Method of manufacture of AAV particles

Abstract

The present disclosure relates to methods of manufacturing compositions of recombinant AAV particles useful for gene therapy using a two-column process. The compositions comprise high amounts of full, active AAV particles and low amounts process and product impurities. The present disclosure also relates to recombinant AAV compositions prepared by methods of the disclosure.

Description

IMPROVED METHOD OF MANUFACTURE OF AAV PARTICLESFIELD OF THE INVENTION

[0001] Methods of manufacturing functional, high-purity AAV capsids for uses including gene therapy.BACKGROUND

[0002] Wild-type Adeno-associated viruses (AAV) are small non-enveloped viruses in the genus Dependoparvovirus . AAV requires a co-infecting helper virus, such as adenovirus, to replicate. The AAV genome is approximately 4.7kB, which inverted terminal repeat (ITR) sequences that flank two genes, cap and rep. The cap gene encodes structural proteins that make up the capsid. The rep gene encodes replicase proteins that are required for AAV replication and packaging. Recombinant AAVs (rAAV) in which a coding sequence for a therapeutic agent replaces the cap and rep genes in the AAV genome can be used as a gene therapy vector.

[0003] AAV has the advantage of a lack of pathogenicity of natural AAV infection. As of 2024 there are now several approved AAV gene therapy products with many more the subject of IND and BLAs. In addition, the FDA continues to closely monitor the development and safety of these products and has recently issued industry guidance. See, eg., https: / / www.fda.gov / media / 170198 / download; see also Briefing Document Cellular, Tissue, and Gene Therapies Advisory Committee (CTGTAC) Meeting #70; Toxicity Risks of Adeno-associated Virus (AAV) Vectors for Gene Therapy (GT), September 2021. https: / / www.fda.gov / media / 151599 / download (“FDA Briefing Document”).

[0004] Clinical AAV compositions for gene therapy have typically contained a portion of empty capsids, which comprise the AAV capsid shell, but lack the nucleic acid encoding the therapeutic product. They may also contain partial capsids, which contain the AAV capsid shell and lack a complete nucleic acid capable of expressing the therapeutic product and may contain contaminating nucleic acid, i.e., that does not contribute to expression of the therapeutic product (for example, plasmid backbone or host-cell DNA). The FDA Briefing Document recognizes that treatment-emergent serious adverse events (TESAEs) have been reported in many studies (about 35% of 149 AAV gene therapy clinical trials). Other product and process impurities may be a source of toxicity as well. The FDA is evaluating and mitigating the risks of AAV vector-basedproduct design and quality. Vector manufacturing is a key consideration when investigating the determinants of toxicities. FDA Briefing Document, p. 15. The presence of empty capsids in clinical formulation is undesirable and a reduction in the number of empty capsids can potentially improve the safety margin of high concentration AAV formulations. FDA Briefing Document, p. 16.

[0005] There is a need to develop improved methods for manufacturing and purifying AAV particles for administration to humans that allow for the production of AAV particle compositions having reduced contamination with empty and / or non-functional capsids, and reduced process and product impurities, coupled with improved yields and process efficiency.SUMMARY OF INVENTION

[0006] Provided herein are methods of manufacturing high purity recombinant AAV particle preparations suitable for therapeutic use in human subjects. The AAV particle preparations prepared according to the invention include AAV particle preparations enriched for full capsids, comprising a therapeutic transgene, that can transduce target cells in the subject and express the therapeutic transgene with minimal process and product impurities, including empty and partial capsids and capsids containing contaminating nucleic acid sequences. Such recombinant AAV particle compositions have reduced toxicity and risk of adverse effects due, at least in part, to the level of purity of the composition.

[0007] The methods described herein are suitable for large-scale manufacture of AAV particle compositions, including for a starting lysate volume of from about 2L to about 1000L, including 10L to 500L, 100L to 500L, 100L to 200L, 100L to lOOOL, 200L to lOOOL and even greater than WOOL. In embodiments, the starting lysate volume is 215L. The methods described herein advantageously result in drug substance with process and product impurity levels and yield comparable to drug substance produced by processes that include purification by cesium gradient on ultracentrifugation even for large scale manufacturing. In addition, the methods disclosed herein are further advantageously efficient such that large scale batches can be processed from lysate to final drug substance in 24 to 48 hours.

[0008] The AAV manufacturing process described herein generally comprises;

[0009] A process for manufacturing a composition of adeno-associated virus composition (AAV), said process comprising; a. providing a cell lysate comprising a first AAV particle composition comprising full AAV capsids, process impurities, including empty and partial AAV capsids, and product impurities, the cell lysate obtained from a culture of AAV producing mammalian cells and optionally clarifying the cell lysate; b. flocculating the optionally clarified cell lysate by sequentially adding to the optionally clarified cell lysate first salt, followed by octanoic acid, followed by addition of formate and allowing the cell lysate to equilibrate with each addition prior to the next addition to form a flocculation mixture buffer; c. forming a separated flocculation mixture by mixing the flocculation mixture and allowing the flocculation mixture to stand for a time sufficient to form a cream layer at the surface of the flocculation mixture buffer; d. separating the flocculation mixture buffer layer below the cream layer and optionally combining it with cationic exchange buffer used to rinse the cream layer to obtain a rinse buffer layer separated from the rinsed cream layer and clarifying by filtration the flocculation mixture buffer optionally combined with the rinse buffer layer to form a second AAV particle composition; e. loading the second AAV particle composition onto a cationic exchange column to capture AAV particles present in material loaded onto the cationic exchange column and washing the cationic exchange column to remove impurities;f. eluting the AAV particles from the cationic exchange column either with a pH gradient or a salt gradient to form an AAV containing cation exchange column eluate to produce a third AAV particle composition; g. if eluting AAV particles by using a salt gradient, reducing the salt concentration of the cation exchange column eluate obtained from eluting the AAV particles with a salt gradient by exchanging the eluate buffer with a low salt buffer using tangential flow filtration to provide an AAV containing anion exchange column loading buffer as the third AAV particle composition; h. loading an anion exchange column with the third AAV particle composition obtained from eluting AAV particles from the cationic exchange column with a pH gradient, or the AAV containing anion exchange column loading buffer of step g, so as to capture the AAV particles present in material loaded onto the anion exchange column; i. washing the anion exchange column with anion exchange buffer to remove impurities; j . eluting the AAV particles from the anion exchange column with a salt gradient to produce an AAV particle containing anion exchange column eluate as a fourth AAV particle composition; k. optionally reducing the conductivity of the AAV containing anion exchange column eluate and loading the resulting reduced conductivity eluate onto an anion exchange column, washing the column with anion exchange buffer and eluting the AAV particles from the anion exchange column with a salt gradient to produce an AAV particle containing eluate as a fourth AAV particle composition; l. exchanging the AAV containing anion exchange eluate from the fourth AAV particle composition from step j or k with a pharmaceutically acceptable buffer using one or more cycles of tangential flow filtration toobtain a fifth AAV particle composition comprising purified AAV particles; m. optionally filtering the fifth AAV particle composition through a sterilizing filter to obtain a drug substance comprising purified AAV particles, provided that the process does not include purification by ultracentrifugation, for example on a cesium or other gradient and results in an AAV drug substance that is sufficiently pure for clinical administration, including drug substances comprising AAV particles at a purity of least 80% full AAV capsids by AUC or CDMS, at least 90% AAV capsid purity by SDS-PAGE or CE-SDS, at least 70% transgene present in the genome in the AAV particle by short read NGS, and / or greater than about 75% DNA reads of the full expression cassette by long read NGS.

[0010] In embodiments, optionally clarifying the cell lysate in step (la) comprises clarifying the cell lysate by filtration. In embodiments, the flocculating step (lb) further comprises optionally adding 6,000 MW PEG to a final concentration of at least about 1% subsequent to the addition of octanoic acid, and prior to the addition of formate.

[0011] In other embodiments, process for manufacturing a composition of adeno-associated virus composition (AAV) comprises: a. Obtaining a first AAV particle composition from AAV producing mammalian host cells comprising less than about 30%, 20%, 10%, 5%, 1%, or 0.1% of host cell DNA and / or host cell protein; b. loading the first AAV particle composition onto a cationic exchange column to capture AAV particles present in material loaded onto the cationic exchange column and washing the cationic exchange column to remove impurities; c. eluting the AAV particles from the cationic exchange column either with a pH gradient or a salt gradient to form an AAV containing cation exchange column eluate to produce a second AAV particle composition;d. if eluting AAV particles by using a salt gradient, reducing the salt concentration of the cation exchange column eluate obtained from eluting the AAV particles with a salt gradient by exchanging the eluate buffer with a low salt buffer using tangential flow filtration to provide an AAV containing anion exchange column loading buffer as the second AAV particle composition; e. loading an anion exchange column with the second AAV particle composition obtained from eluting AAV particles from the cationic exchange column with a pH gradient or the AAV containing anion exchange column loading buffer of step d, so as to capture the AAV particles present in material loaded onto the anion exchange column; f. washing the anion exchange column with anion exchange buffer to remove impurities; g. eluting the AAV particles from the anion exchange column with a salt gradient to produce an AAV particle containing anion exchange column eluate as a third AAV particle composition; h. optionally reducing the conductivity of the AAV containing anion exchange column eluate and loading the resulting reduced conductivity eluate onto an anion exchange column, washing the column with anion exchange buffer and eluting the AAV particles from the anion exchange column with a salt gradient to produce an AAV particle containing eluate as a third AAV particle composition; i. exchanging the AAV containing anion exchange eluate from the third AAV particle composition from step g or h with a pharmaceutically acceptable buffer using one or more cycles of tangential flow filtration to obtain a fourth AAV particle composition comprising purified AAV particles;j . optionally filtering the fourth AAV particle composition through a sterilizing filter to obtain a drug substance comprising purified AAV particles, provided that the process does not include purification by ultracentrifugation, for example on a cesium or other gradient and results in an AAV drug substance that is sufficiently pure for clinical administration, including drug substances comprising AAV particles at a purity of least 80% full AAV capsids by AUC or CDMS, at least 90% AAV capsid purity by SDS-PAGE or CE-SDS, at least 70% transgene present in the genome in the AAV particle by short read NGS, and / or greater than about 75% DNA reads of the full expression cassette by long read NGS.

[0012] In embodiments, the AAV particles are AAV9 particles or an AAV serotype where the capsid has an isoelectric point similar to AAV9. In other embodiments, the AAV particles are AAV8 particles or an AAV serotype where the capsid has an isoelectric point similar to AAV8. In other embodiments, the AAV particles are AAV6 particles or an AAV serotype where the capsid has an isoelectric point similar to AAV6. In other embodiments, the AAV particles are AAV5 particles or an AAV serotype where the capsid has an isoelectric point similar to AAV5.

[0013] In embodiments, the AAV particles are not AAV2 particles. In other embodiments, the AAV particles are not modified to have a different capsid amino acid sequence than the capsid amino acid sequence of a naturally occurring AAV capsid serotype.

[0014] In further embodiments, the AAV particles have single stranded genomes and in other embodiments the AAV are self-complementary. The flocculation and chromatography principles disclosed herein may be further modified or adapted to purify other capsid types.

[0015] In the methods described herein, the AAV particles are purified using a cation exchange(CEX) chromatography column to remove impurities. In embodiments, the CEX chromatography column bed volume is ImL, 4 m , 8 mL, 40 mL, 80 m , 400 mb, 800 mL, 4 L, or 8 L, proportionately sized based upon a bed volume of 800 mL for approximately 200 L to about 250 L of the volume of the initial lysate containing AAV particles, as described below. In embodiments, the CEX chromatography column may have a pore size of greater than 1.1 pm., and in embodiments, is 10 pm, 6 pm, 4 pm or 2 pm. In embodiments, the CEX chromatography column comprises a poly(glycidyl methacrylate-co-ethylene dimethacrylate chromatography resin. Inembodiments, the CEX chromatography column comprises a sulfonate ligand. In embodiments, the AAV particles are eluted from the CEX chromatography column eluting at a rate of from about 6 to about 10 column volumes per minute. In embodiments, the CEX chromatography is using a poly(glycidyl methacrylate-co-ethylene dimethacrylate chromatography resin with a sulfonate ligand and a 6 pm pore size, run at a flow rate of about 6, 7, 8, 9 or 10 column volumes per minute where the column bed volume is approximately 800 mL for a 200 to 250 L lysate or a bed volume proportional to the volume or mass of lysate as described herein.

[0016] In embodiments, the AAV particles are eluted from the cation exchange (CEX) chromatography column with a salt gradient to form an AAV containing cationic exchange column eluate. Alternatively, AAV particles may be eluted from the cationic exchange column with a pH gradient to produce a cationic exchange column eluate containing AAV particles. For example, cation exchange (CEX) chromatography may be conducted using an elution buffer solution that creates a salt gradient ranging from a concentration of about 500 mM NaCl to a final concentration of about 2.5 M NaCl, or, alternatively, an elution buffer solution that creates a pH gradient ranging from a pH of about 3.5 to a final pH of about 9.3 to produce the a cationic exchange column eluate containing AAV particles.

[0017] In embodiments, the AAV particles present in the eluate from the cationic exchange column are further purified using anion exchange (AEX) chromatography conducted under conditions that use a salt gradient to elute the AAV particles from the anion exchange column in the anion exchange eluate (i.e, the fourth AAV particle composition). For example, anion exchange (AEX) chromatography may be conducted using an elution buffer solution that creates a salt gradient ranging from a concentration of about 0 mM NaCl to a final concentration of about 1 M NaCl, to elute the AAV particles and result in an anion exchange eluate containing AAV particles.

[0018] The preparation of drug substance may be accomplished by exchanging the AEX eluate buffer with a pharmaceutically acceptable buffer. Buffers may be exchanged using methods such as tangential flow filtration. Examples of pharmaceutically acceptable buffers include, but are not limited to buffers comprising 10 mM Tris, ImM MgCh, 150 mM NaCl, 0.02% poloxamer 188, at a pH of about 8.0 (the “Formulation Buffer” described below in Examples 1-3). Another example of a pharmaceutically acceptable buffer includes a buffer comprising 20 mM Tris, 150 mM NaCl, 0.02% poloxamer 188 at pH 8.0.

[0019] In embodiments, flocculating the cell lysate involves (i) combining the cell lysate with a solution comprising from about 300 mM to about 600 mM salt (for example, NaCl) and mixing for at least ten minutes, followed by (ii) the addition of a solution containing octanoic acid and mixing for at least ten minutes, followed by (iii) the addition of a solution containing formate and mixing for at least fifteen minutes, thereby forming an aqueous layer comprising full AAV capsids, and a flocculate, or “cream,” layer containing process impurities and product impurities.

[0020] In embodiments, flocculating the cell lysate involves (i) combining the cell lysate with a solution comprising from about 300 mM to about 600 mM salt (for example, NaCl) and mixing for at least ten minutes, followed by (ii) the addition of a solution containing octanoic acid and mixing for at least ten minutes, followed by (iii) the addition of a solution containing polyethylene glycol (PEG), followed by (iv) the addition of a solution containing formate and mixing for at least fifteen minutes, thereby forming an aqueous layer comprising full AAV capsids, and a flocculate, or “cream,” layer containing process impurities and product impurities. The polyethylene glycol (PEG) used in such embodiments may, for example, be 1500 MW or 6000 MW PEG, to a final concentration of up to 0.5%, 0.75%, 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0% or to any experimentally determined final PEG concentration that enhances flocculation. In one embodiment 6000 MW PEG is present in the flocculation mixture at a final concentration of 1%.

[0021] The cell lysate containing AAV particles that serves as the starting material for the methods described herein can be obtained using any known methods. For example, lysate can be obtained by methods comprising: a) culturing mammalian cells comprising nucleotide sequences comprising (1) a gene of interest (GOI) expression cassette comprising a transgene encoding a GOI operably linked to at least one regulatory sequence, flanked by AAV inverted terminal repeat (ITR) sequences; and (2) coding sequences for an AAV Rep, AAV Cap and adenovirus helper factors, under conditions that produce AAV particles comprising i) a nucleic acid, comprising the GOI expression cassette capable of expressing the transgene in a target cell upon transduction of the AAV particle, and ii) an AAV capsid; and b) lysing the mammalian cells to produce a first AAV particle composition that contains full AAV capsids, process impurities, including empty and partial AAV capsids, and product impurities. Cell lysis may be achieved by adding a nonionic surfactant such as, for example, polyoxyethylene (20) sorbitan monolaurate (Tween® 20).

[0022] In embodiments, the AAV capsid is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh74, AAVrhlO, or related engineered capsid. In some embodiments, the nucleic acid is self-complementary. In embodiments, the AAV particles are AAV9 particles or an AAV serotype where the capsid has an isoelectric point similar to AAV9. In other embodiments, the AAV particles are AAV8 particles or an AAV serotype where the capsid has an isoelectric point similar to AAV8. In other embodiments, the AAV particles are AAV6 particles or an AAV serotype where the capsid has an isoelectric point similar to AAV6. In other embodiments, the AAV particles are AAV5 particles or an AAV serotype where the capsid has an isoelectric point similar to AAV5. In some embodiments, the nucleic acid is single stranded. In some embodiments, at least about 80% of the recombinant AAV particles are full capsids relative to total AAV particles as assessed by AUC or CDMS.

[0023] Notably, the AAV purification methods described herein are an improvement over prior art methods that require a cesium ultracentrifugation, and allow for improved purification scalability and efficiency without sacrificing the quality and purity of the final AAV product. In embodiments, entire purification process can be completed in from about 24 to about 48 hours, and the resulting drug substance comprises AAV particles at a pharmaceutically relevant purity of least 80% full AAV capsids by AUC or CDMS, at least 90% AAV capsid purity by SDS-PAGE or CE-SDS, at least 70% transgene present in the genome in the AAV particle by short read NGS, and / or greater than about 75% DNA reads of the full expression cassette by long read NGS.EMBODIMENTS1. A process for manufacturing a composition of adeno-associated virus composition (AAV), said process comprising; a. providing a cell lysate comprising a first AAV particle composition comprising full AAV capsids, process impurities, including empty and partial AAV capsids, and product impurities, the cell lysate obtained from a culture of AAV producing mammalian cells and optionally clarifying the cell lysate; b. flocculating the optionally clarified cell lysate by sequentially adding to the optionally clarified cell lysate first salt, followed by octanoic acid, followed byaddition of formate and allowing the cell lysate to equilibrate with each addition prior to the next addition to form a flocculation mixture buffer; c. forming a separated flocculation mixture by mixing the flocculation mixture and allowing the flocculation mixture to stand for a time sufficient to form a cream layer at the surface of the flocculation mixture buffer; d. separating the flocculation mixture buffer layer below the cream layer and optionally combining it with cationic exchange buffer used to rinse the cream layer to obtain a rinse buffer layer separated from the rinsed cream layer and clarifying by filtration the flocculation mixture buffer optionally combined with the rinse buffer layer to form a second AAV particle composition; e. loading the second AAV particle composition onto a cationic exchange column to capture AAV particles present in material loaded onto the cationic exchange column and washing the cationic exchange column to remove impurities; f. eluting the AAV particles from the cationic exchange column either with a pH gradient or a salt gradient to form an AAV-containing cation exchange column eluate to produce a third AAV particle composition; g. if eluting AAV particles by using a salt gradient, reducing the salt concentration of the cation exchange column eluate obtained from eluting the AAV particles with a salt gradient by exchanging the eluate buffer with a low salt buffer using tangential flow filtration to provide an AAV containing anion exchange column loading buffer as the third AAV particle composition; h. loading an anion exchange column with the third AAV particle composition obtained from eluting AAV particles from the cationic exchange column with a pH gradient, or the AAV containing anion exchange column loading buffer of step g so as to capture the AAV particles present in material loaded onto the anion exchange column;i. washing the anion exchange column with anion exchange buffer to remove impurities; j . eluting the AAV particles from the anion exchange column with a salt gradient to produce an AAV particle-containing anion exchange column eluate as a fourth AAV particle composition; k. optionally reducing the conductivity of the AAV containing anion exchange column eluate and loading the resulting reduced conductivity eluate onto an anion exchange column, washing the column with anion exchange buffer and eluting the AAV particles from the anion exchange column with a salt gradient to produce an AAV particle containing eluate as a fourth AAV particle composition; l. exchanging the AAV containing anion exchange eluate from the fourth AAV particle composition from step j or k with a pharmaceutically acceptable buffer using one or more cycles of tangential flow fdtration to obtain a fifth AAV particle composition comprising purified AAV particles; m. optionally filtering the fifth AAV particle composition through a sterilizing filter to obtain a drug substance comprising purified AAV particles; wherein the process for manufacturing a composition of adeno-associated virus composition (AAV) set forth above does not comprise an ultracentrifugation step; and wherein the process for manufacturing a composition of adeno-associated virus composition (AAV) set forth above results in a drug substance comprising purified AAV particles present at a clinically relevant purity. The method of embodiment 1, wherein optionally clarifying the cell lysate in step (la) comprises clarifying the cell lysate by filtration.The method of embodiment 1, wherein step (lb) further comprises adding 6,000 MW polyethylene glycol (PEG) to a final concentration of about 1% subsequent to the addition of octanoic acid, and prior to the addition of formate.The method of any one of embodiments 1 to 3, wherein the AAV particle comprises an AAV9 capsid, or an AAV capsid with the same isoelectric point as AAV9. The method of any one of embodiments 1 to 3, wherein the AAV particle comprises an AAV8 capsid, or an AAV capsid with the same isoelectric point as AAV8. The method of any one of embodiments 1 to 3, wherein the AAV particle comprises an AAV6 capsid, or an AAV capsid with the same isoelectric point as AAV6. The method of any one of embodiments 1 to 3, wherein the AAV particle comprises an AAV5 capsid, or an AAV capsid with the same isoelectric point as AAV5. The method of any one of embodiments 1 to 3, wherein the AAV particle does not comprise an AAV2 capsid, or an AAV capsid with the same isoelectric point as AAV2. The method of any one of embodiments 1 to 3, wherein the AAV particle does not comprise an AAV capsid having an amino acid sequence that has been modified to have a different amino acid sequence than the amino acid sequence of a naturally occurring AAV capsid. The method according to any one of embodiments 1 to 9, wherein the AAV particle comprises a self-complementary AAV genome. The method of embodiment 10, wherein the ratio of dimer to monomer in the fourth AAV particle composition is about 10 to 1, about 20 to 1, or about 40 to 1. The method of any one of embodiments 1 to 9, wherein the AAV particle comprises a single- stranded AAV genome. The method of any one of embodiments 1 to 12, wherein cell lysate that comprises a first AAV particle composition is obtained by a) culturing mammalian cells comprising nucleotide sequences comprising (1) a gene of interest (GOI) expression cassette comprising a transgene encoding a GOI operably linked to at least one regulatory sequence, flanked by AAV invertedterminal repeat (ITR) sequences; and (2) coding sequences for an AAV Rep, AAV Cap and adenovirus helper factors; the mammalian cells cultured under conditions that produce AAV particles comprising a nucleic acid, comprising the GOI expression cassette capable of expressing the transgene in a target cell upon transduction of the target cell with the AAV particles, packaged within an AAV capsid comprising the nucleic acid; and b) obtaining a lysate by lysing the mammalian cells to produce a first AAV particle composition comprising full AAV capsids containing a gene of interest, process impurities and product impurities. The method of embodiment 13, wherein the mammalian cells cultured according to the method of embodiment 13 are produced by transfecting a population of mammalian cells by contacting the population of mammalian cells with a transfection composition comprising a plasmid encoding the GOI expression cassette flanked by AAV ITRs (pGOI), a plasmid encoding AAV Rep and AAV Cap (pRepCap), and a plasmid encoding adenoviral helper genes (pHelp) under conditions to achieve transfection of the cells with the pGOI plasmid, pRepCap plasmid and pHelp plasmid to obtain the mammalian cells. The method of embodiment 14, wherein the transfection composition further comprises a transfection reagent. The method of embodiment 15, wherein the transfection reagent is FectoVIR®, polycation polyethylenimine (PEI), PEIpro®, AAV-Max, TransIT-VirusGEN® or any other cationic lipid or lipofectamine based reagent. The method of any one of embodiments 13 to 16, wherein the mammalian cells are HEK293 cells. The method of any one of embodiments 13 to 17, wherein prior to step (a) the mammalian cells are cultured and the culture is expanded one or more times. The method of any one of embodiments 13 to 18, wherein the mammalian cells are grown in animal-free culture media.The method of embodiment 19, wherein the animal -free culture media is Expi293™ Expression Medium or Viral Production Medium™ or FreeStyle™ 293 Expression Medium. The method of any one of embodiments 13 to 20, wherein 0.5 to 2 volumes of animal- free culture media is added to the mammalian cells immediately prior to or during the step of transfecting the mammalian cells. The method of any one of embodiments 13 to 21, wherein the AAV Rep is AAV Rep2 and the AAV Cap is Cap9, or encodes an AAV capsid with the same isoelectric point as AAV9. The method of any one of embodiments 13 to 22, wherein step (b) comprises contacting the mammalian cells with non-ionic surfactant. The method of embodiment 23, wherein the non-ionic surfactant is polysorbate 20, and the mammalian cells are further contacted with MgCh. The method of any one of embodiments 1 to 24, wherein the cell lysate comprising a first AAV particle composition is digested with endonuclease prior to flocculating the first AAV particle composition. The method of embodiment 25, wherein the endonuclease is Serratia marcescens nuclease. The method of any one of embodiments 1 to 26, wherein the cell lysate comprising a first AAV particle composition is clarified by filtration prior to flocculating the first AAV composition, and flocculating the first AAV particle composition does not comprise contacting the cell lysate with a solution that contains polyethylene glycol (PEG). The method of embodiment 27, wherein clarifying the cell lysate comprising a first AAV particle composition by filtration results in about a two log reduction in mammalian cell DNA.The method of embodiments 27 or 28, wherein clarifying the cell lysate comprising a first AAV particle composition by filtration comprises the use of a glass fiber filter, PES filter, PVDF filter, cellulose filter or diatomaceous earth filter. The method of any one of embodiments 1 to 29, wherein flocculating the first AAV composition comprises contacting the first AAV particle composition with an anionic surfactant and acidifying the composition to a pH of 2.9 to 4.0. The method of any one of embodiments 1 to 30, wherein flocculating the first AAV composition removes at least 90% of mammalian cell protein. The method of any one of embodiments 1 to 31, wherein the cation exchange column comprises a cation exchange chromatography resin comprising poly(glycidyl methacrylate -co- ethylene dimethacrylate). The method of any one of embodiments 1 to 32, wherein the cation exchange column comprises a cation exchange chromatography resin comprising a sulfonate ligand. The method of any one of embodiments 1 to 33, wherein the cation exchange column has a column bed volume of 800 mL and the volume of the cell lysate comprising a first AAV particle composition is approximately 200 L-250 L, or the column bed volume is similarly proportional to the volume of the cell lysate comprising a first AAV particle composition. The method of any one of embodiments 1 to 34, wherein the cation exchange column has a column pore size of about 2 pm, about 4 pm, about 6 pm, or about 8 pm. The method of any one of embodiments 1 to 35, wherein the cation exchange column comprises a cation exchange chromatography resin having a pore size of about 2 pm. The method of any one of embodiments 1 to 35, wherein the cation exchange column comprises a cation exchange chromatography resin having a pore size of about 6 pm. The method of any one of embodiments 1 to 35 or 37, wherein the cation exchange column comprises a cation exchange chromatography resin comprising poly(glycidylmethacrylate -co- ethylene dimethacrylate), a sulfonate ligand, a bed size of 400 mL, and a 6 pm pore size. The method of any one of embodiments 1 to 38, wherein eluting the AAV particles from the cationic exchange column either with a pH gradient or a salt gradient to form an AAV-containing cation exchange column eluate to produce a third AAV particle composition comprises eluting at rate of from about 6 to about 10 column volumes per minute, or about 6 column volumes per minute, or about 7 column volumes per minute, or about 8 column volumes per minute, or about 9 column volumes per minute, or about 10 column volumes per minute. The method of any one of embodiments 1 to 37 and 39, wherein the anion exchange column has a column bed volume of 800 m and the volume of the cell lysate comprising a first AAV particle composition is approximately 200 L-250 L, or the anion exchange column has a column bed volume that is similarly proportional to the volume of the cell lysate comprising a first AAV particle composition. The method of any one of embodiments 1 to 40, wherein the anion exchange column comprises an anion exchange chromatography resin comprising regenerated macro- porous cellulose with primary amine ligand. The method embodiment 41, wherein the anion exchange column has column bed volume of 75 mL. The method of any one of embodiments 1 to 40, wherein the anion exchange column comprises a cation exchange chromatography resin comprising Poly(glycidyl methacrylate -co-ethylene dimethacrylate). The method of any one of embodiments 1 to 40 or 43, wherein the anion exchange column has a column bed volume of 400 mL. The method of any one of embodiments 1 to 44, wherein the anion exchange (AEX) chromatography resin has a pore size of about 2 pm.The method of any one of embodiments 1 to 44, wherein the AEX chromatography resin has a pore size of about 6 pm. The method of any one of embodiments 1 to 46, wherein the third AAV particle composition is subjected to tangential flow fdtration before it is loaded onto an anion exchange (AEX) chromatography column, or optionally the pH of the third AAV particle composition is adjusted so as to have a pH to allow for capture of AAV particles onto the AEC column before it is loaded onto the anion exchange (AEX) chromatography column. The method of any one of embodiments 1 to 47, wherein the proportion of full AAV capsids and / or partial AAV capsids and / or empty AAV capsids in the fifth AAV particle composition is assessed by AUC or charge detection mass spectroscopy (CDMS), and / or capsid purity is assessed by capillary electrophoresis or SDS-PAGE or ELISA for host cell protein, and / or PCR and / or short read NGS assays and / or long read NGS assays for plasmid and host cell DNA. The method of any of embodiments 1 to 48, wherein the fifth AAV particle composition is filtered through a sterilizing filter to obtain a drug substance comprising purified AAV particles. The method of embodiment 49, wherein the sterilizing filter is a 0.2 pm sterilizing filter. The method of embodiment 49, wherein the sterilizing filter is a 0.22 pm PES filter. The method of embodiment 49, wherein the sterilizing filter is a 0.22 pm PVDF filter. The method of any of embodiments 49 to 52, wherein the drug substance comprises at least 80% full AAV capsids by AUC or CDMS, at least 90% AAV capsid purity by SDS-PAGE or CE-SDS, at least 70% transgene present in the genome in the AAV particle by short read NGS and / or greater than about 75% DNA reads of the full expression cassette by long read NGS.The method of any of embodiments 1 to 53, wherein eluting the AAV particles from the cationic exchange column with a pH gradient comprises a pH gradient ranging from a starting pH of 3.5 to a final pH of 9.3 The method of embodiment 54, wherein the pH gradient comprises elution with a pH 3.5 buffer comprising 20 mM formate. The method of any of embodiments 54 to 55, wherein the pH gradient comprises elution with a pH 3.5 buffer comprising 20 mM formate, 2 M NaCl, and 0.2% poloxamer 188 (P188) at pH 3.5. The method of any of embodiments 1 to 53, wherein eluting the AAV particles from the cationic exchange column with a salt gradient comprises a salt gradient from a 500 mM salt concentration to a final 2 M salt concentration. The method of embodiment 57, wherein the salt is sodium chloride. The method of any of embodiments 1 to 53, wherein eluting the AAV particles from the anion exchange column with a salt gradient comprises a salt gradient from a 0 mM salt concentration to a final 1 M salt concentration. The method of embodiment 59, wherein the salt is sodium chloride. The method of any one of embodiments 13 to 60, wherein culturing mammalian cells comprises growing mammalian cells in a cell culture volume of 50 L, or the mammalian cell lysate is a volume of 50 L. The method of any one of embodiments 13 to 60, wherein culturing mammalian cells comprises growing mammalian cells in a cell culture volume of 215 L, or the mammalian cell lysate is a volume of 215 L. The method of any one of embodiments 13 to 60, wherein culturing mammalian cells comprises growing mammalian cells in a cell culture volume of 500 L, or the mammalian cell lysate is a volume of 500 L.The method of any one of embodiments 13 to 60, wherein culturing mammalian cells comprises growing mammalian cells in a cell culture volume of 1,000 L, or the mammalian cell lysate is a volume of 1,000 L. The method of any one of embodiments 1 to 64, wherein the first AAV particle composition comprises about 1 x 1016to about 5 x 1017AAV particles. The method of any of embodiments 1 to 65, wherein the drug substance comprises (1) at least about 85% full AAV capsids as assessed by analytical ultracentrifugation (AUC) or charge detection mass spectrometry (CDMS); (2) at least about 95% AAV capsid purity as assessed by SDS-PAGE or CE-SDS; (3) less than about 5% empty AAV capsids as assessed by AUC; (4) at least 90% transgene present in the genome in the AAV particle by short read NGS and / or (5) greater than about 80% DNA reads of the full expression cassette by long read NGS. The method of embodiment 66, wherein the drug substance comprises (1) at least about 90% full AAV capsids as assessed by analytical ultracentrifugation (AUC) or charge detection mass spectrometry (CDMS); (2) at least about 98% AAV capsid purity as assessed by SDS-PAGE or CE-SDS; (3) less than about 3% empty AAV capsids as assessed by AUC; and / or (4) at least 95% transgene present in the genome in the AAV particle by short read NGS and / or (5) greater than about 86% DNA reads of the full expression cassette by long read NGS. A drug substance produced according to the method of any of embodiments 1 to 67, wherein drug substance comprises AAV particles comprising (1) a nucleic acid comprising a GOI expression cassette and at least one AAV ITR sequence; and (2) an AAV capsid, wherein a) the recombinant AAV particles are present at a concentration of between about IxlO13and about IxlO14viral vector genomes / mL (vg / mL); b) at least about 80% of the recombinant AAV particles are full AAV capsids as assessed by AUC or CDMS;c) at least about 80% of DNA reads of the full expression cassette by long read NGS; d) the composition has at least about 90% capsid purity as assessed by SDS-PAGE or CE-SDS; e) less than about 2xl05pg residual host cell DNA per 1 x 1013vg is present; and f) less than about 15 ng host cell protein per 1 x 1013vg is present. The drug substance of embodiment 68, wherein less than about 2 ng host cell protein per 1 x 1013vg is present. The drug substance of embodiment 69, wherein less than about 1 ng host cell protein per 1 x 1013vg is present. The drug substance of any of embodiments 68 to 70, wherein the composition comprises at least about 90% full AAV capsids as assessed by analytical ultracentrifugation (AUC) or charge detection mass spectrometry (CDMS). The drug substance of embodiment 71 , wherein the composition comprises at least about 95% full AAV capsids as assessed by analytical ultracentrifugation (AUC) or charge detection mass spectrometry (CDMS). The drug substance of any of embodiments 68 to 72, wherein the composition comprises at least about 95% capsid purity as assessed by SDS-PAGE or CE-SDS. The drug substance embodiment 73, wherein the composition comprises at least about 98% capsid purity as assessed by SDS-PAGE or CE-SDS. The drug substance of any of embodiments 68 to 74, wherein the composition comprises less than about 5% empty AAV capsids as assessed by AUC. The drug substance of embodiment 75, wherein the composition comprises less than about 3% empty AAV capsids as assessed by AUC.The drug substance of any one of embodiments 68 to 76, wherein the composition comprises greater than about 83% DNA reads of the full expression cassette by long read NGS. The drug substance of embodiment 77, wherein the composition comprises greater than about 86% DNA reads of the full expression cassette by long read NGS. The drug substance of any of embodiments 68 to 78, wherein the AAV capsid comprises an AAV9 capsid, or an AAV capsid with the same isoelectric point as AAV9. The drug substance of any of embodiments 68 to 79, wherein the intact AAV capsid has an isoelectric point of about 7.2 to about 7.3, about 7.1 to about 7.4, or about 7.0 to about 7.5. The drug substance of any of embodiments 68 to 80, wherein the nucleic acid is single stranded. The drug substance of any of embodiments 68 to 80, wherein the nucleic acid is self- complementary. The drug substance of embodiment 82, wherein the AAV have a ratio of dimer to monomer of at least about 10 to 1. The drug substance of embodiment 82, wherein the AAV have a ratio of dimer to monomer of at least about 20 to 1. The drug substance of embodiment 82, wherein the AAV have a ratio of dimer to monomer of at least about 40 to 1. The drug substance of any of embodiments 68 to 85, wherein the drug substance contains 5 x 1015to 3 x 1017vg for an initial lysate of volume 215-L. The method of any of embodiments 1 to 86, wherein providing a cell lysate comprising a first AAV particle composition comprising full AAV capsids comprises lysing cellsin the presence of a non-ionic surfactant, optionally, Tween (20) in the presence of one or more nucleases. The method of any one of embodiments 1 to 4, wherein the AAV particle comprises anAAV9 capsid. The method of any one of embodiments 1 to 3 or 5, wherein the AAV particle comprises an AAV8 capsid. The method of any one of embodiments 1 to 3 or 6, wherein the AAV particle comprises an AAV6 capsid. The method of any one of embodiments 1 to 3 or 7, wherein the AAV particle comprises an AAV5 capsid. The method of any one of embodiments 1 to 3 or 8, wherein the AAV particle does not comprise an AAV2 capsid. The method of any one of embodiments 10 to 67, wherein the AAV particle comprises an AAV9 capsid. The method of any one of embodiments 10 to 67, wherein the AAV particle comprises an AAV8 capsid. The method of any one of embodiments 10 to 67, wherein the AAV particle comprises an AAV6 capsid. The method of any one of embodiments 10 to 67, wherein the AAV particle comprises an AAV5 capsid. The method of any one of embodiments 10 to 67, wherein the AAV particle does not comprise an AAV2 capsid. The method of any one of embodiments 10 to 67, wherein the AAV particle does not comprise an AAV capsid having an amino acid sequence that has been modified tohave a different amino acid sequence than the amino acid sequence of a naturally occurring AAV capsid. The drug substance of any one of embodiments 68 to 86, wherein the drug substance comprises AAV particles having an AAV9 capsid. . The drug substance of any one of embodiments 68 to 86, wherein the drug substance comprises AAV particles having an AAV8 capsid. . The drug substance of any one of embodiments 68 to 86, wherein the drug substance comprises AAV particles having an AAV6 capsid. . The drug substance of any one of embodiments 68 to 86, wherein the drug substance comprises AAV particles having an AAV5 capsid. . The drug substance of any one of embodiments 68 to 86, wherein the drug substance comprises AAV particles that do not have an AAV2 capsid. . The method of any one of embodiments 68 to 86, wherein the drug substance comprises AAV particles that do not comprise an AAV capsid having an amino acid sequence that has been modified to have a different amino acid sequence than the amino acid sequence of a naturally occurring AAV capsid. . The method of any one of embodiments 1 to 104, wherein the cell lysate is clarified, and flocculation according to step lb comprises the addition of octanoic acid. . A process for manufacturing a composition of adeno-associated virus composition (AAV), said process comprising; a. Obtaining a first AAV particle composition from a cell lysate from AAV producing mammalian host cells, said first AAV particle composition (i) being the direct product of upstream processing prior to any column processing step, and (ii) comprising less than about 30%, 20%, 10%, 5%, 1%, or 0. 1% of host cell DNA and / or host cell protein;b. loading the first AAV particle composition onto a cationic exchange column to capture AAV particles present in material loaded onto the cationic exchange column and washing the cationic exchange column to remove impurities; c. eluting the AAV particles from the cationic exchange column either with a pH gradient or a salt gradient to form an AAV containing cation exchange column eluate to produce a second AAV particle composition; d. if eluting AAV particles by using a salt gradient, reducing the salt concentration of the cation exchange column eluate obtained from eluting the AAV particles with a salt gradient by exchanging the eluate buffer with a low salt buffer using tangential flow filtration to provide an AAV containing anion exchange column loading buffer as the second AAV particle composition; e. loading an anion exchange column with the second AAV particle composition obtained from eluting AAV particles from the cationic exchange column with a pH gradient, or the AAV containing anion exchange column loading buffer of step g so as to capture the AAV particles present in material loaded onto the anion exchange column; f. washing the anion exchange column with anion exchange buffer to remove impurities; g. eluting the AAV particles from the anion exchange column with a salt gradient to produce an AAV particle containing anion exchange column eluate as a third AAV particle composition; h. optionally reducing the conductivity of the AAV containing anion exchange column eluate and loading the resulting reduced conductivity eluate onto an anion exchange column, washing the column with anion exchange buffer and eluting the AAV particles from the anion exchange column with a salt gradient to produce an AAV particle containing eluate as a third AAV particle composition;i. exchanging the AAV containing anion exchange eluate from the fourth AAV particle composition from step g or h with a pharmaceutically acceptable buffer using one or more cycles of tangential flow filtration to obtain a fourth AAV particle composition comprising purified AAV particles; j . optionally filtering the fourth AAV particle composition through a sterilizing filter to obtain a drug substance comprising purified AAV particles. wherein the process for manufacturing a composition of adeno-associated virus composition (AAV) set forth above does not comprise an ultracentrifugation step; and wherein the process for manufacturing a composition of adeno-associated virus composition (AAV) set forth above results in a drug substance comprising purified AAV particles present at a clinically relevant purity. The method of embodiment 106, wherein the AAV particle comprises an AAV9 capsid, or an AAV capsid with the same isoelectric point as AAV9. The method of embodiment 106, wherein the AAV particle comprises an AAV8 capsid, or an AAV capsid with the same isoelectric point as AAV8. The method embodiment 106, wherein the AAV particle comprises an AAV6 capsid, or an AAV capsid with the same isoelectric point as AAV6. The method of embodiment 106, wherein the AAV particle comprises an AAV5 capsid, or an AAV capsid with the same isoelectric point as AAV5. The method of embodiment 106, wherein the AAV particle does not comprise an AAV2 capsid, or an AAV capsid with the same isoelectric point as AAV2. The method of embodiment 106, wherein the AAV particle does not comprise an AAV capsid having an amino acid sequence that has been modified to have a different amino acid sequence than the amino acid sequence of a naturally occurring AAV capsid.The method according to any one of embodiments 107 to 112, wherein the AAV particle comprises a self-complementary AAV genome. The method of embodiment 113, wherein the ratio of dimer to monomer in the third AAV particle composition is about 10 to 1, about 20 to 1, or about 40 to 1. The method of any one of embodiments 106 to 112, wherein the AAV particle comprises a single-stranded AAV genome. The method of any one of embodiments 106 to 115, wherein obtaining a first AAV particle composition from AAV producing mammalian host cells comprises a) culturing mammalian cells comprising nucleotide sequences comprising (1) a gene of interest (GOI) expression cassette comprising a transgene encoding a GOI operably linked to at least one regulatory sequence, flanked by AAV inverted terminal repeat (ITR) sequences; and (2) coding sequences for an AAV Rep, AAV Cap and adenovirus helper factors; the mammalian cells cultured under conditions that produce AAV particles comprising a nucleic acid, comprising the GOI expression cassette capable of expressing the transgene in a target cell upon transduction of the target cell with the AAV particles, packaged within an AAV capsid comprising the nucleic acid; and b) obtaining a lysate by lysing the mammalian cells to produce a first AAV particle composition comprising full AAV capsids containing a gene of interest, process impurities and product impurities. The method of embodiment 116, wherein the mammalian cells cultured according to the method of embodiment 116 are produced by transfecting a population of mammalian cells by contacting the population of mammalian cells with a transfection composition comprising a plasmid encoding the GOI expression cassette flanked by AAV ITRs (pGOI), a plasmid encoding AAV Rep and AAV Cap (pRepCap), and a plasmid encoding adenoviral helper genes (pHelp) under conditions to achieve transfection of the cells with the pGOI plasmid, pRepCap plasmid and pHelp plasmid to obtain the mammalian cells.The method of embodiment 117, wherein the transfection composition further comprises a transfection reagent. The method of embodiment 118, wherein the transfection reagent is FectoVIR®, polycation polyethylenimine (PEI), PEIpro®, AAV-Max, TransIT-VirusGEN® or any other cationic lipid or lipofectamine based reagent. The method of any one of embodiments 116 to 119, wherein the mammalian cells are HEK293 cells. The method of any one of embodiments 116 to 120, wherein prior to step (a) the mammalian cells are cultured and the culture is expanded one or more times. The method of any one of embodiments 116 to 121, wherein the mammalian cells are grown in animal-free culture media. The method of embodiment 122, wherein the animal-free culture media is Expi293™ Expression Medium or Viral Production Medium™ or FreeStyle™ 293 Expression Medium. The method of any one of embodiments 116 to 123, wherein 0.5 to 2 volumes of animal-free culture media is added to the mammalian cells immediately prior to or during the step of transfecting the mammalian cells. The method of any one of embodiments 116 to 124, wherein the AAV Rep is AAV Rep2 and the AAV Cap is Cap9, or encodes an AAV capsid with the same isoelectric point as AAV9. The method of any one of embodiments 116 to 125, wherein step (b) comprises contacting the mammalian cells with non-ionic surfactant. The method of embodiment 126, wherein the non-ionic surfactant is polysorbate 20, and the mammalian cells are further contacted with MgCh.The method of any one of embodiments 106 to 127, wherein the cation exchange column comprises a cation exchange chromatography resin comprising poly(glycidyl methacrylate -co- ethylene dimethacrylate). The method of any one of embodiments 106 to 128, wherein the cation exchange column comprises a cation exchange chromatography resin comprising a sulfonate ligand. The method of any one of embodiments 106 to 129, wherein the cation exchange column has a column bed volume of 800 mb and the volume of the cell lysate comprising a first AAV particle composition is approximately 200 L-250 L, or wherein the cation exchange column has a column bed volume that is similarly to the volume of the cell lysate comprising a first AAV particle composition. The method of any one of embodiments 106 to 130, wherein the cation exchange column has a column pore size of about 2 pm, about 4 pm, about 6 pm, or about 8 pm. The method of any one of embodiments 106 to 131, wherein the cation exchange column comprises a cation exchange chromatography resin having a pore size of about 2 pm. The method of any one of embodiments 106 to 131, wherein the cation exchange column comprises a cation exchange chromatography resin having a pore size of about 6 pm. The method of any one of embodiments 106 to 131 or 133, wherein the cation exchange column comprises a cation exchange chromatography resin comprising poly(glycidyl methacrylate -co- ethylene dimethacrylate), a sulfonate ligand, a bed size of 400 mL, and a 6 pm pore size. The method of any one of embodiments 106 to 134, wherein eluting the AAV particles from the cationic exchange column either with a pH gradient or a salt gradient to form an AAV-containing cation exchange column eluate to produce a third AAV particle composition comprises eluting at rate of from about 6 to about 10 column volumes perminute, or about 6 column volumes per minute, or about 7 column volumes per minute, or about 8 column volumes per minute, or about 9 column volumes per minute, or about 10 column volumes per minute. The method of any one of embodiments 106 to 133 and 135, wherein the anion exchange column has a column bed volume of 800 mL and the volume of the cell lysate comprising a first AAV particle composition is approximately 200 L-250 L, or the anion exchange column has a column bed volume that is similarly proportional to the volume of the cell lysate comprising a first AAV particle composition. The method of any one of embodiments 106 to 136, wherein the anion exchange column comprises a cation exchange chromatography resin comprising regenerated macro-porous cellulose with primary amine ligand. The method of any one of embodiments 106 to 137, wherein the anion exchange column has a column bed volume of 75 mL. The method of any one of embodiments 106 to 136, wherein the anion exchange column comprises an anion exchange chromatography resin comprising Poly(glycidyl methacrylate -co- ethylene di meth acrylate). The method of any one of embodiments 106 to 136 or 139, wherein the anion exchange column has a column bed volume of 400 mL. The method of any one of embodiments 106 to 140, wherein the anion exchange (AEX) chromatography resin has a pore size of about 2 pm. The method of any one of embodiments 106-140, wherein the AEX chromatography resin has a pore size of about 6 pm. The method of any one of embodiments 106 to 142, wherein the third AAV particle composition is subjected to tangential flow filtration before it is loaded onto an anion exchange (AEX) chromatography column, or optionally the pH of the third AAV particle composition is adjusted so as to have a pH to allow for capture of AAV particlesonto the AEC column before it is loaded onto the anion exchange (AEX) chromatography column. The method of any one of embodiments 106 to 143, wherein the proportion of full AAV capsids and / or partial AAV capsids and / or empty AAV capsids in the fifth AAV particle composition is assessed by AUC or charge detection mass spectroscopy (CDMS), and / or capsid purity is assessed by capillary electrophoresis or SDS-PAGE or ELISA for host cell protein, and / or PCR and / or short read NGS assays and / or long read NGS assays for plasmid and host cell DNA. The method of any of embodiments 106 to 144, wherein the fourth AAV particle composition is filtered through a sterilizing filter to obtain a drug substance comprising purified AAV particles. The method of embodiment 145, wherein the sterilizing filter is a 0.2 pm sterilizing filter. The method of embodiment 145, wherein the sterilizing filter is a 0.22 pm PES filter. The method of embodiment 145, wherein the sterilizing filter is a 0.22 pm PVDF filter. The method of any of embodiments 145 to 148, wherein the drug substance comprises at least 80% full AAV capsids by AUC or CDMS, at least 90% AAV capsid purity by SDS-PAGE or CE-SDS, at least 70% transgene present in the genome in the AAV particle by short read NGS and / or greater than about 75% DNA reads of the full expression cassette by long read NGS. The method of any of embodiments 106 to 149, wherein eluting the AAV particles from the cationic exchange column with a pH gradient comprises a pH gradient ranging from a starting pH of 3.5 to a final pH of 9.3 The method of embodiment 150, wherein the pH gradient comprises elution with a pH 3.5 buffer comprising 20 mM formate.The method of any of embodiments 150 to 151, wherein the pH gradient comprises elution with a pH 3.5 buffer comprising 20 mM formate, 2 M NaCl, and 0.2% poloxamer 188 (P188) at pH 3.5. The method of any of embodiments 106 to 149, wherein eluting the AAV particles from the cationic exchange column with a salt gradient comprises a salt gradient from a 500 mM salt concentration to a final 2 M salt concentration. The method of embodiment 153, wherein the salt is sodium chloride. The method of any of embodiments 106 to 149, wherein eluting the AAV particles from the anion exchange column with a salt gradient comprises a salt gradient from a 0 mM salt concentration to a final 1 M salt concentration. The method of embodiment 155, wherein the salt is sodium chloride. The method of any one of embodiments 116 to 156, wherein culturing mammalian cells comprises growing mammalian cells in a cell culture volume of 50 L, or the mammalian cell lysate is a volume of 50 L. The method of any one of embodiments 116 to 156, wherein culturing mammalian cells comprises growing mammalian cells in a cell culture volume of 215 L, or the mammalian cell lysate is a volume of 215 L. The method of any one of embodiments 116 to 156, wherein culturing mammalian cells comprises growing mammalian cells in a cell culture volume of 500 L, or the mammalian cell lysate is a volume of 500 L. The method of any one of embodiments 116 to 156, wherein culturing mammalian cells comprises growing mammalian cells in a cell culture volume of 1,000 L, or the mammalian cell lysate is a volume of 1,000 L. The method of any one of embodiments 106 to 160, wherein the first AAV particle composition comprises about 1 x 1016to about 5 x 1017AAV particles.The method of any of embodiments 106 to 161, wherein the drug substance comprises (1) at least about 85% full AAV capsids as assessed by analytical ultracentrifugation (AUC) or charge detection mass spectrometry (CDMS); (2) at least about 95% AAV capsid purity as assessed by SDS-PAGE or CE-SDS; (3) less than about 5% empty AAV capsids as assessed by AUC; (4) at least 90% transgene present in the genome in the AAV particle by short read NGS and / or (5) greater than about 80% DNA reads of the full expression cassette by long read NGS. The method of embodiment 162, wherein the drug substance comprises (1) at least about 90% full AAV capsids as assessed by analytical ultracentrifugation (AUC) or charge detection mass spectrometry (CDMS); (2) at least about 98% AAV capsid purity as assessed by SDS-PAGE or CE-SDS; (3) less than about 3% empty AAV capsids as assessed by AUC; and / or (4) at least 95% transgene present in the genome in the AAV particle by short read NGS and / or (5) greater than about 86% DNA reads of the full expression cassette by long read NGS. A drug substance produced according to the method of any of embodiments 106 to 163, wherein drug substance comprises AAV particles comprising (1) a nucleic acid comprising a GOI expression cassette and at least one AAV ITR sequence; and (2) an AAV capsid, wherein a) the recombinant AAV particles are present at a concentration of between about IxlO13and about IxlO14viral vector genomes / mL (vg / mL); b) at least about 80% of the recombinant AAV particles are full AAV capsids as assessed by AUC or CDMS; c) at least about 80% of DNA reads of the full expression cassette by long read NGS; d) the composition has at least about 90% capsid purity as assessed by SDS-PAGE or CE-SDS; e) less than about 2xl05pg residual host cell DNA per I x lO13vg is present; and f) less than about 15 ng host cell protein per I x lO13vg is present.The drug substance of embodiment 164, wherein less than about 2 ng host cell protein per 1 x 1013vg is present. The drug substance of embodiment 165, wherein less than about 1 ng host cell protein per 1 x 1013vg is present. The drug substance of any of embodiments 164 to 166, wherein the composition comprises at least about 90% full AAV capsids as assessed by analytical ultracentrifugation (AUC) or charge detection mass spectrometry (CDMS). The drug substance of embodiment 167, wherein the composition comprises at least about 95% full AAV capsids as assessed by analytical ultracentrifugation (AUC) or charge detection mass spectrometry (CDMS). The drug substance of any of embodiments 164 to 168, wherein the composition comprises at least about 95% capsid purity as assessed by SDS-PAGE or CE-SDS. The drug substance embodiment 169, wherein the composition comprises at least about 98% capsid purity as assessed by SDS-PAGE or CE-SDS. The drug substance of any of embodiments 164 to 170, wherein the composition comprises less than about 5% empty AAV capsids as assessed by AUC. The drug substance of embodiment 171, wherein the composition comprises less than about 3% empty AAV capsids as assessed by AUC. The drug substance of any one of embodiments 164 to 172, wherein the composition comprises greater than about 83% DNA reads of the full expression cassette by long read NGS. The drug substance of embodiment 173, wherein the composition comprises greater than about 86% DNA reads of the full expression cassette by long read NGS.The drug substance of any of embodiments 164 to 174, wherein the AAV capsid comprises an AAV9 capsid, or an AAV capsid with the same isoelectric point as AAV9. The drug substance of any of embodiments 164 to 175, wherein the intact AAV capsid has an isoelectric point of about 7.2 to about 7.3, about 7.1 to about 7.4, or about 7.0 to about 7.5. The drug substance of any of embodiments 164 to 176, wherein the nucleic acid is single stranded. The drug substance of any of embodiments 164 to 176, wherein the nucleic acid is self- complementary. The drug substance of embodiment 178, wherein the AAV have a ratio of dimer to monomer of at least about 10 to 1. The drug substance of embodiment 178, wherein the AAV have a ratio of dimer to monomer of at least about 20 to 1. The drug substance of embodiment 178, wherein the AAV have a ratio of dimer to monomer of at least about 40 to 1. The drug substance of any of embodiments 164 to 181, wherein the first AAV particle composition contains 5 x IO13to 3 x 1017vg in an initial volume 215-L. The method of any of embodiments 106 to 182, wherein providing a first AAV particle composition comprises lysing AAV producing mammalian host cells in the presence of a non-ionic surfactant, optionally, Tween (20) in the presence of one or more nucleases. The method of any one of embodiments 106 to 107, wherein the AAV particle comprises an AAV9 capsid.The method of any one of embodiments 106 or 108, wherein the AAV particle comprises an AAV8 capsid. The method of any one of embodiments 106 or 109, wherein the AAV particle comprises an AAV6 capsid. The method of any one of embodiments 106 or 110, wherein the AAV particle comprises an AAV5 capsid. The method of any one of embodiments 106 or 111, wherein the AAV particle does not comprise an AAV2 capsid. The method of any one of embodiments 113 to 163, wherein the AAV particle comprises an AAV9 capsid. The method of any one of embodiments 113 to 163, wherein the AAV particle comprises an AAV8 capsid. The method of any one of embodiments 113 to 163, wherein the AAV particle comprises an AAV6 capsid. The method of any one of embodiments 113 to 163, wherein the AAV particle comprises an AAV5 capsid. The method of any one of embodiments 113 to 163, wherein the AAV particle does not comprise an AAV2 capsid. The method of any one of embodiments 113 to 163, wherein the AAV particle does not comprise an AAV capsid having an amino acid sequence that has been modified to have a different amino acid sequence than the amino acid sequence of a naturally occurring AAV capsid. The drug substance of any one of embodiments 164 to 182, wherein the drug substance comprises AAV particles having an AAV9 capsid.196. The drug substance of any one of embodiments 164 to 182, wherein the drug substance comprises AAV particles having an AAV8 capsid.197. The drug substance of any one of embodiments 164 to 182, wherein the drug substance comprises AAV particles having an AAV6 capsid.198. The drug substance of any one of embodiments 164 to 182, wherein the drug substance comprises AAV particles having an AAV5 capsid.199. The drug substance of any one of embodiments 164 to 182, wherein the drug substance comprises AAV particles that do not have an AAV2 capsid.200. The method of any one of embodiments 164 to 182, wherein the drug substance comprises AAV particles that do not comprise an AAV capsid having an amino acid sequence that has been modified to have a different amino acid sequence than the amino acid sequence of a naturally occurring AAV capsid.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 shows the results of size exclusion-high performance liquid chromatography (SE-HPLC) conducted on AAV9 capsids containing a GFP reporter construct prepared as set forth in Examples 1-3, in comparison to AAV 9 capsids containing the same GFP reporter construct prepared according to the UC method described in Example 4. The results of this experiment are described in Example 8.DETAILED DESCRIPTION

[0025] Provided herein are methods of manufacturing functional, high-purity AAV capsids for uses including gene therapy. The methods described herein comprise obtaining sufficiently pure starting preparations or processed lysates (for example, by flocculation or other method known in the art or described herein resulting in, for example, less than 20%, 15%, 10%, 5% or 1% host cell protein) of AAV particles to allow for efficient, large-scale purification using cation exchange (CEX) and anion exchange (AEX) chromatography columns, without the need for cesium gradient ultracentrifugation or similar gradient ultracentrifugation methods. The methods described herein also facilitate the use of CEX and AEX chromatography columns of sufficient pore size and flowrate (column volumes per minute) to obtain highly purified AAV particles in an efficient time frame (for example, around 24 to around 48 hours of total manufacturing time). The methods described herein are suitable for large-scale manufacture of AAV particle compositions with starting lysate volumes of from about 2L to about lOOOL, and are capable of achieving AAV drug substance with process and product impurity levels and yield comparable that which can be achieved by cesium gradient ultracentrifugation or similar gradient ultracentrifugation methods.Definitions

[0026] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. For example, “comprising an A, a B, or a C” contemplates and supports embodiments comprising two or more A, two or more B, and two or more C.

[0027] Unless defined otherwise, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments pertain. The preferred materials and methods are described, but it is understood that any methods and materials similar or equivalent to those described can be used in the practice of embodiments. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. In describing and claiming the present invention, the following terminology will be used.

[0028] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the embodiments.

[0029] The term “AAV capsid” refers to the AAV protein shell, i.e., a capsid. It may or may not encapsidate a nucleic acid.

[0030] The terms “AAV particle” or “AAV virion” can be used interchangeably and refer to an infectious non-repl icative virus having an AAV protein shell, i.e., a capsid, encapsidating a nucleic acid. In some embodiments, the AAV particle is active for gene therapy, i.e., upon transduction of an appropriate target cell, a transgene (GOI) in the recombinant genome of the AAV particle is expressed in the target cell (i.e. a recombinant AAV (rAAV)). A “recombinantAAV” (or “AAV”) refers to AAV particles in which a coding sequence for a transgene (GOI) replaces the cap and rep genes in the AAV genome. Expression of the transgene can be measured by either protein expression or a functional assay, for example, as described herein.

[0031] The terms “AAV particle composition” refers to a composition comprising AAV particles and, optionally, other molecules, including contaminants from the manufacturing process, such as pDNA, hcDNA, and HCP. In some embodiments, the term AAV particle composition comprises adjuvants and / or excipients for administration to subjects and / or storage of the AAV particle composition.

[0032] The terms “full AAV capsid” or “full capsid” can be used interchangeably and refer to a recombinant AAV particle having nucleic acid of sufficient size such that the capsids are detected as full by analytical ultracentrifugation (AUC) or charge detection mass spectrometry (CDMS) (approximately 4.5 kb of single stranded DNA) as described herein and, including in embodiments, the nucleic acid required to express the GOI in an expression cassette upon transduction into target cell capable of expressing the GOI in the context of the expression cassette (i.e., the operably linked regulatory elements promote expression of the GOI in the target cell), for example, having sufficient ITR sequences flanking a GOI expression cassette such that the GOI is expressed in a target cell upon transduction. In the case of AAV particles having scDNA, AAV particles having 1 copy of the rAAV nucleic acid which expresses the transgene upon transduction of a target cell (“monomers”) and AAV particles having 2 copies of the rAAV nucleic acid which expresses the transgene upon transduction of a target cell (“dimers”) are both considered to be a full AAV capsid.

[0033] The proportion or percentage of full capsids relative to total capsids (rAAV particles) in a composition can be measured or assessed by AUC or CDMS as described herein. Alternatively, “full capsids” can also be assessed by next generation sequencing methods to determine the identity of the nucleic acid present in the AAV capsid, for example, to determine if the encapsi dated nucleic acid includes the GOI expression cassette flanked by ITR sequences. The ITR sequences may be the full length ITR sequence on both the 3’ or 5’ sides or one of the 3’ or 5’ side of the GOI expression cassette or sufficient length of the ITR sequence such that the GOI is expressed in a target cell upon transduction into the cell under conditions appropriate for GOI expression.

[0034] The term “functional AAV capsid” or “functional capsid” refers to a recombinant AAV capsid or recombinant AAV particle comprising a nucleic acid comprising a GOI expression cassette and ITR sequences wherein the nucleic acid is sufficient to express the GOI upon transduction of a target cell. Functional AAV capsids can be assessed by assays for expression and / or function of the GOI as described herein. In embodiments, whether a capsid is a “functional AAV capsid” or “functional capsid” may be assessed by long read next generation sequencing (“long read NGS”) to determine if the encapsidated nucleic acid comprises a GOI expression cassette and ITR sequences required for transgene expression upon transduction into a target cell, for example, if the nucleic acid comprises the GOI flanked by full length ITR sequences.

[0035] The term “partial capsid” refers to a capsid comprising some nucleic acid, but less than the full GOI expression cassette and ITR sequences required for transgene expression upon transduction into a target cell. Partial capsids may contain nucleic acid contaminants, such as DNA from the transfection plasmids or nucleic acid from the host cell, instead of GOI expression cassette and / or ITR sequences. The term “empty capsid” refers to capsid that does not encapsidate detectable nucleic acid.

[0036] As used herein, the term “cell line” refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. Spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants.

[0037] The term animal-free culture media refers to a composition capable of supporting the growth of a cell culture or cell line and lacking animal-derived products, such as bovine serum albumin (“BSA”). In some embodiments, animal-free culture media is Expi293™ Expression Medium by Gibco™ (https: / / www.thermofisher.com / order / catalog / product / A1435101). In some embodiments, animal-free culture media is Viral Production Medium™ by Gibco™ (https: / / www.thermofisher.com / order / catalog / product / A4817903) In some embodiments, animal- free culture media is FreeStyle™ 293 Expression Medium by Gibco™ (https: / / www.thermofisher.com / order / catalog / product / 12338018). In some embodiments, animal- free culture media is CD 293, or EX-CELL® 420 Serum-Free Medium for Insect Cells by Sigma-Aldrich® (https: / / www.sigmaaldrich.com / US / en / product / sigma / 14420c?srsltid=AfmBOoqab QoKNpD M5K-Jlu3a-XEutpfae-GZlb iNTjoxxOPhp4I9w).

[0038] The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by a promoter to produce a mRNA or other RNA (e.g., siRNA, miRNA) transcription product and / or a protein encoded by the nucleotide sequence.

[0039] As used herein, the term “host cell” refers to any cell that harbors, or is capable of harboring, a substance of interest. Often a host cell can be an animal or mammalian cell (e.g., a non-human primate, rodent, or human cell). In some aspects, the host cell can be a mammalian cell, a yeast cell, a bacterial cell, an insect cell, a plant cell, or a fungal cell. A host cell can be used as a recipient of an AAV helper construct, an AAV plasmid encoding a recombinant AAV genome comprising a transgene, an accessory function vector, or other transfer DNA associated with the production of recombinant AAV (rAAV) particles. The term includes the progeny of the original cell which has been transfected. Thus, a “host cell” as used herein can refer to a cell which has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.

[0040] The term “mammalian cell” refers to cells or a cell line derived from a mammal. Mammalian cells include, for example, HEK293 cells (“293 cells”). 293 cells are readily available through, e.g., the American Type Culture Collection under Accession Number ATCC CRL1573. 293 cells may either be adherent (293 A) or in suspension. See, e.g., USPN 7,445,930. Commercially available 293 cell lines for the production of AAV include AAVpro® 293T cell line by Takara Bio (https: / / www.takarabio.com / products / gene-function / viral-transduction / adeno- associated-virus-(aav) / packaging-systems-and-cells / 293t-cell- line?srsltid=AfmBOoqWEyFp04GeUKEp5og-oxKEP4ZK8Nj683vA8bV678CM23BYmBqb). Examples of additional mammalian cells lines include, but are not limited to, BHK cells, CHO cells, COS cells and HeLa cells.

[0041] The term “insect cell” refers to cells or a cell line derived from an insect. Insect cells lines include, but are not limited to Sf9 cells.

[0042] “Identity” as used herein refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules, or two nucleic acid molecules, such as polynucleotides. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g. , if half (e.g. , five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical. In the case of an insertion or deletion, identity is understood to realign those thereafter which would be identical and is considered to be not identical at the insertion or deletion.

[0043] By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid level to the sequence used for comparison.

[0044] A “nucleic acid,” as used herein, is interchangeable with “polynucleotide” or “a specific sequence of nucleotide or “nucleotide sequences.” These terms refer to a discrete sequence that performs a specific function directly or indirectly in a cell. That function includes encoding a sequence of a gene that is transcribed into mRNA and translated into protein and regulating said transcription (i.e., as a promoter would) and / or translation (i.e., as microRNA would). A nucleic acid inherently has a sequence. In recombinant molecular biology, discrete nucleic acids can be combined. In some embodiments, a nucleic acid that encodes a protein can be ligated to a promoter (which is a nucleic acid), and a cis-acting element of a viral vector (i.e., an inverted-terminal repeat (ITR), which is also a nucleic acid). For convenience, a “nucleic acid” might be used to refer to the discrete elements within the larger nucleic acid, which could be referred to as “a polynucleotide,” “an expression cassette” (i.e., a polynucleotide comprising a promoter and a nucleic acid that encodes a protein), or “a vector” (see definition below).

[0045] “Encoding” refers to the inherent property of a nucleic acid to serve as a template, whether directly (i.e., a sense strand) or indirectly (i.e., an antisense strand) for synthesis of peptide, polypeptides, proteins, or other nucleic acids (i.e., rRNA, tRNA, microRNA). A nucleic acid can “encode” whether it is the sense strand, antisense strand, or a double-stranded segment thereof. The sense strand directly encodes the rRNA, tRNA, microRNA, or mRNA. The mRNA then serves as the template for translation of a peptide, polypeptide, or protein. The anti-sense strand is generally considered to be the reverse complementary sequence and is sometimes called a “noncoding” strand in the art (although for present purposes “non-coding” is a misnomer because the non-coding strand still “encodes” the genetic information by perpetuating it during semiconservative replication by acting as a template for the polymerization of a new, sense strand). Within semi-conservative replication two single strands in double-stranded nucleic acids are separated, and a new strand is polymerized from the information from each of the single- stranded nucleic acids (i.e., single-stranded template), regardless of whether one single-stranded template is the sense strand (e.g., that which is used to transcribe mRNA and thereby, or directly, encode the translate or protein) or the antisense strand. By perpetuating the genetic information, the antisense strand is still encoding the genetic information for, for example, a protein. Accordingly, “a nucleic acid encoding X”, includes sense and antisense sequences or strands whether X is a peptide, a polypeptide, or a protein or X is a sequence that encodes a rRNA, tRNA, microRNA, antisense RNA, etc.

[0046] Further to which, “nucleic acid encoding X,” includes RNA, DNA, and combinations thereof, since nucleic acids are synthesized from transcription, reverse-transcription, and replication, as naturally occurring processes and man-made processes (recombinant biology, molecular biology, etc.).

[0047] With regard to an AAV particle or an AAV virion, the above-noted incorporation of reverse complementary sequences and double-stranded segments into the definition of “a nucleic acid” and the above-noted use of “encoding” as including sense and antisense strands, is intended to incorporate the means by which the AAV vector can introduce an exogenous nucleic acid sequence that encodes nucleic acid or a protein into the cell. It is further intended to incorporate, in some embodiments, processes whereby said introduction results in the expression of said nucleic acid (i.e. miRNA or antisense RNA) or protein.

[0048] Take for example, a nucleic acid encoding a protein, and an AAV vector comprising a nucleic acid encoding said protein. When a typical (i.e. naturally occurring) AAV vector encoding one sense or one antisense strand of the nucleic acid that encodes said protein enters the cell, the inverted-terminal repeats (ITRs) prime the synthesis of a sequence reverse complementary to the sense strand or antisense strand of the nucleic acid that encodes said protein. The polymerization thereby forms a segment of double-stranded DNA comprising the sense and antisense strands, regardless of whether the sense version or antisense version was first introduced to the cell. In this regard, the entire nucleic acid including ITRs and sense and antisense nucleic acids encoding a protein can be one single-stranded DNA, which loops upon itself to form a double-stranded segment, wherein the base-pairs the sense and antisense nucleic acids encoding the protein align.

[0049] From this segment of double-stranded DNA, transcription of mRNA and translation of said protein is achieved from said sense strand of DNA, regardless of whether the AAV vector comprised only the sense strand or only the antisense strand when first entering the cell. In this regard, “an AAV vector comprising a nucleic acid encoding protein X” includes, contemplates, and supports embodiments in which the nucleic acid is the sense strand encoding protein X, the antisense strand encoding protein X, a double-stranded nucleic acid encoding protein X, and a single stranded nucleic acid comprising sense and antisense strands wherein the sense and antisense strands form a segment of double-stranded nucleic acid.

[0050] The term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.

[0051] The terms “final formulation,” and “drug product” as used herein can be used interchangeably and include compositions suitable for administration to mammals, e.g., humans. When the AAV particles of the present disclosure are administered as pharmaceuticals to mammals, e.g., humans, they can be given per se or as a liquid pharmaceutical composition containing, for example, from about 0.005% to about 5% wt / volume of active ingredient in the composition, including about 0.005 to about 1% wt / vol, about 0.005 to about 0.5% wt / vol, about0.01 to about 0.75% wt / vol, or about 0.1 to about 0.5% wt / vol, in combination with a pharmaceutically acceptable carrier. In embodiments, the pharmaceutical composition is a lyophilized composition containing, for example, about 0.1% to about 20% wt / wt, about 0.1% to about 10%, about 0.1% to about 5% or about 1% to about 5% in combination with a pharmaceutically acceptable carrier. The lyophilized composition can be reconstituted prior to administration. The term “drug substance” as used herein refers to a purified rAAV composition that needs only to be filtered and / or have added to it pharmaceutically acceptable excipients, diluents or carriers, to make the final formulation. “Pharmaceutical composition” refers to a composition comprising a drug product that is appropriate for administration to subjects, including human subjects in that excipients or contaminants are physiologically tolerable, on balance, do not detract from the therapeutic activity of the drug product or cause unduly adverse effects.

[0052] The term “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, that would cause a severe adverse reaction or side effect, when administered to a human, depending upon the active ingredient and indication to be treated. In embodiments, as used herein, the term “pharmaceutically acceptable” means approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0053] The terms “promoter” and “regulatory sequence” as used herein, can be used interchangeably and are defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. In some instances, this sequence may be the core promoter and in other instances, this sequence may also include, or be an enhancer alone and / or other regulatory elements which are required for expression of the gene product.

[0054] In certain instances the promoter may comprise enhancer elements, exons, and introns from one or a variety of viruses and animals, and thereby the term “promoter” shall be understood to not be limited to being a non-expressed sequence, nor exclude a non-expressed sequence that is between expressed sequences (i.e. introns), nor be limited to exclude an enhancer alone so long as the combination of sequences used to construct the promoter are capable of initiating the specific transcription of a polynucleotide sequence. Other regulatory elements include polyadenylationsignals and post-transcriptional regulatory elements, such as, for example, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0055] A “target gene” or “gene of interest” (“GOI”) refers to a nucleic acid encoding a target protein to be expressed within a target cell upon entry of the vector carrying the target gene into the cell under conditions such that the operably linked regulatory sequences promote expression of the GOI in the target cell.

[0056] A “vector” is a nucleic acid capable of delivering a target gene to the interior of a cell, and includes not only the expression-region (i.e. a promoter and a nucleic acid encoding a protein or even a nucleic acid), but also some cis-acting genetic component. The cis-acting genetic component provides for packaging within a virion, expression in a cell, replication in a cell, or a combination thereof.

[0057] By way of example, inverted-terminal repeats (ITRs) from adeno-associated viruses (AAVs) constitute a vector when adjoined to the nucleic acid encoding a target protein because the ITRs will provide for the nucleic acid encoding the target protein to be packaged within an AAV virion. ITRs also provide other cis-acting functions for expression of the nucleic acid encoding the target protein in the host cell upon entry of the vector into the host cell. Such cis- acting functions of ITRs include aiding in concatemer formation for genomic insertion; initiation of second strand formation in the case of a single-stranded (ss) AAV (ssAAV) vector; or initiation of replication and transcription in the case of ssAAV and self-complementary (sc) AAV (scAAV) vectors. In this regard, the AAV ITRs can be characterized based on the nucleic acid sequences providing such cis-acting functions from the serotypes of AAVs. That is, an ITR isolated from an AAV2 serotype can be known as an AAV2 ITR, even though the ITR generally does not contribute to the serotype of an AAV.

[0058] “Expression cassette” refers to a nucleic acid comprising a GOI operably linked to regulatory sequences sufficient for expression of the GOI in a target cell. An expression cassette includes a recombinant polynucleotide comprising a nucleic acid that controls expression (j.e. a promoter) and a nucleic acid that encodes the GOI. The GOI includes a nucleic acid that encodes a protein. Generally, the promoter is operatively linked to the nucleic acid that encodes the target protein (or other expression product, such as, but not limited to an siRNA or microRNA) in a manner that is capable of promoting expression of the protein upon entry of the vector into thetarget cell. The Expression Cassette may also include other regulatory elements such as polyadenylation (polyA) signal sequences and other expression elements such as enhancers, introns and post-transcriptional regulatory elements that promote or otherwise increase or decrease expression of the GOI in target cells.

[0059] The terms “separate” and “separation” as used in the present disclosure mean that at least some of a first component is removed from a composition containing at least the first component and a second component. For example, separation of components in a composition can be carried out by chromatography, filtration, ultracentrifugation in a gradient, etc. “Separation” or “separate” does not require that a first component be completely isolated from the second component such that none of the second component is present in a fraction containing the first component, but rather that the resulting fraction or composition is enriched for the first component, i.e, comprises a greater proportion of the first component (by weight or by molar amount) with respect to the second component than in the composition prior to the separation.

[0060] The term “transfection” is used to refer to the uptake of foreign or recombinant DNA by a cell, and a cell has been “transfected” when exogenous DNA has been introduced through the cell membrane. Examples of methods of transfection include Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13: 197. Such techniques can be used to introduce one or more exogenous nucleic acids, such as a plasmid and other nucleic acid molecules, into suitable host cells.

[0061] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.Manufacture and Purification of Recombinant AAV Particles

[0062] Adeno-associated virus (AAV) is a parvovirus which has use as a gene therapy vector. AAV is capable of infecting a variety of human cell types but do not replicate in human cells without expression of adenoviral accessory factors required for viral production. Accordingly, AAV has not been associated with disease in humans. The wild type AAV genome is a single stranded DNA of approximately 4.7 kb encoding the Rep or replication proteins and structural Cap or capsid proteins flanked by inverted terminal repeat (ITR) sequences which form hairpin structures that prime DNA replication upon transduction of the AAV. Recombinant AAV particles comprise a recombinant AAV genome in which an expression cassette, comprising the coding sequence for a gene of interest (GOI) is operably linked to regulatory sequences such as promoters, enhancers, and polyadenylation signals, such that the GOI is expressed in a target cell upon AAV particle transduction of that cell. The recombinant AAV genome has at least one ITR, and in embodiments, two ITRs which flank the expression cassette. In embodiments, one of the ITR sequences may be modified such that the packaged genome is a self-complementary (sc) genome rather than a single stranded (ss) genome. If analyzed by long-read Next Generation Sequencing, as described in Example 10, AAV particles comprising a scDNA genome may have a dimer to monomer ratio of at least about 10 to 1, at least about 20 to 1, or at least about 40 to 1.

[0063] There are multiple AAV serotypes, see, e.g., Gao, J. Virol. 78:6381 (2004), and the serotypes may have different tissue tropisms. The rAAV particles produced by the methods described herein can be any serotype or combination of serotypes. In embodiments, the capsid is from any AAV serotype, the Rep proteins are from any AAV serotype and the ITR sequences are from any AAV serotype, and the serotype of the capsid may differ from that of the Rep protein and / or the ITR sequences. In embodiments, the rAAV particles of the disclosure have a capsid (cap proteins, including VP1, VP2 and VP3) that is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh74, AAVrhlO, or related engineered capsid. See for example, WO2013 / 158879, W02015 / 013313, US 9,169,299, all of which are incorporated by reference in their entirety, for amino acid sequences of these capsids. The term “engineered capsid” or “engineered particle” as used herein, means an AAV having capsid protein which have been recombinantly engineered to include one or more amino acid substitutions, deletions or insertions relative to the amino acid sequence of the capsid proteins found in the wild type AAV. For example, engineered capsids may include, for example, PHP.B(WO 2015 / 038958 by Deverman et al.) and others (see, e.g, WO 2012 / 145601), which are incorporated herein by reference. Engineered capsids may confer enhanced or altered tissue tropism on a particular AAV serotype capsid.

[0064] In some embodiments, the rAAV particles described herein have ITRs from the same serotype as the capsid or are “pseudotyped AAV” particles in which the ITR sequences are from a different serotype than the capsid. In embodiments, the ITR sequences are from AAV2. In some embodiments, the rAAV2, rAAV5, rAAV6, rAAV8 or rAAV9 particles are pseudotyped rAAV2, rAAV5, rAAV6, rAAV8, rAAV9, or related engineered particles. In some embodiments, the rAAV2, rAAV5, rAAV6, rAAV8 or rAAV9 particles are rAAV2 / 2, rAAV2 / 5, rAAV2 / 6, rAAV2 / 8, rAAV2 / 9, or related engineered pseudotyped particles. In some embodiments, the rAAV particles are rAAV8 or rAAV9 particles (e.g., rAAV2 / 8 or rAAV2 / 9 particles). In some embodiments, the rAAV particles are rAAV6 particles (e.g., rAAV2 / 6 particles). In some embodiments, the rAAV particles are rAAV5 particles (e.g., rAAV2 / 5 particles). In some embodiments, the rAAV particles are rAAV9 particles (e.g., rAAV2 / 9 particles). Pseudotyped rAAV particles are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671, 2001).

[0065] The disclosure herein relates to methods of manufacturing and purifying recombinant AAV particles, as well as compositions comprising the purified AAV particles and uses thereof. Manufacture of recombinant AAV particles that can deliver a gene of interest (GOI) to a cell, thereby allowing the GOI to expressed in that cell, are well known in the art. In brief, such methods generally involve culturing cells (“host cells”), including mammalian cells (but also insect cells) that contain the genes encoding the AAV rep and AAV cap proteins and the adenoviral helper factors necessary for AAV production and packaging, and a construct encoding a recombinant AAV genome including a therapeutic gene of interest (or a marker gene, such as a gene encoding GFP) operably linked to an appropriate promoter and other regulatory elements (an “expression cassette”), flanked by AAV ITRs under conditions that produce AAV particles that contain either single stranded or self-complementary nucleic acids able to promote expression of the GOI in target cells upon AAV transduction. The AAV particles are then isolated from the host cells, typically by lysing the host cells, and then purified for therapeutic use. Challenges include the yield of production processes and the purity of the resulting product. Impurities, such as empty capsids or partial capsids, may increase toxicity of the drug product, thereby increasing the potential for adverse effects such as, an immune response. In addition, the presence of emptycapsids or capsids containing nucleic acid not capable of expressing the gene of interest upon transduction in a target cell (for example, partial capsids or capsids containing contaminating nucleic acid) can require an increase in the dose necessary for a therapeutic effect. Other process and product impurities, including the presence of residual host cell DNA or host cell protein, or residual DNA from plasmids utilized during the rAAV production process, may also contribute to adverse effects, and it is therefore desirable to minimize the amount of these impurities in the final drug product formulation. In certain embodiments, the AAV particles are AAV9. In other embodiments, the AAV particles are AAV5 particles or an AAV serotype where the capsid has an isoelectric point similar to AAV5. In other embodiments, the AAV particles are AAV6 particles or an AAV serotype where the capsid has an isoelectric point similar to AAV6. In other embodiments, the AAV particles are AAV8 particles or an AAV serotype where the capsid has an isoelectric point similar to AAV8. In other embodiments, the AAV particles are not AAV2 particles. In other embodiments, the AAV particles are not modified to have a different capsid amino acid sequence than the capsid amino acid sequence of a naturally occurring AAV capsid serotype.

[0066] A method of producing and purifying AAV particles is disclosed herein that results in improved yields and greater purity of the final product, including higher proportions of “full” AAV particle capsids (capsids containing nucleic acids that support the expression of the gene of interest in target cells upon transduction) versus “empty” capsids or “partial / partially filled” capsids (capsids that do not contain a functional recombinant AAV genome and / or have contaminating nucleic acids which are not the expression cassette or ITR nucleic acid (for example, the backbone of the GOI plasmid used to generate the AAV particles may be incorporated into the nucleic acid encapsidated in the process)). Accordingly, provided are methods of purifying AAV particle compositions, including AAV9, AAV8, AAV6, or AAV5 particle compositions, which have greater than about 80%, 85%, 90%, 95% or even 99% capsids with intact AAV genomes relative to the total capsids. Also provided are methods of purifying AAV particle compositions, including AAV9, AAV8, AAV6, or AAV5 particle compositions which have up to about 95%, 98%, or 99% capsids with intact AAV genomes, or undetectable levels of capsids that do not have intact genomes (for example, as determined by long read NGS, as described below in Example 10).

[0067] Also provided are methods of purifying AAV particle compositions whereby the purified AAV particles are of comparable and, in some cases, improved quality in comparison toAAV particles purified using cesium gradient ultracentrifuge methods (for example, as determined by Imaged Capillary Isoelectric Focusing (icIEF) or Capillary Isoelectric Focusing (cIEF) assays, as described below in Example 11 and shown in Table 9). For example, provided are methods of purifying AAV particle compositions wherein the pl of intact purified AAV9 particles as determined by cIEF assay is about 7.0 to about 7.5, about 7.1 to about 7.5, or about 7.2 to about 7.4. Also provided are methods of purifying AAV particle compositions wherein the pl of intact purified AAV particles as determined by cIEF assay is within 1.0 pH units, 0.5 pH units, 0.25 pH units, or 0.1 pH units of the isoelectric point of the specific AAV serotype or engineered capsid. In certain embodiments, the AAV particles are AAV9. In an embodiment, the AAV particle is AAV9, and the resulting drug substance composition comprises AAV9 particles wherein the pl of the intact capsid is about 7.2 to about 7.4 as determined by cIEF.

[0068] Also provided are methods of purifying AAV particle compositions whereby the purified AAV particles are of comparable and, in some cases, higher potency in comparison to AAV particles purified using cesium gradient ultracentrifuge methods (for example, exhibiting from 10% to 50% greater transgene expression upon infection of target cells under conditions that promote expression of the transgene as determined by a functional cell-based assay, as described below in Example 11). In certain embodiments, the AAV particles are AAV9, AAV8, AAV6, or AAV5. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and contains a gene of interest (GOI) GOI construct, and exhibits about 110% to 140% greater expression in HEK293 cells than an AAV9, AAV8, AAV6, or AAV5 particle containing the same GOI construct purified using a cesium gradient ultracentrifuge method. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and contains a GOI construct, and exhibits about 110% to 120%, or about 111% to about 116% greater GOI transgene expression in HEK293-AR cells than an AAV9, AAV8, AAV6, or AAV5 particle containing the same GOI construct purified using a cesium gradient ultracentrifuge method (HEK293-AR cells are engineered to overexpress AAV receptor proteins as described by Pillay S, et al. 2016 Nature 530(7588): 108-112).

[0069] Also provided are methods of purifying AAV particle compositions whereby the purified AAV particles, such as AAV9, AAV8, AAV6, or AAV5 particles, have greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% capsid purity and up to about 95%, 98% or 99% capsid purity or up to undetectable levels of impurities (for example as assessed by capillary electrophoresis-sodium dodecyl sulfate method with a laser-induced fluorescence (CE-SDS (LIF)) or size exclusion-high performance liquid chromatography (SE-HPLC), as described below in Example 8).

[0070] Also provided are methods of purifying AAV particle compositions, including for example AAV9, AAV8, AAV6, or AAV5 particle compositions, that produce final compositions that are highly enriched for full capsids, for example as assessed by analytical ultracentrifugation (AUC) (as described below in Example 6) or charge detection mass spectrometry (CDMS) (as described below in Example 7), or mass photometry (MP) (as described below in Example 7). Also provided are methods of purifying AAV particle compositions, including for example AAV9, AAV8, AAV6, or AAV5 particle compositions, that produce final compositions that are less than about 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% or have undetectable amounts of partial capsids relative to total capsids and less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% or have undetectable amounts of empty capsids relative to total capsids, including as assessed by AUC, CDMS, or MP. For scAAVs, provided are methods in which the drug substance or drug product may have a dimer to monomer ratio as determined by long-read Next Generation Sequencing, as described in Example 10, of at least about 10 to 1, at least about 20 to 1, or at least about 40 to 1.

[0071] The resulting drug substance composition (DC) containing rAAV particles obtained through purification according to the methods described herein has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% full capsids relative to total capsids (for 100%, that is non-full capsids being undetectable) by AUC, CDMS, or MP (and in embodiments, up to about 90%, 95%, 98%, 99% or 100% full capsids by AUC, CDMS, or MP) and / or a capsid purity of at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% by CE-SDS (LIF) or SE-HPLC (with 100% purity meaning impurities are undetectable by the analysis method) (and in embodiments, up to about 95%, 98%, 99% or 100% capsid purity by CE-SDS (LIF) or SE- HPLC). In certain embodiments, the AAV particles are AAV9, AAV8, AAV6, or AAV5. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting drug substance composition contains about 99% to about 99.9%, or about 99.5% to about 99.9% capsid purity as determined by SE-HPLC. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting drug substance composition contains undetectable to about 1% aggregation impurities, or 0.1 % to about 0.5% aggregation impurities, as determined by SE-HPLC.

[0072] In embodiments, the drug substance composition containing rAAV particles obtained through purification according to the methods described herein has less than about 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% partial capsids by AUC, CDMS, or MP analysis (and in embodiments, up to about 10%, 5%, 4%, 3%, 2% or 1% partial capsids by AUC, CDMS, or MP). In embodiments, the drug substance composition has less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% empty capsids by AUC, CDMS, or MP (and in embodiments, up to about 10%, 5%, 4%, 3%, 2% or 1% empty capsids by AUC, CDMS, or MP). For scAAVs, the drug substance product may have a dimer to monomer ratio as determined by long-read Next Generation Sequencing, as described in Example 10, of at least about 10 to 1, at least about 20 to 1, or at least about 40 to 1. In certain embodiments, the AAV particles are AAV9, AAV8, AAV6, or AAV5. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting drug substance composition contains about 2% to about 11% empty capsids, as determined by SV-AUC analysis. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting drug substance composition contains less than about 6% partial capsids, or about 4% to about 5.5% partial capsids, or about 4.4% to about 5.4% partial capsids, as determined by SV- AUC analysis. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting drug substance composition contains about 80% to about 95%, or about 85% to about 93% full capsids, as determined by SV-AUC analysis. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting final drug substance formulation contains about 4% to about 16% empty capsids, as determined by CDMS analysis. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting final drug substance formulation contains about 6% to about 8% partial capsids, as determined by CDMS analysis. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting final drug substance formulation contains about 75% to about 90%, or about 77% to about 88% full capsids, as determined by CDMS analysis. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting drug substance composition contains about 3% to about 17% empty capsids, as determined by MP analysis. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting drug substance composition contains about 7% to about 10% partial capsids, as determined by MP analysis. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting drug substance composition contains about 70% to about 90%, or about 73% to about 87% full capsids, as determined by MP analysis.

[0073] In addition, the resulting drug substance composition has impurities less than about 2 x 105pg, 1 x 105pg, 9 x 104pg, 8 x 104pg, 7 x 104pg, 6 x 104pg, 5 x 104pg, 4 x 104pg, 3 x 104pg, 2 x IO4pg or 1 x 104pg host cell DNA (normalized to 1 x 1013vg) and, in embodiments, less than about 1 x 105pg, 5 x 104pg, l x 104pg or 5 x 103pg of host cell DNA (normalized to 1 x 1013vg); and / or less than about 5%, 4%, 3%, or 2% copies of plasmid DNA per vg, and / or less than about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 ng host cell protein (normalized to 1 x 1013vg) and, in embodiments, not less than about 10, 5, 2, 1, or 0.5 ng, or undetectable ng host cell protein (normalized to 1 x 1013vg). In certain embodiments, the AAV particles are AAV9, AAV8, AAV6, or AAV5. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting drug substance composition contains about 50 ng to about 160 ng, or about 75 ng to about 135 ng, or about 80 ng to about 130 ng of host cell DNA (normalized to 1 x 1013vg), as determined by qcPCR. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting drug substance composition contains about 1.0 ng to about 10 ng, or about 1.5 ng to about 9.2 ng of host cell protein (normalized to 1 x 1013vg), as determined by ELISA. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting drug substance composition contains less than about 3%, 2.5%, 2.4%, or 2.2% of plasmid DNA per vg, as determined by ddPCR.

[0074] In embodiments, the drug substance and / or AAV containing compositions at any stage of the manufacturing process can be assayed for transgene expression and / or activity. For example, determined relative to a reference standard and / or other AAV containing compositions produced during the manufacturing process.

[0075] The process results in recoveries of at least about 5%, 7%, 10% or 15% (and, in embodiments, up to a recovery of about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%) for scDNA capsids, and at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% (and, in embodiments, up to a recovery of about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%) for single-stranded capsids, relative to the rAAV particles in the lysate. In certain embodiments, the AAV particles are AAV9, AAV8, AAV6, or AAV5. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the final recovery is about 50% to about 65%, or about 54% to about 60%, relative to (respectively) the AAV9, AAV8, AAV6, or AAV5 particles in the lysate, as determined by ddPCR. Total number of AAV vector genomes in the final drug substance can be determined based upon the lysate titer, for example, lysate titers of at least about 1 x 1011, 2 xIO11, 3 x IO11, 4 x IO11, 5 x IO11, 6 x IO11, 7 x IO11, 8 x IO11, 9 x IO11, 1 x IO12, 2 x 1012, or 3 x 1012vector genomes per mL (e.g., by ddPCR) in the lysate (and in embodiments, up to about 9 x 1011, 1 x 1012, 2 x 1012, or 3 x 1012vg rAAV particles per mL in the lysate) and calculated as percent recovery relative to lysate vector genomes. This can be calculated using the formula lysate titer (vg / mL) x lysate volume (mL) x recovery (%) = drug substance yield (vg). The recoveries may be achieved for a starting lysate volume of from about 2L to about WOOL, including about 2L, about 5L, about 20L, about 50L, about 100L, about 200L, about 215L, about 500L, about WOOL, WOL to WOOL, or WOL to 500L, or 200 to 500L, or 215 L to 500L. In embodiments, the production process results in total viral production of 5 x W15, 1 x IO16, 2 x IO16, 3 x IO16, 4 x IO16, 5 x W16, 6 x W16, or 7 x IO16, 8 x IO16, 9 x IO16, 1 x IO17, 2 x IO17, or 3 x IO17vg for a 215L starting lysate or a comparable titer normalized to a 215 L lysate. In certain embodiments, the AAV particles are AAV9, AAV8, AAV6, or AAV5. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting drug substance composition contains about 2.0 x W13to about 3.0 x W13, or about 2.2 x W13to about 2.75 xW13AAV9, AAV8, AAV6, or AAV5 vector genomes (respectively), as determined by ddPCR. The process also results in recoveries of viral capsids (as measured by ELISA) of at least about 1 x 1011, 2 x W11, 3 x W11, 4 x 1011, 5 x 1011, 6 x 1011, 7 x W11, 8 x W11, 9 x W11, 1 x W12, 2 x W12, 3 x W124 x W12, 5 x W12, 6 x W12, 7 x W12, 8 x W12, 9 x W12, 1 x W13, 2 x W13, 2.5 x W13, 3.0 x W13, 3.5 x W13, 3.75 x IO13, 4.0 x IO13, 4.5 x IO13, 5.0 x W13, 5.25 x IO13, or 5.5 x IO13, 5.75 x W13, 6 x IO13, 7 x IO13, 8 x IO13, 9 x IO13, or 1 x IO14viral capsids / mL of resulting drug substance. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting drug substance composition contains about 3.5 x W13to about 5.5 x 1013, or about 3.65 x W13to about 5.5 x IO13AAV9, AAV8, AAV6, or AAV5 capsids (particles) / mL (respectively) of resulting drug substance, as determined by ELISA.Production of Recombinant AAV

[0076] The methods disclosed herein relate to the production and purification of AAV particles comprising nucleic acids comprising gene expression cassettes and ITR sequences flanking the expression cassette. Provided are cis plasmid constructs comprising a plasmid backbone for selection and replication in animal, including mammalian cells (and in certain embodiments, insect cells), and an expression cassette flanked by AAV ITRs. The expression cassette comprises the coding sequence for a GOI (in embodiments, a therapeutic protein, siRNA, miRNA, etc. or a detectable marker, such as GFP) that is operably linked to regulatory elements, includingpromoters, enhancers, intron sequences, polyadenylation signals and other elements which promote expression of the GO I, either constitutively, or regulable, or in a tissue-specific manner, in target cells upon transduction with the rAAV particle containing a nucleic acid comprising the expression cassette, and AAV ITR sequences (flanking and sufficient for expression of the GOI upon rAAV transduction). In embodiments, the gene expression cassette is flanked by AAV ITR sequences, including AAV2 ITRs, which may be wild type for producing single stranded AAV genomes in the rAAV particles, or one of the ITR sequences is modified such that the AAV genome is a self-complementary genome. In certain embodiments, the AAV particles are AAV9 particles. In other embodiments, the AAV particles are AAV8 particles. In other embodiments, the AAV particles are AAV6 particles. In other embodiments, the AAV particles are AAV5 particles. In other embodiments, the AAV particles are not AAV2 particles. In other embodiments, the AAV particles are not modified to have a different capsid amino acid sequence than the capsid amino acid sequence of a naturally occurring AAV capsid serotype.

[0077] In some aspects, components to be expressed in the host cell to package a recombinant AAV vector in an AAV capsid, for example an AAV9, and AAV8, an AAV6, or an AAV5 capsid, can be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., recombinant AAV vector, rep sequences, cap sequences, and / or helper functions) can be provided by a stable host cell which has been engineered to contain the nucleotide sequences encoding one or more of the required components in a stable manner, i.e., they are integrated into the host cell genome, such that they are expressed in the host cell. In particular embodiments, provided are host cells comprising the recombinant AAV constructs or plasmids comprising the expression cassette flanked by ITR sequences, a plasmid providing the AAV rep and cap gene sequences, and a construct providing adenoviral helper proteins as needed to produce the recombinant viral particle. For example, a stable host cell may be generated which is derived from HEK 293 cells (including those which contain El helper functions under the control of a constitutive promoter), but which contain the rep and / or cap proteins under the control of inducible promoters. Still other stable host cells may be generated by one of skill in the art.

[0078] The recombinant AAV vector, rep sequences, cap sequences, and helper functions useful for producing the rAAV described herein can be delivered to the packaging host cell using any appropriate genetic element (vector, e.g. plasmid). The selected expression constructs can be delivered by any suitable method, including those described herein. The methods used to constructany of packaging cells disclosed herein are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods of generating rAAV virions are well known. See, e.g., K. Fisher et al, J. Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745.

[0079] Animal, including human, cells used to produce the rAAV particles have the cis GOI construct (including the expression cassette and 1TR sequences) (pGOI), and the Rep and Cap coding sequences (pRepCap) and the coding sequences for the adenoviral helper sequences to support recombinant AAV particle formation (pHelp). In embodiments, the animal cells are transfected with pGOI, pRepCap and pHelp constructs at the same time, as a triple transfection or are transfected in stages. In embodiments, the animal host cells comprise sequences encoding one or more of the adenoviral helper proteins stably integrated into the genome. In other embodiments, the animal cells are packaging cells which comprise and have integrated into their genome for appropriate expression the Rep, Cap and adenoviral helper proteins, or some combination thereof (with the Rep, Cap and adenoviral proteins not expressed by the cells being supplied in trans by transfection of expression constructs encoding those agents). For example, in embodiments, the host cells encode and express the adenovirus El A protein. The packaging cells can be transfected with the pGOI construct to produce host cells for the rAAV production.

[0080] The animal cells are, in embodiments, mammalian cells or insect cells. In embodiments, the cells are human embryonic kidney cells, for example HEK293 cells. Prior to introduction of the constructs, the cells are cultured and expanded. The culture may be by suspension culture or adherent culture using methods and equipment known in the art. In embodiments, the culture is a suspension culture. In embodiments, the cells are cultured in animal free media, including, for example, Expi293™ Expression Medium by Gibco™ (https: / / www.thermofisher.com / order / catalog / product / A1435101), CD 293, or Viral Production Medium™ by Gibco™ (https: / / www.thermofisher.com / order / catalog / product / A4817903), or FreeStyle™ 293 Expression Medium by Gibco™(https: / / www.thermofisher.com / order / catalog / product / 12338018). In embodiments, the cells are maintained in a master cell bank. For use in producing recombinant AAV, cells from the master cell bank are thawed and then expanded through multiple passages, including of increased volume, until the desired cell density and volume is achieved. For example, cells may be initially culturedin approximately 30 ml until target viable cell (vc) density reached and then expanded to a culture volume 2, 3 or 4 fold or more greater and cultured until the target viable cell density is achieved, and then expanded into a volume that is 2, 3, 4, 5, 6 or 10 times greater than the previous culture volume from which the cells are expanded, and so forth, until the target volume and viable cell density are reached. For example, cultures of 2L, 20L, 200L or even larger manufacturing scale volumes of WOOL or more may be achieved with target cell density (for example, as determined by a hemacytometer) of approximately 1 x 106vc / ml, 2 x 106vc / ml, 3 x 106vc / ml, 4 x 106vc / ml or 5 x 106vc / ml. In some embodiments, the target cell density is from about 1.5 x 106vc / ml to about 2.5 x 106vc / ml. Process controls may be implemented to control agitation rate, pH, temperature, and / or dissolved oxygen.

[0081] In some embodiments, once the cells are at the target cell density in the target volume, fresh culture media is added to the cell culture, for example, 0.5 volume of the cell culture volume, 1 volume of the cell culture volume or 1.5 volume of the cell culture volume or 2 volumes of the cell culture volume. For example, for 100L of cell culture, 1 volume (that is, 100L) of fresh cell culture media is added to the culture (for example, a bioreactor). The volume of cell culture media can be added either prior to or concurrently with the transfection composition containing the constructs to be transfected.

[0082] Once cells are at the target volume and density, the cells can be transfected with the appropriate constructs for producing the recombinant AAV particles. In embodiments, the cells are transfected with pGOI construct, pRepCap and pHelp constructs, including with a transfection composition comprising the constructs (plasmids). Optimal ratio, by weight or molarity, of the constructs can be determined by methods known in the art. The optimal ratios may depend upon the pGOI and can be determined for a particular pGOI using methods known in the art. In some embodiments, the host cells comprise and express the adenoviral helper genes and are transfected with the pGOI and the pRepCap constructs. In other embodiments, the host cells comprise and express the Rep and Cap genes and are transfected with the pGOI and the pHelp constructs. In other embodiments, the host cells comprise and express a subset of the adenoviral helper genes, for example the E1A gene, and are transfected with the pGOI, pRepCap and pHelp constructs, where the pHelp construct does not encode the adenoviral helper genes encoded in the host cell.

[0083] The transfection may be aided by adding a transfection reagent to the nucleic acid constructs being transfected into the cells, such as, for example, cationic lipid transfection reagents such as FectoVIR® by Sartorius AG (https: / / shon, sartorius. com / dk / p / fectovir- aav / F ecto VIR A A V) . In other embodiments, the transfection reagent may be polycation polyethylenimine (PEI)-based transfection reagents such as PEIpro® by Sartorius AG (https: / / shop.sartorius.eom / dk / p / peipro / PEIpro), cationic lipid transfection reagents such as AAV- Max by Gib co™ (https: / / www.thermofisher.com / order / catalog / product / A50515? SID=srch-srp- A50515) or TransIT-VirusGEN® by Minis Bio® (https: / / www.mirusbio.com / product / transit- virusgen-transfection-reagent-0-3- ml / ? gad_source= 1 &gclid=EAIaIQobChMI3 OPL957Jj AMVFLhaBRl Xj qNEAAYAS AAEgJg BfD BwE), or any other PEI, cationic lipid, or lipofectamine based reagents. The amount of transfection agent can be determined according to methods known in the art. In embodiments, the transfection agent, including FectoVIR®, is added to the constructs to be transfected at a weight ratio of approximately 1 transfection agent: 1 weight of all DNA constructs to be transfected, or a weight ratio of approximately 1.5: 1, 2:1, 1 : 1.5, 1 :2, etc. In embodiments, the transfection occurs over about 15 minutes, about 30 minutes, about 45 minutes, or about 60 minutes or more.

[0084] After the transfection step, the transfected cells are incubated in the bioreactor for a time and under conditions appropriate for production of the rAAV particles, z.e., AAV capsids containing nucleic acid comprising the expression cassette flanked by AAV ITRs or, in embodiments, enough of one ITR or two flanking ITRs sufficient to support expression of the GOI after transduction in a target cell. The incubation time for production of the rAAV particles may be about 1 day, about 2 days, about 3 days, about 4 days or about 5 days. In embodiments, the transfected cells are incubated for about 3 days. The optimal incubation time can be determined by methods known in the art, for example, by ddPCR, tAAV and pDNA assessments after different incubation times.Recombinant AAV Harvesting

[0085] The rAAV particles may be released, harvested or isolated from the cell culture by any method known in the art in preparation for downstream processing of the rAAV particles. Examples of ways in which the cells may be lysed include freeze / thaw cycles, solid shearing on a French press or Hughes press, hypotonic solution lysis with water or citric buffer, liquid shear witha homogenizer or microfluidizer, sonication, or detergent lysis, such as with non-ionic detergents, sodium dodecyl sulfate or ethyl trimethyl ammonium bromide. Alternatively, the rAAV particles may be released and separated from the host cells without disruption of the host cells with hypertonic media.

[0086] In an embodiment, the cells are lysed by treatment with a non-ionic detergent, including polysorbate 20 (TWEEN® 20). Other non-ionic detergents that can be used are Triton®X-100, bile salts such as cholates, zwitterionic detergents such as CHAPS, and NP-40. Optionally, MgCh is also added to the cell culture during lysis. In embodiments, about 0.25%, 0.5%, 1%. 1.5% or 2% culture volume of the Tween 20 is added to the culture, in embodiments about 0.5% is added. In embodiments, MgCh is added to achieve a concentration of about 0.5 mM, 1 mM, 1 .5 mM or 2 mM. In an embodiment, the culture is lysed with 0.5% culture volume of Tween 20 and 2 mM MgCh. The cell culture and lysis mixture are agitated under appropriate conditions for a period of time sufficient to result in cell lysis, for example, until cell viability is less than or equal to about 15%, 10% or 5% or 1%. The culture may also undergo endonuclease digestion to break down the host cell nucleic acid either concurrently with the lysis step or prior to it or subsequent to it. Endonuclease digestion may be carried out with any appropriate endonuclease or combination of endonucleases that break down DNA and / or RNA. In embodiments the endonuclease is a genetically engineered endonuclease from Serratia marcescens (e.g., Benzonase® or Denarase®) or any other appropriate endonuclease. In embodiments, 50 U / mL Benzonase® is added to digest the host cell DNA and / or RNA.

[0087] The cell lysate that serves as the starting material for the AAV purification methods described herein can be obtained using any method for preparing AAV known in the art. For example, lysate can be obtained by methods comprising: a) culturing mammalian cells comprising nucleotide sequences comprising (1) a gene of interest (GOI) expression cassette comprising a transgene encoding a GOI operably linked to at least one regulatory sequence, flanked by AAV inverted terminal repeat (ITR) sequences; and (2) coding sequences for an AAV Rep, AAV Cap and adenovirus helper factors, under conditions that produce AAV particles comprising i) a nucleic acid, comprising the GOI expression cassette capable of expressing the transgene in a target cell upon transduction of the AAV particle, and ii) an AAV capsid; and b) lysing the mammalian cells to produce a first AAV particle composition that contains full AAV capsids, process impurities, including empty and partial AAV capsids, and product impurities.

[0088] The methods described herein are suitable for large-scale manufacture of AAV particle compositions, including for a starting lysate volume of from about 2L to about OOL, including 10L to 500L, 100L to 500L, 100L to 200L, 100L to WOOL, 200L to WOOL and even greater than OOL. In certain embodiments, the AAV particle composition comprises AAV9, AAV8, AAV6, or AAV5 particles. In embodiments, the starting lysate volume is 215L. The methods described herein advantageously result in AAV particle drug substance or drug product with process and product impurity levels and yield comparable to AAV particle drug substance or drug product produced by processes that include purification by cesium gradient on ultracentrifugation even for large scale manufacturing. For example, provided are methods of purifying AAV particle compositions whereby the yield of AAV particle drug substance is about 1 to about 2.5 times greater, or about 1.5 to about 2.0 times greater, or about 1.6 to about 1.8 times greater than the yield of AAV particles purified using cesium gradient ultracentrifuge methods. In certain embodiments, the AAV particle drug substance comprises AAV9 particles. In other embodiments, the AAV particle drug substance comprises AAV8 particles. In other embodiments, the AAV particle drug substance comprises AAV6 particles. In other embodiments, the AAV particle drug substance comprises AAV5 particles In other embodiments, the AAV particle drug substance does not comprise AAV2 particles. In other embodiments, the AAV particle drug substance does not comprise AAV particles that are modified to have a different capsid amino acid sequence than the capsid amino acid sequence of a naturally occurring AAV capsid serotype. In addition, the methods developed by the present inventors are further advantageously efficient such that large scale batches can be processed from lysate to final AAV particle drug substance in 24 to 48 hours.Purification of Recombinant AAV Particles

[0089] The AAV purification methods described herein generally involve (i) providing a cell lysate, from HEK293 cells for example, that comprises a first AAV particle composition that contains full AAV capsids, process impurities, and product impurities including empty and partial AAV capsids; (ii) flocculating the cell lysate to form an aqueous layer comprising AAV particles, and a flocculate layer containing process impurities and product impurities, isolating the liquid layer containing AAV particles from the flocculate layer, and clarifying the liquid layer containing AAV particles by filtration to produce a second AAV particle composition; (iii) further purifying the second AAV particle composition using cation exchange (CEX) chromatography to produce a third AAV particle composition; (iv) further purifying the third AAV particle composition usinganion exchange (AEX) chromatography to produce a fourth AAV particle composition; (v) further purifying the fourth AAV particle composition by subjecting it to tangential flow filtration (TFF) to produce a fifth AAV particle composition; and (vi) performing a bioburden-reduction step using a sterilizing filter to produce an AAV particle drug substance. In certain embodiments, purifying the second AAV particle composition using cation exchange (CEX) chromatography is followed by tangential flow filtration (TFF) to produce the third AAV particle composition prior to anion exchange (AEX) chromatography. In certain embodiments, step (iv) involves two successive rounds of AEX chromatography to produce the fourth AAV particle composition. In certain embodiments, the fifth AAV particle composition is further purified (for example, through a 0.2 pm sterilizing filter, such as a 0.22 pm PES or PVDF filter) to produce a drug substance containing purified AAV particles. In embodiments, the AAV particles are AAV9 particles, other AAV serotypes or engineered pseudotypes. In embodiments, the AAV capsid is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh74, AAVrhlO, or related engineered capsid. In embodiments, the AAV particles are an AAV type where the capsid has an isoelectric point similar to AAV9, AAV8, AAV6, or AAV5. In further embodiments, the AAV particles have single stranded genomes and in other embodiments the AAV are self-complementary.

[0090] In certain embodiments, purifying the second AAV particle composition, including for example AAV9, AAV8, AAV6, or AAV5 particle compositions for this and subsequent steps, using cation exchange (CEX) chromatography is conducted under conditions that create a salt or pH gradient to produce the third AAV particle composition. For example, cation exchange (CEX) chromatography may be conducted using an elution buffer set that creates a salt gradient ranging from a concentration of about 500 mM NaCl to a final concentration of about 2.5 M NaCl, or, alternatively, an elution buffer set that creates a pH gradient ranging from a pH of about 3.5 to a final pH of about 9.3 to produce the third AAV particle composition. In embodiments, the CEX chromatography resin is poly(glycidyl methacrylate -co- ethylene dimethacrylate) with sulfonate ligand (e.g., CIMmultus SO3) with a pore size of 2 pm, 4 pm or 6 pm, and, in embodiments has a 6pm pore size. The bed size is proportional to the starting lysate volume or mass, for example, an 800 mL bed volume column is used for an approximately 200 L to 250 L, including 215 L, culture or lysate, and run at 6-10 column volumes per minute, including about 6 column volumes per minute, about 7 column volumes per minute, about 8 column volumes per minute, about 9 columnvolumes per minute, or about 10 column volumes per minute, including during loading and / or elution.

[0091] In certain embodiments, purifying the third AAV particle composition, including AAV9, AAV8, AAV6, or AAV5 particles for example, using anion exchange (AEX) chromatography is conducted under conditions that create a salt gradient to produce the fourth AAV particle composition. For example, anion exchange (AEX) chromatography may be conducted using an elution buffer set that creates a salt gradient ranging from a concentration of about 0 mM NaCl to a final concentration of about 1 M NaCl, to produce the fourth AAV particle composition.

[0092] In certain embodiments, flocculating the cell lysate involves (i) combining the cell lysate with a solution containing salt (for example, NaCl) and mixing for at least five minutes, followed by (ii) the addition of a solution containing octanoic acid and mixing for at least five minutes, to achieve a salt concentration of from about 300 mM to about 600 mM, and an octanoic acid concentration of about 0.5%, 1.0%, 1.5%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, or any octanoic concentration experimentally determined to enhance flocculation, followed by (iii) the addition of a solution containing formate to achieve a target pH of about 3.3 to about 3.7, and mixing for at least five minutes, thereby forming an aqueous layer comprising full AAV capsids, and a flocculate layer containing process impurities and product impurities. In certain embodiments the AAV capsids, are AAV9, AAV8, AAV6, or AAV 5 capsids and the cells are HEK293 cells.

[0093] In other embodiments, flocculating the cell lysate involves (i) combining the cell lysate with a solution containing salt (for example, NaCl) and mixing for at least five minutes, followed by (ii) the addition of a solution containing octanoic acid and mixing for at least five minutes, followed by (iii) the addition of a solution containing polyethyelene glycol (PEG) and mixing for at least five minutes, to achieve a salt concentration of from about 300 mM to about 600 mM, an octanoic acid concentration of about 0.5%, 1.0%, 1.5%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, or any octanoic concentration experimentally determined to enhance flocculation, and a PEG concentration of up to about 0.5%, 0.75%, 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, followed by (iv) the addition of a solution containing formate to achieve a target pH of about 3.3 to about 3.7, and mixing for at least ten minutes, thereby forming an aqueous layer comprising full AAVcapsids, and a flocculate layer containing process impurities and product impurities. The polyethylene glycol (PEG) solution used in such embodiments may, for example, 1500 MW or 6000 MW. In certain embodiments the cell lysate is from HEK293 cells and the AAV capsids are AAV9, AAV8, AAV6, or AAV5 capsids.

[0094] In one embodiment, the purification process is conducted as set forth below in Example 1.

[0095] In another embodiment, the purification process is conducted as set forth below in Example 2.

[0096] In another embodiment, the purification process is conducted as set forth below in Example 3.Lysate Clarification

[0097] In embodiments, the host cell lysate comprising AAV particles for purification is clarified to remove initial contaminants and product impurities. The cell lysate in an embodiment of the invention is obtained by incubating the cell culture in the presence of a non-ionic surfactant, such as, for example, polyoxyethylene (20) sorbitan monolaurate (Tween 20) and in some embodiments present in the lysate at from about 0.1 to 1% culture volume, 0.25 to 0.75% culture volume, or at about 0.5% culture volume. Any strategy for clarification of host cell lysate (for example, filtration or centrifugation) may be utilized. In embodiments, the host cell lysate comprising AAV particles for purification is clarified by passing the lysate through a depth filter prior to further processing. A depth filter has a porous filtration medium which retains particles throughout the medium and advantageously can hold a large volume of particles compared to filters retaining particles on the surface of the filtration medium. Depth filters may be made from any materials known in the art for depth filtration, for example cellulose fibers, diatomaceous earth, or glass fibers, that are of the appropriate size and volume for the material to be filtered. In some embodiments, the flow of lysate through the filter may be enhanced using centrifugation, or positive or negative pressure. In certain embodiments, the depth filter is a glass-fiber filter, for example a 0.5 pm filter, a 0.65 pm filter, a 1.0 pm filter or a 1.2 pm filter, for example, a Meissner or Sartorius or any other glass-fiber filter and appropriate buffered saline solution, such as, but not limited to, 20 mM Tris, 120 mM NaCl, pH 7.4, is added during or after the filtration step. In certain embodiments the cell lysate is from HEK 293 cells and the AAV particles are AAV9,AAV8, AAV6, or AAV 5 capsid particles. In other embodiments, the AAV particles are not AAV2 particles. In other embodiments, the AAV particles are not modified to have a different capsid amino acid sequence than the capsid amino acid sequence of a naturally occurring AAV capsid serotype.

[0098] In embodiments, about 50 L of lysate is clarified with 6 m2of Meissner 1.0 pm pore glass-fiber filter (Protec Capsule Filter, HB, Sterile, GR2RF1-5NCC2) at 25 LMH (liters / m2 / hour) and chased with 15 kg of Lysis Clarification Buffer (20 mM Tris, 120 mM NaCl at pH 7.4).

[0099] In embodiments, about 200 L of lysate is clarified with 18m2of Meissner 1.0 pm pore glass-fiber filter (Protec Capsule Filter, HB, Sterile, GR2RF1-5NCC2) at 25 LMH (liters / m2 / hour) and chased with 15 kg of Lysis Clarification Buffer (20 mM Tris, 120 mM NaCl at pH 7.4).

[0100] In embodiments, when clarifying the cell lysate with a glass-fiber filter, the cell lysate is passed through the glass-fiber filter at from about 7 to about 14 L / m2.

[0101] In embodiments of the method, the lysate is not processed through a depth filter prior to the flocculation or other separation or purification step.Flocculation

[0102] To remove host cell impurities such as protein and nucleic acid (both DNA and RNA), the lysate or clarified (filtered) lysate is further processed by a flocculation and, in embodiments, a clarification step. In embodiments, the lysate or clarified lysate is flocculated or precipitated by acidification, for example, acidified to a pH of about 2.9 to 4.0, in embodiments, to a pH of about 3.0 to 4.0, or to a pH of about 3.3 to 3.7, or to a pH of about 3.0 to 3.7, or to a pH of about 3.3 to 4.0. The solution may be acidified with any appropriate acid and, in embodiments, formic acid or formate is added until the pH is within the desired range. Other acids that may be used to acidify the lysate or clarified lysate include citric acid, acetic acid, phosphoric acid, and various amino acids.

[0103] In embodiments, in addition to the acidification, an anionic surfactant (e.g. a fatty acid) is also added to the lysate or clarified lysate during the flocculation step (in embodiments after or prior to acidification, and / or the addition of polyethylene glycol (PEG), and / or the addition of a salt solution, and / or the addition of formate, as further described herein). For example, fatty acids may be a saturated or unsaturated unbranched-chain fatty acids or bile salts like sodium cholate orsodium deoxycholate. Fatty acids may also include saturated fatty acids of 5 carbon, 6 carbon, 7 carbon, 8 carbon, 9 carbon or 10 carbon chains. In embodiments, saturated fatty acids of 7, 8, or 9 carbons are used. In embodiments, the lysate or clarified lysate is flocculated by addition of a fatty acid, in embodiments at about 0.5%, about 1%, about 2%, about 3% or about 4%, and acidified to a pH range of about 2.9 to about 4.0, in embodiments, to a pH range of about 3.0 to about 4.0, or to a pH range of about 3.3 to about 3.7, or to a pH range of about 3.0 to 3.7, or to a pH range of about 3.3 to 4.0. In certain embodiments, octanoic acid may be combined with the lysate or clarified lysate during the flocculation step (in embodiments after or prior to acidification and / or the addition of polyethylene glycol (PEG), and / or the addition of a salt solution, and / or the addition of formate, as further described herein). Thus, in some embodiments, the flocculation step may include adding octanoic acid to a final concentration of about 2% octanoic acid. In other embodiments, the flocculation step may include adding octanoic acid to a final octanoic acid concentration of up to 0.5%, 1.0%, 1.5%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, or to any experimentally determined final octanoic concentration that enhances flocculation. In certain embodiments the lysate, or clarified cell lysate, is obtained from HEK293 cells producing AAV9, AAV8, AAV6, or AAV5 particles.

[0104] In certain embodiments, a salt solution may be combined with the lysate or clarified lysate during the flocculation step (in embodiments after or prior to acidification, and / or the addition of surfactants such as octanoic acid, and / or the addition of polyethylene glycol (PEG), and / or the addition of formate, as further described herein), and / or the addition of formate, as further described herein). Thus, in some embodiments, the flocculation step may include the addition of a salt solution (for example, NaCl) to a final salt concentration of 300 mM to about 500 mM, or from about 200 mM to about 600 mM, or from about 100 mM to 800 mM, or to any experimentally determined final salt concentration that enhances flocculation. In certain embodiments the lysate, or clarified cell lysate, is obtained from HEK293 cells producing AAV9, AAV8, AAV6, or AAV 5 particles.

[0105] In certain embodiments polyethylene glycol (PEG) is also added to the lysate or clarified lysate during the flocculation step (in embodiments after or prior to acidification, and / or the addition of surfactants such as octanoic acid, and / or the addition of a salt solution, and / or the addition of formate, as further described herein). Thus, in some embodiments, the flocculation step may include the addition a 1500 MW or 6000 MW PEG solution to a concentration of 1% PEG.In other embodiments, the flocculation step may include adding a 6000 MW PEG solution to a final concentration of up to 0.5%, 0.75%, 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0% or to any experimentally determined final 6000 MW PEG concentration that enhances flocculation. In certain embodiments the lysate, or clarified cell lysate is obtained from HEK293 cells producing AAV9, AAV8, AAV6, or AAV5 particles.

[0106] In certain embodiments, formate is also added to the lysate or clarified lysate during the flocculation step (in embodiments after or prior to acidification, and / or the addition of surfactants such as octanoic acid, and / or the addition of polyethylene glycol (PEG), and / or the addition of a salt solution, as further described herein). Thus, in some embodiments, the flocculation step may include the addition of about 1 M formate at pH 2.5 to a target pH of 3.5. In other embodiments, the flocculation step may include the addition of up to 0.25 M, 0.5 M, 0.75 M, 1.25 M, 1.5 M, 1.75 M, or 2.0 M formate to a target pH of up to 4.0, 4.5, or to any experimentally determined final pH that enhances flocculation. In certain embodiments the lysate, or clarified cell lysate is obtained from HEK293 cells producing AAV9, AAV8, AAV6, or AAV5 particles.

[0107] In certain embodiments, flocculating the cell lysate involves (i) combining the cell lysate with a solution containing from about 300 mM to about 600 mM salt (for example, NaCl) and mixing for at least ten minutes, followed by (ii) the addition of a solution containing octanoic acid and mixing for at least ten minutes, followed by (iii) the addition of a solution containing formate and mixing for at least fifteen minutes, thereby forming an aqueous layer comprising full, empty, and partial AAV capsids, and a flocculate layer containing process impurities. In certain embodiments the lysate, or clarified cell lysate is obtained from HEK293 cells producing AAV9, AAV8, AAV6, or AAV5 particles.

[0108] In other embodiments, flocculating the cell lysate involves (i) contacting the cell lysate with a solution containing from about 300 mM to about 500 mM salt (for example, NaCl) and mixing for at least ten minutes, followed by (ii) the addition of a solution containing octanoic acid and mixing for at least ten minutes, followed by (iii) the addition of a solution containing polyethylene glycol (PEG) solution, followed by (iv) the addition of a solution containing formate and mixing for at least fifteen minutes, thereby forming an aqueous layer comprising full AAV capsids, and a flocculate layer containing process impurities and product impurities. Thepolyethylene glycol (PEG) solution used in such embodiments may, for example, be 6000 MW PEG. In certain embodiments the lysate, or clarified cell lysate is obtained from HEK293 cells producing AAV9, AAV8, AAV6, or AAV5 particles.

[0109] Once flocculant has formed (for example after about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes of agitation, about 60 minutes, or 120 minutes depending on scale), the flocculant is allowed to form a separate layer (the “cream layer”) from the liquid layer, for example after a 30 minute incubation (or 15, 20, or 25 minutes or longer, including 45 minutes, 60 minutes, or 120 minutes) without agitation. The liquid layer, which contains recombinant AAV particles, may be separated from the cream layer by any method known in the art, for example, by transferring the aqueous buffer liquid layer to a separate container without transferring the cream layer material. Removal may be by allowing the liquid buffer layer to flow out of the bottom of the flocculant container or vessel. In addition, the cream layer may be washed and the liquid aqueous buffer layer from the wash transferred to the liquid layer. The liquid aqueous buffer layer may be further clarified by filtration, including, for example, through a glass fiber filter, including 0.45 pm, 0.5 pm, 0.65 pm, 1.0 pm, 1.2 pm, 2 pm, 3 pm, 4 pm or 5 pm filters of appropriate size. In certain embodiments the lysate, or clarified cell lysate is obtained from HEK293 cells producing AAV9, AAV8, AAV6, or AAV5 particles.

[0110] In certain embodiments, clarified cell lysate is flocculated in a 100-L single-use mixer (SUM, Thermo Scientific, HyPerforma Single-use Mixer) by addition of 4 M NaCl and then mixing at 200 RPM for at least 10 minutes, followed by the addition of octanoic acid and then mixing for at least 10 additional minutes, such that the flocculation mixture has a salt concentration of 500 mM NaCl, and the octanoic acid concentration is 2% octanoic acid. The flocculation mixture is then acidified by the addition of 1 M formate at pH 2.5 to a target pH of 3.5 while mixing for at least 15 minutes. The flocculated mixture is allowed to stand for at least 30 minutes without mixing during which time a cream layer separated from the buffer layer and floated to the top of the solution. The buffer layer is pumped out from the bottom of the single-use mixer (SUM) through 6 m2of Meissner 0.5 pm pore glass-fiber filter (Protec Capsule Filter, HB, Sterile, GR2RF0.5-5NCC2) at 25 LMH. The remaining cream layer in the SUM is rinsed with 20% of the flocculate weight (i.e., the weight of the lysate plus the weight of the NaCl, octanoic acid, and formate added above) of CEX Buffer A (20 mM formate, 0.5 MNaCl, 0.2% pol oxamer 188 (Pl 88) at pH 3.5) at pH 3.5) and mixed at 80 RPM for 10 minutes (the “cream rinse”). The cream rinse isallowed to stand without mixing for at least 30 minutes. The buffer layer of the cream rinse is then pumped through the same set of 0.5 pm fdters at 25 liters per m2filter area per hour (“LMH”) and followed by the addition of 15 kg of CEX Buffer A through the same set of 0.5 pm filters at 25 LMH (the “filter chase”). The cream-rinse and filter-chase buffers are collected in the same bag as the initial clarified flocculate buffer layer to form the clarified flocculate that is also the CEX Load. The combined pool is mixed at 215 RPM for 5 minutes before sampling. In certain embodiments the lysate, or clarified cell lysate is obtained from HEK293 cells producing AAV9, AAV8, AAV6, or AAV5 particles.

[0111] In certain embodiments, unclarified cell lysate is warmed to 33.1 °C (range 30 to 37°C) and transferred to a 100-L single-use mixer (SUM, Thermo Scientific, HyPerforma Single-use Mixer), and flocculated by addition of 4 M NaCl and then mixing at 200 RPM for at least 10 minutes, followed by the addition of octanoic acid and then mixing for at least 10 additional minutes, followed by addition of a 40% 6000 MW PEG solution and then mixing for at least 10 additional minutes, such that the flocculation mixture has a salt concentration of 500 mM NaCl, the octanoic acid concentration is 2% octanoic acid, and the 6000 MW PEG concentration is 1%. The flocculation mixture is then acidified by the addition of 1 M formate at pH 2.5 to a target pH of 3.5 while mixing for at least 15 minutes. The flocculated mixture is allowed to stand for at least 30 minutes without mixing during which time a cream layer is allowed to separate from the buffer layer and floated to the top of the solution. The buffer layer is pumped out from the bottom of the single-use mixer (SUM) through 6 m2of Meissner 0.5 pm pore glass-fiber filter (Protec Capsule Filter, HB, Sterile, GR2RF0.5-5NCC2) at 25 LMH. The remaining cream layer in the SUM is rinsed with 20% of the flocculate weight (i.e., the weight of the lysate plus the weight of the NaCl, octanoic acid, and formate added above) of CEX Buffer A (20 mM formate, 0.5 M NaCl, 0.2% P188 at pH 3.5) and mixed at 80 RPM for 10 minutes. The cream rinse is allowed to stand without mixing for at least 30 minutes. The buffer layer of the cream rinse is then pumped through the same set of 0.5 pm filters at 25 liters per m2filter area per hour (“LMH”) and followed by the addition of 15 kg of CEX Buffer A through the same set of 0.5 pm filters at 25 LMH (the “filter chase”). The cream-rinse and filter-chase buffers are collected in the same bag as the initial clarified flocculate buffer layer to form the clarified flocculate that was also the CEX Load. The combined pool is then mixed at 215 RPM for 5 minutes before sampling. In certain embodimentsthe lysate, or clarified cell lysate is obtained from HEK293 cells producing AAV9, AAV8, AAV6, or AAV5 particles.

[0112] The flocculation step, in some embodiments with the clarification step, results in a about a one log reduction, 1.5 log reduction, 2 log reduction, 2.5 log reduction or 3 log reduction (including by about 70%, 80%, 90%, 95%, 99% or 99.9%) in host cell DNA and / or host cell protein. The reduction in host cell DNA or host cell protein may be normalized to a set number of rAAV particles in the preparation before and after the flocculation step (for example, normalized to 1 x 1013vg detected). See e.g. Example 5 and Table 2. Residual host cell protein and DNA levels may be assessed by any method known in the art. For example, levels of host cell DNA may be assessed by PCR, ddPCR, and / or qPCR (see, e.g., Example 5, Table 2, columns 2, 5, and 6) and host cell protein levels may be quantitated by an ELISA assay for host cell (for example, HEK293 cell) protein (see, e.g., Example 5, Table 2, columns 3 and 4). In certain embodiments the lysate, or clarified cell lysate is obtained from HEK293 cells producing AAV9, AAV8, AAV6, or AAV5 particles.

[0113] In embodiments, the one or two log reduction in host cell DNA and / or host cell protein due to the flocculation step facilitates a subsequent cation exchange chromatography step using a resin with a pore size of greater than 1.1pm, for example, 2 pm or greater, 4 pm or greater or to about 10 pm, and, in embodiments, 6 pm or greater, or to about 8 pm or 10 pm, including 2 pm, 4 pm, 6 pm, or 8 pm cation exchange resin (for example, a CIMmultus SO3 resin having a porosity of 6 pm) in an appropriate column bed volume. In certain embodiments the lysate, or clarified cell lysate is obtained from HEK293 cells producing AAV9, AAV8, AAV6, or AAV5 particles.

[0114] In embodiments, the flocculation step is performed at room temperature, or above room temperature (from about 30 °C to about 35 °C for above room temperature). In embodiments, other purification steps described herein (e.g., cation exchange (CEX) chromatography, anion exchange (AEX) chromatography, and / or tangential flow filtration (TFF)) are performed at room temperature or above room temperature (from about 30 °C to about 35 °C for above room temperature). In certain embodiments the lysate, or clarified cell lysate is obtained from HEK293 cells producing AAV9, AAV8, AAV6, or AAV5 particles.Cation Exchange (CEX) Chromatography

[0115] In embodiments, the clarified composition from the flocculation step, or alternatively an AAV particle composition that (i) is the direct product of upstream processing prior to any column processing step, and (ii) comprises less than about 30%, 20%, 10%, 5%, 1%, or 0.1% of host cell DNA and / or host cell protein, is processed using cation exchange (CEX) chromatography. Any strategy for reducing the volume of the composition containing the AAV particles on a CEX chromatography column may be utilized, and CEX chromatography columns comprising various resins, flow capacities, and pore sizes can be used. In embodiments, the cation exchange monoliths has a pore size of greater than 1.1 pm., and in embodiments, is 10 pm, 6 pm, 4 pm or 2 pm. In embodiments, the CEX resin is made from poly(glycidyl methacrylate -co- ethylene dimethacrylate), including with a sulfonate ligand; for example, the CEX chromatography column used is a CIMmultus S03-400 mL (or 800 mL), 6-pm pore column, BIA Separations 814.6157-6 (https: / / shop.sartorius.com / us / p / cimmultus-so3-400-ml-cgmp-compliant-monolithic-column-6- m / 924,6157-6#; https: / / shop, sartorius. com / dk / p / cimmultus-oligo-dtl 8-c6-linker-800-ml - monolithic-column-2-m / 811, 1218-2). In certain embodiments the clarified composition from the flocculation step is obtained from HEK293 cells and the AAV particles are AAV9, AAV8, AAV6, or AAV5 particles.

[0116] In embodiments, the recombinant AAV particles are eluted from the column with a gradient of increasing salt concentration. In embodiments, the initial salt concentration is .2 M or 0.5 M NaCl with an increase as a salt gradient to 2 M NaCl. In embodiments, the column bed volume can be 1 mL, 4 mL, 8 mL, 40 mL, 80 mL, 400 mL, 800 mL, 4 L, or 8 L depending upon the starting lysate volume. In embodiments the flow rate is 6, 7, 8, 9 or 10 column volumes (CV) / min. Eluate may be monitored for UV280 or UV260 absorbance. For example, in embodiments with an 800 mL bed volume column, fractions are collected from UV280 inflection from baseline, such as 0.15 AU and continuing until the UV2 0 returns to baseline. Other embodiments include, from inflection from baseline to 1.5 to 4 column volumes (CV). In certain embodiments, the initial gradient buffer is 20 mM formate, 0.2% poloxamer 188, pH 3.5 with 0.5 M NaCl and the high salt buffer to form the gradient is 20 mM formate, 0.2% poloxamer 188, pH 3.5 with 2 M NaCl. In certain embodiments the recombinant AAV particles are AAV9, AAV8, AAV6, or AAV5 particles.

[0117] In embodiments, column bed volume of the cation exchange column is proportionately sized to the volume of the initial lysate containing the AAV particles based upon a bed volume of 800 mL for approximately about 200 L to about 250 L of the initial lysate, processed or unprocessed, containing AAV particles. For example, a cation exchange column with a bed volume of 800 mL can be used where the initial lysate containing AAV particles is about 200 L to about 250 L. Alternatively, a cation exchange column with a bed volume of 1 mL can be used where the initial lysate containing AAV particles is about 250 mL to about 313 mL. Alternatively, a cation exchange column with a bed volume of 8 mL can be used where the initial lysate containing AAV particles is about 2 L to about 2.5 L. Alternatively, a cation exchange column with a bed volume of 40 mL can be used where the initial lysate containing AAV particles is about 10 L to about 12.5 L. Alternatively, a cation exchange column with a bed volume of 80 mL can be used where the initial lysate containing AAV particles is about 20 L to about 25 L. Alternatively, a cation exchange column with a bed volume of 400 mL can be used where the initial lysate containing AAV particles is about 100 L to about 125 L. Alternatively, a cation exchange column with a bed volume of 4 L can be used where the initial lysate containing AAV particles is about 1,000 L to about 1,250 L. Alternatively, a cation exchange column with a bed volume of 8 L can be used where the initial lysate containing AAV particles is about 2,000 L to about 2,500 L.

[0118] In embodiments, cation exchange (CEX) chromatography is conducted under conditions that create a salt or pH gradient. For example, cation exchange (CEX) chromatography may be conducted using an elution buffer solution that creates a salt gradient ranging from a concentration of about 200 mMNaCl to a final concentration of about 2 M NaCl, or, alternatively, an elution buffer solution that creates a pH gradient ranging from a pH of about 3.5 to a final pH of about 9.3 to produce the third AAV particle composition. In certain embodiments the recombinant AAV particles are AAV9, AAV8, AAV6, or AAV5 particles.

[0119] In certain embodiments, column exchange (CEX) chromatography is carried out as follows. Clarified flocculate from approximately 50 L to 100 L of lysate containing AAV particles is loaded onto a 400-mL CEX monolith column (CIMmultus S03-400 mL, 6-pm pore column, BIA Separations 814.6157-6) at 2.8 L / min (7 CV / min) at less than 3.9 bar. The column is washed for 20 column volumes (“CV”) of CEX Buffer A prior to initiating the elution salt gradient. AAV particles are eluted at 1.2 L / min (3 CV / min) with a gradient from 0% to 100% CEX Buffer B (20mM formate, 2 M NaCl, 0.2% poloxamer 188 (P188) at pH 3.5) over 20 CV (elution may conducted at a slower rate to allow for manual sample collection, but elution could also be conducted at about 6 to about 10 column volumes per minute where manual sample collection is not needed). The peak vector fraction is collected manually beginning with a sudden rise in absorbance at 260 nm for about 3.2 CV, when the absorbance at 260 nm returned to nearly baseline on the downward slope of the peak. The pH of the peak fraction is adjusted to 8.0 + / - 0.5 by addition of 1 M Tris at pH 9.0 to produce a Neutralized CEX Eluate (N. CEX Eluate) containing AAV particles, which may be stored at 2-8 °C prior to further processing. In certain embodiments the clarified composition from the flocculation step is obtained from HEK293 cells and the AAV particles are AAV9, AAV8, AAV6, or AAV5 particles.

[0120] In certain embodiments, column exchange (CEX) chromatography is carried out as follows. A 400-mL CEX monolith column (CIMmultus S03-400 mL, 6-pm pore column, BIA Separations 814.6157-6) (or a column with a bed volume appropriate for the amount of starting material as taught herein) is equilibrated with CEX Buffer Al (20 mM formate, 0.5 M NaCl, 0.2% Pl 88 at pH 3.5). The clarified flocculate (CEX Load) from approximately 50 L to 100 L of lysate containing AAV particles is loaded onto a 400-mL CEX monolith column at 2.8 L / min (7 CV / min) with at less than 3.9 bar. The column is washed for 30 CV with CEX Buffer Al following loading. The column is then washed with the no-salt CEX Buffer A2 (20 mM formate, 0.2% Pl 88 at pH 3.5) for 10 CV prior to initiating the pH elution gradient. AAV vector is eluted at 1.2 L / min (3 CV / min) with a pH gradient from 100 % CEX Buffer A2 (pH 3.5) to 100% CEX Buffer Bl (20 mM BTP, 0.2% Pl 88 at pH 9.3) over 20 CV (elution may be conducted at a slower rate to allow for manual sample collection, but elution could also be conducted at about 6 to about 10 column volumes per minute where manual sample collection is not needed). The vector peak fraction is collected manually beginning with a sudden rise in absorbance at 260 and 280 nm (about 60 mAU) and ending when the absorbance at 260 nm returned to about 60 mAU on the downward slope of the peak. The pH of the peak vector eluate fraction is about 8.1. The CEX Eluate may be stored at 2-8 °C prior to further processing. In certain embodiments the clarified composition from the flocculation step is obtained from HEK293 cells and the AAV particles present in the clarified flocculate are AAV9, AAV8, AAV6, or AAV5 particles.

[0121] In embodiments, cation exchange chromatography results in a about a one log reduction, 2 log reduction or 3 log reduction (including by about 70%, 80%, 90%, 95%, 99% or99.9%) in host cell protein (that is in comparison to the material loaded onto the cation exchange column, the clarified flocculate). The reduction in host cell protein may be normalized to a set number of rAAV particles in the preparation before and / or after the flocculation step (for example, normalized to 1 x 1013vg detected). See, e.g. Example 4 and Table 1. Residual host cell protein levels may be assessed by any method known in the art. For example, host cell protein levels may be quantitated by an ELISA assay for host cell (HEK293 cells protein (see, e.g., Example 5, Table 2, columns 3 and 4)). In certain embodiments AAV9, AAV8, AAV6, or AAV5 particles are purified using the cation exchange chromatography described herein.Anion Exchange (AEX) Chromatography

[0122] Any strategy for purifying the composition containing the AAV particles on an AEX chromatography column may be utilized, and AEX chromatography columns comprising various resins, flow capacities, and pore sizes can be used. In embodiments, poly(glycidyl methacrylate - co- ethylene dimethacrylate) AEX columns may be used; for example the CIMmultus QA-400 mL, 2-pm pore column, BIA Separations 814.5113-2 (https: / / shop.sartorius.com / us / p / cimmultus- qa-400-ml-monolithic-column-2-m / 814,5113-2). In embodiments, cross-linked, regenerated macro-porous cellulose with primary amine ligand AEX columns may be used; for example, the 75mL Sartobind STIC® PA filter (Sartorius) (https: / / shop.sartorius.com / usZp / sartobind-stic- pa / M_Sartobind_STIC_PA). In certain embodiments AAV9, AAV8, AAV6, or AAV5 particles are further purified using anion exchange chromatography.

[0123] In embodiments, column bed volume of the anion exchange column is proportionately sized to the volume of the initial lysate containing the AAV particles based upon a bed volume of 800 mL for approximately 200 L to about 250 L of the initial lysate, processed or unprocessed, containing AAV particles. For example, an anion exchange column with a bed volume of 800 mL can be used where the initial lysate containing AAV particles is about 200 L to about 250 L. Alternatively, an anion exchange column with a bed volume of 1 mL can be used where the initial lysate containing AAV particles is about 250 mL to about 313 mL. Alternatively, an anion exchange column with a bed volume of 8 mL can be used where the initial lysate containing AAV particles is about 2 L to about 2.5 L. Alternatively, an anion exchange column with a bed volume of 40 mL can be used where the initial lysate containing AAV particles is about 10 L to about 12.5 L. Alternatively, an anion exchange column with a bed volume of 80 mL can be used where theinitial lysate containing AAV particles is about 20 L to about 25 L. Alternatively, an anion exchange column with a bed volume of 400 mb can be used where the initial lysate containing AAV particles is about 100 L to about 125 L. Alternatively, an anion exchange column with a bed volume of 4 L can be used where the initial lysate containing AAV particles is about 1,000 L to about 1,250 L. Alternatively, an anion exchange column with a bed volume of 8 L can be used where the initial lysate containing AAV particles is about 2,000 L to about 2,500 L.

[0124] In embodiments, anion exchange (AEX) chromatography is conducted under conditions that use a salt gradient to elute the AAV particles, including for example AAV9, AAV8, AAV6, or AAV5 particles, from the anion exchange column in the anion exchange eluate. For example, anion exchange (AEX) chromatography may be conducted using an elution buffer solution that creates a salt gradient ranging from a concentration of about 0 mM NaCl to a final concentration of about 1 M NaCl, to elute the AAV particles and results in an anion exchange eluate containing AAV particles.

[0125] In some embodiments (for example, as described below in Example 1), the cation exchange (CEX) chromatography eluate containing AAV particles, for example AAV9, AAV8, AAV6, or AAV5 particles, is neutralized and then further purified by tangential flow filtration (TFF) to provide the starting material for further processing using anion exchange (AEX) chromatography. In certain embodiments, the pH of the cation exchange (CEX) chromatography eluate containing AAV particles, including for example AAV9 particles, is adjusted to 8.0 + / - 0.5 by addition of 1 M Tris at pH 9.0 to produce the Neutralized CEX Eluate (N. CEX Eluate), and the N. CEX Eluate is held overnight at 2-8 °C. The N. CEX Eluate is then buffer exchanged into AEX Buffer A (20 mM Bis-Tris Propane (BTP), 1% sorbitol, 0.2% Pl 88 at pH 9.3) using a KrosFlo KR2i tangential flow system with a 0.1 m2HyStream, 100 kDa cassette. The N. CEX Eluate is concentrated 3-fold by ultrafiltration (UF) at a feed rate of 400 mL / min and a transmembrane pressure (TMP) target of 5 psi (5 to 10 psi range). The UF retentate is diafiltered for eight diavolumes at the same pump rate and TMP as the UF step. The retentate is recirculated with the permeate port closed and collected prior to flushing the system with three times the system hold-up volume. The flush is pooled with the primary retentate to form the TFF1 retentate pool (AEX Load). The TFF1 retentate pool (AEX Load) is loaded onto a 400-mL AEX monolith column (CIMmultus QA-400 mL, 2-pm pore column, BIA Separations 814.5113-2) at 0.4 L / min (1 CV / min) at less than 3.9 bar. The column is washed for 30 CV with AEX Buffer A (20 mMBis-Tris Propane (BTP), 1% sorbitol, 0.2% P188 at pH 9.3) following loading prior to initiating the elution gradient. AAV vector, including for example AAV9, AAV8, AAV6, or AAV5 vector, is eluted at 0.4 L / min (1 CV / min) with a gradient from 9% to 100% AEX Buffer B (20 mM BTP, 132 mMNaCl, 1% sorbitol, 0.2% Pl 88 at pH 9.3) over 30 CV. The peak vector fraction is collected manually beginning with a sudden rise in absorbance at 280 and 260 nm until the 260 nm absorbance crossed below the 280 nm absorbance on the downward slope of the peak. The peak fraction is about 8 CV. The pH of the peak fraction is adjusted to a pH of about 7.8 by addition of 1 M Tris at pH 6.0 to produce a Neutralized AEX Eluate (N. AEX Eluate). The N. AEX Eluate may be held overnight at 2-8 °C.

[0126] In embodiments, cation exchange (CEX) chromatography eluate containing AAV particles (as described above), including for example AAV9, AAV8, AAV6, or AAV5 particles, provides the starting material for further processing using anion exchange (AEX) chromatography, without an intervening tangential flow filtration (TFF) step. In certain embodiments (for example, as described below in Examples 2 and 3), anion exchange (AEX) chromatography to further purify the AAV particles, including for example AAV9, AAV8, AAV6, or AAV5 particles, recovered in the CEX eluate (without an intervening tangential filtration (TFF) step) is carried out as follows. The pH of the CEX eluate is adjusted to pH 9.3 by a 2% (w / w) addition of 1 M BTP at pH 10.0. The pH adjusted CEX eluate is loaded without dilution onto the 400-mL AEX monolith column (CIMmultus QA-400 m , 2-p.m pore column, BIA Separations 814.5113-2) that has been preequilibrated with AEX Buffer A (20 mM BTP, 1% sorbitol, 0.2% P188 at pH 9.3) at 0.4 L / min (1 CV / min) at less than 3.9 bar. The column is washed for 30 CV with AEX Buffer A prior to initiating the elution gradient. AAV vector was eluted at 0.4 L / min (1 CV / min) with a 30 CV gradient from 9% to 100% AEX Buffer B (20 mM BTP, 132 mM NaCl, 1% sorbitol, 0.2% P188 at pH 9.3). The peak vector fraction is collected manually beginning with a sudden rise in absorbance at 260 and 280 nm until the 260 nm absorbance crossed below the 280 nm absorbance on the downward slope of the peak. The peak fraction is about 10 CV. The pH of the peak fraction is adjusted to a pH of about 8.0 by addition of 1 M Tris at pH 6.0 to produce a Neutralized AEX Eluate (N. AEX Eluate).

[0127] In certain embodiments (for example, as described below in Example 3), the N. AEX Eluate (prepared as described above) is subjected to a second round of anion exchange (AEX) chromatography to further purify the AAV particle composition, including for example an AAV9,AAV8, AAV6, or AAV5 particle composition. In certain embodiments, this second round of anion exchange (AEX) chromatography is carried out as follows. N. AEX Intermediate is diluted approximately 8-fold with seven times the eluate weight of AEX Buffer A to bring the conductivity down to less than 2 mS / cm (for example about 1.6 mS / cm) and a pH of 9.4. The diluted AEX eluate is loaded onto a QA column equilibrated with AEX Buffer A for a second AEX purification cycle under the same loading, wash, and elution conditions listed above for the first AEX cycle. This second round of AEX purification further reduces host-cell protein and enriches for full AAV capsids with minimal loss of full AAV capsids. The pH of the peak fraction is adjusted to a pH of about 8.0 by addition of 1 M Tris at pH 6.0 to produce the Neutralized AEX Eluate (N. AEX Eluate).Tangential Flow Filtration (TFF)

[0128] In some embodiments, tangential flow filtration (TFF) is used to concentrate the AAV particle composition, remove impurities, and / or transfer the AAV particle composition into a buffer appropriate for further processing. In some embodiments, tangential flow filtration (TFF) is performed prior to final filtration for bioburden reduction to produce a drug substance containing purified AAV particles. In some embodiments, tangential flow filtration (TFF) is also performed as an intermediary purification step (for example, between a cation exchange (CEX) chromatography step and an anion exchange (AEX) chromatography step). In certain embodiments TFF is used to concentrate and remove impurities from an AAV9, AAV8, AAV6, or AAV5 particle composition.

[0129] When processed using tangential flow filtration (TFF), the bulk of the AAV particle composition flows tangentially across the surface of the filter, minimizing membrane fouling while maintaining a high filtration rate. TFF systems are known in the art and commercially available systems are sold by, for example, Millipore, Repligen, Sartorius and Pall. The filter matrix can be any appropriate matrix known in the art, for example, regenerated cellulose or PES, with a lOOkDa MW cutoff. The transmembrane pressure, pump and feed rates can be any appropriate rates known in the art, for example, TMP of 5 to 10 psig and a flow rate of 4-6 L / min / m2filter area.

[0130] In some embodiments (for example, as described below in each of Examples 1, 2, and 3) tangential flow filtration (TFF) is performed prior to final filtration for bioburden reduction to produce a drug substance containing purified AAV particles, such as for example AAV9, AAV8,AAV6, or AAV5 particles. In certain embodiments, this tangential flow filtration (TFF step) is carried out as follows. N. AEX Eluate containing AAV particles (prepared as described herein) is buffer exchanged into Formulation Buffer (10 mM Tris, ImM MgCh, 150 mM NaCl, 0.02% poloxamer 188, at a pH of about 8.0) using the KrosFlo KR2i tangential flow system with a 0.1 m2 Hy Stream, 100 kDa cassette. The N. AEX Eluate is concentrated about 38-fold by ultrafiltration (UF) at a feed rate of 400 mL / min and a TMP target of 5 psi (5 to 10 psi range). The UF retentate is diafiltered for eight diavolumes at the same pump rate and TMP as the UF step. The retentate is recirculated sampled for vector concentration determination by Nanodrop optical density assay. The retentate vector concentration is used to determine if further concentration or dilution is needed prior to dilution with the final system chase buffer.

[0131] In some embodiments (for example, as described below in Example 1), and those using AAV9, AAV8, AAV6, or AAV5 particles for example, tangential flow filtration (TFF) may also be performed between the cation exchange (CEX) chromatography step and the anion exchange (AEX) chromatography step. In certain embodiments, this tangential flow filtration (TFF step) is carried out as follows. N. CEX Eluate (prepared as described herein) is buffer exchanged into AEX Buffer A (20 mM Bis-Tris Propane (BTP), 1% sorbitol, 0.2% Pl 88 at pH 9.3) using a KrosFlo KR2i tangential flow system with a 0.1 m2Hy Stream, 100 kDa cassette. The N. CEX Eluate is concentrated 3-fold by ultrafiltration (UF) at a feed rate of 400 mL / min and a transmembrane pressure (TMP) target of 5 psi (5 to 10 psi range). The UF retentate is diafiltered for eight diavolumes at the same pump rate and TMP as the UF step. The retentate is recirculated with the permeate port closed and collected prior to flushing the system with three times the system holdup volume. The flush is pooled with the primary retentate to form a composition containing AAV particles that may be subjected to further processing.

[0132] In some embodiments tangential flow filtration (TFF) is used to concentrate the AAV particle composition, remove impurities and transfer the AAV particle composition into a buffer appropriate for the final formulation. In some embodiments, that buffer is 10 mM Tris, 1 mM MgCb, 150 mM NaCl, and 0.02% poloxamer 188, pH 8.0. The AAV particles may be AAV9, AAV8, AAV6, or AAV5 particles.Filtration / Drug Substance Production

[0133] In some embodiments, the AAV particle composition, including for example an AAV9, AAV8, AAV6, or AAV5 particle composition, processed as described herein is fdtered through a 0.2 pm sterilizing filter, such as a 0.22 pm PES or PVDF filter to produce a drug substance containing AAV particles with reduced bioburden. In some embodiments, the AAV particle composition is filtered using a peristaltic pump.Final Formulation

[0134] Provided are formulations of the rAAV drug substance or drug product manufactured by the methods described herein that meet yield and purity limitations. After formulation, the drug product may be sterile filtered and filled into containers suitable for long term storage. These containers may include glass, cyclic olefin polymer (COP), cyclic olefin copolymer (COC), or similar vials using an inert layer, such as organosilicate, deposited on the interior of the vial. Once filled and sealed, the drug product may be stored at suitable temperatures including frozen at < - 80°C, < -60°C, < -40°C, or < -20°C or liquid at 2 - 8 °C, 15 - 25 °C, or 2 - 25 °C. In certain embodiments the drug substance or drug product comprises an rAAV9, rAAV8, rAAV6, or rAAV5 composition.

[0135] In some embodiments, the final formulation has greater than about 80% full capsids of the total capsids in the formulation as measured by AUC. In some embodiments, the final formulation has greater than about 85% full capsids as measured by AUC. In some embodiments, the final formulation has greater than about 90% full capsids as measured by AUC. In some embodiments, the final formulation has greater than about 91% full capsids as measured by AUC. In some embodiments, the final formulation has greater than about 92% full capsids as measured by AUC. In some embodiments, the final formulation has greater than about 93% full capsids as measured by AUC. In some embodiments, the final formulation has greater than about 94% full capsids as measured by AUC. In some embodiments, the final formulation has greater than about 95% full capsids as measured by AUC. In some embodiments, the final formulation has greater than about 96% full capsids as measured by AUC. In some embodiments, the final formulation has greater than about 97% full capsids as measured by AUC. In some embodiments, the final formulation has greater than about 98% full capsids as measured by AUC. In embodiments, the final drug substance formulation is no more than about 99% full capsids or the capsids that are notfull capsids are undetectable by AUC. In some embodiments, the final formulation has up to about 90%, 95%, 98%, 99% or 100% full capsids as measured by AUC. In certain embodiments, the final formulation comprises AAV9, AAV8, AAV6, or AAV5 particles. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting drug substance composition contains about 80% to about 95%, or about 85% to about 93% full capsids, as determined by SV- AUC analysis. In embodiments in which the drug substance is an scAAV, and may have a dimer to monomer ratio as determined by long-read Next Generation Sequencing, as described in Example 10, of at least about 10 to 1, at least about 20 to 1, or at least about 40 to 1.

[0136] In some embodiments, the final formulation has greater than about 70% full capsids (of the total capsids in the formulation) as measured by CDMS. In some embodiments, the final formulation has greater than about 85% full capsids as measured by CDMS. In some embodiments, the final formulation has greater than about 90% full capsids as measured by CDMS. In some embodiments, the final formulation has greater than about 91% full capsids as measured by CDMS. In some embodiments, the final formulation has greater than about 92% full capsids as measured by CDMS. In some embodiments, the final formulation has greater than about 93% full capsids as measured by CDMS. In some embodiments, the final formulation has greater than about 94% full capsids as measured by CDMS. In some embodiments, the final formulation has greater than about 95% full capsids as measured by CDMS. In some embodiments, the final formulation has greater than about 96% full capsids as measured by CDMS. In some embodiments, the final formulation has greater than about 97% full capsids as measured by CDMS. In some embodiments, the final formulation has greater than about 98% full capsids as measured by CDMS. In some embodiments, the final formulation has greater than about 99% full capsids as measured by CDMS. In embodiments, the final drug substance formulation is no more than about 99% full capsids or the capsids that are not full capsids are undetectable by CDMS. In some embodiments, the final formulation has up to about 90%, 95%, 98%, 99% or 100% full capsids as measured by CDMS. In certain embodiments, the final drug substance formulation comprises AAV9, AAV8, AAV6, or AAV5 particles. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting final drug substance formulation contains about 75% to about 90%, or about 77% to about 88% full capsids, as determined by CDMS.

[0137] In some embodiments, the final formulation has greater than about 70% full capsids (of the total capsids in the formulation) as measured by mass photometry (MP). In some embodiments,the final formulation has greater than about 80% full capsids as measured by MP. In some embodiments, the final formulation has greater than about 90% full capsids as measured by MP. In some embodiments, the final formulation has greater than about 91% full capsids as measured by MP. In some embodiments, the final formulation has greater than about 92% full capsids as measured by MP. In some embodiments, the final formulation has greater than about 93% full capsids as measured by MP. In some embodiments, the final formulation has greater than about 94% full capsids as measured by MP. In some embodiments, the final formulation has greater than about 95% full capsids as measured by MP. In some embodiments, the final formulation has greater than about 96% full capsids as measured by MP. In some embodiments, the final formulation has greater than about 97% full capsids as measured by MP. In some embodiments, the final formulation has greater than about 98% full capsids as measured by MP. In some embodiments, the final formulation has greater than about 99% full capsids as measured by MP. In embodiments, the final drug substance formulation is no more than about 99% full capsids or the capsids that are not full capsids are undetectable by MP. In some embodiments, the final formulation has up to about 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% full capsids as measured by MP. In certain embodiments, the AAV particles are AAV9, AAV8, AAV6, or AAV5. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting drug substance composition contains about 70% to about 90%, or about 73% to about 89% full capsids, as determined by MP.

[0138] In some embodiments, the final formulation has less than about 11% empty capsids (of the total capsids in the formulation) as measured by AUC. In some embodiments, the final formulation has less than about 9% empty capsids as measured by AUC. In some embodiments, the final formulation has less than about 8% empty capsids as measured by AUC. In some embodiments, the final formulation has less than about 7% empty capsids as measured by AUC. In some embodiments, the final formulation has less than about 6% empty capsids as measured by AUC. In some embodiments, the final formulation has less than about 5% empty capsids as measured by AUC. In some embodiments, the final formulation has less than about 4% empty capsids as measured by AUC. In some embodiments, the final formulation has less than about 3% empty capsids as measured by AUC. In some embodiments, the final formulation has less than about 2% empty capsids as measured by AUC. In some embodiments, the final formulation has less than about 1% empty capsids as measured by AUC. In embodiments, the final formulationhas an undetectable amount of empty capsids by AUC. In embodiments, the final formulation has up to about 10%, 5%, 4%, 3%, 2% or 1% empty capsids by AUC. In certain embodiments, the final formulation comprises AAV9, AAV8, AAV6, or AAV5 particles. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting drug substance composition contains about 2% to about 11% empty capsids, as determined by SV-AUC.

[0139] In some embodiments, the final formulation has less than about 20% empty capsids (of the total capsids in the formulation) as measured by CDMS. In some embodiments, the final formulation has less than about 9% empty capsids as measured by CDMS. In some embodiments, the final formulation has less than about 8% empty capsids as measured by CDMS. In some embodiments, the final formulation has less than about 7% empty capsids as measured by CDMS. In some embodiments, the final formulation has less than about 6% empty capsids as measured by CDMS. In some embodiments, the final formulation has less than about 5% empty capsids as measured by CDMS. In some embodiments, the final formulation has less than about 4% empty capsids as measured by CDMS. In some embodiments, the final formulation has less than about 3% empty capsids as measured by CDMS. In some embodiments, the final formulation has less than about 2% empty capsids as measured by CDMS. In some embodiments, the final formulation has less than about 1% empty capsids as measured by CDMS. In some embodiments, the final formulation has less than about 0.8% empty capsids as measured by CDMS. In some embodiments, the final formulation has less than about 0.6% empty capsids as measured by CDMS. In some embodiments, the final formulation has less than about 0.5% empty capsids as measured by CDMS. In some embodiments, the final formulation has less than about 0.4% empty capsids as measured by CDMS. In some embodiments, the final formulation has less than about 0.3% empty capsids as measured by CDMS. In embodiments, the final formulation has undetectable amounts of empty capsids by CDMS. In embodiments, the final formulation has up to about 10%, 5%, 4%, 3%, 2% or 1% empty capsids by CDMS. In certain embodiments, the final formulation comprises AAV9, AAV8, AAV6, or AAV5 particles. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting final drug substance formulation contains about 4% to about 16% empty capsids, as determined by CDMS.

[0140] In some embodiments, the final formulation has less than about 20% empty capsids (of the total capsids in the formulation) as measured by MP. In some embodiments, the final formulation has less than about 9% empty capsids as measured by MP. In some embodiments, thefinal formulation has less than about 8% empty capsids as measured by MP. In some embodiments, the final formulation has less than about 7% empty capsids as measured by MP. In some embodiments, the final formulation has less than about 6% empty capsids as measured by MP. In some embodiments, the final formulation has less than about 5% empty capsids as measured by MP. In some embodiments, the final formulation has less than about 4% empty capsids as measured by MP. In some embodiments, the final formulation has less than about 3% empty capsids as measured by MP. In some embodiments, the final formulation has less than about 2% empty capsids as measured by MP. In some embodiments, the final formulation has less than about 1% empty capsids as measured by MP. In some embodiments, the final formulation has less than about 0.8% empty capsids as measured by MP. In some embodiments, the final formulation has less than about 0.6% empty capsids as measured by MP. In some embodiments, the final formulation has less than about 0.5% empty capsids as measured by MP. In some embodiments, the final formulation has less than about 0.4% empty capsids as measured by MP. In some embodiments, the final formulation has less than about 0.3% empty capsids as measured by MP. In embodiments, the final formulation has undetectable amounts of empty capsids by MP. In embodiments, the final formulation has up to about 10%, 5%, 4%, 3%, 2% or 1% empty capsids by MP. In certain embodiments, the AAV particles are AAV9, AAV8, AAV6, or AAV5. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting drug substance composition contains about 3% to about 17.5% empty capsids, as determined by MP.

[0141] In some embodiments, the final formulation has less than about 20% partial capsids (of the total capsids in the formulation) as measured by AUC. In some embodiments, the final formulation has less than about 15% partial capsids as measured by AUC. In some embodiments, the final formulation has less than about 10% partial capsids as measured by AUC. In some embodiments, the final formulation has less than about 5% partial capsids as measured by AUC. In some embodiments, the final formulation has less than about 4% partial capsids as measured by AUC. In some embodiments, the final formulation has less than about 3% partial capsids as measured by AUC. In some embodiments, the final formulation has less than about 2% partial capsids as measured by AUC. In some embodiments, the final formulation has less than about 1% partial capsids as measured by AUC. In embodiments, the final formulation has undetectable amounts of partial capsids as measured by AUC. In embodiments, the final formulation has up to about 10%, 5%, 4%, 3%, 2% or 1% partial capsids by AUC. In certain embodiments, the finalformulation comprises AAV9, AAV8, AAV6, or AAV5 particles. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting drug substance composition contains about 4% to about 5.5%, or about 4.5% to about 5.4% partial capsids, as determined by SV-AUC.

[0142] In some embodiments, the final formulation has less than about 20% partial capsids (of the total capsids in the formulation) as measured by CDMS. In some embodiments, the final formulation has less than about 15% partial capsids as measured by CDMS. In some embodiments, the final formulation has less than about 10% partial capsids as measured by CDMS. In some embodiments, the final formulation has less than about 5% partial capsids as measured by CDMS. In some embodiments, the final formulation has less than about 4% partial capsids as measured by CDMS. In some embodiments, the final formulation has less than about 3% partial capsids as measured by CDMS. In some embodiments, the final formulation has less than about 2% partial capsids as measured by CDMS. In some embodiments, the final formulation has less than about 1% partial capsids as measured by CDMS. In embodiments, the final formulation has undetectable amounts of partial capsids as measured by CDMS. In embodiments, the final formulation has up to about 10%, 5%, 4%, 3%, 2% or 1% partial capsids by CDMS. In certain embodiments, the final formulation comprises AAV9, AAV8, AAV6, or AAV5 particles. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting final drug substance formulation contains about 5% to about 9% partial capsids, or about 6% to about 8% partial capsids, as determined by CDMS.

[0143] In some embodiments, the final formulation has less than about 20% partial capsids (of the total capsids in the formulation) as measured by MP. In some embodiments, the final formulation has less than about 15% partial capsids as measured by MP. In some embodiments, the final formulation has less than about 10% partial capsids as measured by MP. In some embodiments, the final formulation has less than about 5% partial capsids as measured by MP. In some embodiments, the final formulation has less than about 4% partial capsids as measured by MP. In some embodiments, the final formulation has less than about 3% partial capsids as measured by MP. In some embodiments, the final formulation has less than about 2% partial capsids as measured by MP. In some embodiments, the final formulation has less than about 1% partial capsids as measured by MP. In embodiments, the final formulation has undetectable amounts of partial capsids as measured by MP. In embodiments, the final formulation has up to about 10%, 5%, 4%, 3%, 2% or 1% partial capsids by MP. In certain embodiments, the AAVparticles are AAV9, AAV8, AAV6, or AAV5. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting drug substance composition contains about 7% to about 10% partial capsids, or about 7.5% to about 10% partial capsids as determined by MP.

[0144] In some embodiments, the final formulation has greater than about 90% capsid purity as measured by SDS-PAGE or capillary electrophoresis-sodium dodecyl sulfate with laser induced fluorescence (CE-SDS LIF). In some embodiments, the final formulation has greater than about 92% capsid purity as measured by SDS-PAGE or CE-SDS LIF. In some embodiments, the final formulation has greater than about 93% capsid purity as measured by SDS-PAGE or CE-SDS LIF. In some embodiments, the final formulation has greater than about 94% capsid purity as measured by SDS-PAGE or CE-SDS LIF. In some embodiments, the final formulation has greater than about 95% capsid purity as measured by SDS-PAGE or CE-SDS LIF. In some embodiments, the final formulation has greater than about 96% capsid purity as measured by SDS-PAGE or CE-SDS LIF. In some embodiments, the final formulation has greater than about 97% capsid purity as measured by SDS-PAGE or CE-SDS LIF. In some embodiments, the final formulation has greater than about 98% capsid purity as measured by SDS-PAGE or CE-SDS LIF. In some embodiments, the final formulation has greater than about 99% capsid purity as measured by SDS-PAGE or CE-SDS LIF. In some embodiments, the final formulation has greater than about 99.5% capsid purity as measured by SDS-PAGE or CE-SDS LIF. In some embodiments, the final formulation has greater than about 99.8% capsid purity as measured by SDS-PAGE or CE-SDS LIF. In some embodiments, the final formulation has greater than about 99.9% capsid purity as measured by SDS-PAGE or CE-SDS LIF. In embodiments the final formulation has up to about 95%, 99% or 99.9% capsid purity by SDS-PAGE or CE-SDS LIF (or impurities are undetectable by SDS-PAGE or CE-SDS LIF). In embodiments, the final formulation has up to about 10%, 5%, 4%, 3%, 2% or 1% process or product impurities as measured by SDS-PAGE or CE-SDS LIF. In certain embodiments, the final formulation comprises AAV9, AAV8, AAV6, or AAV5 particles. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting final drug substance formulation contains about 98% to about 99.9%, or about 98.5% to about 99.5%, or about 98.5% to about 99.1% capsid purity, as measured by CE-SDS-LIF.

[0145] In some embodiments, the final formulation has greater than about 70% DNA reads of the transgene by short-read NGS. In some embodiments, the final formulation has greater than about 75% DNA reads of the transgene by short-read NGS. In some embodiments, the finalformulation has greater than about 80% DNA reads of the transgene by short-read NGS. In some embodiments, the final formulation has greater than about 85% DNA reads of the transgene by short-read NGS. In some embodiments, the final formulation has greater than about 90% DNA reads of the transgene by short-read NGS. In some embodiments, the final formulation has greater than about 95% DNA reads of the transgene by short-read NGS. In some embodiments, the final formulation has greater than about 98% DNA reads of the transgene by short-read NGS. In some embodiments, the final formulation has greater than about 99% DNA reads of the transgene by short-read NGS. In embodiments, the final formulation has up to about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% DNA reads of the transgene by short-read NGS. In certain embodiments, the final formulation comprises AAV9, AAV8, AAV6, or AAV5 particles. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting final drug substance formulation contains about 95% to about 98%, or about 96% DNA reads of the transgene by shortread NGS. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting final drug substance formulation contains about 2.5% to about 3.5%, or about 2.7% to about 3.4% of packaging plasmid backbone DNA reads by short-read NGS. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting final drug substance formulation contains about 0.2% to about 0.5%, or about 0.3% to about 0.43% reads of Rep / Cap and pHelp DNA reads by short-read NGS. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting final drug substance formulation contains about 0.4% to about 1.25%, or about 0.5% to about 1.22% of host cell DNA reads by short-read NGS.

[0146] In some embodiments, long-read next generation sequencing (NGS) is used to confirm the integrity and identity of the polynucleotide packaged in the rAAV particles in the harvested band material, particularly to assess whether the packaged nucleic acid includes the GOI expression cassette. “Full expression cassette” means the regulatory elements and GOI between and at least one or both ITR sequence or enough that the GOI is expressed. . In some embodiments, the final formulation has greater than about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% DNA reads of the full expression cassette by long read NGS. In embodiments, the final formulation has up to about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% DNA reads of the full expression cassette by long read NGS. In certain embodiments, the final formulation comprises AAV9, AAV8, AAV6, or AAV5 particles.In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting final drug substance formulation contains about 82% to about 85%, or about 83.8% to about 84.4% of full expression cassette DNA reads by long-read NGS. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting final drug substance formulation contains about 6.5% to about 9.0%, or about 6.9% to about 8.5% of partial expression cassette DNA reads by long-read NGS. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting final drug substance formulation contains about 5.0% to about 6.5%, or about 5.3% to about 6.4% of packaging plasmid DNA reads by long-read NGS. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting final drug substance formulation contains about 0.5% to about 0.9%, or about 0.6% to about 0.85% of Rep / Cap and pHelp DNA reads by long-read NGS. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting final drug substance formulation contains about 0.5% to about 0.75%, or about 0.56% to about 0.71% of host cell DNA reads by long-read NGS.

[0147] In some embodiments, the final formulation has less than about 2 x 105pg hcDNA / I x 1013vg. In some embodiments, the final formulation has less than about 1 x 105pg hcDNA / I x 1013vg. In some embodiments, the final formulation has less than about 5 x 104pg hcDNA / I x 1013vg. In some embodiments, the final formulation has less than about 1 x 104pg hcDNA / I x 1013vg. In some embodiments, the level of host cell DNA in the final formulation is undetectable. In some embodiments, the final formulation has up to about 2 x 105, 1 x 105, 5 x 104or 1 x 104pg hcDNA / I x 1013vg. In certain embodiments, the final formulation comprises AAV9, AAV8, AAV6, or AAV5 particles. In certain embodiments, the AAV particles are AAV9, AAV8, AAV6, or AAV5. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting drug substance composition contains about 0.5 x 103pg to about 1.5 x 103pg, or about 0.75 x 105pg to about 1.35 x 105pg, or about 0.8 x 105pg to about 1.25 x 105pg of hcDNA (normalized to 1 x 1013vg), as determined by qcPCR.

[0148] In some embodiments, the final formulation has less than about 15 ng HCP / I x 1013vg. In some embodiments, the final formulation has less than about 10 ng HCP / I x 1013vg. In some embodiments, the final formulation has less than about 9 ng HCP / I x 1013vg. In some embodiments, the final formulation has less than about 8 ng HCP / I x 1013vg. In some embodiments, the final formulation has less than about 7 ng HCP / I x 1013vg. In some embodiments, the final formulation has less than about 6 ng HCP / I x 1013vg. In someembodiments, the final formulation has less than about 5 ng HCP / I x 1013vg. In some embodiments, the final formulation has less than about 4 ng HCP / I x 1013vg. In some embodiments, the final formulation has less than about 3 ng HCP / I x 1013vg. In some embodiments, the final formulation has less than about 2 ng HCP / I x 1013vg. In some embodiments, the final formulation has less than about 1 ng HCP / I x 1013vg. In some embodiments, the amount of HCP in the final formulation is undetectable by methods described herein. In some embodiments, the final formulation has up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 15 ng HCP / I x 1013vg. In certain embodiments, the final formulation comprises AAV9, AAV8, AAV6, or AAV5 particles. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting drug substance composition contains from undetectable to about 10 ng, or about 1.0 ng to about 9.5 ng, or about 1 .5 ng to about 9.2 ng of HCP(normalized to 1 x 1013vg), as determined by ELISA.

[0149] In some embodiments, the recovery for scDNA AAV in the drug substance is greater than about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 27%, 30%, 32%, 35%, 40%, 45% or 50% of the AAV particles in the lysate. In some embodiments, the recovery for ssDNA AAV in the drug substance is greater than about 10%, 15%, 20%, 25%, 27%, 30%, 32%, 35%, 40%, 45% or 50% of the amount of AAV particles present in the lysate. In embodiments, the recoveries are up to about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of the AAV particles (scDNA) present in the lysate. In embodiments, the recoveries are up to about 10%, 15%, 20%, 25%, 27%, 30%, 32%, 35%, 40%, 45%, or 50% of the AAV particles (ssDNA) present in the lysate. In certain embodiments, the drug substance comprises AAV9, AAV8, AAV6, or AAV5 particles.

[0150] Total number of AAV vector genomes in the final drug substance can be determined based upon the lysate titer, for example, lysate titers of at least about 1 x 1011, 2 x 1011, 3 x 1011, 4 x 1011, 5 x 1011, 6 x 1011, 7 x 1011, 8 x 1011, 9 x 1011, 1 x 1012, 2 x 1012, or 3 x 1012vector genomes per mL (e.g., by ddPCR) in the lysate (and in embodiments, up to about 9 x 1011, 1 x 1012, 2 x 1012, or 3 x 1012vg rAAV particles per mL in the lysate) and calculated percent recovery relative to lysate vector genomes. This can be calculated using the formula lysate titer (vg / mL) x lysate volume (mL) x recovery (%) = drug substance yield (vg). The recoveries may be achieved for a starting lysate volume of from about 2L to about 1000L, including about 2L, about 5L, about 20L, about 50L, about 100L, about 200L, about 215L, about 500L, about lOOOL, 100L to OOL, or100L to 500L, or 200 to 500L, or 215 L or 500L. Total vector genome yields may also be calculated based upon unit volume of starting culture or lysate, for example, IL volume. In embodiments, the production process results in total viral production of 5 x IO13, 1 x 1016, 2 x 1016, 3 x 1016, 4 x 1016, 5 x 1016, 6 x 1016, or 7 x 1016vg for a 215L starting lysate or a comparable titer normalized to a 215 L lysate. In certain embodiments, the final drug substance comprises AAV9, AAV8, AAV6, or AAV5 particles. In an embodiment, the AAV particle is AAV9, AAV8, AAV6, or AAV5, and the resulting drug substance composition contains about 2.0 x 1013to about 3.0 x 1013, or about 2.2 x 1013to about 2.75 xlO13AAV9, AAV8, AAV6, or AAV5 vector genomes, as determined by ddPCR.

[0151] In some aspects, rAAV virion compositions can be formulated to reduce aggregation of rAAV virions in the composition, particularly where high rAAV virion concentrations are present (e.g., about 5xl013vg / ml or more depending on the serotype). Methods for reducing aggregation of rAAVs include, for example, addition of surfactants, pH adjustment, salt concentration adjustment, etc. (See, e.g., Wright FR, et al., Molecular Therapy (2005) 12, 171- 178.) In certain embodiments, the rAAV virion composition comprises AAV9, AAV8, AAV6, or AAV5 particles.

[0152] In some aspects, these formulations can contain at least about 0.005% of the active compound or more, although the percentage of the active ingredient(s) in a liquid formulation may, of course, be varied and can be conveniently be between about 0.005% and about 5% or about 5% or more of the volume of the total formulation. In some embodiments, the percentage of active compound can be between about 0.005% and about 1% wt / vol., between about 0.005% and about 0.5% wt / vol., between about 0.1% and about 0.75% wt / vol., or between about 0.01% and about 0.5% wt / vol., of the weight per volume of the total formulation. In embodiments, the percentage of active ingredient(s) in a lyophilized formulation before reconstitution for administration (wt active ingredient / wt total formulation) may be between about 0.1% to about 20% wt / wt, about 0.1% to about 10% wt / wt, about 0.1% to about 5% wt / wt or about 1% to 5% wt / wt. Naturally, the amount of active compound in each therapeutically-useful composition can be prepared in such a way that a suitable dosage can be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelflife, as well as other pharmacological considerations can be contemplated by one skilled in the art of preparingsuch pharmaceutical formulations, and as such, a variety of dosages and treatment regimens can be desirable.

[0153] In some aspects, it will be desirable to deliver the rAAV virions in suitably formulated pharmaceutical compositions as disclosed herein either subcutaneously, intrapancreatically, intranasally, parenterally, intravenously, intramuscularly, intrathecally, intraperitoneally, or intracerebroventricularly. In some aspects, the administration modalities as described in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 can be used to deliver rAAVs.

[0154] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases the form can be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, di saccharide, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, isotonic agents, for example, sugars or sodium chloride can be included. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0155] For administration of an injectable aqueous solution, for example, the solution can be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions can be suitable for intravenous, intramuscular, subcutaneous, intracerebroventricular, and intraperitoneal administration. In this connection, a sterile aqueous medium can be employed. For example, one dosage can be dissolved in 1 mL of isotonic NaCl solution and either added to 1000 mL of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington’s Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject. The person responsible for administration will, in any event, determine the appropriate dose for the subject.

[0156] Sterile injectable solutions can be prepared by incorporating the active rAAV virion, including AAV9, AAV8, AAV6, or AAV5 compositions for example, in the required amount in the appropriate solvent with various of the other ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions can be prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation can be vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0157] The rAAV virion compositions, including AAV9, AAV8, AAV6, or AAV5 compositions for example, disclosed herein can be also be formulated in a neutral or salt form. Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which can be formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine, and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations can be easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.

[0158] In embodiments, the drug substance, including AAV9, AAV8, AAV6, or AAV5 compositions for example, is formulated in a storage buffer or formulation buffer of 10 mM Tris, ImM MgCh, 150 mM NaCl, 0.02% poloxamer 188, at a pH of about 8.0.

[0159] As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. Supplementary active ingredients canalso be incorporated into the compositions. The phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a subject.

[0160] Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present disclosure into suitable host cells. In particular, the rAAV vector delivered transgenes can be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.

[0161] In some aspects, the methods can include administering one or more additional therapeutic agents to a subject who has been administered an rAAV or pharmaceutical composition as described herein.

[0162] In some aspects, administering the rAAV virions described to a subject promotes expression of GOI by 10-fold compared to a control. In some aspects, administering the rAAV virions described herein to a subject promotes expression of GOI by 5-fold to 100-fold compared to control (e.g., 5-fold to 10-fold, 10-fold to 15-fold, 10-fold to 20-fold, 15-fold to 25-fold, 20-fold to 30-fold, 25-fold to 35-fold, 30-fold to 40-fold, 35-fold to 45-fold, 40-fold to 60-fold, 50-fold to 75-fold, 60-fold to 80-fold, 75-fold to 100-fold compared to an untreated control or to the subject prior to the administration). In certain embodiments, administering the rAAV virions described to a subject comprises the delivery of rAAV9, rAAV8, rAAV6, or rAAV5 particles.

[0163] In some aspects, administering the rAAV virions described herein to a subject promotes expression of the GOI in a subject (e.g., promotes expression of the GOI in the CNS of a subject) by between a 5% and 200% increase (e.g., 5-50%, 25-75%, 50-100%, 75-125%, 100-200%, or 100-150% etc.) compared to a control subject or the subject prior to the administration. In certain embodiments, administering the rAAV virions described to a subject comprises the delivery of rAAV9, rAAV8, rAAV6, or rAAV5 particles.

[0164] As used herein, the term “treating” refers to the application or administration of a composition (e.g, an isolated nucleic acid or rAAV as described herein) to a subject who has a disease or disorder associated with low levels of GOI protein expression, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward a disease.

[0165] Alleviating a disease associated with low levels of GOI protein expression includes delaying the development or progression of the disease, or reducing disease severity. Alleviating the disease does not necessarily require curative results. As used therein, “delaying” the development of a disease means to defer, hinder, slow, retard, stabilize, and / or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and / or individuals being treated. A method that “delays” or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and / or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.

[0166] In particular, administration of the rAAV virion described herein to a human subject suffering from GOI protein deficiency will within 10 weeks, 1 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks, 50 weeks or 1 year after the administration will result in reduction in one or more biomarkers or hallmarks of the disease. In certain embodiments, administering the rAAV virions described to a subject comprises the delivery of rAAV9 , rAAV8, rAAV6, or rAAV5 particles.

[0167] As used herein, “Development” or “progression” of a disease means initial manifestations and / or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that can be undetectable. As used herein the terms development or progression refer to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein “onset” or “occurrence” of a disease can be associated with low levels of target protein expression.

[0168] In some aspects, the rAAV virions disclosed herein can be administered in sufficient amounts to transfect the cells of a desired tissue and to provide sufficient levels of gene transfer and expression without undue adverse effects.

[0169] In some embodiments, the administration, treating, contacting, or effective amount comprises at least, or no more than, about 104, 10?, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, or 1015virions / mL of the AAV. In certain embodiments, the administration, treating, contacting,or effective amount of the AAV comprises administration, treating, contacting, or effective amount of rAAV9, rAAV8, rAAV6, or rAAV5 particles.

[0170] As used herein, “immunosuppressed” or “immunosuppression” refers to a decrease in the activation or efficacy of an immune response in a subject. Immunosuppression can be induced in a subject using one or more (e.g., multiple, such as 2, 3, 4, 5, or more) agents, including, but not limited to, rituximab, methylprednisolone, prednisolone, sirolimus, immunoglobulin injection, prednisone, methotrexate, and any combination thereof.

[0171] In some aspects, methods disclosed herein can further comprise the step of inducing immunosuppression (e.g., administering one or more immunosuppressive agents) in a subject prior to the subject being administered an rAAV virion (e.g., an rAAV virion or pharmaceutical composition as disclosed herein). In some aspects, a subject can be immunosuppressed (e.g., immunosuppression is induced in the subject) between about 30 days and about 0 days (e.g., any time between 30 days until administration of the rAAV virion, inclusive) prior to administration of the rAAV virion to the subject. In some aspects, the subject can be pretreated with immune suppression agent (e.g., rituximab, sirolimus, and / or prednisone) for at least 7 days.

[0172] In some aspects, immunosuppression of a subject maintained during and / or after administration of a rAAV virion or pharmaceutical composition. In some aspects, a subject can be immunosuppressed (e.g., administered one or more immunosuppressants) for between 1 day and 1 year after administration of the rAAV virion or pharmaceutical composition.Kits

[0173] Disclosed herein are kits comprising any of the reagents, equipment, and components necessary to conduct the AAV purification methods described herein. In some aspects, any of the agents disclosed herein can be assembled into pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic, diagnostic or research applications. A kit can include one or more containers housing the components of the disclosure and instructions for use. Specifically, such kits may include one or more agents described herein, along with instructions describing the intended application and the proper use of these agents. In some aspects, the agents in a kit can be in a pharmaceutical formulation and dosage suitable for a particular application and for a methodof administration of the agents. Kits for research purposes can contain the components in appropriate concentrations or quantities for running various experiments.

[0174] Also disclosed herein are kits for producing rAAV virions, including AAV9, AAV8, AAV6, or AAV5 virions or particles. In some aspects, the kit can comprise a container housing an isolated nucleic acid encoding a target protein or a portion thereof. In some aspects, the kits can further comprise instructions for producing the rAAV virion. In some aspects, the kit further comprises at least one container housing a recombinant AAV vector, wherein the recombinant AAV vector comprises a transgene.

[0175] In some aspects, the kits can comprise a container housing a recombinant AAV particle as described supra. In some aspects, the kits can further comprises a container housing a pharmaceutically acceptable carrier. For example, a kit can comprise one container housing a rAAV virion and a second container housing a buffer suitable for injection of the rAAV virion into a subject. In some aspects, the container can be a syringe.

[0176] In some aspects, the kits can be designed to facilitate use of the methods described herein by researchers and can take many forms. Each of the compositions of the kit, where applicable, may be provided in liquid form (e.g., in solution), or in solid form, (e.g., a dry powder). In some aspects, some of the compositions can be constitutable or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water or a cell culture medium), which may or may not be provided with the kit. As used herein, “instructions” can define a component of instruction and / or promotion, and typically involve written instructions on or associated with packaging of the disclosure. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions can be associated with the kit, for example, audiovisual (e.g. , videotape, DVD, etc.), internet, and / or web-based communications, etc. The written instructions can be in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which instructions can also reflect approval by the agency of manufacture, use or sale for animal administration.

[0177] The kits disclosed herein can also contain any one or more of the components described herein in one or more containers. In some aspects, the kits can include instructions for mixing one or more components of the kit and / or isolating and mixing a sample and applying to a subject. Thekits can include a container housing agents described herein. The agents can be in the form of a liquid, gel or solid (powder). The agents can be prepared sterilely, packaged in syringe and shipped refrigerated. Alternatively, it can be housed in a vial or other container for storage. A second container can have other agents prepared sterilely. Alternatively the kits can include the active agents premixed and shipped in a syringe, vial, tube, or other container. The kits can have one or more or all of the components required to administer the agents to an animal, such as a syringe, topical application devices, or iv needle tubing and bag, particularly in the case of the kits for producing specific somatic animal models.

[0178] In some aspects the kits can include the buffers and reagents to carry out the procedures described herein. Non-limiting examples of such buffers and reagents may include salt, such as NaCl solutions; octanoic acid solutions, PEG powder or solution, CEX and AEX materials, flocculation mixer and tubing, other materials, buffers or reagents as useful, and instructions for use.

[0179] In some aspects, the method disclosed herein can involve transfecting cells with total cellular DNAs isolated from the tissues that potentially harbor proviral AAV genomes at very low abundance and supplementing with helper virus function (e.g., adenovirus) to trigger and / or boost AAV rep and cap gene transcription in the transfected cell. In some aspects, RNA from the transfected cells can provide a template for RT-PCR amplification of cDNA and the detection of novel AAVs. In cases where cells are transfected with total cellular DNAs isolated from the tissues that potentially harbor proviral AAV genomes, it is often desirable to supplement the cells with factors that promote AAV gene transcription. For example, the cells can also be infected with a helper virus, such as an Adenovirus or a Herpes Virus. In some aspects, the helper functions can be provided by an adenovirus. The adenovirus can be a wild-type adenovirus, and can be of human or non-human origin, for example, non-human primate (NHP) origin. Similarly, adenoviruses known to infect non-human animals (e.g., chimpanzees, mouse) can also be employed in the methods of the disclosure (See, e.g., U.S. Pat. No. 6,083,716). In addition to wild-type adenoviruses, recombinant viruses or non-viral vectors (e.g., plasmids, episomes, etc.) carrying the necessary helper functions can be utilized. Such recombinant viruses are known in the art and may be prepared according to published techniques. See, e.g., U.S. Pat. No. 5,871,982 and U.S. Pat. No. 6,251 ,677, which describe a hybrid Ad / AAV virus. A variety of adenovirus strains are available from the American Type Culture Collection, Manassas, Va., or available by request froma variety of commercial and institutional sources. Further, the sequences of many such strains are available from a variety of databases including, e.g., PubMed and GenBank.

[0180] Cells can also be transfected with a vector (e.g., helper vector) which provides helper functions to the AAV. The vector providing helper functions can provide adenovirus functions, including, e.g., Ela, Elb, E2a, E4ORF6. The sequences of adenovirus gene providing these functions can be obtained from any known adenovirus serotype, such as serotypes 2, 3, 4, 7, 12 and 40, and further including any of the presently identified human types known in the art. Thus, in some aspects, the methods involve transfecting the cell with a vector expressing one or more genes necessary for AAV replication, AAV gene transcription, and / or AAV packaging.

[0181] In some aspects, an isolated capsid gene can be used to construct and package recombinant AAV vectors, using methods well known in the art, to determine functional characteristics associated with the novel capsid protein encoded by the gene. For example, isolated capsid genes can be used to construct and package recombinant AAV (rAAV) vectors comprising a reporter gene (e.g., B-Galactosidase, GFP, Luciferase, etc.). The rAAV vector can then be delivered to an animal (e.g., mouse) and the tissue targeting properties of the isolated capsid gene can be determined by examining the expression of the reporter gene in various tissues (e.g., heart, liver, kidneys) of the animal. Other methods for characterizing isolated capsid genes are disclosed herein and still others are well known in the art.

[0182] The kits disclosed can have a variety of forms, such as a blister pouch, a shrink wrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, or a similar pouch or tray form, with the accessories loosely packed within the pouch, one or more tubes, containers, a box or a bag. The kits can be sterilized after the accessories are added, thereby allowing the individual accessories in the container to be otherwise unwrapped. The kits can be sterilized using any appropriate sterilization techniques, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art. The kits can also include other components, depending on the specific application, for example, containers, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves, a support for the agents prior to administration etc.

[0183] The instructions included within the kit can involve methods for detecting a latent AAV in a cell. In addition, kits of the disclosure can include, instructions, a negative and / or positivecontrol, containers, diluents and buffers for the sample, sample preparation tubes and a printed or electronic table of reference AAV sequence for sequence comparisons.EXAMPLES

[0184] The following examples are provided to illustrate embodiments of the claimed invention, and are not intended to limit the scope of the claimed invention to these specific examples. Persons of ordinary skill in the art can apply the disclosures and teachings presented herein to develop other embodiments without undue experimentation, and with a likelihood of success. All such embodiments disclosed in the examples below are considered part of the claimed invention.Example 1

[0185] HEK-293 cells were cultured in the presence of nucleotide sequences comprising (1) a gene expression cassette comprising a transgene encoding a GFP reporter gene operably linked to at least one regulatory sequence, flanked by AAV inverted terminal repeat (ITR) sequences; and (2) coding sequences for an AAV Rep, AAV Cap and adenovirus helper factors; to produce AAV particles comprising a nucleic acid, comprising the GFP reporter gene expression cassette capable of expressing the GFP reporter protein in a target cell upon transduction of the target cell with the AAV particles, packaged within an AAV capsid comprising the nucleic acid. The HEK-293 cells were then lysed according to methods known in the art to produce a cell lysate comprising full AAV capsids, process impurities and product impurities (alternatively referred to as the “first AAV particle composition” in the detailed description above).

[0186] Fifty -two kilograms of the cell lysate produced as described above were then processed as disclosed herein to drug substance (DS) by clarification, flocculation, clarification, cationexchange chromatography (CEX), tangential -flow filtration (TFF) buffer exchange, anion- exchange chromatography (AEX), TFF buffer exchange into formulation buffer, and filtration through a 0.2 pm filter for bioburden reduction to generate a purified drug substance (DS).

[0187] Lysate Clarification: The lysate was clarified with 6 m2of Meissner 1.0 pm pore glass-fiber filter (Protec Capsule Filter, HB, Sterile, GR2RF1-5NCC2) at 25 LMH (liters / m2 / hour) and chased with 15 kg of Lysis Clarification Buffer (20 mM Tris, 120 mM NaCl at pH 7.4).

[0188] Flocculation: The clarified lysate was then flocculated in a 100-L single-use mixer (SUM, Thermo Scientific, HyPerforma Single-use Mixer) by addition of 4 M NaCl and then mixing at 200 RPM for at least 10 minutes, followed by the addition of octanoic acid and then mixing for at least 10 additional minutes, such that the flocculation mixture had a salt concentration of 500 mM NaCl, and the octanoic acid concentration was 2% octanoic acid. The flocculation mixture was then acidified by the addition of 1 M formate at pH 2.5 to a target pH of 3.5 while mixing for at least 15 minutes. The flocculated mixture was allowed to stand for at least 30 minutes without mixing during which time a cream layer separated from the buffer layer and floated to the top of the solution.

[0189] The buffer layer was pumped out from the bottom of the single-use mixer (SUM) through 6 m2of Meissner 0.5 pm pore glass-fiber filter (Protec Capsule Filter, HB, Sterile, GR2RF0.5-5NCC2) at 25 LMH. The remaining cream layer in the SUM was rinsed with 20% of the flocculate weight (i.e., the weight of the lysate plus the weight of the NaCl, octanoic acid, and formate added above) of CEX Buffer A (20 mM formate, 0.5 MNaCl, 0.2% pol oxamer 188 (Pl 88) at pH 3.5) and mixed at 80 RPM for 10 minutes (the “cream rinse”). The cream rinse was allowed to stand without mixing for at least 30 minutes. The buffer layer of the cream rinse was then pumped through the same set of 0.5 pm filters at 25 liters per m2filter area per hour (“LMH”) and followed by the addition of 15 kg of CEX Buffer A through the same set of 0.5 pm filters at 25 LMH (the “filter chase”). The cream-rinse and filter-chase buffers were collected in the same bag as the initial clarified flocculate buffer layer to form the clarified flocculate that was also the CEX Load (alternatively referred to as the “second AAV particle composition” in the detailed description above). The combined pool was mixed at 215 RPM for 5 minutes before sampling.

[0190] CEX Chromatography: The clarified flocculate (CEX Load) (alternatively referred to as the “second AAV particle composition” in the detailed description above) was loaded onto a 400-mL CEX monolith column (CIMmultus S03-400 mL, 6-pm pore column, BIA Separations 814.6157-6) at 2.8 L / min (7 CV / min) at less than 3.9 bar. The column was washed for 20 column volumes (“CV”) of CEX Buffer A prior to initiating the elution gradient. AAV vector was eluted at 1.2 L / min (3 CV / min) with a gradient from 0% to 100% CEX Buffer B (20 mM formate, 2 M NaCl, 0.2% poloxamer 188 (P 188) at pH 3.5) over 20 CV (for this Example, elution was conducted at a slower rate to allow for manual sample collection, but elution could also be conducted at about 6 to about 10 column volumes per minute where sample collection is not needed). The peak vectorfraction was collected manually beginning with a sudden rise in absorbance at 260 nm for about 3.2 CV, when the absorbance at 260 nm returned to nearly baseline on the downward slope of the peak. The pH of the peak fraction was adjusted to 8.0 + / - 0.5 by addition of 1 M Tris at pH 9.0 to produce the Neutralized CEX Eluate (N. CEX Eluate) (alternatively referred to as the “third AAV particle composition” in the detailed description above). The N. CEX Eluate was held overnight at 2-8 °C.

[0191] TFF1 Buffer Exchange: The N. CEX Eluate (alternatively referred to as the “third AAV particle composition” in the detailed description above) was buffer exchanged into AEX Buffer A (20 mM Bis-Tris Propane (BTP), 1% sorbitol, 0.2% Pl 88 at pH 9.3) using a KrosFlo KR2i tangential flow system with a 0.1 m2HyStream, 100 kDa cassette. The N. CEX Eluate was concentrated 3 -fold by ultrafdtration (UF) at a feed rate of 400 mL / min and a transmembrane pressure (TMP) target of 5 psi (5 to 10 psi range). The UF retentate was diafiltered for eight diavolumes at the same pump rate and TMP as the UF step. The retentate was recirculated with the permeate port closed and collected prior to flushing the system with three times the system hold-up volume. The flush was pooled with the primary retentate to form the TFF1 retentate pool (AEX Load).

[0192] AEX Chromatography: The TFF1 retentate pool (AEX Load) was loaded onto a 400- mL AEX monolith column (CIMmultus QA-400 mL, 2-pm pore column, BIA Separations 814.5113-2) at 0.4 L / min (1 CV / min) at less than 3.9 bar. The column was washed for 30 CV with AEX Buffer A (20 mM Bis-Tris Propane (BTP), 1% sorbitol, 0.2% P188 at pH 9.3) following loading prior to initiating the elution gradient. AAV vector was eluted at 0.4 L / min (1 CV / min) with a gradient from 9% to 100% AEX Buffer B (20 mM BTP, 132 mM NaCl, 1% sorbitol, 0.2% P188 at pH 9.3) over 30 CV. The peak vector fraction was collected manually beginning with a sudden rise in absorbance at 280 and 260 nm until the 260 nm absorbance crossed below the 280 nm absorbance on the downward slope of the peak. The peak fraction was about 8 CV. The pH of the peak fraction was adjusted to a pH of about 7.8 by addition of 1 M Tris at pH 6.0 to produce the Neutralized AEX Eluate (N. AEX Eluate) (alternatively referred to as the “fourth AAV particle composition” in the detailed description above). The N. AEX Eluate was held overnight at 2-8 °C.

[0193] TFF1 Buffer Exchange: The N. AEX Eluate (alternatively referred to as the “fourth AAV particle composition” in the detailed description above) was buffer exchanged intoFormulation Buffer (20 mM Tris, 150 mM NaCl, 0.02% P188 at pH 8.0) using the KrosFlo KR2i tangential flow system with a 0.1 m2 Hy Stream, 100 kDa cassette. The N. AEX Eluate was concentrated about 38-fold by ultrafiltration (UF) at a feed rate of 400 mL / min and a TMP target of 5 psi (5 to 10 psi range). The UF retentate was diafiltered for eight diavolumes at the same pump rate and TMP as the UF step. The retentate (alternatively referred to as the “fifth AAV particle composition” in the detailed description above) was recirculated sampled for vector concentration determination by Nanodrop optical density assay. The retentate vector concentration was used to determine if further concentration or dilution was needed prior to dilution with the final system chase buffer.

[0194] Drug Substance Production: The retentate was then filtered through a 0.2 um membrane filter for bioburden reduction to produce the drug substance. The drug substance was sampled and frozen at less than -60 °C.Example 2

[0195] HEK-293 cells were cultured in the presence of nucleotide sequences comprising (1) a gene expression cassette comprising a transgene encoding a GFP reporter gene operably linked to at least one regulatory sequence, flanked by AAV inverted terminal repeat (ITR) sequences; and (2) coding sequences for an AAV Rep, AAV Cap and adenovirus helper factors; to produce AAV particles comprising a nucleic acid, comprising the GFP reporter gene expression cassette capable of expressing the GFP reporter protein in a target cell upon transduction of the target cell with the AAV particles, packaged within an AAV capsid comprising the nucleic acid. The HEK-293 cells were then lysed according to methods known in the art to produce a cell lysate comprising full AAV capsids, process impurities and product impurities (alternatively referred to as the “first AAV particle composition” in the detailed description above).

[0196] Fifty -four kilograms of the cell lysate produced as described above was then processed to drug substance without lysate clarification. The unclarified lysate was processed to drug substance (DS) by flocculation of the un-clarified lysate, flocculate clarification, cation-exchange chromatography (CEX), dilution of the CEX Eluate prior to anion-exchange chromatography (AEX), TFF buffer exchange into formulation buffer, and filtration through a 0.2 pm bioburden reduction filter to generate a purified drug substance (DS).

[0197] Note that there was no TFF step between the CEX and AEX steps described in Example 2 because the vector was eluted from the CEX column using a pH gradient with very little conductivity. Instead, the pH of the undiluted CEX Eluate was adjusted to 9.3 prior to loading directly onto the AEX column.

[0198] Lysate Flocculation: The unclarified cell lysate (alternatively referred to as the “first AAV particle composition” in the detailed description above) was warmed to 33.1 0C (range 30 to 37°C) and transferred to a 100-L single-use mixer (SUM, Thermo Scientific, HyPerforma Single-use Mixer), and flocculated by addition of 4 M NaCl and then mixing at 200 RPM for at least 10 minutes, followed by the addition of octanoic acid and then mixing for at least 10 additional minutes, followed by addition of a 40% 6000 MW PEG solution and then mixing for at least 10 additional minutes, such that the flocculation mixture had a salt concentration of 500 mM NaCl, the octanoic acid concentration was 2% octanoic acid, and the 6000 MW PEG concentration was 1%. The flocculation mixture was then acidified by the addition of 1 M formate at pH 2.5 to a target pH of 3.5 while mixing for at least 15 minutes. The flocculated mixture was allowed to stand for at least 30 minutes without mixing during which time a cream layer separated from the buffer layer and floated to the top of the solution.

[0199] The buffer layer was pumped out from the bottom of the single-use mixer (SUM) through 6 m2of Meissner 0.5 pm pore glass-fiber filter (Protec Capsule Filter, HB, Sterile, GR2RF0.5-5NCC2) at 25 LMH. The remaining cream layer in the SUM was rinsed with 20% of the flocculate weight (i.e., the weight of the lysate plus the weight of the NaCl, octanoic acid, and formate added above) of CEX Buffer A (20 mM formate, 0.5 M NaCl, 0.2% P188 at pH 3.5) and mixed at 80 RPM for 10 minutes. The cream rinse was allowed to stand without mixing for at least 30 minutes. The buffer layer of the cream rinse was then pumped through the same set of 0.5 pm filters at 25 liters per m2filter area per hour (“LMH”) and followed by the addition of 15 kg of CEX Buffer A through the same set of 0.5 pm filters at 25 LMH (the “filter chase”). The creamrinse and filter-chase buffers were collected in the same bag as the initial clarified flocculate buffer layer to form the clarified flocculate that was also the CEX Load (alternatively referred to as the “second AAV particle composition” in the detailed description above). The combined pool was mixed at 215 RPM for 5 minutes before sampling.

[0200] CEX Chromatography: The 400-mL CEX monolith column (CIMmultus S03-400 mL, 6-pm pore column, BIA Separations 814.6157-6) was equilibrated with CEX Buffer Al (20 mM formate, 0.5 M NaCl, 0.2% P188 at pH 3.5). The clarified flocculate (CEX Load) (alternatively referred to as the “second AAV particle composition” in the detailed description above) was loaded onto a 400-mL CEX monolith column at 2.8 L / min (7 CV / min) at less than 3.9 bar. The column was washed for 30 CV with CEX Buffer Al following loading. The column was then washed with the no-salt CEX Buffer A2 (20 mM formate, 0.2% P188 at pH 3.5) for 10 CV prior to initiating the pH elution gradient. AAV vector was eluted at 1.2 L / min (3 CV / min) with a pH gradient from 100 % CEX Buffer A2 (pH 3.5) to 100% CEX Buffer Bl (20 mM BTP, 0.2% P188 at pH 9.3) over 20 CV (for this Example, elution was conducted at a slower rate to allow for manual sample collection, but elution could also be conducted at about 6 to about 10 column volumes per minute where manual sample collection is not needed). The pH gradient was followed by a 10 CV hold with CEX Buffer Bl and then stepped to 100% high-salt CEX Buffer B2 (20 mM BTP, 1 M NaCl, 0.2% P188 at pH 9.3) for 20 CV. The vector peak fraction was collected manually beginning with a sudden rise in absorbance at 260 and 280 nm (about 60 mAU) and ending when the absorbance at 260 nm returned to about 60 mAU on the downward slope of the peak. The pH of the peak vector eluate fraction was 8.1. The CEX Eluate (alternatively referred to as the “third AAV particle composition” in the detailed description above) was held overnight at 2-8 °C.

[0201] TFF1 Buffer Exchange: No TFF step is required to adjust the CEX Eluate for loading onto the AEX column.

[0202] AEX Chromatography: The pH of the CEX eluate (alternatively referred to as the “third AAV particle composition” in the detailed description above) was adjusted to pH 9.3 by a 2% (w / w) addition of 1 M BTP at pH 10.0. The pH adjusted CEX eluate was loaded without dilution onto the 400-mL AEX monolith column (CIMmultus QA-400 mL, 2-pm pore column, BIA Separations 814.5113-2) that had been pre-equilibrated with AEX Buffer A (20 mM BTP, 1% sorbitol, 0.2% P188 at pH 9.3) at 0.4 L / min (1 CV / min) at less than 3.9 bar. AAV vector was eluted at 0.4 L / min (1 CV / min) with a 30 CV gradient from 9% to 100% AEX Buffer B (20 mM BTP, 132 mM NaCl, 1% sorbitol, 0.2% P188 at pH 9.3). The peak vector fraction was collected manually beginning with a sudden rise in absorbance at 260 and 280 nm until the 260 nm absorbance crossed below the 280 nm absorbance on the downward slope of the peak. The peak fraction was about 10 CV. The pH of the peak fraction was adjusted to a pH of about 8.0 byaddition of 1 M Tris at pH 6.0 to produce the Neutralized AEX Eluate (N. AEX Eluate) (alternatively referred to as the “fourth AAV particle composition” in the detailed description above).

[0203] Note that about half the volume of the N. AEX Eluate above was then processed further as described below in the remainder of Example 2 while the remainder of the volume of the N. AEX Eluate served as the starting point for the successive purification procedures described below in Example 3.

[0204] TFF1 Buffer Exchange: The N. AEX Eluate (alternatively referred to as the “fourth AAV particle composition” in the detailed description above) was buffer exchanged into Formulation Buffer (20 mM Tris, 150 mM NaCl, 0.02% P188 at pH 8.0) using the KrosFlo KR2i tangential flow system with a 0.1 m2 Hy Stream, 100 kDa cassette. The N. AEX Eluate was concentrated about 13 -fold by ultrafiltration (UF) at a feed rate of 400 mL / min and a TMP target of 5.0 psi (5.0 to 10.0 psi range). The UF retentate was diafiltered for greater than or equal to eight diavolumes at the same pump rate and TMP as the UF step. The retentate (alternatively referred to as the “fifth AAV particle composition” in the detailed description above) was recirculated and sampled for vector concentration determination by Nanodrop optical density assay. The retentate vector concentration was used to determine if further concentration or dilution was needed prior to dilution with the final system chase buffer.

[0205] Drug Substance Production: The retentate was then filtered through a 0.2 um membrane filter for bioburden reduction to produce the drug substance. The drug substance was sampled and frozen at less than -60 °C.Example 3

[0206] The starting material used for the purification procedures described in this Example 3 was about one-half of the volume of the N. AEX Eluate (alternatively referred to as the “fourth AAV particle composition” in the detailed description above) that was reserved as described in Example 2 above. Therefore, the Example 3 process includes all prior steps from lysis to N. AEX Eluate described in Example 2.

[0207] The N. AEX Intermediate (from Example 2) was diluted approximately 8-fold with seven times the eluate weight of AEX Buffer A to bring the conductivity down to less than 2mS / cm (Actual value was 1.6 mS / cm) and a pH of 9.4. The diluted AEX eluate was loaded onto a QA column equilibrated with AEX Buffer A for a second AEX purification cycle under the same loading, wash, and elution conditions listed above for the first AEX cycle. This two-cycle AEX process is the standard AEX process for this AEX embodiment to further reduce host-cell protein and enrich for full AAV capsids with minimal loss of full AAV capsids. The pH of the peak fraction was adjusted to a pH of about 8.0 by addition of 1 M Tris at pH 6.0 to produce the Neutralized AEX Eluate (N. AEX Eluate).

[0208] TFF1 Buffer Exchange: The second-cycle N. AEX Eluate was buffer exchanged into Formulation Buffer (20 mM Tris, 150 mM NaCl, 0.02% P188 at pH 8.0) using a KrosFlo KR2i system with a 0.1 m2HyStream, 100 kDa cassette. The N. AEX Eluate was concentrated about 13-fold by ultrafiltration (UF) at a feed rate of 400 mL / min and a TMP target of 5.0 psi (5.0 to 10.0 psi range). The UF retentate was diafiltered for greater than or equal to eight diavolumes at the same pump rate and TMP as the UF step. The retentate (alternatively referred to as the “fifth AAV particle composition” in the detailed description above) was recirculated sampled for vector concentration determination by Nanodrop optical density assay. The retentate vector concentration was used to determine if further concentration or dilution was needed prior to dilution with the final system chase buffer.

[0209] Drug Substance Production: The retentate was then filtered through a 0.2 um membrane filter for bioburden reduction to produce the drug substance. The drug substance was sampled and frozen at less than -60 °C.Example 4 — Viral Recovery

[0210] Recovery of AAV9 capsids containing the GFP reporter construct prepared as set forth above in Examples 1-3 is compared to recovery of AAV9 capsids containing the same GFP reporter construct after purification using methods that involved ultracentrifugation of the AAV particle composition on a cesium chloride (CsCl) gradient and harvesting a band containing “full” AAV capsids (“UC method”). In brief, to prepare the AAV particle composition using the UC method, lysate from mammalian cells used to produce AAV9 capsids containing the GFP reporter construct according to comparator methods is clarified by filtration through a glass-fiber filter, flocculated with addition of 4 M NaCl to 500 mM NaCl, addition of anionic surfactant to a concentration of 2%, and acidified to pH 3.5 by addition of 1 M formate at pH 2.5. The flocculateis clarified by filtration though a glass-fiber filter. The clarified flocculate is loaded onto a CEX column and eluted as CEX eluate. The CEX eluate is diluted and passed through an AEX filter in flow-through mode such that the AAV particles passed through the filter while residual HCP was bound to the filter. The AEX filtrate is concentrated by TFF and diafiltered to buffer exchange into ultracentrifuge (UC) buffer containing 3 M CsCl. The TFF retentate is loaded into UC tubes and spun overnight to separate the various density bands. The product band is extracted from the UC tubes and pooled prior to TFF buffer exchange and concentration. The TFF retentate pool is filtered to produce the drug substance.

[0211] Genomic titers are quantitatively determined using a ddPCR method. Samples are treated with DNase to remove any non-encapsi dated DNA before the capsids are lysed to release the encapsidated DNA for amplification during the ddPCR assay. Through the use of primers and probes that are specific to the GFP reporter gene, the released encapsidated DNA is quantified by ddPCR. Results are shown below in Table 1 and FIG. 1. Table 1 shows the amount of AAV particles, normalized for starting lysate volume, at different stages of the purification processTable 1. Recovery of AAV Capsids1. Recovery of CEX Eluate for Example 1 was determined to be 152%, which is assumed to be the result of an error in sampling and analyzing that particular sample.2. Examples 1-3 did not involve this AEX Filtration step.3. Examples 2-3 did not involve this initial TFF Pool step.4. Examples 1-3 did not involve this UC Product Pool step.Example 5 — Viral Titer and Purity

[0212] Titer and purity of AAV9 capsids containing the GFP reporter construct prepared as set forth above in Examples 1-3 are compared to titer and purity of AAV9 capsids containing the same GFP reporter construct prepared according to the UC method described above in Example 4.

[0213] Viral Genome per mL (vg / mL) is quantitatively determined using a ddPCR method. In brief, samples are treated with DNase to remove any non-encapsidated DNA before the capsids are lysed to release the encapsidated DNA. Through the use of primers and probes that are specific to the GFP reporter gene, the encapsidated DNA is quantified by ddPCR. The results for drug substances are shown below in column 2 of Table 2.

[0214] Viral Capsids (a measure of total AAV titer) are quantitatively determined using a commercially available ELISA kit. In brief, the Progen AAV9 Xpress Titration ELISA kit is AAV9 specific and uses a microplate with capture antibodies that detect a conformational epitope not present on unassembled AAV9 capsid proteins. Samples are diluted in assay buffer to achieve a target sample concentration on the order of 109UV AAV / mL, which is then used to prepare three serial dilutions that will all be within the range of the method (e.g., 5-fold, 10-fold, and 20-fold dilution). Three serial dilutions are plated in triplicate. Once the proteins are selectively captured, biotin-conjugated anti-AAV9 antibodies are added followed by the addition of Streptavidin-HRP, resulting in the formation of a complex that is detected colorimetrically when 3, 3’, 4,4’- tetramethylbenzidine (TMB) is added. The reaction is stopped using a stop buffer and the result is read using a plate reader at 450 nm. The AAV9 capsid concentration in capsids / mL is determined based on the response relative to the response of standards fit to a four-parameter curve. The assay range is defined as 1.6* 107capsids / mL to 2.9* 108capsids / mL, with a minimum dilution of 250-fold. The results from all three sample dilution levels must be within the range of the method prior to correcting for dilution. The results for drug substances are shown below in column 3 of Table 2.

[0215] The amount of residual HEK293 host cell protein (HCP) is determined using a commercially available ELISA kit. In brief, the Cygnus Technologies HEK293 HCP ELISA Kit quantifies HEK293 HCPs with a horseradish peroxidase (HRP)-labeled anti-HEK293 antibody in microtiter strips, which results in the formulation of a sandwich complex of solid phase antibody- HCP-enzyme-labeled antibody. After washing to remove any unbound reactants, TMB is addedto cause a color change. The reaction is stopped by the addition of acid, after which the color change is measured spectrophotometrically at 450 nm. The amount of hydrolyzed substrate is directly proportional to the concentration of HCPs present. The concentration is determined in comparison to a standard curve fit to a 4-parameter logistic curve. The results for drug substances are shown below in column 4 of Table 2.

[0216] The amount of host cell DNA is determined using a quantitative PCR (qcPCR) method. In brief, the amount of residual HEK293 host cell DNA is determined using commercially- available sample preparation and quantitative qPCR detection kits. The PrepSEQ residual DNA Sample Preparation Kit extracts host cell DNA through the use of chemical lysis and magnetic beads. Once extracted, the Thermo Fisher Scientific resDNASEQ™ Quantitative HEK293 DNA Kit is used to quantitatively determine the amount of HEK293 host cell DNA using a qPCR readout. The results for drug substances are shown below in column 5 of Table 2.

[0217] The amount of residual plasmid DNA (“pDNA”) is determined using a ddPCR method specific to the kanamycin resistance (KanR) gene region present in the transgene plasmid, AAV2 Rep and AAV9 Cap (RC9) plasmid, and helper plasmid. Samples are diluted into the operating range of the method and heated during thermal cycling to lyse the AAV capsids, resulting in the release of encapsidated plasmid DNA into solution for quantitation of total plasmid DNA by ddPCR. The results are shown below in column 6 of Table 2.Table 2. Titer And Purity of AAV Capsids at Drug Substance1. Below limit of quantitation of 4 ng / mL.Example 6 — Amount of Empty, Partial, or Full AAV9-GFP capsid as Determined by Sedimentation Velocity - Analytical Ultracentrifugation (SV-AUC) Analysis

[0218] The amount of empty, partial, or full AAV9 capsids containing the GFP reporter construct prepared as set forth above in Examples 1-3 are compared to the amount of empty, partial, or full AAV9 capsids containing the same GFP reporter construct prepared according to the UC method described above in Example 4. In brief, the relative amounts of empty, intermediate, and full capsid particles are determined by the SV-AUC technique, combined with SEDFIT analysis and ChemStation integration, to determine the sedimentation behavior of AAV samples. The data are used to derive (1) the distribution profiles of all species present in the sample, (2) the sedimentation coefficient values (S-values), and (3) the relative abundance of empty, intermediate, and full capsid particles. The percentage of empty, partial, and full capsids present in the initial CEX eluate for the various processes is shown below in columns 2, 3, and 4 of Table 3. The percentage of empty, partial, and full capsids present in the drug substances (DS) are shown below in columns 5, 6, and 7 of Table 3.Table 3. Relative Amount of Empty, Partial, or Full AAV9-GFP capsids as Determined by SV-AUC AnalysisExample 7 — Amount of Empty, Partial, or Full AAV9-GFP capsid as Determined byCharge Detection Mass Spectrometry (CDMS) and Mass Photometry (MP) Analysis

[0219] Charge detection mass spectrometry (CDMS) is used to determine the molecular weight and relative abundance of different capsid species present after AAV9 capsids containing the GFP reporter construct are prepared as set forth above in Examples 1-3, as compared to AAV9 capsids containing the same GFP reporter construct prepared according to the UC methoddescribed above in Example 4. In brief, samples are diluted and injected into a charge detection mass spectrometer, to determine the mass and charge of individual molecules. The number of particles are plotted as a function of mass in MDa. This distribution provides the relative abundance and molecular weight of each of the species present in the sample. The percentage of empty, partial, and full capsids as determined by CDMS for drug substances (DS) are shown below columns 2, 3, and 4 of Table 4.

[0220] Mass Photometry (MP) is also used to determine the molecular weight and relative abundance of different capsid species present after AAV9 capsids containing the GFP reporter construct are prepared as set forth above in Examples 1-3, as compared to AAV9 capsids containing the same GFP reporter construct prepared according to the UC method described above in Example 4. In brief, drug substance (DS) samples were analyzed with a Refeyn SamuxMP mass photometer, and measurements are taken at a wavelength of 488 nm. The analysis is conducted in a room where the temperature is maintained at around 20°C + / - 2°C. Samples are diluted to about 1 x 1011particles / mL with buffer containing 20 mM Tris, 150 mM NaCl, 2 mMMgC12, and 0.01% poloxamer 188 at pH 7.5. Ten microliters of sample are added to one of the wells of a 6-well slide and analyzed for about 60 seconds. This dilution targets about 500 to 1500 total counts of empty, partial, and full capsids per analysis. Windows were set based on empty and full peak positions and widths, accounting for mass (kDa) of the gene of interest (here, GFP). The counts for each sample are then assigned to empty, partial, and full capsid ranges to calculate the percentage of these species in the drug substance samples. The percentage of empty, partial, and full capsids as determined by MP for drug substances (DS) prepared as described above are shown below columns 5, 6, and 7 of Table 4.Table 4. Relative Amount of Empty, Partial, or Full AAV9-GFP capsids as Determined by Charge Detection Mass Spectrometry (CDMS) and Mass Photometry (MP) AnalysisExample 8-Aggregation and Protein Purity

[0221] Non-reduced capillary electrophoresis-sodium dodecyl sulfate laser-induced fluorescence (CE-SDS (LIF)) and size exclusion-high performance liquid chromatography (SE- HPLC) are used (respectively) to determine capsid purity and aggregation for AAV9 capsids containing the GFP reporter construct prepared as set forth above in Examples 1-3, in comparison to AAV 9 capsids containing the same GFP reporter construct prepared according to the UC method described above in Example 4.

[0222] Capsid purity is determined by using a non-reduced capillary electrophoresis-sodium dodecyl sulfate method with a laser-induced fluorescence (CE-SDS (LIF)) detector. In brief, capsid proteins are isolated and denatured under non-reducing conditions, complexed with SDS, and labeled with a fluorescent dye. These labeled protein-SDS complexes are separated by size when passing through the gel sieving matrix within a capillary by an applied electric field. The proteins are detected using a solid-state laser with an excitation wavelength of 488 nm and an emission filter of 600 nm. The percent purity is determined based on the corrected peak areas of viral protein 1 (VP1), viral protein 2 (VP2), and viral protein 3 (VP3) relative to the entire corrected peak area for all peaks in an injection. CE-SDS (LIF) results for drug substances from the various AAV purification methods are are shown below in column 2 of Table 5.

[0223] Aggregation of AAV capsid is determined by size exclusion-high performance liquid chromatography (SE-HPLC). In brief, samples are injected under isocratic conditions onto the SE-HPLC column and the individual sample components, consisting of monomeric and higher order viral capsid aggregates and fragements of viral capsids, are separated based on their respective molecular weights, with the larger components eluting first followed by the smaller components. The sample components are detected with a fluorescence light detector as they elute from the column, where light with a wavelength of 280 nm is used to excite fluorophores intrinsic to the sample, primarily tryptophan amino acid residues, and then the light emitted by the sample with a wavelength of 330 nm is detected and quantified. This assay was conducted on AAV9capsids containing the GFP reporter construct prepared as set forth above in Examples 1-3, in comparison to AAV 9 capsids containing the same GFP reporter construct prepared according to the UC method described above in Example 4, and the results are shown in FIG. 1. The fractions of aggregate viral capsids (high molecular weight, HMW), monomeric viral capsids, and fragments of viral capsids and host cell proteins (low molecular weight, LMW) are then calculated by summing the peak areas of the individual peaks that contribute to the aggregate, monomer, and fragment peak groups and dividing by the total peak area. SE-HPLC percent aggregation for the drug substances are shown below in column 3 of Table 5 and the SE-HPLC percent purity of monomers for the drug substances are shown below in column 4 of Table 5 for the various AAV purification methods.Table 5. Aggregation and Protein PurityExample 9 — DNA Quantity by Short-Read NGS

[0224] Short-read next-generation sequencing (“short-read NGS”) is used to measure the relative quantity of DNA from various DNA sources for AAV9 capsids prepared as set forth above in Examples 1-3, in comparison to AAV9 capsids prepared according to the (“UC”) purification method described above in Example 4. In brief, to perform short-read Next Generation Sequencing (NGS) by Illumina, DNA is extracted from AAV samples and testing is performed by Nanodrop to assess A260 / A280 and A260 / A230 ratios to verify the quality of the DNA starting material. Extracted DNA is assayed on the Agilent D5000 ScreenTape System to ensure recovery of expected DNA size to verify the quality of the DNA starting material. A sequencing library is prepared from extracted DNA by fragmentation of DNA and addition of specialized adapters toboth ends of DNA fragments. These adapters contain complementary sequences that allow DNA fragments to bind to flow cells used in the sequencing process. Samples can be multiplexed through the use of barcoded adapters. Libraries are loaded onto a flow cell and DNA fragments are amplified. In a process called sequencing by synthesis, chemically modified nucleotides bind to the DNA template that contain a fluorescent tag and reversible terminator that will block the incorporation of the next base. The fluorescent signal indicates which nucleotide has been added, then the terminator is cleaved so the next base can bind. This process is repeated to sequence the entire DNA fragment. Sequence results are compared against reference files representing all potential sources of DNA to determine sequence variants present in the DNA population and relative abundance / quantity of DNA fragments mapping to provided reference files. The results of short-read NGS analysis of the CEX eluate are shown below in Table 6. The results of short-read NGS of the drug substance are shown below in Table 7.Table 6. DNA Quality by Short Read NGS (CEX Eluate)1. As described above, half the volume of the N. AEX Eluate produced as described in Example 2 was further processed as described in Example 2 while the other half of the volume of the N. AEX Eluate eluate served as the starting point for the additional AEX purification and additional processing described in Example 3. As such, Short Read NGS results at the CEX Eluate stage are identical for Examples 2 and 3.Table 7. DNA Quality by Short Read NGS (Drug Substance)Example 10 — DNA Quality by Long-Read NGS

[0225] Long-read next-generation sequencing (“long-read NGS”) is used to measure the proportion of DNA sequence reads from varous DNA sources AAV9 capsids prepared as set forth above in Examples 1-3, in comparison to AAV9 capsids prepared according to the (“UC”) purification method described above in Example 4. In brief, for long-read NGS by PacBio, DNA is extracted from AAV samples and testing is performed by Nanodrop to assess A260 / A280 and A260 / A230 ratios to verify the quality of the DNA starting material. Extracted DNA is assayed on the Agilent D5000 ScreenTape System to ensure recovery of expected DNA size to verify the quality of the quality of the DNA starting material. Extracted DNA is assayed on the Agilent D5000 ScreenTape System to ensure recovery of expected DNA size. Recovered DNA is then repaired to produce blunt ends to be used for the ligation of barcoded adapters to each side of the molecules, resulting in a single stranded circular DNA configuration. Primers specific to the introduced adapter sequences are used to produce rolling circle amplification that results in many copies of the amplicon on a single DNA molecule. In a process called “sequencing by synthesis,” chemically modified nucleotides bind to the DNA template that contain a fluorescent tag. The fluorescent signal indicates which nucleotide has been added and is read in real time until the entire DNA molecule has been sequenced several times. Since many amplicons are sequenced from the same starting molecule, sequencing errors can be eliminated by producing a consensus sequence from the individual sub-reads. The consensus sequence can then be assessed to determine DNA sequences present in the AAV sample with high accuracy. Because this workflow does not prepare a fragmented DNA library, DNA molecule length and sequence of DNA in the capsids can be assessed across large DNA molecules. Following generation of raw data, sequencing files areanalyzed using an AAV script developed by PacBio to determine genomic integrity. The frequence of various categories of DNA reads mapping to DNA starting materials by long-read NGS for drug substances are shown below in Table 8.Table 8. DNA Quality by Long-Read NGS (Drug Substance)1. Full-Functional = full expression cassette capable of expressing transgene.Example 11 — Potency of Purified AAV as Measured by icIEF Analysis and a Cell-Based GFP Fluorescence Assay

[0226] Imaged Capillary Isoelectric Focusing (icIEF), Capillary Isoelectric Focusing (cIEF), and cell-based GFP fluorescence assays are performed to determine the charge characteristics and potency of AAV9 capsids containing the GFP reporter construct prepared as set forth above in Examples 1-3, in comparison to AAV 9 capsids containing the same GFP reporter construct prepared according to the UC method described above in Example 4.

[0227] Charge heterogeneity is determined by icIEF, which separates proteins based on charge differences using a pH gradient inside a capillary. In the presence of a pH gradient and under an electric field, a protein will migrate toward its isoelectric point (pl, the pH at which the molecule has no net charge). When the protein reaches its pl, its movement will stabilize within the electric field. AAV samples are tested under reduced conditions after heat denaturing using the Protein Simple Maurice icIEF system, where the distribution of proteins within the capillary is monitored in real-time with whole-column imaging detection. The pl value of the sample is determined by calibrating to known pl markers. The relative abundance of resolved peaks is quantified using Waters Empower chromatography software. The method provides information on chargeheterogeneity of the capsid proteins, which can be correlated with the actual potency of purified AAV preparations. The results of this icIEF analysis of AAV9 capsids containing the GFP reporter construct processed as set forth above in Examples 1-3, as well as AAV9 capsids containing the same GFP reporter construct prepared according to the UC method described above in Example 4, are shown below in columns 2-5 of Table 9.

[0228] The isoelectric point (pl) of intact capsids is determined by capillary isoelectric focusing (cIEF), which separates capsids based on charge differences using a pH gradient inside a capillary. In the presence of a pH gradient and under an electric field, a capsid will migrate toward its isoelectric point (pl, the pH at which the molecule has no net charge). When the intact capsid reaches its pl, its movement will stabilize within the electric field. Intact capsid pls were determined using isoelectric focusing in a pH 3-10 gradient using a neutral capillary on a SciEx PA800 system. The pl value of a sample is determined by calibrating with known pl markers. The method provides the overall intact capsid pl which can be correlated with the potency of purified AAV preparations as seen during accelerated stability studies of AAV9. The results of this intactcapsid pl analysis of AAV9 capsids containing the GFP reporter construct processed as set forth above in Examples 1-3, as well as AAV9 capsids containing the same GFP reporter construct prepared according to the UC method described above in Example 4, are shown below in column 6 of Table 9.

[0229] A cell-based GFP fluorescence assay is also used to determine the actual potency of the various AAV preparations. In brief, HEK293 or HEK293-AR cells (HEK293-AR cells are engineered to overexpress AAV receptor proteins as described by Pillay S, et al. 2016 Nature 530(7588): 108-112) are transduced with AAV9 capsids containing the GFP reporter construct processed as set forth above in Examples 1-3, or with AAV9 capsids containing the same GFP reporter construct prepared according to the UC method described above in Example 4. 68-76 hours after transduction, GFP fluorescence is assayed using a microplate reader at 480 nm excitation and 525 nm emission. Results are expressed relative to the 525 nm emission of the UC capsid samples run on each plate. The results of this analysis are shown below in columns 7-8 of Table 9.Table 9. Quality of Purified AAV Drug Substance as Measured by icTEF Analysis and Cell-Based GFP Fluorescence Assay1. icIEF monitors deamidation via acid forms of capsid proteins. Deaminated capsid proteins correlate with loss of bioactivity.2. Potency of AAV9 capsids containing the GFP reporter construct prepared as set forth above in Examples 1-3 is expressed as a percentage of the potency of AAV 9 capsids containing the same GFP reporter construct prepared according to the UC method described above in Example 4.REFERENCES

[0230] All publications, patent applications, patents, and other references mentioned herein (e.g., sequence database reference numbers) are incorporated by reference in their entirety. For example, all GenBank, Unigene, and Entrez sequences referred to herein, e.g., in any Table herein, are incorporated by reference. Unless otherwise specified, the sequence accession numbers specified herein, including in any Table herein, refer to the database entries current as of the filing date of this application. When one gene or protein references a plurality of sequence accession numbers, all of the sequence variants are encompassed.

Claims

CLAIMSWe claim:

1. A process for manufacturing a composition of adeno-associated virus composition (AAV), said process comprising; a. providing a cell lysate comprising a first AAV particle composition comprising full AAV capsids, process impurities, including empty and partial AAV capsids, and product impurities, the cell lysate obtained from a culture of AAV producing mammalian cells and optionally clarifying the cell lysate; b. flocculating the optionally clarified cell lysate by sequentially adding to the optionally clarified cell lysate first salt, followed by octanoic acid, followed by addition of formate and allowing the cell lysate to equilibrate with each addition prior to the next addition to form a flocculation mixture buffer; c. forming a separated flocculation mixture by mixing the flocculation mixture and allowing the flocculation mixture to stand for a time sufficient to form a cream layer at the surface of the flocculation mixture buffer; d. separating the flocculation mixture buffer layer below the cream layer and optionally combining it with cationic exchange buffer used to rinse the cream layer to obtain a rinse buffer layer separated from the rinsed cream layer and clarifying by filtration the flocculation mixture buffer optionally combined with the rinse buffer layer to form a second AAV particle composition; e. loading the second AAV particle composition onto a cationic exchange column to capture AAV particles present in material loaded onto the cationic exchange column and washing the cationic exchange column to remove impurities; f. eluting the AAV particles from the cationic exchange column either with a pH gradient or a salt gradient to form an AAV-containing cation exchange column eluate to produce a third AAV particle composition;g. if eluting AAV particles by using a salt gradient, reducing the salt concentration of the cation exchange column eluate obtained from eluting the AAV particles with a salt gradient by exchanging the eluate buffer with a low salt buffer using tangential flow filtration to provide an AAV containing anion exchange column loading buffer as the third AAV particle composition; h. loading an anion exchange column with the third AAV particle composition obtained from eluting AAV particles from the cationic exchange column with a pH gradient, or the AAV containing anion exchange column loading buffer of step g so as to capture the AAV particles present in material loaded onto the anion exchange column; i. washing the anion exchange column with anion exchange buffer to remove impurities; j . eluting the AAV particles from the anion exchange column with a salt gradient to produce an AAV particle-containing anion exchange column eluate as a fourth AAV particle composition; k. optionally reducing the conductivity of the AAV containing anion exchange column eluate and loading the resulting reduced conductivity eluate onto an anion exchange column, washing the column with anion exchange buffer and eluting the AAV particles from the anion exchange column with a salt gradient to produce an AAV particle containing eluate as a fourth AAV particle composition; l. exchanging the AAV containing anion exchange eluate from the fourth AAV particle composition from step j or k with a pharmaceutically acceptable buffer using one or more cycles of tangential flow filtration to obtain a fifth AAV particle composition comprising purified AAV particles; m. optionally filtering the fifth AAV particle composition through a sterilizing filter to obtain a drug substance comprising purified AAV particles;wherein the process for manufacturing a composition of adeno-associated virus composition (AAV) set forth above does not comprise an ultracentrifugation step; and wherein the process for manufacturing a composition of adeno-associated virus composition (AAV) set forth above results in a drug substance comprising purified AAV particles present at a clinically relevant purity.

2. The method of claim 1, wherein optionally clarifying the cell lysate in step (la) comprises clarifying the cell lysate by filtration.

3. The method of claim 1, wherein step (lb) further comprises adding 6,000 MW polyethylene glycol (PEG) to a final concentration of about 1% subsequent to the addition of octanoic acid, and prior to the addition of formate.

4. The method of any one of claims 1 to 3, wherein the AAV particle comprises an AAV9 capsid, or an AAV capsid with the same isoelectric point as AAV9.

5. The method of any one of claims 1 to 3, wherein the AAV particle comprises an AAV8 capsid, or an AAV capsid with the same isoelectric point as AAV8.

6. The method of any one of claims 1 to 3, wherein the AAV particle comprises an AAV6 capsid, or an AAV capsid with the same isoelectric point as AAV6.

7. The method of any one of claims 1 to 3, wherein the AAV particle comprises an AAV5 capsid, or an AAV capsid with the same isoelectric point as AAV5.

8. The method of any one of claims 1 to 3, wherein the AAV particle does not comprise an AAV2 capsid, or an AAV capsid with the same isoelectric point as AAV2.

9. The method of any one of claims 1 to 3, wherein the AAV particle does not comprise an AAV capsid having an amino acid sequence that has been modified to have a different amino acid sequence than the amino acid sequence of a naturally occurring AAV capsid.

10. The method according to any one of claims 1 to 9, wherein the AAV particle comprises a self-complementary AAV genome.

11. The method of claim 10, wherein the ratio of dimer to monomer in the fourth AAV particle composition is about 10 to 1, about 20 to 1, or about 40 to 1.

12. The method of any one of claims 1 to 9, wherein the AAV particle comprises a singlestranded AAV genome.

13. The method of any one of claims 1 to 12, wherein cell lysate that comprises a first AAV particle composition is obtained by a) culturing mammalian cells comprising nucleotide sequences comprising (1) a gene of interest (GOI) expression cassette comprising a transgene encoding a GOI operably linked to at least one regulatory sequence, flanked by AAV inverted terminal repeat (ITR) sequences; and (2) coding sequences for an AAV Rep, AAV Cap and adenovirus helper factors; the mammalian cells cultured under conditions that produce AAV particles comprising a nucleic acid, comprising the GOI expression cassette capable of expressing the transgene in a target cell upon transduction of the target cell with the AAV particles, packaged within an AAV capsid comprising the nucleic acid; and b) obtaining a lysate by lysing the mammalian cells to produce a first AAV particle composition comprising full AAV capsids containing a gene of interest, process impurities and product impurities.

14. The method of claim 13, wherein the mammalian cells cultured according to the method of claim 13 are produced by transfecting a population of mammalian cells by contacting the population of mammalian cells with a transfection composition comprising a plasmid encoding the GOI expression cassette flanked by AAV ITRs (pGOI), a plasmid encoding AAV Rep and AAV Cap (pRepCap), and a plasmid encoding adenoviral helper genes (pHelp) under conditions to achieve transfection of the cells with the pGOI plasmid, pRepCap plasmid and pHelp plasmid to obtain the mammalian cells.

15. The method of claim 14, wherein the transfection composition further comprises a transfection reagent.

16. The method of claim 15, wherein the transfection reagent is FectoVIR®, polycation polyethylenimine (PEI), PEIpro®, AAV-Max, TransIT-VirusGEN® or any other cationic lipid or lipofectamine based reagent.

17. The method of any one of claims 13 to 16, wherein the mammalian cells are HEK293 cells.

18. The method of any one of claims 13 to 17, wherein prior to step (a) the mammalian cells are cultured and the culture is expanded one or more times.

19. The method of any one of claims 13 to 18, wherein the mammalian cells are grown in animal-free culture media.

20. The method of claim 19, wherein the animal-free culture media is Expi293™ Expression Medium or Viral Production Medium™ or FreeStyle™ 293 Expression Medium.

21. The method of any one of claims 13 to 20, wherein 0.5 to 2 volumes of animal-free culture media is added to the mammalian cells immediately prior to or during the step of transfecting the mammalian cells.

22. The method of any one of claims 13 to 21, wherein the AAV Rep is AAV Rep2 and the AAV Cap is Cap9, or encodes an AAV capsid with the same isoelectric point as AAV9.

23. The method of any one of claims 13 to 22, wherein step (b) comprises contacting the mammalian cells with non-ionic surfactant.

24. The method of claim 23, wherein the non-ionic surfactant is polysorbate 20, and the mammalian cells are further contacted with MgCh.

25. The method of any one of claims 1 to 24, wherein the cell lysate comprising a first AAV particle composition is digested with endonuclease prior to flocculating the first AAV particle composition.

26. The method of claim 25, wherein the endonuclease is Serratia marcescens nuclease.

27. The method of any one of claims 1 to 26, wherein the cell lysate comprising a first AAV particle composition is clarified by filtration prior to flocculating the first AAV composition, and flocculating the first AAV particle composition does not comprise contacting the cell lysate with a solution that contains polyethylene glycol (PEG).

28. The method of claim 27, wherein clarifying the cell lysate comprising a first AAV particle composition by filtration results in about a two log reduction in mammalian cell DNA.

29. The method of claims 27 or 28, wherein clarifying the cell lysate comprising a first AAV particle composition by filtration comprises the use of a glass fiber filter, PES filter, PVDF filter, cellulose filter or diatomaceous earth filter.

30. The method of any one of claims 1 to 29, wherein flocculating the first AAV composition comprises contacting the first AAV particle composition with an anionic surfactant and acidifying the composition to a pH of 2.9 to 4.0.

31. The method of any one of claims 1 to 30, wherein flocculating the first AAV composition removes at least 90% of mammalian cell protein.

32. The method of any one of claims 1 to 31, wherein the cation exchange column comprises a cation exchange chromatography resin comprising poly(glycidyl methacrylate -co- ethylene dimethacrylate).

33. The method of any one of claims 1 to 32, wherein the cation exchange column comprises a cation exchange chromatography resin comprising a sulfonate ligand.

34. The method of any one of claims 1 to 33, wherein the cation exchange column has a column bed volume of 800 mL and the volume of the cell lysate comprising a firstAAV particle composition is approximately 200 L-250 L, or the column bed volume is similarly proportional to the volume of the cell lysate comprising a first AAV particle composition.

35. The method of any one of claims 1 to 34, wherein the cation exchange column has a column pore size of about 2 pm, about 4 pm, about 6 pm, or about 8 pm.

36. The method of any one of claims 1 to 35, wherein the cation exchange column comprises a cation exchange chromatography resin having a pore size of about 2 pm.

37. The method of any one of claims 1 to 35, wherein the cation exchange column comprises a cation exchange chromatography resin having a pore size of about 6 pm.

38. The method of any one of claims 1 to 35 or 37, wherein the cation exchange column comprises a cation exchange chromatography resin comprising poly(glycidyl methacrylate -co- ethylene dimethacrylate), a sulfonate ligand, a bed size of 400 mL, and a 6 pm pore size.

39. The method of any one of claims 1 to 38, wherein eluting the AAV particles from the cationic exchange column either with a pH gradient or a salt gradient to form an AAV- containing cation exchange column eluate to produce a third AAV particle composition comprises eluting at rate of from about 6 to about 10 column volumes per minute.

40. The method of any one of claims 1 to 37 and 39, wherein the anion exchange column has a column bed volume of 800 mL and the volume of the cell lysate comprising a first AAV particle composition is approximately 200 L-250 L, or the anion exchange column has a column bed volume that is similarly proportional to the volume of the cell lysate comprising a first AAV particle composition.

41. The method of any one of claims 1 to 40, wherein the anion exchange column comprises an anion exchange chromatography resin comprising regenerated macro- porous cellulose with primary amine ligand.

42. The method claim 41, wherein the anion exchange column has column bed volume of43. The method of any one of claims 1 to 40, wherein the anion exchange column comprises a cation exchange chromatography resin comprising Poly(glycidyl methacrylate -co-ethylene dimethacrylate).

44. The method of any one of claims 1 to 40 or 43, wherein the anion exchange column has a column bed volume of 400 mL.

45. The method of any one of claims 1 to 44, wherein the anion exchange (AEX) chromatography resin has a pore size of about 2 pm.

46. The method of any one of claims 1 to 44, wherein the AEX chromatography resin has a pore size of about 6 pm.

47. The method of any one of claims 1 to 46, wherein the third AAV particle composition is subjected to tangential flow fdtration before it is loaded onto an anion exchange (AEX) chromatography column, or optionally the pH of the third AAV particle composition is adjusted so as to have a pH to allow for capture of AAV particles onto the AEC column before it is loaded onto the anion exchange (AEX) chromatography column.

48. The method of any one of claims 1 to 47, wherein the proportion of full AAV capsids and / or partial AAV capsids and / or empty AAV capsids in the fifth AAV particle composition is assessed by AUC or charge detection mass spectroscopy (CDMS), and / or capsid purity is assessed by capillary electrophoresis or SDS-PAGE or ELISA for host cell protein, and / or PCR and / or short read NGS assays and / or long read NGS assays for plasmid and host cell DNA.

49. The method of any of claims 1 to 48, wherein the fifth AAV particle composition is filtered through a sterilizing filter to obtain a drug substance comprising purified AAV particles.

50. The method of claim 49, wherein the sterilizing filter is a 0.2 pm sterilizing filter.

51. The method of claim 49, wherein the sterilizing filter is a 0.22 pm PES filter.

52. The method of claim 49, wherein the sterilizing filter is a 0.22 m PVDF filter.

53. The method of any of claims 49 to 52, wherein the drug substance comprises at least 80% full AAV capsids by AUC or CDMS, at least 90% AAV capsid purity by SDS- PAGE or CE-SDS, at least 70% transgene present in the genome in the AAV particle by short read NGS and / or greater than about 75% DNA reads of the full expression cassette by long read NGS.

54. The method of any of claims 1 to 53, wherein eluting the AAV particles from the cationic exchange column with a pH gradient comprises a pH gradient ranging from a starting pH of 3.5 to a final pH of 9.

355. The method of claim 54, wherein the pH gradient comprises elution with a pH 3.5 buffer comprising 20 mM formate.

56. The method of any of claims 54 to 55, wherein the pH gradient comprises elution with a pH 3.5 buffer comprising 20 mM formate, 2 M NaCl, and 0.2% poloxamer 188 (P188) at pH 3.5.

57. The method of any of claims 1 to 53, wherein eluting the AAV particles from the cationic exchange column with a salt gradient comprises a salt gradient from a 500 mM salt concentration to a final 2 M salt concentration.

58. The method of claim 57, wherein the salt is sodium chloride.

59. The method of any of claims 1 to 53, wherein eluting the AAV particles from the anion exchange column with a salt gradient comprises a salt gradient from a 0 mM salt concentration to a final 1 M salt concentration.

60. The method of claim 59, wherein the salt is sodium chloride.

61. The method of any one of claims 13 to 60, wherein culturing mammalian cells comprises growing mammalian cells in a cell culture volume of 50 L, or the mammalian cell lysate is a volume of 50 L.

62. The method of any one of claims 13 to 60, wherein culturing mammalian cells comprises growing mammalian cells in a cell culture volume of 215 L, or the mammalian cell lysate is a volume of 215 L.

63. The method of any one of claims 13 to 60, wherein culturing mammalian cells comprises growing mammalian cells in a cell culture volume of 500 L, or the mammalian cell lysate is a volume of 500 L.

64. The method of any one of claims 13 to 60, wherein culturing mammalian cells comprises growing mammalian cells in a cell culture volume of 1,000 L, or the mammalian cell lysate is a volume of 1,000 L.

65. The method of any one of claims 1 to 64, wherein the first AAV particle composition comprises about 1 x 1016to about 5 x 1017AAV particles.

66. The method of any of claims 1 to 65, wherein the drug substance comprises (1) at least about 85% full AAV capsids as assessed by analytical ultracentrifugation (AUC) or charge detection mass spectrometry (CDMS); (2) at least about 95% AAV capsid purity as assessed by SDS-PAGE or CE-SDS; (3) less than about 5% empty AAV capsids as assessed by AUC; (4) at least 90% transgene present in the genome in the AAV particle by short read NGS and / or (5) greater than about 80% DNA reads of the full expression cassette by long read NGS.

67. The method of claim 66, wherein the drug substance comprises (1) at least about 90% full AAV capsids as assessed by analytical ultracentrifugation (AUC) or charge detection mass spectrometry (CDMS); (2) at least about 98% AAV capsid purity as assessed by SDS-PAGE or CE-SDS; (3) less than about 3% empty AAV capsids as assessed by AUC; and / or (4) at least 95% transgene present in the genome in the AAV particle by short read NGS and / or (5) greater than about 86% DNA reads of the full expression cassette by long read NGS.

68. A drug substance produced according to the method of any of claims 1 to 67, wherein drug substance comprises AAV particles comprising (1) a nucleic acid comprising aGOI expression cassette and at least one AAV ITR sequence; and (2) an AAV capsid, wherein a) the recombinant AAV particles are present at a concentration of between about IxlO13and about IxlO14viral vector genomes / mL (vg / mL); b) at least about 80% of the recombinant AAV particles are full AAV capsids as assessed by AUC or CDMS; c) at least about 80% of DNA reads of the full expression cassette by long read NGS; d) the composition has at least about 90% capsid purity as assessed by SDS-PAGE or CE-SDS; e) less than about 2xl05pg residual host cell DNA per I x lO13vg is present; and f) less than about 15 ng host cell protein per 1 x 1013vg is present.

69. The drug substance of claim 68, wherein less than about 2 ng host cell protein per 1 x 1013vg is present.

70. The drug substance of claim 69, wherein less than about 1 ng host cell protein per 1 x 1013vg is present.

71. The drug substance of any of claims 68 to 70, wherein the composition comprises at least about 90% full AAV capsids as assessed by analytical ultracentrifugation (AUC) or charge detection mass spectrometry (CDMS).

72. The drug substance of claim 71, wherein the composition comprises at least about 95% full AAV capsids as assessed by analytical ultracentrifugation (AUC) or charge detection mass spectrometry (CDMS).

73. The drug substance of any of claims 68 to 72, wherein the composition comprises at least about 95% capsid purity as assessed by SDS-PAGE or CE-SDS.

74. The drug substance claim 73, wherein the composition comprises at least about 98% capsid purity as assessed by SDS-PAGE or CE-SDS.

75. The drug substance of any of claims 68 to 74, wherein the composition comprises less than about 5% empty AAV capsids as assessed by AUC.

76. The drug substance of claim 75, wherein the composition comprises less than about 3% empty AAV capsids as assessed by AUC.

77. The drug substance of any one of claims 68 to 76, wherein the composition comprises greater than about 83% DNA reads of the full expression cassette by long read NGS.

78. The drug substance of claim 77, wherein the composition comprises greater than about 86% DNA reads of the full expression cassette by long read NGS.

79. The drug substance of any of claims 68 to 78, wherein the AAV capsid comprises an AAV9 capsid, or an AAV capsid with the same isoelectric point as AAV9.

80. The drug substance of any of claims 68 to 79, wherein the intact AAV capsid has an isoelectric point of about 7.2 to about 7.3, about 7.1 to about 7.4, or about 7.0 to about 7.5.

81. The drug substance of any of claims 68 to 80, wherein the nucleic acid is single stranded.

82. The drug substance of any of claims 68 to 80, wherein the nucleic acid is self- complementary.

83. The drug substance of claim 82, wherein the AAV have a ratio of dimer to monomer of at least about 10 to 1.

84. The drug substance of claim 82, wherein the AAV have a ratio of dimer to monomer of at least about 20 to 1.

85. The drug substance of claim 82, wherein the AAV have a ratio of dimer to monomer of at least about 40 to 1.

86. The drug substance of any of claims 68 to 85, wherein the drug substance contains 5 x 1015to 3 x 1017vg for an initial lysate of volume 215-L.

87. The method of any of claims 1 to 86, wherein providing a cell lysate comprising a first AAV particle composition comprising full AAV capsids comprises lysing cells in the presence of a non-ionic surfactant, optionally, Tween (20) in the presence of one or more nucleases.

88. The method of any one of claims 1 to 4, wherein the AAV particle comprises an AAV9 capsid.

89. The method of any one of claims 1 to 3 or 5, wherein the AAV particle comprises an AAV8 capsid.

90. The method of any one of claims 1 to 3 or 6, wherein the AAV particle comprises an AAV6 capsid.

91. The method of any one of claims 1 to 3 or 7, wherein the AAV particle comprises an AAV5 capsid.

92. The method of any one of claims 1 to 3 or 8, wherein the AAV particle does not comprise an AAV2 capsid.

93. The method of any one of claims 10 to 67, wherein the AAV particle comprises anAAV9 capsid.

94. The method of any one of claims 10 to 67, wherein the AAV particle comprises anAAV8 capsid.

95. The method of any one of claims 10 to 67, wherein the AAV particle comprises anAAV6 capsid.

96. The method of any one of claims 10 to 67, wherein the AAV particle comprises anAAV5 capsid.

97. The method of any one of claims 10 to 67, wherein the AAV particle does not comprise an AAV2 capsid.

98. The method of any one of claims 10 to 67, wherein the AAV particle does not comprise an AAV capsid having an amino acid sequence that has been modified to have a different amino acid sequence than the amino acid sequence of a naturally occurring AAV capsid.

99. The drug substance of any one of claims 68 to 86, wherein the drug substance comprises AAV particles having an AAV9 capsid.

100. The drug substance of any one of claims 68 to 86, wherein the drug substance comprises AAV particles having an AAV8 capsid.

101. The drug substance of any one of claims 68 to 86, wherein the drug substance comprises AAV particles having an AAV6 capsid.

102. The drug substance of any one of claims 68 to 86, wherein the drug substance comprises AAV particles having an AAV5 capsid.

103. The drug substance of any one of claims 68 to 86, wherein the drug substance comprises AAV particles that do not have an AAV2 capsid.

104. The method of any one of claims 68 to 86, wherein the drug substance comprises AAV particles that do not comprise an AAV capsid having an amino acid sequence that has been modified to have a different amino acid sequence than the amino acid sequence of a naturally occurring AAV capsid.

105. The method of any one of claims 1 to 104, wherein the cell lysate is clarified, and flocculation according to step lb comprises the addition of octanoic acid.

106. A process for manufacturing a composition of adeno-associated virus composition (AAV), said process comprising;a. Obtaining a first AAV particle composition from a cell lysate from AAV producing mammalian host cells, said first AAV particle composition (i) being the direct product of upstream processing prior to any column processing step, and (ii) comprising less than about 30%, 20%, 10%, 5%, 1%, or 0.1% of host cell DNA and / or host cell protein; b. loading the first AAV particle composition onto a cationic exchange column to capture AAV particles present in material loaded onto the cationic exchange column and washing the cationic exchange column to remove impurities; c. eluting the AAV particles from the cationic exchange column either with a pH gradient or a salt gradient to form an AAV containing cation exchange column eluate to produce a second AAV particle composition; d. if eluting AAV particles by using a salt gradient, reducing the salt concentration of the cation exchange column eluate obtained from eluting the AAV particles with a salt gradient by exchanging the eluate buffer with a low salt buffer using tangential flow filtration to provide an AAV containing anion exchange column loading buffer as the second AAV particle composition; e. loading an anion exchange column with the second AAV particle composition obtained from eluting AAV particles from the cationic exchange column with a pH gradient, or the AAV containing anion exchange column loading buffer of step g so as to capture the AAV particles present in material loaded onto the anion exchange column; f. washing the anion exchange column with anion exchange buffer to remove impurities; g. eluting the AAV particles from the anion exchange column with a salt gradient to produce an AAV particle containing anion exchange column eluate as a third AAV particle composition;h. optionally reducing the conductivity of the AAV containing anion exchange column eluate and loading the resulting reduced conductivity eluate onto an anion exchange column, washing the column with anion exchange buffer and eluting the AAV particles from the anion exchange column with a salt gradient to produce an AAV particle containing eluate as a third AAV particle composition; i. exchanging the AAV containing anion exchange eluate from the fourth AAV particle composition from step g or h with a pharmaceutically acceptable buffer using one or more cycles of tangential flow filtration to obtain a fourth AAV particle composition comprising purified AAV particles; j . optionally filtering the fourth AAV particle composition through a sterilizing filter to obtain a drug substance comprising purified AAV particles. wherein the process for manufacturing a composition of adeno-associated virus composition (AAV) set forth above does not comprise an ultracentrifugation step; and wherein the process for manufacturing a composition of adeno-associated virus composition (AAV) set forth above results in a drug substance comprising purified AAV particles present at a clinically relevant purity.

107. The method of claim 106, wherein the AAV particle comprises an AAV9 capsid, or an AAV capsid with the same isoelectric point as AAV9.

108. The method of claim 106, wherein the AAV particle comprises an AAV8 capsid, or an AAV capsid with the same isoelectric point as AAV8.

109. The method claim 106, wherein the AAV particle comprises an AAV6 capsid, or an AAV capsid with the same isoelectric point as AAV6.

110. The method of claim 106, wherein the AAV particle comprises an AAV5 capsid, or an AAV capsid with the same isoelectric point as AAV5.

111. The method of claim 106, wherein the AAV particle does not comprise an AAV2 capsid, or an AAV capsid with the same isoelectric point as AAV2.

112. The method of claim 106, wherein the AAV particle does not comprise an AAV capsid having an amino acid sequence that has been modified to have a different amino acid sequence than the amino acid sequence of a naturally occurring AAV capsid.

113. The method according to any one of claims 107 to 112, wherein the AAV particle comprises a self-complementary AAV genome.

114. The method of claim 113, wherein the ratio of dimer to monomer in the third AAV particle composition is about 10 to 1, about 20 to 1, or about 40 to 1.

115. The method of any one of claims 106 to 112, wherein the AAV particle comprises a single- stranded AAV genome.

116. The method of any one of claims 106 to 115, wherein obtaining a first AAV particle composition from AAV producing mammalian host cells comprises a) culturing mammalian cells comprising nucleotide sequences comprising (1) a gene of interest (GOI) expression cassette comprising a transgene encoding a GOI operably linked to at least one regulatory sequence, flanked by AAV inverted terminal repeat (ITR) sequences; and (2) coding sequences for an AAV Rep, AAV Cap and adenovirus helper factors; the mammalian cells cultured under conditions that produce AAV particles comprising a nucleic acid, comprising the GOI expression cassette capable of expressing the transgene in a target cell upon transduction of the target cell with the AAV particles, packaged within an AAV capsid comprising the nucleic acid; and b) obtaining a lysate by lysing the mammalian cells to produce a first AAV particle composition comprising full AAV capsids containing a gene of interest, process impurities and product impurities.

117. The method of claim 116, wherein the mammalian cells cultured according to the method of claim 116 are produced by transfecting a population of mammalian cells by contacting the population of mammalian cells with a transfection composition comprising a plasmid encoding the GOI expression cassette flanked by AAV ITRs (pGOI), a plasmid encoding AAV Rep and AAV Cap (pRepCap), and a plasmidencoding adenoviral helper genes (pHelp) under conditions to achieve transfection of the cells with the pGOI plasmid, pRepCap plasmid and pHelp plasmid to obtain the mammalian cells.

118. The method of claim 117, wherein the transfection composition further comprises a transfection reagent.

119. The method of claim 118, wherein the transfection reagent is FectoVIR®, polycation polyethylenimine (PEI), PEIpro®, AAV-Max, TransIT-VirusGEN® or any other cationic lipid or lipofectamine based reagent.

120. The method of any one of claims 116 to 119, wherein the mammalian cells are HEK293 cells.

121. The method of any one of claims 116 to 120, wherein prior to step (a) the mammalian cells are cultured and the culture is expanded one or more times.

122. The method of any one of claims 116 to 121, wherein the mammalian cells are grown in animal-free culture media.

123. The method of claim 122, wherein the animal-free culture media is Expi293™ Expression Medium or Viral Production Medium™ or FreeStyle™ 293 Expression Medium.

124. The method of any one of claims 116 to 123, wherein 0.5 to 2 volumes of animal-free culture media is added to the mammalian cells immediately prior to or during the step of transfecting the mammalian cells.

125. The method of any one of claims 116 to 124, wherein the AAV Rep is AAV Rep2 and the AAV Cap is Cap9, or encodes an AAV capsid with the same isoelectric point as AAV9.

126. The method of any one of claims 116 to 125, wherein step (b) comprises contacting the mammalian cells with non-ionic surfactant.

127. The method of claim 126, wherein the non-ionic surfactant is polysorbate 20, and the mammalian cells are further contacted with MgCh.

128. The method of any one of claims 106 to 127, wherein the cation exchange column comprises a cation exchange chromatography resin comprising poly(glycidyl methacrylate -co- ethylene dimethacrylate).

129. The method of any one of claims 106 to 128, wherein the cation exchange column comprises a cation exchange chromatography resin comprising a sulfonate ligand.

130. The method of any one of claims 106 to 129, wherein the cation exchange column has a column bed volume of 800 mb and the volume of the cell lysate comprising a first AAV particle composition is approximately 200 L-250 L, or wherein the cation exchange column has a column bed volume that is similarly to the volume of the cell lysate comprising a first AAV particle composition.

131. The method of any one of claims 106 to 130, wherein the cation exchange column has a column pore size of about 2 pm, about 4 pm, about 6 pm, or about 8 pm.

132. The method of any one of claims 106 to 131, wherein the cation exchange column comprises a cation exchange chromatography resin having a pore size of about 2 pm.

133. The method of any one of claims 106 to 131, wherein the cation exchange column comprises a cation exchange chromatography resin having a pore size of about 6 pm.

134. The method of any one of claims 106 to 131 or 133, wherein the cation exchange column comprises a cation exchange chromatography resin comprising poly(glycidyl methacrylate -co- ethylene dimethacrylate), a sulfonate ligand, a bed size of 400 mb, and a 6 pm pore size.

135. The method of any one of claims 106 to 134, wherein eluting the AAV particles from the cationic exchange column either with a pH gradient or a salt gradient to form an AAV-containing cation exchange column eluate to produce a third AAV particle composition comprises eluting at rate of from about 6 to about 10 column volumes per minute.

136. The method of any one of claims 106 to 133 and 135, wherein the anion exchange column has a column bed volume of 800 mL and the volume of the cell lysate comprising a first AAV particle composition is approximately 200 L-250 L, or the anion exchange column has a column bed volume that is similarly proportional to the volume of the cell lysate comprising a first AAV particle composition.

137. The method of any one of claims 106 to 136, wherein the anion exchange column comprises a cation exchange chromatography resin comprising regenerated macro- porous cellulose with primary amine ligand.

138. The method of any one of claims 106 to 137, wherein the anion exchange column has a column bed volume of 75 mL.

139. The method of any one of claims 106 to 136, wherein the anion exchange column comprises an anion exchange chromatography resin comprising Poly(glycidyl methacrylate -co- ethylene dimethacrylate).

140. The method of any one of claims 106 to 136 or 139, wherein the anion exchange column has a column bed volume of 400 mL.

141. The method of any one of claims 106 to 140, wherein the anion exchange (AEX) chromatography resin has a pore size of about 2 pm.

142. The method of any one of claims 106-140, wherein the AEX chromatography resin has a pore size of about 6 pm.

143. The method of any one of claims 106 to 142, wherein the third AAV particle composition is subjected to tangential flow filtration before it is loaded onto an anion exchange (AEX) chromatography column, or optionally the pH of the third AAV particle composition is adjusted so as to have a pH to allow for capture of AAV particles onto the AEC column before it is loaded onto the anion exchange (AEX) chromatography column.

144. The method of any one of claims 106 to 143, wherein the proportion of full AAV capsids and / or partial AAV capsids and / or empty AAV capsids in the fifth AAVparticle composition is assessed by AUC or charge detection mass spectroscopy (CDMS), and / or capsid purity is assessed by capillary electrophoresis or SDS-PAGE or ELISA for host cell protein, and / or PCR and / or short read NGS assays and / or long read NGS assays for plasmid and host cell DNA.

145. The method of any of claims 106 to 144, wherein the fourth AAV particle composition is fdtered through a sterilizing filter to obtain a drug substance comprising purified AAV particles.

146. The method of claim 145, wherein the sterilizing filter is a 0.2 pm sterilizing filter.

147. The method of claim 145, wherein the sterilizing filter is a 0.22 pm PES filter.

148. The method of claim 145, wherein the sterilizing filter is a 0.22 pm PVDF filter.

149. The method of any of claims 145 to 148, wherein the drug substance comprises at least80% full AAV capsids by AUC or CDMS, at least 90% AAV capsid purity by SDS- PAGE or CE-SDS, at least 70% transgene present in the genome in the AAV particle by short read NGS and / or greater than about 75% DNA reads of the full expression cassette by long read NGS.

150. The method of any of claims 106 to 149, wherein eluting the AAV particles from the cationic exchange column with a pH gradient comprises a pH gradient ranging from a starting pH of 3.5 to a final pH of 9.3151. The method of claim 150, wherein the pH gradient comprises elution with a pH 3.5 buffer comprising 20 mM formate.

152. The method of any of claims 150 to 151, wherein the pH gradient comprises elution with a pH 3.5 buffer comprising 20 mM formate, 2 M NaCl, and 0.2% poloxamer 188 (Pl 88) at pH 3.5.

153. The method of any of claims 106 to 149, wherein eluting the AAV particles from the cationic exchange column with a salt gradient comprises a salt gradient from a 500 mM salt concentration to a final 2 M salt concentration.

154. The method of claim 153, wherein the salt is sodium chloride.

155. The method of any of claims 106 to 149, wherein eluting the AAV particles from the anion exchange column with a salt gradient comprises a salt gradient from a 0 mM salt concentration to a final 1 M salt concentration.

156. The method of claim 155, wherein the salt is sodium chloride.

157. The method of any one of claims 116 to 156, wherein culturing mammalian cells comprises growing mammalian cells in a cell culture volume of 50 L, or the mammalian cell lysate is a volume of 50 L.

158. The method of any one of claims 116 to 156, wherein culturing mammalian cells comprises growing mammalian cells in a cell culture volume of 215 L, or the mammalian cell lysate is a volume of 215 L.

159. The method of any one of claims 116 to 156, wherein culturing mammalian cells comprises growing mammalian cells in a cell culture volume of 500 L, or the mammalian cell lysate is a volume of 500 L.

160. The method of any one of claims 116 to 156, wherein culturing mammalian cells comprises growing mammalian cells in a cell culture volume of 1,000 L, or the mammalian cell lysate is a volume of 1,000 L.

161. The method of any one of claims 106 to 160, wherein the first AAV particle composition comprises about 1 x 1016to about 5 x 1017AAV particles.

162. The method of any of claims 106 to 161, wherein the drug substance comprises (1) at least about 85% full AAV capsids as assessed by analytical ultracentrifugation (AUC) or charge detection mass spectrometry (CDMS); (2) at least about 95% AAV capsid purity as assessed by SDS-PAGE or CE-SDS; (3) less than about 5% empty AAV capsids as assessed by AUC; (4) at least 90% transgene present in the genome in the AAV particle by short read NGS and / or (5) greater than about 80% DNA reads of the full expression cassette by long read NGS.

163. The method of claim 162, wherein the drug substance comprises (1) at least about 90% full AAV capsids as assessed by analytical ultracentrifugation (AUC) or charge detection mass spectrometry (CDMS); (2) at least about 98% AAV capsid purity as assessed by SDS-PAGE or CE-SDS; (3) less than about 3% empty AAV capsids as assessed by AUC; and / or (4) at least 95% transgene present in the genome in the AAV particle by short read NGS and / or (5) greater than about 86% DNA reads of the full expression cassette by long read NGS.

164. A drug substance produced according to the method of any of claims 106 to 163, wherein drug substance comprises AAV particles comprising (1) a nucleic acid comprising a GOI expression cassette and at least one AAV ITR sequence; and (2) an AAV capsid, wherein a) the recombinant AAV particles are present at a concentration of between about IxlO13and about IxlO14viral vector genomes / mL (vg / mL); b) at least about 80% of the recombinant AAV particles are full AAV capsids as assessed by AUC or CDMS; c) at least about 80% of DNA reads of the full expression cassette by long read NGS; d) the composition has at least about 90% capsid purity as assessed by SDS-PAGE or CE-SDS; e) less than about 2xl05pg residual host cell DNA per 1 x 1013vg is present; and f) less than about 15 ng host cell protein per I x lO13vg is present.

165. The drug substance of claim 164, wherein less than about 2 ng host cell protein per 1 x 1013vg is present.

166. The drug substance of claim 165, wherein less than about 1 ng host cell protein per 1 x 1013vg is present.

167. The drug substance of any of claims 164 to 166, wherein the composition comprises at least about 90% full AAV capsids as assessed by analytical ultracentrifugation (AUC) or charge detection mass spectrometry (CDMS).

168. The drug substance of claim 167, wherein the composition comprises at least about 95% full AAV capsids as assessed by analytical ultracentrifugation (AUC) or charge detection mass spectrometry (CDMS).

169. The drug substance of any of claims 164 to 168, wherein the composition comprises at least about 95% capsid purity as assessed by SDS PAGE or CE-SDS.

170. The drug substance claim 169, wherein the composition comprises at least about 98% capsid purity as assessed by SDS-PAGE or CE-SDS.

171. The drug substance of any of claims 164 to 170, wherein the composition comprises less than about 5% empty AAV capsids as assessed by AUC.

172. The drug substance of claim 171, wherein the composition comprises less than about 3% empty AAV capsids as assessed by AUC.

173. The drug substance of any one of claims 164 to 172, wherein the composition comprises greater than about 83% DNA reads of the full expression cassette by long read NGS.

174. The drug substance of claim 173, wherein the composition comprises greater than about 86% DNA reads of the full expression cassette by long read NGS.

175. The drug substance of any of claims 164 to 174, wherein the AAV capsid comprises an AAV9 capsid, or an AAV capsid with the same isoelectric point as AAV9.

176. The drug substance of any of claims 164 to 175, wherein the intact AAV capsid has an isoelectric point of about 7.2 to about 7.3, about 7.1 to about 7.4, or about 7.0 to about 7.5.

177. The drug substance of any of claims 164 to 176, wherein the nucleic acid is single stranded.

178. The drug substance of any of claims 164 to 176, wherein the nucleic acid is self- complementary.

179. The drug substance of claim 178, wherein the AAV have a ratio of dimer to monomer of at least about 10 to 1.

180. The drug substance of claim 178, wherein the AAV have a ratio of dimer to monomer of at least about 20 to 1.

181. The drug substance of claim 178, wherein the AAV have a ratio of dimer to monomer of at least about 40 to 1.

182. The drug substance of any of claims 164 to 181, wherein the first AAV particle composition contains 5 x IO13to 3 x 1017vg in an initial volume 215-L.

183. The method of any of claims 106 to 182, wherein providing a first AAV particle composition comprises lysing AAV producing mammalian host cells in the presence of a non-ionic surfactant, optionally, Tween (20) in the presence of one or more nucleases.

184. The method of any one of claims 106 to 107, wherein the AAV particle comprises an AAV9 capsid.

185. The method of any one of claims 106 or 108, wherein the AAV particle comprises an AAV8 capsid.

186. The method of any one of claims 106 or 109, wherein the AAV particle comprises an AAV6 capsid.

187. The method of any one of claims 106 or 110, wherein the AAV particle comprises an AAV5 capsid.

188. The method of any one of claims 106 or 111, wherein the AAV particle does not comprise an AAV2 capsid.

189. The method of any one of claims 113 to 163, wherein the AAV particle comprises an AAV9 capsid.

190. The method of any one of claims 113 to 163, wherein the AAV particle comprises an AAV8 capsid.

191. The method of any one of claims 113 to 163, wherein the AAV particle comprises an AAV6 capsid.

192. The method of any one of claims 113 to 163, wherein the AAV particle comprises an AAV5 capsid.

193. The method of any one of claims 113 to 163, wherein the AAV particle does not comprise an AAV2 capsid.

194. The method of any one of claims 113 to 163, wherein the AAV particle does not comprise an AAV capsid having an amino acid sequence that has been modified to have a different amino acid sequence than the amino acid sequence of a naturally occurring AAV capsid.

195. The drug substance of any one of claims 164 to 182, wherein the drug substance comprises AAV particles having an AAV9 capsid.

196. The drug substance of any one of claims 164 to 182, wherein the drug substance comprises AAV particles having an AAV8 capsid.

197. The drug substance of any one of claims 164 to 182, wherein the drug substance comprises AAV particles having an AAV6 capsid.

198. The drug substance of any one of claims 164 to 182, wherein the drug substance comprises AAV particles having an AAV5 capsid.

199. The drug substance of any one of claims 164 to 182, wherein the drug substance comprises AAV particles that do not have an AAV2 capsid.

200. The method of any one of claims 164 to 182, wherein the drug substance comprises AAV particles that do not comprise an AAV capsid having an amino acid sequence that has been modified to have a different amino acid sequence than the amino acid sequence of a naturally occurring AAV capsid.

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