006 / 068888 (see, e.g., SEQ ID NO:1 and and 3-6), WO 2006 / 110689 (see, for example, SEQ ID NOs: 5-38) , WO 2009 / 104964 (e.g., SEQ ID NOs: 1 to 5, 7, 9, 20, 22, 24 and 31), WO 2010 / 127097 (see, e.g., SEQ ID NOs: 5-38) ), and WO 2015 / 191508 (see, for example, SEQ ID NOs: 80-294), and U.S. Patent Publication No. 20150023924 (see, e.g., SEQ ID NOs: 1, 5-10). At least one nucleotide sequence encoding the VP1, VP2, and / or VP3 sequences of the AAV capsid disclosed in At least 80% identical, e.g., 85%, 85%, 87%, 88%, 89%, 90% %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99 .5%, i.e., up to 100% identical capsid proteins.

[0578] Nucleic acid sequences of AAV-based viral vectors, as well as recombinant AAV and AAV capsids. Methods for preparing the amide are described, for example, in U.S. Pat. Nos. 7,282,199 and 7,906,111. Same No. 8,524,446, Same No. 8,999,678, Same No. 8,628,966, Same No. No. 8,927,514, No. 8,734,809, No. 9,284,357, No. No. 9,409,953, No. 9,169,299, No. 9,193,956, No. 9 Nos. 4,58517 and 9,587,282, U.S. Patent Application Publication No. 2015 / 037 No. 4803, No. 2015 / 0126588, No. 2017 / 0067908, No. 2013 / 0224836, 2016 / 0215024, 2017 / 005 No. 1257, International Patent Application No. PCT / US2015 / 034799, and International Patent Application No. PCT / EP No. 2015 / 053335, WO 2003 / 052051, WO 2005 / 0333 21, WO 03 / 042397, WO 2006 / 068888, WO 2006 / 1 10689, WO 2009 / 104964, WO 2010 / 127097, and WO No. 2015 / 191508, and U.S. Patent Publication No. 20150023924. It is being done.

[0579] The methods provided are suitable for use in the production of recombinant AAV encoding a transgene. In some embodiments, provided herein is a nucleic acid encoding an anti-VEGF Fab. In certain embodiments, provided herein is an AAV viral vector. The virus vector is an AAV8-based vector encoding anti-VEGF Fab. In an exemplary embodiment, provided herein is an AAV encoding ranibizumab. 8. In some embodiments, provided herein are viral vectors that utilize , an AAV viral vector encoding iduronidase (IDUA). In one embodiment, provided herein is a method for producing a virulent vector using AAV9 encoding IDUA. In some embodiments, provided herein is a vector encoding iduronic acid 2 - an AAV viral vector encoding a sulfatase (IDS). In one embodiment, provided herein is an AAV9-based virus that encodes an IDS. In some embodiments, provided herein is a vector that is a low density lipoprotein (LDLP) vector. In certain embodiments, the AAV viral vector encodes a soluble leukocyte receptor (LDLR). In the present specification, a virus using AAV8 encoding LDLR is provided. In some embodiments, provided herein are tripeptidyl peptidyl vectors. It is an AAV viral vector that encodes the transgenic tyrosine phosphate 1 (TPP1) protein. In an embodiment, provided herein is a method for the production of a medicament for the treatment of cancer using AAV9 encoding TPP. It is a viral vector.

[0580] In further embodiments, the rAAV particles comprise pseudotyped rAAV particles. In particular, the pseudotyped AAV is a pseudotyped AAV of rAAV2 / 8 or rAAV2 / 9. Methods for making and using rAAV particles are known in the art (e.g., Duan et al. al., J. Virol., 75:7662-7671 (2001);Halb ert et al., J. Virol., 74:1524-1532 (200 0);Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, (2001).

[0581] In further embodiments, the rAAV particles comprise a mixture of two or more AAV capsid serotypes. In some embodiments, the capsid comprises a capsid protein. AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV 8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14 , AAV-15 and AAV-16, rAAV.rh8, rAAV.rh10, rAAV. rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV .hu37, rAAV.Anc80, rAAV.Anc80L65, rAAV.7m8, rAAV.PHP.B, rAAV2.5, rAAV2tYF, rAAV3B, rAAV. LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4 , AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV .HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV. From HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 It is a chimera of two or more AAV capsid proteins from a selected AAV serotype.

[0582] In certain embodiments, single-stranded AAV (ssAAV) can be used. In certain embodiments, self-complementary vectors, such as scAAV, are used. (For example, Wu, 2007, Human Gene Therapy, 1 8(2):171-82, McCarty et al, 2001, Gene T herapy, Vol. 8, Number 16, Pages 1248-12 54; and U.S. Patent Nos. 6,596,535, 7,125,717, and 6,125,717. See US Pat. No. 7,456,683, each of which is incorporated herein by reference in its entirety. (Incorporated herein by reference).

[0583] In some embodiments, the rAAV particles include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV17, AAV18, AAV19, AAV20, AAV21, AAV22, AAV23, AAV24, AAV25, AV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV -12, AAV-13, AAV-14, AAV-15 and AAV-16, rAAV.rh 8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh7 4, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.A nc80L65, rAAV.7m8, rAAV.PHP.B, rAAV2.5, rAAV 2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV. HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC 11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC a capsid protein from an AAV serotype selected from AAV.HSC16, AAV.HSC17, or AAV.HSC18 or a derivative, modification, or pseudotype thereof. The offspring may be, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV 7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, A AV-14, AAV-15 and AAV-16, rAAV.rh8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4- 1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, rAAV .7m8, rAAV.PHP.B, rAAV2.5, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV .HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC 8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12 , AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HS VP1, VP2, and / or VP3 sequences of AAV capsid serotypes selected from C16 At least 80% identical to, e.g., 85%, 85%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99%, 99.5%, etc., i.e., up to 100% identical, including capsid proteins nothing.

[0584] In some embodiments, the rAAV particles comprise an AAV selected from AAV-8 or AAV-9. In some embodiments, the rAAV particles comprise capsid proteins derived from an AV capsid serotype. The offspring have an AAV capsid serotype of AAV-8. has the AAV capsid serotype of AAV-9.

[0585] In some embodiments, the rAAV particles contain AAV-8 or AAV-9 capsid proteins. In some embodiments, the capsid protein may be a derivative, modification, or pseudotype of the capsid protein. The rAAV particles contain AAV-8 capsid proteins VP1, VP2, and / or VP At least 80% identical to the three sequences, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, AAV-8 capsids that are 98%, 99%, 99.5%, etc., i.e. up to 100% identical It has proteins, including capsid proteins.

[0586] In some embodiments, the rAAV particles are derived from, or modified with, the AAV-9 capsid protein. In some embodiments, the rAAV particles include capsid proteins that are pseudotyped or pseudotyped. for the VP1, VP2, and / or VP3 sequences of the AAV-9 capsid protein, At least 80% identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, i.e., up to 100% identical AAV-9 capsid protein It contains the capsid protein.

[0587] In a further embodiment, the rAAV particle comprises a mosaic capsid. V particles are composed of a mixture of viral capsid proteins from different AAV serotypes In some embodiments, the rAAV particles are selected from AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV- 10, rAAVrh10, AAV-11, AAV-12, AAV-13, AAV-14, A monoclonal antibody comprising a capsid protein of a serotype selected from AAV-15 and AAV-16. In some embodiments, the rAAV particles comprise an AAV-1, AAV-2, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV A clone comprising a capsid protein of a serotype selected from AAVrh.8, AAVrh.10, and AAVrh.11. Contains iccapsid.

[0588] In further embodiments, the rAAV particles comprise pseudotyped rAAV particles. In accordance with the present invention, a pseudotyped rAAV particle comprises (a) a nucleic acid vector comprising AAV ITRs; and (b) an AAV vector comprising AAV ITRs. AVx (e.g. AAV-1, AAV-3, AAV-4, AAV-5, AAV-6, AA V-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV Capsid proteins derived from AAV-13, AAV-14, AAV-15, and AAV-16 In a further embodiment, the rAAV particle comprises an AAV capsid. 1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV 9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV- 15 and AAV-16, rAAV.rh8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37 , rAAV.Anc80, rAAV.Anc80L65, rAAV.7m8, rAAV. PHP.B, rAAV2.5, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV. HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9 , AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13 AAV.HSC14, AAV.HSC15, and AAV.HSC16. The present invention also includes pseudotyped rAAV particles composed of capsid proteins of AV serotypes. In this embodiment, the rAAV particles are pseudotyped to contain AAV-8 capsid proteins. In a further embodiment, the rAAV particle comprises an AAV-9 capsid protein. In some embodiments, the pseudotyped rAAV particles are pseudotyped rAAV8 or rAAV 9 particles are rAAV2 / 8 or rAAV2 / 9 pseudotyped particles. Its preparation and use are known in the art (e.g., Duan et al., J. Virol., 75:7662-7671 (2001);Halbert et a l., J. Virol., 74:1524-1532 (2000);Zolot Ukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 1 0:3075-3081, (2001).

[0589] In further embodiments, the rAAV particles comprise a mixture of two or more AAV capsid serotypes. In some embodiments, the rAAV particle comprises a capsid that includes a capsid protein that is a marker. The AAV-8 capsid protein and the AAV1, AAV2, AAV3, AAV4, and AA V5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV- 12, AAV-13, AAV-14, AAV-15 and AAV-16, rAAV.rh8 , rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74 , rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.An c80L65, rAAV.7m8, rAAV.PHP.B, rAAV2.5, rAAV2 tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, A AV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.H SC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC1 1, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC1 5, and one or more AAVs from an AAV serotype selected from AAV.HSC16. In some embodiments, the AAV capsid protein is a chimera with a capsid protein. In this study, rAAV particles consist of AAV-8 capsid proteins and AAV-1, AAV-2, and AAV-3 proteins. AV-5, AAV-6, AAV-7, AAV-9, AAV-10, rAAVrh10, A One or more from AAV serotypes selected from AAVrh.8, AAVrh.10, and AAVrh.11 The AAV capsid protein is a chimera with the AAV capsid protein of In some embodiments, the rAAV particles contain the AAV-9 capsid protein and the AAV1, AAV2 , AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV1 0, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AA V-16, rAAV.rh8, rAAV.rh10, rAAV.rh20, rAAV.r h39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, rAAV. Anc80, rAAV.Anc80L65, rAAV.7m8, rAAV.PHP.B, rAAV2.5, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HS C1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, A AV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.H SC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.H One or more selected from AAV.HSC14, AAV.HSC15, and AAV.HSC16 AAV capsid proteins that are chimeric with the capsid proteins of the AAV capsid serotypes In some embodiments, the rAAV particles comprise an AAV-9 capsid protein and an AAV-2 protein. AV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AA One or more AAV species selected from AAVrh.V9, AAVrh.8, and AAVrh.10 The AAV capsid protein is a chimera with a capsid protein of any of the AAV serotypes.

[0590] 6.5 rAAV particle isolation method In some embodiments, the present disclosure provides an isolated recombinant adeno-associated virus as disclosed herein. A method for producing a formulation comprising (rAAV) particles is provided, the method comprising: (a) removing impurities (e.g., rAAV) particles; (b) isolating rAAV particles from a feed comprising the rAAV production culture; and formulating the AAV particles using the methods disclosed herein to produce the formulation; In some embodiments, the isolated recombinant adeno-associated virus (rAAV) disclosed herein The method for producing a formulation containing rAAV particles includes (a) removing impurities (e.g., rAAV production cultures) from the (b) isolating the rAAV particles from the supply; and (b) determining the genomic titer of the isolated rAAV particles. and (c) determining whether the isolated rAAV particles are and preparing the formulation.

[0591] In some embodiments, the present disclosure further comprises an isolated recombinant adeno-associated A method for producing a pharmaceutical unit dosage form of a formulation comprising viral (rAAV) particles is provided, the method comprising: , isolating rAAV particles from a feed containing impurities (e.g., rAAV production cultures) The genomic titer of the isolated rAAV particles is determined, and the isolated rAAV particles are In some embodiments, the present invention provides a method for the preparation of a pharmaceutical composition comprising the steps of: Pharmaceutical units of formulations containing isolated recombinant adeno-associated virus (rAAV) particles disclosed herein The method for producing the dosage form comprises extracting rAAV from a feed containing impurities (e.g., rAAV production cultures). and isolating rAAV particles and isolating the isolated rAAV particles using the methods disclosed herein. and formulating the compound.

[0592] The isolated rAAV particles are isolated using methods known in the art. In some embodiments, the method for isolating rAAV particles can be further processed downstream, e.g. Harvesting the cell culture, and clarifying the harvested cell culture (e.g., by centrifugation or depth filtration). , tangential flow filtration, affinity chromatography, anion exchange chromatography chromatography, cation exchange chromatography, size exclusion chromatography, Aqueous interaction chromatography, hydroxylapatite chromatography, sterile filtration In some embodiments, downstream processing includes at least two, at least three, at least four, at least five, or or at least six of the following: harvesting the cell culture, clarification of the harvested cell culture (e.g., by centrifugation or depth filtration), tangential flow filtration, affinity chromatography Raffin, anion exchange chromatography, cation exchange chromatography, size Exclusion chromatography, hydrophobic interaction chromatography, hydroxylapatite In some embodiments, downstream processing includes harvesting the cell culture. Collection, clarification of harvested cell cultures (e.g., by depth filtration), sterile filtration, tangent Flow Filtration, Affinity Chromatography, and Anion Exchange Chromatography In some embodiments, downstream processing includes clarification of the harvested cell culture, sterile filtration, , tangential flow filtration, affinity chromatography, and anion exchange In some embodiments, downstream processing includes chromatography of the harvested cell culture. Clarification by layer filtration, sterile filtration, tangential flow filtration, affinity chromatography In some embodiments, the collected Clarification of the cell culture comprises sterile filtration. In some embodiments, downstream processing comprises centrifugation. In some embodiments, the rAAV particles contain the capsid protein of the AAV-8 serotype. In some embodiments, the rAAV particles comprise the capsid protein of the AAV-9 serotype. Includes.

[0593] In some embodiments, the method of isolating rAAV particles includes harvesting a cell culture, extracting the harvested cell culture, and Clarification of the product (e.g. by depth filtration), primary sterile filtration, primary tangential flow filtration filtration, affinity chromatography, monolith anion exchange chromatography, In some embodiments, the rAA The isolation method for V particles involves clarification of the harvested cell culture, first sterile filtration, and then first tangential filtration. Flow Filtration, Affinity Chromatography, Monolith Anion Exchange Chromatograph In some embodiments, the method includes a first tangential flow filtration, a second sterilizing filtration, and a second sterile filtration. The isolation method for rAAV particles involves first clarification by depth filtration of the harvested cell culture, then sterile filtration. , First Tangential Flow Filtration, Affinity Chromatography, Monolith Aniline The procedure included ion exchange chromatography, a second tangential flow filtration, and a second sterile filtration. In some embodiments, the method does not include centrifugation. Clarification of the cell culture comprises sterile filtration. In some embodiments, the rAAV particles are In some embodiments, the rAAV particles contain capsid proteins of AAV- Contains capsid proteins of 9 serotypes.

[0594] Numerous methods are known in the art for producing rAAV particles, including The results are useful for transfection, generation of stable cell lines, and expression of adenovirus-AAV hybrids, hepatitis C virus (HCV), and hepatitis B virus (HV). Pesvirus-AAV hybrids and baculovirus-AAV hybrids The rAAV virus particles are used to produce infectious hybrid viruses. All rAAV production cultures require the following: (1) a stable host cell; Such cells include, for example, HeLa, A549, or HEK293 cells and their derivatives. Human cell lines such as HEK293T cells and HEK293F cells, Vero cells, etc. Any mammalian cell line or insect-derived cell line, such as SF-9 in the case of the baculovirus production system, (2) a suitable helper virus function, which may be a wild-type or mutant adenovirus viruses (e.g., temperature-sensitive adenoviruses), herpes viruses, baculoviruses, or by a plasmid construct that provides helper functions; (3) the rep (4) the AAV ITR sequences flanked by the nucleotide sequence of ... (5) a suitable transgene to support rAAV production Media and Media Components. Suitable media known in the art may be used for the production of rAAV vectors. Such a medium can be used without particular limitation, for example, modified Eagle medium (MEM), Hyclone Labora Tories and JRH media, and the media described in U.S. Pat. No. 6,723,551. and Sf-900 II SFM medium as described in U.S. Pat. No. 6,723,551. Nos. 6,313,933 and 6,313,325, which are incorporated herein by reference in their entirety.

[0595] rAAV production cultures are grown under a variety of conditions appropriate to the particular host cell utilized (over a wide temperature range). They can be routinely grown over a range of temperatures, for varying lengths of time, etc. As is known in the art, rAAV production cultures include attachment-dependent cultures, The culture may be grown in any suitable attachment-dependent container, such as roller bottles, hollow fiber filters, Cultivation in microcarriers, packed bed or fluidized bed bioreactors, etc. As the rAAV vector production culture, a host cell suitable for suspension culture, such as H eLa, HEK293, Vero, and derivatives thereof, as well as SF-9 cells. These cells can be cultured in a variety of ways, for example in spinners. Flasks, stirred tank bioreactors, and disposable systems such as the Wave bag system A number of suspension cultures for the production of rAAV particles are known in the art. Such cultures include those described in, for example, U.S. Pat. Nos. 6,995,006 and 9,788,792. No. 3,826, and the cultures disclosed in U.S. Patent Application Publication No. 20120122155. each of which is incorporated herein by reference in its entirety.

[0596] In some embodiments, the method for producing rAAV particles includes the step of: providing a cell culture comprising a histone deacetylase (HDAC); adding an inhibitor to a final concentration of about 0.1 mM to about 20 mM; and In some embodiments, the method further comprises maintaining the HV particles under conditions that permit production of the HV particles. The DAC inhibitor comprises a short chain fatty acid or a salt thereof. In some embodiments, the HDAC inhibitor butyrates (e.g., sodium butyrate), valproates (e.g., sodium valproate) ), a propionate salt (e.g., sodium propionate), or a combination thereof include.

[0597] In some embodiments, the rAAV particles are described in U.S. PCT International Publication No. WO2020 / 0338. No. 42, published on February 13, 2020, titled “SCALABLE METHOD FOR Produced as disclosed in “RECOMBINANT AAV PRODUCTION” No. 6,399,433, which is incorporated herein by reference in its entirety.

[0598] Recombinant AAV particles are prepared by harvesting or from a production culture containing the host cells. can be harvested from rAAV production cultures by collection of spent media, with the exception When the medium is harvested, the cells are incubated for 1 h at 4°C for 2 h at 4°C, inducing the release of rAAV particles from intact host cells into the medium. provided that the cells are cultured under conditions known in the art to induce recombination. AAV particles can be harvested from rAAV production cultures by lysis of the host cells of the production culture. Suitable methods for lysing cells are known in the art, and such methods may be used in For example, multiple freeze-thaw cycles, sonication, microfluidization, and interface This includes treatment with chemicals such as activators and / or proteases.

[0599] At the time of harvest, rAAV production cultures may contain one or more of the following: (1) host cell proteins, (2) host cell DNA, (3) plasmid DNA, (4) ) helper virus, (5) helper virus protein, (6) helper virus DNA A, and (7) medium components. Medium components include, for example, serum proteins, amino acids, trans The rAAV production culture further contains the production Product-related impurities, e.g., inactive vectors, empty viral capsids, aggregated viral particles Contains progeny or capsids, misfolded viral capsids, and disassembled viral particles There is a possibility that.

[0600] In some embodiments, the rAAV production culture harvest is clarified to remove host cell debris. In some embodiments, the production culture harvest is filtered through a series of depth filters. Clarification can be accomplished by a variety of other standard techniques known in the art, such as centrifugation. or any cellulose acetate filter known in the art with a pore size of 0.2 mm or greater. In some embodiments, clarification of the harvested cell culture may be accomplished by filtration. In some embodiments, the production culture harvest is clarified by centrifugation. In some embodiments, clarification of the cell culture does not include centrifugation.

[0601] In some embodiments, the harvested cell culture is clarified using filtration. In some embodiments, clarification of the harvested cell culture comprises depth filtration. Clarification of the concentrated cell culture further includes depth filtration and sterile filtration. The harvested cell cultures were then filtered through a filter train (F) containing one or more different filtration media. In some embodiments, the eluate is clarified using a filter train. In some embodiments, the filter train comprises one or more depth filtration media. In some embodiments, the filter train includes two depth filtration media. In some embodiments, the filter train comprises a sterile filtration media. The filter train includes two depth filtration media and a sterile filtration media. The depth filtration filter medium is a porous depth filtration filter. The filter train is equipped with Clarisolve® 20MS, Millistak+ In some embodiments, the filter media comprises: The filter train consists of Clarisolve® 20MS, Millistak+ (registered trademark) COHC, and Sartopore (registered trademark) 2 XLG 0.2 μm In some embodiments, the harvested cell culture is pretreated before being filtered through a depth filtration filter. In some embodiments, the pretreatment comprises adding salt to the harvested cell culture. In some embodiments, the pretreatment comprises adding a chemical flocculant to the harvested cell culture. In some embodiments, the harvested cell culture is contacted with a depth filtration filter. Not pre-processed before.

[0602] In some embodiments, the production culture harvest is a culture medium comprising ... A1 issue, published on November 8, 2019, name “SCALABLE CLARIFICATI ON PROCESS FOR RECOMBINANT AAV PRODUCTIO The mixture was clarified by filtration as disclosed in "N" et al., which is incorporated herein by reference in its entirety. and is incorporated herein by reference.

[0603] In some embodiments, the rAAV production culture harvest is purified using a nuclease (e.g., Benzo nase®) or endonucleases (e.g., Serratia martensii cescens endonuclease) to separate the macromolecules present in the production culture. Nuclease or endonuclease digestion is performed using methods known in the art. For example, nuclease digestion can be performed using Benzo The final concentration of nase® was 1-2.5 units / ml, and the temperature range was room temperature to 37°C. It takes place over a period of 0 minutes to several hours.

[0604] Sterile filtration includes filtration using a sterilizing grade filter media. For sterilizing grade filter media, the sterilizing grade filter media is 0.2 or 0.22 μm pore size filters. In some embodiments, the sterilizing grade filter media is polyethersulfone (P In some embodiments, the sterilizing grade filter media comprises polyvinylidene ether (PSE). In some embodiments, the sterilizing grade filter media includes In some embodiments, the sterilizing grade filter media is , with a hydrophilic heterogeneous bilayer design of a 0.8 μm pre-filter and a 0.2 μm final filter membrane. In some embodiments, the sterilizing grade filter media includes a 1.2 μm pre-filter and In some embodiments, the filter has a hydrophilic heterogeneous bilayer design of 0.2 μm final filter membrane. The bacterial grade filter media is a 0.2 or 0.22 μm pore size filter. In one embodiment, the sterilizing grade filter media is a 0.2 μm pore size filter. In some embodiments, the sterilizing grade filter media is Sartopore (registered trademark). 2 XLG 0.2μm, Durapore™ PVDF membrane 0.45μm, Or Sartoguard® PES 1.2μm + 0.2μm nominal pore size combination In some embodiments, the sterilizing grade filter media is a Sart Opore®2 XLG 0.2 μm.

[0605] In some embodiments, the clarified feed is filtered by tangential flow filtration ("TFF"). Once concentrated, the product is transferred to a chromatography medium, e.g., affinity chromatography. Large-scale concentration of viruses using TFF ultrafiltration was reported by Paul et al. al., Human Gene Therapy 4:609-615 (1993) The TFF concentration of the clarified feed is performed by concentrating the clarified feed in a technically manageable volume. It allows for the chromatography of materials without the need for lengthy recirculation times. In some embodiments, the clarification feed is The clarified supply is concentrated at least 2-fold to at least 10-fold. The feed is concentrated at least 10-fold to at least 20-fold. The feed is concentrated by at least 20 times to at least 50 times. The clarified feed is concentrated by approximately 20-fold. As one of ordinary skill in the art will appreciate, TFF is a method for the purification of small molecules. Impurities (e.g., cell culture media containing media components, serum albumin, or other serum proteins) It may also be used to remove nutrient contaminants and form a clarified feed by diafiltration. In some embodiments, the clarified feed is subjected to diafiltration to remove small molecule impurities. In some embodiments, diafiltration is performed with about 3 to about 10 diafiltration volumes of buffer. In some embodiments, the diafiltration includes the use of about 5 diafiltration volumes of buffer. As one of ordinary skill in the art would also appreciate, TFF is a useful tool in the purification process. It can also be used in any process where it is desirable to exchange the buffer before the next process. In some embodiments, rAAV is isolated from the clarified feed disclosed herein. A method includes the use of TFF to exchange the buffer.

[0606] Isolating rAAV particles from the composition using affinity chromatography. In some embodiments, affinity chromatography can be used to clarify the The rAAV particles are isolated from the purified feed. In some embodiments, affinity chromatography is used. rAA from the clarified feed subjected to tangential flow filtration using a Isolate the V particles. Suitable affinity chromatography media are known in the art. Examples of such media include, but are not limited to, AVB Sepharose (trademark), P OROS(TM) CaptureSelect(TM) AAVX affinity resin, POROS (TM) CaptureSelect (TM) AAV9 affinity resin, and POROS (TM) Examples of suitable AAV8 affinity resins include CaptureSelect™ AAV8 affinity resins. Thus, the affinity chromatography medium is POROS™ Capture eSelect™ AAV9 affinity resin. In some embodiments, the affinity - Chromatography media is POROS™ CaptureSelect™ In some embodiments, the affinity chromatography medium is an AAV8 affinity resin. The material is POROS™ CaptureSelect™ AAVX affinity resin. do.

[0607] Isolating rAAV particles from the composition using anion exchange chromatography In some embodiments, affinity chromatography can be followed by anion exchange. Anion exchange chromatography is used as the final concentration and purification step. Chromatographic media are known in the art, and such media include, without limitation, , Unosphere Q (Biorad, Hercules, Calif.), and N Charged amino or imino resins, such as POROS 50 PI, or any DEA E, TMAE, tertiary or quaternary amines, or PEI-based resins as known in the art. (U.S. Patent No. 6,989,264;Brument et al., Mol. Th erapy 6(5):678-686 (2002);Gao et al., Hu m. Gene Therapy 11:2079-2091 (2000)) In some embodiments, the anion exchange chromatography medium comprises a quaternary amine. In some embodiments, the anion exchange medium is a monolithic anion exchange chromatography medium. In some embodiments, the monolithic anion exchange chromatography medium is Glycidyl methacrylate-ethylene dimethacrylate or styrene-divinylbenzene copolymer In some embodiments, the monolithic anion exchange chromatography medium comprises a C IMmultus(TM)QA-1 Advanced Composite Colu mn (quaternary amine), CIMmultus(TM) DEAE-1 Advanced Composite Column (Diethylamino), CIM® QA Di sk (quaternary amines), CIM® DEAE, and CIM® EDA Disk(ethylenediamino). In some embodiments, the monolith The anion exchange chromatography medium is CIMmultus™ QA-1 Ad vanced Composite Column (quaternary amine). Therefore, the monolithic anion exchange chromatography media is CIM® QA Disk (quaternary amine). In some embodiments, anion exchange chromatography - The medium is CIM QA (BIA Separations, Slovenia) In some embodiments, the anion exchange chromatography medium is BIA CIM (registered trademark). The column volume is 80 mL. However, appropriate ions are added so that the rAAV remains bound to the resin and impurities are washed away. Strong washing buffers can be identified and are not considered to be impurities introduced by upstream purification steps. Examples of the possible causes include, but are not limited to,

[0608] In some embodiments, anion exchange chromatography is carried out according to the method described in PCT International Published Application No. WO 2005 / 023666. 2019 / 241535, published on December 19, 2019, titled “Anion Exch ange Chromatography for Recombinant AAV The method was performed according to the method disclosed in "Production of Fluorescent Microbes," which is incorporated herein by reference in its entirety. No. 6,393,436, which is incorporated herein by reference.

[0609] In some embodiments, the method for isolating rAAV particles comprises providing a composition comprising the isolated rAAV particles. The vector genome titer, capsid titer, and / or ratio of full to empty capsids are determined. In some embodiments, the vector genome titer is determined by quantitative PCR (qPCR). R) or by digital PCR (dPCR) or droplet digital PCR (ddPCR) In some embodiments, the capsid titer is determined by serotype-specific ELISA. In some embodiments, the ratio of full to empty capsids is determined by analytical ultracentrifugation (AUC) or Identified by transmission electron microscopy (TEM).

[0610] In some embodiments, vector genome titer, capsid titer, and / or full-antibody capsid titer are assayed. The ratio of side is determined spectrophotometrically, for example by measuring the absorbance of the composition at 260 nm, and The composition is identified by measuring the absorbance at 280 nm of the composition. In some embodiments, the rAAV particles are not denatured prior to measuring the absorbance of the composition. In some embodiments, the rAAV particles are denatured prior to measuring the absorbance of the composition. In this case, the absorbance of the composition at 260 nm and 280 nm is determined using a spectrophotometer. In some embodiments, the absorbance of the composition at 260 nm and 280 nm is measured using HPLC. In some embodiments, the absorbance is the peak absorbance. Several methods for measuring absorbance at 280 nm and 280 nm are known in the art. Determine the vector genome titer and capsid titer of compositions containing recombinant rAAV particles The method is described in PCT International Publication No. WO2019212922A1, published November 7, 2019. , named “Systems and methods of spectrophotom etry for the determination of genome con tent, capsid content and full / empty rati os of adeno-associated virus particles” No. 6,399,633, which is incorporated herein by reference in its entirety.

[0611] 6.6 Kit In some embodiments, the present disclosure provides a method for treating a disease or condition comprising administering isolated recombinant AAV particles. The compound can be used to treat a disease or disorder in a subject in need of treatment for the disorder. In some embodiments, the kit includes a formulation that can be used to administer the drug to a patient. The present invention provides a method for preparing a pharmaceutical composition comprising administering to one or more containers a small amount of a formulation comprising the rAAV particles disclosed herein. In some embodiments, the kit comprises at least one formulation of isolated recombinant AAV particles. The present invention relates to a method for treating a disease or disorder in a subject in need of such treatment, the method comprising administering It contains all the components necessary and / or sufficient to perform the therapy. However, the formulations containing the rAAV particles disclosed herein can be used in established kit formats. The methods can be easily combined into one kit, and such kit formats are well known in the art.

[0612] The present disclosure provided in this specification can be easily understood and the present disclosure can be practically effective. For purposes of illustration, certain embodiments will now be described by way of the following non-limiting examples. do.

[0613] 6.7 Functional characteristics In certain embodiments, the formulations or stable formulations described herein are administered intravenously. administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., microinjection with a microneedle) via suprachoroidal drug delivery devices such as retinal stenosis, or via transvitreal approaches (surgical procedures) Intravitreal injection, juxtascleral injection, intravitreal injection, subconjunctival injection, intraretinal injection, retinal injection, and plexus injection via the suprachoroidal space Intrathecal administration (e.g., a catheter that can be inserted into the suprachoroidal space and advanced toward the posterior pole) A surgical procedure via a subretinal drug delivery device equipped with a microneedle to inject into the subretinal space. ), and / or posterior juxtascleral depot surgery (e.g., inserting the tip for direct attachment to the scleral surface) via a juxtascleral drug delivery device with a cannula that can be maintained There are.

[0614] In certain embodiments, the pharmaceutical composition can be administered intravenously, subcutaneously, by intramuscular injection, or via injection. Choroidal injection (e.g., using a suprachoroidal drug delivery device such as a microinjector with a microneedle) via a chair), subretinal injection via a transvitreal approach (surgical procedure), juxtascleral injection, vitreous injection Intraconjunctival administration, subconjunctival administration, intraretinal administration, subretinal administration via the suprachoroidal space (e.g., A subretinal drug delivery device comprising a catheter that can be inserted into the retina and advanced toward the posterior pole. surgical techniques, in which a microneedle is used to inject into the subretinal space, and / or posterior juxtascleral debridement Posterior surgery (e.g., a cannula that can be inserted with its tip to maintain direct attachment to the scleral surface) The drug has a desired density suitable for use in a juxtascleral drug delivery device equipped with a

[0615] In certain embodiments, the pharmaceutical composition can be administered intravenously, subcutaneously, by intramuscular injection, or via injection. Choroidal injection (e.g., using a suprachoroidal drug delivery device such as a microinjector with a microneedle) via a chair), juxtascleral administration, intravitreal administration, subconjunctival administration, intraretinal administration, transvitreal approach (surgical procedure), subretinal injection via the suprachoroidal space (e.g., A subretinal drug delivery device comprising a catheter that can be inserted into the retina and advanced toward the posterior pole. surgical techniques, in which a microneedle is used to inject into the subretinal space, and / or posterior juxtascleral debridement Posterior surgery (e.g., a cannula that can be inserted with its tip to maintain direct attachment to the scleral surface) The desired viscosity is suitable for administration via a juxtascleral drug delivery device equipped with a

[0616] In certain embodiments, recombinant adeno-associated virus (rAAV) particles in a stable formulation The results showed that the rAAV particles exhibited a significantly increased affinity to the lyophilization or reconstitution process compared to the same rAAV particles in a reference formulation. Stability is at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about %, approx. 20%, approx. 25%, approx. 30%, approx. 35%, approx. 40%, approx. 45%, approx. 50%, approx. 10 0%, about 2 times, about 3 times, about 5 times, about 10 times, about 100 times, or about 1000 times higher. In certain embodiments, the stability of rAAV particles can be assessed using the assays disclosed in Section 6.8. It is identified by a (singular or plural)

[0617] In certain embodiments, the rAAV particles in the stable formulation contain the same rAAAV as in the reference formulation. Compared to V particles, the infectivity is at least about 2%, 5%, 7%, 10%, 12%, About 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, Approximately 50%, approximately 100%, approximately 2 times, approximately 3 times, approximately 5 times, approximately 10 times, approximately 100 times, or approximately 10 In certain embodiments, the viral infectivity of the rAAV particles is 6.80 times higher. In certain embodiments, the method is characterized by the assay(s) disclosed in the section. In certain embodiments, infectivity is measured before or after lyophilization. Infecting activity is measured before and after reconstitution of the lyophilized formulation.

[0618] In certain embodiments, the rAAV particles in the stable formulation contain the same rAAAV as in the reference formulation. Compared to V particles, aggregation is at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, approximately 100%, approximately 2x, approximately 3x, approximately 5x, approximately 10x, approximately 100x, or approximately 100 In certain embodiments, rAAV particle aggregation is reduced by 100% as disclosed in Section 6.8. In certain embodiments, the agglutination is identified by an assay or assays. is measured before or after lyophilization. In certain embodiments, aggregation is measured after lyophilization. Measured before or after reconstitution of the dry formulation.

[0619] In certain embodiments, the stable formulation is lyophilized prior to storage.

[0620] In certain embodiments, the stable formulation is reconstituted after storage.

[0621] In certain embodiments, the formulation is maintained at -80°C, -70°C, -20°C, 4°C, 20°C. , stored at 25°C, 30°C, 35°C, 37°C, or 40°C.

[0622] In certain embodiments, the formulation may be administered for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, approx. 10 months, approx. 11 months, 12 months, approx. 15 months, approx. 18 months, approx. 24 months, approx. In certain embodiments, the composition is stored for about 2 years, about 3 years, about 4 years, or about 5 years. The rAAV particles in the stable formulation can be used to inactivate the formulation over a period of time, e.g., about one week, about two weeks, , about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months , about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about After storage for 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years, The stability is at least about 2 times greater than that of the same rAAV particles in a reference formulation stored under the same conditions. %, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, About 30%, about 35%, about 40%, about 45%, about 50%, about 100%, about 2 times, about 3 times, about 5-fold, about 10-fold, about 100-fold, or about 1000-fold higher. The stability of rAAV particles over a period of time can be assessed using the assays disclosed in Section 6.8. The term "substitute" is used to refer to a specific area or areas.

[0623] In certain embodiments, the rAAV particles in a stable formulation can be stable over a period of time. , for example, at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, About 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months , about 11 months, 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, Stability compared to the same rAAV particles in a reference formulation after storage for approximately 4 or 5 years However, at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 100%, About 2 times, about 3 times, about 5 times, about 10 times, about 100 times, or about 1000 times higher. In one embodiment, the stability of the rAAV particles over a period of time is determined using methods disclosed in Section 6.8. The antibody is identified by an assay or assays that

[0624] In certain embodiments, the rAAV particles in the stable formulation contain the same rAAAV as in the reference formulation. Compared to V particles, the in vitro relative potency (IVRP) is at least about 2% and at least about 5% , about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, Approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 100%, approximately 2 times, approximately 3 times, approximately 5 times, approximately 1 0 fold, about 100 fold, or about 1000 fold higher. The in vitro relative potency (IVRP) of the compound was determined using the assays disclosed in Chapter 6.8 (singular In certain embodiments, the IVRP is identified by a lyophilized In certain embodiments, the IVRP is measured before or after reconstitution of a lyophilized formulation. Measured before or after construction.

[0625] In certain embodiments, the rAAV particles in the stable formulation contain the same rAAAV as in the reference formulation. Free DNA is at least about 2%, about 5%, about 7%, about 10%, about 12%, or about 16% relative to V particles. %, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45 %, about 50%, about 100%, about 2x, about 3x, about 5x, about 10x, about 100x, or about In certain embodiments, the free DNA of the rAAV particles is 6. In certain embodiments, the method is characterized by the assay(s) disclosed in Section 8. In certain embodiments, the free DNA is measured before or after lyophilization. In this study, free DNA was measured before or after reconstitution of the lyophilized formulation.

[0626] In certain embodiments, the rAAV particles in the stable formulation contain the same rAAAV as in the reference formulation. rAAV genome release is at least about 2%, about 5%, about 7%, about 10% compared to V particles. , about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40% , about 45%, about 50%, about 100%, about 2 times, about 3 times, about 5 times, about 10 times, about 100 times, or about 1000-fold less. In certain embodiments, rAAV genome release is A is identified by measuring the relative fluorescence in the presence of a specific fluorescent stain. In one embodiment, rAAV genome release can be measured using the assay (s) disclosed in Section 6.8. In certain embodiments, the rAAV genome release is determined by lyophilization. In certain embodiments, rAAV genome release is measured before or after freezing. Measured before or after reconstitution of the dry formulation.

[0627] In certain embodiments, the rAAV particles in a stable formulation can be stable over a period of time. For example, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, About 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months Months, approx. 12 months, approx. 15 months, approx. 18 months, approx. 24 months, approx. 2 years, approx. 3 years, approx. 4 years or after about 5 years of storage, the dimensional change is at most about 20%, about 15%, about 10%, about 8%, %, about 5%, about 4%, about 3%, about 2%, or about 1%. The size of the rAAV particles can be determined by the assay(s) disclosed in Section 6.8. In certain embodiments, the dimensions are measured before or after a freeze-thaw cycle. It is determined.

[0628] In certain embodiments, the rAAV particles in a stable formulation can be stable over a period of time. , for example, at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, About 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months , about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years After storage for about 4 years or about 5 years, the dimensional changes were at most 20%, 15%, 10%, In certain embodiments, the rA The size of the AV particles is determined by the assay(s) disclosed in Section 6.8. In certain embodiments, the dimensions are measured before or after lyophilization. In certain embodiments, the dimensions are measured before or after reconstitution of the lyophilized formulation.

[0629] In certain embodiments, the rAAV particles in the stable formulation can be stored at -80° C., -70° C., - When stored at 20℃, 4℃, 20℃, 25℃, 30℃, 35℃, 37℃, or 40℃ , stability is at least about 2%, about 5%, about 7%, or more than the same rAAV particles in a reference formulation. %, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35 %, approx. 40%, approx. 45%, approx. 50%, approx. 100%, approx. 2x, approx. 3x, approx. 5x, approx. 10x, In certain embodiments, the stability of the rAAV particles is about 100-fold or about 1000-fold higher. The assay is determined by the assay(s) disclosed in Section 6.8.

[0630] In certain embodiments, the rAAV particles in the stable formulation can be stored at -80° C., -70° C., - 20℃, 4℃, 20℃, 25℃, 30℃, 35℃, 37℃, or 40℃ for a period of time For example, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about When stored for 4 or approximately 5 years, the infectivity was significantly increased compared to the same rAAV particles in a reference formulation. But at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2x, 3x, 5x, 10x, 10 In certain embodiments, the viral load of the rAAV particles is Infectivity is determined by the assay(s) disclosed in Section 6.8.

[0631] In certain embodiments, the rAAV particles in the stable formulation can be stored at -80° C., -70° C., - 20℃, 4℃, 20℃, 25℃, 30℃, 35℃, 37℃, or 40℃ for a period of time For example, at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 1 0 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about compared to the same rAAV particles in a reference formulation when stored for 3, ~4, or ~5 years. and the infectiousness is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, or 20%. , 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2x, 3x, 5x, 1 0, 100, or 1000 times higher. Viral infectivity is determined by the assay(s) disclosed in Section 6.8. do.

[0632] In certain embodiments, the rAAV particles in the stable formulation can be stored at -80° C., -70° C., - 20℃, 4℃, 20℃, 25℃, 30℃, 35℃, 37℃, or 40℃ for a period of time For example, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about When stored for 4 or approximately 5 years, there was less aggregation compared to the same rAAV particles in a reference formulation. ,At least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 3 0%, 35%, 40%, 45%, 50%, 100%, 2x, 3x, 5x, 10x, 100 In certain embodiments, rAAV particle aggregation is reduced by 1000 or 1000 times. Identified by the assay(s) disclosed in Section 6.8.

[0633] In certain embodiments, the rAAV particles in the stable formulation can be stored at -80° C., -70° C., - 20℃, 4℃, 20℃, 25℃, 30℃, 35℃, 37℃, or 40℃ for a period of time For example, at least about 11 weeks, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, When stored for approximately 3, 4, or 5 years, the rAAV particles were All samples were analyzed to determine whether aggregation was at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, or 20%. , 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2x, 3x, 5x, 1 0, 100, or 1000 times less. Aggregation of is identified by the assay(s) disclosed in Section 6.8.

[0634] In certain embodiments, the rAAV particles in the stable formulation can be stored at -80° C., -70° C., - 20℃, 4℃, 20℃, 25℃, 30℃, 35℃, 37℃, or 40℃ for a period of time For example, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about Stability compared to the same rAAV particles in a reference formulation when stored for 4 or approximately 5 years But at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2x, 3x, 5x, 10x, 10 0 fold, or 1000 fold higher. In certain embodiments, the rAAV particles Stability over a period of time is determined by the assay(s) disclosed in Section 6.8. .

[0635] In certain embodimen...

Claims

1. Recombinant adeno-associated virus (rAAV) particles; a) Tris, b) sugar, and c) a salt that does not crystallize during freezing of the formulation; A stable formulation comprising: The formulation is suitable for lyophilization; and The formulation, wherein the rAAV particles comprise AAV9 capsid proteins.

2. 2. The formulation of claim 1, wherein the salt has an ionic strength of 30 mM or greater.

3. The formulation of claim 1 or 2, wherein the formulation has an ionic strength of less than 200 mM.

4. The formulation of any one of claims 1 to 3, wherein the salt has an ionic strength of from 30 mM to 100 mM.

5. The formulation comprises: a) 1 mM to 50 mM Tris, or b) Tris at 1 mM to 30 mM, 1 mM to 20 mM, 5 mM to 30 mM, 5 mM to 20 mM, 10 mM to 30 mM, 10 mM to 20 mM, or 20 mM to 50 mM, or c) 1 mM, 2 mM, 3 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, or 40 mM Tris The formulation according to any one of claims 1 to 4, comprising:

6. The formulation of claim 5, comprising 5 mM Tris.

7. The formulation of claim 1, wherein the salt is a multivalent salt containing one or more multiply charged ions.

8. The formulation of claim 1, wherein the salt is a citrate or sulfate.

9. 9. The formulation of any one of claims 1 to 8, wherein the salt is sodium citrate, sodium sulfate, ammonium sulfate, magnesium sulfate, or a combination thereof.

10. 10. The formulation of claim 1, wherein the formulation comprises 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 120 mM, 140 mM, 150 mM, or 200 mM sodium sulfate.

11. The pH is, a) 6.5-8.0, b) 7.2 to 7.8, c) 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8; d) 7.5, or e) 7.1 The formulation according to any one of claims 1 to 10, wherein

12. a) 50 mM to 400 mM sugar; b) 50 mM to 350 mM, 50 mM to 300 mM, 50 mM to 250 mM, 50 mM to 200 mM, or 50 mM to 150 mM sugar; c) 100 mM to 400 mM, 150 mM to 400 mM, 200 mM to 400 mM, 250 mM to 400 mM, or 300 mM to 400 mM sugar; d) 100 mM to 300 mM, 150 mM to 250 mM, 200 mM to 300 mM, or 250 mM to 350 mM sugar, or e) 50 mM, 100 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, 300 mM, or 350 mM sugar.

12. The formulation of claim 1 , comprising:

13. The formulation of claim 12, comprising 190 mM to 230 mM, 170 mM to 250 mM, or 250 mM to 270 mM sugar.

14. The formulation of claim 13, comprising 210 mM sugar.

15. a) the sugar is a non-reducing sugar, or b) the sugar is a reducing sugar; 15. A formulation according to any one of claims 1 to 14.

16. The formulation of claim 15, wherein the non-reducing sugar is selected from the group consisting of sucrose, trehalose, and raffinose; or the reducing sugar is selected from the group consisting of glucose, fructose, mannose, galactose, and lactose.

17. 17. The formulation of any one of claims 1 to 16, comprising sucrose.

18. a) less than 100 mM sodium citrate; b) 10 mM to 100 mM sodium citrate; c) 10 mM to 100 mM, 20 mM to 100 mM, 30 mM to 100 mM, 40 mM to 100 mM, 50 mM to 100 mM, 10 mM to 80 mM, 10 mM to 60 mM, 10 mM to 50 mM, 10 mM to 40 mM, or 10 mM to 30 mM sodium citrate; d) 10 mM to 50 mM, 20 mM to 60 mM, 30 mM to 70 mM, or 10 mM to 30 mM sodium citrate, or e) 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, or 60 mM sodium citrate 18. The formulation of any one of claims 1 to 17, comprising:

19. The formulation of claim 18, comprising 20 mM sodium citrate.

20. moreover, a) 0.0005% to 0.01% of a non-ionic surfactant, or b) 0.002% non-ionic surfactant 20. The formulation of any one of claims 1 to 19, comprising:

21. 21. The formulation of claim 20, wherein the non-ionic surfactant comprises poloxamer 188, poloxamer 407, polysorbate 80, polysorbate 20, Pluronic F-68, or BRIJ 35.

22. 22. The formulation of any one of claims 1 to 21, further comprising a stabilizer selected from the group consisting of glycerol or sorbitol.

23. The formulation comprises: a) 0.1% to 5% glycerol; b) 0.1% to 2% glycerol, or c) 0.25% to 2% glycerol 23. The formulation of claim 22, comprising:

24. a) the formulation does not contain mannitol, or b) the formulation comprises less than 10 mM, 20 mM, 50 mM, 100 mM or 150 mM mannitol; 24. A formulation according to any one of claims 1 to 23.

25. A. a) 1 mM to 25 mM Tris; b) 50 mM to 400 mM sugar; c) 10 mM to 100 mM sodium citrate, and d) 0.0005% to 0.01% of a non-ionic surfactant and having a pH of 7.2 to 7.8; or B. a) 2 mM to 10 mM Tris; b) 150 mM to 250 mM sugar; c) 10 mM to 20 mM sodium citrate, and d) 0.001% to 0.005% of a non-ionic surfactant and having a pH of 7.2 to 7.8; 25. A formulation according to any one of claims 1 to 24.

26. A. a) 5 mM Tris; b) 210 mM sugar; c) 20 mM sodium citrate, and d) 0.002% non-ionic surfactant and having a pH of 7.5; B. a) 5 mM Tris; b) 210 mM sucrose; c) 20 mM sodium citrate, and d) 0.002% (w / v) Poloxamer 188 Contains or C. a) 5 mM Tris; b) 210 mM sucrose; c) 20 mM sodium citrate; d) 0.002% (w / v) poloxamer 188, and e) 0.25% (w / v) glycerol Contains or D. a) 5 mM Tris; b) 210 mM sucrose; c) 20 mM sodium citrate; d) 0.002% (w / v) poloxamer 188, and e) 0.5% (w / v) sorbitol Contains or E. a) 5 mM Tris; b) 30 mM sodium sulfate; c) 263 mM sucrose, and d) 0.005% (w / v) Poloxamer 188 26. The formulation of claim 25, comprising:

27. A. rAAV particles between 1.0E+11 genome copies / mL (GC / mL) and 1.0E+15 GC / mL, or B. rAAV particles of 1.0E+11GC / mL, 1.0E+12GC / mL, 1.0E+13GC / mL, 1.0E+14GC / mL, or 1.0E+15GC / mL 27. The formulation of any one of claims 1 to 26, comprising:

28. The remaining moisture content is a) 1% to 7%, b) 1% to 7%, 2% to 7%, 3% to 7%, 4% to 7%, 5% to 7%, 1% to 6%, 1% to 5%, 1% to 4%, or 1% to 3%, c) 3% to 7%, 3% to 6%, or 3% to 5%, d) 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, or 6%; e) 1% to 2%, f) 1.5%, 1.4%, 1.3%, 1.2%, or 1.1%, or g) 1% 28. The formulation of any one of claims 1 to 27, wherein

29. a) the glass transition temperature (Tg) of a lyophilized cake of said formulation is greater than 35° C.; or b) the glass transition temperature (Tg') of the maximum freeze-concentrated solution of said formulation is greater than -40°C; 29. A formulation according to any one of claims 1 to 28.

30. a) the % relative potency of the rAAV particles is at least 60%, at least 70%, or at least 80% after storage of the formulation at room temperature for 3 months, where the reference rAAV particles are stored in DPBS with 0.001% poloxamer 188 buffer at -70°C; b) the % relative potency of the rAAV particles is at least 60%, at least 70%, or at least 80% after storage of the formulation at room temperature for 6 months, where the reference rAAV particles are stored at -70°C in Dulbecco's Phosphate Buffered Saline (DPBS) with 0.001% Poloxamer 188 buffer; c) the % relative potency of the rAAV particles is at least 30%, at least 40%, at least 50%, or at least 60% after one week of storage of the formulation at 35° C., where the reference rAAV particles are stored in DPBS with 0.001% poloxamer 188 buffer at −70° C.; d) the % relative potency of the rAAV particles is at least 30%, at least 40%, at least 50%, or at least 60% after storage of the formulation for 2 weeks at 35° C., where the reference rAAV particles are stored in DPBS with 0.001% poloxamer 188 buffer at −70° C.; or e) the % relative potency of the rAAV particles is at least 30%, at least 40%, at least 50%, or at least 60% after storage of the formulation at 35° C. for 4 weeks, where the reference rAAV particles are stored at −70° C. in DPBS with 0.001% poloxamer 188 buffer; 30. A formulation according to any one of claims 1 to 29.

31. 31. The formulation of any one of claims 1 to 30, wherein the formulation is for lyophilization prior to storage.

32. The formulation of claim 31, wherein the formulation is for reconstitution after storage.

33. A. the % relative potency of the rAAV particles immediately after lyophilization is at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%; B. The level of rAAV particle aggregation of the formulation is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% when compared to the level of rAAV particle aggregation in a reference formulation; or C. a) the stability of the formulation is assessed by vector genome content or viral titer assay, and the formulation has at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200% more genome content after storing the formulation for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, or 3 years, when compared to the genome content of a reference formulation stored under the same conditions; b) the stability of the formulation is assessed by measuring the relative potency of the formulation, wherein the formulation has a relative potency that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200% greater after storing the formulation for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, or 3 years, when compared to the genome content of a reference formulation stored under the same conditions; c) the stability of the formulation is assessed by loss of infectivity, and the formulation exhibits at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200% less loss in infectivity after storage of the formulation for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, or 3 years, when compared to the loss in infectivity of a reference formulation stored under the same conditions; or d) the stability of the formulation is assessed by rAAV genome release, which is determined by measuring relative fluorescence in the presence of a DNA-specific fluorescent stain, and the formulation has a relative fluorescence level that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200% lower after storing the formulation for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, or 3 years, when compared to the relative fluorescence level of a reference formulation stored under the same conditions; The reference formulation is (i) DPBS with 0.001% Poloxamer 188 buffer; (ii) a sugar-free formulation, or (iii) Plasticizer-free formulations 33. The formulation of any one of claims 1 to 32, wherein

34. a) the formulation is stored at −80° C., −70° C., −20° C., 4° C., 20° C., 25° C., 30° C., 35° C., or 40° C.; b) the formulation is frozen at a temperature of −20° C. in the process of lyophilization; c) the frozen formulation maintains a pH between pH 6 and pH 9 when frozen at −20° C.; d) the frozen formulation, when frozen to -20°C, maintains a pH value within plus or minus one unit of the pre-freezing pH value; or e) the formulation is a stabilized aqueous formulation of rAAV for lyophilization; 34. A formulation according to any one of claims 1 to 33.

35. 35. A method for producing a stable formulation comprising recombinant adeno-associated virus (rAAV) particles, the method comprising mixing rAAV particles with Tris, sugar, and salt of a formulation described in any one of claims 1 to 34, thereby producing the formulation comprising rAAV.

36. 1. A method for reducing rAAV genome release from rAAV particles, comprising: a) producing a formulation according to any one of claims 1 to 34, wherein rAAV genome release from the rAAV particles after three freeze-thaw cycles is reduced compared to rAAV genome release in a formulation that does not contain sugar; or b) producing a formulation according to any one of claims 1 to 34, wherein rAAV genome release from the rAAV particles after lyophilization and reconstitution is reduced compared to rAAV genome release in a formulation that does not contain sugar.

37. 35. A method for producing a stable lyophilized formulation of an rAAV product, comprising subjecting a pre-lyophilized formulation to lyophilization, wherein the pre-lyophilized formulation is one described in any one of claims 1 to 34.

38. 35. The formulation of any one of claims 1 to 34 for use in a method of treating or preventing a disease, the formulation being for lyophilization and reconstitution prior to administration to a subject in need thereof.

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