AAV vector column purification methods

Abstract

To provide purification, production and manufacturing methods for recombinant adeno-associated viral (rAAV) vector particles.SOLUTION: Purification, production and manufacturing methods for rAAV vector particles include at least two column chromatography steps. The column chromatography steps include, for example, cation exchange chromatography, anion exchange chromatography, size exclusion chromatography and / or AAV affinity chromatography alone or in combination and in any order.SELECTED DRAWING: Figure 1

Description

[Background technology]

[0001]

[0002] Gene delivery is a promising method for the treatment of acquired and inherited diseases. Several virus-based systems for gene transfer have been described, including adeno-associated virus (AAV)-based systems.

[0002]

[0003] AAV is a helper-dependent DNA parvovirus belonging to the Dependovirus genus. AAV requires the function of a helper virus, such as adenovirus, herpesvirus, or vaccinia, to cause productive infection. In the absence of helper virus function, AAV establishes a latent state by inserting its genome into the chromosome of host cells. Subsequent infection with the helper virus rescues the integrated viral genome, which can then replicate to produce infectious AAV progeny.

[0003]

[0004] AAV has a broad host range and can replicate in cells from any species in the presence of a suitable helper virus. For example, human AAV can replicate in canine cells co-infected with a canine adenovirus. AAV has never been associated with any human or animal disease and does not appear to adversely affect the biology of host cells upon integration.

[0004]

[0005] AAV vectors can be engineered to carry heterologous nucleic acid sequences of interest (e.g., selected genes encoding therapeutic proteins, inhibitory nucleic acids, e.g., antisense molecules, ribozymes, miRNAs, etc.) by deleting the entire or partial internal portion of the AAV genome and inserting the nucleic acid sequence of interest between the ITRs. The ITRs still function in such vectors to allow replication and packaging of the rAAV containing the heterologous nucleic acid sequence of interest. The heterologous nucleic acid sequence is also typically linked to a promoter sequence that can drive expression of the nucleic acid in the target cells of the patient. Termination signals, such as polyadenylation sites, can also be included in the vector.

[0005]

[0006] The construction of infectious recombinant AAV (rAAV) vectors has been described in several publications, see, e.g., U.S. Patent Nos. 5,173,414 and 5,139,941; WO 92 / 01070 and WO 93 / 03769; Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, BJ (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129; and Kotin, RM (1994) Human Gene Therapy 5:793-801.

[0006]

[0007] Recombinant adeno-associated virus (AAV) vectors have shown good therapeutic promise in several early clinical trials by multiple groups. Development of this new class of biological product toward approval will involve improvements in vector characterization and quality control methods, including a better understanding of how vector design and manufacturing process parameters affect the impurity profile in clinical-grade vectors.

[0007]

[0008] An important objective in designing rAAV production and purification systems is to implement strategies to minimize / control the occurrence of production-related impurities, such as vector-related impurities including proteins, nucleic acids, and wild-type / pseudo-wild-type AAV species (wtAAV), as well as residual AAV encapsidated DNA impurities. Removal of impurities in AAV vectors is complicated due to the way in which rAAV vectors are produced. In one production process, rAAV vectors are produced by a transient transfection process using three plasmids. A significant amount of plasmid DNA is introduced into cells to produce rAAV vectors. In addition, when the rAAV vectors are released from the producing cells, the cellular proteins and nucleic acids are also released. Given that the rAAV vectors only account for about 1% of the biomass, it is a great challenge to purify rAAV vectors to a level of purity that can be used as a clinical human gene therapy product. (Smith PH Wright JF. Qu G. et al. 2003, Mo. Therapy, 7:8348; Chadeuf G. et al., Mo. Therapy 2005, 12:744. Report from the CHMP Gene Therapy Expert Group Meeting. European Medicines Agency EMEA / CHMP 2005, 183989 / 2004).

[0008]

[0009] Development of a manufacturing process to purify recombinant AAV as a product to treat human disease should achieve the following objectives: 1) consistent vector purity, titer, and safety; 2) feasibility of scalable manufacturing process; and 3) acceptable manufacturing costs. Current "industry standard" scalable AAV vector purification processes do not adequately achieve removal of impurities important to meet the first objective listed above (consistent vector purity, titer, and safety). Moreover, impurities cannot be adequately removed using current industry standard scalable purification processes because: 1) development of purification processes for viral products such as recombinant AAV for applications other than vaccines (where an immune response is typically sought rather than avoided) is relatively new; 2) many groups involved in developing scalable purification processes for AAV vectors have been unaware of high levels of vector-associated impurities and / or have assumed that such impurities do not contribute to clinically significant vector immunogenicity; and 3) it is technically challenging to develop a scalable purification process suitable for industrial-scale manufacturing of rAAV vectors. Summary of the Invention

[0009]

[0010] The present invention provides a method for purifying and producing recombinant adeno-associated virus (rAAV) vector particles. The method of the present invention comprises at least two column chromatography steps.

[0010]

[0011] In one embodiment, the method includes the steps of: (a) harvesting cells and / or cell culture supernatant comprising rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) lysing the harvest produced in step (a) or the concentrated harvest produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; and (e) optionally, (f) subjecting the nucleic acid-reduced lysate produced in step (d), the clarified lysate produced in step (e), or the diluted and clarified lysate produced in step (e) to cation exchange column chromatography to produce a column eluate comprised of rAAV vector particles, thereby freeing the rAAV vector particles from protein impurities or other production / process related impurities. (g) subjecting the column eluate produced in step (f) or the diluted column eluate to anion exchange chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating the rAAV vector particles from protein impurities or production / process related impurities, and optionally concentrating the second column eluate to produce a concentrated second column eluate; and (h) subjecting the second column eluate or the diluted column eluate produced in step (g) to anion exchange chromatography to produce a concentrated second column eluate. (i) subjecting the concentrated second column eluate produced in step (h) to size exclusion column chromatography (SEC) to produce a third column eluate comprised of rAAV vector particles, thereby separating the rAAV vector particles from protein impurities or production / process related impurities, and optionally concentrating the third column eluate to produce a concentrated third column eluate; and (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles.

[0011]

[0012] In another embodiment, the method includes the steps of: (a) harvesting cells and / or cell culture supernatant containing the rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce an enriched harvest; (c) lysing the harvest produced in step (a) or the enriched harvest produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; and (e) optionally, (f) subjecting the nucleic acid-reduced lysate produced in step (d), the clarified lysate produced in step (e), or the diluted and clarified lysate produced in step (e) to cation exchange column chromatography to produce a column eluate comprised of rAAV vector particles, thereby freeing the rAAV vector particles from protein impurities or other production / process related impurities. (g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography (SEC) to produce a second column eluate comprised of rAAV vector particles, thereby separating the rAAV vector particles from protein impurities or other production / process related impurities, and optionally diluting the second column eluate to produce a concentrated second column eluate; and (h) subjecting the column eluate produced in step (g) or the concentrated column eluate to size exclusion column chromatography (SEC) to produce a second column eluate comprised of rAAV vector particles, thereby separating the rAAV vector particles from protein impurities or other production / process related impurities, and optionally diluting the second column eluate to produce a concentrated second column eluate. (i) subjecting the second column eluate or the diluted second column eluate to anion exchange chromatography to produce a third column eluate comprised of rAAV vector particles, thereby separating the rAAV vector particles from protein impurities production / process related impurities, and optionally diluting the third column eluate to produce a diluted third column eluate; and (i) filtering the third column eluate or concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles.

[0012]

[0013] In a further embodiment, the method includes the steps of: (a) harvesting cells and / or cell culture supernatant comprising rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) lysing the harvest produced in step (a) or the concentrated harvest produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) optionally filtering the nucleic acid-reduced lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted and clarified lysate; and (f) filtering the nucleic acid-reduced lysate of step (d), the clarified lysate of step (e), or the diluted and clarified lysate produced in step (e). (g) subjecting the column eluate produced in step (f) or the diluted column eluate to anion exchange chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating the rAAV vector particles from protein impurities or other production / process related impurities, and optionally concentrating the second column eluate to produce a concentrated second column eluate; and (h) filtering the second column eluate produced in step (g) or the concentrated second column eluate, thereby producing purified rAAV vector particles.

[0013]

[0014] In additional embodiments, the method includes the steps of: (a) harvesting cells and / or cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce an enriched harvest; (c) lysing the harvest produced in step (a) or the enriched harvest produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; and (e) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate. (f) optionally filtering the nucleic acid-reduced lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; and (f) subjecting the nucleic acid-reduced lysate produced in step (d), or the clarified lysate or diluted clarified lysate produced in step (e), to AAV affinity column chromatography to produce a column eluate comprised of rAAV vector particles, thereby isolating the rAAV vector particles from protein impurities or other production / process impurities. (g) subjecting the column eluate produced in step (f) or the diluted column eluate to anion exchange chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating the rAAV vector particles from protein impurities or other production / process related impurities, and optionally concentrating the second column eluate to produce a concentrated second column eluate; (h) optionally subjecting the second column eluate produced in step (g) or the concentrated second column eluate to size exclusion column chromatography (SEC) to produce a third column eluate comprised of rAAV vector particles, thereby separating the rAAV vector particles from protein impurities or other production / process related impurities, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the second column eluate produced in step (g) or the diluted second column eluate,or filtering the third column eluate or concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles.

[0014]

[0015] In yet another embodiment, the method comprises the steps of: (a) harvesting cells and / or cell culture supernatant comprising rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce an enriched harvest; (c) lysing the harvest produced in step (a) or the enriched harvest produced in step (b) to produce a lysate; and (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate. (e) optionally filtering the nucleic acid-reduced lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; and (f) subjecting the nucleic acid-reduced lysate produced in step (d), or the clarified lysate or diluted clarified lysate produced in step (e), to AAV affinity column chromatography to produce a column eluate comprised of rAAV vector particles, thereby isolating the rAAV vector particles from protein impurities or other manufacturing / process impurities. (g) subjecting the column eluate produced in step (f) or the concentrated column eluate to size exclusion column chromatography (SEC) to produce a second column eluate comprised of rAAV vector particles, thereby separating the rAAV vector particles from protein impurities or other production / process related impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) optionally subjecting the second column eluate produced in step (g) or the diluted second column eluate to anion exchange chromatography to produce a third column eluate comprised of rAAV vector particles, thereby separating the rAAV vector particles from protein impurities or other production / process related impurities, and optionally diluting the third column eluate to produce a diluted third column eluate; (i) filtering the second column eluate produced in step (g) or the diluted second column eluate,or filtering the third column eluate or concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles.

[0015]

[0016] In a particular embodiment of the method of the invention, the concentration in step (b) and / or step (f) and / or step (g) and / or step (h) is by UF / DF, e.g. by Tangential Flow Filtration (TFF).

[0016]

[0017] In certain embodiments of the methods of the invention, the concentration in step (b) reduces the volume of the recovered cells and cell culture supernatant by about 2- to 20-fold.

[0017]

[0018] In certain embodiments of the methods of the invention, the concentration in step (f) and / or step (g) and / or step (h) reduces the volume of the column eluate by about 5-20 fold.

[0018]

[0019] In certain aspects of the methods of the invention, lysis of the harvest produced in step (a) or the concentrated harvest produced in step (b) is by physical or chemical means. Non-limiting examples of physical means include microfluidization and homogenization. Non-limiting examples of chemical means include detergents. Detergents include non-ionic and ionic detergents. A non-limiting example of a non-ionic detergent includes Triton X-100. Non-limiting examples of detergent concentrations are between about 0.1 and 1.0%, inclusive.

[0019]

[0020] In certain aspects of the methods of the invention, step (d) comprises treating with a nuclease, thereby reducing contaminating nucleic acids. Non-limiting examples of nucleases include benzonase.

[0020]

[0021] In certain aspects of the methods of the invention, the filtration of the clarified lysate or diluted clarified lysate in step (e) is through a filter. Non-limiting examples of filters are those having a pore diameter of between about 0.1 and 10.0 microns, inclusive.

[0021]

[0022] In certain embodiments of the methods of the invention, dilution of the clarified lysate in step (e) is with an aqueous phosphate buffer, an aqueous acetate buffer, or an aqueous Tris buffer. Non-limiting examples of the pH of the solution are between about 4.0 and 7.4, inclusive. Non-limiting examples of the pH of the Tris solution are greater than 7.5, e.g., between about 8.0 and 9.0, inclusive.

[0022]

[0023] In certain embodiments of the method of the invention, dilution of the column eluate in step (f) or the second column eluate in step (g) is with an aqueous phosphate buffer, an aqueous acetate buffer, or an aqueous Tris buffer. Non-limiting examples of the pH of the solution are between about 4.0 and 7.4, inclusive. Non-limiting examples of the pH of the Tris solution are greater than 7.5, e.g., between about 8.0 and 9.0, inclusive.

[0023]

[0024] In certain aspects of the methods of the invention, the rAAV vector particles resulting from step (i) are formulated with a surfactant to produce an AAV vector formulation.

[0024]

[0025] In certain aspects of the methods of the invention, the anion exchange column chromatography of steps (f), (g) and / or (h) comprises polyethylene glycol (PEG) conditioned column chromatography.

[0025]

[0026] In certain aspects of the methods of the invention, the anion exchange column chromatography of steps (g) and / or (h) is washed with a PEG solution prior to elution of the rAAV vector particles from the column.

[0026]

[0027] In certain embodiments of the methods of the present invention, the PEG has an average molecular weight in the range of about 1,000 to 80,000 g / mol, inclusive.

[0027]

[0028] In certain embodiments of the methods of the present invention, PEG is at a concentration of about 4% to about 10%, inclusive.

[0028]

[0029] In certain aspects of the methods of the invention, the anion exchange column in steps (g) and / or (h) is washed with an aqueous detergent solution prior to elution of the rAAV vector particles from the column.

[0029]

[0030] In certain aspects of the methods of the invention, the cation exchange column in step (f) is washed with a detergent solution prior to elution of the rAAV vector particles from the column.

[0030]

[0031] In certain embodiments of the methods of the present invention, the PEG solution and / or detergent solution comprises an aqueous TrisCl / NaCl buffer, an aqueous phosphate / NaCl buffer, or an aqueous acetate / NaCl buffer.

[0031]

[0032] In certain embodiments of the methods of the invention, NaCl in the buffer or solution ranges from about 20 to 300 mM NaCl, inclusive, or from about 50 to 250 mM NaCl, inclusive.

[0032]

[0033] In certain aspects of the methods of the present invention, the surfactant comprises a cationic or anionic surfactant.

[0033]

[0034] In certain embodiments of the methods of the present invention, the surfactant comprises a surfactant having a 12 carbon chain.

[0034]

[0035] In certain embodiments of the methods of the present invention, the surfactant comprises dodecyltrimethylammonium chloride (DTAC) or sarkosyl.

[0035]

[0036] In certain aspects of the methods of the invention, the rAAV vector particles are eluted from the anion exchange column of steps (f), (g), and / or (h) with an aqueous TrisCl / NaCl buffer.

[0036]

[0037] In certain embodiments of the methods of the invention, the TrisCl / NaCl buffer comprises 100 to 400 mM NaCl, inclusive, optionally at a pH ranging from about 7.5 to about 9.0, inclusive.

[0037]

[0038] In certain aspects of the methods of the invention, the anion exchange column in steps (f), (g) and / or (h) is washed with an aqueous TrisCl / NaCl buffer solution.

[0038]

[0039] In certain embodiments of the methods of the present invention, NaCl in the aqueous TrisCl / NaCl buffer ranges from about 75 to 125 mM, inclusive.

[0039]

[0040] In certain embodiments of the methods of the present invention, the aqueous TrisCl / NaCl buffer has a pH of about 7.5 to about 9.0, inclusive.

[0040]

[0041] In certain aspects of the methods of the invention, the anion exchange column of steps (f), (g) and / or (h) is washed one or more times to reduce the amount of empty AAV capsids in the second or third column eluate.

[0041]

[0042] In certain embodiments of the methods of the invention, washing of the anion exchange column removes AAV empty capsids from the column prior to and / or in place of rAAV removal, thereby reducing the amount of AAV empty capsids in the second or third column eluates.

[0042]

[0043] In certain embodiments of the methods of the invention, washing of the anion exchange column removes at least about 50% of the total AAV empty capsids from the column prior to and / or in place of rAAV removal, thereby reducing the amount of AAV empty capsids in the second or third column eluate by about 50%.

[0043]

[0044] In certain embodiments of the methods of the present invention, the NaCl in the aqueous TrisCl / NaCl buffer ranges from about 110 to 120 mM, inclusive.

[0044]

[0045] In certain embodiments of the methods of the present invention, the ratio and / or amount of eluted rAAV vector particles and AAV empty capsids is controlled by the wash buffer.

[0045]

[0046] In certain embodiments of the methods of the invention, the vector particles are eluted from the cation exchange column in step (f) in an aqueous phosphate / NaCl buffer or an aqueous acetate / NaCl buffer. Non-limiting examples of NaCl concentrations in the buffer range from about 125 to 500 mM NaCl, inclusive. Non-limiting examples of pH of the buffer range from about 5.5 to about 7.5, inclusive.

[0046]

[0047] In certain aspects of the methods of the invention, the anion exchange columns of steps (f), (g) and / or (h) contain quaternary ammonium functional groups, such as quaternized polyethyleneimine.

[0047]

[0048] In certain embodiments of the methods of the invention, the size exclusion column (SEC) of steps (g) and / or (h) has a separation / fractionation range (molecular weight) of about 10,000 to about 600,000, inclusive.

[0048]

[0049] In certain aspects of the methods of the invention, the cation exchange column in step (f) comprises a sulfonic acid or functional group, such as sulfopropyl.

[0049]

[0050] In certain embodiments of the methods of the present invention, the AAV affinity column comprises a protein or ligand that binds to the capsid protein of AAV. Non-limiting examples of proteins include antibodies that bind to the capsid protein of AAV. More specific non-limiting examples include single chain llama antibodies that bind to the capsid protein of AAV.

[0050]

[0051] In certain aspects of the methods of the invention, the methods exclude the step of cesium chloride gradient ultracentrifugation.

[0051]

[0052] In certain aspects of the methods of the invention, the rAAV vector particle comprises a transgene encoding a nucleic acid selected from the group consisting of an siRNA, an antisense molecule, an miRNA, a ribozyme, and an shRNA.

[0052]

[0053] In certain embodiments of the methods of the invention, the rAAV vector particles are capable of expressing any of the following hormones: insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor alpha (TGF), erythropoietin (EPO), erythropoietin (EPO), erythropoietin-binding protein 1 (ERP), erythropoietin-binding protein 2 (ERP), erythropoietin-binding protein 3 (ERP), erythropoietin-binding protein 4 (ERP), erythropoietin-binding protein 5 (ERP), erythropoietin-binding protein 6 (ERP), erythropoietin-binding protein 7 (ERP), erythropoietin-binding protein 8 (ERP), erythropoietin-binding protein 9 (ERP), erythropoietin-binding protein 1 ... α), platelet-derived growth factor (PDGF), insulin-like growth factors I and II (IGF-I and IGF-II), TGFβ, activin, inhibin, bone morphogenetic proteins (BMPs), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, and tyrosine hydroxylase.

[0053]

[0054] In certain aspects of the methods of the invention, the rAAV vector particle comprises a transgene encoding a gene product selected from the group consisting of thrombopoietin (TPO), interleukins (IL1-IL-17), monocyte chemotactic protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factors alpha and beta, interferons alpha, beta, and gamma, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD, and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I and class II MHC molecules.

[0054]

[0055] In certain embodiments of the methods of the invention, the rAAV vector particles are capable of expressing any of the following enzymes: carbamoyl synthetase I, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor V, factor VIII, factor IX, cystatin beta-synthase, branched-chain keto acid decarboxylase, albumin, isoforms, ribozymes ... The transgenes include those encoding proteins useful for correcting inborn errors of metabolism selected from the group consisting of valeryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, RPE65, H-protein, T-protein, cystic fibrosis transmembrane conductance regulator (CFTR) sequence, and dystrophin cDNA sequence.

[0055]

[0056] In certain embodiments of the methods of the invention, the rAAV vector particle comprises a transgene encoding factor VIII or factor IX.

[0056]

[0057] In certain aspects of the methods of the invention, the methods recover about 50-90% of all rAAV vector particles from the harvest produced in step (a) or the concentrated harvest in step (b).

[0057]

[0058] In certain aspects of the methods of the invention, the methods produce rAAV vector particles that have greater purity than rAAV vector particles produced or purified by a single round of AAV affinity column purification.

[0058]

[0059] In certain aspects of the method of the present invention, steps (c) and (d) are carried out substantially simultaneously.

[0059]

[0060] In certain aspects of the methods of the invention, NaCl is adjusted after step (c) but before step (f) to be in the range of about 100-400 mM NaCl, inclusive, or in the range of about 140-300 mM NaCl, inclusive.

[0060]

[0061] In certain aspects of the methods of the invention, the rAAV vector particle is derived from an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10 and Rh74.

[0061]

[0062] In certain aspects of the methods of the invention, the rAAV vector particle comprises a capsid sequence having 70% or greater identity to the capsid sequence of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, Rh74, SEQ ID NO:1, or SEQ ID NO:2.

[0062]

[0063] In certain aspects of the methods of the invention, the rAAV vector particle comprises an ITR sequence having 70% or greater identity to the ITR sequence of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, or Rh74.

[0063]

[0064] In certain aspects of the methods of the invention, the cells are suspension or adherent cells.

[0064]

[0065] In certain embodiments of the methods of the present invention, the cell is a mammalian cell. Non-limiting examples include HEK cells, such as HEK-293 cells.

[0065]

[0066] In certain aspects of the methods of the invention, the methods are carried out using any one or more columns, conditions, concentrations, molarity, volumes, capacity, flow rates, pressures, materials, temperatures, pH, or steps described in any of Examples 1-3.

[0066]

[0067] In certain aspects of the methods of the invention, cell lysis and / or preparation prior to column purification as described herein is performed by any one or more of the conditions, concentrations, molarity, volumes, capacities, flow rates, pressures, materials, temperatures, pH, or steps described in Example 4. [Brief description of the drawings]

[0067] [Figure 1] FIG. 13 shows CEX (Poros 50HS) → AEX (Poros 50HQ)>UF>SEC (Superdex 200 prep grade) column chromatography of a starting rAAV harvest volume of 500-600 ml, which can be scaled up to larger volumes for substantially increased rAAV production (e.g., 1.2 L or greater). [Diagram 2] FIG. 13 shows a CEX (Poros 50HS) → AEX (Poros 50HQ) column chromatography of a starting rAAV harvest volume of 500-600 ml, which can be scaled up to larger volumes for substantially increased rAAV production (e.g., 1.2 L or greater). [Diagram 3] FIG. 13 shows CEX (Poros 50HS) → UF>SEC (Superdex 200 prep grade)>AEX (Poros 50HQ) column chromatography of a starting rAAV harvest volume of 500-600 ml, which can be scaled up to larger volumes for substantially increased rAAV production (e.g., 1.2 L or greater). [Figure 4] Figure 1 shows affinity (AVB Sepharose HP) → AEX (Poros 50HQ) column chromatography of a starting rAAV harvest volume of 500-600 ml, which can be scaled up to larger volumes for substantially increased rAAV production (e.g., 1.2 L or greater). Following low pH elution from the affinity column (approximately 7.5 mL eluate), 4.5 mL of high pH neutralization solution is added, followed by approximately 120 mL of 5 mM TrisCl (pH 8.5) / 40 mM NaCl solution, bringing the total load volume of the AEX (Poros 50HQ) column to approximately 132 mL. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068]

[0072] The present invention provides a method for the purification and production of recombinant adeno-associated virus (AAV) vectors (rAAV) that can be scaled up to large scale. For example, suspensions are cultured to volumes of 5, 10, 10-20, 20-50, 50-100, 100-200 liters or more. The present invention provides a method for the purification and production of recombinant adeno-associated virus (AAV) vectors (rAAV) that can be applied to a wide variety of AAV serotypes / capsid variants. The method of the present invention used for the purification or production of rAAV vectors includes the removal of process impurities and impurities associated with production. The method of the present invention includes a unique combination of chromatography and process steps that provide the potential for large scale purification of rAAV vectors of many different serotypes / pseudotypes.

[0069]

[0073] Impurities include impurities associated with AAV vector production, including proteins, nucleic acids (e.g., DNA), cellular components, e.g., intracellular and membrane components, that are impurities distinct from the AAV vector. The term "production or process related impurities" refers to any components that are released during the AAV purification and production process and are not true rAAV particles.

[0070]

[0074] A bona fide rAAV vector refers to a rAAV vector particle that contains a heterologous nucleic acid (e.g., a transgene) that can infect a target cell. The phrase excludes empty AAV capsids, AAV vectors that lack a complete insert in the packaged genome, or AAV vectors that contain contaminating host cell nucleic acid. In one embodiment, a bona fide rAAV vector refers to a rAAV vector particle that also lacks contaminating plasmid sequences in the packaged vector genome.

[0071]

[0075] "Empty capsid" and "empty particle" refer to an AAV particle or virion that contains an AAV capsid shell but lacks, in whole or in part, a genome containing a heterologous nucleic acid sequence flanked on one or both sides by AAV ITRs. Such empty capsids do not function to transfer heterologous nucleic acid sequences into a host cell or cell within an organism.

[0072]

[0076] The term "vector" refers to small carriers of nucleic acid molecules, plasmids, viruses (e.g., rAAV vectors), or other vehicles that can be manipulated by inserting or incorporating nucleic acid. Vectors can be used for genetic engineering (i.e., "cloning vectors") to introduce / transfer polynucleotides into cells and to transcribe or translate the inserted polynucleotides in cells. An "expression vector" is a vector that contains a gene or nucleic acid sequence with regulatory regions necessary for expression in a host cell. A vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous nucleic acid sequence, expression control elements (e.g., promoters, enhancers), introns, inverted terminal repeats (ITRs), optional selectable markers, polyadenylation signals.

[0073]

[0077] rAAV vectors are derived from adeno-associated viruses. AAV vectors are useful as gene therapy vectors because they can introduce nucleic acid / genetic material into cells, so that the nucleic acid / genetic material can be maintained in cells. Because AAV is not associated with pathogenic disease in humans, rAAV vectors can deliver heterologous nucleic acid sequences (e.g., therapeutic proteins and agents) to human patients without causing substantial AAV pathogenesis or disease.

[0074]

[0078] The term "recombinant" as modified genes of vectors such as rAAV vectors, and as modified gene sequences such as recombinant polynucleotides and polypeptides, generally means that the composition has been manipulated (i.e., altered) in a manner that does not occur in nature. A particular example of a recombinant AAV vector would be when a nucleic acid not normally present in the wild-type AAV genome is inserted into the genome of the virus. An example would be when a nucleic acid (e.g., a gene) encoding a therapeutic protein or polynucleotide sequence is cloned into a vector with or without the 5', 3' and / or intron regions with which the gene is normally associated in the AAV genome. As used herein, the term "recombinant" is not necessarily used with respect to AAV vectors, as well as sequences such as polynucleotides, although recombinant forms including AAV vectors, polynucleotides, etc. are expressly included despite any such omissions.

[0075]

[0079] A "rAAV vector" is derived from the wild-type genome of a virus such as AAV by using molecular methods to remove the wild-type genome from the AAV genome and replace it with a non-native (heterologous) nucleic acid, such as a nucleic acid encoding a therapeutic protein or polynucleotide sequence. Typically, for AAV, one or both inverted terminal repeat (ITR) sequences of the AAV genome are retained in the rAAV vector. rAAV is clearly distinguished from the AAV genome because all or part of the AAV genome has been replaced with a non-native sequence, e.g., a heterologous nucleic acid encoding a therapeutic protein or polynucleotide sequence, with respect to the nucleic acid of the AAV genome. The incorporation of a non-native sequence therefore defines the AAV as a "recombinant" AAV vector, which can be referred to as a "rAAV vector."

[0076]

[0080] Recombinant AAV vector sequences can be packaged and referred to herein as "particles" for subsequent infection (transduction) of cells ex vivo, in vitro or in vivo. When recombinant vector sequences are encapsidated or packaged in an AAV particle, the particle can also be referred to as a "rAAV" or "rAAV particle" or "rAAV virion." Such rAAV, rAAV particles and rAAV virions include proteins that encapsidate or package the vector genome. As a specific example, in the case of AAV, capsid proteins are included.

[0077]

[0081] Vector "genome" refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsidated to form the rAAV particle. When a recombinant plasmid is used to construct or produce a recombinant AAV vector, the AAV vector genome does not include the portion of the "plasmid" that does not correspond to the vector genome sequence of the recombinant plasmid. The non-vector genome portion of the recombinant plasmid is referred to as the "plasmid backbone", which is important for the processes required for plasmid cloning and amplification, propagation and recombinant virus production, but is not itself packaged or encapsidated into the rAAV particle. Thus, vector "genome" refers to the nucleic acid that is packaged or encapsidated by the rAAV.

[0078]

[0082] "AAV helper functions" refers to coding sequences (proteins) from AAV that can be expressed to provide AAV gene products and AAV vectors that function automatically in trans for productive AAV replication and packaging. Thus, AAV helper functions include AAV open reading frames (ORFs), including rep and cap, as well as others, such as AAPs for certain AAV serotypes. Rep expression products have been shown to have many functions, including, among others: recognition, binding, and nicking of the AAV origin of DNA replication; DNA helicase activity; and regulation of transcription from AAV (or other heterologous) promoters. The cap expression product (capsid) provides the necessary packaging functions. AAV helper functions are used to compensate for AAV functions missing from the AAV vector genome in trans.

[0079]

[0083] "AAV helper construct" generally refers to a nucleic acid sequence that includes a nucleotide sequence that provides an AAV function that is deleted from an AAV vector to be used to produce a transducing AAV vector for delivering a nucleic acid sequence of interest, for example, for gene therapy to a subject. AAV helper constructs are usually used to transiently express AAV rep and / or cap genes to compensate for missing AAV functions required for AAV vector replication. Helper constructs generally lack AAV ITRs and cannot replicate or package themselves. AAV helper constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. Several AAV helper constructs have been described, such as plasmids pAAV / Ad and pIM29+45, which encode both Rep and Cap expression products (see, e.g., Samulsk et al. (1989) J. Virol. 63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945). Many other vectors have been described that encode Rep and / or Cap expression products (see, e.g., U.S. Patent Nos. 5,139,941 and 6,376,237).

[0080]

[0084] The term "accessory functions" refers to non-AAV derived viral and / or cellular functions that AAV depends on for replication. The term includes proteins and RNAs required for AAV replication, including portions involved in activation of AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products, and packaging of AAV capsids. Viral-based accessory functions can be derived from any known helper virus, such as adenovirus, herpesvirus (other than herpes simplex virus type 1), and vaccinia virus.

[0081]

[0085] "Accessory function vector" generally refers to a nucleic acid molecule that includes a polynucleotide sequence that provides accessory functions. Such sequences can be on the accessory function vector and transfected into a suitable host cell. The accessory function vector can support rAAV virion production in a host cell. The accessory function vector can be in the form of a plasmid, phage, transposon, or cosmid. In addition, not all adenovirus genes are required for accessory functions. For example, adenovirus mutants that are incapable of DNA replication and late gene synthesis have been reported to be permissive for AAV replication (Ito et al., (1970) J. Gen. Virol. 9:243; Ishibashi et al., (1971) Virology 45:317). Similarly, mutants in the E2B and E3 regions have been shown to support AAV replication, indicating that the E2B and E3 regions are probably not involved in providing accessory functions (Carter et al., (1983) Virology 126:505). Adenoviruses defective in the E1 region or lacking the E4 region are unable to support AAV replication. Thus, the E1A and E4 regions appear to be required for AAV replication, either directly or indirectly (Laughlin et al., (1982) J. Virol. 41:868; Janik et al., (1981) Proc. Natl. Acad. Sci. USA 78:1925; Carter et al., (1983) Virology 126:505).Other characterized adenovirus mutants include: E1B (Laughlin et al. (1982), supra; Janik et al. (1981), supra; Ostrove et al. (1980) Virology 104:502); E2A (Handa et al. (1975) J. Gen. Virol. 29:239; Strauss et al. (1976) J. Virol. 17:140; Myers et al. (1980) J. Virol. 35:665; Jay et al. (1981) Proc. Natl. Acad. Sci. USA 78:2927; Myers et al. (1981) J. Biol. Chem. 256:567); E2B (Carter, Adeno-Associated Virus Helper in I CRC Handbook of Parvoviruses (ed. P. Tijssen, 1990) Functions); E3 (Carter et al. (1983), supra); and E4 (Carter et al. (1983), supra; Carter (1995)). Studies of accessory functions provided by adenoviruses with mutations in the E1B coding region have yielded conflicting results, although E1B55k may be required for AAV virion production, while E1B19k is not (Samulski et al., (1988) J. Virol. 62:206-210). In addition, WO 97 / 17458 and Matshushita et al., (1998) Gene Therapy 5:938-945 describe accessory function vectors encoding various adenoviral genes. Exemplary accessory function vectors include the adenovirus VA RNA coding region, the adenovirus E4 ORF6 coding region, the adenovirus E2A 72 kD coding region, the adenovirus E1A coding region, and the adenovirus E1B region lacking the complete E1B55k coding region. Such accessory function vectors are described, for example, in WO 01 / 83797.

[0082]

[0086] As used herein, the term "serotype" is a distinction used to refer to an AAV that has a capsid that is serologically distinct from other AAV serotypes. Serological distinctiveness is determined based on the lack of cross-reactivity between antibodies comparing one AAV to another. Differences in cross-reactivity are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in AAV serotypes in VP1, VP2, and / or VP3 sequences).

[0083]

[0087] Based on the traditional definition, a serotype means that the virus of interest has been tested for neutralizing activity against sera specific for all existing and characterized serotypes, and no antibodies were found to neutralize the virus of interest. As more naturally occurring virus isolates are discovered and / or capsid mutants arise, there may or may not be serological differences from any of the currently existing serotypes. Thus, if a new virus (e.g., AAV) does not have serological differences, it is a subgroup or variant of the corresponding serotype. In many cases, serological testing for neutralizing activity must be further performed on mutant viruses with altered capsid sequences to determine whether they are of another serotype according to the traditional definition of serotype. Thus, for convenience and to avoid repetition, the term "serotype" refers broadly to both serologically distinct viruses (e.g., AAV) and non-serologically distinct viruses (e.g., AAV) that may be within a subgroup or variant of a given serotype.

[0084]

[0088] rAAV vectors include any virus strain or serotype. As a non-limiting example, rAAV plasmid or vector genome or particle (capsid) can be based on any AAV serotype, such as AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, etc. Such vectors can be based on the same strain or serotype (or subgroup or variant), or can be different from each other. As a non-limiting example, rAAV plasmid or vector genome or particle (capsid) based on the genome of one serotype can be identical in one or more of the capsid proteins that package the vector. In addition, a rAAV plasmid or vector genome can be based on a serotype genome of AAV (e.g., AAV2) that is distinct from one or more of the capsid proteins that package the vector genome, where at least one of the three capsid proteins can be, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, SEQ ID NO:1 or SEQ ID NO:2, or variants thereof. rAAV vectors therefore contain gene / protein sequences identical to those characteristic for particular serotypes, as well as mixed serotypes.

[0085]

[0089] In various exemplary embodiments, the rAAV vector comprises or consists of a capsid sequence that is at least 70% or more identical (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) to the capsid protein of one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, SEQ ID NO:1, or SEQ ID NO:2. In various exemplary embodiments, the rAAV vector comprises or consists of a sequence at least 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) identical to one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, or Rh74 ITR(s).

[0086]

[0090] rAAV, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, SEQ ID NO:1 and SEQ ID NO:2, as well as mutant, hybrid and chimeric sequences, can be constructed using recombinant techniques known to those skilled in the art to contain one or more heterologous polynucleotide sequences (transgenes) flanked by one or more functional AAV ITR sequences. Such vectors have one or more of the wild-type AAV genes deleted in whole or in part, but retain at least one functional adjacent ITR sequence(s) as necessary for rescuing, replicating and packaging the recombinant vector into rAAV vector particles. The rAAV vector genome therefore contains the sequences necessary for replication and packaging (e.g., functional ITR sequences) in cis configuration.

[0087]

[0091] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to all forms of nucleic acid, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids include genomic DNA, cDNA and antisense DNA, and spliced ​​or unspliced ​​mRNA, rRNA, tRNA and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), small or short interfering (si)RNA, trans-splicing RNA, or antisense RNA). Nucleic acids include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. Nucleic acids can be single-, double-, or triplexed, linear or circular, and of any length. In discussing nucleic acids, the sequence or structure of a particular polynucleotide may be described herein according to the convention of providing the sequence in the 5' to 3' direction.

[0088]

[0092] A "heterologous" nucleic acid sequence refers to a polynucleotide inserted into an AAV plasmid or vector for the purpose of vector-mediated transfer / delivery of the polynucleotide into a cell. A heterologous nucleic acid sequence is distinct from, i.e., non-naturally occurring with respect to, the AAV nucleic acid. Once transferred / delivered into a cell, the heterologous nucleic acid sequence contained within the vector may be expressed (e.g., transcribed and translated, if appropriate). Alternatively, the heterologous polynucleotide contained within the transferred / delivered vector in the cell may not be expressed. Although the term "heterologous" is not necessarily used herein in reference to nucleic acid sequences and polynucleotides, reference to a nucleic acid sequence or polynucleotide is intended to include heterologous nucleic acid sequences and polynucleotides, despite the omission, even in the absence of a "heterologous" modifying gene.

[0089]

[0093] "Polypeptides," "proteins," and "peptides" encoded by a "nucleic acid sequence" consequently include naturally occurring proteins, as well as functional subsequences, modified forms, or sequence variants, so long as the modified forms or variants retain some functionality of the native full-length protein. Such polypeptides, proteins, and peptides encoded by nucleic acid sequences can, but need not, be identical to endogenous proteins that are defective or poorly or incompletely expressed in the mammal being treated.

[0090]

[0094] "Transgene" is used conveniently herein to refer to a nucleic acid (e.g., heterologous) that is intended or introduced into a cell or organism. A transgene includes any nucleic acid, such as a heterologous nucleic acid that encodes a therapeutic protein or polynucleotide sequence.

[0091]

[0095] In a cell carrying a transgene, the transgene has been introduced / transferred by a plasmid or AAV vector, "transduction" or "transfection" of the cell. The terms "transduction" and "transfect" refer to the introduction of a molecule, such as a nucleic acid, into a host cell (e.g., HEK293) or cell of an organism. The transgene may or may not be integrated into the genomic nucleic acid of the recipient cell. If the introduced nucleic acid is integrated into the nucleic acid (genomic DNA) of the recipient cell or organism, the introduced nucleic acid can be stably maintained in that cell or organism and further passed on to or inherited by the descendant cells or organisms of the cell of the recipient cell or organism.

[0092]

[0096] "Host cell" refers to, for example, microorganisms, yeast cells, insect cells, and mammalian cells that can be or have been used as recipients of AAV vector plasmids, AAV helper constructs, accessory function vectors, or other transfer DNA. The term includes the progeny of the original transfected cell. Thus, "host cell" generally refers to a cell that has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parent cell may not necessarily be completely identical in morphology or genomic or total DNA complement to the original parent due to natural, accidental, or deliberate mutations. Exemplary host cells include human embryonic kidney (HEK) cells, such as HEK293.

[0093]

[0097] A "transduced cell" is a cell into which a transgene has been introduced. Thus, a "transduced" cell refers to the genetic change in a cell following incorporation of an exogenous molecule, e.g., a nucleic acid (e.g., a transgene), into the cell. Thus, a "transduced" cell is a cell into which an exogenous nucleic acid has been introduced, or its progeny. The cell(s) can be propagated (cultured) and the cell expresses the introduced protein or transcribes the nucleic acid, or produces a vector, such as rAAV. For gene therapy uses and methods, the transduced cell can be present in a subject.

[0094]

[0098] As used herein, the term "stable" or "stably integrated" in reference to a cell means that a nucleic acid sequence, such as a selectable marker or heterologous nucleic acid sequence, or a plasmid or vector, is inserted into a chromosome (e.g., by homologous recombination, non-homologous end joining, transfection, etc.) or is maintained extrachromosomally in a recipient cell or host organism and remains in the chromosome or is maintained extrachromosomally for a period of time. In the case of cultured cells, a nucleic acid sequence, such as a heterologous nucleic acid sequence, inserted into a chromosome, or a plasmid or vector, can be maintained over the course of multiple cell passages.

[0095]

[0099] "Cell line" refers to a population of cells capable of continuous or long-term growth and division under suitable culture conditions in vitro. A cell line can be, but need not be, a population of clones derived from a single progenitor cell. In cell lines, spontaneous or induced changes can occur in the karyotype during storage or transfer of such clonal populations, as well as during long-term passage in tissue culture. Thus, progeny cells derived from a cell line may not be strictly identical to the ancestral cells or cultures. An exemplary cell line applicable to the purification method of the present invention is HEK293.

[0096]

[0100] "Expression control element" refers to a nucleic acid sequence(s) that influences the expression of an operably linked nucleic acid. Control elements include expression control elements such as promoters and enhancers described herein. rAAV vectors can include one or more "expression control elements". Typically, such elements are included to facilitate transcription and, if appropriate, translation of the appropriate heterologous polynucleotide (e.g., promoters, enhancers, splicing signals for introns, maintaining the correct reading frame of the gene to allow in-frame translation of mRNA, and stop codons, etc.). Such elements typically act in cis and are referred to as "cis-acting" elements, but may also act in trans.

[0097]

[0101] Expression control can be affected at the level of transcription, translation, splicing, message stability, etc. Typically, expression control elements that regulate transcription are juxtaposed near the 5' end (i.e., "upstream") of the transcribed nucleic acid. Expression control elements can also be located at the 3' end (i.e., "downstream") of the transcribed sequence or within the transcript (e.g., in an intron). Expression control elements can be located adjacent to the transcribed sequence or even at a significant distance (e.g., 1-10, 10-25, 25-50, 50-100, 100-500, or more nucleotides from the polynucleotide). Nevertheless, due to the length limitations of the rAAV vector, expression control elements are typically within 1-1000 nucleotides of the transcribed nucleic acid.

[0098]

[0102] Functionally, expression of an operably linked nucleic acid can be at least partially controlled by an element (e.g., a promoter) that regulates transcription of the nucleic acid and, where appropriate, translation of the transcript. A specific example of an expression control element is a promoter, which is usually located 5' to the sequence to be transcribed. A promoter typically increases the amount of expression from an operably linked nucleic acid compared to the amount expressed in the absence of the promoter.

[0099]

[0103] As used herein, "enhancer" can refer to a sequence located adjacent to a nucleic acid sequence, such as a selectable marker or a heterologous nucleic acid sequence. Enhancer elements are typically located upstream of a promoter element, but can also function and be located downstream or internal to the sequence. Thus, enhancer elements can be located upstream or downstream, for example, within 100, 200, or 300 or more base pairs of a heterologous nucleic acid encoding a selectable marker and / or a therapeutic protein or polynucleotide sequence. Enhancer elements typically increase expression of an operably linked nucleic acid beyond that caused by a promoter element.

[0100]

[0104] The term "operably linked" means that regulatory sequences required for expression of a nucleic acid sequence are appropriately positioned relative to the sequence so as to effect expression of the nucleic acid sequence. This same definition is sometimes applied to the arrangement of nucleic acid sequences and transcriptional control elements (e.g., promoters, enhancers, and termination elements) in an expression vector, e.g., a rAAV vector.

[0101]

[0105] In the example of an expression control element operably linked to a nucleic acid, the relationship is such that the control element regulates expression of the nucleic acid. More specifically, for example, two DNA sequences that are operably linked means that the two DNA sequences are positioned in such a relationship (either cis or trans) that at least one of the DNA sequences can have a physiological effect on the other sequence.

[0102]

[0106] Thus, additional elements for a vector include, but are not limited to, expression control elements (e.g., promoters / enhancers), transcription termination signals or stop codons, 5' or 3' untranslated regions (e.g., polyadenylation (polyA) sequences) present adjacent to sequences such as the AAV ITR sequences, or one or more copies of an intron.

[0103]

[0107] Further elements include, for example, filler or stuffer polynucleotide sequences, for example, to improve packaging and reduce the presence of contaminating nucleic acids. AAV vectors typically accept DNA insertions, generally having a size range of about 4 kb to about 5.2 kb, or slightly more. Thus, for shorter sequences, a stuffer or filler is included to adjust the length to near or to the normal size of the viral genomic sequence that is acceptable for packaging of the vector into rAAV particles. In various embodiments, the filler / stuffer nucleic acid sequence is a non-translated (non-protein coding) segment of nucleic acid. For nucleic acid sequences less than 4.7 Kb, the filler or stuffer polynucleotide sequence has a length that, when combined with the sequence (e.g., inserted into a vector), has a total length of between about 3.0-5.5 Kb, or between about 4.0-5.0 Kb, or between about 4.3-4.8 Kb.

[0104]

[0108] A "therapeutic protein" is, in one embodiment, a peptide or protein that can alleviate or relieve a symptom resulting from an insufficient amount, absence, or deficiency of a protein in a cell or subject. A "therapeutic" protein encoded by a transgene can provide a benefit to the subject, e.g., correct a genetic defect, correct a deficiency (of expression or function) of a gene, etc.

[0105]

[0109] Non-limiting examples of heterologous nucleic acids encoding gene products (e.g., therapeutic proteins) useful according to the present invention include those that may be used in the treatment of diseases or disorders including, but not limited to, "blood congestion" or blood clotting disorders, such as hemophilia A, hemophilia A patients with inhibitory antibodies, hemophilia B, deficiencies of coagulation factors VII, VIII, IX and X, XI, V, XII, II, von Willebrand factor, combined FV / FVIII deficiency, thalassemia, vitamin K epoxide reductase C1 deficiency, gamma-carboxylase deficiency; bleeding associated with anemia, trauma, injury, thrombosis, thrombocytopenia, stroke, coagulation disorders, disseminated intravascular coagulation (DIC); hyperanticoagulation associated with heparin, low molecular weight heparins, pentasaccharides, warfarin, small molecule antithrombotic drugs (i.e., FXa inhibitors); and platelet disorders, such as Bernard-Slier syndrome, Glanzmann thrombasthenia, and storage pool deficiency.

[0106]

[0110] Nucleic acid molecules, vectors, such as cloning vectors, expression vectors (e.g., vector genomes), and plasmids, can be prepared using recombinant DNA techniques. The availability of nucleotide sequence information allows for the preparation of nucleic acid molecules by various means. For example, heterologous nucleic acid encoding factor IX (FIX), including vectors or plasmids, can be produced by cell expression or in vitro translation and chemical synthesis techniques using a variety of standard cloning, recombinant DNA techniques. The purity of the polynucleotide can be determined by sequencing, gel electrophoresis, and the like. For example, nucleic acids can be isolated using hybridization or computer-based database screening techniques. Such techniques include, but are not limited to, (1) hybridization of genomic DNA or cDNA libraries with probes to detect homologous nucleotide sequences; (2) antibody screening to detect polypeptides with shared structural features, e.g., using expression libraries; (3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers that can anneal to the nucleic acid sequence of interest; (4) computer searches of sequence databases for related sequences; and (5) differential screening of subtracted nucleic acid libraries.

[0107]

[0111] The term "isolated," when used as a modifier of a composition, means that the composition is made by the hand of man or is completely or at least partially separated from its naturally occurring in vivo environment. Generally, an isolated composition is substantially free of one or more materials that are normally associated with it in nature, such as one or more proteins, nucleic acids, lipids, carbohydrates, cell membranes.

[0108]

[0112] With respect to proteins, the terms "isolated protein" or "isolated and purified protein" are sometimes used herein. This term primarily refers to a protein produced by expression of a nucleic acid molecule. Alternatively, this term may refer to a protein that has been sufficiently separated from other proteins with which it is naturally associated so that it exists in "substantially pure" form.

[0109]

[0113] The term "isolated" does not exclude combinations produced by the hand of man, such as recombinant rAAV and pharmaceutical formulations. The term "isolated" also does not exclude alternative physical forms of the composition, such as hybrid / chimeric, multimeric / oligomeric, modified (e.g., phosphorylated, glycosylated, lipid-modified) or derivatized forms, or forms expressed in a host cell produced by the hand of man.

[0110]

[0114] The term "substantially pure" refers to a preparation that contains at least 50-60% by weight of the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). The preparation may contain at least 75%, or about 90-99%, by weight of the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g., chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, etc.).

[0111]

[0115] The phrase "consisting essentially of," when referring to a particular nucleotide or amino acid sequence, means a sequence having the properties of the given sequence. For example, when used in reference to an amino acid sequence, the phrase includes the sequence itself and molecular modifications that do not affect the basic and novel properties of the sequence.

[0112]

[0116] Methods for generating rAAV virions are known in the art, such as: transfection using an AAV vector and an AAV helper sequence in conjunction with co-infection with an AAV helper virus (e.g., adenovirus, herpesvirus, or vaccinia virus), or transfection using a recombinant AAV vector, an AAV helper vector, and an accessory function vector. Non-limiting methods for generating rAAV virions are described, for example, in U.S. Patent Nos. 6,001,650 and 6,004,797, International Application PCT / US16 / 64414 (published as WO 2017 / 096039), and U.S. Provisional Patent Applications Nos. 62 / 516,432 and 62 / 531,626. After production of recombinant rAAV vectors (i.e., generation of vectors in cell culture systems), rAAV virions can be obtained from host cells and cell culture supernatants and purified as described herein.

[0113]

[0117] As an initial step, typically, the host cells producing rAAV virions can be harvested, optionally in combination with harvesting the cell culture supernatant (medium) in which the host cells (suspension or adherent) producing rAAV virions were grown. In the methods herein, the harvested cells and optionally the cell culture supernatant can be used as is or purified, as appropriate. Furthermore, if infection is used to express accessory functions, residual helper virus can be inactivated. For example, adenovirus can be inactivated by heating to a temperature of about 60° C., for example, for 20 minutes or more, thereby inactivating only the helper virus, since AAV is heat stable but the helper adenovirus is heat labile.

[0114]

[0118] The harvested cells and / or supernatant are lysed by disrupting the cells, e.g., by chemical or physical means, e.g., detergents, microfluidization, and / or homogenization, to release the rAAV particles. A nuclease, such as benzonase, can also be added simultaneously during or after cell lysis to degrade contaminating DNA. Typically, the resulting lysate is clarified to remove cellular debris, e.g., by filtration, centrifugation, etc., to produce a clarified cell lysate. In certain instances, the lysate is filtered using a micron-sized pore filter (e.g., a filter with a pore size of 0.1-10.0 μm, e.g., a filter with a pore size of 0.45 μm and / or 0.2 μm) to produce a clarified lysate.

[0115]

[0119] The lysate (optionally clarified) contains AAV particles (both authentic rAAV vectors and AAV empty capsids) and impurities associated with the AAV vector manufacturing / process, such as soluble cellular components from the host cells, which may include cellular proteins, lipids, and / or nucleic acids, among others, as well as cell culture media components. The optionally clarified lysate is then subjected to an additional purification step using chromatography to purify the AAV particles (including authentic rAAV vectors) from impurities. The clarified lysate may be diluted or concentrated with an appropriate buffer prior to the first step of chromatography.

[0116]

[0120] As disclosed herein, after cell lysis, optional clarification, and optional dilution or concentration, multiple sequential chromatographic steps are used to purify the rAAV particles. Such methods typically exclude a cesium chloride gradient ultracentrifugation step.

[0117]

[0121] As disclosed herein, the first chromatography step can be cation exchange chromatography or anion exchange chromatography. If the first chromatography step is cation exchange chromatography, the second chromatography step can be anion exchange chromatography or size exclusion chromatography (SEC). Thus, in one rAAV purification method, purification is by cation exchange chromatography followed by anion exchange chromatography.

[0118]

[0122] Alternatively, if the first chromatography step is cation exchange chromatography, the second chromatography step can be size exclusion chromatography (SEC). Thus, in another rAAV purification method, purification is by cation exchange chromatography followed by size exclusion chromatography (SEC).

[0119]

[0123] As also disclosed herein, the first chromatography step can be affinity chromatography. If the first chromatography step is affinity chromatography, the second chromatography step can be anion exchange chromatography. Thus, in a further rAAV purification method, purification is performed by affinity chromatography followed by anion exchange chromatography.

[0120]

[0124] Optionally, a third chromatography step can be added to the above chromatography steps. Typically, the optional third chromatography step follows a cation exchange, anion exchange, size exclusion or affinity chromatography.

[0121]

[0125] Thus, in an additional rAAV purification method, purification is by cation exchange chromatography, followed by anion exchange chromatography, followed by size exclusion chromatography (SEC), and in yet another rAAV purification method, purification is by cation exchange chromatography, followed by size exclusion chromatography (SEC), followed by anion exchange chromatography.

[0122]

[0126] In yet another additional rAAV purification method, purification is by affinity chromatography followed by anion exchange chromatography followed by size exclusion chromatography (SEC). In yet another rAAV purification method, purification is by affinity chromatography followed by size exclusion chromatography (SEC) followed by anion exchange chromatography.

[0123]

[0127] Cation exchange chromatography serves to separate AAV particles from cells and other components present in the clarified lysate and / or column eluate from size exclusion chromatography. Examples of strong cation exchange resins capable of binding rAAV particles over a wide pH range include, but are not limited to, any sulfonic acid-based resin as indicated by the presence of sulfonate functional groups, including aryl and alkyl substituted sulfonates, e.g., sulfopropyl or sulfoethyl resins. Representative matrices include, but are not limited to, POROS HS, POROS HS50, POROS XS, POROS SP, and POROS S (strong cation exchangers, available from Thermo Fisher Scientific, Inc., Waltham, Massachusetts). Additional examples include Capto S, Capto S ImpAct, Capto S ImpRes (strong cation exchangers, available from GE Healthcare, Marlborough, Massachusetts), as well as the commercially available DOWEX®, AMBERLITE®, and AMBERLYST® families of resins, available from Aldrich Chemical Company, Milliwaukee, Wisconsin. Weak cation exchange resins include, but are not limited to, any carboxylic acid-based resin. Exemplary cation exchange resins also include carboxymethyl (CM), phospho (based on phosphoric acid functionality), methylsulfonate (S), and sulfopropyl (SP) resins.

[0124]

[0128] Anion exchange chromatography serves to separate AAV particles from proteins, cells and other components present in the column eluate from the clarified lysate and / or size exclusion chromatography. Anion exchange chromatography can also be used to control the amount of empty AAV capsids in the eluate. For example, an anion exchange column with bound rAAV vector can be washed with a moderate concentration (e.g., about 100-125 mM, e.g., 110-115 mM) of NaCl, and a portion of the empty capsids can be eluted in the flow-through without substantial elution of the rAAV vector. The rAAV vector bound to the anion exchange column can then be eluted using a higher concentration of NaCl (e.g., about 130-300 mM NaCl), thereby producing a column eluate with reduced or depleted amounts of empty AAV capsids and proportionally increased amounts of rAAV.

[0125]

[0129] Exemplary anion exchange resins include, but are not limited to, polyamine resin-based resins and other resins. Examples of strong anion exchange resins include resins generally based on quaternized nitrogen atoms, including, but not limited to, quaternary ammonium salt resins such as trialkylbenzylammonium resins. Suitable exchange chromatography includes MACRO PREP Q (strong anion exchanger, available from BioRad, Hercules, CA); UNOSPHERE Q (strong anion exchanger, available from BioRad, Hercules, CA); POROS 50HQ (strong anion exchanger, available from Applied Biosystems, Foster City, CA); POROS XQ (strong anion exchanger, available from Applied Biosystems, Foster City, CA); POROS 50D (weak anion exchanger, available from Applied Biosystems, Foster City, CA); POROS 50PI (weak anion exchanger, available from Applied Biosystems, Foster City, CA); Capto Q, Capto XQ, Capto Q ImpRes, and SOURCE 30Q (strong anion exchanger, available from GE healthcare, Marlborough, MA); DEAE SEPHAROSE (weak anion exchanger, available from Amersham Exemplary anion exchange resins include, but are not limited to, Q SEPHAROSE (strong anion exchanger, available from Amersham Biosciences, Piscataway, NJ). Additional exemplary anion exchange resins include aminoethyl (AE), diethylaminoethyl (DEAE), diethylaminopropyl (DEPE), and quaternary aminoethyl (QAE).

[0126]

[0130] Chromatographic media such as cation exchange, anion exchange, size exclusion and affinity can be equilibrated, washed and eluted with a variety of buffers under a variety of conditions such as pH and buffer volume. The following is intended to describe certain non-limiting examples, but is not intended to limit the invention.

[0127]

[0131] Cation exchange chromatography can be equilibrated using standard buffers according to the manufacturer's specifications. For example, the chromatography medium can be equilibrated with 5-100 mM, or 10-50 mM, e.g., 10-30 mM, phosphate buffer, and sodium chloride. After equilibration, the sample is then loaded. The chromatography medium is then washed at least once or more, e.g., 2-10 times. Elution from the chromatography medium is at least once with a high salt buffer, but elution can also be with two or more buffers of the same or higher salt concentration.

[0128]

[0132] Typical equilibration buffers and solutions for washing and elution for cation exchange chromatography are of a suitable pH from about pH 3 to pH 8, more typically from about pH 4 to pH 7.5, such as pH 6.0-6.5, 6.5-7.0, 7.0-7.5, or any pH in or between the above mentioned ranges, for example 7.0, 7.1, 7.2, 7.3 or 7.4.

[0129]

[0133] Suitable equilibration buffers and solutions for washing and elution for cation exchange columns are known in the art and are generally anionic, including but not limited to buffers using the following buffering ions: phosphate, acetate, citrate, borate, or sulfate.

[0130]

[0134] In one embodiment, the cation exchange chromatography medium is first equilibrated, the sample is applied, and washed with a low salt concentration, e.g., 10-150 mM NaCl, e.g., 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 60-125 mM, or any concentration in or between these ranges, e.g., 100 mM.

[0131]

[0135] Following the first wash, the chromatographic medium can also be treated with a higher salt concentration, e.g., a higher NaCl concentration, or another buffer of higher ionic strength, to elute impurities. After additional impurities have been eluted from the column, the ionic strength of the buffer can be increased using a salt, such as NaCl, KCl, sulfate, formate, or acetate, to elute the rAAV particles, and collected. In one embodiment, elution is performed at a high salt concentration, e.g., 200-500 mM NaCl, or any concentration or range, such as 250 mM, 300 mM, 350 mM, or 400 mM.

[0132]

[0136] Additional components may be included in the equilibration buffers and solutions for washing and elution. For example, a wash buffer for cation exchange chromatography may contain an anionic detergent such as sarkosyl (e.g., 1-10 mM), and a wash buffer for anion exchange chromatography may contain a cationic detergent such as dodecyltrimethylammonium chloride (e.g., 1-10 mM).

[0133]

[0137] Typical equilibration buffers and solutions for washing and elution for anion exchange chromatography are suitable at a pH of about pH 7.5 to pH 12, more typically about pH 8.0 to pH 10, even more typically about pH 8.0 to pH 9.0, for example pH 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0.

[0134]

[0138] Suitable equilibration buffers and solutions for washing and elution for anion exchange columns are generally cationic or zwitterionic in nature. Such buffers include, but are not limited to, buffers having the following buffers: N-methylpiperazine; piperazine; bis-tris; bis-trispropane; triethanolamine; tris; N-methyldiethanolamine; 1,3-diaminopropane; ethanolamine; acetic acid, and the like. To elute the sample, the ionic strength of the starting buffer is increased using salts such as NaCl, KCl, sulfate, formate, or acetate. Such equilibration buffers and solutions for washing and elution can have about 5-100 mM, more typically about 10-50 mM of the aforementioned buffers.

[0135]

[0139] In one embodiment, the anion exchange chromatography medium is first equilibrated, the sample is applied, and washed with a low salt concentration, e.g., 50-150 mM NaCl, e.g., 50-60, 60-70, 70-80, 80-90, 90-100, 100-100 mM, or any concentration in or between these ranges. Following the first wash, the chromatography medium can also be treated with a higher salt concentration, e.g., a higher NaCl concentration, or with another buffer of stronger ionic strength, to elute impurities such as empty capsids of AAV. One example for use as the second buffer is a Tris-based buffer with a NaCl concentration of about 110 mM to 125 mM, or any concentration in or between the ranges mentioned above.

[0136]

[0140] After additional impurities have been eluted from the column, the AAV particles can be recovered by elution with a higher concentration of salt. One example for an elution buffer is a Tris-based buffer having a NaCl concentration of 125 mM or more, for example, 125-150 mM, 150-200 mM, or 200-250 MM NaCl, or any concentration within these aforementioned ranges or therein.

[0137]

[0141] The wash solution for the anion exchange chromatography medium may contain polyethylene glycol (PEG). This is referred to as polyethylene glycol (PEG) conditioned column chromatography. The PEG wash solution may be applied to the anion exchange chromatography medium prior to elution of the AAV vector particles.

[0138]

[0142] Typically, the PEG in such a cleaning solution has an average molecular weight in the range of about 1,000 to 80,000 g / mol, inclusive. Typical amounts of PEG in such cleaning solutions range from about 0.1% to about 20% PEG or any amount within the ranges mentioned above, or from about 1% to about 10% PEG or any amount within the ranges mentioned above.

[0139]

[0143] Size exclusion chromatography (SEC) media can be equilibrated according to manufacturer's specifications using standard buffers. For example, the chromatography media can be equilibrated with, for example, about 1-5 mM, 5-50 mM, or 5-25 mM phosphate buffer and, for example, about 50-100 mM, 100-150 mM, 150-200 mM, 200-250 mM, 250-300 mM, or 300-400 mM NaCl, or any amount within these aforementioned ranges or therein.

[0140]

[0144] After equilibration, the sample is then loaded. The flow-through containing the rAAV particles is then collected. An additional volume of buffer (e.g., phosphate buffer) can be added for rAAV particle recovery based on the amount of chromatography media and / or column size.

[0141]

[0145] In certain embodiments, the size exclusion chromatography media has a separation range (molecular weight) of between about 10,000 and 600,000 inclusive. Specific resins (media) suitable for size exclusion chromatography include, but are not limited to, porous cellulose particles or beads, cross-linked agarose (Sepharose, GE Healthcare, Marlborough, Mass.), cross-linked dextran (Sephadex, GE Healthcare, Marlborough, Mass.), styrene-divinylbenzene (Dianon HP-20), polyacrylamide (Bio Gel), methacrylics (Toyopearl), and controlled pore glass.

[0142]

[0146] Affinity columns are typically composed of a ligand linked or conjugated to a substrate. Particular examples of ligands include antibodies that bind to AAV. Such substrates include sepharose and other materials typically used in such affinity purification applications and can be made or are commercially available (e.g., AVB Sepharose™ High Performance, GE healthcare, Marlborough, Massachusetts).

[0143]

[0147] Suitable equilibration buffers for affinity columns as well as solutions for washing and elution are typically Tris or acetate based. For example, the affinity chromatography medium can be equilibrated with, for example, about 1-5 mM, 5-50 mM, or 5-20 mM Tris buffer and, for example, about 50-100 mM, 100-150 mM, 150-200 mM, 200-250 mM, 250-300 mM, or any amount of NaCl in or within these aforementioned ranges.

[0144]

[0148] Typical equilibration buffers for affinity chromatography have a pH of about pH 7.5 to pH 9.0, more typically about pH 8.0 to pH 8.5, and even more typically a pH such as pH 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5.

[0145]

[0149] After equilibration, the sample is then loaded. The rAAV particles are then eluted from the affinity column by lowering the pH of the buffer to less than 7.0. The elution buffer can be acetate-based, and typically has a pH less than 5.0, more typically less than 4.0, such as less than 3.0, and more particularly between about 2.0 and 3.0, or any pH within these aforementioned ranges or ranges.

[0146]

[0150] The volumes of buffers for equilibration, washing, and elution can be based on the amount of chromatography media and / or column size to achieve rAAV particle recovery, with typical volumes being 1-10 times the volume of the column.

[0147]

[0151] The column eluate is collected after elution(s) / passage through each of the chromatographic steps. AAV can be detected in the fractions using standard techniques, such as monitoring UV absorbance at 260 and 280 nm.

[0148]

[0152] The use of cation or anion exchange chromatography media, the nature of the media used (i.e., strong or weak ion exchanger), as well as the salt concentration, buffer used, and pH conditions can vary based on the AAV capsid (i.e., the serotype or pseudotype of the AAV capsid). Although AAV capsid structures typically share characteristics such as size and shape, capsids can have different amino acid sequences, resulting in subtle differences in molecular topology and surface charge distribution. Thus, capsid sequence variants are expected to be amenable to purification by the methods of the present invention, and related methods can be determined in a systematic manner using chromatography media and buffer screening studies to determine if different conditions are used for AAV capsid variants for rAAV particle purification.

[0149]

[0153] The eluate containing rAAV particles from any of the cation exchange, anion exchange, size exclusion, and / or affinity chromatography steps described herein can be efficiently concentrated by UF / DF if desired. The volume reduction can be controlled by one of skill in the art. In certain non-limiting examples, the volume reduction achieved is between about 1-30 fold, inclusive. Thus, a 1-fold reduction reduces the volume by half, e.g., 1000 ml is concentrated to 500 ml. A 10-fold reduction reduces the volume by a factor of 10, e.g., 2000 ml is concentrated to 200 ml. A 20-fold reduction reduces the volume by a factor of 20, e.g., 2000 ml is concentrated to 100 ml. A 30-fold reduction reduces the volume by a factor of 30, e.g., 2000 ml is concentrated to 66.67 ml.

[0150]

[0154] A non-limiting example of UF / DF is tangential flow filtration (TFF), e.g., hollow fiber membranes with a nominal pore size corresponding to a molecular weight cutoff of 100 kDa, which allows preparation when large amounts of AAV vector are present in larger volumes of eluate.

[0151]

[0155] Cell lysates and column eluates containing rAAV particles from any of the cation exchange, anion exchange, size exclusion, or affinity chromatography steps described herein can be diluted, if desired. Typical dilutions range from 25-100%, 1-2x, 2-5x, or volumes or amounts within these aforementioned ranges or ranges.

[0152]

[0156] The methods of the invention achieve substantial recovery of rAAV particles. For example, the methods of the invention achieve recovery of about 40-70% of the total rAAV vector particles from the harvested host cells and host cell culture supernatant. In another example, the rAAV particles are present in the final (e.g., third column) eluate at a concentration of about 100 mg / mL. The rAAV vector particles are present in the final (e.g., third column) eluate at a concentration of about 10 per mL. 10 ~10 11 More than 10 particles per mL 11 ~10 12 Particles, 10 per mL 12 ~10 13 The concentration of the particles may be present.

[0153]

[0157] Alternatively, if the rAAV vector particle concentration is lower, the purified rAAV particles can be concentrated. For example, the purified AAV particles can be concentrated by ultrafiltration / diafiltration (e.g., TFF). If a higher concentration of vector is desired, the purified AAV particles can be concentrated by ultrafiltration / diafiltration (e.g., TFF) to a concentration of 10 per mL. 12 ~10 13 More than 10 particles per mL 13 ~10 14 It can be concentrated to above particles or even higher.

[0154]

[0158] In other embodiments, rAAV particles with a packaged genome (i.e., true rAAV vector particles) are "substantially free" of AAV encapsidated nucleic acid impurities when at least about 30% or more of the virions present are rAAV particles with a packaged genome (i.e., true rAAV vector particles). Production of rAAV particles with a packaged genome (i.e., true rAAV vector particles) that is substantially free of AAV encapsidated nucleic acid impurities can be about 40% to about 20% or less, about 20% to about 10% or less, about 10% to about 5% or less, about 5% to about 1% or less, or less of the product that contains AAV encapsidated nucleic acid impurities.

[0155]

[0159] Methods for determining the infectious titer of an AAV vector containing a transgene are known in the art (see, e.g., Zhen et al. (2004) Hum. Gene Ther. (2004) 15:709). Methods for assaying empty capsids and AAV vector particles with packaged genomes are known (see, e.g., Grimm et al., Gene Therapy (1999) 6:1322-1330; Sommer et al., Molec. Ther. (2003) 7:122-128).

[0156]

[0160] To determine the presence or amount of disassembled / denatured capsid, the purified AAV can be subjected to SDS-polyacrylamide gel electrophoresis consisting of any gel capable of separating the three capsid proteins, for example, a gradient gel, and then run the gel until the sample is separated, and the gel can be blotted onto a nylon or nitrocellulose membrane. An anti-AAV capsid antibody is then used as the primary antibody that binds to the denatured capsid protein (see, for example, Wobus et al., J. Virol. (2000) 74:9281-9293). A secondary antibody that binds to the primary antibody contains a means for detecting the primary antibody. The binding between the primary and secondary antibodies is detected semi-quantitatively to determine the amount of capsid. Another method can be analytical HPLC with a SEC column or analytical ultracentrifugation.

[0157]

[0161] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0158]

[0162] All patent applications, publications, patents, and other references, GenBank citations, and ATCC citations cited herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control.

[0159]

[0163] All features disclosed herein may be combined in any combination. Each feature disclosed herein may also be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, the disclosed features (e.g., nucleic acid sequences, vectors, rAAV vectors, etc.) are examples of a class of equivalent or similar features.

[0160]

[0164] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "an AAV vector" or "AAV particle" includes a plurality of such AAV vectors and AAV particles, and reference to "a cell" or "host cell" includes a plurality of cells and host cells.

[0161]

[0165] As used herein, the term "about" means a value that is within 10% (plus or minus) of a reference value.

[0162]

[0166] As used herein, all numerical values ​​or ranges of numbers include integers within such ranges and fractions or integers of values ​​within the ranges, unless the context clearly indicates otherwise. Thus, by way of example, reference to 80% or greater identity includes 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% etc., as well as 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, etc., 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, etc.

[0163]

[0167] Reference to an integer with greater than or less than includes any number greater than or less than the referenced number, respectively. Thus, for example, reference to less than 100 includes 99, 98, 97, etc., all the way down to the number 1; less than 10 includes 9, 8, 7, etc., all the way down to the number 1.

[0164]

[0168] As used herein, all numerical values ​​or ranges are inclusive. Moreover, all numerical values ​​or ranges include fractions of values ​​and integers within such ranges, and fractions of integers within such ranges, unless the context clearly indicates otherwise. Thus, by way of example, reference to a numerical range such as 1-10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 1.1, 1.2, 1.3, 1.4, 1.5, etc. Thus, reference to a range of 1-50 includes up to and including 50, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., and 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc., and so forth.

[0165]

[0169] Reference to a series of ranges includes ranges combining the boundary values ​​of the different ranges within that series. Thus, by way of example, a series of ranges, e.g., 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 75, 75 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 400, 400 to 500, 500 to 750, 750 to 1,000, 1,000 to 1,500, 1,500 to 2,000, 2,000 to 2,500, References to 0, 2,500-3,000, 3,000-3,500, 3,500-4,000, 4,000-4,500, 4,500-5,000, 5,500-6,000, 6,000-7,000, 7,000-8,000, or 8,000-9,000 include ranges such as 10-50, 50-100, 100-1,000, 1,000-3,000, 2,000-4,000 etc.

[0166]

[0170] The present invention has been generally disclosed herein using positive language, and numerous embodiments and aspects have been described. The present invention also specifically includes embodiments in which certain subject matter, such as substances or materials, method steps and conditions, protocols, or procedures, are excluded in whole or in part. For example, in certain embodiments or aspects of the present invention, materials and / or method steps are excluded. Thus, even if the present invention is not generally expressed herein as not including, aspects that are not expressly excluded by the present invention are nevertheless disclosed herein.

[0167]

[0171] Several embodiments of the present invention have been described. Nevertheless, those skilled in the art can make various changes and modifications to the present invention to adapt it to various applications and conditions without departing from the spirit and scope of the present invention. Thus, the following examples are intended to illustrate but not to limit the scope of the invention as claimed. EXAMPLES

[0168] Example 1 Exemplary First Column, Cation Exchange or Affinity 1.1. Cation, Poros50 HS (or Poros XS from ThermoFisher). Preconditioning, equilibration, loading material, washing and elution 1.1.1. List of Exemplary Buffers 1.1.1.1. Buffers, pH, conductivity a) Prerequisite: Water for injection b) 3x Dilution Buffer: 60mM Na phosphate, pH 7.3 c) Equilibration buffer: 20mM Na phosphate, pH 7.3, 100mM NaCl d) Wash buffer 1 and 3: 20 mM Na phosphate, pH 7.3, 100 mM NaCl e) Wash buffer 2: 20 mM Na phosphate, pH 7.3, 100 mM NaCl, detergent (5 mM), e.g. Sarkosyl f) Elution buffer: 20 mM Na phosphate, pH 7.3, 300 mM NaCl (can be more than 300 mM, e.g. 300-400, e.g. 400 mM NaCl). g) Volume of elution buffer (e.g., collect eluate after a given pre-peak volume) or ODA 280 Increase in baseline ODA 280 The eluate is collected based on either a 1 mAu increase in h) Column stripping solution: high salt, e.g., 1M NaCl i) Sterilizing solution: 1M NaCl, 1N NaOH j) Stock solution: Approximately 22.5% ethanol (e.g. 18-22.5%) 1.1.2. Exemplary column preparation 1.1.2.1. Prerequisites Downflow (4CV) WFI 1.1.2.2. Equilibration Downflow (4CV) Equilibration Buffer 1.1.3. Exemplary Purification 1.1.3.1. Load material downflow (25 CV) sample 1.1.3.2. Wash 1 Downflow (4 CV) Wash 1 Buffer 1.1.3.3. Wash 2 Downflow (6 CV) Wash 2 Buffer 1.1.3.4. Wash 3 Downflow (8 CV) Buffer from Wash 3 1.1.4. Exemplary Block (Wash) Volume(s) Determined by Scale, Column Size 1.1.5. Binding capacity: 2.5-3x loading (Poros50 HS), with or without UF / DF. 1.1.6. Example flow rates: 150cm / h~300cm / h 1.1.7. Load capacity: 10L or more 1.1.8. Expected Recovery: 60-90% 1.2. Affinity, AVB-Sepharose HP 1.2.1. List of buffer solutions 1.2.1.1. Volume of each buffer solution (pH, conductivity) block 1.2.1.2. Exemplary Buffers / Solutions a) Equilibration buffer: 20mM Tris, pH8.0, 250mM NaCl b) Elution buffer: 10 mM Na acetate, pH 2.5, 250 mM NaCl c) Neutralization buffer: 1M TrisCl, pH 10.5 d) Disinfectant solution: PAB (120 mM phosphoric acid, 167 mM acetic acid, 2.2% benzyl alcohol) e) Stock solution: Approximately 22.5% ethanol (18-22.5%) 1.2.2. Exemplary flow rates: approximately 150 cm / h or greater 1.2.3. Expected recovery: 70-90% (AAV virion capsid), 15-70% (vector genome)

[0169] Example 2 Exemplary Second Column, Anion Exchange or Size Exclusion 1.3. Anion, Poros 50HQ (Poros XQ from ThermoFisher). Used for dual functions: preconditioning, equilibration, loading material, washing and elution (further purification and empty / solid vector ratio control) 1.3.1. List of Exemplary Buffers 1.3.1.1. Buffer solution, pH, conductivity, and volume of each block a) Prerequisite: Water for injection b) 3x (or 4x) dilution buffer: about 60 mM (or about 80 mM) Tris, pH 8.5 (e.g., after dilution, the loading material is in the range of 10-50 mM Tris, pH 8.0-8.5, 100 mM NaCl) c) Equilibration buffer (and wash 1 buffer): 20 mM Tris, pH 8.5, 100 mM NaCl d) Wash 2 Buffer (for removal of AAV empty capsids): 20 mM Tris, pH 8.5, 115 mM NaCl e) Elution buffer: 20 mM Tris, pH 8.5, NaCl≧120 mM, e.g., 200 mM NaCl f) Column stripping buffer: 1M NaCl g) Disinfectant solution: 1N NaOH h) Stock solution: Approximately 22.5% ethanol (e.g. 18-22.5%) 1.3.2. Exemplary column preparation 1.3.2.1. Prerequisites Downflow (5CV) WFI 1.3.2.2. Equilibration Downflow (4CV) Equilibration Buffer 1.3.3. Exemplary Purification 1.3.3.1. Load Material Downflow (50 CV) CEX Eluate and Dilution 1.3.3.2. Wash 1 Downflow (5 CV) Equilibration Buffer 1.3.3.3. Elution 1 (empty capsid removal) Downflow (3 CV) Elution buffer 1 1.3.3.4. Elution 2 (solid AAV vector recovery) Downflow (3 CV) Elution buffer 2 1.3.4. Example flow rates: 150cm / h~300cm / h 1.4. Size Exclusion, SEC (e.g., Superdex 200 preparative grade from GE healthcare), optional 1.4.1. List of Exemplary Buffers 1.4.1.1. Buffer solution, pH, conductivity, and volume of each block a) Prerequisite: Water for injection b) Equilibration wash and elution buffer: 10 mM Na phosphate, pH 7.2, 150-300 mM NaCl (higher NaCl, e.g., 300 mM, should improve recovery of AAV vector (vg). c) Volume of elution buffer (e.g., collect eluate after a given pre-peak volume) or ODA 280 Increase in baseline ODA 280 The eluate is collected based on either a 1 mAu increase in d) Disinfectant solution: 0.5N NaOH e) Stock solution: 22.5% ethanol (e.g. 18-22.5%) 1.4.2. Exemplary column preparation 1.4.2.1. Prerequisites Downflow (2CV) WFI 1.4.2.2. Equilibration Downflow (2CV) Equilibration Buffer (PBS300) 1.4.3. Exemplary Purification 1.4.3.1. Load material downflow (0.05CV) 1.4.3.2. Elution Downflow (1.5 CV) Equilibration Buffer (dPBS) 1.4.4. Exemplary loading capacity: ≦5% of column volume 1.4.5. Example flow rate: 45cm / h 1.4.6. Elution buffer volume or ODA 280 The eluate is collected based on either

[0170] Example 3 Exemplary third columns: size exclusion or anion exchange. The third column is optional and may not be required if an affinity column (e.g., AVB-Sepharose HP) is the first column. The third column used will also be based on the second column used (SEC>HQ, or HQ>SEC, etc.). 1.5. Size Exclusion, SEC (e.g., Superdex 200 preparative grade from GE healthcare), optional 1.5.1. Exemplary Buffer List 1.5.1.1. Buffer solution, pH, conductivity, and volume of each block a) Prerequisite: Water for injection b) Equilibration and running buffer: 10 mM Na-P, pH 7.2, 150-300 mM NaCl (higher NaCl, e.g., 300 mM, should improve recovery of AAV vector (vg). c) Volume of elution buffer (e.g., collect eluate after a given pre-peak volume) or ODA 280 Increase in baseline ODA 280 The eluate is collected based on either a 1 mAu increase in d) Disinfectant solution: 0.5N NaOH e) Stock solution: 22.5% ethanol 1.5.2. Exemplary column preparation 1.5.2.1. Prerequisites Downflow (2CV) WFI 1.5.2.2. Equilibration Downflow (2CV) Equilibration Buffer (PBS300) 1.5.3. Exemplary Purification 1.5.3.1. Load material downflow (0.05CV) 1.5.3.2. Elution Downflow (1.5 CV) Equilibration Buffer (dPBS) 1.5.4. Exemplary loading capacity: ≦5% of column volume 1.5.5. Example flow rate: 45cm / h 1.5.6. Elution buffer volume or ODA 280 The eluate is collected based on either 1.6. Anionic, Poros 50HQ (Poros XQ from ThermoFisher) for dual function (further rAAV purification and empty / solid rAAV vector ratio control). Preconditioning, equilibration, loading material, washing and elution 1.6.1. List of Exemplary Buffers 1.6.1.1. Buffer solution, pH, conductivity, and volume of each block a) Prerequisite: Water for injection b) 3x (or 4x) dilution buffer: 60 mM (or 80 mM) Tris, pH 8.5 (e.g., after dilution, the loading material will be in the range of 10-50 mM Tris, pH 8.0-8.5, 100 mM NaCl) c) Equilibration buffer (and wash 1 buffer): 20 mM Tris, pH 8.5, 100 mM NaCl d) Wash 2 Buffer (for removal of AAV empty capsids): 20 mM Tris, pH 8.5, 115 mM NaCl e) Elution buffer: 20 mM Tris, pH 8.5, NaCl≧120 mM, e.g., 200 mM NaCl f) Column stripping buffer: 1M NaCl g) Disinfectant solution: 1N NaOH h) Stock solution: 22.5% ethanol (e.g. 18-22.5%) 1.6.2. Exemplary column preparation 1.6.2.1. Prerequisites Downflow (5CV) WFI 1.6.2.2. Equilibration Downflow (4CV) Equilibration Buffer 1.6.3. Exemplary Purification 1.6.3.1. Load Material Downflow (50 CV) CEX Eluate and Dilution 1.6.3.2. Wash 1 Downflow (5 CV) Equilibration Buffer 1.6.3.3. Elution 1 (empty capsid removal) Downflow (3 CV) Elution buffer 1 1.6.3.4. Elution 2 (solid AAV vector recovery) Downflow (3 CV) Elution buffer 2 1.6.4. Example flow rates: 150cm / h~300cm / h

[0171] Example 4 Exemplary cell lysis and preparation prior to column purification. 1. Cell lysis, chemical or physical 1.1 Exemplary Chemical Dissolution Methods 1.6.5. Triton X-100 or equivalent non-ionic detergent i. Final concentration, 0.1%~1% ii. Incubation time, up to 1 hour iii. The impeller agitation speed is typically around 400-600 rpm (or higher). iv. The incubation temperature is about 25 to 37°C (e.g., 37°C). Benzonase i. Final concentration, ≥ 50U / mL, e.g., 100U / mL ii. The treatment can be simultaneous or sequential with the surfactant. iii. The MgCl2 concentration is 1.0-5.0 mM (e.g., 2 mM). 1.1.3. Additional Salts to Help Recover AAV Vectors During or After the Filtration Step II. The final concentration of NaCl is 200-400 mM (e.g., 300 mM). III. NaCl, other salt equivalents to NaCl can also be used 1.7. Exemplary Physical Lysis Methods (Microfluidization, Homogenization, etc.) 1.7.1. Working conditions (based on 10 L of adherent cell culture that can be scaled up to larger volumes) i. Pressure ≦ approx. 5,000 psi ii. Temperature, approximately 18~25℃ 1.7.2. Pretreatment and chase buffers i. Buffers, pH, conductivity It all depends on the first column loading conditions; in most cases, the DF buffer is the same or an equivalent buffer used for equilibration on the first column. If HS is the first column, PBS can be the common buffer.) For benzonase treatment, both pH and conductivity should be within the range of benzonase processing conditions (e.g., approximately pH 6.5-8.5, conductivity >15 mS / cm). Typical benzonase amounts for DNA digestion are 100-200 U / mL. 2 mM MgCl2 should be added for proper digestion. ii. Volume: The pretreated volume must be greater than the residual liquid volume of the system (in most cases, 3 times the residual liquid volume). : The tracking volume is 5-10% of the sample volume. 2. Tangential Flow Filtration (TFF, also known as UF / DF), optional 2.1. TFF with Hollow Fiber Cartridges 2.1.1. For 10L cell culture i. TFF is performed prior to cell lysis. ii. For 10L, use GE hollow fiber cartridge UFP-100-C-9A (1.2m 2 ) and other filters iii. Capacity. Preliminary data indicates that 20L should work with the same UFP-100-C-9A hollow fiber cartridge. iv. TFF before or after cell lysis. TFR before cell lysis. v. Maximum pressure. TMP. ≥ 5 psig before cell lysis (5-10 psi after cell lysis) vi. Buffer for pretreatment or tracing. (In case of cationic HS) For the first column, 20 mM phosphate buffer, pH 7-7.5 / 100-150 mM NaCl is a suitable buffer, (In case of anionic HQ) For the first column, 20 mM Tris, pH 7.5-8.5 / 100-150 mM NaCl is a suitable buffer. 2.1.2. Ultrafiltration to concentrate purified AAV vector intermediates before the SEC column i. TFF to concentrate the anion exchange (Poros 50 HQ) eluate to approximately 5% of the SEC column volume ii. Material: Poros 50HQ eluate 0.75 column volumes iii. Configuration: hollow fiber UFP-C-100-4MA (for a starting volume of 10 L) iv. Flushing volume (residual liquid volume): approx. 45 mL v. Equilibrium buffer: PBS300 (10mM Na-P, pH7.2 / 300mM NaCl) vi. Disinfection buffer: 1N NaOH vii. The same conditions as for adherent cell culture can be applied. viii. TFF can be performed either before or after cell lysis. 2.1.3. TFF for final formulation and removal of possible small molecular weight impurities i. Material: Poros50 HQ eluate (or SEC Vector Peak) ii. Configuration: Hollow fiber UFP-C-100-4MA (for a starting volume of 10 L) iii. Flushing volume (residual liquid volume): approx. 45mL iv. Equilibrium buffer: PBS180 (10mM Na-P, pH7.3 / 180mM NaCl) v. Diafiltration buffer: PBS180 (10mM Na-P, pH7.3 / 180mM NaCl) vi. Disinfection buffer: 1N NaOH vii. Target concentration: 1.0E+13vg / mL viii. Diafiltration: 12x UF target volume (5.0E12vg / mL) 2.2. Alternating Tangential Flow (ATF) or TFF with either spiral wound membrane modules or flat plate modules is an alternative to TFF with hollow fiber cartridges 3. Clarification and filtration 3.1. Depth Filter i. Filter options include Clarisolve 20MS (0.5-20 μM), Sartoclear DL series (10-1 μM) or Millistak COHC or DOHC (0.65-8 μM) ii. Capacity is 1L / 25cm 2 It could be. iii. Maximum flow rate, approximately 250mL / min / 25cm 2 (10mL / min / cm 2 ) iv. Maximum working pressure: approx. 32 psig v. Adjustment buffer: Approximately PBS 300 may be appropriate vi. Number of filtration steps (1 or 2, coarse then fine filter): Possibly a single step: coarse filter 3.2. Millipore SHC (0.5 / 0.2μM) or Sartopore 2 (0.45 / 0.2μM, Sartorius) i. Capacity approx. 500mL / 500cm 2 (0.2% Triton X-100 helps a little, 10-20% increase) ii. Maximum flow rate approximately 1000mL / min / 500cm 2 (2mL / min / cm 2 ) iii. Maximum working pressure: approx. 35 psig iv. An example for the adjustment buffer is PBS300 v. Final step Capacity may be limited (approximately 55maxi Sartopore 2 (1.8m 2 ) is required; if this is a second filtration step, the capacity may be increased 3.3. Exemplary 10 L-scale adherent cell culture i. Sartopore 2 MaxiCaps (1.2m 2) can be used for a 10 L culture volume ii. This same filter can be used for a 20L culture volume. iii. The conductivity of the pretreatment and chase buffers may be around 15-30 mS / cm to prevent possible interactions between the AAV vector and the membrane.

[0172] Example 5 1.0 Desired development criteria for 1.2 L and larger volumes of rAAV vector manufacturing based on processes developed for 500-600 ml rAAV harvest volumes 1.1 rAAV vector (e.g., LK03-FVIII) harvested from 1.2 L bioreactor suspension culture [Table 1] 1.2 Purification of rAAV Vectors from 1.2 L Bioreactor Suspension Cultures [Table 2]

[0173] Example 6 2.0 Harvest of rAAV Vectors (e.g., LK03-FVIII) from 1.2 L Bioreactor Suspension Cultures 2.1 Process flow diagram 2.1.1 Below is an exemplary process flow diagram for the recovery portion of the downstream processing of rAAV vectors. [Table 3] 2.2 Process flow description 2.2.1 The following is a description of an exemplary process flow for the recovery portion of the downstream processing of rAAV vectors: [Table 4] 2.3 Parameters changed during the development of the recovery process 2.3.1 The following parameters will be evaluated for development of the rAAV vector downstream processing recovery portion: [Table 5]

[0174] Example 7 As disclosed herein, the lysis methods, number of columns, and type of columns can be selected and used in various permutations. 4.0 Chromatography Process for 1.2L Bioreactor Suspension Culture 4.1 Parameters Varying During the rAAV Vector (e.g., LK03-FVIII) Chromatography Process Development Study 4.1.1 The following options will be developed for the chromatography portion of downstream processing of rAAV vectors: [Table 6] 4.2 Exemplary non-limiting column purification sequences (A-E): A) First column, cation (Poros 50HS) → second column, anion (e.g., Poros 50HQ) B) 1st column, cation (Poros 50HS) → 2nd column, anion (e.g., Poros 50HQ) → 3rd column, size exclusion (e.g., Superdex 200PG); C) First column, cation (Poros 50HS) → second column, size exclusion (e.g., Superdex 200PG) → third column, anion (e.g., Poros 50HQ) D) First column, affinity (AVB Sepharose HP) → Second column, anion (e.g., Poros 50HQ) → Third column (optional), size exclusion (e.g., Superdex 200PG) E) First column, affinity (AVB Sepharose HP) → Second column, size exclusion (e.g., Superdex 200PG) → Third column (optional), anion (e.g., Poros 50HQ)

[0175] Example 8 Representative AAV capsid (VP1) protein. AAV-SPK VP1 capsid (SEQ ID NO:1) 1 MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLD 61 KGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ 121 AKKRVLEPLGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDS 181 ESVPDPQPIGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRV 241 ITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQ 301 RLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSA 361 HQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFED 421 VPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNW 481 LPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSS 541 GVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNS 601 QGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADP 661 PTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTE 721 GTYSEPRPIGTRYLTRNL AAV-LK03 VP1 capsid (SEQ ID NO:2) MAADGYLPDWLEDNLSEGIREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRPL

[0176]

[0172] The present invention may have the following aspects. 1. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and / or cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) lysing the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) optionally filtering the nucleic acid-reduced lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the nucleic acid-reduced lysate from step (d), the clarified lysate from step (e), or the diluted clarified lysate produced in step (e) to cation exchange column chromatography to produce a column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities or other production / process related impurities, and optionally diluting the column eluate to produce a diluted column eluate; (g) subjecting the column eluate or the diluted column eluate produced in step (f) to anion exchange chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities or production / process related impurities, and optionally concentrating the second column eluate to produce a concentrated second column eluate; (h) subjecting the second column eluate or the concentrated second column eluate produced in step (g) to size exclusion column chromatography (SEC) to produce a third column eluate comprised of rAAV vector particles, thereby separating the rAAV vector particles from protein impurities or production / process related impurities, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles. The method includes: 2. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and / or cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) lysing the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) optionally filtering the nucleic acid-reduced lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the nucleic acid-reduced lysate from step (d), the clarified lysate from step (e), or the diluted and clarified lysate produced in step (e) to cation exchange column chromatography to produce a column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities or other production / process related impurities, and optionally concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography (SEC) to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities or other production / process related impurities, and optionally diluting the second column eluate to produce a concentrated second column eluate; (h) subjecting the second column eluate or the diluted second column eluate produced in step (g) to anion exchange chromatography to produce a third column eluate comprised of rAAV vector particles, thereby separating the rAAV vector particles from protein impurities production / process related impurities, and optionally diluting the third column eluate to produce a diluted third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles. The method includes: 3. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and / or cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) lysing the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) optionally filtering the nucleic acid-reduced lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the nucleic acid-reduced lysate from step (d), the clarified lysate from step (e), or the diluted clarified lysate produced in step (e) to cation exchange column chromatography to produce a column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities or other production / process related impurities, and optionally diluting the column eluate to produce a diluted column eluate; (g) subjecting the column eluate or the diluted column eluate produced in step (f) to anion exchange chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating the rAAV vector particles from production / process related impurities, and optionally concentrating the second column eluate to produce a concentrated second column eluate; (h) filtering the second column eluate or the concentrated second column eluate produced in step (g), thereby producing purified rAAV vector particles. The method includes: 4. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and / or cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) lysing the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) optionally filtering the nucleic acid-reduced lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the nucleic acid-reduced lysate from step (d), or the clarified lysate or diluted clarified lysate produced in step (e), to AAV affinity column chromatography to produce a column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities or other production / process related impurities, and optionally diluting the column eluate to produce a diluted column eluate; (g) subjecting the column eluate or the diluted column eluate produced in step (f) to anion exchange chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating the rAAV vector particles from protein impurities or other production / process related impurities, and optionally concentrating the second column eluate to produce a concentrated second column eluate; (h) optionally subjecting the second column eluate or the concentrated second column eluate produced in step (g) to size exclusion column chromatography (SEC) to produce a third column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities or other production / process related impurities, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the second column eluate or the diluted second column eluate produced in step (g), or filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles. The method includes: 5. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and / or cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) lysing the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) optionally filtering the nucleic acid-reduced lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the nucleic acid-reduced lysate from step (d), or the clarified lysate or diluted clarified lysate produced in step (e), to AAV affinity column chromatography to produce a column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities or other production / process related impurities, and optionally concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography (SEC) to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities or other production / process related impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) optionally subjecting the second column eluate produced in step (g) or the diluted second column eluate to anion exchange chromatography to produce a third column eluate comprised of rAAV vector particles, thereby separating the rAAV vector particles from protein impurities or other production / process related impurities, and optionally diluting the third column eluate to produce a diluted third column eluate; (i) filtering the second column eluate or the diluted second column eluate produced in step (g), or filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles. The method includes: 6. 6. The method according to any one of items 1 to 5, wherein said concentration of step (b) and / or step (f) and / or step (g) and / or step (h) is by UF / DF, optionally by Tangential Flow Filtration (TFF). 7. 7. The method according to any one of items 1 to 6, wherein the concentration in step (b) reduces the volume of the recovered cells and cell culture supernatant by about 2- to 20-fold. 8. 8. The method according to any one of items 1 to 7, wherein the concentration of step (f) and / or step (g) and / or step (h) reduces the volume of the column eluate by about 5 to 20 times. 9. 9. The method according to any one of items 1 to 8, wherein the dissolution of the harvest produced in step (a) or the concentrated harvest produced in step (b) is by physical or chemical means. 10. 10. The method of claim 9, wherein the physical means comprises microfluidization or homogenization. 11. 10. The method of claim 9, wherein the chemical means comprises a detergent, optionally a non-ionic detergent, such as Triton X-100, optionally at a concentration of between about 0.1 and 1.0%, inclusive. 12. 12. The method of any one of items 1 to 11, wherein step (d) comprises treating with a nuclease, thereby reducing contaminating nucleic acids. 13. 13. The method of claim 12, wherein the nuclease comprises benzonase. 14. 14. The method of any one of items 1 to 13, wherein the filtration of the clarified lysate or the diluted clarified lysate in step (e) is through a filter having a pore diameter of between about 0.1 and 10.0 microns, inclusive. 15. 15. The method according to any one of items 1 to 14, wherein the dilution of the clarified lysate in step (e) is with an aqueous phosphate buffer, an aqueous acetate buffer or an aqueous Tris buffer. 16. 16. The method according to any one of items 1 to 15, wherein the dilution of the column eluate of step (f) or the second column eluate of step (g) is with an aqueous phosphate buffer, an aqueous acetate buffer or an aqueous Tris buffer. 17. 17. The method according to item 15 or 16, wherein the aqueous phosphate buffer or acetate buffer has a pH between about 4.0 and 7.4, inclusive. 18. 17. The method according to item 15 or 16, wherein the aqueous Tris buffer solution has a pH of greater than 7.5, optionally between about 8.0 and 9.0 inclusive. 19. 19. The method of any one of items 1 to 18, wherein the rAAV vector particles resulting from step (i) are formulated with a surfactant to produce an AAV vector formulation. 20. 20. The method according to any one of items 1 to 19, wherein the anion exchange column chromatography of steps (f), (g) and / or (h) comprises polyethylene glycol (PEG) conditioned column chromatography. twenty one. 21. The method of item 20, wherein the anion exchange column chromatography of steps (g) and / or (h) comprises washing the column with a PEG solution prior to elution of the rAAV vector particles from the column. twenty two. 22. The method according to item 20 or 21, wherein the PEG has an average molecular weight in the range of about 1,000 to 80,000 g / mol, inclusive. twenty three. 23. The method according to any one of items 20 to 22, wherein the PEG is at a concentration of about 4% to about 10%, inclusive. twenty four. 24. The method of any one of items 1 to 23, wherein the anion exchange column in steps (g) and / or (h) comprises washing the column with an aqueous detergent solution prior to elution of the rAAV vector particles from the column. twenty five. 25. The method of any one of items 1 to 24, wherein the cation exchange column in step (f) comprises washing the column with a detergent solution prior to elution of the rAAV vector particles from the column. 26. 26. The method according to any one of items 21 to 25, wherein the PEG solution and / or the surfactant solution comprises a TrisCl / NaCl aqueous buffer solution, a phosphate / NaCl aqueous buffer solution, or an acetate / NaCl aqueous buffer solution. 27. 27. The method of claim 26, wherein the NaCl buffer comprises a range of between about 20 and 300 mM NaCl, inclusive, or between about 50 and 250 mM NaCl, inclusive. 28. 27. The method according to any one of items 24 to 26, wherein the surfactant comprises a cationic or anionic surfactant. 29. 29. The method according to any one of items 24 to 28, wherein the surfactant comprises a surfactant having a chain of 12 carbons. 30. 30. The method according to any one of items 24 to 29, wherein the surfactant comprises dodecyltrimethylammonium chloride (DTAC) or sarkosyl. 31. 31. The method of any one of items 1 to 30, wherein the rAAV vector particles are eluted from the anion exchange column of steps (f), (g) and / or (h) with an aqueous TrisCl / NaCl buffer. 32. 32. The method of claim 31, wherein the TrisCl / NaCl buffer comprises 100 to 400 mM NaCl, inclusive, and optionally has a pH in the range of about 7.5 to about 9.0, inclusive. 33. 33. The method according to any one of items 1 to 32, wherein the anion exchange column in steps (f), (g) and / or (h) is washed with an aqueous TrisCl / NaCl buffer. 34. 34. The method of claim 33, wherein the NaCl in the aqueous TrisCl / NaCl buffer is in the range of about 75 to 125 mM NaCl, inclusive. 35. 34. The method according to any one of items 31 to 33, wherein the aqueous TrisCl / NaCl buffer solution has a pH of about 7.5 to about 9.0, inclusive. 36. 40. The method of any one of items 1 to 39, wherein the anion exchange column of steps (f), (g) and / or (h) is washed one or more times to reduce the amount of AAV empty capsids in the second or third column eluate. 37. 37. The method of claim 33 or 36, wherein washing of the anion exchange column removes AAV empty capsids from the column prior to and / or in place of rAAV removal, thereby reducing the amount of AAV empty capsids in the second or third column eluate. 38. 37. The method of claim 33 or 36, wherein washing the anion exchange column removes at least about 50% of total AAV empty capsids from the column prior to rAAV removal and / or in place of rAAV, thereby reducing the amount of AAV empty capsids in the second or third column eluate by about 50%. 39. 39. The method according to any one of items 33 or 36 to 38, wherein the NaCl in the aqueous TrisCl / NaCl buffer is in the range of about 110 to 120 mM NaCl, inclusive. 40. 41. The method of any one of items 33 to 40, wherein the ratio and / or amount of the eluted rAAV vector particles and AAV empty capsids is controlled by the wash buffer. 41. 42. The method of any one of items 1 to 41, wherein the rAAV vector particles are eluted from the cation exchange column in step (f) into an aqueous phosphate / NaCl buffer or an aqueous acetate / NaCl buffer. 42. 42. The method of claim 41, wherein the aqueous phosphate / NaCl or acetate / NaCl buffer solution has a concentration in the range of about 125 to 500 mM NaCl, inclusive. 43. Item 42. The method according to item 41, wherein the aqueous phosphate / NaCl or acetate / NaCl buffer has a pH in the range of about 5.5 to about 7.5, inclusive. 44. 44. The method according to any one of the preceding claims, wherein the anion exchange column of steps (f), (g) and / or (h) comprises a quaternary ammonium functional group, such as a quaternized polyethyleneimine. 45. 45. The method according to any one of items 1 to 44, wherein the size exclusion column (SEC) of step (g) and / or (h) has a separation / fractionation range (molecular weight) of about 10,000 to about 600,000, inclusive. 46. 46. ​​The method according to any one of items 1 to 45, wherein the cation exchange column in step (f) contains a functional group such as sulfonic acid or sulfopropyl. 47. 47. The method according to any one of items 3 to 46, wherein the AAV affinity column comprises a protein or ligand that binds to a capsid protein of AAV. 48. 48. The method of claim 47, wherein the protein comprises an antibody that binds to a capsid protein of AAV. 49. 49. The method of claim 48, wherein the antibody that binds to the capsid protein of AAV comprises a single chain antibody from a llama (camelidae). 50. 50. The method according to any one of items 1 to 49, wherein the step of cesium chloride gradient ultracentrifugation is excluded. 51. 51. The method of any one of items 1 to 50, wherein the rAAV vector particle comprises a transgene encoding a nucleic acid selected from the group consisting of an siRNA, an antisense molecule, an miRNA, a ribozyme, and an shRNA. 52. The rAAV vector particle is capable of expressing any of the following hormones: insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor alpha (TGFα), platelet derived growth factor (PDGF), erythropoietin (ERG ... 51. The method of any one of items 1 to 50, comprising a transgene encoding a gene product selected from the group consisting of: insulin-like growth factors I and II (IGF-I and IGF-II), TGFβ, activin, inhibin, bone morphogenetic proteins (BMPs), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, and tyrosine hydroxylase. 53. 51. The method of any one of items 1 to 50, wherein the rAAV vector particle comprises a transgene encoding a gene product selected from the group consisting of thrombopoietin (TPO), interleukins (IL1-IL-17), monocyte chemotactic protein, leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor, Fas ligand, tumor necrosis factors alpha and beta, interferons alpha, beta, and gamma, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD, and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I and class II MHC molecules. 54. The rAAV vector particle is capable of expressing any one of the following: carbamoyl synthetase I, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor V, factor VIII, factor IX, cystathione beta-synthase, branched-chain keto acid decarboxylase, albumin, isovaleryl-coA dehydrogenase 51. The method of any one of items 1 to 50, comprising a transgene encoding a protein useful for correction of an inborn error of metabolism selected from the group consisting of: propionyl CoA carboxylase, methylmalonyl CoA mutase, glutaryl CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, RPE65, H-protein, T-protein, cystic fibrosis transmembrane conductance regulator (CFTR) sequence, and dystrophin cDNA sequence. 55. 55. The method of any one of items 1 to 54, wherein the rAAV vector particle comprises a transgene encoding factor VIII or factor IX. 56. 56. The method of any one of items 1 to 55, wherein about 50-90% of total rAAV vector particles are recovered from the harvest produced in step (a) or the concentrated harvest produced in step (b). 57. 57. The method of any one of items 1 to 56, which produces rAAV vector particles having a higher purity than rAAV vector particles produced or purified by a single AAV affinity column purification. 58. 58. The method according to any one of items 1 to 57, wherein steps (c) and (d) are carried out substantially simultaneously. 59. 59. The method according to any one of items 1 to 58, wherein after step (c) but before step (f), NaCl is adjusted to be in the range of about 100 to 400 mM NaCl, inclusive, or in the range of about 140 to 300 mM NaCl, inclusive. 60. 60. The method of any one of items 1 to 59, wherein the rAAV vector particle is derived from an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10 and Rh74. 61. 61. The method of any one of items 1 to 60, wherein the rAAV vector particle comprises a capsid sequence having 70% or greater identity to the capsid sequence of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, Rh74, SEQ ID NO: 1, or SEQ ID NO: 2. 62. 62. The method of any one of items 1 to 61, wherein the rAAV vector particle comprises an ITR sequence having 70% or more identity to an ITR sequence of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, or Rh74. 63. 63. The method of any one of items 1 to 62, wherein the cells comprise suspension or adherent cells. 64. 64. The method of any one of items 1 to 63, wherein the cell comprises a mammalian cell. 65. 65. The method of any one of items 1 to 64, wherein the cells comprise HEK-293 cells. 66. 66. The method according to any one of items 1 to 65, carried out by any one or more columns, conditions, concentrations, molarity, volumes, capacities, flow rates, pressures, materials, temperatures, pH, or steps according to any one of examples 1 to 3. 67. 67. The method according to any one of items 1 to 66, wherein the cell lysis and / or preparation prior to column purification is carried out by any one or more of the conditions, concentrations, molarity, volumes, capacities, flow rates, pressures, materials, temperatures, pH, or steps described in Example 4.

[0177] [Related Applications]

[0001] This patent application claims the benefit of U.S. Application No. 62 / 527,633, filed June 30, 2017; U.S. Application No. 62 / 531,744, filed July 12, 2017; and U.S. Application No. 62 / 567,905, filed October 4, 2017. The entire contents of the foregoing applications are incorporated herein by reference, including all text, tables, figures, and sequences.

Claims

[Claim 1] 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and / or cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) dissolving the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) optionally filtering the nucleic acid-depleted lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the nucleic acid-reduced lysate from step (d), the clarified lysate from step (e), or the diluted clarified lysate produced in step (e) to cation exchange column chromatography to produce a column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities or other production / process related impurities, and optionally diluting the column eluate to produce a diluted column eluate; (g) subjecting the column eluate or the diluted column eluate produced in step (f) to anion exchange chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities or production / process related impurities, and optionally concentrating the second column eluate to produce a concentrated second column eluate; (h) subjecting the second column eluate or the concentrated second column eluate produced in step (g) to size exclusion column chromatography (SEC) to produce a third column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities or production / process related impurities, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles; The method includes:

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