Condensed AAV Refined

Abstract

The present disclosure provides methods for purifying adeno-associated virus (AAV) particles by introducing an aggregation step into large-scale manufacturing processes, thereby improving the purity and yield of AAV particles.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 425,998, filed November 16, 2022, entitled "FLOCCULATION AAV PURIFICATION," the contents of which are incorporated herein by reference in their entirety.

[0002] The present invention relates to a method for purifying recombinant AAV particles for use in gene therapy. [Background technology]

[0003] During typical gene therapy drug manufacturing, viral vectors are produced in cell culture and isolated from the cultured cells harvested in a process involving cell lysis.The isolated viral vector preparation contains impurities from the manufacturing process, including cellular material released during cell lysis.The impurities can cause instability of the viral vector and also impose a heavy burden on downstream purification processes. Summary of the Invention [Problem to be solved by the invention]

[0004] Therefore, there is a need to improve current manufacturing processes for viral vectors used in gene therapy. [Means for solving the problem]

[0005] This application provides methods and compositions for purifying recombinant adeno-associated virus (rAAV) particles from cell culture. In some aspects, the methods and compositions are useful for large-scale production of rAAV for use in gene therapy, and can enhance the purity and stability of rAAV compositions.

[0006] In some embodiments, the rAAV particles are isolated from a cell culture containing the rAAV particles using a process comprising: a) contacting an rAAV preparation obtained from the cell culture with an acidic glycine solution under conditions sufficient to promote aggregation of cellular material present in the rAAV preparation; and b) separating the rAAV particles from the aggregated cellular material.

[0007] 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, the lysate is obtained using a chemical lysis technique. In some embodiments, the rAAV preparation is obtained using a nucleic acid degradation technique. For example, in some embodiments, the rAAV preparation is obtained by a process comprising contacting a cell lysate containing rAAV particles with a nuclease.

[0008] In some embodiments, the pH of the acidic glycine solution used to aggregate cellular material is less than 4. In some embodiments, the pH of the acidic glycine solution is about 2.5. In some embodiments, a 1-3 M acidic glycine solution is added to the rAAV preparation. In some embodiments, a 2 M acidic glycine solution at pH 2.5 is added to the rAAV preparation.

[0009] In some embodiments, the acidic glycine solution is added to the rAAV preparation at a volume of 5-10%. In some embodiments, the acidic glycine solution is added to the rAAV preparation at a volume of 8%. In some embodiments, the volume of acidic glycine solution is added to the rAAV preparation within a 10 minute period. In some embodiments, the volume of acidic glycine solution is added to the rAAV preparation within a 5 minute period.

[0010] In some embodiments, the rAAV preparation is mixed with the added acidic glycine solution using an agitation speed of 30-150 RPM, hi some embodiments, the agitation speed is 100 RPM.

[0011] In some embodiments, the volume of the rAAV preparation is between 2 and 500 L. In some embodiments, the volume of the AAV preparation is 5 L, 50 L, or 500 L.

[0012] In some embodiments, the volume of the rAAV preparation is about 5 L and the stirring rate is about 90-110 RPM, e.g., about 100 RPM. In some embodiments, the volume of the rAAV preparation is about 50 L and the stirring rate is about 50-75 RPM, e.g., about 63 RPM. In some embodiments, the volume of the AAV preparation is about 500 L and the stirring rate is about 30-50 RPM, e.g., about 42 RPM.

[0013] In some embodiments, the product of a) has a pH of 3 to 5 after the addition of glycine (e.g., after the addition of 5 to 10%, e.g., 8%, by volume of 2 M glycine at pH 2.5). In some embodiments, the pH of the product of a) is around 4 (e.g., about 4).

[0014] In some embodiments, the mixture of the rAAV preparation of a) and the acidic glycine solution is allowed to stand in a container for 10 to 60 minutes (e.g., at room temperature) to promote aggregation of the cellular material. In some embodiments, the mixture is allowed to stand for 15 to 45 minutes. In some embodiments, the mixture is allowed to stand for about 30 minutes. In some embodiments, the aggregated material from a) is resuspended prior to separating the rAAV particles from the aggregated cellular material.

[0015] 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 by filtration. In some embodiments, the filtration is depth filtration.

[0016] In some embodiments, glycine is the only pH-lowering agent used to flocculate the cellular material. However, in some embodiments, alternative or additional flocculating agents can be used. In some embodiments, the alternative or additional flocculating agent is a pH-lowering agent or a cationic polymer. In some embodiments, the alternative or additional pH-lowering agent can include citric acid, phosphoric acid, and / or caprylic acid. In some embodiments, the cationic polymer is polyethyleneimine (PEI) or polydiallyldimethylammonium chloride (pDADMAC).

[0017] In some embodiments, a lysing agent, such as a detergent, can be used in conjunction with the flocculating agent.

[0018] In some embodiments, the rAAV particles comprise capsid proteins of an 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.

[0019] In some embodiments, the rAAV particles comprise a recombinant nucleic acid (e.g., a recombinant AAV genome) that includes 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.

[0020] In some embodiments, the rAAV particles are further purified, e.g., after clarification of the rAAV preparation, using, e.g., one or more affinity, ion exchange chromatography, and / or hydrophobic interaction chromatography steps.

[0021] In some embodiments, the rAAV particles are added to a pharmaceutically acceptable solution (e.g., after one or more purification steps).

[0022] The application also provides compositions comprising the rAAV particles and methods for administering the rAAV particles to a subject (e.g., a human subject having a condition that the therapeutic RNA and / or protein can help treat).

[0023] These and other embodiments are described in the following detailed description and examples, along with the figures. [Brief explanation of the drawings]

[0024] [Figure 1A-1B]

[0023] Figure 1A shows a schematic of the aggregation procedure, and Figure 1B shows a non-limiting embodiment of an aggregation step incorporated into a large-scale rAAV manufacturing process. [Figure 2A] Figure 2A shows a flocculant screening glass. Figure 2A shows a non-limiting example of host cell protein (HCP) reduction after flocculation under different flocculant conditions. [Figure 2B] Figure 2B shows an agglutinant screening glass. Figure 2B shows a non-limiting example of rAAV titer recovery after aggregation. [Figure 3A-3B] Non-limiting examples of depth filtration performance with and without flocculation are shown in Figure 3A. HCP reduction after depth filtration is shown in Figure 3A, and throughput improvement after flocculation is shown in Figure 3B. [Figure 4A] Figure 4 shows ultrafiltration / diafiltration (UFDF) performance with and without aggregation. Figure 4A shows UFDF performance where aggregated material has double the flux with no detectable flux decay compared to that with non-aggregated material. [Figure 4B] Figure 4B shows ultrafiltration / diafiltration (UFDF) performance with and without flocculation. Figure 4B shows that the run time with flocculating material is two times faster than the run time without flocculating material. [Figure 5A] Figures 5A and 5B show the effect of aggregation on capture chromatography performance with (A) and without (B) aggregation. [Figure 5B]Figures 5A and 5B show the effect of aggregation on capture chromatography performance with (A) and without (B) aggregation. [Figure 5C] Figure 5C shows the effect of aggregation on the performance of capture chromatography, and Figure 5C shows host cell DNA reduction in affinity eluates with and without aggregation. [Figure 6A] The polishing chromatographic performance with (A) and without (B) flocculation is shown. The average product size before and after dilution is shown below, demonstrating the stability of the product. [Figure 6B] The polishing chromatographic performance with (A) and without (B) flocculation is shown. The average product size before and after dilution is shown below, demonstrating the stability of the product. [Figure 7A] Complete capsid enrichment in polishing chromatography with aggregated (A) and non-aggregated (B) material is shown. The highlighted areas of the chromatogram indicate the enriched region. The table below shows the percentage of complete capsid enrichment after the polishing step. [Figure 7B] Complete capsid enrichment in polishing chromatography with aggregated (A) and non-aggregated (B) material is shown. The highlighted areas of the chromatogram indicate the enriched region. The table below shows the percentage of complete capsid enrichment after the polishing step. [Figure 8] The stability of the product after UFDF agglomerated (triangles) or non-agglomerated (circles) material is shown. [Figure 9] 1 shows recovery of AAV from the producer cell line platform after aggregation. [Figure 10] 1 shows host cell DNA reduction after aggregation of non-limiting producer cell line platforms. [Figure 11A] The number of affinity resin cycles for purifying non-aggregated material (A) and aggregated material (B) are shown. [Figure 11B] The number of affinity resin cycles for purifying non-aggregated material (A) and aggregated material (B) is shown. [Figure 12]This shows that the AAV product becomes more stable after aggregation treatment in the recovery step. [Figure 13] Figure 1 shows the stability of the product during a low pH hold. No loss of AAV titer was observed upon low pH aggregation. [Figure 14] Figure 1 shows that the aggregated material does not show an increase in turbidity after heat inactivation. DETAILED DESCRIPTION OF THE INVENTION

[0025] Recombinant adeno-associated virus (rAAV) vectors are useful in gene therapy for delivering therapeutic genes to patient cells and tissues. rAAV particles typically contain a recombinant nucleic acid encapsulated within an AAV capsid protein to form an rAAV particle that can be administered to a subject. The recombinant nucleic acid (e.g., a recombinant AAV genome) typically contains 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 protein can be a naturally occurring capsid of a different AAV serotype. 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 protein contains one or more amino acid substitutions compared to a naturally occurring capsid protein.

[0026] Various manufacturing techniques can be used to produce 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, AAV capsid proteins, 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 plates). 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 producer cells.

[0027] Isolated rAAV preparations made from large-scale cultivation processes typically contain contaminants, including host cell material, that can interfere with the purification process and / or destabilize the purified rAAV.

[0028] In some embodiments, aspects of the present application relate to the incorporation of an aggregation step in rAAV manufacturing procedures. In some embodiments, an acidic solution is added to an rAAV preparation under conditions that promote effective removal of host cell material (e.g., host cell proteins). In some embodiments, the acidic solution is mixed with the cell preparation in an amount and for a time sufficient to effectively remove the host cell material. 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 producer cells. In some embodiments, the acidic solution is mixed with the cell preparation after cell lysis. In some embodiments, the acidic solution is not a triprotonic acidic solution. In some embodiments, the acidic solution is an acidic glycine solution.

[0029] In some embodiments, the cell preparation is at a density suitable for rAAV recovery. In some embodiments, the cell preparation is at a density of about 0.5-12 x 10 6 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 cells / mL. 6 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 cells / mL. 6 with a density of 100 cells / mL.

[0030] FIG. 1A shows a non-limiting example of a procedure for adding a flocculating agent (e.g., an acidic solution) to a cell preparation containing rAAV particles. In some embodiments, the flocculating agent is an acidic glycine solution. In some embodiments, the flocculating agent is a solution with a pH of 4 or less, a solution with a pH of 3 or less, or a solution with a pH of 2 or less. In some embodiments, the flocculating agent is a solution with 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 flocculating agent can be added to a vessel (e.g., a bioreactor) containing a mixing device (e.g., an impeller). In some embodiments, the appropriate speed of the impeller can be determined using one or more of equations (1), (2), and / or (3) set forth in Example 1.

[0031] In some embodiments, the cell preparation comprises a cell culture. In some embodiments, the cell preparation comprises a resuspended cell pellet. 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 producer cells. In some embodiments, an acidic solution is mixed with the cell preparation after cell lysis. In some embodiments, the cell preparation is a cell harvest.

[0032] Figure 1B shows a non-limiting example of a procedure for isolating rAAV particles from cell culture. In some embodiments, an aggregation step (e.g., using an acidic glycine solution) is incorporated after the cell lysis and nuclease steps, as shown in Figure 1B, and before subsequent clarification and additional purification steps. However, in some embodiments, the aggregation step can be incorporated 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 in place of cell lysis and / or nuclease addition. In some embodiments, cell lysis comprises mechanical lysis, liquid homogenization, sonication, freeze / thaw cycles, or chemical lysis. In some embodiments, chemical lysis conditions comprise 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).

[0033] In some embodiments, the methods for agglomerating cellular material are adapted for large-scale culture and isolation processes, providing surprising improvements over existing methods. In some embodiments, large-scale cultures include cultures of greater than 1 L, greater than 10 L, greater than 25 L, greater than 50 L, greater than 100 L, greater than 250 L, or greater than 500 L. In some embodiments, large-scale cultures include cultures of 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, implementation of the agglomeration process described herein at process scale efficiently removes impurities, resulting in a 4- to 5-fold reduction in host cell protein (HCP) for downstream purification processes.

[0034] In some embodiments, a method useful on a process scale involves purifying recombinant adeno-associated virus (rAAV) particles from a cell culture containing the rAAV particles by contacting the rAAV preparation obtained from the cell culture with an acidic solution (e.g., an acidic glycine solution, a citric acid solution (also called "citric acid"), a caprylic acid solution) under conditions sufficient to promote aggregation of cellular 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., a chemical lysate) containing rAAV particles. In some embodiments, the rAAV preparation is contacted with a nuclease (e.g., after lysis and before aggregation). However, in some embodiments, no nuclease is added prior to aggregation.

[0035] In some embodiments, the pH of the acidic solution is less than 4 (e.g., about 2.5). In some embodiments, sufficient acid is added to lower the pH of the rAAV preparation to about 2-4 (e.g., about 2-4), about 3-4, about 3-5, about 4-5, about 2.5-3.5, about 2.5-4.5, or about 3.5-5.5 (e.g., about pH 4). In some embodiments, the acidic solution is about 0.5 M, about 1 M, about 2 M, about 3 M, about 4 M, about 5 M, about 6 M, about 7 M, about 8 M, about 9 M, or about 10 M. In some embodiments, a 2 M acidic 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 acidic solution (e.g., a 2 M acidic glycine solution).

[0036] In some embodiments, the acidic solution is an acidic glycine solution. In some embodiments, the pH of the wash solution is less than 4 (e.g., about 2.5). In some embodiments, sufficient acidic glycine is added to lower the pH of the rAAV preparation to about 2-4 (e.g., about 2-4), about 3-4, about 3-5, about 4-5, about 2.5-3.5, about 2.5-4.5, or about 3.5-5.5 (e.g., about pH 4). In some embodiments, the acidic glycine solution is about 1 M, about 2 M, about 3 M, about 4 M, about 5 M, about 6 M, about 7 M, about 8 M, about 9 M, or about 10 M. In some embodiments, a 2 M acidic glycine solution at pH 2.5 is added to the rAAV preparation.

[0037] In some embodiments, the acidic solution is a citric acid solution. In some embodiments, the pH of the citric acid solution is less than 4 (e.g., about 2.5). In some embodiments, sufficient citric acid is added to lower the pH of the rAAV preparation to about 2-4 (e.g., 2-4), about 3-4, about 3-5, about 4-5, about 2.5-3.5, about 2.5-4.5, or about 3.5-5.5 (e.g., about pH 4). In some embodiments, the citric acid solution is about 1 M, about 2 M, about 3 M, about 4 M, about 5 M, about 6 M, about 7 M, about 8 M, about 9 M, or about 10 M. In some embodiments, a 2 M citric acid solution at pH 2.5 is added to the rAAV preparation.

[0038] In some embodiments, an acidic solution (e.g., an acidic glycine solution) is added to the rAAV preparation at a volume of 5-10% (e.g., around 8%). In some embodiments, the acidic solution is added to the rAAV preparation at a volume of 1-10%, 1-5%, 2-9%, 3-8%, 4-7%, 5-9%, or 4-8%. In some embodiments, the acidic solution is added to the rAAV preparation over a period of about 10 minutes (e.g., within a period of about 5 minutes (e.g., about 5 minutes)). In some embodiments, the rAAV preparation is mixed with the added acidic solution using a stirring speed of about 30-150 RPM (e.g., about 30-150 RPM). In some embodiments, the rAAV preparation is mixed with the added acidic solution using a stirring speed of about 50-150 RPM (e.g., about 50-150 RPM). In some embodiments, a stirring speed of around 90-110 RPM (e.g., about 90-110 RPM) (e.g., about 100 RPM) is used for approximately 5 L of rAAV preparation. In some embodiments, a stirring speed of around 30-200 RPM or 90-200 RPM (e.g., about 30-200 RPM or 90-200 RPM) is used for approximately 5 L of rAAV preparation. In some embodiments, a stirring speed of around 50-75 RPM (e.g., about 50-75 RPM) (e.g., about 63 RPM) is used for approximately 50 L of rAAV preparation. In some embodiments, a stirring speed of around 50-100 or 50-150 RPM (e.g., about 50-100 or 50-150 RPM) is used for approximately 50 L of rAAV preparation. In some embodiments, an agitation rate of around 30-50 RPM (e.g., about 42 RPM) is used for approximately 500 L of rAAV preparation. In some embodiments, an agitation rate of around 30-100 RPM is used for approximately 500 L of rAAV preparation. In some embodiments, the agitation rate is adjusted to achieve a power / volume (P / V) ratio of around 2-5 (e.g., about 2-5). In some embodiments, the agitation rate is adjusted to achieve a P / V ratio of around 4.7. In some embodiments, the agitation rate is adjusted to achieve a P / V ratio of around 3.1.In some embodiments, the mixture of the rAAV preparation and an acidic solution (e.g., an acidic glycine solution) is allowed to stand in a container for 10 to 60 minutes (e.g., at room temperature) to promote aggregation of cellular material prior to subsequent purification steps. In some embodiments, the mixture is allowed to stand for 15 to 45 minutes (e.g., a holding time of 15 to 45 minutes). In some embodiments, the mixture is allowed to stand for about 30 minutes (e.g., a holding time of 30 minutes). In some embodiments, the mixture is allowed to stand 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 allowed to stand for 30 minutes to 4 hours, 30 minutes to 10 hours, 10 minutes to 5 hours, 20 minutes to 6 hours, 10 minutes to 4 hours, or 1 hour to 4 hours. In some embodiments, the mixture of the rAAV preparation and acidic solution (e.g., acidic glycine solution) is stirred slowly (e.g., at 30-150 rpm) in a vessel for 10-60 minutes (e.g., at room temperature) to promote aggregation of cellular material prior to subsequent purification steps. In some embodiments, the mixture is stirred slowly (e.g., at 30-150 rpm) for 15-45 minutes. In some embodiments, the mixture is stirred slowly (e.g., at 30-150 rpm) for approximately 30 minutes. In some embodiments, the mixture is stirred slowly (e.g., at 30-150 rpm) for up to 10 hours; in some embodiments, the mixture is stirred slowly (e.g., at 30-150 rpm) for 30 minutes to 4 hours, 30 minutes to 10 hours, 10 minutes to 5 hours, 20 minutes to 6 hours, 10 minutes to 4 hours, or 1 hour to 4 hours. In some embodiments, aggregated material from these is resuspended prior to subsequent purification (e.g., prior to one or more clarification steps). Thus, in some embodiments, the flocculation mixture is clarified without intervening resuspension. In some embodiments, the flocculation mixture is resuspended prior to clarification. In some embodiments, the clarification is by filtration. In some embodiments, the filtration is depth filtration.

[0039] In some embodiments, the method is carried out at room temperature, hi some embodiments, the method is carried out at 10-40 C, e.g., 15-35 C, 15-20 C, 20-25 C, or 25-30 C.

[0040] In some embodiments, the methods comprise contacting the rAAV preparation with a flocculating agent (e.g., acidic glycine). In some embodiments, the methods comprise contacting the rAAV preparation with glycine. In some embodiments, the methods comprise contacting the rAAV preparation with an alternative or additional flocculating agent, e.g., a cationic polymer such as polyethyleneimine (PEI) or polydiallyldimethylammonium chloride (pDADMAC), and / or an alternative or additional pH-lowering agent, e.g., citric acid, phosphoric acid, and / or caprylic acid, and / or an alternative or additional lysing agent, e.g., a detergent. In some embodiments, the detergent is Triton, PS20 (tween 20), or other detergent.

[0041] The present disclosure also provides compositions comprising AAV particles produced by the methods described herein. In some embodiments, rAAV preparations after aggregation and before subsequent purification steps are more stable than corresponding preparations without aggregation. In some embodiments, the aggregated rAAV preparations can be held (e.g., for up to two weeks or longer). In some embodiments, one or more aggregated rAAV preparations can be held, for example, for one to two weeks or longer, and then combined for subsequent purification steps.

[0042] Recombinant AAV Naturally occurring AAV capsid proteins can be used to produce rAAV for gene therapy. Different naturally occurring AAVs have different characteristics (e.g., different tissue tropism) and can be used for different indications. AAVs are 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. 6381-6388; 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 Forgone Therapy Using AAV Vectors. Hum Gene Ther. 2010. 21(6): pp. 704-704). AAVs are useful as viral vectors. Many serotypes exist, each with different tissue-type tropism (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), allowing specific tissues to be preferentially targeted with appropriate pseudotyping. Some serotypes, such as serotypes 8, 9, and rh10, transduce mammalian tissue.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): p. 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): p. 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): p. 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): p. 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): p. 59-65.AAV9 has been demonstrated to cross the blood-brain barrier, which is inaccessible to many viral vectors and biologics (see Foust, KD, et al., Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol, 2009, 27(1):59-65; and Rahim, AA, 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):3505-18). Certain AAVs carry payloads of 4.7–5.0 kb, including the viral inverted terminal repeats (ITRs) required in cis 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, JY, P D Dan, and R A Frizzell, Quantitative analysis of the packaging capacity of recombinant adeno-associated viruses. Hum Gene Ther, 1996. 7(17): pp. 2101-12.

[0043] In some embodiments, the rAAV may comprise one or more variant AAV capsid proteins having one or more amino acid substitutions compared to a naturally occurring AAV capsid protein.

[0044] Thus, in some embodiments, the rAAV particles comprise 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 particles comprise a hybrid capsid protein derived from any combination of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 capsid proteins.

[0045] In some embodiments, the methods described herein are beneficial to manufacturing processes. These advantages include, but are not limited to, a greater than 10-fold reduction in host cell impurities without the use of endonucleases, efficient filtration without flux attenuation, and a 5-fold longer affinity resin life cycle due to lower impurities in the loading material. Furthermore, the slight charge difference between the three capsid types allows for finer resolution and better enrichment of intact capsids due to less interference from impurities. By controlling impurity levels in the upstream feedstream, rAAV viral vectors exhibit improved stability with minimal aggregation at low conductivity, further improving process recovery. With a cleaner feedstream, downstream intermediates achieve more than 10-fold lower turbidity values ​​with maintained rAAV titers, enabling easier filtration and manufacturing robustness. Aggregation has been successfully proven as an innovative rAAV manufacturing technology for multiple AAV serotypes. Its implementation in an rAAV process platform not only provides superior product quality and significant benefits for downstream recovery, but also significant cost savings in rAAV manufacturing.

[0046] These and other aspects are illustrated by the following non-limiting examples. [Example]

[0047] Example 1 As shown in Figures 1A and 1B, the pH of the cell culture harvest was lowered to pH 4 using 2 M glycine acid 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 a power / volume ratio of 4.7 in others. Similar calculations of power / volume ratios (e.g., 2-5) are expected to work similarly. The cell lysis and digestion step was performed in conjunction with the purification of AAV produced using the triple transfection method but is optional for AAV produced from producer 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 with a 1-minute hold time for pH reading, and the remainder of the glycine was pumped in until a target pH of 4 was reached. The pump speed was calculated by limiting the acid addition to a 10-minute addition period. Once the pH was reached, the flocculated material was allowed to stand (e.g., without stirring) for 30 minutes to allow for large particle formation. The two phases (precipitate and supernatant) were remixed and loaded into a Clarisolve depth filter. The filtrate was neutralized immediately after depth filtration by adding 5% V / V of 2 M Tris buffer.

[0048] The aggregation efficiency can be evaluated using the following equation (1):

number

[0049] where C0 is the initial impurity level, which is assumed to be the same under the same cell density and lysis conditions, and t F is the aggregation time for the impurities to precipitate and form large particles, and C F is the flocculant dosage, N is the impeller agitation speed, which can also affect the mixing efficiency during flocculant addition and the size of the precipitate, and P / V is the mean turbulent kinetic energy dissipation rate ε avewhere ρ is the power input, ρ is the target pH of the bioreactor after flocculation, and T is the temperature of the bioreactor. Cr is the impurity level after flocculation, which can be a criterion for evaluating 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).

number

[0050] To perform flocculation at process scale, the scale-up rule was based on the same flocculation efficiency. Considering the target pH and T, the flocculation efficiency is determined by a constant P / V ratio, flocculation time (t F ) and coagulation input (C F The agitation rate was determined by the geometric shape of the bioreactor, as shown in equation (2). Taking into account the impeller diameters in the large-scale (DL) and small-scale (DS), the agitation rate in the large-scale (NL) was determined by equation (3) to maintain the same flocculation efficiency during scale-up.

[0051] Example 2 An AAV purification process was developed to purify AAV particles from cell culture and enrich AAV preparations for complete AAV particles (e.g., containing the recombinant AAV genome) versus empty AAV particles (e.g., containing capsid proteins but no encapsulated nucleic acid).

[0052] An updated purification process was developed that uses 2 M glycine acid pH 2.5 as a flocculation buffer to introduce flocculation after DNA digestion, lowering the post-lysis harvest to a pH of 4. In some embodiments, the flocculation procedure involves developing a target agitation rate, a target pump speed at which the acid is pumped, and / or reaching and holding a target pH for a target retention time. This process was developed using AAV9 as an example. This updated purification process was surprisingly effective. It was characterized by several improvements, including a 4-5-fold reduction in HCP / DNA in the harvest step, higher throughput in clarification, and a more stable and higher-yielding AAV product.

[0053] As shown in Figures 2A and 2B, four different acids (citric acid, phosphoric acid, glycine, and caprylic acid) and two different cationic polymers, polyethyleneimine (PEI) and polydiallyldimethylammonium chloride (pDADMAC), were used as flocculants during harvesting. The post-flocculation titer and host cell protein reduction were studied after using different flocculants. HCP levels were measured at pH 4, which was more effective than pH 4.5 and pH 5, resulting in a 4- to 5-fold HCP reduction (Figure 2A). Citric acid and pDADMAC also provided detectable levels of HCP reduction. According to the post-flocculation titers in Figure 2B, there was no apparent titer loss across multiple flocculants. Higher titers were observed when glycine acid was used to flocculate the cell harvest.

[0054] Further analysis was performed using acidic glycine-based flocculation at pH 4. The flocculated material was clarified using a Clarisolve depth filter. Compared to the HCP levels in the clarified filtrate without flocculation, the HCP levels in the clarified filtrate with flocculation were more than 10-fold lower (Figure 3A). Depth filter performance also improved after flocculation. This is noteworthy because fine particles with an average size of 2 μm at throughput easily block the depth filter, significantly reducing throughput. The flocculation process resulted in the aggregation of fine particles into larger particles, increasing depth filter throughput from 31 to 92.5 L / m without increasing inlet pressure. 2 The condition improved to 100% (Figure 3B).

[0055] Therefore, glycine can be used to lower the harvest pH 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.

[0056] In some embodiments, performing a flocculation procedure can improve the performance of one or more purification steps and / or can improve the quality and stability of the product.

[0057] Example 3 To determine the effect of aggregation on downstream aspects of AAV purification, aggregated and non-aggregated material was purified using ultrafiltration / diafiltration, capture chromatography, and polishing chromatography.

[0058] Ultrafiltration / Diafiltration The performance of UFDF / DF with and without flocculation is compared in Figures 4A and 4B. Figure 4A shows that UFDF performance with flocculated material has double the flux without any obvious flux decay compared to that with non-flocculated material. Figure 4B shows that the run time with flocculated material is twice as fast as that without flocculated material. This allows for a shift to higher throughput when using flocculated material, potentially saving on the cost of supplies on UFDF filters. Due to the lower impurity (HCl / HCl) levels in the TFF load after flocculation, UFDF performed much better, with higher flux, shorter run time, and a much cleaner pool after concentration.

[0059] Capture chromatography The effect of aggregation on capture chromatography (affinity column) is shown in Figures 5A, 5B, and 5C. Without aggregation, a very high UV signal in the flow-through is observed (Figure 5B), indicating a high level of impurities in the affinity load. After aggregation, the UV signal in the flow-through drops from 2000 mAU to less than 100 mAU (Figure 5A), indicating very low impurities in the affinity load. The low level of impurities improves the efficiency of column binding and capture recovery. Host cell DNA in the affinity eluate with and without aggregation is also shown in Figure 5C. Host cell DNA is more than 10-fold lower after aggregation compared to the affinity eluate without aggregation. This is further evidence that aggregation significantly removes impurities and improves product quality.

[0060] Polishing Chromatography The advantages over polishing chromatography are shown in Figures 6A-6B. The aggregated material shows a single peak (Figure 6A), while the non-aggregated material shows multiple peaks without concentration. The multiple peak pattern in Figure 6B indicates that several impurity-related species may be present, making it difficult to separate intact vectors from empty vectors. Impurities can also induce product aggregation, resulting in a Z-avg greater than 30 nm, especially at low conductivities. The Z-avg values ​​in the table show that after dilution, the aggregated material remained at 30 nm with excellent stability, while the average particle size for the non-aggregated material increased significantly.

[0061] Complete vector enrichment The enrichment of intact vectors after polishing chromatography is shown in Figure 7A. The material with aggregation shows that intact capsids are enriched from 18% to 50.3% to the right of the main peak. However, the material without aggregation in Figure 7B shows multiple peaks, with enrichment occurring only in the central section of the peak. This result indicates that the fewer impurities associated with the product, the easier it is for the polishing step to isolate intact capsids with higher enrichment and higher recovery.

[0062] Product stability The stability of the product with and without aggregation is shown in Figure 8. The post-UFDF material was kept at room temperature, and the turbidity was measured after different storage periods. Without wishing to be bound by theory, unstable material forms aggregates and precipitates, resulting in an increase in turbidity values. The post-aggregation material exhibits a stable turbidity profile over half a month, with all values ​​below 20 NTU, while the turbidity of the material without aggregation treatment increases dramatically from 150 to over 500 NTU within 10 days. The aggregation step can highly stabilize the product because impurities can associate with the viral vector product and induce aggregation during storage.

[0063] Reusing affinity resin Affinity resins are another item that significantly impacts the cost of goods in AAV process development. Reusing affinity resins while maintaining the same purification capacity can significantly reduce manufacturing costs.

[0064] The cell lysate material with and without aggregation was loaded onto an affinity column. For the non-aggregated material, a high content of impurities in the loading material could clog the column or jeopardize its lifespan. Figures 11A and 11B show the affinity resin cycle times for purifying the non-aggregated material (A) and aggregated material (B).

[0065] As shown in Figure 11A, the pre-column pressure increased significantly after five cycles, making it impossible to reuse the column for multiple cycles. By flocculating during the recovery step, very clean material was loaded onto the affinity chromatography column. In Figure 11B, the same resin can be used for up to 20 cycles without any impact on yield and product quality. The low impurity load in the affinity load ensures that the affinity column has a higher potential for obtaining better purification performance, with more column cycles and reduced costs.

[0066] AAV gathering AAV assembly behavior at low conductivity is a major technical challenge for gene therapy process development. The underlying cause of this behavior remains unclear, 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. Presence of trace amounts of nucleic acids or host cell proteins in the process can induce significant product aggregation at low conductivity. Mitigating AAV assembly at low conductivity offers significant benefits to polishing chromatography and improves manufacturing robustness. Figure 12 shows AAV assembly before and after aggregation in the process. The material from runs 1–4 was not treated with aggregation, while the material from runs 5–8 was treated with glycine acid in the recovery process. All eight runs were treated with endonuclease. Keeping the rest of the unit operation the same, high conductivity was measured after affinity chromatography. The aggregation level can be obtained by the average hydrodynamic diameter (Z-avg). AAV product aggregated up to 400 nm at low conductivity without aggregation. For runs 5-8, the flocculation step during recovery controlled impurity levels, resulting in a smaller amount of impurities entering the downstream flow. By removing the impurities, less aggregation was observed at lower conductivities, indicating improved product stability upon incorporation of a flocculation step as described herein.

[0067] conclusion Flocculation using acid precipitation for AAV purification demonstrates significant reduction of HCP and HC DNA for downstream purification. Glycine is 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 are defined to ensure robust flocculation efficiency across multiple large-scale production runs. The benefits of implementing flocculation are demonstrated herein in terms of better product quality and stability, shorter UFDF run times, and higher intact viral vector concentration and yield. This demonstrated method holds promise for other AAV purification processes to ensure process robustness and better product quality performance.

[0068] Example 4 Non-limiting embodiments of the aggregation methods described herein were tested with different rAAV serotypes produced from an acidic cell line (PCL), which were different from the rAAV serotypes tested in Examples 1-3, which were produced using a triple transfection method.

[0069] -Flocculant screening Glycine and citric acid were used at different pH levels to treat cell culture harvest material containing the PCL-based rAAV serotypes being tested, and the effect of acid type and pH conditions on impurity removal was evaluated. The results are shown in Figure 9. Both glycine and citric acid used as flocculating agents have minimal adverse effects on the recovery rate of PCL-based rAAV serotype cell culture harvest. Therefore, different buffers can be used to lower the pH from pH 7.5 or pH 8 to an acidic pH, with pH being the driving force for flocculation. Depending on which acid will be used in subsequent purification steps, different acids can be selected as flocculating reagents for treating the cell culture harvest, allowing flexibility in the selectivity of the flocculating reagent.

[0070] Removal of host cell DNA Host cell DNA (HC DNA) concentrations were measured under different aggregation conditions for PCL-based rAAV serotypes. In parallel, cell culture material was treated with an endonuclease, and HC DNA concentrations were quantified as a control. Endonucleases are expensive enzymes, and their use therefore significantly impacts the cost of goods for gene therapy process development. Replacing endonucleases while maintaining the same level of HC DNA reduction would be an ideal situation. To examine the level of HC DNA reduction, acid-based aggregation was directly applied to cell cultures without endonuclease digestion. Compared to endonuclease digestion, host cell DNA levels were significantly reduced after acid treatment, from 9806 to less than 1000 ng / mL (Figure 10). The aggregation method provides much cleaner upstream material, offering several advantages for downstream purification and manufacturing robustness. In addition, an endonuclease-free process also contributes to significantly reduced manufacturing costs.

[0071] Product stability during low pH hold Cell culture harvests containing PCL-based rAAV particles were adjusted to pH 4 using an acid buffer for aggregation and incubated for different periods (0.5, 1, 2, 3, and 4 hours). Recovery results showed that AAV was stable at pH 4 for at least 4 hours without titer loss. A slight increase in titer, ranging from approximately 10% to 20%, was observed at various time points. The results are shown in Figure 13.

[0072] heat inactivation In the producer cell line (PCL) platform, Ad5 needs to be introduced into the cell culture harvest and removed downstream. Heating to a temperature that can inactivate Ad5 while maintaining AAV activity is one of the major strategies for viral clearance. However, during heat inactivation, impurities (e.g., host cell DNA and proteins) degrade and aggregate, causing a significant increase in turbidity. High turbidity poses many challenges for downstream processing, including filtration clogging and product loss. As seen in Figure 14, aggregated material shows little or no increase in turbidity after heat inactivation, thus avoiding the difficulties imposed by high turbidity.

[0073] equivalent While several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions and / or obtaining one or more of the results and / or advantages described herein, and each such variation and / or modification is deemed to be within the scope of the embodiments of the present invention described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the specific application or applications for which the teachings of the present invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Accordingly, the above-described embodiments are presented by way of example only, and it should be understood that, within the scope of the appended claims and their equivalents, embodiments of the present disclosure are directed to each individual 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 is included within the inventive scope of the present disclosure, provided that such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0074] All definitions and terms used herein should be understood to govern by dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0075] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, and in some cases may include the entire document.

[0076] The indefinite articles "a" and "an," as used in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0077] The term "and / or," as used herein in the specification and claims, means "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same manner, i.e., "one or more" of the conjoined elements. Other elements other than the elements specifically identified by the "and / or" clause may optionally be present, whether related or unrelated to those elements identified. Thus, as a non-limiting example, with reference to "A and / or B," when used in conjunction with open-ended language such as "comprising," for example, in one embodiment, it may refer to only A (optionally including elements other than B); in another embodiment, it may refer to only B (optionally including elements other than A); in yet other embodiments, it may refer to both A and B (optionally including other elements).

[0078] As used in this specification and the claims, "or" should 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" should be construed as inclusive, i.e., including at least one but also more than one of a number or list of elements, and optionally including additional unlisted items. "Only one of" or "exactly one of," or, when used in the claims, "consisting of," refers to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein should be construed to indicate exclusive alternatives (i.e., "one or the other, but not both") only when preceded by exclusive terms such as "either," "one of," "only one," or "exactly one." When used in the claims, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law.

[0079] As used in this specification and claims, the phrase "at least one," in reference 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 every element specifically listed in the list of elements, and not excluding combinations of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to the elements 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") may, for example, in one embodiment, refer to at least one (optionally multiple) A's (optionally including elements other than B) in the absence of B; in another embodiment, it may refer to at least one (optionally multiple) B's (optionally including elements other than A) in the absence of A; in yet another embodiment, it may refer to at least one (optionally multiple) A's and at least one (optionally multiple) B's (optionally including other elements), etc.

[0080] It should also be understood that, unless expressly indicated to the contrary, in methods including multiple steps or acts claimed herein, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are described.

[0081] In the claims and the above specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," and "composed of" are to be understood to be open-ended, i.e., meaning including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in Section 2111.03 of the United States Patent Office Manual of Patent Examining Procedures. It should be understood that embodiments described herein using an open-ended transitional phrase (e.g., "comprising") are also contemplated in alternative embodiments as "consisting of" and "consisting essentially of" the features recited by the open-ended transitional phrase. For example, if the disclosure describes "a composition comprising A and B," the disclosure also contemplates the alternative embodiments "a composition consisting of A and B" and "a composition consisting essentially of A and B."

Claims

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

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

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

4. The method of any one of claims 1 to 3, 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 to 4, wherein the pH of the acidic solution is less than 4.

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

5.

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

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

9. 9. The method of claim 8, wherein the acidic solution is added to the rAAV preparation at 8% volume.

10. 10. The method of any one of claims 1 to 9, wherein the acidic solution is added to the rAAV preparation within a period of 10 minutes.

11. 11. The method of any one of claims 1 to 10, wherein the acidic solution is added to the rAAV preparation within a period of 5 minutes.

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

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

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

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

16. 16. The method of 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. 16. The method of 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. 16. The method of any one of claims 1 to 15, wherein the volume of the rAAV preparation is 500 L and the stirring speed is about 30-50 RPM or 42 RPM.

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

20. 20. The method of claim 19, wherein the pH is around or about 4.

21. 21. The method of any one of claims 1 to 20, wherein the mixture of the rAAV preparation and the acidic solution of a) is allowed to stand in a container for 10 to 60 minutes to promote aggregation of the cellular material.

22. 22. The method of claim 21, wherein the mixture is allowed to stand for 15 to 45 minutes.

23. 23. The method of claim 22, wherein the mixture is allowed to stand for at least 30 minutes.

24. 21. The method of any one of claims 1 to 20, wherein the aggregated material from a) is resuspended prior to separating the AAV particles from the aggregated cellular material.

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

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

27. 27. The method of claim 25 or 26, wherein the clarification is by filtration.

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

29. 29. The method of any one of claims 1 to 28, 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 thereof, or wherein the rAAV particles comprise a hybrid capsid.

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

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

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

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

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

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

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

37. 1. A method for purifying rAAV particles from a cell culture containing rAAV particles, the method comprising: a) contacting a rAAV preparation obtained from a cell culture with an aggregating agent under conditions sufficient to promote aggregation of cellular material present in the rAAV preparation; b) separating the rAAV particles from the aggregated cellular material.

38. 38. The method of claim 37, wherein the flocculant is a pH-lowering agent or a cationic polymer.

39. 39. The method of claim 38, wherein the pH-lowering agent comprises citric acid, phosphoric acid, glycine, and / or caprylic acid.

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

41. 1. A method for purifying rAAV particles from a cell culture containing rAAV particles, the method comprising: a) contacting a rAAV preparation obtained from a cell culture with an aggregating agent under conditions sufficient to promote aggregation of cellular material present in the rAAV preparation; b) separating the rAAV particles from the aggregated cellular material; 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. 42. The method of claim 41, wherein the rAAV particles in the cell culture are produced using a PCL method.

43. 43. The method of claim 41 or 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 thereof, or wherein the rAAV particles comprise a hybrid capsid.

44. The cell culture is 0.5 to 12 x 10 ６ 44. The method of any one of claims 1 to 43, comprising a density of cells / mL.

45. The cell culture is 0.5 to 2 x 10 ６ 45. The method of claim 44, comprising a density of cells / mL.

46. The cell culture is 11 to 12 x 10 ６ 45. The method of claim 44, comprising a density of cells / mL.

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