AAV Vector Column Purification Method

Abstract

Described herein are methods for purifying rAAV particles, and in particular for purifying complete rAAV particles from rAAV preparations that contain both complete and incomplete rAAV particles.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 209,680, filed June 11, 2021, and U.S. Provisional Patent Application No. 63 / 366,094, filed June 9, 2022, the disclosures of which are incorporated herein by reference.

[0002] This application relates to methods for purifying recombinant adeno-associated virus (rAAV) particles. More particularly, this application relates to methods for purifying complete rAAV particles from preparations containing both complete and non-complete rAAV particles. [Background technology]

[0003] Gene delivery is a promising method for treating acquired and inherited diseases. A number of virus-based systems for gene transfer have been reported, including adeno-associated virus (AAV)-based systems. AAV is a helper-dependent DNA parvovirus belonging to the Dependovirus genus, which requires helper virus functions, such as adenovirus, herpesvirus, or vaccinia, to produce a productive infection. AAV has a broad host range and can replicate in the cells of any species in the presence of a suitable helper virus. AAV has not been associated with any human or animal disease and is not believed to have any deleterious effects on the biology of the host cell upon integration. AAV vectors can be engineered to carry a heterologous nucleic acid sequence of interest (e.g., a selected gene encoding a therapeutic protein, an antisense molecule, a ribozyme, an miRNA, or other nucleic acid) by deleting an internal portion of the AAV genome in whole or in part and inserting the heterologous nucleic acid sequence of interest between the inverted terminal repeats (ITRs). The ITRs remain functional in such vectors and allow for the replication and packaging of recombinant adeno-associated viruses (rAAV) containing the heterologous nucleic acid sequence of interest. The heterologous nucleic acid sequence is also typically linked to a promoter sequence capable of driving nucleic acid expression in the patient's target cells. Termination signals, such as polyadenylation sites, may also be included in the vector. The rAAV genomic DNA is packaged into the viral capsid, a protein shell that contains a mixture of three capsid proteins (VP1, VP2, and VP3) arranged in icosahedral symmetry. Recombinant adeno-associated viruses (rAAV) have shown great therapeutic promise in several early-phase clinical trials by multiple groups. Development of this new class of biologics toward approval will involve improved vector characterization and quality control methods, including a better understanding of how vector design and manufacturing process parameters affect the impurity profile of clinical-grade vectors.

[0004] One of the challenges with rAAV production is the formation of "incomplete" rAAV particles that do not contain complete genetic material. Incomplete rAAV particles, as used herein, refers to a set of particles or variants, including "empty" and "partial" particles. "Partial" particles, as used herein, refer to rAAV particles that have some genetic material, but not the complete genetic material of complete particles. Doubts have arisen about the impact of incomplete particles (including empty and partial particles) on the clinical safety and efficacy of rAAV-mediated gene expression, necessitating the development of purification methods to remove or separate these species from complete particles. Given the structural similarity between these types of rAAV particles, it remains difficult to develop a robust and scalable purification method to efficiently separate incomplete and complete AAV particles. rAAV particles differ in the presence and length of single-stranded DNA genomes in rAAV. Various techniques have been developed to separate complete rAAV particles from incomplete particles. However, these techniques often result in low purity and / or low yield of designed complete rAAV particles.

[0005] Thus, there remains a need for the development of new systems and methods for purifying complete rAAV particles from incomplete particles in high purity and / or high yield, including purification from empty or partial particles. Summary of the Invention

[0006] The present application relates to methods and systems for purifying complete recombinant adeno-associated virus (rAAV) particles from rAAV preparations that contain complete rAAV particles as well as incomplete particles, which may include empty and / or partial particles, using column chromatography techniques. In one basic aspect, the present application provides a method for purifying complete recombinant adeno-associated virus (rAAV) particles, comprising: (a) providing an rAAV preparation comprising complete and incomplete rAAV particles; (b) loading the rAAV preparation in a loading buffer onto a column containing a chromatography medium, wherein complete rAAV particles have a higher binding affinity for the chromatography medium than incomplete particles; and (c) eluting the intact rAAV particles bound to the chromatography medium with an elution buffer to obtain a purified preparation. The present invention relates to a method comprising the steps of:

[0007] In some embodiments, the imperfect particles include empty particles. In some embodiments, an incomplete particle comprises a partial particle. In some embodiments, incomplete particles include empty particles and partial particles. In some embodiments, imperfect particles do not bind to the chromatography medium and pass through the column. In some embodiments, the subparticles do not bind to the chromatography medium and pass through the column. In some embodiments, the amount of complete and empty rAAV particles applied to the column exceeds the binding capacity of the chromatography medium, and empty particles bound to the chromatography medium are displaced by the complete rAAV particles into the loading flow-through from the column. In some embodiments, the amount of complete and incomplete rAAV particles applied to the column exceeds the binding capacity of the chromatography medium, and empty particles bound to the chromatography medium are displaced by the partial and complete rAAV particles into the loading flow-through from the column.

[0008] In some embodiments, the amount of complete and incomplete rAAV particles applied to the column exceeds the binding capacity of the chromatography medium, and empty and partial particles bound to the chromatography medium are displaced by complete rAAV particles into the loading flow-through from the column. In some embodiments, the chromatography medium is an ion exchange column chromatography medium, preferably an anion exchange chromatography medium. In some embodiments, the column chromatography media is selected from the group consisting of Poros50HQ, Poros50D, Poros50PI, Capto ImpRes Q, and Poros XQ, preferably Poros XQ. In some embodiments, the column chromatography media is a monolith, such as a CIMmultus™ QA monolithic column. In some embodiments, the loading buffer comprises at least one buffer selected from the group consisting of Tris, Bis-tris, Bis-tris propane, Tris acetate, ethanolamine, and phosphate. In some embodiments, the loading buffer contains K(I), Li(I), Ca(II), Mg(II), Cu(II), Ba(II), Co(II), Ni(II), Mn(II), Zn(II), Cd(II), Pb(II), Fe(III), Fe(II), Na(I), and NH4 + The anion component of the salt is not critical. In some embodiments, the loading buffer has a pH of about 6-10, preferably 8-9.

[0009] In some embodiments, the elution buffer comprises at least one buffer selected from the group consisting of Tris, Bis-tris, Bis-tris propane, Tris acetate, ethanolamine, and phosphate. In some embodiments, the elution buffer contains K(I), Li(I), Ca(II), Mg(II), Cu(II), Ba(II), Co(II), Ni(II), Mn(II), Zn(II), Cd(II), Pb(II), Fe(III), Fe(II), Na(I), and NH4 + The anion component of the salt is not critical. In some embodiments, the elution buffer has a pH of about 6-10, preferably 8-9.

[0010] In some embodiments, the yield of purified intact rAAV particles is 70% or greater, preferably 80% or greater, more preferably 90% or greater, and most preferably 95% or greater. In some embodiments, the purified preparation is substantially free of incomplete particles. In other embodiments, the purified preparation comprises an increased ratio of complete rAAV particles to incomplete particles than the rAAV preparation. Preferably, the ratio of complete rAAV particles to incomplete particles in the purified preparation is 9:1 or more, e.g., 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, or 50:1 or more, or any ratio therebetween, more preferably 49:1 or more. In some embodiments, the complete rAAV particle comprises a transgene encoding a polypeptide or a nucleic acid selected from the group consisting of an siRNA, an antisense molecule, an miRNA, a ribozyme, and an shRNA. In some embodiments, the rAAV particles comprise a capsid derived from an AAV serotype. In another embodiment, the rAAV particles comprise capsids derived from genetically engineered capsids capable of binding to an ion exchange chromatography medium.

[0011] In another basic aspect, the present application provides a method for purifying complete recombinant adeno-associated virus (rAAV) particles, comprising: (a) providing an rAAV preparation comprising complete and incomplete rAAV particles; (b) loading a first batch of the rAAV preparation in a loading buffer onto a first column comprising a first chromatography medium, wherein the complete rAAV particles have a higher binding affinity for the first chromatography medium than the incomplete particles, and the amount of complete rAAV particles and incomplete particles applied to the first column exceeds the binding capacity of the first chromatography medium, and the incomplete particles bound to the first chromatography medium are displaced by the complete rAAV particles into a first loading flow-through from the first column; (c) loading the first loading flow-through onto a second column comprising a second chromatographic medium to obtain a partially loaded (i.e., not fully saturated) second column, preferably the second chromatographic medium being of the same type as the first chromatographic medium; (d) optionally washing the first column with a wash buffer to obtain a washed first column; (e) after step (c), or after washing step (d) if performed, bypassing the second column and eluting the complete rAAV particles bound to the first chromatography medium with an elution buffer to obtain a first eluate from the first column and an eluted first column, the first eluate comprising an increased ratio of complete to incomplete rAAV particles; (f) loading a second batch of the rAAV preparation in loading buffer onto the partially loaded second column, wherein the amount of complete and incomplete rAAV particles applied to the second column exceeds the binding capacity of the second chromatography medium, and the incomplete particles bound to the second chromatography medium are displaced by the complete rAAV particles into the second loading flow-through from the second column; (g) loading the second loading flow-through onto the eluted first column to obtain a partially loaded first column; (h) optionally washing the second column with a wash buffer to obtain a washed second column; (i) after step (g), or after washing step (h), if performed, bypassing the first column and eluting the complete rAAV particles bound to the second chromatography medium with an elution buffer to obtain a second eluate and an eluted second column, the second eluate comprising an increased ratio of complete to incomplete rAAV particles; and (j) combining the first eluate and the second eluate to produce a purified preparation. The present invention relates to a method comprising the steps of:

[0012] In some embodiments, the imperfect particles include empty particles. In some embodiments, an incomplete particle comprises a partial particle. In some embodiments, incomplete particles include both empty particles and partial particles. In some embodiments, when the incomplete particles include both empty and partial particles, the second eluate of step (i) contains an increased ratio of complete and partial rAAV particles to empty rAAV particles. In some embodiments, more than two columns can be used, with two being the minimum. In a three-column setup, complete particles are enriched in the first column, partial particles, if present, are enriched in the second column, and empty particles are enriched in the third column. In some embodiments, if impurities or aggregates are present in the preparation, the impurities or aggregates may bind to the first column with higher affinity than complete particles. In such embodiments, complete particles will be enriched in the subsequent columns. In some embodiments, the first chromatographic medium and / or the second chromatographic medium is an ion exchange column chromatographic medium, preferably an anion exchange column chromatographic medium.

[0013] In some embodiments, the second column is partially loaded after loading the first flow-through. In some embodiments, steps (b) through (i) are performed in one cycle or multiple cycles. In some embodiments, prior to the next loading cycle, the eluted column may be subjected to subsequent steps that are beneficial or necessary to maintain a consistent column binding capacity throughout the cycle, including, but not limited to, stripping the column, cleaning and / or sanitizing the column, and / or re-equilibrating the column. In some embodiments, the first or second column chromatography media is selected from the group consisting of Poros50HQ, Poros50D, Poros50PI, Capto ImpRes Q, and Poros XQ, preferably Poros XQ. In some embodiments, the column chromatography media is a monolith, such as a CIMmultus™ QA monolithic column.

[0014] In some embodiments, the loading buffer comprises at least one buffer selected from the group consisting of Tris, Bis-tris, Bis-tris propane, Tris acetate, ethanolamine, and phosphate. In some embodiments, the loading buffer contains K(I), Li(I), Ca(II), Mg(II), Cu(II), Ba(II), Co(II), Ni(II), Mn(II), Zn(II), Cd(II), Pb(II), Fe(III), Fe(II), Na(I), and NH4 + The anion component of the salt is not critical. In some embodiments, the loading buffer has a pH of about 6-10, preferably 8-9. In some embodiments, the loading buffer comprises at least one detergent. In some embodiments, the detergent in the loading buffer is selected from the group consisting of poloxamer 188, polysorbate 80, polysorbate 20, NP-40, Triton X-100, and Triton CG-110. In some embodiments, the concentration of the detergent in the loading buffer is between 0.0001% and 0.1%.

[0015] In some embodiments, the elution buffer comprises at least one buffer selected from the group consisting of Tris, Bis-tris, Bis-tris propane, Tris acetate, ethanolamine, and phosphate. In some embodiments, the elution buffer contains K(I), Li(I), Ca(II), Mg(II), Cu(II), Ba(II), Co(II), Ni(II), Mn(II), Zn(II), Cd(II), Pb(II), Fe(III), Fe(II), Na(I), and NH4 + The anion component of the salt is not critical. In some embodiments, the elution buffer has a pH of about 6-10, preferably 8-9. In some embodiments, the elution buffer comprises at least one detergent. In some embodiments, the detergent in the elution buffer is selected from the group consisting of poloxamer 188, polysorbate 80, polysorbate 20, NP-40, Triton X-100, and Triton CG-110.

[0016] In some embodiments, the concentration of the detergent in the elution buffer is between 0.0001% and 0.1%. In some embodiments, the yield of purified intact rAAV particles is 70% or greater, preferably 80% or greater, more preferably 90% or greater, and most preferably 95% or greater. In some embodiments, the purified preparation is substantially free of incomplete particles. In other embodiments, the purified preparation comprises an increased ratio of complete rAAV particles to incomplete particles than the rAAV preparation. Preferably, the ratio of complete particles to incomplete particles in the purified preparation is 9:1 or more, e.g., 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, or 50:1 or more, or any ratio therebetween, more preferably 49:1 or more. In some embodiments, the complete rAAV particle comprises a transgene encoding a polypeptide, a nucleic acid encoding a protein or transcribed into a transcript of interest, or a nucleic acid selected from the group consisting of an siRNA, an antisense molecule, an miRNA, a ribozyme, and an shRNA.

[0017] In some embodiments, the rAAV particles are prepared using the methods described in Pulicherla et al., Mol. Ther., 19(6):1070-1078 (2011) (which describes, among other things, AAV9 variants, including AAV9.47); U.S. Pat. Nos. 7,906,111 (which describes, among other things, AAV9(hu14)); 10,532,111 (which describes, among other things, NP59); 10,738,087 (which describes, among other things, Anc-80); 9,169,299 (which describes "LK03"); 9,840,719 (which describes "RHM4-1"); 7,749,492; 7,588,772 (which describes "DJ" and "DJ8"); and 9,587,282. The capsids include those derived from one or more AAVs selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, Rh8, Rh10, Rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ, DJ8, NP59, Anc-80, and variants thereof, including variants of the AAV capsids set forth in the specification, and in WO 2012 / 145601, WO 2013 / 158879, WO 2015 / 013313, WO 2018 / 156654, and U.S. Patent Application Publication No. 2013 / 0059732. All of these documents are incorporated herein by reference in their entirety.

[0018] In another basic aspect, the present application provides a method for purifying complete recombinant adeno-associated virus (rAAV) particles, comprising: (a) providing an rAAV preparation comprising complete and incomplete rAAV particles; (b) loading the rAAV preparation in a loading buffer onto a column containing a chromatography medium, the loading buffer containing CaCl2, and wherein the intact rAAV particles bind to the chromatography medium; and (c) eluting the intact rAAV particles bound to the chromatography medium with an elution buffer to obtain a purified preparation, the elution buffer optionally containing CaCl. The present invention relates to a method comprising the steps of:

[0019] In some embodiments, the chromatography medium is an ion exchange column chromatography medium, preferably an anion exchange chromatography medium. In some embodiments, the column chromatography media is selected from the group consisting of Poros50HQ, Poros50D, Poros50PI, Capto ImpRes Q, and Poros XQ, preferably Poros XQ. In some embodiments, the loading buffer comprises at least one detergent. In some embodiments, the detergent in the loading buffer is selected from the group consisting of poloxamer 188, polysorbate 80, polysorbate 20, NP-40, Triton X-100, and Triton CG-110. In some embodiments, the concentration of the detergent in the loading buffer is between 0.0001% and 0.1%.

[0020] In some embodiments, the loading buffer comprises at least one buffer selected from the group consisting of Tris, Bis-tris, Bis-tris propane, Tris acetate, ethanolamine, and phosphate. In some embodiments, the elution buffer comprises at least one buffer selected from the group consisting of Tris, Bis-Tris, Bis-Tris propane, Tris acetate, ethanolamine, and phosphate. In some embodiments, the loading buffer contains K(I), Li(I), Ca(II), Mg(II), Cu(II), Ba(II), Co(II), Ni(II), Mn(II), Zn(II), Cd(II), Pb(II), Fe(III), Fe(II), Na(I), and NH4 + The compound comprises at least one salt of a cation selected from the group consisting of: In some embodiments, the elution buffer contains K(I), Li(I), Ca(II), Mg(II), Cu(II), Ba(II), Co(II), Ni(II), Mn(II), Zn(II), Cd(II), Pb(II), Fe(III), Fe(II), Na(I), and NH4 + The compound comprises at least one salt of a cation selected from the group consisting of:

[0021] In some embodiments, the loading buffer contains about 0-10 mM CaCl2, preferably 0.1-2.5 mM CaCl2. In some embodiments, the elution buffer comprises about 0.1-20 mM CaCl2, preferably 5-10 mM CaCl2. In some embodiments, the loading buffer comprises about 0-100 mM LiCl, preferably 0-75 mM LiCl. In some embodiments, the elution buffer comprises about 0-200 mM LiCl, preferably 0-150 mM LiCl. In some embodiments, the loading buffer comprises about 0-10 mM CuCl2, preferably 0.1-3 mM CuCl2. In some embodiments, the elution buffer comprises about 0-10 mM CuCl2, preferably 0-3 mM CuCl2. In some embodiments, the loading buffer further comprises NaCl and / or MgCl 2 .

[0022] In some embodiments, the loading buffer has a pH of about 6-10, preferably 8-9. In some embodiments, the elution buffer further comprises NaCl and / or MgCl 2 . In some embodiments, the elution buffer comprises at least one detergent. In some embodiments, the detergent in the elution buffer is selected from the group consisting of poloxamer 188, polysorbate 80, polysorbate 20, NP-40, Triton X-100, and Triton CG-110. In some embodiments, the concentration of the detergent in the elution buffer is between 0.0001% and 0.1%. In some embodiments, the elution buffer has a pH of about 6-10, preferably 8-9.

[0023] In some embodiments, the yield of purified intact rAAV particles is 70% or greater, preferably 80% or greater, more preferably 90% or greater, and most preferably 95% or greater. In some embodiments, the purified preparation is substantially free of incomplete particles. In other embodiments, the purified preparation comprises an increased ratio of complete rAAV particles to incomplete particles than the rAAV preparation. Preferably, the ratio of complete particles to incomplete particles in the purified preparation is 9:1 or more, e.g., 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, or 50:1 or more, or any ratio therebetween, more preferably 49:1 or more. In some embodiments, the complete rAAV particle comprises a transgene encoding a polypeptide or a nucleic acid selected from the group consisting of an siRNA, an antisense molecule, an miRNA, a ribozyme, and an shRNA.

[0024] In some embodiments, the rAAV particles are prepared using the methods described in Pulicherla et al., Mol. Ther., 19(6):1070-1078 (2011) (which describes, among other things, AAV9 variants, including AAV9.47), U.S. Pat. Nos. 7,906,111 (which describes, among other things, AAV9(hu14)), 10,532,111 (which describes, among other things, NP59), 10,738,087 (which describes, among other things, Anc-80), 9,169,299 (which describes "LK03"), 9,840,719 (which describes "RHM4-1"), 7,749,492, 7,588,772 (which describes "DJ" and "DJ8"), 9,5 and variants thereof, including variants of the AAV capsid set forth in WO 2012 / 145601, WO 2013 / 158879, WO 2015 / 013313, WO 2018 / 156654, and U.S. Patent Publication No. 2013 / 0059732. All of these documents are incorporated herein by reference in their entirety.

[0025] In one basic aspect, the present application provides a method for purifying partial rAAV particles, comprising: (a) providing an incomplete rAAV preparation comprising empty and partial particles; (b) loading the incomplete rAAV preparation in a loading buffer onto a column containing a chromatography medium, wherein the partial rAAV particles have a higher binding affinity for the chromatography medium than the empty particles; and (c) eluting the partial rAAV particles bound to the chromatography medium with an elution buffer to obtain a purified preparation. The present invention relates to a method comprising the steps of:

[0026] In some embodiments, the amount of partial rAAV particles and empty rAAV particles applied to the column exceeds the binding capacity of the chromatography medium, and empty particles bound to the chromatography medium are displaced by the partial rAAV particles into the loading flow-through from the column. In some embodiments, the chromatography medium is an ion exchange column chromatography medium, preferably an anion exchange chromatography medium. In some embodiments, the column chromatography media is selected from the group consisting of Poros50HQ, Poros50D, Poros50PI, Capto ImpRes Q, and Poros XQ, preferably Poros XQ. In some embodiments, the column chromatography media is a monolith, such as a CIMmultus™ QA monolithic column.

[0027] In some embodiments, the loading buffer comprises at least one buffer selected from the group consisting of Tris, Bis-tris, Bis-tris propane, Tris acetate, ethanolamine, and phosphate. In some embodiments, the loading buffer contains K(I), Li(I), Ca(II), Mg(II), Cu(II), Ba(II), Co(II), Ni(II), Mn(II), Zn(II), Cd(II), Pb(II), Fe(III), Fe(II), Na(I), and NH4 + The anion component of the salt is not critical. In some embodiments, the loading buffer has a pH of about 6-10, preferably 8-9. In some embodiments, the loading buffer comprises at least one detergent. In some embodiments, the detergent in the loading buffer is selected from the group consisting of poloxamer 188, polysorbate 80, polysorbate 20, NP-40, Triton X-100, and Triton CG-110. In some embodiments, the concentration of the detergent in the loading buffer is between 0.0001% and 0.1%.

[0028] In some embodiments, the elution buffer comprises at least one buffer selected from the group consisting of Tris, Bis-tris, Bis-tris propane, Tris acetate, ethanolamine, and phosphate. In some embodiments, the elution buffer contains K(I), Li(I), Ca(II), Mg(II), Cu(II), Ba(II), Co(II), Ni(II), Mn(II), Zn(II), Cd(II), Pb(II), Fe(III), Fe(II), Na(I), and NH4 + The anion component of the salt is not critical. In some embodiments, the elution buffer has a pH of about 6-10, preferably 8-9. In some embodiments, the elution buffer comprises at least one detergent. In some embodiments, the detergent in the elution buffer is selected from the group consisting of poloxamer 188, polysorbate 80, polysorbate 20, NP-40, Triton X-100, and Triton CG-110.

[0029] In some embodiments, the concentration of the detergent in the elution buffer is between 0.0001% and 0.1%. In some embodiments, the purified preparation is substantially free of empty particles. In other embodiments, the purified preparation comprises an increased ratio of partial rAAV particles to empty particles than the rAAV preparation. Preferably, the ratio of partial rAAV particles to empty particles in the purified preparation is 9:1 or greater, e.g., 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, or 50:1 or greater, or any ratio therebetween, more preferably 49:1 or greater. In some embodiments, the rAAV particles comprise a capsid derived from an AAV serotype. In another embodiment, the rAAV particles comprise capsids derived from genetically engineered capsids capable of binding to an ion exchange chromatography medium.

[0030] In some embodiments, the rAAV particles are prepared using the methods described in Pulicherla et al., Mol. Ther., 19(6):1070-1078 (2011) (which describes, among other things, AAV9 variants, including AAV9.47), U.S. Pat. Nos. 7,906,111 (which describes, among other things, AAV9(hu14)), 10,532,111 (which describes, among other things, NP59), 10,738,087 (which describes, among other things, Anc-80), 9,169,299 (which describes "LK03"), 9,840,719 (which describes "RHM4-1"), 7,749,492, 7,588,772 (which describes "DJ" and "DJ8"), 9,5 and variants thereof, including variants of the AAV capsid set forth in WO 2012 / 145601, WO 2013 / 158879, WO 2015 / 013313, WO 2018 / 156654, and U.S. Patent Publication No. 2013 / 0059732. All of these documents are incorporated herein by reference in their entirety.

[0031] In one basic aspect, the present application provides a method for purifying empty rAAV particles, comprising: (a) providing an rAAV preparation comprising empty rAAV particles and at least one of complete rAAV particles and partial rAAV particles; (b) loading the rAAV preparation in a loading buffer onto a column containing a chromatography medium, wherein the empty rAAV particles have a higher binding affinity for the chromatography medium than the full or partial particles, and the amount of the empty rAAV particles and at least one of the full and partial particles applied to the column exceeds the binding capacity of the chromatography medium, and at least one of the full and partial particles bound to the chromatography medium is displaced by the empty rAAV particles into the flow-through from the column; and (c) eluting the empty rAAV particles bound to the chromatography medium with an elution buffer to obtain a purified preparation. The present invention relates to a method comprising the steps of:

[0032] In some embodiments, the amount of empty rAAV particles and full and partial particles applied to the column exceeds the binding capacity of the chromatography medium, and at least one of the full and partial particles bound to the chromatography medium is displaced by the empty rAAV particles into the loading flow-through from the column. In some embodiments, the rAAV preparation comprises whole particles. In some embodiments, the rAAV preparation comprises partial particles. In some embodiments, the rAAV preparation contains both complete and partial particles. In some embodiments, the chromatography medium is an ion exchange column chromatography medium, preferably an anion exchange chromatography medium. In some embodiments, the column chromatography media is selected from the group consisting of Poros50HQ, Poros50D, Poros50PI, Capto ImpRes Q, and Poros XQ, preferably Poros XQ.

[0033] In some embodiments, the column chromatography media is a monolith, such as a CIMmultus™ QA monolithic column. In some embodiments, the loading buffer comprises at least one buffer selected from the group consisting of Tris, Bis-tris, Bis-tris propane, Tris acetate, ethanolamine, and phosphate. In some embodiments, the loading buffer contains K(I), Li(I), Ca(II), Mg(II), Cu(II), Ba(II), Co(II), Ni(II), Mn(II), Zn(II), Cd(II), Pb(II), Fe(III), Fe(II), Na(I), and NH4 + The anion component of the salt is not critical. In some embodiments, the loading buffer has a pH of about 6-10, preferably 8-9.

[0034] In some embodiments, the loading buffer comprises at least one detergent. In some embodiments, the detergent in the loading buffer is selected from the group consisting of poloxamer 188, polysorbate 80, polysorbate 20, NP-40, Triton X-100, and Triton CG-110. In some embodiments, the concentration of the detergent in the loading buffer is between 0.0001% and 0.1%. In some embodiments, the elution buffer comprises at least one buffer selected from the group consisting of Tris, Bis-tris, Bis-tris propane, Tris acetate, ethanolamine, and phosphate. In some embodiments, the elution buffer contains K(I), Li(I), Ca(II), Mg(II), Cu(II), Ba(II), Co(II), Ni(II), Mn(II), Zn(II), Cd(II), Pb(II), Fe(III), Fe(II), Na(I), and NH4 + The anion component of the salt is not critical.

[0035] In some embodiments, the elution buffer has a pH of about 6-10, preferably 8-9. In some embodiments, the elution buffer comprises at least one detergent. In some embodiments, the detergent in the elution buffer is selected from the group consisting of poloxamer 188, polysorbate 80, polysorbate 20, NP-40, Triton X-100, and Triton CG-110. In some embodiments, the concentration of the detergent in the elution buffer is between 0.0001% and 0.1%.

[0036] In some embodiments, the purified preparation is substantially free of empty particles. In other embodiments, the purified preparation comprises an increased ratio of partial rAAV particles to empty particles than the rAAV preparation. Preferably, the ratio of partial rAAV particles to empty particles in the purified preparation is 3:1 or greater, e.g., 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, or 7.5:1 or greater, or any ratio therebetween, more preferably 4:1 or greater. In some embodiments, the rAAV particles comprise a capsid derived from an AAV serotype. In another embodiment, the rAAV particles comprise capsids derived from genetically engineered capsids capable of binding to an ion exchange chromatography medium.

[0037] In some embodiments, the rAAV particles are prepared using the methods described in Pulicherla et al., Mol. Ther., 19(6):1070-1078 (2011) (which describes, among other things, AAV9 variants, including AAV9.47), U.S. Pat. Nos. 7,906,111 (which describes, among other things, AAV9(hu14)), 10,532,111 (which describes, among other things, NP59), 10,738,087 (which describes, among other things, Anc-80), 9,169,299 (which describes "LK03"), 9,840,719 (which describes "RHM4-1"), 7,749,492, 7,588,772 (which describes "DJ" and "DJ8"), 9,5 and variants thereof, including variants of the AAV capsid set forth in WO 2012 / 145601, WO 2013 / 158879, WO 2015 / 013313, WO 2018 / 156654, and U.S. Patent Publication No. 2013 / 0059732. All of these documents are incorporated herein by reference in their entirety.

[0038] The details of one or more embodiments of the invention are set forth in the description below. Other features and advantages will become apparent from the following detailed description and the appended claims. The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. It is to be understood that the invention is not limited to the precise embodiments shown in the drawings. [Brief description of the drawings]

[0039] [Figure 1A-1C]Separation of complete and incomplete rAAV particles on Poros 50HQ resin at three different pH values: pH 8.0 (Figure 1A), pH 8.6 (Figure 1B), and pH 9.25 (Figure 1C). [Figures 2A-2D] Separation of complete and incomplete rAAV particles on Poros 50D resin is shown using different buffers at different pH: pH 8.6 and 50 mM Tris (Figure 2A), pH 8.6 and 25 mM Tris (Figure 2B), pH 8.0 and 25 mM Tris (Figure 2C), and pH 9.25 and 25 mM Tris (Figure 1D). [Figure 3A-3E] Separation of complete and incomplete rAAV particles on Poros 50PI resin using different binding strengths at different pH values: pH 8.0, no NaCl (Figure 3A), pH 8.6, no NaCl (Figure 3B), pH 8.6 and 30 mM NaCl (Figure 3C), pH 9.2, no NaCl (Figure 4C), and pH 9.2 and 30 mM NaCl (Figure 3E). [Figure 4A-4C] Separation of complete and incomplete rAAV particles on Capto ImpRes Q resin is shown using different buffers with different pH values: pH 8.0 and 50 mM Tris, no NaCl (Figure 4A), pH 8.6 and 25 mM Tris, no NaCl (Figure 4B), and pH 9.0 and 25 mM Tris, no NaCl (Figure 4C). [Diagram 5] 1 shows that separation of complete and incomplete rAAV particles on Poros XQ resin was best at pH 8.75 compared to pH 8 and pH 9.25. [Figure 6] 1 shows the effect of binding salt (1.6, 5, and 6.8 mS / cm) on the separation of complete and incomplete rAAV particles on Poros XQ resin. [Figure 7] 1 shows the effect of flow rate on the separation of complete and incomplete rAAV particles on Poros XQ resin. [Figure 8A-8B] FIG. 8A shows the HPLC analysis of the sample at low load, and FIG. 8B shows the HPLC analysis of the sample at breakthrough load. [Figure 9A-9D]The effect of salt on binding strength under breakthrough conditions is shown: 45 mM NaCl (FIG. 9A), 60 mM NaCl (FIG. 9B), 75 mM NaCl (FIG. 9C), and 90 mM NaCl (FIG. 9D). [Figure 10A-10D] Step elution at different binding strengths with salts: 60 mM NaCl (FIG. 10A), 75 mM NaCl (FIG. 10B), 90 mM NaCl (FIG. 10C), and 120 mM NaCl (FIG. 10D). [Figure 11A-11B] FIG. 11B shows that the sample was loaded onto the PXQ resin in 50 mM Tris pH 8.5 buffer with 60 mM NaCl. Elution was performed with 300 mM NaCl, 50 mM Tris pH 8.5 buffer using a linear gradient. In FIG. 11B, more sample was loaded onto the column than in FIG. 11A. These figures show that displacement chromatography separates complete rAAV particles from incomplete particles. FIG. 11B further shows that displacement became more pronounced with more sample loaded, increasing product purity to nearly 100% with a yield of >90%. [Figure 12] 12 shows that the sample was loaded onto the PXQ resin in 50 mM Tris pH 8.5 buffer with 10 mM MgCl2. Elution was performed with 300 mM NaCl, 50 mM Tris pH 8.5 buffer. FIG. 12 shows that the use of a high concentration of 10 mM MgCl2 for the loaded sample removes incomplete particles during loading. [Figure 13] 1 shows that the sample was loaded onto the PXQ resin in 50 mM Tris pH 8.5 buffer with 2.5 mM CaCl2. Elution was performed with 300 mM NaCl, 50 mM Tris pH 8.5 buffer. FIG. 13 shows that the addition of CaCl2 to the load removes incomplete particles during loading. This method is more robust compared to the method in FIG. 12 due to the larger conductivity difference between complete and incomplete particles (>4 mS / cm). [Figure 14A-14B]14 shows that the sample was loaded onto the PXQ resin with 50 mM Tris pH 8.5 buffer with 1-1.5 mM CaCl2 + 2.5 mM MgCl2 + 20 mM NaCl. Elution was performed with 10 mM CaCl2, 2.5 mM MgCl2, 20 mM NaCl, 50 mM Tris pH 8.5 buffer. These figures show the robustness of CaCl2 concentration. Figure 14A is loading with 1.5 mM CaCl2 and Figure 14B is loading with 1 mM CaCl2. This method resulted in 100% removal of incomplete particles without any loss of complete particles even when the CaCl2 concentration was changed by 50% and even when other additives were present. Furthermore, elution with a CaCl2 gradient does not elute complete particles until 5 mM CaCl2 is applied. This shows a robust difference of 4 mM CaCl2 between incomplete and complete collection. [Fig. 14C-14D] The same conditions as in FIG. 14B are shown together with analytical ultracentrifugation (AUC) analysis of the drug substance shown in FIG. 14D. [Figure 15] Chromatogram of sample loaded onto PXQ resin in 50 mM Tris pH 8.5. The column was washed with 50 mM Tris pH 8.5 buffer with 1 mM CaCl2 + 2.5 mM MgCl2. This figure shows that when CaCl2 was not added to the sample load, both yield and purity were compromised in purifying complete particles from complete particles. In addition, it was difficult to remove all of the complete particles bound to the column by washing. Furthermore, in subsequent elutions with increasing CaCl2 concentrations, the bound complete particles were not stable on the resin and new impurities were generated, which in turn compromised the final purity. [Figure 16] 1 shows a scheme for carrying out cycle displacement chromatography to separate complete rAAV particles from incomplete particles. [Figure 17]The chromatogram shows the sample was loaded onto the PXQ resin in 50 mM Tris pH 8.5 buffer with 58 mM LiCl. The column was eluted with 50 mM Tris pH 8.5 + 120 mM LiCl buffer. Under these conditions, the conductivity difference between incomplete and complete elution is 0.2 mS / cm. [Figure 18] FIG. 1 shows a scheme for carrying out cycle displacement chromatography to separate complete rAAV particles from incomplete particles using additives in at least the loading buffer, and preferably in the wash and elution buffers. [Figure 19A-19B] Removal of incomplete particles during sample loading and recovery of complete particles by decreasing pH using gradient or step elution are shown. Loading sample buffer is 50 mM Tris, 2 mM MgCl2, 2 mM CaCl2, 20 mM NaCl, pH 8.5. Elution with a decreasing pH gradient using 20 mM BisTris-30 mM acetate, 2 mM MgCl2, 2 mM CaCl2, pH 6.0 is shown in Figure 19A. Elution with a pH step method using 20 mM BisTris-30 mM acetate, 2 mM MgCl2, 2 mM CaCl2, pH 7.0 and pH 6.0 is shown in Figure 19B. [Figure 20A-20B] Addition of (NH4)2SO4 to the sample load shows that incomplete particles flow through during loading (Figure 20B). Samples were loaded onto the PXQ resin in 100 mM Tris pH 8.5 buffer with 0.0002% poloxamer 188, 20 mM NaCl, 20 mM (NH4)2SO4, and 2.0 mM MgCl2. The elution buffer is 50 mM Tris pH 8.5 with 0.0002% poloxamer 188 and 300 mM NaCl. In the absence of (NH4)2SO4 (shown in the inset), incomplete particles were not removed during sample loading at the same buffer conductivity of 7 mS / cm (Figure 20A). [Figure 21]It is shown that the addition of (NH4)2SO4 to the sample load results in incomplete particle flow-through during sample loading onto the monolith column. The sample was loaded onto the BIA 1.3 μm monolith column in 50 mM Tris pH 8.5 buffer with 0.0002% poloxamer 188, 20 mM NaCl, 20 mM (NH4)2SO4, and 2.0 mM MgCl2. The elution buffer is 50 mM Tris pH 8.5 buffer with 0.0002% poloxamer 188 and 200 mM NaCl. [Fig. 22A-22B] It has been shown that adding CuCl2 to the sample load results in incomplete particle flow-through during sample loading. Samples were loaded onto the PXQ resin in 50 mM Tris pH 8.5 buffer with 0.0002% poloxamer 188, 20 mM NaCl, 15 mM (NH4)2SO4, 2.0 mM MgCl2, and 1.5 mM CuCl2. The elution buffer is 180 mM sodium phosphate pH 7.2 buffer with 0.0002% poloxamer 188 and 2 mM MgCl2. [Fig. 22C-22E] It is shown that the addition of CuCl2 to the sample load leads to an increase in the resolution between incomplete particles (left-most peak) and complete particles (second peak from the left) at the analytical scale. In addition, the addition of CuCl2 leads to a significant reduction in some product variants (peaks after the second peak). This indicates that the addition of CaCl2 to the load removes incomplete particles during loading. [Figure 22F] We show that Cu(II), among other ions, leads to the greatest increase in resolution between partial and complete peaks. Samples are in 50 mM Tris pH 8.5 with 0.5-2 mM of the indicated ions. [Figures 23A-23E]Addition of CuCl2 allows removal of incomplete particles during loading, indicating that some degree of resolution between partial and complete particles can be achieved. rAAV preparations were loaded onto the PXQ resin in 50 mM Tris pH 8.5 buffer with 0.0002% poloxamer 188, 20 mM NaCl, 15 mM NH4SO4, 2.0 mM MgCl2, and 1.5 mM CuCl2. Bound particles were stepwise eluted with 50 mM sodium acetate pH 6.0 buffer with 0.0002% poloxamer 188 and two different amounts of NaCl. Elution peak 1 is obtained at 50 mM NaCl and elution peak 2 is obtained at 200 mM NaCl (Figures 23A-23B). Figures 23C-23E show the sedimentation coefficient distribution of the eluate peaks using analytical ultracentrifugation. As shown in Table 1 below, peak 1 contains both partial particles (40.1%) and complete particles (49.3%), while peak 2 is enriched primarily in complete particles (69.8%) with a lower percentage of partial particles (15.5%) and with incomplete particles (15.5%) removed. [Table 1] [Fig. 24A-24G]24A-24D show the use of a three-column displacement chromatography method to enrich for rAAV particle variants. The rAAV preparation was loaded onto three PXQ columns connected in series. The loading buffer was 50 mM Tris pH 8.5, 75 mM NaCl, and 2 mM MgCl2. The three columns were eluted sequentially using 200 mM NaCl, 50 mM Tris buffer pH 8.5 with 0.0002% poloxamer 188 and 2 mM MgCl2 (see Figures 24A-24B for preparative chromatograms). The collected fractions were analyzed on an IEX column using UPLC. As shown in Figures 24C-24D, the empty particles flowed through the columns and the majority of the empty particles were removed during loading. In addition, analytical IEX chromatograms show that the first column is enriched for the strongest binding particles (longest retention time, FIG. 24E), while the second and third columns are enriched for the second strongest and weakest binding particles, respectively (FIGS. 24F-G). The first and second column eluates are enriched for intact particles. This demonstrates the use of displacement chromatography to enrich for different particle variants in a three-column setup. [Fig. 25A-25D]Use of a two-column displacement chromatography method to enrich for intact particles is shown. The rAAV preparation was loaded onto two PXQ columns connected in series. The loading buffer was 50 mM Tris pH 8.5, 10 mM NaCl, 2.5 mM MgCl2, and 1 mM CaCl2. Elution was performed sequentially from column 1 and column 2 with 50 mM Tris pH 8.5, 200 mM NaCl, 2.5 mM MgCl2, and 0.0002% poloxamer 188 (see Figures 25A-25B for preparative chromatograms). Collected fractions were analyzed using UPLC. As shown in Figure 25C, empty particles were washed out during loading. Impurities in the rAAV preparation bound to the PXQ resin with higher affinity than intact particles. As a result, these impurities were reduced in the preparation loaded onto column 2, and intact particles were the species with the highest affinity for binding to column 2. Upon elution, the column 2 eluate is more enriched for complete particles. Thus, as shown in Figure 25D (UPLC analysis), the column 2 elution peak shows better enrichment for complete particles than column 1. [Figures 26A-26D] Figure 26 shows the use of a three-column displacement chromatography method to enrich for partial and complete particles. The rAAV preparation was loaded onto three PXQ columns connected in series. The loading buffer was 50 mM Tris pH 8.5, 20 mM NaCl, 15 mM ammonium sulfate, 2 mM MgCl2, and 1.5 mM CaCl2. Elution was performed sequentially from columns 1, 2, and 3 with 50 mM Tris pH 8.5, 200 mM NaCl, 2.5 mM MgCl2, and 0.0002% poloxamer 188 (see Figures 26A-26B for preparative chromatograms). Figures 26C-26D show the characterization of the flow-through and eluate by analytical ion exchange chromatography and analytical ultracentrifugation (AUC). Empty particles flowed out during loading (Figure 26C). The main eluate peak from column 1 is enriched in complete particles, and the main eluate peak from column 3 is enriched in partial particles, while column 2 contains both partial and complete particles (Figure 26D). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] Throughout the Background and specification, various publications, articles, and patents are cited or described, and each such reference is incorporated herein by reference in its entirety. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of such forms part of the prior art with respect to any invention(s) disclosed or claimed. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains, unless otherwise defined. Otherwise, certain terms used herein have the meanings set forth herein. All patents, published patent applications, and publications cited herein are incorporated by reference as if fully set forth herein. It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0041] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprising" can be replaced with the terms "containing" or "including" and, as used herein, can sometimes be replaced with the term "having."

[0042] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Whenever used herein in the context of an aspect or embodiment of the invention, any of the preceding terms "comprising," "containing," "including," and "having" can be replaced with the terms "consisting of" or "consisting essentially of" to modify the scope of the disclosure.

[0043] As used herein, the term "about" when used in conjunction with a numerical value refers to any numerical value within ±10%, such as within ±5% or ±1% of the reference numerical value. For example, a pH of about 5.0 means any pH between 4.5 and 5.5, inclusive. As used herein, the connector term "and / or" between multiple described elements is understood to include both individual options and combinations of options. For example, when two elements are joined by "and / or", the first option refers to the applicability of the first element, but not the second element. The second option refers to the applicability of the second element, but not the first element. The third option refers to the applicability of the first element and the second element. Any one of these options is understood to fall within the meaning and thus meets the requirements of the term "and / or" as used herein. The simultaneous application of more than one option is also understood to fall within the meaning and thus meets the requirements of the term "and / or".

[0044] The term "vector" refers to any small carrier of a nucleic acid molecule, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, or other vehicle that can be manipulated by insertion or integration of a nucleic acid. Vectors can be used for genetic manipulation (i.e., "cloning vectors"), introduction / transfer of polynucleotides into cells, and transcription or translation of the inserted polynucleotide in cells. An "expression vector" is a vector that contains a gene or nucleic acid sequence with the necessary regulatory regions required 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 contains additional elements such as heterologous nucleic acid sequences, expression control elements (e.g., promoters, enhancers), introns, inverted terminal repeats (ITRs), optional selectable markers, polyadenylation signals, etc.

[0045] The term "adeno-associated virus (AAV) vector" or "AAV vector" is intended to mean a vector that is capable of expressing an AAV molecule, such as a human adeno-associated virus (HAV) vector, or a human adeno-associated virus (HAV) vector. The term "adeno-associated virus (AAV) vector" or "AAV vector" may be used in a variety of applications, including those described in Pulicherla et al., Mol. Ther., vol. 19(6): 1070-1078 (2011) (which describes, among other things, AAV9 variants, including AAV9.47); U.S. Pat. No. 7,906,111 (which describes, among other things, AAV9(hu14)); U.S. Pat. No. 10,532,111; No. 10,738,087 (among others, NP59 is mentioned), No. 9,169,299 (where "LK03" is mentioned), No. 9,840,719 (where "RHM4-1" is mentioned), No. 7,749,492, No. 7,588,772 (where "DJ" and "DJ8" are mentioned), No. 9,587 AAV capsid variants, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV15, AAV16, AAV17, AAV18, AAV19, AAV20, AAV20, AAV30, AAV31, AAV32, AAV40, AAV41, AAV42, AAV43, AAV44, AAV45, AAV50, AAV51, AAV52, AAV53, AAV54, AAV55, AAV66, AAV67, AAV68, AAV69, AAV70, AAV71, AAV72, AAV73, AAV74, AAV75, AAV76, AAV77, AAV78, AAV79, AAV81, AAV82, AAV83, AAV84, AAV85, AAV86, AAV86, AAV87, AAV88, AAV89, AAV91, AAV92, AAV93, AAV94, AAV95, AAV96, AAV97, AAV98, AAV99, AAV10, AAV100, AAV101, AAV102, AAV103, AAV104, AAV105, AAV106, AAV107, AAV108, AAV109, AAV110, AAV111, AAV112, AAV113, AAV114, AAV115, AAV115, AAV120, AAV121, AAV122, AAV123, AAV124, AAV125, AAV126, AAV127, It refers to vectors derived from adeno-associated virus serotypes, including AAV serotypes such as AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 9.47, 9(hu14), 10, 11, 12, 8, 10, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115 Functional ITR sequences are necessary for rescue, replication, and packaging of AAV virions.Thus, an AAV vector is defined herein as comprising at least those sequences required in cis for viral replication and packaging (e.g., functional ITRs). The ITRs need not be wild-type nucleotide sequences, but may be altered, for example, by insertion, deletion, or substitution of nucleotides, so long as the sequences provide for functional rescue, replication, and packaging.

[0046] The term "AAV virion" refers to a viral particle, such as a wild-type (wt) AAV virus particle, that comprises a linear, single-stranded nucleic acid genome associated with an AAV capsid protein coat. Recombinant adeno-associated virus (rAAV vector) is derived from adeno-associated virus. 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 the cells. Because AAV is not associated with human pathogenic disease, rAAV vectors can deliver heterologous nucleic acid sequences (e.g., therapeutic proteins and agents) to human patients without causing substantial AAV pathogenesis or disease.

[0047] The term "recombinant" as a modifier of vectors, such as recombinant adeno-associated virus (rAAV) vectors, and sequences, such as recombinant polynucleotides and polypeptides, means that a composition has been manipulated (i.e., genetically engineered) in a manner that does not generally occur in nature. An example of a rAAV vector may be one in which a nucleic acid not normally present in the wild-type AAV genome is inserted into the viral genome. For example, a nucleic acid (e.g., a gene) encoding a therapeutic protein or polynucleotide sequence is cloned into the vector with or without the 5', 3', and / or intron regions with which the gene is normally associated in the AAV genome. The term "recombinant" is not always used herein for AAV vectors and sequences, such as polynucleotides, but recombinant forms including AAV vectors, polynucleotides, and the like, are expressly included, despite any such omission.

[0048] rAAV vectors are 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-natural (heterologous) nucleic acid, such as a nucleic acid encoding a therapeutic protein or nucleic acid molecule of interest. Typically, in the case of AAV, one or both inverted terminal repeat (ITR) sequences of the AAV genome are retained in the rAAV vector. The rAAV genome is distinct from the AAV genome because all or part of the AAV genome has been replaced with a sequence that is non-natural with respect to the AAV genome nucleic acid, such as a heterologous nucleic acid encoding a therapeutic protein or polynucleotide sequence. Thus, the incorporation of a non-natural sequence defines the AAV as a "recombinant" AAV vector, which can be referred to as a "rAAV vector." The recombinant AAV vector sequence can be packaged, which is referred to herein as a "particle" for subsequent infection (transduction) of cells ex vivo, in vitro, or in vivo.

[0049] The terms "recombinant AAV virion", "rAAV virion", "AAV vector particle", "complete rAAV capsid", "complete rAAV particle", "complete capsid" and "complete particle" each refer to an infectious replication-defective virus that contains an AAV protein shell in which a nucleic acid molecule containing a heterologous nucleotide sequence of interest flanked on one or both sides by AAV ITRs is encapsulated. Complete rAAV particles are produced in a suitable host cell that has the AAV vector, AAV helper functions, and sequences that specify accessory functions introduced therein. In this way, the host cell is capable of encoding the AAV polypeptides necessary to package the AAV vector (containing the recombinant nucleotide sequence of interest) into an infectious recombinant virion particle for subsequent gene delivery.

[0050] The terms "incomplete capsid" and "incomplete particle", as used herein, each refer to an AAV particle or virion that includes an AAV particle shell that includes a heterologous nucleic acid sequence flanked on one or both sides by AAV ITRs, but lacks a complete nucleic acid molecule. Such incomplete particles do not transfer a complete heterologous nucleic acid sequence into a host cell or into cells within an organism. These incomplete particles include variants with different lengths or amounts of incomplete genetic material. Incomplete particles that lack or have no sufficient genetic material to be detected as having any genetic material by analytical methods (e.g., UPLC and AUC) are referred to as "empty" particles. Incomplete particles that have some genetic material but enough genetic material to be detected by analytical methods as less than a complete particle are referred to as "partial" particles. The incomplete genetic material may be intact or fragmented.

[0051] Any analytical method known in the art can be used to quantify complete and incomplete particles, including determining the ratio of complete to incomplete or incomplete to complete particles. For example, such methods may be, but are not limited to, physical titer calculation; A260&A280 absorbance; analytical anion exchange chromatography (e.g., UPLC); multi-angle light scattering; analytical ultracentrifugation (AUC); cryogenic electron microscopy (Cryo-EM); or charge detection mass spectrometry (CDMS). To illustrate quantitative evaluation, several different methods, UPLC, A260&A280 absorbance, and AUC, are presented in this disclosure.

[0052] Vector "genome" refers to the portion of the recombinant sequence that is ultimately packaged or encapsulated 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. This non-vector genome portion of the recombinant plasmid is called the "plasmid backbone" and is important for cloning and amplification of the plasmid, a step required for propagation and recombinant virus production, but is not itself packaged or encapsulated into the rAAV particle. Thus, vector "genome" refers to the nucleic acid that is packaged or encapsulated by the rAAV.

[0053] The term "AAV helper functions" refers to AAV-derived coding sequences (proteins) that, when expressed, can provide AAV gene products and AAV vectors, which then function 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 assembly activating proteins (AAPs) of certain AAV serotypes. Rep expression products have been shown to have multiple functions, including, among others, recognition, binding, and nicking of AAV origins of DNA replication; DNA helicase activity; and modulation of transcription from AAV (or other heterologous) promoters. Cap expression products (capsids) provide the necessary packaging functions. AAV helper functions are used to complement AAV functions in trans that are missing from the AAV vector genome.

[0054] The term "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 that is to be used to produce a transducing AAV vector for delivering a nucleic acid sequence of interest, for example, by gene therapy to a subject. AAV helper constructs are generally used to provide transient expression of AAV rep and / or cap genes to complement missing AAV functions required for AAV vector replication. Helper constructs generally lack AAV ITRs and cannot replicate or package themselves. AAV helper constructs may be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. A number of AAV helper constructs have been reported, such as the plasmids pAAV / Ad and pIM29+45, which encode both Rep and Cap expression products (see, e.g., Samulski et al. (1989) J. Virol. 63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945). A number of other vectors have been reported that encode Rep and / or Cap expression products (see, e.g., U.S. Pat. Nos. 5,139,941 and 6,376,237).

[0055] The term "accessory functions" refers to non-AAV derived viral and / or cellular functions on which AAV depends for replication. This 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 AAV capsid packaging. Viral-based accessory functions may be derived from any of the known helper viruses, such as adenovirus, herpesvirus (other than herpes simplex virus type 1), and vaccinia virus.

[0056] "Accessory function vector" generally refers to a nucleic acid molecule that includes a polynucleotide sequence that provides an accessory function. Such a sequence may be present in an accessory function vector and transfected into a suitable host cell. The accessory function vector is capable of supporting rAAV virion production in a host cell. The accessory function vector may be in the form of a plasmid, phage, transposon, or cosmid. In addition, accessory functions do not require a full complement of adenovirus genes. 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 lacking the E1 region or lacking the E4 region are unable to support AAV replication. Thus, the E1A and E4 regions are thought 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 EIB (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 Functions, I CRC Handbook of Parvoviruses (ed. P. Tijssen, 1990); 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 EIB coding region have yielded conflicting results, although EIB55k may be required for AAV virion production, whereas EIB19k 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 adenovirus 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 EIB region lacking an intact E1B55k coding region. Such accessory function vectors are described, for example, in WO 01 / 83797.

[0057] As used herein, the term "serotype" is a distinction used to refer to an AAV that has a serologically distinct capsid from other AAV serotypes. Serological distinctiveness is determined based on the lack of cross-reactivity between antibodies to one AAV compared to another AAV. Differences in cross-reactivity are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes).

[0058] Under the traditional definition, a serotype means that the virus of interest is tested against all existing characterized serotype-specific sera for neutralizing activity and no antibodies are found that 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 with any of the currently existing serotypes. Thus, if a new virus (e.g., AAV) does not show serological differences, the new virus (e.g., AAV) will be a subgroup or variant of the corresponding serotype. In many cases, serological testing for neutralizing activity has not yet been performed on mutant viruses with capsid sequence modifications to determine whether they are separate serotypes 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) as well as viruses (e.g., AAV) that are not serologically distinct and may exist within a subgroup or variant of a given serotype.

[0059] rAAV vectors include any viral strain or serotype. As non-limiting examples, rAAV plasmid or vector genomes or particles (capsids) may be prepared as described in Pulicherla et al., Mol. Ther., vol. 19(no. 6) pp. 1070-1078 (2011) (wherein, among other things, AAV9 variants are described, including AAV9.47); U.S. Pat. Nos. 7,906,111 (wherein, among other things, AAV9(hu14) is described); 10,532,111 (wherein, among other things, NP59 is described); 10,738,087 (wherein, among other things, Anc-80 is described); 9,169,299 (wherein, “LK03”); 9,840,719 (wherein, “RHM4-1”); 7,749,492; 7,588,772 (wherein, “DJ” and “DJ8”); For example, but not limited to, variants of the AAV capsid set forth in WO 9,587,282, WO 2012 / 145601, WO 2013 / 158879, WO 2015 / 013313, WO 2018 / 156654, and U.S. Patent Application Publication No. 2013 / 0059732, The vectors may be based on any AAV serotype, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, Rh8, Rh10, Rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ, DJ8, NP59, Anc-80, and variants thereof. All of these documents are incorporated herein by reference in their entirety. All of the above AAV descriptions, including sequence information, are incorporated by reference in their entirety. Such vectors may be based on the same strain or serotype (or subgroup or variant), or may be different from each other. As a non-limiting example, rAAV plasmid or vector genomes or particles (capsids) based on one serotype genome may be identical in one or more of the capsid proteins that package the vector.Additionally, a rAAV plasmid or vector genome may be based on a different AAV (e.g., AAV2) serotype genome than one or more of the capsid proteins that package the vector genome, where at least one of the three capsid proteins is a serotype of AAV, as described in, but not limited to, Pulicherla et al., Mol. Ther., 19(6):1070-1078 (2011) (wherein, among other things, AAV9 variants are described, including AAV9.47), U.S. Pat. No. 7,906,111 (wherein, among other things, AAV9(hu14) is described), U.S. Pat. No. 10,532,111 (wherein, among other things, NP59 is described), U.S. Pat. No. 10,738,087 (wherein, among other things, Anc-80 is described), U.S. Pat. No. 9,169,299 (wherein, "LK03" is described), and U.S. Pat. No. 9,840,719 (wherein, "RHM4-1" is described). No. 7,749,492, No. 7,588,772 (wherein "DJ" and "DJ8" are mentioned), No. 9,587,282, and WO 2012 / 145601, WO 2013 / 158879, WO 2015 / 013313, WO 2018 / 156654, U.S. Patent Application Publication No. 2013 / 0059 The rAAV vector may be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, Rh8, Rh10, Rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ, DJ8, NP59, Anc-80, and variants thereof, including variants of the AAV capsids shown in the 732 specification. All of these documents are incorporated by reference in their entirety. Thus, the rAAV vector contains gene / protein sequences identical to those characteristic of specific serotypes as well as mixed serotypes. The various embodiments are applicable to any rAAV or AAV capsid from any source, so long as the capsid is capable of binding to an ion exchange chromatography column.

[0060] In various exemplary embodiments, the rAAV vector is an AAV vector as described in Pulicherla et al., Mol. Ther., vol. 19(6) pp. 1070-1078 (2011) (which describes, among other things, AAV9 variants, including AAV9.47); U.S. Pat. No. 7,906,111 (which describes, among other things, AAV9(hu14)); U.S. Pat. No. 10,532,111 (which describes, among other things, NP59); U.S. Pat. No. 10,532,111 (which describes, among other things, NP59); No. 738,087 (in which Anc-80 is described, among others), No. 9,169,299 (in which "LK03" is described), No. 9,840,719 (in which "RHM4-1" is described), No. 7,749,492, No. 7,588,772 (in which "DJ" and "DJ8" are described), No. 9,587,282, and WO 2012 / 145601. AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), including variants of the AAV capsid shown in WO 2013 / 158879, WO 2015 / 013313, WO 2018 / 156654, and U.S. Patent Application Publication No. 2013 / 0059732; or consisting of a capsid sequence that is at least 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) identical to one or more capsid proteins of AAV10, AAV11, AAV12, Rh8, Rh10, Rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ, DJ8, NP59, Anc-80, and variants thereof, all of which are incorporated by reference in their entireties.In various exemplary embodiments, the rAAV vector is a vector as described in Pulicherla et al., Mol. Ther., 19(6):1070-1078 (2011) (which describes, among other things, AAV9 variants, including AAV9.47), U.S. Pat. Nos. 7,906,111 (which describes, among other things, AAV9(hu14)), 10,532,111 (which describes, among other things, NP59), 10,738,087 (which describes, among other things, Anc-80), 9,169,299 (which describes "LK03"), 9,840,719 (which describes "RHM4-1"), 7,749 AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAV9.492, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAV9.47, AAV9(hu14), ....47, AAV9(hu14), AAV11, AAV12, AAV9.47, AAV9.47, AAV9(hu14), AAV11, AAV12, AAV9.47, AAV9.47, AAV9(hu14), AAV11, AAV12, AAV9.47, AAV9.47, AAV9.47, AAV9.47, AAV9.47, AAV9.47, AAV9.47, AAV9. or consisting of a sequence that is at least 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) identical to one or more ITRs of Rh8, Rh10, Rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ, DJ8, NP59, Anc-80, and variants thereof, all of which are incorporated herein by reference in their entireties.

[0061] See, but not limited to, Pulicherla et al., Mol. Ther., vol. 19(6) pp. 1070-1078 (2011) (which describes, among other things, AAV9 variants, including AAV9.47); U.S. Pat. No. 7,906,111 (which describes, among other things, AAV9(hu14)); U.S. Pat. No. 10,532,111 (which describes, among other things, NP59); U.S. Pat. No. 10,738,087 (which describes, among other things, NP59); No. 9,169,299 (wherein Anc-80 is described, among others), No. 9,840,719 (wherein "RHM4-1" is described), No. 7,749,492, No. 7,588,772 (wherein "DJ" and "DJ8" are described), No. 9,587,282, and WO 2012 / 145601, WO 2012 / 145602, WO 2012 / 145603, WO 2012 / 145604, WO 2012 / 145605, WO 2012 / 145606, WO 2012 / 145607, WO 2012 / 145609 ... AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, including variants of the AAV capsid set forth in WO 2013 / 158879, WO 2015 / 013313, WO 2018 / 156654, and U.S. Patent Application Publication No. 2013 / 0059732, all of which are incorporated by reference in their entireties. rAAVs, including AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, Rh8, Rh10, Rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ, DJ8, NP59, Anc-80, and variants thereof, as well as variants, hybrids, 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 are deleted in whole or in part of one or more wild-type AAV genes, but retain at least one functional flanking ITR sequence required for rescue, replication, and packaging of the recombinant vector into rAAV vector particles. Thus, the rAAV vector genome will contain sequences (e.g., functional ITR sequences) required in cis for replication and packaging.

[0062] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein and 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, as well as 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 polynucleotides, synthetic polynucleotides, and polynucleotides that have been intentionally modified or altered. Nucleic acids may be single-stranded, double-stranded, or triple-stranded, linear or circular, and of any length. Where a nucleic acid is discussed, 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.

[0063] A "heterologous" nucleic acid sequence refers to a polynucleotide inserted into an AAV plasmid or vector for vector-mediated transfer / delivery of the polynucleotide into a cell. A heterologous nucleic acid sequence is distinct from, i.e., non-native to, the AAV nucleic acid. Once transferred / delivered into a cell, the heterologous nucleic acid sequence contained within the vector can be expressed (e.g., transcribed and translated, if appropriate). Alternatively, the heterologous polynucleotide contained within the vector transferred / delivered into a cell does not need to be expressed.

[0064] "Polypeptides," "proteins," and "peptides" encoded by a "nucleic acid sequence" include the full-length native sequence as in the naturally occurring protein, as well as functional subsequences, modifications, or sequence variants, so long as the subsequence, modification, or variant retains some degree of function of the full-length native protein. Such polypeptides, proteins, and peptides encoded by nucleic acid sequences may, but need not, be identical to endogenous proteins that are missing, or whose expression is insufficient, or deficient in the mammal being treated. "Transgene" is used herein for convenience to refer to a nucleic acid (e.g., heterologous) that is intended to be or has been 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.

[0065] In a cell carrying a transgene, the transgene has been introduced / transferred by "transduction" or "transfection" of the cell with a plasmid or AAV vector. The terms "transduce" 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. Once integrated into the nucleic acid (genomic DNA) of a recipient cell or organism, the introduced nucleic acid can be stably maintained in the cell or organism and further inherited or inherited by progeny cells or progeny organisms of the recipient cell or organism's cells. "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 cell that has been transfected. 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 in genomic DNA 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.

[0066] A "therapeutic protein," as used herein, is a peptide or protein that can alleviate or reduce symptoms resulting from an insufficient amount, absence, or deficiency of a protein in a cell or subject. A "therapeutic" protein encoded by a transgene can confer a benefit to a subject, such as, for example, correction of a genetic defect, correction of a genetic (expression or function) deficiency. Non-limiting examples of heterologous nucleic acids encoding gene products (e.g., therapeutic proteins) useful according to the present invention include those that can be used to treat diseases or disorders, including, but not limited to, the following: "Hemostasis" or blood clotting disorders, e.g., 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 CI deficiency, gamma-carboxylase deficiency; anemia, bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC); excessive anticoagulation associated with heparin, low molecular weight heparins, pentasaccharides, warfarin, small molecule antithrombotic agents (i.e., FXa inhibitors); and platelet disorders such as Bernard-Soulier syndrome, Glanzman thromblastemia, and storage pool deficiency.

[0067] In certain embodiments, the disease or disorder affects or originates in the central nervous system (CNS). In certain embodiments, the disease is a neurodegenerative disease. In certain embodiments, the CNS or neurodegenerative disease is Alzheimer's disease, Huntington's disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy's disease, polyglutamine repeat disease, or Parkinson's disease. In certain embodiments, the CNS or neurodegenerative disease is a polyglutamine repeat disease. In certain embodiments, the polyglutamine repeat disease is spinocerebellar ataxia (SCA1, SCA2, SCA3, SCA6, SCA7, or SCA17).

[0068] In certain embodiments, the heterologous nucleic acid encodes a protein selected from the group consisting of: GAA (acid alpha-glucosidase) for the treatment of Pompe disease; ATP7B (copper transporting ATPase 2) for the treatment of Wilson disease; alpha galactosidase for the treatment of Fabry disease; ASS1 (argininosuccinate synthase) for the treatment of citrullinemia type 1; beta-glucocerebrosidase for the treatment of Gaucher disease type 1; beta-hexosaminidase A for the treatment of Tisachs disease; SERPING1 (C1 protease inhibitor or C1 esterase inhibitor) for the treatment of hereditary angioedema (HAE), also known as C1 inhibitor deficiency types I and II; and glucose-6-phosphatase for the treatment of glycogen storage disease type I (GSDI).

[0069] In certain embodiments, the heterologous nucleic acid encodes: CFTR (cystic fibrosis transmembrane conductance regulator), blood clotting (clotting) factors (such as factor XIII, factor IX, factor VIII, factor X, factor VII, factor VIIa, protein C, etc.), gain-of-function blood clotting factors, antibodies, retinal pigment epithelium-specific 65 kDa protein (RPE65), erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, β-globin, α-globin, spectrin, α-antitrypsin, adenosine deaminase. (ADA), metal transporters (ATP7A or ATP7), sulfamidase, enzymes involved in lysosomal storage diseases (ARSA), hypoxanthine guanine phosphoribosyltransferase, beta-25 glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain ketoacid dehydrogenase, hormones, growth factors, insulin-like growth factor 1 or 2, platelet-derived growth factor, epidermal growth factor, nerve growth factor, neurotrophic factor-3 and -4, brain-derived neurotrophic factor, glial-derived growth factor, transforming growth factor alpha and beta, Cytokines, α-interferon, β-interferon, interferon-γ, interleukin-2, interleukin-4, interleukin-12, granulocyte-macrophage colony-stimulating factor, lymphotoxin, suicide gene product, herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor necrosis factor, drug resistance proteins, tumor suppressor proteins (e.g., p53, Rb, Wt-1, NF1, von Hippel-Lindau (VHL), adenomatous polyposis coli (A PC), peptides with immunomodulatory properties, tolerogenic or immunogenic peptides or proteins T-regitope or hCDR1, insulin, glucokinase, guanylate cyclase 2D (LCA-GUCY2D), Rab escort protein 1 (choroideremia), LCA5 (LCA-Lebercilin), ornithine ketoacid aminotransferase (gyrate atrophy), retinoschisin 1 (X-linked retinoschisis), USH1C (Usher syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP),MERTK (AR type RP: retinitis pigmentosa), DFNB1 (connexin 26 deafness), ACHM2, 3, and 4 (color blindness), PKD-1 or PKD-2 (polycystic kidney disease), TPP1, CLN2, sulfatase, N-acetylglucosamine-1-phosphate transferase, cathepsin A, GM2-AP, NPC1, VPC2, sphingolipid activating protein, one or more zinc finger nucleases for genome editing, or one or more donor sequences used as repair templates for genome editing.

[0070] Nucleic acid molecules, vectors such as cloning vectors, expression vectors (e.g., vector genomes), and plasmids can be prepared using recombinant DNA technology methods. 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 using a variety of standard cloning, recombinant DNA techniques, by cellular expression or in vitro translation, and chemical synthesis techniques. The purity of the polynucleotide can be determined by sequencing and 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 with probes to detect homologous nucleotide sequences with genomic DNA or cDNA libraries; (2) antibody screening to detect polypeptides with common structural features, for example, using expression libraries; (3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to the nucleic acid sequence of interest; (4) computer searches of sequence databases for related sequences; and (5) differential screening of subtraction nucleic acid libraries.

[0071] Methods known in the art for producing rAAV virions include, for example, transfection using an AAV vector and AAV helper sequences in conjunction with co-infection with one AAV helper virus (e.g., adenovirus, herpesvirus, or vaccinia virus), or transfection with a recombinant AAV vector, an AAV helper vector, and an accessory function vector. Non-limiting methods for producing 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 recombinant rAAV vector production (i.e., vector production in a cell culture system), rAAV virions can be obtained from host cells and cell culture supernatants and then purified as described herein.

[0072] Method for purification of intact rAAV particles In one basic aspect, the present application provides a method for purifying complete recombinant adeno-associated virus (rAAV) particles, comprising: (a) providing an rAAV preparation comprising complete and incomplete rAAV particles; (b) loading the rAAV preparation in a loading buffer onto a column containing a chromatography medium, wherein complete rAAV particles have a higher binding affinity for the chromatography medium than incomplete particles; and (c) eluting the intact rAAV particles bound to the chromatography medium with an elution buffer to obtain a purified preparation containing an increased ratio of intact rAAV particles to incomplete particles. The present invention relates to a method comprising the steps of:

[0073] According to embodiments of the present application, the rAAV preparation may be any mixture including complete rAAV particles, and incomplete particles, including empty and partial particles, and variants thereof. The rAAV preparation may also include a mixture of various incomplete particles (e.g., empty and partial particles) for further separation. For example, the rAAV preparation may be a cell lysate, a processed cell lysate, a supernatant, or a previously purified preparation. The rAAV preparation useful for the methods of the present application may be obtained using any method for collecting rAAV known in the art in light of the present disclosure. For example, the cell lysate may be obtained by disrupting or lysing cells and removing cell debris by centrifugation, microfluidization, and / or depth filtration. The cell lysate may be used directly or may be further processed or stored before use in the methods of the present application.

[0074] Typically, the cell lysate is clarified, such as by filtration and centrifugation, to remove cell debris, resulting in a clarified cell lysate. The lysate (optionally clarified) contains complete rAAV particles, incomplete particles, and other rAAV vector production / process-related impurities, such as soluble cellular components of the host cells, which may include, among others, cellular proteins, lipids, and / or nucleic acids, as well as cell culture medium components. The clarified lysate can then be optionally subjected to additional purification steps to remove other process-related impurities by any method known in the art. The resulting processed lysate may be diluted or concentrated with an appropriate buffer prior to use in the methods of the present application.

[0075] In some embodiments, the imperfect particles include empty particles. In some embodiments, an incomplete particle comprises a partial particle. In some embodiments, incomplete particles include both empty particles and partial particles. In some embodiments, the chromatographic medium is an ion exchange column chromatographic medium, preferably an anion exchange chromatographic medium. The anion exchange chromatographic medium used in the method may be a strong anion exchange resin or a weak anion exchange resin. Preferably, the anion exchange chromatographic medium comprises an anion exchange ligand such as a proprietary quaternary amine, quaternized polyethyleneimine, polyethyleneimine, or dimethylaminopropyl. More preferably, the anion exchange chromatographic medium is selected from a weak anion exchange resin (e.g., Poros50D, Poros50PI) or a strong anion exchange resin (e.g., Poros XQ, Poros50HQ). Other examples of anion exchange chromatographic media include, but are not limited to, DEAE Sepharose FF, Q-Sepharose (HP and FF), QSepharose FF (low and high substitution), Capto Q, Q XP, Source30Q and 15Q, Fractogel DEAE, and MPHQ. Other examples of anion exchange chromatographic media include monoliths such as CIMmultus™ QA monolithic columns.

[0076] In some embodiments, the chromatography medium is an ion exchange column chromatography medium, preferably an anion exchange chromatography medium. In some embodiments, the column chromatography media is selected from the group consisting of Poros50HQ, Poros50D, Poros50PI, Capto ImpRes Q, and Poros XQ, preferably Poros XQ. In some embodiments, the column chromatography media is a monolith, such as a CIMmultus™ QA monolithic column. In some embodiments, multiple chromatographic media are used. In some embodiments, when multiple chromatographic media are used, the media are the same. In some embodiments, ion exchange chromatography media is used in conjunction with (meaning before or after) affinity chromatography media such as AVB Sepharose™ High Performance (GE Healthcare, Marlborough, Massachusetts), or size exclusion chromatography such as Superdex200 (GE Healthcare).

[0077] In some embodiments, when the rAAV preparation is loaded in step (b) onto a column containing a chromatography medium with a loading buffer, only complete rAAV particles bind to the chromatography medium; incomplete particles do not bind to the chromatography medium and flow through the column. In some embodiments, the flow-through from the column includes empty particles. In some embodiments, the flow-through from the column includes partial particles. In some embodiments, the flow-through from the column includes both complete rAAV particles and empty particles. In some embodiments, the flow-through from the column includes both complete rAAV particles and partial particles. In some embodiments, the flow-through from the column includes both complete rAAV particles and incomplete particles, including empty particles and partial particles.

[0078] In some embodiments, when the rAAV preparation in the loading buffer is applied to a column containing a chromatography medium in step (b), both complete rAAV particles and incomplete particles, including variant-like particles, bind to the chromatography medium, but the complete rAAV particles have a higher binding affinity for the chromatography medium than the incomplete particles, and the amount of complete and incomplete particles applied to the column exceeds the binding capacity of the chromatography medium, and the incomplete particles bound to the chromatography medium are displaced by the complete rAAV particles into the loading flow-through from the column. The loading flow-through from the column contains both complete and incomplete particles. In certain embodiments, some incomplete particles (e.g., empty particles and / or partial particles) bind to the chromatography medium but can be eluted with a wash buffer prior to elution of the complete rAAV particles with an elution buffer.

[0079] Without wishing to be bound by theory, it is believed that separation of complete and incomplete AAV particles occurs during loading due to a displacement phenomenon. When an rAAV preparation containing a mixture of particles is first applied to the column, all particles bind to available sites on the column. As available sites are occupied toward the inlet of the column, complete rAAV particles with a higher affinity for the chromatographic medium displace bound incomplete particles with a lower affinity for the chromatographic medium, resulting in enrichment of complete particles in the upper portion of the column. In the process, incomplete particles bind to the downstream chromatographic medium. As more of the mixture is applied to the column, more complete rAAV particles will bind to the upstream chromatographic medium, displacing the bound incomplete particles and sweeping the incomplete particles further downstream. In certain embodiments, after sample loading is complete, there are no incomplete particles bound to the chromatographic medium, or the amount of incomplete particles bound to the chromatographic medium is reduced compared to the amount of complete rAAV. The displacement of incomplete particles by complete particles is a result of their different affinities for the chromatographic medium, and thus enhancing the contrast in their affinities can improve separation. The affinity of a particle to bind to a chromatographic medium is determined by factors such as the rAAV particle, the resin, and the environment, including buffer conditions (salt and pH). Similarly, this reasoning also applies to partial particles, if the incomplete rAAV particles include partial particles. As variants of the incomplete particle population, partial particles can bind to a chromatographic medium with higher affinity than other incomplete particles, especially empty particles, but lower affinity than complete particles, and thus both complete and partial particles can displace other incomplete particles. By enhancing the contrast of the affinity of complete and partial particles to the chromatographic medium in addition to other incomplete particles, the separation of partial particles from complete particles can be further improved. Also similarly, if the incomplete rAAV particles include empty particles, the method of the present disclosure can be used to improve the separation of empty particles from complete and other incomplete particles.

[0080] The benefit of displacement is realized when breakthrough occurs in the column and the binding capacity of the column chromatography media is exceeded. As the chromatography media resin continues to exceed its saturation locally, competition between complete and incomplete particles occurs, leading to the displacement of bound incomplete particles by the incoming complete particles. The benefits of substitution can be similarly applied to the separation of other rAAV impurities and product variants, such as partially filled capsids with truncated transgenes, empty or partial or full capsid variants with post-translational modifications, or fragments or aggregates, based on differing affinities for chromatographic media.

[0081] In some embodiments, when the incomplete particles include both empty and partial particles, all of the incomplete particles bind to available sites in the column, and when an available site is occupied, the partial particles, which have a relatively higher affinity for the chromatography medium than the empty particles, displace the bound empty particles. Chromatographic media, such as anion exchange resins, can be equilibrated, washed, and eluted with a variety of buffers under a variety of conditions, such as pH and buffer volume. The following are intended to illustrate certain non-limiting examples, but are not intended to limit the invention. Anion exchange chromatography can be equilibrated using standard buffers according to the manufacturer's specifications. After equilibration, the sample is then loaded. The chromatographic medium is then washed at least once or more times, for example with 2-10 column volumes. Elution from the chromatographic medium is with 2-20 column volumes of high salt buffer.

[0082] According to an embodiment of the present application, equilibration buffers and solutions for washing and elution of anion exchange chromatography are suitable at a pH of about pH 6.0 to pH 12. In addition, suitable equilibration buffers and solutions for washing and elution of anion exchange columns are generally cationic or zwitterionic in nature. Such buffers include, but are not limited to, buffers having the following buffering agents: N-methylpiperazine; piperazine; Bis-Tris; Bis-Tris propane; triethanolamine; Tris; Tris acetate; N-methyldiethanolamine; 1,3-diaminopropane; ethanolamine; and acetic acid. To elute the sample, the ionic strength of the starting buffer is increased using salts such as NaCl, KCl, MgCl2, CaCl2, sulfates, formates, or acetates. In some embodiments, the loading buffer comprises at least one buffer selected from the group consisting of Tris, Bis-tris, Bis-tris propane, Tris acetate, ethanolamine, and phosphate. In certain embodiments, the loading buffer comprises a selected buffer at a concentration of about 20-50 mM, for example, 20 mM, 30 mM, 40 mM, 50 mM, 100 mM, or any concentration therebetween. In a preferred embodiment, the loading buffer comprises 20-50 mM Tris.

[0083] In some embodiments, the loading buffer contains K(I), Li(I), Ca(II), Mg(II), Cu(II), Ba(II), Co(II), Ni(II), Mn(II), Zn(II), Cd(II), Pb(II), Fe(III), Fe(II), Na(I), and NH4 + For example, the loading buffer may contain multiple salts selected from the group consisting of NaCl, MgCl2, and CaCl2. In a preferred embodiment, the loading buffer contains NaCl, MgCl2, CuCl2, LiCl, and CaCl2. The anion component to the salt is not critical and no particular anion is preferred.

[0084] In some embodiments, the loading buffer contains K(I), Li(I), Ca(II), Mg(II), Cu(II), Ba(II), Co(II), Ni(II), Mn(II), Zn(II), Cd(II), Pb(II), Fe(III), Fe(II), Na(I), and NH4 + The compound comprises at least one salt of a cation selected from the group consisting of: In some embodiments, the loading buffer contains K(I), Li(I), Ca(II), Mg(II), Cu(II), Ba(II), Co(II), Ni(II), Mn(II), Zn(II), Cd(II), Pb(II), Fe(III), Fe(II), Na(I), and NH4 + In a preferred embodiment, the loading buffer comprises at least one salt of a cation selected from the group consisting of: , wherein only complete rAAV particles bind to the chromatography medium, and incomplete particles do not bind to the chromatography medium and pass through the column. In a preferred embodiment, the loading buffer comprises at least CaCl2.

[0085] In some embodiments, the loading buffer comprises a sodium salt at about 10-100 mM, e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mM, or any concentration therebetween. In preferred embodiments, the sodium salt concentration is about 20-60 mM, e.g., 20, 25, 30, 35, 40, 45, 50, 55, 60 mM, or any concentration therebetween. In some embodiments, the loading buffer comprises a magnesium salt at a concentration of about 0-20 mM, e.g., 0, 5, 10, 15, 20 mM, or any concentration therebetween. In preferred embodiments, the magnesium salt concentration is about 1-10 mM, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mM, or any concentration therebetween.

[0086] In some embodiments, the loading buffer comprises a lithium salt at a concentration of about 0-100 mM, e.g., 0, 20, 30, 40, 50, 60, 70, 80, 90, 100 mM, or any concentration therebetween. In preferred embodiments, the lithium salt concentration is about 0-75 mM, e.g., 0, 15, 25, 35, 45, 55, 65, 75 mM, or any concentration therebetween. In some embodiments, the loading buffer comprises a calcium salt at a concentration of about 0-10 mM, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mM, or any concentration therebetween. In preferred embodiments, the calcium salt concentration is about 0.1-2.5 mM, e.g., 0.1, 0.5, 1.0, 1.5, 2.0, 2.5 mM, or any concentration therebetween. In some embodiments, the loading buffer comprises a copper salt at a concentration of about 0-5 mM, e.g., 0, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 mM, or any concentration therebetween. In preferred embodiments, the copper salt concentration is about 0.1-2.5 mM, e.g., 0.1, 0.5, 1.0, 1.5, 2.0, 2.5 mM, or any concentration therebetween. In some embodiments, the loading buffer comprises an ammonium salt at about 5-100 mM, e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 mM, or any concentration therebetween. In preferred embodiments, the concentration of ammonium is about 10-50 mM, e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50 mM, or any concentration therebetween.

[0087] In some embodiments, the loading buffer contains K(I), Li(I), Ca(II), Mg(II), Cu(II), Ba(II), Co(II), Ni(II), Mn(II), Zn(II), Cd(II), Pb(II), Fe(III), Fe(II), Na(I), and NH4 + The compound comprises at least one salt of a cation selected from the group consisting of: In some embodiments, the loading buffer has a pH of about 7-10, such as 7, 7.5, 8, 8.5, 9, 9.5, 10, or any pH therebetween, preferably a pH of about 8-9. In certain embodiments, the loading buffer preferably comprises about 20-60 mM NaCl, 1-5 mM MgCl2, 0.1-2.5 mM CaCl2 in Tris, more preferably in 20-50 mM Tris.

[0088] In certain embodiments, the loading buffer comprises about 20-60 mM NaCl, 10-30 mM (NH4)2SO4, 1-5 mM MgCl2, 0.1-2.5 mM CuCl2, preferably in Tris, more preferably in 20-100 mM Tris. In some embodiments, the loading buffer comprises at least one detergent. In some embodiments, the detergent in the loading buffer is selected from the group consisting of poloxamer 188, polysorbate 80, polysorbate 20, NP-40, Triton X-100, and Triton CG-110. In some embodiments, the concentration of the detergent in the loading buffer is between 0.0001% and 0.1%.

[0089] After the loading step (b), the rAAV preparation in a loading buffer is loaded onto a column containing a chromatography medium, where complete rAAV particles have a higher binding affinity for the chromatography medium than incomplete particles, and the complete rAAV particles bind to the chromatography medium. Optionally, the column may be washed with a wash buffer prior to elution. For example, the wash buffer may have an increased salt concentration and / or an increased elution pH compared to the loading conditions to remove incomplete particles bound to the chromatography medium. The complete particles bound to the chromatographic medium are then eluted in a purer form, for example using an increased salt concentration compared to the loading conditions and / or an adjusted pH for elution. Adjusting the pH can enhance the separation between complete and incomplete particles. Preferably, elution of complete particles is achieved using only a salt gradient without changing the pH. In some embodiments, the elution buffer comprises at least one buffer selected from the group consisting of Tris, Bis-tris, Bis-tris propane, Tris acetate, ethanolamine, and phosphate. In certain embodiments, the elution buffer comprises a selected buffer at a concentration of about 20-70 mM, for example, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, or any concentration therebetween. In a preferred embodiment, the elution buffer comprises 40-60 mM Tris.

[0090] In some embodiments, the elution buffer contains K(I), Li(I), Ca(II), Mg(II), Cu(II), Ba(II), Co(II), Ni(II), Mn(II), Zn(II), Cd(II), Pb(II), Fe(III), Fe(II), Na(I), and NH4 + For example, the elution buffer may preferably contain one or more salts selected from the group consisting of NaCl, MgCl2, LiCl, CuCl2, and CaCl2. The anion content of the salt is not critical. In some embodiments, the elution buffer contains K(I), Li(I), Ca(II), Mg(II), Cu(II), Ba(II), Co(II), Ni(II), Mn(II), Zn(II), Cd(II), Pb(II), Fe(III), Fe(II), Na(I), and NH4 + The compound comprises at least one salt of a cation selected from the group consisting of:

[0091] In some embodiments, the elution buffer comprises NaCl at a concentration of about 0-1000 mM, e.g., 0, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mM, or any concentration therebetween. In preferred embodiments, the concentration of NaCl is about 20-300 mM, e.g., 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300 mM, or any concentration therebetween. In some embodiments, the elution buffer comprises MgCl2 at a concentration of about 0-30 mM, e.g., 0, 5, 10, 15, 20, 25, 30 mM, or any concentration therebetween. In preferred embodiments, the concentration of MgCl2 is about 2-15 mM, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 mM, or any concentration therebetween.

[0092] In some embodiments, the elution buffer is about 0-200 mM LiCl, e.g., 0, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 mM, or any concentration therebetween. In preferred embodiments, the LiCl concentration is about 0-150 mM, e.g., 0, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 mM, or any concentration therebetween. In some embodiments, the elution buffer comprises CaCl2 at a concentration of about 0.1-20 mM, e.g., 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20 mM, or any concentration therebetween. In preferred embodiments, the concentration of CaCl2 is about 5-10 mM, e.g., 5, 6, 7, 8, 9, 10 mM, or any concentration therebetween. In some embodiments, the elution buffer comprises CuCl2 at a concentration of about 0-10 mM, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mM, or any concentration therebetween. In preferred embodiments, the concentration of CuCl2 is about 0-3 mM, e.g., 0, 1, 2, 3 mM, or any concentration therebetween.

[0093] In some embodiments, the elution buffer comprises (NH4)2SO4 at a concentration of about 5-100 mM, e.g., 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mM, or any concentration therebetween. In preferred embodiments, the concentration of (NH4)2SO4 is about 10-50 mM, e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50 mM, or any concentration therebetween. In some embodiments, the elution buffer has a pH of about 6-10, such as 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or any pH therebetween, preferably a pH of about 7-9. In certain embodiments, the elution buffer comprises about 20-150 mM NaCl, preferably in Tris, more preferably in 40-60 mM Tris. In some embodiments, the elution buffer comprises at least one detergent. In some embodiments, the detergent in the elution buffer is selected from the group consisting of poloxamer 188, polysorbate 80, polysorbate 20, NP-40, Triton X-100, and Triton CG-110. In some embodiments, the concentration of the detergent in the elution buffer is between 0.0001% and 0.1%.

[0094] In another basic aspect, the present application provides a method for purifying complete recombinant adeno-associated virus (rAAV) particles, comprising: (a) providing an rAAV preparation comprising complete and incomplete rAAV particles; (b) loading a first batch of the rAAV preparation in a loading buffer onto a first column comprising a first chromatography medium, wherein the complete rAAV particles have a higher binding affinity for the first chromatography medium than the incomplete particles, and the amount of complete rAAV particles and incomplete particles applied to the first column exceeds the binding capacity of the first chromatography medium, and the incomplete particles bound to the first chromatography medium are displaced by the complete rAAV particles into a first loading flow-through from the first column; (c) loading the first loading flow-through onto a second column comprising a second chromatographic medium to obtain a loaded or partially loaded second column and a first flow-through from the second column, the second chromatographic medium being of the same type as the first chromatographic medium; (d) optionally washing the first column with a wash buffer to obtain a washed first column; (e) after step (c), or after washing step (d) if performed, bypassing the second column and eluting the complete rAAV particles bound to the first chromatography medium with an elution buffer to obtain a first eluate from the first column and an eluted first column, the first eluate comprising an increased ratio of complete to incomplete rAAV particles; (f) optionally, if the second column is not saturated (i.e., loaded) after the first column flow-through, a second batch of the rAAV preparation in loading buffer may be applied to the partially loaded second column, where the amount of complete rAAV particles and incomplete particles applied to the second column exceeds the binding capacity of the second chromatography medium, and the incomplete particles bound to the second chromatography medium are displaced by complete rAAV particles into the second loading flow-through from the second column; (g) if step (f) is performed, between steps (d) and (e), loading the second loading flow-through from the second column onto the washed first column to obtain a second loaded first column, and optionally washing the second loaded first column before proceeding to elution in step (e); (h) optionally washing the second column with a wash buffer to obtain a washed second column; (i) after step (g), or after washing step (h), if performed, bypassing the first column and eluting the complete rAAV particles bound to the second chromatography medium with an elution buffer to obtain a second eluate and an eluted second column, the second eluate comprising an increased ratio of complete to incomplete rAAV particles; and (j) combining the first eluate and the second eluate to produce a purified preparation of intact rAAV particles. The present invention relates to a method comprising the steps of:

[0095] In some embodiments, the incomplete particles include empty particles. In some embodiments, an incomplete particle comprises a partial particle. In some embodiments, incomplete particles include both empty particles and partial particles. In some embodiments, the first chromatographic medium and / or the second chromatographic medium is an ion exchange column chromatographic medium, preferably an anion exchange column chromatographic medium. In some embodiments, the two-column purification method is set up and operated according to the implementation scheme shown in Figure 12. Steps (a) and (b) of the two-column method are similar to the first two steps of the one-column displacement chromatography method. After these two steps, complete rAAV particles bind to the first chromatography medium, and the first loading flow-through from the first column contains both complete and incomplete rAAV particles. In step (f), a second batch of the rAAV preparation in loading buffer is applied to the second column partially loaded in step (c). The amount of the second batch of rAAV preparation may be the same as the first batch or may be different from the first batch. Preferably, the amount of the second batch is the same as the first batch.

[0096] According to an embodiment of the present application, the amount of complete and incomplete rAAV particles applied to the second column in step (f), including the amount from the first loading flow-through and the amount from the second batch of rAAV preparation, exceeds the binding capacity of the second chromatography medium. During loading of the second batch onto the partially loaded second column, incomplete particles bound to the second chromatography medium are displaced by complete rAAV particles into the second loading flow-through from the second column. Thereafter, in step (g), the second loading flow-through from the second column is loaded onto the previously washed or eluted first column. Then, after step (g), the first column is partially loaded, such that the amount of complete and incomplete rAAV particles in the second loading flow-through from the second column does not now exceed the binding capacity of the first chromatography medium.

[0097] Steps (b)-(i) can be performed in one or multiple cycles depending on the amount of rAAV preparation. The term "one cycle" as used herein refers to performing steps (b)-(i) consecutively one time. The term "multiple cycles" as used herein refers to performing steps (b)-(i) consecutively more than one time. When steps (b) through (i) are performed in multiple cycles, the "first batch" in the repeating step (b) refers to a new "first batch" of rAAV preparation for that cycle number, and the "second batch" in the repeating step (f) refers to a new "second batch" of rAAV preparation for that same cycle.

[0098] In some embodiments, when the incomplete particles include both empty and partial particles, the second eluate of step (i) contains an increased ratio of complete and partial rAAV particles to empty rAAV particles. In some embodiments, when multiple cycles are performed, the eluted column may be subjected to subsequent steps prior to the next loading cycle that are beneficial or necessary to maintain a consistent column binding capacity throughout the cycles, including, but not limited to, column stripping, column cleaning and / or sanitization, and / or column re-equilibration. In some embodiments, the loading buffer comprises at least one buffer selected from the group consisting of Tris, Bis-tris, Bis-tris propane, Tris acetate, ethanolamine, and phosphate. In certain embodiments, the loading buffer comprises a selected buffer at a concentration of about 20-50 mM, for example, 20 mM, 30 mM, 40 mM, 50 mM, or any concentration therebetween. In a preferred embodiment, the loading buffer comprises 20-50 mM Tris.

[0099] In some embodiments, the loading buffer contains 0-200 mM of K(I), Li(I), Ca(II), Mg(II), Cu(II), Ba(II), Co(II), Ni(II), Mn(II), Zn(II), Cd(II), Pb(II), Fe(III), Fe(II), Na(I), and NH4 + The loading buffer preferably comprises at least one salt of a cation selected from the group consisting of: NaCl, MgCl2, LiCl, CuCl2, and CaCl2. The anion component to the salt is not determinative and no particular anion is preferred. For example, the loading buffer may preferably comprise one or more salts selected from the group consisting of NaCl, MgCl2, LiCl, CuCl2, and CaCl2. In some embodiments, the loading buffer comprises a sodium salt at about 10-100 mM, e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mM, or any concentration therebetween. In preferred embodiments, the sodium salt concentration is about 20-60 mM, e.g., 20, 25, 30, 35, 40, 45, 50, 55, 60 mM, or any concentration therebetween.

[0100] In some embodiments, the loading buffer comprises a magnesium salt at a concentration of about 0-20 mM, e.g., 0, 5, 10, 15, 20 mM, or any concentration therebetween. In preferred embodiments, the magnesium salt concentration is about 1-10 mM, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mM, or any concentration therebetween. In some embodiments, the loading buffer comprises a lithium salt at a concentration of about 0-100 mM, e.g., 0, 20, 30, 40, 50, 60, 70, 80, 90, 100 mM, or any concentration therebetween. In preferred embodiments, the lithium salt concentration is about 0-75 mM, e.g., 0, 15, 25, 35, 45, 55, 65, 75 mM, or any concentration therebetween.

[0101] In some embodiments, the loading buffer comprises a calcium salt at a concentration of about 0-10 mM, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mM, or any concentration therebetween. In preferred embodiments, the calcium salt concentration is about 0.1-2.5 mM, e.g., 0.1, 0.5, 1.0, 1.5, 2.0, 2.5 mM, or any concentration therebetween. In some embodiments, the loading buffer comprises a copper salt at a concentration of about 0-5 mM, e.g., 0, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 mM, or any concentration therebetween. In preferred embodiments, the copper salt concentration is about 0.1-2.5 mM, e.g., 0.1, 0.5, 1.0, 1.5, 2.0, 2.5 mM, or any concentration therebetween. In some embodiments, the loading buffer comprises an ammonium salt at about 5-100 mM, e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 mM, or any concentration therebetween. In preferred embodiments, the concentration of ammonium is about 10-50 mM, e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50 mM, or any concentration therebetween.

[0102] In some embodiments, the loading buffer comprises at least one detergent. In some embodiments, the detergent in the loading buffer is selected from the group consisting of poloxamer 188, polysorbate 80, polysorbate 20, NP-40, Triton X-100, and Triton CG-110. In some embodiments, the concentration of the detergent in the loading buffer is between 0.0001% and 0.1%. In some embodiments, the loading buffer has a pH of about 6-10, e.g., 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or any pH therebetween, preferably a pH of about 8-9. In certain embodiments, the loading buffer comprises about 20-60 mM NaCl, 1-5 mM MgCl2, 0.1-2.5 mM CuCl2, preferably in Tris, more preferably in 20-50 mM Tris.

[0103] In certain embodiments, the loading buffer comprises about 20-60 mM NaCl, 50-30 mM (NH4)2SO4, 1-5 mM MgCl2, 0.1-3 mM CuCl2, preferably in Tris, more preferably in 20-50 mM Tris, with 0.00005-0.01% poloxamer 188, more preferably 0.0002-0.001% poloxamer 188. After the loading step, complete particles bind to the chromatographic medium and incomplete particles exit the column into the flow-through. Optionally, the column may be washed with a suitable wash buffer prior to elution. For example, the wash buffer may have an increased salt concentration and / or an increased elution pH compared to the loading conditions to remove incomplete particles bound to the chromatographic medium.

[0104] The intact particles bound to the chromatographic medium are then subsequently eluted in a purer form, for example using an increased salt concentration and / or an adjusted pH for elution compared to the loading conditions. Adjusting the pH can allow for enhanced separation. Preferably, elution of the intact particles is achieved using only a salt gradient without increasing the pH. In some embodiments, the elution buffer comprises at least one buffer selected from the group consisting of Tris, Bis-tris, Bis-tris propane, Tris acetate, ethanolamine, and phosphate. In certain embodiments, the loading buffer comprises a selected buffer at a concentration of about 20-70 mM, for example, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, or any concentration therebetween. In a preferred embodiment, the loading buffer comprises 40-60 mM Tris.

[0105] In some embodiments, the elution buffer contains K(I), Li(I), Ca(II), Mg(II), Cu(II), Ba(II), Co(II), Ni(II), Mn(II), Zn(II), Cd(II), Pb(II), Fe(III), Fe(II), Na(I), and NH4 +For example, the elution buffer may preferably contain one or more salts selected from the group consisting of NaCl, MgCl2, LiCl, CuCl2, and CaCl2. The anion content of the salt is not critical. In some embodiments, the elution buffer contains K(I), Li(I), Ca(II), Mg(II), Cu(II), Ba(II), Co(II), Ni(II), Mn(II), Zn(II), Cd(II), Pb(II), Fe(III), Fe(II), Na(I), and NH4 + For example, the loading buffer may preferably comprise one or more salts selected from the group consisting of NaCl, MgCl2, LiCl, CuCl2, and CaCl2.

[0106] In some embodiments, the elution buffer comprises a sodium salt at about 0-1000 mM, such as 0, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mM, or any concentration therebetween. In preferred embodiments, the sodium salt concentration is about 20-300 mM, such as 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300 mM, or any concentration therebetween. In some embodiments, the elution buffer comprises a magnesium salt at about 0-30 mM, e.g., 0, 5, 10, 15, 20, 25, 30 mM, or any concentration therebetween. In preferred embodiments, the magnesium salt concentration is about 2-15 mM, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 mM, or any concentration therebetween.

[0107] In some embodiments, the elution buffer comprises a lithium salt at a concentration of about 0-200 mM, e.g., 0, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 mM, or any concentration therebetween. In preferred embodiments, the lithium salt concentration is about 0-150 mM, e.g., 0, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 mM, or any concentration therebetween. In some embodiments, the elution buffer comprises a calcium salt at a concentration of about 0.1-20 mM, e.g., 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20 mM, or any concentration therebetween. In preferred embodiments, the calcium salt concentration is about 5-10 mM, e.g., 5, 6, 7, 8, 9, 10 mM, or any concentration therebetween.

[0108] In some embodiments, the elution buffer comprises a copper salt at a concentration of about 0-5 mM, e.g., 0, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 mM, or any concentration therebetween. In preferred embodiments, the copper salt concentration is about 0.1-2.5 mM, e.g., 0.1, 0.5, 1.0, 1.5, 2.0, 2.5 mM, or any concentration therebetween. In some embodiments, the elution buffer comprises an ammonium salt at about 5-100 mM, e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 mM, or any concentration therebetween. In preferred embodiments, the concentration of ammonium is about 10-50 mM, e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50 mM, or any concentration therebetween. In some embodiments, the elution buffer has a pH of about 6-10, such as 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or any pH therebetween, preferably a pH of about 8-9.

[0109] In certain embodiments, the elution buffer comprises about 20-150 mM NaCl, preferably in Tris, more preferably in 40-60 mM Tris. In some embodiments, the elution buffer comprises at least one detergent. In some embodiments, the detergent in the elution buffer is selected from the group consisting of poloxamer 188, polysorbate 80, polysorbate 20, NP-40, Triton X-100, and Triton CG-110. In some embodiments, the concentration of the detergent in the elution buffer is between 0.0001% and 0.1%.

[0110] In some embodiments, the method uses at least two columns, e.g., three columns, for purification of intact rAAV particles. In particular, the method does not include steps (g)-(j), but (k) after step (f), loading the second loading flow-through from the second column onto a third column comprising a third chromatographic medium to obtain a loaded or partially loaded third column, preferably the third chromatographic medium being of the same type as the first chromatographic medium; (l) optionally washing the second column with a wash buffer to obtain a washed second column; (m) after step (k), or after washing step (l), if performed, bypassing the first column and eluting the complete rAAV particles bound to the second chromatography medium with an elution buffer to obtain a second eluate and an eluted second column, the second eluate comprising an increased ratio of complete to incomplete rAAV particles; (n) optionally, if the third column is not saturated (i.e., loaded) after the second column flow-through, a third batch of the rAAV preparation in loading buffer may be applied to the partially loaded third column, where the amount of complete rAAV particles and incomplete particles applied to the third column exceeds the binding capacity of the third chromatography medium, and the incomplete particles bound to the third chromatography medium are displaced by complete rAAV particles into the third loading flow-through from the third column; (o) if step (n) is performed, between steps (d) and (e), loading the third loaded flow-through from the third column onto a washed first column to obtain a second loaded first column, and optionally washing the second loaded first column before proceeding with elution in step (e); (p) optionally washing the third column with a wash buffer to obtain a washed third column; (q) bypassing the first column and the second column and eluting the complete rAAV particles bound to the third chromatography medium with an elution buffer to obtain a third eluate and an eluted third column, the third eluate comprising an increased ratio of complete to incomplete rAAV particles; and (r) combining the first eluate, the second eluate, and the third eluate to generate a purified preparation of intact rAAV particles. Further includes.

[0111] In some embodiments, when the incomplete particles include both empty and partial particles, the complete particles are enriched in a first column, the partial particles are enriched in a second column, and the empty particles are enriched in a third column.

[0112] To the inventors' knowledge, there is no substitution chromatography method for purifying complete rAAV particles from incomplete rAAV particles. Compared to existing purification methods for purifying complete particles, such as conventional anion exchange chromatography, the purity of complete particles after the method of the present application is significantly increased. In some embodiments, the purified preparation is substantially free of incomplete particles, more particularly, substantially free of empty particles and / or partial particles. In other embodiments, the purified preparation contains an increased ratio of complete rAAV particles to incomplete particles than the rAAV preparation. Preferably, the ratio of complete rAAV particles to incomplete particles in the purified preparation is 9:1 or more, e.g., 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, or 50:1 or more, or any ratio therebetween, more preferably 49:1 or more.

[0113] According to embodiments of the present application, the ratio of complete rAAV particles to incomplete particles in a purified preparation can be calculated by the number of complete rAAV particles and incomplete particles. In some embodiments, the ratio is derived from a calibration curve based on the molar concentrations of complete rAAV particles and incomplete particles. In some embodiments, the ratio is calculated based on the number of complete rAAV particles and incomplete particles.

[0114] In some embodiments, the ratio of complete to incomplete particles in the purified complete rAAV particles is 9:1 or greater, preferably 49:1 or greater. The displacement chromatography method of the present application can also achieve high yields / recoveries of purified complete particles. In some embodiments, the yield of purified complete rAAV particles is 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more. The term "yield" as used herein refers to the percentage or ratio of complete rAAV particles in the purified preparation relative to the complete rAAV particles in the initial rAAV preparation. There are multiple ways to calculate the yield. One way to calculate the yield is percent (%) = (amount of complete rAAV particles in the purified preparation) / (amount of complete rAAV particles in the initial rAAV preparation) x 100. Another way to calculate the yield percentage is to divide the number of transgene copies in the purified preparation by the number of transgene copies in the initial preparation and multiply by 100.

[0115] In another basic aspect, the present application provides a method for purifying complete recombinant adeno-associated virus (rAAV) particles, comprising: (a) providing an rAAV preparation comprising complete and incomplete rAAV particles; (b) loading the rAAV preparation in a loading buffer onto a column containing a chromatography medium, the loading buffer containing CaCl2, and wherein the intact rAAV particles bind to the chromatography medium; (c) eluting the intact rAAV particles bound to the chromatography medium with an elution buffer to obtain a purified preparation, the elution buffer optionally containing CaCl. The present invention relates to a method comprising the steps of:

[0116] In some embodiments, the chromatography medium is an ion exchange column chromatography medium, preferably an anion exchange chromatography medium. In some embodiments, the column chromatography media is selected from the group consisting of Poros HQ, Poros PD, polyethyleneimine (PI), Capto ImpRes Q, and Poros XQ, preferably Poros XQ. In some embodiments, the amount of complete rAAV particles and incomplete particles applied to the column does not exceed the binding capacity of the chromatography medium.

[0117] In some embodiments, the amount of complete and incomplete rAAV particles applied to the column exceeds the binding capacity of the chromatography medium, and incomplete particles bound to the first chromatography medium are displaced by complete rAAV particles into the first loading flow-through from the first column. In some embodiments, the incomplete particles include empty particles. In some embodiments, an incomplete particle comprises a partial particle. In some embodiments, incomplete particles include both empty particles and partial particles.

[0118] According to an embodiment of the present application, K(I), Li(I), Ca(II), Mg(II), Cu(II), Ba(II), Co(II), Ni(II), Mn(II), Zn(II), Cd(II), Pb(II), Fe(III), Fe(II), Na(I), and NH4 + By adding a salt of a cation selected from the group consisting of to the loading buffer, the difference in affinity for the chromatography medium can be increased such that more complete rAAV particles bind to the chromatography medium and fewer incomplete particles bind to the chromatography medium. Thus, the loading flow-through from the column contains fewer complete particles, thus increasing the recovery of complete particles. The anion content of a given salt is not critical.

[0119] In some embodiments, the loading buffer comprises a calcium salt at a concentration of about 0-10 mM, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mM, or any concentration therebetween. In preferred embodiments, the calcium salt concentration is about 0.1-2.5 mM, e.g., 0.1, 0.5, 1.0, 1.5, 2.0, 2.5 mM, or any concentration therebetween. In some embodiments, the loading buffer comprises a lithium salt at a concentration of about 0-100 mM, e.g., 0, 20, 30, 40, 50, 60, 70, 80, 90, 100 mM, or any concentration therebetween. In preferred embodiments, the lithium salt concentration is about 0-75 mM, e.g., 0, 15, 25, 35, 45, 55, 65, 75 mM, or any concentration therebetween. In some embodiments, the loading buffer comprises at least one buffer selected from the group consisting of Tris, Bis-tris, Bis-tris propane, Tris acetate, ethanolamine, and phosphate. In certain embodiments, the loading buffer comprises a selected buffer at a concentration of about 20-50 mM, such as 20 mM, 30 mM, 40 mM, 50 mM, or any concentration therebetween. In a preferred embodiment, the loading buffer comprises 20-50 mM Tris.

[0120] In some embodiments, the loading buffer further comprises a sodium salt and / or a magnesium salt, hi a preferred embodiment, the loading buffer further comprises both a sodium salt and a magnesium salt. In some embodiments, the loading buffer comprises a sodium salt at about 10-100 mM, e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mM, or any concentration therebetween. In preferred embodiments, the sodium salt concentration is about 20-60 mM, e.g., 20, 25, 30, 35, 40, 45, 50, 55, 60 mM, or any concentration therebetween. In some embodiments, the loading buffer comprises a magnesium salt at a concentration of about 0-20 mM, e.g., 0, 5, 10, 15, 20 mM, or any concentration therebetween. In preferred embodiments, the magnesium salt concentration is about 1-10 mM, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mM, or any concentration therebetween.

[0121] In some embodiments, the loading buffer has a pH of about 6-10, e.g., 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or any pH therebetween, preferably a pH of about 8-9. In certain embodiments, the loading buffer preferably comprises about 20-60 mM NaCl, 1-5 mM MgCl2, 0.1-2.5 mM CaCl2 in Tris, more preferably in 20-50 mM Tris. In some embodiments, the loading buffer comprises at least one detergent. In some embodiments, the detergent in the loading buffer is selected from the group consisting of poloxamer 188, polysorbate 80, polysorbate 20, NP-40, Triton X-100, and Triton CG-110. In some embodiments, the concentration of the detergent in the loading buffer is between 0.0001% and 0.1%.

[0122] After the loading step, complete particles bind to the chromatographic medium and incomplete particles exit the column into the flow-through. Optionally, the column may be washed with a suitable wash buffer. For example, the wash buffer may have an increased salt concentration and / or an adjusted pH for elution compared to the loading conditions to remove incomplete particles bound to the chromatographic medium. The complete particles bound to the chromatography medium are then subsequently eluted in a purer form, for example, using an increased salt concentration and / or an adjusted pH for elution compared to the loading conditions. Adjusting the pH can increase the separation between complete and incomplete rAAV particles. Preferably, elution of complete particles is achieved using only a salt gradient without increasing the pH. In some embodiments, the elution buffer comprises at least one buffer selected from the group consisting of Tris, Bis-tris, Bis-tris propane, Tris acetate, ethanolamine, and phosphate.

[0123] In some embodiments, the elution buffer contains K(I), Li(I), Ca(II), Mg(II), Cu(II), Ba(II), Co(II), Ni(II), Mn(II), Zn(II), Cd(II), Pb(II), Fe(III), Fe(II), Na(I), and NH4 +For example, the elution buffer may preferably contain one or more salts selected from the group consisting of NaCl, MgCl2, LiCl, CuCl2, and CaCl2. The anion content of the salt is not critical. In some embodiments, the elution buffer contains K(I), Li(I), Ca(II), Mg(II), Cu(II), Ba(II), Co(II), Ni(II), Mn(II), Zn(II), Cd(II), Pb(II), Fe(III), Fe(II), Na(I), and NH4 + For example, the loading buffer may preferably comprise one or more salts selected from the group consisting of NaCl, MgCl2, LiCl, CuCl2, and CaCl2.

[0124] In some embodiments, the elution buffer comprises a calcium salt at a concentration of about 0.1-20 mM, e.g., 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20 mM, or any concentration therebetween. In preferred embodiments, the calcium salt concentration is about 5-10 mM, e.g., 5, 6, 7, 8, 9, 10 mM, or any concentration therebetween. In some embodiments, the elution buffer comprises a lithium salt at a concentration of about 0-200 mM, e.g., 0, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 mM, or any concentration therebetween. In preferred embodiments, the lithium salt concentration is about 0-150 mM, e.g., 0, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 mM, or any concentration therebetween.

[0125] In some embodiments, the elution buffer comprises at least one buffer selected from the group consisting of Tris, Bis-tris, Bis-tris propane, Tris acetate, and phosphate. In certain embodiments, the loading buffer comprises a selected buffer at a concentration of about 20-70 mM, for example, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, or any concentration therebetween. In a preferred embodiment, the loading buffer comprises 40-60 mM Tris. In some embodiments, the elution buffer comprises at least one detergent. In some embodiments, the detergent in the elution buffer is selected from the group consisting of poloxamer 188, polysorbate 80, polysorbate 20, NP-40, Triton X-100, and Triton CG-110. In some embodiments, the concentration of the detergent in the elution buffer is between 0.0001% and 0.1%. In some embodiments, the elution buffer further comprises a sodium salt and / or a magnesium salt. In a preferred embodiment, the loading buffer further comprises both a sodium salt and a magnesium salt. The anionic component of the salt is not critical.

[0126] In some embodiments, the elution buffer comprises a sodium salt at about 0-1000 mM, e.g., 0, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mM, or any concentration therebetween. In preferred embodiments, the sodium salt concentration is about 20-150 mM, e.g., 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 mM, or any concentration therebetween. In some embodiments, the elution buffer comprises a magnesium salt at about 0-30 mM, e.g., 0, 5, 10, 15, 20, 25, 30 mM, or any concentration therebetween. In preferred embodiments, the magnesium salt concentration is about 2-15 mM, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 mM, or any concentration therebetween. In some embodiments, the elution buffer has a pH of about 6-10, such as 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or any pH therebetween, preferably a pH of about 8-9.

[0127] In certain embodiments, the elution buffer comprises about 20-150 mM NaCl, preferably in Tris, more preferably in 40-60 mM Tris. In some embodiments, the purified preparation is substantially free of incomplete particles. In other embodiments, the purified preparation comprises an increased ratio of complete rAAV particles to incomplete particles than the rAAV preparation. Preferably, the ratio of complete rAAV particles to incomplete particles in the purified preparation is 9:1 or greater, e.g., 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, or 50:1 or greater, or any ratio therebetween, more preferably 49:1 or greater. According to embodiments of the present application, the ratio of complete rAAV particles to incomplete particles in a purified preparation can be calculated by the number of complete rAAV particles and incomplete particles. In some embodiments, the ratio is derived from a calibration curve based on the molar concentrations of complete rAAV particles and incomplete particles. In some embodiments, the ratio is calculated based on the number of complete rAAV particles and incomplete particles.

[0128] In some embodiments, the ratio of complete to incomplete particles in the purified complete rAAV particles is 9:1 or greater, preferably 49:1 or greater. In some embodiments, the yield of purified complete rAAV particles is 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more. The term "yield" as used herein refers to the percentage of complete rAAV particles in the purified preparation relative to the complete rAAV particles in the initial rAAV preparation. For example, yield (%) = (amount of complete rAAV particles in the purified preparation) / (amount of complete rAAV particles in the initial rAAV preparation), as a percentage. The following embodiments apply to each of the basic aspects disclosed herein, including those described above. In some embodiments, the complete rAAV particle comprises a transgene encoding a polypeptide, a nucleic acid encoding a protein or transcribed into a transcript of interest, or a nucleic acid selected from the group consisting of an siRNA, an antisense molecule, an miRNA, a ribozyme, and an shRNA.

[0129] Various embodiments disclosed herein are applicable to any rAAV or AAV capsid, particle, impurity, or aggregate capable of binding to an ion exchange chromatography column, regardless of the source or serotype of the capsid. Because the methods of the present disclosure are applicable to any AAV or rAAV capsid capable of binding to an ion exchange chromatography column, the capsid may be from any source, for example, human, avian, bovine, canine, equine, primate, non-primate, ovine, or derivatives thereof.

[0130] In some embodiments, the rAAV particles comprise AAV capsids having peptide modifications, such as cell-targeting peptides, as described in Pulicherla et al., Mol. Ther., 19(6):1070-1078 (2011) (which describes, among other things, AAV9 variants, including AAV9.47); U.S. Pat. No. 7,906,111 (which describes, among other things, AAV9(hu14)); U.S. Pat. No. 10,532,623; ,111 (in which NP59 is described), 10,738,087 (in which Anc-80 is described), 9,169,299 (in which "LK03" is described), 9,840,719 (in which "RHM4-1" is described), 7,749,492, 7,588,772 (in which "DJ" and "DJ8" are described), 9,587, 282, and variants of the AAV capsid set forth in WO 2012 / 145601, WO 2013 / 158879, WO 2015 / 013313, WO 2018 / 156654, and U.S. Patent Application Publication No. 2013 / 0059732, all of which are incorporated herein by reference in their entireties. The vectors are derived from one or more AAVs selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, Rh8, Rh10, Rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ, DJ8, NP59, Anc-80, and variants thereof, including those derived from the vectors.

[0131] In some embodiments, the complete rAAV particle comprises a transgene encoding a gene product selected from the group consisting of 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 (TGFa), platelet-derived growth factor (PDGF), insulin growth factor I and II (IGF-I and IGF-II), TGFp, 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.

[0132] In some embodiments, the complete rAAV 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.

[0133] In some embodiments, the complete rAAV particle contains 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, cystathione beta synthase, branched-chain keto acid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylase, ribozyme, ribozyme, ribozyme-binding protein (RIK), ... carboxylate), hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, RPE65, H-protein, T-protein, cystic fibrosis transmembrane conductance regulator (CFTR) sequence, and dystrophin cDNA sequence.

[0134] In some embodiments, the complete rAAV particles contain transgenes encoding factors VIII and IX. The complete rAAV particles of interest to be purified by the methods according to the embodiments of the present application can be produced in host cells. 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 producing rAAV virions (suspension or adherence) were cultivated. The harvested cells and optionally the cell culture supernatant can be used as is or concentrated as required in the methods herein. Furthermore, when infection is used to express accessory functions, residual helper virus can be inactivated. For example, adenovirus can be inactivated by heating, for example, to a temperature of approximately 60° C. for 20 minutes or more, thereby inactivating only the helper virus, since AAV is heat stable, whereas the helper adenovirus is heat labile.

[0135] The harvested cells and / or supernatant are lysed by disrupting the cells by chemical or physical means, such as, for example, detergents, microfluidization, and / or homogenization, to release the rAAV particles. A nuclease, such as benzonase, may be added simultaneously with or subsequent to cell lysis to degrade contaminating DNA. Typically, the resulting lysate is clarified by filtration, centrifugation, or the like to remove cell debris, resulting in a clarified cell lysate. In certain instances, the lysate is filtered through a micron diameter pore size filter (e.g., a 0.1-10.0 μm pore size filter, such as a 0.45 μm and / or 0.2 μm pore size filter) to produce a clarified lysate.

[0136] The lysate (optionally clarified) contains rAAV particles (complete rAAV particles and AAV incomplete particles) and soluble cellular components derived from the host cells, which may include AAV vector production / process related impurities, such as cellular proteins, lipids, and / or nucleic acids, among others, as well as cell culture media components. If necessary, the clarified lysate is then subjected to an additional purification step to purify the complete AAV particles from impurities using chromatography. The clarified lysate may be diluted or concentrated with an appropriate buffer prior to the chromatographic methods of the present application. EXAMPLES

[0137] Example 1 Resin screening to separate complete and incomplete rAAV particles In this example, various resins were screened to identify the best for separating complete and incomplete rAAV particles. Methods and Materials The sample contained incomplete and complete RHM4-1 rAAV particles in a ratio of 2.4:1 (i.e., 70% impurities / 30% product). The column size was 0.353 mL 3 mm x 50 mm. Resin screening was performed using pulse loading (10% of binding capacity) and a residence time of 1 minute. During screening, three binding strengths with three pH values ​​(8.0, 8.6, and 9.25) and salt (6.8, 5, and 1.6 mS / cm) were tested. The loading buffer was 50 mM Tris or 25 mM Tris. The flow-through from the column was analyzed by high performance liquid chromatography (HPLC) to determine the separation of complete and incomplete particles.

[0138] result Poros 50HQ resin (Thermo Scientific; POROS™ - Waltham, Massachusetts): This resin is based on quaternized polyethyleneimine functional groups with some weak anion exchange (AEX) functionality. As shown in Figures 1A-1C, the separation of complete and incomplete particles on Poros 50HQ resin was not significantly different at all three pH values ​​in 50 mM Tris and 30 mM NaCl, with the separation at pH 8.6 being only slightly better compared to the other two pH values. Poros 50D resin: This resin is based on dimethylaminopropyl functional groups. As shown in Figures 2A-2D, binding of rAAV particles to Poros 50D resin was poor under screening conditions and decreased with increasing pH. Various loading buffers were used in the figures: pH 8.6 and 50 mM Tris (Figure 2A), pH 8.6 and 25 mM Tris (Figure 2B), pH 8.0 and 25 mM Tris (Figure 2C), and pH 9.25 and 25 mM Tris (Figure 2D).

[0139] Poros50PI resin: This resin is based on polyethyleneimine functional groups. As shown in Figures 3A-3E, the binding of rAAV particles to Poros50PI resin decreased with increasing pH. Note that the addition of NaCl also slightly improved the resolution. In these figures, Poros50PI resin at different pH and salt concentrations is evaluated: pH 8.0, no NaCl (Figure 3A), pH 8.6, no NaCl (Figure 3B), pH 8.6 and 30 mM NaCl (Figure 3C), pH 9.2, no NaCl (Figure 4C), pH 9.2 and 30 mM NaCl (Figure 3E). Capto ImpRes Q resin: This resin uses an agarose matrix based on Capto™ (Cytiva Life Sciences-Marlborough, MA) with ionic group ligands. As shown in Figures 4A-4C, binding of rAAV particles to Capto ImpRes Q resin increased with increasing pH. However, under screening conditions, separation of complete and incomplete particles was poor with Capto ImpRes Q resin. In these figures, Capto ImpRes Q resin is evaluated with different buffers at different pH values: pH 8.0 and 50 nM Tris, no NaCl (Figure 4A), pH 8.6 and 25 mM Tris, no NaCl (Figure 4B), and pH 9.0 and 30 mM Tris, no NaCl (Figure 4C).

[0140] Poros XQ resin: This resin is based on a unique quaternary amine functional group. Screening showed that Poros XQ resin is best for separating complete and incomplete particles. As shown in Figure 5, separation was best at pH 8.75 compared to other pH values ​​(pH 8 and 9.25). As shown in Figure 6, binding salt also affected separation. In addition, as shown in Figure 7, separation was similar under different flow rates (84 cm / h, 150 cm / h, and 300 cm / h) when performed at pH 8.75.

[0141] Example 2 Optimization of displacement chromatography using Poros XQ resin. In this example, different conditions were screened to identify optimal conditions for the displacement chromatographic separation of complete RHM4-1 rAAV particles from incomplete particles on Poros XQ resin. Figure 8A shows that when the sample was loaded in 30 mM Tris, pH 8.6, and the loading amount was low, there were two product peaks in the HPLC analysis of the flow-through. Of the two peaks, the ratio of incomplete to complete particles (E / F) was 9.9 for peak P1, whereas the E / F ratio for peak P2 was 0.8. This indicates that the affinity of incomplete particles to bind to the Poros XQ resin was weaker than that of complete particles to bind to the resin, and therefore the incomplete particles eluted earlier than the complete particles. In contrast, when the amount of sample loaded exceeded the binding capacity, the E / F ratio in the flow-through (FT) was 2.4 at breakthrough, as shown in Figure 8B. Figure 8B shows that the addition of 30 mM NaCl to the load was not sufficient to promote displacement. Thus, the E / F ratio of the flow-through is the same as that of the load. Next, we screened different binding strengths at different salt concentrations to identify the optimal salt concentration for better separation of incomplete and complete particles under displacement column chromatography. As shown in Figures 9A and 9B, increasing the NaCl concentration in the loading buffer from 45 mM to 60 mM, respectively, increased the E / F ratio in the flow-through from 3.4 to 229, indicating a greater displacement of incomplete particles by complete particles at higher salt concentrations. At the same time, the purity of product peak P2 was improved from E / F=1 to E / F=0.7.

[0142] However, further increasing the salt concentration to 75 mM and 90 mM did not further enhance displacement because it reduced the overall affinity of the sample for the resin, as shown in Figures 9C and 9D, respectively. Thus, Figures 9A-9D show that optimal displacement occurred at intermediate binding strengths, where the difference in binding affinity between imperfect and complete particles was most pronounced. More generally, these results indicate that salt-tunable binding strengths can be used as a clue to facilitate displacement of imperfect particles, thereby improving resolution. In addition, step elution conditions were also tested to determine the salt range in the elution buffer. The loading buffer used in the test contained 50 mM Tris pH 8.5 and 30 mM NaCl. As shown in Figures 10A-10D, step elution worked with all tested elution buffers with NaCl concentrations of 60 mM, 75 mM, 90 mM, and 120 mM, respectively.

[0143] In this example, further displacement was also studied. As shown in Figures 11A-11B, when more sample was loaded beyond the breakthrough load, further displacement occurred, and the incomplete particles bound to the bottom part of the column were also displaced by complete particles and were present in the flow-through. Figure 11B showed that further displacement improved the purity of rAAV in the purified preparation to nearly 100%. In addition, further displacement chromatography also yielded purified complete particles at a high yield, higher than 90%.

[0144] Example 3 Use of CaCl2 as an additive in sample loading and washing buffers In a study of the separation of complete and incomplete RHM4-1 rAAV particles using Poros XQ resin, product losses were observed in displacement chromatography purification. For example, in a breakthrough run, when the loading buffer was 50 mM Tris / 60 mM NaCl, pH 8.5, the recovery yield was 89.81% for the single-column method and 74.92% for the proposed two-column method. The losses were primarily due to the presence of complete particles in the flow-through. To improve resolution and reduce the amount of complete rAAV particles in the flow-through, and therefore increase the recovery yield, the addition of additives to the sample loading or wash buffers was investigated. As shown in FIG. 12, the addition of MgCl2 to the sample load was first tested. Using MgCl2 as an additive, the resolution could be improved. However, in the subsequent washing steps, the concentration of MgCl2 had to be increased to wash away the incomplete particles bound to the column. The addition of MgCl2 to the sample load was found to be not robust even under non-binding conditions, since the difference in conductivity between the loading and elution buffers was only about 1 mS / cm.

[0145] Other additives were also tested. CaCl2 was identified as the additive that provided the best flow-through for purification with Poros XQ resin. As shown in FIG. 13, the addition of CaCl2 improved the separation of incomplete and complete rAAV particles. In addition, with CaCl2, the difference in conductivity between the loading and elution buffers was robust, e.g., approximately 4 mS / cm. It was also found that both yield and product purity were compromised in the purification of complete rAAV particles from incomplete particles when CaCl2 was not added to the sample load. As shown in Figure 15, when no additives other than NaCl were used in the sample load and the wash buffer contained 1 mM CaCl2 and 2.5 mM MgCl2, it was difficult to remove all of the incomplete particles bound to the column by washing. Furthermore, during subsequent elution with increasing CaCl2 concentrations (Figure 15), the bound incomplete particles were unstable on the resin and new impurities were likely generated, which in turn affected product purity. Thus, the inventors have surprisingly found that the addition of CaCl2 to the sample load, preferably to the wash and elution buffers, results in optimal separation and improved yields.

[0146] Example 4 Use of LiCl as an additive in sample loading and washing buffers Another additive tested for purification on Poros XQ resin was lithium chloride. As shown in Figure 17, the addition of LiCl improved the separation between incomplete and complete RHM4-1 rAAV particles. In addition, with LiCl, the difference in conductivity between the loading and elution buffers was approximately 0.2 mS / cm.

[0147] Example 5 Enhanced separation and yield in displacement chromatography by adding CaCl2 to sample load and wash buffers Improved separation of incomplete and complete RHM4-1 rAAV particles by using additives such as CaCl2 in the loading buffer and optionally the wash and elution buffers, as shown in Examples 3 and 4, can also be used in conjunction with the displacement chromatography method shown in Example 2. These methods, when combined as shown in Figure 18, will result in optimal separation and yield of complete versus incomplete rAAV particles.

[0148] Example 6 Enhancement of separation by additives and pH reduction The improved separation of incomplete and complete RHM4-1 rAAV particles by using additives such as CaCl2 in the loading buffer and optionally the wash and elution buffers, as shown in Examples 3 and 4, can be enhanced by decreasing the pH using gradient or step elution, as shown in Figure 19.

[0149] Example 7 Enhancement of separation by additive (NH4)2SO4 Improved separation of empty and full RHM4-1 rAAV particles on PXQ resin can be achieved by using (NH4)2SO4 as an additive in the loading buffer, as shown in Figures 20A-20B.

[0150] Example 8 Enhancement of separation by additive (NH4)2SO4 Improved separation of empty and complete RHM4-1 rAAV particles on the BIA monolith resin can be achieved by using (NH4)2SO4 as an additive in the loading buffer, as shown in Figure 21.

[0151] Example 9 Enhancement of separation by CuCl2 additive Improved separation of empty and full RHM4-1 rAAV particles on PXQ resin can be achieved by using CuCl2 as an additive in the loading buffer, as shown in Figures 22A-22E.

[0152] Example 10 Enhanced separation of partial particles with CuCl2 and elution with lower pH and increasing salt Enhanced separation of empty, partial, and full LK03 rAAV particles by using additives such as CuCl2 in the loading buffer can be enhanced by gradient or step elution with increasing salt at lower pH, as shown in Figures 23A-23E. Example 11 Improved separation of partial particles using multiple columns The improved separation of empty, partial, and full LK03 rAAV particles observed using two columns can be enhanced by using at least three columns, as shown in Figures 24-26. This method can be used to remove impurities and aggregates that have a stronger affinity for the column than the full particles, as shown in Figure 25. In this two-column setup, the second column is enriched for full particles because aggregates and impurities that have a stronger affinity for the column than the full particles bind to the first column. In a multiple column setup, the first column enriches for the strongest binding particles (highest retention time), and the subsequent columns enrich for the next strongest binding particles. Once the column to which the target rAAV particles or impurities have the greatest affinity has been determined, the methods of the present disclosure can be used to purify the desired rAAV particles or impurities. This example further illustrates the use of the methods of the present disclosure to enrich for different particle variants in a multiple column setup.

[0153] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that modifications can be made to the above-described embodiments without departing from the broad inventive concept thereof. For example, the inventive concept includes separating partial particles from empty particles, or separating impurities that have a stronger affinity for the resin than full particles, as shown in Figure 25, and enriching full particles in a second column as opposed to a first column. It is therefore understood that the invention is not limited to the specific embodiments disclosed, but is intended to cover modifications within the spirit and scope of the invention as defined by the appended claims.

Claims

1. A method for purifying fully recombinant adeno-associated virus (rAAV) particles, comprising: (a) preparing an rAAV preparation comprising fully rAAV particles and incomplete rAAV particles; (b) loading the rAAV preparation in a loading buffer onto a column containing a chromatography medium, wherein the fully rAAV particles have a higher binding affinity for the chromatography medium than the incomplete particles, and the amounts of the fully rAAV particles and the incomplete particles applied to the column exceed the binding capacity of the chromatography medium, and the incomplete particles bound to the chromatography medium are displaced by the fully rAAV particles and enter from the column into the flow-through; and (c) eluting the fully rAAV particles bound to the chromatography medium with an elution buffer to obtain a purified preparation. A method comprising the above steps.

2. The method according to claim 1, wherein the chromatography medium is an ion exchange column chromatography medium, preferably an anion exchange chromatography medium.

3. The column chromatography medium is selected from the group consisting of Poros 50 HQ, Poros 50 D, Poros 50 PI, Capto ImpRes Q, CIMmultus TM QA monolithic column, and Poros XQ, preferably Poros XQ. The method according to claim 2.

4. A method for purifying fully recombinant adeno-associated virus (rAAV) particles, comprising: (a) preparing an rAAV preparation comprising the fully rAAV particles and incomplete particles; (b) loading a first batch of the rAAV preparation in the loading buffer onto a first column comprising a first chromatography medium, wherein the full rAAV particles have a higher binding affinity for the first chromatography medium than the incomplete particles, and the amounts of the full rAAV particles and the incomplete particles applied to the first column exceed the binding capacity of the first chromatography medium, and the incomplete particles bound to the first chromatography medium are displaced by the full rAAV particles and enter the first load flow-through from the first column, (c) loading the first load flow-through onto a second column comprising a second chromatography medium to obtain a loaded or partially loaded second column, wherein the second chromatography medium is of the same type as the first chromatography medium, (d) optionally, washing the first column with a washing buffer to obtain a washed first column, (e) eluting the full rAAV particles bound to the first chromatography medium with an elution buffer to obtain a first eluate from the first column and an eluted first column, wherein the first eluate comprises an increased ratio of the full rAAV particles to the incomplete rAAV particles, (f) optionally, applying a second batch of the rAAV preparation in the loading buffer to the at least partially loaded second column, wherein the amounts of the full rAAV particles and the incomplete particles applied to the second column exceed the binding capacity of the second chromatography medium, and the incomplete particles bound to the second chromatography medium are displaced by the full rAAV particles and enter the second load flow-through from the second column, (g) optionally, after step (e), loading the load flow-through from the second column onto the eluted first column to obtain a second loaded first column, and optionally, washing the second loaded first column before performing the elution in step (e), (h) Optionally, washing the second column with a washing buffer to obtain a washed second column; (i) Eluting the fully rAAV particles bound to the second chromatography medium with an elution buffer to obtain a second eluate and an eluted second column, wherein the second eluate comprises an increased ratio of the fully rAAV particles to the incomplete rAAV particles; and (j) Combining the first eluate and the second eluate to produce a purified preparation of fully rAAV particles A method comprising.

5. The method according to claim 4, wherein the first chromatography medium and / or the second chromatography medium is an ion exchange column chromatography medium, preferably an anion exchange column chromatography medium.

6. The method according to claim 4, wherein steps (b) to (i) are carried out in one or more cycles.

7. The method according to claim 4, wherein the second column is partially loaded after step (c).

8. The first column chromatography medium is selected from the group consisting of Poros 50HQ, Poros 50D, Poros 50PI, Capto ImpRes Q, CIMmultus TM QA monolithic column, and Poros XQ, preferably Poros XQ, the method according to claim 5.

9. A method for purifying fully recombinant adeno-associated virus (rAAV) particles, comprising: (a) preparing an rAAV preparation comprising the fully rAAV particles and incomplete particles; (b) loading a first batch of the rAAV preparation in the loading buffer onto a first column comprising a first chromatography medium, wherein the full rAAV particles have a higher binding affinity for the first chromatography medium than the incomplete particles, and the amounts of the full rAAV particles and the incomplete particles applied to the first column exceed the binding capacity of the first chromatography medium, and the incomplete particles bound to the first chromatography medium are displaced by the full rAAV particles and enter from the first column into a first load flow-through; (c) loading the first load flow-through onto a second column comprising a second chromatography medium to obtain a loaded or partially loaded second column, wherein the second chromatography medium is of the same type as the first chromatography medium; (d) optionally, washing the first column with a washing buffer to obtain a washed first column; (e) eluting the full rAAV particles bound to the first chromatography medium with an elution buffer to obtain a first eluate from the first column and an eluted first column, wherein the first eluate comprises an increased ratio of the full rAAV particles to the incomplete rAAV particles; (f) optionally, applying a second batch of the rAAV preparation in the loading buffer to the at least partially loaded second column, wherein the amounts of the full rAAV particles and the incomplete particles applied to the second column exceed the binding capacity of the second chromatography medium, and the incomplete particles bound to the second chromatography medium are displaced by the full rAAV particles and enter from the second column into a second load flow-through; (g) loading the second load flow-through from the second column onto a third column comprising a third chromatography medium to obtain a loaded or partially loaded third column, preferably, the third chromatography medium is of the same type as the first chromatography medium; (h) optionally, washing the second column with a wash buffer to obtain a washed second column; (i) after step (k), or if a washing step (l) is performed, after the washing step (l), bypassing the first column and eluting the fully rAAV particles bound to the second chromatography medium with an elution buffer to obtain a second eluate and an eluted second column, the second eluate comprising an increased ratio of the fully rAAV particles to the incomplete rAAV particles; (j) optionally, if the third column is not saturated (i.e., loaded) after the second column flow-through, a third batch of the rAAV preparation in a loading buffer may be applied to the partially loaded third column, the amount of the fully rAAV particles and the incomplete particles applied to the third column exceeding the binding capacity of the third chromatography medium, the incomplete particles bound to the third chromatography medium being displaced by the fully rAAV particles and entering from the third column into a third load flow-through; (k) if step (n) is performed, between steps (d) and (e), loading the third load flow-through from the third column onto the washed first column to obtain a second loaded first column, and optionally, washing the second loaded first column before performing elution in step (e); (l) optionally, washing the third column with a wash buffer to obtain a washed third column; (m) bypassing the first column and the second column and eluting the fully rAAV particles bound to the third chromatography medium with an elution buffer to obtain a third eluate and an eluted third column, wherein the third eluate comprises an increased ratio of the fully rAAV particles to the incomplete rAAV particles, and (n) combining the first eluate, the second eluate, and the third eluate to produce a purified preparation of fully rAAV particles A method comprising.

10. The method according to claim 9, wherein the first chromatography medium and / or the second chromatography medium and / or the third chromatography medium is an ion exchange column chromatography medium, preferably an anion exchange column chromatography medium.

11. The method according to claim 9, wherein steps (b) to (m) are carried out in one or more cycles.

12. The method according to claim 9, wherein the second column is partially loaded after step (c).

13. The method according to claim 9, wherein the third column is partially loaded after step (g).

14. The first column chromatography medium is selected from the group consisting of Poros 50 HQ, Poros 50 D, Poros 50 PI, Capto ImpRes Q, CIMmultus TM QA monolithic column, and Poros XQ, preferably Poros XQ, the method according to claim 9.

15. The method according to any one of claims 1 to 14, wherein the incomplete particles comprise empty particles and / or partial particles, preferably both empty particles and partial particles.

16. The method according to any one of claims 1 to 14, wherein the loading buffer contains at least one buffer selected from the group consisting of Tris, Bis-tris, Bis-tris propane, Tris acetate, ethanolamine, and phosphate.

17. The loading buffer contains at least one salt of a cation selected from the group consisting of K(I), Li(I), Ca(II), Mg(II), Cu(II), Ba(II), Co(II), Ni(II), Mn(II), Zn(II), Cd(II), Pb(II), Fe(III), Fe(II), Na(I), and NH 4 + The method according to any one of claims 1 to 14.

18. The loading buffer is a) about 10 to 100 mM of a sodium salt, preferably 20 to 60 mM NaCl; b) about 0 to 20 mM of a magnesium salt, preferably 1 to 10 mM MgCl2; c) about 0 to 10 mM of a calcium salt, preferably 0.1 to 2.5 mM CaCl2; d) about 0 to 100 mM of a lithium salt, preferably 0 to 75 mM LiCl; e) about 0 to 5 mM of a copper salt, preferably 0.1 to 2.5 mM CuCl2; or f) about 5 to 100 mM of an ammonium salt, preferably 10 to 50 mM (NH4)2SO4, and the method according to any one of claims 1 to 14.

19. The loading buffer is about 20 to 60 mM NaCl, 20 to 50 mM Tris, 1 to 5 mM MgCl 2 , 0 to 75 mM LiCl, 0 to 10 mM CuCl 2 , and / or 0.1 to 2.5 mM CaCl 2The method according to any one of claims 1 to 14, comprising, or containing about 20 to 60 mM NaCl, 10 to 30 mM (NH4)2SO4, 1 to 5 mM MgCl2, 0.1 to 2.5 mM CuCl2, preferably in Tris, more preferably in 20 to 100 mM Tris.

20. The method according to any one of claims 1 to 14, wherein the loading buffer has a pH of about 6 to 10.

21. The method according to any one of claims 1 to 14, wherein the loading buffer contains at least one surfactant selected from the group consisting of poloxamer 188, polysorbate 80, polysorbate 20, NP-40, Triton X-100, and Triton CG-110.

22. The method according to claim 21, wherein the concentration of the surfactant in the loading buffer is 0.0001% to 0.1%.

23. The elution buffer contains at least one salt of a cation selected from the group consisting of K(I), Li(I), Ca(II), Mg(II), Cu(II), Ba(II), Co(II), Ni(II), Mn(II), Zn(II), Cd(II), Pb(II), Fe(III), Fe(II), Na(I), and NH 4 + The method according to any one of claims 1 to 14.

24. The elution buffer is a) about 0 to 1000 mM NaCl, preferably 20 to 300 mM NaCl; b) about 0 to 30 mM MgCl2, preferably 2 to 15 mM MgCl2; c) about 0 to 200 mM LiCl, preferably 0 to 150 mM LiCl; d) about 0.1 to 20 mM CaCl2, preferably 5 to 10 mM CaCl2; e) about 0 to 10 mM CuCl2, preferably 0 to 3 mM CuCl2; or (f) The method according to any one of claims 1 to 14, comprising an ammonium salt of about 5 to 100 mM, preferably 10 to 50 mM (NH4)2SO4.

25. The elution buffer contains at least one buffer selected from the group consisting of Tris, Bis-tris, Bis-tris propane, Tris acetate, ethanolamine, and phosphate, and the method according to any one of claims 1 to 14.

26. The elution buffer contains about 20 to 200 mM NaCl, preferably in a Tris buffer, and the method according to any one of claims 1 to 14.

27. The impurities in the rAAV preparation bind to the first column with a higher affinity than the complete rAAV particles, and the method according to claim 9.

28. A method for purifying fully recombinant adeno-associated virus (rAAV) particles, comprising: (a) preparing an rAAV preparation containing the complete rAAV particles and incomplete particles; (b) loading the rAAV preparation in a loading buffer onto a column containing a chromatography medium, the loading buffer containing CaCl 2 and the complete rAAV particles binding to the chromatography medium; and (c) eluting the complete rAAV particles bound to the chromatography medium with an elution buffer to obtain a purified preparation, the elution buffer containing CaCl 2 and the step A method comprising the steps.

29. The chromatography medium is an ion exchange column chromatography medium, preferably an anion exchange chromatography medium, and the method according to claim 28.

30. The column chromatography medium is selected from the group consisting of Poros 50 HQ, Poros 50 D, Poros 50 PI, Capto ImprRes Q, CIMmultus TM QA monolithic column, and Poros XQ, and preferably is Poros XQ, the method according to claim 29.

31. The loading buffer contains at least one salt of a cation selected from the group consisting of K(I), Li(I), Ca(II), Mg(II), Cu(II), Ba(II), Co(II), Ni(II), Mn(II), Zn(II), Cd(II), Pb(II), Fe(III), Fe(II), Na(I), and NH 4 + the method according to claim 28.

32. The loading buffer is a) about 0 to 10 mM CaCl 2 preferably, 0.1 to 2.5 mM CaCl 2 ; b) about 10 to 100 mM NaCl, preferably 20 to 60 mM NaCl; c) about 0 to 20 mM MgCl 2, preferably 1 to 10 mM MgCl 2; d) about 0 to 100 mM LiCl, preferably 0 to 75 mM LiCl; e) about 0 to 5 mM of a copper salt, preferably 0.1 to 2.5 mM CuCl 2; or f) about 5 to 100 mM of an ammonium salt, preferably 10 to 50 mM (NH 4 ) 2 SO 4, the method according to claim 28.

33. The loading buffer contains at least one buffer selected from the group consisting of Tris, Bis-tris, Bis-tris propane, Tris acetate, ethanolamine, and phosphate, the method according to any one of claims 28 to 32.

34. The loading buffer is about 20 to 60 mM NaCl, 1 to 5 mM MgCl 2, 0 to 75 mM LiCl, 0.1 to 2.5 mM CaCl 2 is contained, preferably in 20 to 50 mM Tris, or about 20 to 60 mM NaCl, 10 to 30 mM (NH4)2SO4, 1 to 5 mM MgCl2, 0.1 to 2.5 mM CuCl2 is contained, preferably in Tris, more preferably in 20 to 100 mM Tris, the method according to any one of claims 28 to 32.

35. The loading buffer has a pH of about 6 to 10, preferably 8 to 9, the method according to any one of claims 28 to 32.

36. The elution buffer is K(I), Li(I), Ca(II), Mg(II), Cu(II), Ba(II), Co(II), Ni(II), Mn(II), Zn(II), Cd(II), Pb(II), Fe(III), Fe(II), Na(I), and NH 4 + contains at least one salt of a cation selected from the group consisting of, the method according to any one of claims 28 to 32.

37. The elution buffer is a) about 0.1 to 20 mM CaCl 2 , preferably 5 to 10 mM CaCl 2 ; b) about 0 to 1000 mM NaCl, preferably 20 to 300 mM NaCl; c) about 0 to 30 mM MgCl2, preferably 2 to 15 mM MgCl2; or d) about 0 to 200 mM LiCl, preferably 0 to 150 mM LiCl, the method according to any one of claims 28 to 32.

38. The elution buffer contains at least one buffer selected from the group consisting of Tris, Bis-tris, Bis-tris propane, Tris acetate, ethanolamine, and phosphate, the method according to any one of claims 28 to 32.

39. The elution buffer contains about 20 to 200 mM NaCl, preferably in 40 to 60 mM Tris buffer, according to the method of any one of claims 28 to 32.

40. The loading buffer and / or the elution buffer contains at least one surfactant selected from the group consisting of poloxamer 188, polysorbate 80, polysorbate 20, NP-40, Triton X-100, and Triton CG-110, according to the method of any one of claims 28 to 32.

41. The concentration of the surfactant in the loading buffer and / or the elution buffer is 0.0001% to 0.1%, according to the method of claim 40.

42. The elution buffer has a pH of about 6 to 10, preferably 8 to 9, according to the method of any one of claims 1, 5, 10 or 28.

43. The yield of purified complete rAAV particles is 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more, according to the method of any one of claims 1, 5, 10 or 28.

44. The ratio of the complete rAAV particles to the previous incomplete particles in the purified preparation is 9:1 or more, preferably 49:1 or more, according to the method of any one of claims 1, 5, 10 or 28.

45. The incomplete particles include empty particles and / or partial particles, preferably both empty particles and partial particles, according to the method of any one of claims 1, 5, 10 or 28.

46. A method for purifying partial rAAV particles, (a) preparing an incomplete rAAV preparation containing empty particles and partial particles, (b) loading the incomplete rAAV preparation in a loading buffer onto a column comprising a chromatography medium, wherein the partial rAAV particles have a higher binding affinity to the chromatography medium than the empty particles, and (c) eluting the partial rAAV particles bound to the chromatography medium with an elution buffer to obtain a purified preparation A method comprising: **Claim 47** The method according to any one of claims 1, 5, 10, 28 or 46, wherein the complete rAAV particles comprise a transgene encoding a polypeptide or a nucleic acid selected from the group consisting of siRNA, antisense molecule, miRNA, ribozyme, and shRNA. **Claim 48** A method for purifying empty recombinant adeno-associated virus (rAAV) particles, comprising: (a) preparing an rAAV preparation comprising the empty rAAV particles and at least one of complete rAAV particles and partial rAAV particles; (b) loading the rAAV preparation in a loading buffer onto a column comprising a chromatography medium, wherein the empty rAAV particles have a higher binding affinity to the chromatography medium than the complete particles or the partial particles, and the amount of the empty rAAV particles applied to the column and at least one of the complete particles and the partial particles exceeds the binding capacity of the chromatography medium, and at least one of the complete particles and the partial particles bound to the chromatography medium is displaced by the empty rAAV particles and enters the flow-through from the column, and (c) eluting the empty rAAV particles bound to the chromatography medium with an elution buffer to obtain a purified preparation A method comprising: **Claim 49** The method according to any one of claims 1, 5, 10, 28, 46 or 48, wherein the rAAV particles comprise a capsid derived from one or more AAVs selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, Rh8, Rh10, Rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ, DJ8, NP59, Anc-80, and variants thereof.

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