Flocculated AAV purification

Abstract

The present disclosure provides a method of purifying adeno-associated virus (AAV) particles by introducing a flocculant step into a large scale manufacturing process, thereby improving the purity and yield of AAV particles.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 425,998, filed on November 16, 2022, titled "Flocculative AAV Purification", the content of which is hereby incorporated by reference in its entirety. Technical Field

[0003] The present invention relates to methods for purifying recombinant AAV particles for use in gene therapy. Background Art

[0004] In a typical gene therapy manufacturing process, a viral vector is produced in a cell culture and isolated from the harvested cultured cells in a process involving a cell lysis step. The isolated viral vector preparation contains impurities from the manufacturing process, including cellular material released during the cell lysis process. Such impurities may cause instability of the viral vector and also pose a significant burden on downstream purification steps.

[0005] Accordingly, there is a need to improve current methods for manufacturing viral vectors for gene therapy. Summary of the Invention

[0006] This application provides methods and compositions for purifying recombinant adeno - associated virus (rAAV) particles from cell cultures. In some aspects, the methods and compositions can be used for large - scale manufacturing of rAAV for gene therapy and can increase the purity and stability of rAAV compositions.

[0007] In some embodiments, an rAAV particle is isolated from a cell culture containing rAAV particles using a process comprising: a) contacting the rAAV preparation with a glycine solution in conditions sufficient to promote flocculation of cellular material present in the rAAV preparation obtained from the cell culture; and b) separating the rAAV particles from the flocculated cellular material.

[0008] In some embodiments, the rAAV preparation is a cell culture harvest containing rAAV particles. In some embodiments, the rAAV preparation is a cell culture lysate obtained from the cell culture harvest. In some embodiments, chemical lysis techniques are used to obtain the lysate. In some embodiments, nucleic acid degradation techniques are used to obtain the rAAV preparation. For example, in some embodiments, the rAAV preparation is obtained in a process comprising contacting a cell lysate containing rAAV particles with a nuclease.

[0009] In some embodiments, the pH of the acid glycine solution for flocculating cell material is below 4. In some embodiments, the pH of the acid glycine solution is about 2.5. In some embodiments, a 1-3M acid glycine solution is added to the rAAV preparation. In some embodiments, a 2M acid glycine solution at pH 2.5 is added to the rAAV preparation.

[0010] In some embodiments, the acid glycine solution is added to the rAAV preparation at a volume of 5%-10%. In some embodiments, the acid glycine solution is added to the rAAV preparation at a volume of 8%. In some embodiments, the volume of the acid glycine solution is added to the rAAV preparation over a 10-minute period. In some embodiments, the volume of the acid glycine solution is added to the rAAV preparation over a 5-minute period.

[0011] In some embodiments, the rAAV preparation is mixed with the added acid glycine solution at a stirring speed of 30-150 RPM. In some embodiments, the stirring speed is 100 RPM.

[0012] In some embodiments, the volume of the rAAV preparation is 2-500 L. In some embodiments, the volume of the AAV preparation is 5 L, 50 L, or 500 L.

[0013] In some embodiments, the volume of the rAAV preparation is about 5 L, and the stirring speed is about 90-110 RPM, such as about 100 RPM. In some embodiments, the volume of the rAAV preparation is about 50 L, and the stirring speed is about 50-75 RPM, such as about 63 RPM. In some embodiments, the volume of the AAV preparation is about 500 L, and the stirring speed is about 30-50 RPM, such as about 42 RPM.

[0014] In some embodiments, after adding glycine (e.g., after adding 5%-10%, such as 8% volume of 2M glycine at pH 2.5), the product of a) has a pH of 3-5. In some embodiments, the pH of the product of a) is approximately (e.g., about) 4.

[0015] In some embodiments, the mixture of the rAAV preparation of a) and the acid glycine solution is kept stationary in a container for 10-60 minutes (e.g., at room temperature) to promote flocculation of the cell material. In some embodiments, the mixture is kept stationary for 15-45 minutes. In some embodiments, the mixture is kept stationary for about 30 minutes. In some embodiments, the flocculated material from a) is resuspended before separating the rAAV particles from the flocculated cell material.

[0016] In some embodiments, the product of a) is clarified. In some embodiments, the resuspended product of a) is clarified. In some embodiments, the clarification is carried out by filtration. In some embodiments, the filtration is depth filtration.

[0017] In some embodiments, glycine is the only pH-lowering agent used for flocculating cell material. However, in some embodiments, alternative or additional flocculants may be used. In some embodiments, the alternative or additional flocculant is a pH-lowering agent or a cationic polymer. In some embodiments, the alternative or additional pH-lowering agent may include citric acid, phosphoric acid, and / or octanoic acid. In some embodiments, the cationic polymer is polyethyleneimine (PEI) or polydiallyldimethylammonium chloride (pDADMAC).

[0018] In some embodiments, a lysis agent, such as a detergent, can be used together with the flocculant.

[0019] In some embodiments, the rAAV particles comprise a capsid protein of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 serotype, or a variant thereof. In some embodiments, the rAAV particles are rAAV9 particles.

[0020] In some embodiments, the rAAV particles comprise a recombinant nucleic acid (e.g., a recombinant AAV genome) comprising a recombinant gene of interest flanked by AAV inverted terminal repeats (ITRs). In some embodiments, the gene of interest encodes a therapeutic RNA or protein.

[0021] In some embodiments, the rAAV particles are further purified, e.g., using one or more affinity, ion exchange chromatography, and / or hydrophobic interaction chromatography steps, e.g., after clarification of the rAAV preparation.

[0022] In some embodiments, the rAAV particles (e.g., after one or more purification steps) are added to a pharmaceutically acceptable solution.

[0023] This application also provides a composition comprising rAAV particles, and a method of administering the rAAV particles to a subject (e.g., a human subject suffering from a condition that can be helped by a therapeutic RNA and / or protein).

[0024] These and other embodiments are described in the following detailed description and examples together with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1A and Figure 1B illustrate non - limiting embodiments of a harvesting procedure using flocculation at the scale of the manufacturing process. Figure 1A Shows a schematic diagram of the flocculation procedure. Figure 1B Displays non - limiting embodiments of the flocculation step incorporated into a large - scale rAAV manufacturing process.

[0026] Figure 2A and Figure 2B Shows a flocculant screening graph. Figure 2A Shows non - limiting examples of the reduction of host cell protein (HCP) after flocculation under different flocculant conditions. Figure 2B Shows non - limiting examples of the rAAV titer recovery rate after flocculation.

[0027] Figure 3A and Figure 3B Shows non - limiting examples of the depth filtration performance with and without flocculation. Figure 3A shows the reduction of HCP after depth filtration, and Figure 3B shows the improvement of throughput after flocculation.

[0028] Figure 4A and Figure 4B Shows the ultrafiltration / diafiltration (UFDF) performance with and without flocculation. Figure 4A Shows that the UFDF performance in the case of flocculated material has a double flux and no detectable flux decay compared to the UFDF performance in the case of non - flocculated material. Figure 4B Shows that the running time in the case of flocculated material is twice as fast as that in the case of non - flocculated material.

[0029] Figures 5A - 5C Shows the effect of flocculation on the performance of capture chromatography. Figure 5A and Figure 5B Shows the capture chromatography performance in (A) the case with flocculation and in (B) the case without flocculation. Figure 5C Shows the reduction of host cell DNA in the affinity eluate with and without flocculation.

[0030] Figure 6A and Figure 6B Shows the performance of polishing chromatography in (A) the case with flocculation and in (B) the case without flocculation. The average product size before and after dilution is shown below to indicate product stability.

[0031] Figure 7A and Figure 7BShows the complete capsid enrichment of the refined chromatography in the case of (A) flocculated material and in the case of (B) non-flocculated material. The highlighted regions in the chromatogram show the enrichment regions. The following table shows the percentage of complete capsid enrichment after the refining step.

[0032] Figure 8 Shows the post-UFDF product stability of flocculated (triangles) or non-flocculated (circles) material.

[0033] Figure 9 Shows the AAV recovery from the production cell line platform after flocculation.

[0034] Figure 10 Shows the reduction of host cell DNA after flocculation for a non-limiting production cell line platform.

[0035] Figure 11A and Figure 11B Shows the number of affinity resin cycles for purifying non-flocculated material (A) and flocculated material (B).

[0036] Figure 12 Shows that the AAV product becomes more stable after flocculation treatment in the harvest step.

[0037] Figure 13 Shows the product stability during low pH hold. No loss of AAV titer was observed during low pH flocculation.

[0038] Figure 14 Shows that the flocculated material shows no increase in turbidity after heat inactivation. Detailed Description

[0039] Recombinant adeno-associated virus (rAAV) vectors can be used in gene therapy to deliver therapeutic genes to patient cells and tissues. rAAV particles typically contain recombinant nucleic acid, which is encapsulated within AAV capsid proteins to form rAAV particles that can be administered to a subject. The recombinant nucleic acid (e.g., recombinant AAV genome) typically includes a heterologous gene of interest (e.g., encoding a therapeutic nucleic acid and / or protein) flanked by AAV inverted terminal repeat (ITR) sequences. The AAV capsid proteins can be naturally occurring capsids of different AAV serotypes. For example, different AAV serotypes have different tissue tropisms and can be used to target different tissue types and associated diseases. In some embodiments, the AAV capsid proteins contain one or more amino acid substitutions relative to the naturally occurring capsid proteins.

[0040] Different manufacturing techniques can be used to generate rAAV particles. Typically, rAAV particles are assembled in host cells in culture (e.g., in a bioreactor or other cell culture vessel). One or more nucleic acids encoding a recombinant AAV genome, an AAV capsid protein, and / or one or more Rep and helper genes are expressed in the host cells. The host cells are grown in culture (e.g., in suspension culture or on a plate). The assembled rAAV is then isolated from the cell culture. The host cells can be mammalian cells, insect cells, or other cell types. In some embodiments, the host cells are production cells.

[0041] Isolated rAAV preparations prepared by large-scale culture processes typically contain contaminating materials, including host cell materials, which may interfere with the purification process and / or destabilize the purified rAAV.

[0042] In some embodiments, aspects of the present application relate to incorporating a flocculation step in an rAAV manufacturing procedure. In some embodiments, an acid solution is added to the rAAV preparation under conditions that promote efficient removal of host cell materials (e.g., host cell proteins). In some embodiments, the acid solution is mixed with a sufficient amount of cell preparation for a time sufficient to effectively remove host cell materials. In some embodiments, the cell preparation comprises a plurality of cells for producing rAAV. In some embodiments, the cell preparation comprises a plurality of triple-transfected cells. In some embodiments, the cell preparation comprises a plurality of production cells. In some embodiments, the acid solution is mixed with the cell preparation after cell lysis. In some embodiments, the acid solution is not a triprotic acid solution. In some embodiments, the acid solution is an acid glycine solution.

[0043] In some embodiments, the density of the cell preparation is suitable for rAAV harvest. In some embodiments, the cell preparation has a density of about 0.5 - 12 x 10 6 cells / mL. In some embodiments, the cell preparation has a density of about 0.5 - 2, about 2 - 4, about 4 - 6, about 6 - 8, about 8 - 10, or about 10 - 12 x 10 6 cells / mL. In some embodiments, the cell preparation has a density of 0.5 - 1, 2 - 3, 3 - 4, 4 - 5, 5 - 6, 6 - 7, 7 - 8, 8 - 9, 9 - 10, or 11 - 12 x 10 6 cells / mL.

[0044] Figure 1ADisclosed are non-limiting examples of procedures for adding a flocculant (e.g., an acid solution) to a cell preparation containing rAAV particles. In some embodiments, the flocculant is an acid glycine solution. In some embodiments, the flocculant is a solution having a pH of 4 or lower, a pH of 3 or lower, or a pH of 2 or lower. In some embodiments, the flocculant is a solution having a pH of about 4, about 3.5, about 3, about 2.5, about 2, about 1.5, or about 1. In some embodiments, the flocculant can be added to a container (e.g., a bioreactor) including a mixing device (e.g., an impeller). In some embodiments, the appropriate speed of the impeller can be determined using one or more of the equations (1), (2), and / or (3) listed in Example 1.

[0045] In some embodiments, the cell preparation contains a cell culture. In some embodiments, the cell preparation contains a resuspended cell pellet. In some embodiments, the cell preparation contains a plurality of cells for producing rAAV. In some embodiments, the cell preparation contains a plurality of triple-transfected cells. In some embodiments, the cell preparation contains a plurality of production cells. In some embodiments, after cell lysis, an acid solution is mixed with the cell preparation. In some embodiments, the cell preparation is a cell harvest.

[0046] Figure 1B Disclosed are non-limiting examples of procedures for separating rAAV particles from a cell culture. In some embodiments, as Figure 1B shown, a flocculation step (e.g., using an acid glycine solution) is introduced after the cell lysis and nuclease steps and before subsequent clarification and additional purification steps. However, in some embodiments, the flocculation step can be combined before the cell lysis and nuclease steps, between the cell lysis and nuclease steps, simultaneously with the cell lysis and / or nuclease steps, and / or as an alternative to the addition of the cell lysis and / or nuclease. In some embodiments, cell lysis includes mechanical lysis, liquid homogenization, sonication, freeze / thaw cycles, or chemical lysis. In some embodiments, the chemical lysis conditions include treatment with a detergent such as Tween 20 or Triton X-100. In some embodiments, the nuclease is an endonuclease. In some embodiments, the nuclease is or includes Benzonase (Merck, an endonuclease derived from Serratia marcesens, optionally expressed in Escherichia Coli). In some embodiments, the nuclease is M-SAN HQ (nuclease; ArcticZymes).

[0047] In some embodiments, the method of flocculating cell material is suitable for large-scale culture and separation processes and provides surprising improvements over existing methods. In some embodiments, large-scale culture includes cultures of more than 1 L, more than 10 L, more than 25 L, more than 50 L, more than 100 L, more than 250 L, or more than 500 L. In some embodiments, large-scale culture includes 1 - 10 L, 10 - 25 L, 25 - 50 L, 50 - 100 L, 100 - 500 L, or 500 - 1000 L. In some embodiments, subsequent processing steps are significantly more efficient (e.g., shorter processing times and higher yields). In some embodiments, the resulting rAAV product is more stable. For example, in some embodiments, introducing the flocculation process described in the present application at the process scale efficiently removes impurities and results in a 4- to 5-fold reduction in host cell protein (HCP) for the downstream purification process.

[0048] In some embodiments, methods applicable at the process scale include purifying recombinant adeno-associated virus (rAAV) particles from a cell culture containing rAAV particles by contacting an rAAV preparation obtained from the cell culture with an acid solution (e.g., acid glycine solution, citric acid solution (also referred to as “citric acid”), octanoic acid solution) under conditions sufficient to promote flocculation of the cell material present in the rAAV preparation prior to subsequent purification of the rAAV. In some embodiments, the rAAV preparation is a cell culture harvest containing rAAV particles. In some embodiments, the rAAV preparation is a cell culture lysate (e.g., chemical lysate) containing rAAV particles. In some embodiments, the rAAV preparation is contacted with a nuclease (e.g., after lysis and prior to flocculation). However, in some embodiments, no nuclease is added prior to flocculation.

[0049] In some embodiments, the pH of the acid solution is below 4 (e.g., about 2.5). In some embodiments, sufficient acid is added to reduce the pH of the rAAV preparation to about (e.g., approximately) 2 - 4, about 3 - 4, about 3 - 5, about 4 - 5, about 2.5 - 3.5, about 2.5 - 4.5, about 3.5 - 5.5 (e.g., about pH 4). In some embodiments, the acid solution is a about 0.5 M solution, about 1 M solution, about 2 M solution, about 3 M solution, about 4 M solution, about 5 M solution, about 6 M solution, about 7 M solution, about 8 M solution, about 9 M solution, or about 10 M solution. In some embodiments, a 2 M acid solution at pH 2.5 is added to the rAAV preparation. In some embodiments, the pH of the rAAV preparation is adjusted to about pH 4 by adding an acid solution (e.g., 2 M acid glycine solution).

[0050] In some embodiments, the acid solution is a glycine acid solution. In some embodiments, the pH of the glycine acid solution is below 4 (e.g., about 2.5). In some embodiments, sufficient glycine acid is added to reduce the pH of the rAAV formulation to approximately (e.g., about) 2-4, about 3-4, about 3-5, about 4-5, about 2.5-3.5, about 2.5-4.5, about 3.5-5.5 (e.g., about pH 4). In some embodiments, the glycine acid solution is a about 1M solution, about 2M solution, about 3M solution, about 4M solution, about 5M solution, about 6M solution, about 7M solution, about 8M solution, about 9M solution, or about 10M solution. In some embodiments, a 2M glycine acid solution at pH 2.5 is added to the rAAV formulation.

[0051] In some embodiments, the acid solution is a citric acid solution. In some embodiments, the pH of the citric acid solution is below 4 (e.g., about 2.5). In some embodiments, sufficient citric acid is added to reduce the pH of the rAAV formulation to approximately (e.g., about) 2-4, about 3-4, about 3-5, about 4-5, about 2.5-3.5, about 2.5-4.5, about 3.5-5.5 (e.g., about pH 4). In some embodiments, the citric acid solution is a about 1M solution, about 2M solution, about 3M solution, about 4M solution, about 5M solution, about 6M solution, about 7M solution, about 8M solution, about 9M solution, or about 10M solution. In some embodiments, a 2M citric acid solution at pH 2.5 is added to the rAAV formulation.

[0052] In some embodiments, an acid solution (e.g., acid glycine solution) is added to the rAAV formulation at a volume of 5%-10% (e.g., approximately 8%). In some embodiments, the acid solution is added to the rAAV formulation at a volume of 1%-10%, 1%-5%, 2%-9%, 3%-8%, 4%-7%, 5%-9%, or 4%-8%. In some embodiments, the acid solution is added to the rAAV formulation over a period of approximately 10 minutes (e.g., over a period of approximately (e.g., about) 5 minutes). In some embodiments, the rAAV formulation is mixed with the added acid solution using a stirring speed of approximately (e.g., about) 30-150 RPM. In some embodiments, the rAAV formulation is mixed with the added acid solution using a stirring speed of approximately (e.g., about) 50-150 RPM. In some embodiments, for an rAAV formulation of approximately 5 L, a stirring speed of approximately (e.g., about) 90-110 RPM (e.g., about 100 RPM) is used. In some embodiments, for an rAAV formulation of approximately 5 L, a stirring speed of approximately (e.g., about) 30-200 RPM or 90-200 RPM is used. In some embodiments, for an rAAV formulation of approximately 50 L, a stirring speed of approximately (e.g., about) 50-75 RPM (e.g., about 63 RPM) is used. In some embodiments, for an rAAV formulation of approximately 50 L, a stirring speed of approximately (e.g., about) 50-100 or 50-150 RPM is used. In some embodiments, for an rAAV formulation of approximately 500 L, a stirring speed of approximately 30-50 RPM (e.g., about 42 RPM) is used. In some embodiments, for an rAAV formulation of approximately 500 L, a stirring speed of approximately 30-100 is used. In some embodiments, the stirring speed is adjusted to achieve a power / volume (P / V) ratio of approximately (e.g., about) 2-5. In some embodiments, the stirring speed is adjusted to achieve a P / V ratio of approximately 4.7. In some embodiments, the stirring speed is adjusted to achieve a P / V ratio of approximately 3.1. In some embodiments, before subsequent purification steps, the mixture of the rAAV formulation and the acid solution (e.g., acid glycine solution) is kept stationary in a container for 10-60 minutes (e.g., at room temperature) to promote flocculation of cell material. In some embodiments, the mixture is kept stationary for 15-45 minutes (e.g., the holding time is 15-45 minutes). In some embodiments, the mixture is kept stationary for about 30 minutes (e.g., the holding time is 30 minutes).In some embodiments, the mixture is kept static for up to 10 hours, up to 12 hours, up to 14 hours, up to 16 hours, up to 18 hours, up to 20 hours, up to 22 hours, or up to 24 hours; in some embodiments, the mixture is kept static between 30 minutes and 4 hours, between 30 minutes and 10 hours, between 10 minutes and 5 hours, between 20 minutes and 6 hours, between 10 minutes and 4 hours, or between 1 hour and 4 hours. In some embodiments, before a subsequent purification step, the mixture of the rAAV preparation and an acid solution (e.g., acid glycine solution) is slowly stirred (e.g., at 30 - 150 rpm) for 10 - 60 minutes (e.g., at room temperature) in a container to promote flocculation of the cell material. In some embodiments, the mixture is slowly stirred (e.g., at 30 - 150 rpm) for 15 - 45 minutes. In some embodiments, the mixture is slowly stirred (e.g., at 30 - 150 rpm) for about 30 minutes. In some embodiments, the mixture is slowly stirred (e.g., at 30 - 150 rpm) for up to 10 hours; in some embodiments, the mixture is slowly stirred (e.g., at 30 - 150 rpm) between 30 minutes and 4 hours, between 30 minutes and 10 hours, between 10 minutes and 5 hours, between 20 minutes and 6 hours, between 10 minutes and 4 hours, or between 1 hour and 4 hours. In some embodiments, before subsequent purification (e.g., before one or more clarification steps), the flocculated material is resuspended. Thus, in some embodiments, the flocculated mixture is clarified without an intermediate resuspension. In some embodiments, the flocculated mixture is resuspended before clarification. In some embodiments, clarification is carried out by filtration. In some embodiments, the filtration is depth filtration.

[0053] In some embodiments, the method is carried out at room temperature. In some embodiments, the method is carried out at 10°C - 40°C, such as 15°C - 35°C, 15°C - 20°C, 20°C - 25°C, or 25°C - 30°C.

[0054] In some embodiments, the method includes contacting the rAAV preparation with a flocculant (e.g., acid glycine). In some embodiments, the method includes contacting the rAAV preparation with glycine. In some embodiments, the method includes contacting the rAAV preparation with: alternative or additional flocculants, such as cationic polymers, such as polyethyleneimine (PEI) or polydiallyldimethylammonium chloride (pDADMAC), etc., and / or alternative or additional pH lowering agents, such as citric acid, phosphoric acid, and / or octanoic acid, and / or alternative or additional lysis agents, such as detergents. In some embodiments, the detergent is Triton, PS20 (tween20), or other detergents.

[0055] The present disclosure also provides compositions comprising AAV particles produced by the methods described herein. In some embodiments, an rAAV preparation after flocculation but before subsequent purification steps is more stable than the corresponding preparation without flocculation. In some embodiments, the post-flocculation rAAV preparation can be held (e.g., for up to 2 weeks or longer). In some embodiments, one or more post-flocculation rAAV preparations can be held, e.g., for 1 - 2 weeks or longer, and then combined for subsequent purification steps.

[0056] Recombinant AAV

[0057] Naturally occurring AAV capsid proteins can be used to generate rAAV for gene therapy. Different naturally occurring AAVs have different characteristics (including, for example, different tissue tropisms) and can be used for different indications. AAV is highly prevalent in the human population (see Gao, G. et al., Clades of Adeno-associated viruses are widely disseminated in human tissues J Virol. 2004. 78(12): pp. 6381-8, and Boutin, S. et al., Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population, implications for gene therapy using AAV vectors. Hum Gene Ther. 2010. 21(6): pp. 704-12) and can be used as viral vectors. There are many serotypes, each with a different tropism for tissue types (see Zincarelli, C. et al., Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther, 2008. 16(6): pp. 1073-80), which allows for preferential targeting of specific tissues with appropriate pseudotyping. Some serotypes, such as serotypes 8, 9, and rh10, transduce the mammalian body.See Zincarelli, C. et al., Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther, 2008. 16(6): pp. 1073-80, Inagaki, K. et al., Robust systemic transduction with AAV9 vectors in mice: efficient global cardiac gene transfer superior to that of AAV8. Mol Ther, 2006. 14(1): pp. 45-53, Keeler, A.M. et al., Long-term correction of very long-chain acyl-coA dehydrogenase deficiency in mice using AAV9 gene therapy. Mol Ther, 2012. 20(6): pp. 1131-8, Gray, S.J. et al., Preclinical differences of intravascular AAV9 delivery to neurons and glia: a comparative study of adult mice and nonhuman primates. Mol Ther, 2011. 19(6): pp. 1058-69, Okada, H. et al., Robust Long-term Transduction of Common Marmoset Neuromuscular Tissue With rAAV1 and rAAV9. Mol Ther Nucleic Acids, 2013. 2: p. e95, and Foust, K.D. et al., Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol, 2009. 27(1): pp. 59-65.It has been demonstrated that AAV9 crosses the blood-brain barrier (see Foust, K.D. et al., Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol, 2009. 27(1): pp. 59-65, and Rahim, A.A. et al., Intravenous administration of AAV2 / 9 to the fetal and neonatal mouse leads to differential targeting of CNS cell types and extensive transduction of the nervous system. FASEB J, 2011. 25(10): pp. 3505-18), which is inaccessible to many viral vectors and biologics. The payload of certain AAVs is 4.7 - 5.0 kb (including the viral inverted terminal repeats (ITRs), which are cis-essential for viral packaging). See Wu, Z., H. Yang and P. Colosi, Effect of genome size on AAV vector packaging. Mol Ther, 2010. 18(1): pp. 80-6 and Dong, J.Y., P.D. Fan and R.A. Frizzell, Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Hum Gene Ther, 1996. 7(17): pp. 2101-12.

[0058] In some embodiments, the rAAV can include one or more variant AAV capsid proteins having one or more amino acid substitutions relative to a naturally occurring AAV capsid protein.

[0059] Thus, in some embodiments, the rAAV particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 capsid protein, or an amino acid sequence variant thereof. In some embodiments, the rAAV particle comprises a hybrid capsid protein derived from any combination of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 capsid proteins.

[0060] In some embodiments, the methods described herein are beneficial for manufacturing processes. These benefits include, but are not limited to, a greater than 10-fold reduction in host cell impurities without the use of endonucleases, efficient filtration with no flux decline, and a 5-fold higher affinity resin life cycle due to low impurities in the loading material. Additionally, due to less interference from impurities, the subtle charge differences between the three capsids enable higher resolution and better enrichment of the intact capsids. By controlling the impurity level of the upstream feed stream, the rAAV viral vector exhibits improved stability and minimal aggregation at low conductivity, which further enhances the process recovery rate. In the case of a cleaner feed stream, the downstream intermediate reaches a turbidity value that is more than 10-fold lower and the rAAV titer is maintained, enabling easy filtration and manufacturing robustness. Flocculation has been successfully demonstrated as an innovative rAAV manufacturing technology for multiple AAV serotypes. Implementation in the rAAV process platform not only results in excellent product quality, significant benefits to downstream recovery, but also substantial cost savings in rAAV manufacturing.

[0061] These and other aspects are illustrated by the following non-limiting examples.

[0062] Example

[0063] Example 1

[0064] As Figure 1A and Figure 1B shown, the pH of the cell culture harvest was reduced to pH 4 using 2M glycine at pH 2.5. After cell lysis and digestion, the impeller speed was reduced to match a power / volume ratio of 3.1 in some experiments and 4.7 in some experiments. Similar calculations of power / volume ratio (e.g., 2 - 5) are expected to behave similarly. The cell lysis and digestion steps were performed in the context of the purification of AAV produced using the triple transfection method, but are optional for AAV produced from production cell lines. The flocculation buffer was pumped into the bioreactor through a dip tube near the impeller. There were two acid addition steps, with a total target volume of 8% of the cell culture harvest volume. First, 80% of the target volume was added, held for one minute for pH reading, and the remaining glycine was pumped in until the target pH of 4 was reached. The pumping rate was calculated by limiting the acid addition to a 10-minute addition period. When the pH was reached, the flocculated material was kept static (e.g., not stirred) for 30 minutes to allow large particles to form. The two phases (precipitate and supernatant) were remixed and loaded onto a Clarisolve depth filter. Immediately after depth filtration, the filtrate was neutralized by adding 5% V / V 2M Tris buffer.

[0065] The following equation (1) can be used to evaluate the flocculation efficiency:

[0066]

[0067] where C0 is the initial impurity level, and it is assumed that the impurity level is the same under the same cell density and the same lysis conditions. t F is the flocculation time for precipitating impurities and forming large particles, C F is the dosage of the flocculant, N is the impeller stirring speed, which can affect the mixing efficiency during the addition of the flocculant and also the size of the precipitate. P / V is the power input, approaching the average turbulent kinetic energy dissipation ε 平均 , pH is the target pH in the bioreactor after flocculation, and T is the temperature in the bioreactor. Cr is the impurity level after flocculation, and this impurity level can be the criterion for evaluating the flocculation efficiency. As shown in equation (2), the P / V ratio is proportional to the impeller type, configuration, spacing (Np), impeller speed (N), liquid density (ρ), and impeller diameter (D).

[0068]

[0069] To achieve flocculation on a process scale, the scale-up rule is based on the same flocculation efficiency. Given the target pH and T, the flocculation efficiency is based on a constant P / V ratio, flocculation time (t F ) and flocculation dosage (C F / C0). As shown in equation (2), the stirring speed is determined by the geometry of the bioreactor. Given the impeller diameters in large scale (DL) and small scale (DS), the stirring speed in large scale NL is determined by equation (3) to maintain the same flocculation efficiency during the scale-up process.

[0070] Example 2

[0071] An AAV purification process has been developed to purify AAV particles from cell cultures and enrich AAV preparations of intact AAV particles (e.g., containing a recombinant AAV genome) relative to empty AAV particles (e.g., containing capsid proteins but no encapsulated nucleic acid).

[0072] An improved purification process was developed. The method uses 2M glycine at pH 2.5 as a flocculant buffer after DNA digestion to lower the lysed harvest to pH 4 to introduce flocculation. In some embodiments, the flocculation procedure involves forming a target agitation speed, a target pump rate for pumping in acid, and / or reaching a target pH and maintaining that pH for a target hold time. This process was developed using AAV9 as an example. This improved purification process is surprisingly effective. It is characterized by several improvements, including: a 4- to 5-fold reduction in HCP / DNA in the harvest step, a higher clarification throughput, and a more stable and higher-yield AAV product.

[0073] As Figure 2A and Figure 2B shown, four different acids (citric acid, phosphoric acid, glycine, and octanoic acid) and two different cationic polymers, polyethyleneimine (PEI) or poly(diallyldimethylammonium chloride) (pDADMAC), were used as flocculants during harvest. After using different flocculants, the post-flocculation titer and host cell protein reduction were studied. The HCP levels indicated that pH 4 was more effective than pH 4.5 and pH 5 and provided a 4- to 5-fold reduction in HCP ( Figure 2A ). Citric acid and pDADMAC also provided detectable levels of HCP reduction. According to Figure 2B the post-flocculation titers in

[0074] no significant titer loss was observed among the various flocculants. Higher titers were observed when glycine was used to flocculate the cell harvest. Figure 3A ) When using acid-glycine-based flocculation at pH 4 for further analysis. The flocculated material was clarified using a Clarisolve depth filter. The HCP level in the clarified filtrate after flocculation was more than 10-fold lower than the HCP level in the clarified filtrate without flocculation ( 2 ). After flocculation, the depth filter performance was also improved. Fine particles with an average size of 2 µm in the throughput were notable because they easily clog the depth filter and significantly reduce the throughput. As a result of the flocculation process, the fine particles aggregated into large particles, which increased the throughput of the depth filter from 31 to 92.5 L / m Figure 3B ) without increasing the inlet pressure.

[0075] Therefore, glycine can be used to lower the pH of the harvest and precipitate impurities. The acid addition rate, agitation in the bioreactor during acid addition, and scale-up rules were defined to ensure robust flocculation efficiency in multiple large-scale manufacturing processes.

[0076] In some embodiments, implementing a flocculation procedure can improve the performance of one or more purification stages and / or improve product quality and stability.

[0077] Example 3

[0078] To determine the impact of flocculation on downstream aspects of AAV purification, flocculated and non-flocculated material were purified using ultrafiltration / diafiltration, capture chromatography, and polishing chromatography.

[0079] Ultrafiltration / diafiltration

[0080] exist Figure 4A and Figure 4B The ultrafiltration / diafiltration performance with and without flocculation was compared in . Figure 4A The UFDF performance with flocculated material is shown to have double the flux and no significant flux decay compared to the UFDF performance with non-flocculated material. Figure 4B It is shown that the run time in the case of flocculated material is twice as fast as that in the case of non-flocculated material. This can translate into higher throughput and potential savings in material costs on the UFDF filter when using flocculated material. Due to the low level of impurities (HCL / HC DNA) in the TFF load after flocculation, the UFDF performs much better with higher flux, shorter run times and much cleaner pools after concentration.

[0081] Capture chromatography

[0082] Figure 5A , Figure 5B and Figure 5C The effect of flocculation on capture chromatography (affinity column) is shown in . In the absence of flocculation, a very high UV signal was observed in the flow-through ( Figure 5B ), which means high impurity level in the affinity load. After flocculation, the UV signal in the flow-through decreased from 2000mAU to less than 100mAU ( Figure 5A ), indicating very low impurities in the affinity load. Low impurity levels improve the efficiency of column binding and capture recovery. Figure 5C Also shown in Figure 1 is the host cell DNA in the affinity eluate with and without flocculation. Compared to the affinity eluate without flocculation, the host cell DNA is more than 10 times lower after flocculation. This is another evidence that flocculation significantly removes impurities and improves product quality.

[0083] Refining chromatography

[0084] Figures 6A - 6B The benefit of polishing chromatography is shown. The flocculated material showed a single peak ( Figure 6A) while the material without flocculation shows multiple peaks and no enrichment. Figure 6B The multi-peak pattern in Figure 6B means that there may be several impurity-related substances and makes it difficult to separate the intact vector from the empty vector. Impurities may also induce product aggregation and make the Z-avg greater than 30 nm, especially in the case of low conductivity. The z-avg in the table shows that after dilution, the flocculated material retains 30 nm and has excellent stability, while the average particle size of the non-flocculated material increases significantly.

[0085] Intact vector concentration

[0086] Figure 7A The enrichment of the intact vector after refined chromatography is shown in Figure 7A . The material in the case of flocculation shows that the intact capsid is enriched from 18% to 50.3% on the right side of the main peak. However, Figure 7B the material without flocculation in Figure 7B shows multiple peaks, and enrichment only occurs in the middle part of the peaks. This result proves that in the case of fewer impurities related to the product, the refined step is easier to separate the intact capsid with a higher enrichment degree and a higher recovery rate.

[0087] Product stability

[0088] Figure 8 The product stability with and without flocculation is shown in Figure 8 . The material after UFDF is kept at room temperature, and the turbidity is measured after keeping for different time periods. Without being bound by theory, unstable materials will form aggregates and precipitate, resulting in an increase in the turbidity value. The flocculated material shows a stable turbidity profile for more than half a month, with all values below 20 NTU, while the turbidity of the non-flocculated material treated without a flocculant increases sharply from 150 to more than 500 NTU within 10 days. Since impurities can be related to the viral vector product and induce aggregation during storage, the flocculation step can highly stabilize the product.

[0089] Reuse of affinity resin

[0090] Affinity resin is another item that has a huge impact on the material cost in AAV process development. Recycling the affinity resin while having the same purification capacity can significantly reduce the manufacturing cost.

[0091] The cell lysate materials with and without flocculation are loaded onto the affinity column. For the non-flocculated material, the high impurity content in the loaded material has the potential to clog the column or endanger the column life. Figure 11A and Figure 11B show the number of affinity resin cycles used to purify the non-flocculated material (A) and the flocculated material (B).

[0092] As Figure 11AAs shown, the pre-column pressure increased significantly after 5 cycles, making it impossible for the column to be reused for multiple cycles. With flocculation during the harvest step, much cleaner material was loaded onto the affinity chromatography column. In Figure 11B , the same resin can be used up to 20 cycles without any impact on yield and product quality. The lower impurity burden in the affinity load ensures that the affinity column has a higher potential to achieve better purification performance with more column cycles and cost savings.

[0093] AAV aggregation

[0094] The AAV aggregation behavior at low conductivity is a major technical challenge in the development of gene therapy processes. The root cause of this behavior is not yet clear, and several hypotheses are under investigation. One widely discussed hypothesis is that this behavior is related to the impurity profile in the process buffer matrix. Trace amounts of nucleic acids or host cell proteins in the process may induce significant product aggregation at low conductivity. Mitigating AAV aggregation at low conductivity has great benefits for polishing chromatography and improving manufacturing robustness. Figure 12 Shows AAV aggregation before and after flocculation in the process. The materials in Runs 1-4 were not treated with flocculation, while the materials in Runs 5-8 were treated with glycine during the harvest process. All eight runs were treated with endonuclease. The remaining unit operations were kept the same, and high conductivity was measured after affinity chromatography. The aggregation level can be obtained by averaging the hydrodynamic diameter (Z-avg). The AAV product aggregated up to 400 nm at low conductivity without flocculation. For Runs 5-8, the flocculation step at harvest controlled the impurity level and provided less impurity entering downstream. Due to the removal of impurities, less aggregation was observed at low conductivity, indicating enhanced product stability after introducing the flocculation step as described herein.

[0095] Conclusion

[0096] Using acid precipitation for flocculation in AAV purification showed a significant reduction in HCP and HC DNA for downstream purification. Glycine was used to lower the pH of the harvest and precipitate impurities. The acid addition rate, agitation in the bioreactor during acid addition, and scale-up rules have been defined to ensure robust flocculation efficiency in multiple large-scale manufacturing. The benefits of achieving flocculation have been shown in terms of better product quality and stability, shorter UFDF run times, higher enrichment and yield of intact viral vectors in this article. This method demonstrated has the potential to be used in other AAV purification processes to ensure process robustness and better product quality performance.

[0097] Example 4

[0098] Non-limiting embodiments of the flocculation method described herein were tested on different rAAV serotypes produced from a production cell line (PCL). The rAAV serotypes are different from the rAAV serotypes tested in Examples 1-3 produced using the triple transfection method.

[0099] - Flocculant screening

[0100] Cell culture harvest material containing the tested PCL-based rAAV serotypes was treated with glycine and citric acid at different pHs, and the effects of acid type and pH conditions on impurity removal were evaluated. The results are shown in Figure 9 . Both glycine and citric acid used as flocculants had a minimal negative impact on the recovery of cell culture of PCL-based rAAV serotypes, and thus the pH could be reduced from pH 7.5 or pH 8 to acidic using different buffers, and the pH is the driving force for flocculation. Depending on which acid is used in subsequent purification steps, different acids can be selected as flocculants to treat cell culture harvests, which gives flexibility in the selectivity of flocculant reagents.

[0101] Host cell DNA removal

[0102] For PCL-based rAAV serotypes, host cell DNA (HC DNA) concentrations were measured under different flocculation conditions. In parallel, cell culture material was treated with an endonuclease, and the HC DNA concentration was quantified as a control. The endonuclease is an expensive enzyme, and thus its use has a huge impact on the material cost of gene therapy process development. It is ideal to maintain the same level of HC DNA reduction while substituting the endonuclease. Without endonuclease digestion, flocculation using an acid was directly applied to the cell culture to study the level of HC DNA reduction. Compared with endonuclease digestion, the host cell DNA level decreased significantly from 9806 to below 1000 ng / mL after acid treatment ( Figure 10 ). The flocculation method provides a much cleaner upstream material and brings many benefits to downstream purification and manufacturing robustness. In addition, the endonuclease-free process also helps to significantly reduce the manufacturing cost.

[0103] Product stability during low pH hold

[0104] Cell culture harvests containing PCL-based rAAV particles were adjusted to pH 4 with an acid buffer for flocculation and incubated for different time periods (0.5, 1, 2, 3, 4 hours). The percentage recovery results showed that AAV was stable for at least 4 hours at pH 4 incubation without titer loss. A slight increase in titer was observed at different time points, ranging from about 10% to 20%. The results are shown in Figure 13 .

[0105] Heat inactivation

[0106] In a production cell line (PCL) platform, Ad5 is introduced into cell culture harvests and needs to be removed downstream. Heating to a temperature that inactivates Ad5 while maintaining AAV activity is a major strategy for virus clearance. However, during heat inactivation, impurities (e.g., host cell DNA and proteins) degrade and aggregate, resulting in a substantial increase in turbidity. High turbidity poses many challenges to downstream processing, including filter clogging and product loss. As Figure 14 shown, the flocculated material shows little or no increase in turbidity after heat inactivation, thus avoiding the difficulties associated with high turbidity.

[0107] Equivalent embodiments

[0108] Although several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision various other devices and / or structures and / or one or more of the advantages described herein for performing the functions and / or obtaining the results, and each such variation and / or modification is considered to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and the actual parameters, dimensions, materials, and / or configurations will depend on one or more specific applications of the teachings of this invention. Those skilled in the art will recognize or be able to determine many equivalents of the inventive embodiments described herein using only routine experimentation. Accordingly, it should be understood that the foregoing embodiments are presented by way of example only, and that within the scope of the appended claims and their equivalents, the inventive embodiments may be practiced in a manner different from that specifically described and claimed. The inventive embodiments of this disclosure are directed to each separate feature, system, article, material, kit, and / or method described herein. Additionally, any combination of two or more such features, systems, articles, materials, kits, and / or methods that are not mutually inconsistent is included within the scope of the invention of this disclosure.

[0109] All definitions defined and used herein should be understood to be prior to dictionary definitions, definitions in incorporated documents by reference, and / or ordinary meanings of the defined terms.

[0110] All references, patents, and patent applications disclosed herein are incorporated by reference for the subject matter for which each is cited, and in some cases they may cover the entire document.

[0111] Unless the contrary is clearly indicated, the indefinite articles "a" and "an" as used herein in the specification and claims shall be understood to mean "at least one".

[0112] As used herein in the specification and claims, the phrase "and / or" shall be understood to mean "either or both" of the elements so combined, i.e., elements that in some cases coexist and in other cases separate exist. Multiple elements listed with "and / or" shall be construed in the same manner, i.e., "one or more" of the elements so combined. In addition to the elements specifically identified by the "and / or" clause, other elements may optionally exist, whether or not related to those specifically identified. Thus, as a non-limiting example, when used in conjunction with open-ended language such as "comprising", a reference to "A and / or B" may in one embodiment refer only to A (optionally including elements other than B); in another embodiment only to B (optionally including elements other than A); in yet another embodiment to both A and B (optionally including other elements); and so on.

[0113] As used herein in the specification and claims, "or" shall be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted inclusively, i.e., including at least one, but also including more than one of several elements or the list of elements, and optionally, additional unlisted items. Only terms that clearly indicate the contrary, such as "only one of... " or "exactly one of... " or when used in a claim "consisting of... ", will refer to including exactly one of several elements or the list of elements. In general, the term "or" as used herein shall be interpreted as indicating an exclusive alternative (i.e., "one or the other, but not both") only when followed by an exclusive term such as "either", "one of... ", "only one of... " or "exactly one of... ". When used in a claim, "consisting essentially of... " shall have its ordinary meaning as used in the field of patent law.

[0114] As used herein in the specification and claims, the phrase "at least one" with respect to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each of the specifically listed elements in the list of elements, and not excluding any combinations of elements in the list of elements. This definition also allows that elements other than those specifically recited in the list of elements referred to in the phrase "at least one" may optionally be present, whether or not related to those specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B" or equivalently "at least one of A and / or B") can in one embodiment mean at least one, optionally including more than one A, with no B present (and optionally including elements other than B); in another embodiment mean at least one B, optionally including more than one B, with no A present (and optionally including elements other than A); in yet another embodiment mean at least one A, optionally including more than one A, and at least one B, optionally including more than one B (and optionally including other elements); and so forth.

[0115] It should also be understood that, unless explicitly indicated to the contrary, in any method claimed herein that includes more than one step or act, the order of the steps or acts of the method need not be limited to the recited order of the steps or acts of the method.

[0116] In the claims as well as in the above specification, all transitional phrases such as "comprising", "including", "carrying", "having", "containing", "involving", "holding", "consisting of", etc., should be understood to be open-ended, i.e., meaning including but not limited to. As set forth in Section 2111.03 of the Patent Examination Procedure Manual of the United States Patent and Trademark Office, only the transitional phrases "consisting of" and "consisting essentially of" should be closed or semi-closed transitional phrases, respectively. It should be understood that embodiments described in this document using open-ended transitional phrases (e.g., "including") are also considered in alternative embodiments as "consisting of the features described by the open-ended transitional phrase" and "consisting essentially of the features described by the open-ended transitional phrase". For example, if the present disclosure describes "a composition comprising A and B", the present disclosure also contemplates alternative embodiments "a composition consisting of A and B" and "a composition consisting essentially of A and B".

Claims

1. A method for purifying recombinant adeno-associated virus (rAAV) particles from a cell culture containing the rAAV particles, the method comprising: a) contacting the rAAV preparation with an acid solution under conditions sufficient to promote flocculation of the cellular material present in the rAAV preparation obtained from the cell culture; and b) separating the rAAV particles from the flocculated cellular material, optionally wherein the acid solution comprises an acid glycine solution.

2. The method according to claim 1, wherein the rAAV preparation is a cell culture harvest containing rAAV particles.

3. The method according to claim 1, wherein the rAAV preparation is a cell culture lysate containing rAAV particles.

4. The method according to any one of claims 1-3, the method further comprising contacting the rAAV preparation with a nuclease and / or subjecting the preparation to cell lysis.

5. The method according to any one of claims 1-4, wherein the pH of the acid solution is below 4.

6. The method according to any one of claims 1-5, wherein the pH of the acid solution is about 2.

5.

7. The method according to any one of claims 1-6, wherein a 2M acid solution at pH 2.5 is added to the rAAV preparation.

8. The method according to any one of claims 1-7, wherein the acid solution is added to the rAAV preparation in a volume of 5%-10%.

9. The method according to claim 8, wherein the acid solution is added to the rAAV preparation in a volume of 8%.

10. The method according to any one of claims 1-9, wherein the acid solution is added to the rAAV preparation over a period of 10 minutes.

11. The method according to any one of claims 1-10, wherein the acid solution is added to the rAAV preparation over a period of 5 minutes.

12. The method according to any one of claims 1-11, wherein the rAAV preparation is mixed with the added acid solution using a stirring speed of 30-150 RPM.

13. The method according to claim 12, wherein the stirring speed is 100 RPM.

14. The method according to any one of claims 1-13, wherein the volume of the rAAV preparation is 2-500 L.

15. The method according to claim 14, wherein the volume is 5 L, 50 L or 500 L.

16. The method according to any one of claims 1-15, wherein the volume of the rAAV preparation is 5 L, and the stirring speed is about 90-110 RPM or 100 RPM.

17. The method according to any one of claims 1-15, wherein the volume of the rAAV preparation is 50 L, and the stirring speed is about 50-75 RPM or 63 RPM.

18. The method according to any one of claims 1-15, wherein the volume of the rAAV preparation is 500 L, and the stirring speed is about 30-50 RPM or 42 RPM.

19. The method according to any one of claims 1-18, wherein the product of a) has a pH of 3-5.

20. The method according to claim 19, wherein the pH is about 4 or approximately 4.

21. The method according to any one of claims 1-20, wherein the mixture of the rAAV preparation of a) and the acid solution is kept static in a container for 10-60 minutes to promote flocculation of the cell material.

22. The method according to claim 21, wherein the mixture is kept static for 15-45 minutes.

23. The method according to claim 22, wherein the mixture is kept static for at least 30 minutes.

24. The method according to any one of claims 1-20, wherein the flocculated material from a) is resuspended before separating the AAV particles from the flocculated cell material.

25. The method according to any one of claims 1-24, wherein the product of a) is clarified.

26. The method according to claim 24, wherein the resuspended product of a) is clarified.

27. The method according to claim 25 or 26, wherein the clarification is carried out by filtration.

28. The method according to claim 27, wherein the filtration is depth filtration.

29. The method according to any one of claims 1-28, wherein the rAAV particles are recombinant AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 particles, their derivatives and / or combinations, or wherein the rAAV particles comprise a hybrid capsid.

30. The method according to any one of claims 1-29, wherein the rAAV particles are rAAV9 particles.

31. The method according to any one of claims 1-30, wherein the rAAV particles are added to a pharmaceutically acceptable solution.

32. The method according to any one of claims 1-31, wherein a plurality of the rAAV particles encapsulate a recombinant nucleic acid, the recombinant nucleic acid comprising a gene of interest flanked by AAV ITRs.

33. The method according to any one of claims 1-31, wherein the rAAV particles are produced using a triple transfection method.

34. The method according to any one of claims 1-31, wherein the rAAV particles are produced using a producer cell line (PCL) method.

35. A composition comprising rAAV particles produced by the method according to any one of claims 1-34.

36. A method comprising administering to a subject the composition according to claim 35.

37. A method for purifying rAAV particles from a cell culture comprising rAAV particles, the method comprising: a) contacting the rAAV preparation with a flocculant under conditions sufficient to promote flocculation of the cell material present in the rAAV preparation obtained from the cell culture; and b) separating the rAAV particles from the flocculated cell material.

38. The method according to claim 37, wherein the flocculant is a pH reducing agent or a cationic polymer.

39. The method according to claim 38, wherein the pH reducing agent comprises citric acid, phosphoric acid, glycine, and / or octanoic acid.

40. The method according to claim 37, wherein the cationic polymer is polyethyleneimine (PEI) or polydiallyldimethylammonium chloride (pDADMAC).

41. A method for purifying rAAV particles from a cell culture comprising rAAV particles, the method comprising: a) contacting the rAAV preparation with a flocculant under conditions sufficient to promote flocculation of the cell material present in the rAAV preparation obtained from the cell culture; and b) separating the rAAV particles from the flocculated cell material, wherein the method does not include the steps of: (i) contacting the rAAV preparation with an endonuclease and / or (ii) subjecting the rAAV preparation to cell lysis.

42. The method according to claim 41, wherein the rAAV particles in the cell culture are produced using the PCL method.

43. The method according to claim 41 or claim 42, wherein the rAAV particles are recombinant AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 particles, derivatives thereof, and / or combinations, or wherein the rAAV particles comprise a hybrid capsid.

44. The method according to any one of the preceding claims, wherein the density of the cell culture is 0.5 - 12 x 10 6 cells / mL.

45. The method according to claim 44, wherein the density of the cell culture is 0.5 - 2 x 10 6 cells / mL.

46. The method according to claim 44, wherein the density of the cell culture is 11-12 x 10 6 cells / mL.

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