CN117794629A

CN117794629A - Column purification method of AAV vector

Info

Publication number: CN117794629A
Application number: CN202280054838.1A
Authority: CN
Inventors: O·卡纳尔; V·库马; 金米
Original assignee: Spark Medical Co ltd
Current assignee: Spark Medical Co ltd
Priority date: 2021-06-11
Filing date: 2022-06-10
Publication date: 2024-03-29

Abstract

Described herein are methods for purifying rAAV particles, particularly for purifying intact rAAV particles from rAAV formulations comprising both intact and non-intact particles.

Description

Column purification method of AAV vector

Cross Reference to Related Applications

The present application claims priority from U.S. provisional patent application No. 63/209,680 filed on day 11 6 of 2021 and from U.S. provisional patent application No. 63/366,094 filed on day 9 of 2022, the disclosures of which are incorporated herein by reference.

Technical Field

The present application relates to methods for purifying recombinant adeno-associated virus (rAAV) particles. More specifically, the present application relates to methods for purifying intact rAAV particles from a formulation comprising both intact rAAV particles and non-intact rAAV particles.

Background

Gene delivery is a promising approach to treat acquired and inherited diseases. Many virus-based systems have been described for gene transfer purposes, including adeno-associated virus (AAV) based systems.

AAV is a helper-dependent DNA parvovirus belonging to the genus dependovirus. AAV requires helper viral functions, such as adenovirus, herpes virus, or vaccinia virus, in order for productive infection to occur.

AAV has a broad host range and is capable of replication in cells from any species in the presence of a suitable helper virus. AAV is not associated with any human or animal disease and does not appear to adversely affect the biological properties of the host cell after integration.

AAV vectors can be engineered to carry a heterologous nucleic acid sequence of interest (e.g., a nucleic acid encoding a selected gene of a therapeutic protein, such as an antisense molecule, ribozyme, miRNA, etc.) by deleting an internal portion of the AAV genome in whole or in part and inserting the heterologous nucleic acid sequence of interest between Inverted Terminal Repeats (ITRs). ITRs remain functional in vectors that allow replication and packaging of recombinant adeno-associated viruses (rAAVs) containing heterologous nucleic acid sequences of interest. The heterologous nucleic acid sequence is also typically linked to a promoter sequence capable of driving expression of the nucleic acid in the target cell of the patient. Termination signals (e.g., polyadenylation sites) may also be included in the vector. The rAAV genomic DNA is packaged in a viral capsid, which is a protein capsid containing a mixture of three capsid proteins (VP 1, VP2, and VP 3) arranged in icosahedral symmetry.

Recombinant adeno-associated virus (rAAV) shows excellent therapeutic promise in several early clinical trials in multiple groups. Development of this new type of biologic towards approval would involve improvements in the vector characterization and quality control methods, including a better understanding of how vector design and manufacturing process parameters affect impurity profiles in clinical grade vectors.

One of the challenges associated with the production of rAAV is the formation of "incomplete" rAAV particles that do not contain intact genetic material. As used herein, non-intact rAAV particles refer to a range of particles or variants including "empty" particles and "partial" particles. "partial" particles herein refer to rAAV particles that have some genetic material but not complete genetic material as in complete particles. The problem with the impact of non-intact particles (including empty and partial particles) on the clinical safety and efficacy of rAAV-mediated gene expression necessitates the development of purification methods to remove or isolate these species from the intact particles. Given the structural similarity between these types of rAAV particles, developing a robust and scalable purification method that efficiently separates non-intact AAV particles from intact AAV particles remains a challenge. These rAAV particles differ in the presence and length of the single stranded DNA genome in the rAAV. Different techniques have been developed to separate intact rAAV particles from non-intact particles. However, these techniques typically provide the designed whole rAAV particles in low purity and/or low yield.

Thus, there remains a need to develop new systems and methods for purifying whole rAAV particles from non-whole particles (including from empty particles or partial particles) in high purity and/or in high yield.

Disclosure of Invention

The present application relates to methods and systems for purifying intact recombinant adeno-associated virus (rAAV) particles from rAAV formulations comprising intact rAAV particles and non-intact particles, which may include empty particles and/or partial particles, using column chromatography techniques.

In one general aspect, the present application relates to a method for purifying whole recombinant adeno-associated virus (rAAV) particles, the method comprising:

(a) Providing a rAAV formulation comprising the intact rAAV particle and the non-intact particle;

(b) Loading the rAAV formulation in a loading buffer into a column comprising a chromatographic medium, wherein the intact rAAV particle has a higher binding affinity to the chromatographic medium than the non-intact particle;

and

(c) Eluting the intact rAAV particles bound to the chromatographic medium with an elution buffer to obtain a purified preparation.

In some embodiments, the non-intact particles comprise empty particles.

In some embodiments, the non-intact particles comprise a portion of particles.

In some embodiments, the non-intact particles include both empty particles and partial particles.

In some embodiments, the non-intact particles do not bind to the chromatographic medium and flow through the column.

In some embodiments, the portion of the particles are not bound to the chromatographic medium and flow through the column.

In some embodiments, the amount of intact rAAV particles and empty rAAV particles applied to the column exceeds the binding capacity of the chromatographic medium, such that empty particles bound to the chromatographic medium are replaced by intact rAAV particles into a loading flow through (flowthrough) from the column.

In some embodiments, the amount of intact rAAV particles and non-intact rAAV particles applied to the column exceeds the binding capacity of the chromatographic medium such that empty particles bound to the chromatographic medium are replaced by partial rAAV particles and intact rAAV particles into a loaded flow-through from the column.

In some embodiments, the amount of intact rAAV particles and non-intact rAAV particles applied to the column exceeds the binding capacity of the chromatographic medium, such that empty particles and portions of particles bound to the chromatographic medium are replaced by intact rAAV particles into the loaded flow-through from the column.

In some embodiments, the chromatographic medium is an ion exchange column chromatographic medium, preferably an anion exchange chromatographic medium.

In some embodiments, the column chromatography medium is selected from the group consisting of Poros 50HQ, poros 50D, poros PI, capto ImpRes Q, and Poros XQ, preferably Poros XQ.

In some embodiments, the column chromatography medium is as CIMmultus ^TM Integral (monoliths) of QA monolithic columns.

In some embodiments, the loading buffer comprises at least one buffer selected from the group consisting of: tris, bis-Tris propane, tris acetate, ethanolamine and phosphate.

In some embodiments, the loading buffer comprises 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 anionic component of the salt is not critical.

In some embodiments, the pH of the loading buffer is about 6-10, preferably 8-9.

In some embodiments, the elution buffer comprises at least one buffer selected from the group consisting of: tris, bis-Tris propane, tris acetate, ethanolamine and phosphate.

In some embodiments, the elution buffer comprises 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 anionic component of the salt is not critical.

In some embodiments, the pH of the elution buffer is about 6-10, preferably 8-9.

In some embodiments, the yield of the purified whole rAAV particle is no less than 70%, preferably no less than 80%, more preferably no less than 90%, and most preferably no less than 95%.

In some embodiments, the purified formulation is substantially free of the non-intact particles. In other embodiments, the purified preparation has an increased ratio of the intact rAAV particles to the non-intact particles compared to the rAAV preparation. Preferably, the ratio of intact rAAV particles to non-intact particles in the purified formulation is no less than 9:1, such as no less than 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, or 50:1, or any ratio in between, more preferably no less than 49:1.

In some embodiments, the intact rAAV particle comprises a transgene encoding a polypeptide or a nucleic acid selected from the group consisting of: siRNA, antisense molecules, miRNA, ribozymes, and shRNA.

In some embodiments, the rAAV particle comprises a capsid derived from an AAV serotype.

In another embodiment, the rAAV particle comprises a capsid derived from an engineered capsid capable of binding to an ion exchange chromatography medium.

In another general aspect, the present application relates to a method for purifying whole recombinant adeno-associated virus (rAAV) particles, the method comprising:

(a) Providing a rAAV formulation comprising intact rAAV particles and non-intact particles;

(b) Loading a first batch of the rAAV formulation in a loading buffer into a first column comprising a first chromatographic medium, wherein the intact rAAV particles have a higher binding affinity to the first chromatographic medium than the non-intact particles, and the amount of the intact rAAV particles and the non-intact particles applied to the first column exceeds the binding capacity of the first chromatographic medium such that the non-intact particles bound to the first chromatographic medium are displaced by the intact rAAV particles to a first loading cargo from the first column;

(c) Loading the first loaded carrier to a second column comprising a second chromatographic medium to obtain a partially loaded (i.e. incompletely saturated) second column, preferably the second chromatographic medium is of the same type as the first chromatographic medium;

(d) Optionally washing the first column with a washing buffer to obtain a washed first column;

(e) Bypassing the second column after the step (c) or after the washing step (d) if the washing step (d) is performed, and eluting the intact rAAV particles bound to the first chromatographic medium with an elution buffer to obtain a first eluate from the first column and an eluted first column, wherein the first eluate has an increased ratio of intact rAAV particles to non-intact rAAV particles;

(f) Loading a second batch of rAAV formulation in the loading buffer to a partially loaded second column, wherein the amount of intact rAAV particles and non-intact particles applied to the second column exceeds the binding capacity of the second chromatographic medium such that non-intact particles bound to the second chromatographic medium are replaced by intact rAAV particles into a second loading flow-through from the second column;

(g) Loading the second loading permeate into 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) Bypassing the first column after step (g) or after washing step (h) if washing step (h) is performed, and eluting the intact rAAV particles bound to the second chromatographic medium with an elution buffer to obtain a second eluate and an eluted second column, wherein the second eluate has an increased ratio of intact rAAV particles to non-intact rAAV particles; and

(j) Combining the first eluate and the second eluate to produce a purified preparation.

In some embodiments, the non-intact particles comprise empty particles.

In some embodiments, the non-intact particles comprise a portion of particles.

In some embodiments, when the non-intact particles include both empty particles and partial particles, the second eluate in step (i) has an increased ratio of intact rAAV particles and partial rAAV particles to empty rAAV particles.

In some embodiments, more than two columns may be used, and two are minimal. In a three column arrangement, whole particles are enriched in the first column, while part of the particles are enriched in the second column when present, and empty particles are enriched in the third column. In some embodiments, when an impurity or aggregate is present in the formulation, the impurity or aggregate may bind the first column with greater affinity than the intact particle. In these embodiments, the intact particles will be enriched on one or more subsequent columns.

In some embodiments, the first chromatography medium and/or the second chromatography medium is an ion exchange column chromatography medium, preferably an anion exchange chromatography medium.

In some embodiments, the second column is partially loaded after loading the first circulation.

In some embodiments, the steps (b) through (i) are performed in one cycle or in multiple cycles.

In some embodiments, the eluted column may undergo subsequent steps that are beneficial or necessary to maintain consistent column binding capacity throughout the cycle, prior to the next cycle of loading. For example, such steps include, but are not limited to, stripping the column, cleaning and/or sterilizing the column, and/or rebalancing the column.

In some embodiments, the first or second column chromatography medium is selected from the group consisting of Poros 50HQ, poros 50D, poros PI, capto ImpRes Q, and Poros XQ, preferably Poros XQ.

In some embodiments, the loading buffer comprises at least one surfactant.

In some embodiments, the surfactant 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 surfactant in the loading buffer is between 0.0001% and 0.1%.

In some embodiments, the elution buffer comprises at least one surfactant.

In some embodiments, the surfactant 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 surfactant in the elution buffer is between 0.0001% and 0.1%.

In some embodiments, the purified formulation is substantially free of the non-intact particles. In other embodiments, the purified preparation has an increased ratio of the intact rAAV particles to the non-intact particles compared to the rAAV preparation. Preferably, the ratio of intact particles to non-intact particles in the purified formulation is no less than 9:1, such as no less than 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1 or 50:1, or any ratio in between, more preferably no less than 49:1.

In some embodiments, the intact 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: siRNA, antisense molecules, miRNA, ribozymes, and shRNA.

In some embodiments, the rAAV particle comprises a capsid derived from one or more AAV selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9 (hu 14), AAV10, AAV11, AAV12, rh8, rh10, rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ8, NP59, anc-80 and variants thereof, including AAV capsid variants set forth in: pulcherla et al mol. Ter., 19 (6) 1070-1078 (2011) (describing AAV9 variants, including in particular AAV 9.47), U.S. patent No. 7,906,111 (describing in particular AAV9 (hu 14)), 10,532,111 (describing in particular NP 59), 10,738,087 (describing in particular Anc-80), 9,169,299 (describing "LK 03"), 9,840,719 (describing "RHM 4-1"), 7,749,492, 7,588,772 (describing "DJ" and "DJ 8"), 9,587,282, and patent applications WO 2012/145601, WO 2013/158879, WO 2015/01353, WO 2018/156654, US2013/0059732, all of which are incorporated herein by reference in their entirety.

(b) Loading the rAAV formulation in a loading buffer to a chromatographic mediumIn the column, wherein the loading buffer comprises CaCl ₂ And the intact rAAV particle is bound to the chromatographic medium;

and

(c) Eluting the intact rAAV particles bound to the chromatographic medium with an elution buffer to obtain a purified preparation, wherein the elution buffer optionally comprises CaCl ₂ 。

In some embodiments, the loading buffer comprises at least one surfactant.

In some embodiments, the loading buffer comprises a 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 ⁺ 。

In some embodiments, the elution buffer comprises a 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 ⁺ 。

In some embodiments, the loading buffer comprises about 0-10mM CaCl ₂ Preferably 0.1-2.5mM CaCl ₂ 。

In some embodiments, the elution buffer comprises about 0.1-20mM CaCl ₂ Preferably 5-10mM CaCl ₂ 。

In some embodiments, the loading buffer comprises about 0-100mM LiCl, preferably 0-75mM LiCl.

In some embodiments, the elution buffer comprises about 0-200mM LiCl, preferably 0-150mM LiCl.

In some embodiments, the loading buffer comprises about 0-10mM CuCl ₂ Preferably 0.1-3mM CuCl ₂ 。

In some embodiments, the elution buffer comprises about 0-10mM CuCl ₂ Preferably 0-3mM CuCl ₂ 。

In some embodiments, the loading buffer further comprises NaCl and/or MgCl ₂ 。

In some embodiments, the elution buffer further comprises NaCl and/or MgCl ₂ 。

In some embodiments, the elution buffer comprises at least one surfactant.

In some embodiments, the purified formulation is substantially free of the non-intact particles. In other embodiments, the purified preparation has an increased ratio of the intact rAAV particles to the non-intact particles compared to the rAAV preparation. Preferably, the ratio of intact particles to non-intact particles in the purified formulation is not less than 9:1, such as not less than 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1 or 50:1, or any ratio in between, preferably not less than 49:1.

In some embodiments, the rAAV particle is derived from an AAV selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9 (hu 14), AAV10, AAV11, AAV12, rh8, rh10, rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ8, NP59, anc-80 and variants thereof, including AAV capsid variants set forth in: pulcherla et al mol. Ter., 19 (6) 1070-1078 (2011) (describing AAV9 variants, including in particular AAV 9.47), U.S. patent No. 7,906,111 (describing in particular AAV9 (hu 14)), 10,532,111 (describing in particular NP 59), 10,738,087 (describing in particular Anc-80), 9,169,299 (describing "LK 03"), 9,840,719 (describing "RHM 4-1"), 7,749,492, 7,588,772 (describing "DJ" and "DJ 8"), 9,587,282, and patent applications WO 2012/145601, WO 2013/158879, WO 2015/01353, WO 2018/156654, US2013/0059732, all of which are incorporated herein by reference in their entirety.

In one general aspect, the present application relates to a method for purifying a portion of a rAAV particle, the method comprising:

(a) Non-intact rAAV formulations comprising empty particles and partial particles are provided;

(b) Loading the incomplete rAAV formulation in a loading buffer into a column comprising chromatographic media,

wherein the partial rAAV particle has a higher binding affinity to the chromatographic medium than the empty particle; and

(c) Eluting the fraction of rAAV particles bound to the chromatographic medium with an elution buffer to obtain a purified preparation.

In some embodiments, the amount of partial rAAV particles and empty rAAV particles applied to the column exceeds the binding capacity of the chromatographic medium such that empty particles bound to the chromatographic medium are replaced by partial rAAV particles into the loaded flow-through from the column.

In some embodiments, the loading buffer comprises at least one surfactant.

In some embodiments, the elution buffer comprises at least one surfactant.

In some embodiments, the purified formulation is substantially free of the empty particles. In other embodiments, the purified preparation has an increased ratio of the partial rAAV particles to the empty particles compared to the rAAV preparation. Preferably, the ratio of partial rAAV particles to empty particles in the purified formulation is no less than 9:1, such as no less than 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, or 50:1, or any ratio in between, more preferably no less than 49:1.

In one general aspect, the present application relates to a method for purifying an empty rAAV particle, the method comprising:

(a) Providing a rAAV formulation comprising at least one of a complete rAAV particle and a partial rAAV particle, and the empty rAAV particle;

(b) Loading the rAAV formulation in a loading buffer into a column comprising a chromatographic medium, wherein the empty rAAV particle has a higher binding affinity to the chromatographic medium than the whole particle or a portion of the particle and at least one of the whole particle and the portion of the particle applied to the column,

And the amount of the empty rAAV particles exceeds the binding capacity of the chromatographic medium such that at least one of the intact particles and a portion of the particles bound to the chromatographic medium are displaced by the empty rAAV particles into a flow-through from the column; and

(c) Eluting the empty rAAV particles bound to the chromatographic medium with an elution buffer to obtain a purified preparation.

In some embodiments, the amount of at least one of the intact particle and the partial particle applied to the column, and the empty particle, exceeds the binding capacity of the chromatographic medium such that at least one of the intact particle and the partial particle bound to the chromatographic medium is displaced by the empty rAAV particle into a loaded flow-through from the column.

In some embodiments, the rAAV formulation comprises intact particles.

In some embodiments, the rAAV formulation comprises a portion of the particle.

In some embodiments, the rAAV formulation comprises both whole particles and partial particles.

In some embodiments, the loading buffer comprises at least one surfactant.

In some embodiments, the elution buffer comprises at least one surfactant.

In some embodiments, the purified formulation is substantially free of the empty particles. In other embodiments, the purified preparation has an increased ratio of the partial rAAV particles to the empty particles compared to the rAAV preparation. Preferably, the ratio of partial rAAV particles to empty particles in the purified formulation is no less than 3:1, such as no less than 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 any ratio in between, more preferably no less than 4:1.

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.

Drawings

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 should be understood that the invention is not limited to the precise embodiments shown in the drawings.

FIGS. 1A-1C illustrate the separation of intact rAAV particles from non-intact particles on a Poros 50HQ resin at three different pH values: pH 8.0 (fig. 1A), pH8.6 (fig. 1B), and pH 9.25 (fig. 1C).

FIGS. 2A-2D show the separation of intact rAAV particles and non-intact particles on a Poros 50D resin at the following different pH values and different buffers: pH8.6 and 50mM Tris (FIG. 2A), pH8.6 and 25mM Tris (FIG. 2B), pH 8.0 and 25mM Tris (FIG. 2C), and pH 9.25 and 25mM Tris (FIG. 1D).

Figures 3A-3E show the separation of intact rAAV particles and non-intact particles on Poros 50PI resin at different pH values and different binding strengths as follows: pH 8.0 NaCl-free (FIG. 3A), pH8.6 NaCl-free (FIG. 3B), pH8.6 and 30mM NaCl (FIG. 3C), pH 9.2 NaCl-free (FIG. 4C) and pH 9.2 and 30mM NaCl (FIG. 3E).

FIGS. 4A-4C illustrate the separation of intact rAAV particles and non-intact particles on Capto ImpRes Q resin with different buffers at different pH values: pH 8.0 and 50mM Tris-NaCl free (FIG. 4A), pH8.6 and 25mM Tris-NaCl free (FIG. 4B), pH 9.0 and 25mM Tris-NaCl free (FIG. 4C).

Figure 5 shows that separation of intact rAAV particles and non-intact particles on Poros XQ resin is optimal at pH 8.75 compared to pH8 and pH 9.25.

FIG. 6 shows the effect of binding salts (1.6, 5 and 6.8 mS/cm) on the separation of intact rAAV particles and non-intact particles on a Poros XQ resin.

FIG. 7 shows the effect of flow rate on the separation of intact rAAV particles and non-intact particles on a Poros XQ resin.

Fig. 8A shows HPLC analysis of low loaded samples, and fig. 8B shows HPLC analysis of penetration (break-through) loaded samples.

Fig. 9A-9D illustrate the effect of binding strength under penetrating conditions with the following salts: 45mM NaCl (FIG. 9A), 60mM NaCl (FIG. 9B), 75mM NaCl (FIG. 9C) and 90mM NaCl (FIG. 9D).

Fig. 10A-10D show step elution for different binding strengths with the following salts: 60mM NaCl (FIG. 10A), 75mM NaCl (FIG. 10B), 90mM NaCl (FIG. 10C) and 120mM NaCl (FIG. 10D).

FIGS. 11A-11B show samples loaded onto PXQ resin in 50mM Tris pH 8.5 buffer with 60mM NaCl. Elution was accomplished with 300mM NaCl 50mM Tris pH 8.5 buffer using a linear gradient. In fig. 11B more sample is loaded onto the column than in fig. 11A. These figures demonstrate that displacement chromatography separates intact rAAV particles from non-intact particles. Figure 11B further demonstrates that displacement is more pronounced as more samples are loaded, product purity increases to nearly 100% and yields of > 90%.

FIG. 12 shows the reaction with 10mM MgCl ₂ Samples loaded onto PXQ resin in 50mm Tris pH 8.5 buffer. Elution was performed with 300mM NaCl 50mM Tris pH 8.5 buffer. FIG. 12 shows the use of MgCl at a high concentration of 10mM in the loaded sample during loading ₂ And removing the non-intact particles.

FIG. 13 shows the toolWith 2.5mM CaCl ₂ Samples loaded onto PXQ resin in 50mm Tris pH 8.5 buffer. Elution was performed with 300mM NaCl 50mM Tris pH 8.5 buffer. FIG. 13 illustrates CaCl addition to the loader during loading ₂ And removing the non-intact particles. Compared with the method in FIG. 12, the method has larger conductivity difference between the intact particles and the non-intact particles >4 mS/cm) and more robust.

FIGS. 14A-14B illustrate the use of a solution having 1 to 1.5mM CaCl ₂ +2.5mM MgCl ₂ Samples loaded onto PXQ resin in 50mM Tris pH 8.5 buffer of +20mM NaCl. With 10mM CaCl ₂ 、2.5mM MgCl ₂ 20mM NaCl 50mM Tris pH 8.5 buffer was used for elution. These figures show the CaCl targets ₂ Robustness of concentration. FIG. 14A is a sample of 1.5mM CaCl in the loading material ₂ FIG. 14B is a sample of 1mM CaCl ₂ . Even CaCl ₂ The concentration was varied by 50% and in the presence of other additives the process also produced 100% removal of non-intact particles without any loss of intact particles. In addition, caCl is used ₂ During gradient elution, intact particles were not eluted until 5mM CaCl was applied ₂ Showing 4mM CaCl between incomplete and complete collection ₂ Is a strong difference in (a). Fig. 14C-14D show the same conditions as fig. 14B, but the drug product shown in fig. 14D was analyzed by Analytical Ultracentrifugation (AUC).

FIG. 15 shows a chromatogram of a sample loaded onto PXQ resin in 50mM Tris pH 8.5. The column was run with a column with 1mM CaCl ₂ +2.5mM MgCl2 in 50mM Tris pH 8.5 buffer. The figure demonstrates that if no CaCl was added to the sample load ₂ Both yield and purity are compromised when purifying intact particles from non-intact particles. In addition, it is difficult to remove all non-intact particles bound to the column via washing. Furthermore, in use increased CaCl ₂ In subsequent elution of the concentration, the bound non-intact particles are unstable on the resin and new impurities are generated, which in turn impair the final purity.

Figure 16 illustrates an implementation of cyclic displacement chromatography for separating intact rAAV particles from non-intact particles.

FIG. 17 shows the chromatogram of a sample loaded onto PXQ resin in 50mM Tris pH 8.5 buffer with 58mM LiCl. The column was eluted with 50mM Tris pH 8.5+120mM LiCl buffer. The conductivity difference in the incomplete elution and the complete elution under this condition was 0.2mS/cm.

Figure 18 illustrates an implementation of cyclic displacement chromatography for separating intact rAAV particles from non-intact particles using at least additives in the loading buffer and preferably in the wash and elution buffers.

Figures 19A-19B illustrate the removal of incomplete particles and recovery of complete particles during sample loading using a decreasing pH with a gradient elution or a stepwise elution. The loading sample buffer was 50mM Tris, 2mM MgCl ₂ 、2mM CaCl ₂ 20mM NaCl, pH 8.5. With decreasing pH gradient with 20mM BisTris-30mM acetate, 2mM MgCl ₂ 、2mM CaCl ₂ Elution at pH 6.0 is shown in FIG. 19A. With pH step and 20mM BisTris-30mM acetate, 2mM MgCl ₂ 、2mM CaCl ₂ Elution at pH 7.0 and pH 6.0 is shown in FIG. 19B.

FIGS. 20A-20B illustrate the application of (NH) during loading ₄ ) ₂ SO ₄ Addition to the sample load resulted in the flow-through of non-intact particles (fig. 20B). The samples were incubated with poloxamer 188 at 0.0002%, naCl at 20mM, 20mM (NH) ₄ ) ₂ SO ₄ And 2.0mM MgCl ₂ Is loaded onto the PXQ resin in a 100mM Tris pH 8.5 buffer. The elution buffer was 50mM Tris pH 8.5 buffer with 0.0002% poloxamer 188 and 300mM NaCl. In the absence (NH) ₄ ) ₂ SO ₄ In the case shown in the inset, incomplete particles were not removed during sample loading at the same buffer conductivity of 7mS/cm (fig. 20A).

FIG. 21 shows that during sample loading of the monolithic column, the (NH ₄ ) ₂ SO ₄ The addition to the sample load resulted in the flow-through of non-intact particles. The samples were incubated with poloxamer 188 at 0.0002%, naCl at 20mM, 20mM (NH) ₄ ) ₂ SO ₄ And 2.0mM MgCl ₂ The solution was loaded onto BIA 1.3 μm monolith in 50mM Tris pH 8.5 buffer.The elution buffer was 50mM Tris pH 8.5 buffer with 0.0002% poloxamer 188 and 200mM NaCl

FIGS. 22A-22B illustrate CuCl during sample loading ₂ The addition to the sample load resulted in the flow-through of non-intact particles. The samples were incubated with poloxamer 188 at 0.0002%, naCl at 20mM, 15mM (NH) ₄ ) ₂ SO ₄ 、2.0mM MgCl ₂ And 1.5mM CuCl ₂ Is loaded onto PXQ resin in 50mM Tris pH8.5 buffer. The elution buffer was 0.0002% poloxamer 188 and 2mM MgCl ₂ 180mM sodium phosphate pH 7.2 buffer. FIGS. 22C-22E illustrate the application of CuCl ₂ The addition to the sample load resulted in an increased degree of separation between non-intact particles (leftmost peak) and intact particles (lefttwo peaks) at the analytical scale. In addition, cuCl is added ₂ Resulting in a significant reduction of some product variants (peaks after the second peak). This demonstrates that CuCl is to be loaded during loading ₂ The addition removes non-intact particles in the loading. Fig. 22F shows that Cu (II) ions resulted in the highest increase in the degree of separation between incomplete peaks and complete peaks compared to other ions. The samples were in 50mM Tris pH8.5 with 0.5-2mM designated ions.

FIGS. 23A-23E illustrate the addition of CuCl ₂ In the case of (a), incomplete particles can be removed during loading and a certain degree of separation between partial particles and complete particles can be achieved. The rAAV preparation is prepared in a solution with 0.0002% poloxamer 188, 20mM NaCl and 15mM NH ₄ SO ₄ 、2.0mM MgCl ₂ And 1.5mM CuCl ₂ Is loaded into PXQ resin in 50mM Tris pH8.5 buffer. The bound particles were eluted in a stepwise manner with 50mM sodium acetate pH 6.0 buffer with 0.0002% poloxamer 188 and two different amounts of NaCl. Elution peak 1 was obtained with 50mM NaCl, while elution peak 2 was obtained with 200mM NaCl (FIGS. 23A-23B). Fig. 23C-23E show the sedimentation coefficient distribution of the eluate peak using analytical ultracentrifugation. As shown in table 1 below, peak 1 contained a fraction of particles (40.1%) and intact particles (49.3%), while peak 2 was mainly rich in intact particles (69.8%) with a lower percentage of fraction particles (15.5%) and removed non-intact particles (15.5%).

TABLE 1 sedimentation coefficient distribution

FIGS. 24A-24G illustrate enrichment of rAAV particle variants using a three column displacement chromatography method. The rAAV formulation was loaded onto three PXQ columns connected in series. The loading buffer was 50mM Tris pH 8.5, 75mM NaCl and 2mM MgCl ₂ . Use of a drug with 0.0002% poloxamer 188 and 2mM MgCl ₂ The three columns were eluted sequentially at pH 8.5 (preparative chromatograms are shown in FIGS. 24A-24B) in the buffer of 200mM NaCl 50mM Tris. The collected fractions were analyzed on IEX columns using UPLC. As shown in fig. 24C-24D, the empty particle stream is threaded through the column and most of the empty particles are removed during loading. In addition, the analytical IEX chromatogram shows that the first column is enriched for the strongest binding particles (highest retention time; FIG. 24E), while the second and third columns are enriched for the second and weakest binding particles, respectively (FIGS. 24F-24G). The first column and second column elute enriched in intact particles. This demonstrates the enrichment of different particle variants using displacement chromatography on a three column setup.

FIGS. 25A-25B illustrate enrichment of intact particles using a two-column displacement chromatography method. The rAAV formulation was loaded onto two PXQ columns connected in series. The loading buffer was 50mM Tris pH 8.5, 10mM NaCl, 2.5mM MgCl ₂ And 1mM CaCl ₂ . From columns 1 and 2, 50mM Tris pH 8.5, 200mM NaCl, 2.5mM MgCl was used ₂ And 0.0002% poloxamer 188 were eluted sequentially (preparative chromatograms see fig. 25A-25B). The fractions collected were analyzed using UPLC. As shown in fig. 25C, the empty particles flow through during loading. Impurities in the rAAV formulation bind to the PXQ resin with higher affinity than intact particles. Thus, these impurities are reduced in the formulation loaded onto column 2, and the whole particles are the species with the greatest affinity for binding to column 2. After elution, the column 2 eluate is more enriched in intact particles. The column 2 elution peak therefore exhibited better complete particle enrichment than column 1, as shown in figure 25D (UPLC analysis).

FIGS. 26A-26D illustrate enrichment using a three column displacement chromatography methodPartial particles and complete particles. The rAAV formulation was loaded onto three PXQ columns connected in series. The loading buffer was 50mM Tris pH 8.5, 20mM NaCl, 15mM ammonium sulfate, 2mM MgCl ₂ And 1.5mM CuCl ₂ . From columns 1, 2 and 3, 50mM Tris pH 8.5, 200mM NaCl, 2.5mM MgCl was used ₂ And 0.0002% poloxamer 188 were eluted sequentially (preparative chromatograms see fig. 26A-26B). Fig. 26C-26D show the characteristics of the runthrough and eluate by analytical ion exchange chromatography and Analytical Ultracentrifugation (AUC). During loading the empty particles flow through (fig. 26C). The main eluate peak from column 1 is rich in intact particles and the main eluate peak from column 3 is rich in partial particles, while column 2 contains both partial particles and intact particles (fig. 26D).

Detailed Description

Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is incorporated by reference herein in its entirety. The 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 these matters form part of the prior art base with respect to any invention disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Otherwise, certain terms used herein have the meaning as set forth in the specification. All patents, published patent applications, and publications cited herein are incorporated by reference as if fully set forth herein. It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "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. The term "comprising" as used herein may be replaced with the term "containing" or "including" or sometimes with the term "having" as used herein.

As used herein, "consisting of … …" does not include any elements, steps or components not specified in the claimed elements. As used herein, "consisting essentially of … …" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claims. Any of the above-described terms "comprising," "including," "comprising," and "having" whenever used in the context of an aspect or embodiment of the present invention may be substituted with the term "consisting of … …" or "consisting essentially of … …" to alter the scope of this disclosure.

As used herein, the term "about" when used in connection with a number refers to any number within 10% (e.g., ±5% or ±1%) of the reference number. For example, a pH of about 5.0 means any pH from 4.5 to 5.5 (inclusive).

As used herein, the connection term "and/or" between a plurality of recited elements is understood to encompass both individual and combined options. For example, where two elements are connected by an "and/or," a first option means that the first element applies and the second element does not. The second option means that the second element is applicable and the first element is not applicable. The third option means that the first element and the second element are applicable together. Any of these options is understood to fall within the meaning and therefore meets the requirements of the term "and/or" as used herein. More than one of the options applies simultaneously also being understood as falling within the meaning, thus fulfilling the requirement of the term "and/or".

The term "vector" refers to any small vector of a nucleic acid molecule, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, or other vector that can be manipulated by insertion or incorporation of a nucleic acid. Vectors can be used for gene manipulation (i.e., a "cloning vector") to introduce/transfer polynucleotides into cells and to transcribe or translate inserted polynucleotides in cells. An "expression vector" is a vector containing a gene or nucleic acid sequence having the necessary regulatory regions required for expression in a host cell. The vector nucleic acid sequence typically contains at least an origin of replication for propagation in the cell and optionally 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.

The term "adeno-associated virus (AAV) vector" or "AAV vector" refers to a vector derived from an adeno-associated virus serotype, including, but not limited to, AAV serotypes such as: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9 (hu 14), AAV10, AAV11, AAV12, rh8, rh10, rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ8, NP59, anc-80 and variants thereof, including AAV capsid variants set forth in: pulcherla et al mol. Ter., 19 (6) 1070-1078 (2011) (describing AAV9 variants, including in particular AAV 9.47), U.S. patent No. 7,906,111 (describing in particular AAV9 (hu 14)), 10,532,111 (describing in particular NP 59), 10,738,087 (describing in particular Anc-80), 9,169,299 (describing "LK 03"), 9,840,719 (describing "RHM 4-1"), 7,749,492, 7,588,772 (describing "DJ" and "DJ 8"), 9,587,282, and patent applications WO 2012/145601, WO 2013/158879, WO 2015/01353, WO 2018/156654, US2013/0059732, all of which are incorporated herein by reference in their entirety. In addition, AAV vectors include AAV engineered for tissue tropism, stability, and transduction efficiency. In AAV vectors, one or more of the AAV wild type genes (preferably replication (rep) and/or capsid (cap) genes) may be deleted in whole or in part, but functional flanking ITR sequences are retained. Functional ITR sequences are necessary for rescue, replication and packaging of AAV virions. Thus, AAV vectors are defined herein to include at least those sequences (e.g., functional ITRs) required for replication and packaging of the virus in cis. ITRs need not be wild-type nucleotide sequences and may be altered, for example, by nucleotide insertions, deletions or substitutions, so long as the sequence provides functional rescue, replication and packaging.

The term "AAV virion" refers to a viral particle (e.g., a wild-type (wt) AAV viral particle) comprising a linear, single stranded nucleic acid genome associated with a coat of AAV capsid proteins.

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 such that the nucleic acid/genetic material can be maintained in the cells. Because AAV is not associated with pathogenic diseases in humans, rAAV vectors are capable of delivering heterologous nucleic acid sequences (e.g., therapeutic proteins and agents) to human patients without causing significant AAV morbidity or disease.

The term "recombinant" as a modifier of a vector (e.g., a recombinant adeno-associated virus (rAAV) vector) and a modifier of a sequence (e.g., a recombinant polynucleotide and polypeptide) means that the composition is manipulated (i.e., engineered) in a manner that is not normally found in nature. An example of a rAAV vector may be a vector in which nucleic acid that is not normally present in the wild-type AAV genome is inserted within the viral genome. For example, a nucleic acid (e.g., a gene) encoding a therapeutic protein or polynucleotide sequence is cloned into the AAV genome in a vector, with or without 5', 3' and/or intron regions typically associated with the gene. Although the term "recombinant" is not always used herein with respect to AAV vectors and sequences such as polynucleotides, recombinant forms including AAV vectors, polynucleotides, and the like are expressly included despite any such omissions.

rAAV vectors are produced from the wild-type genome of a virus (e.g., AAV) by: the wild-type genome is removed from the AAV genome using molecular methods and replaced with a non-native (heterologous) nucleic acid (e.g., a nucleic acid encoding a therapeutic protein or a nucleic acid molecule of interest). Typically, for AAV, one or both Inverted Terminal Repeat (ITR) sequences of the AAV genome remain in the rAAV vector. The rAAV genome is distinguished from the AAV genome in that all or part of the AAV genome has been replaced with a non-native sequence relative to AAV genomic nucleic acid, such as by a heterologous nucleic acid or heterologous polynucleotide sequence encoding a therapeutic protein. Thus, the incorporation of non-native sequences defines AAV as a "recombinant" AAV vector, which may be referred to as a "rAAV vector. Recombinant AAV vector sequences may be packaged, referred to herein as "particles," for subsequent infection (transduction) of cells ex vivo, in vitro, or in vivo.

The terms "recombinant AAV virions", "rAAV virions", "AAV vector particles", "intact rAAV capsids", "intact rAAV particles", "intact capsids" and "intact particles" as used herein each refer to an infectious replication defective virus comprising an AAV protein capsid encapsulating a nucleic acid molecule comprising a heterologous nucleotide sequence of interest flanked on one or both sides by AAV ITRs. The intact rAAV particles are produced in a suitable host cell into which sequences specifying AAV vectors, AAV helper functions, and accessory functions have been introduced. In this way, the host cell is enabled to encode AAV polypeptides that are required for packaging AAV vectors (containing recombinant nucleotide sequences of interest) into infectious recombinant virion particles for subsequent gene delivery.

As used herein, the terms "incomplete capsid" and "incomplete particle" each refer to an AAV particle or virion comprising an AAV particle shell but lacking an intact nucleic acid molecule comprising a heterologous nucleic acid sequence flanked on one or both sides by AAV ITRs. Such non-intact particles do not transfer the intact heterologous nucleic acid sequence into one or more host cells within the organism. These incomplete particles include variants with different lengths or amounts of incomplete genetic material. Non-intact particles that lack sufficient genetic material or that have not been detected by analytical methods (e.g., UPLC and AUC) as having any genetic material at all are referred to as "empty" particles. Non-intact particles having sufficient genetic material to be detected by analytical methods as having some genetic material but less than intact particles are referred to as "partial" particles. Incomplete genetic material may be intact or fragmented.

Any analytical method known in the art may be used to quantify intact particles and non-intact particles, including determining the ratio of intact particles to non-intact particles or the ratio of non-intact particles to intact particles. For example, such methods may be, but are not limited to, physical titer calculations; a260 and 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 assessment, several different methods (UPLC, a260 and a280 absorbance and AUC) have been shown in this disclosure.

The vector "genome" refers to the portion of the recombinant sequence that is ultimately packaged or encapsulated to form a rAAV particle. In the case of recombinant plasmids used to construct or make recombinant AAV vectors, the AAV vector genome does not include a "plasmid" portion of the vector genome sequence that does not correspond to the recombinant plasmid. This non-vector genomic portion of the recombinant plasmid, termed the "plasmid backbone", is important for cloning and amplification of the plasmid (the method required for propagation and recombinant virus production), but is not itself packaged or encapsulated into rAAV particles. Thus, a vector "genome" refers to a nucleic acid packaged or encapsulated by a rAAV.

The term "AAV helper functions" refers to AAV-derived coding sequences (proteins) that can be expressed to provide AAV gene products and AAV vectors that in turn function in trans for productive AAV replication and packaging. Thus, AAV helper functions include AAV Open Reading Frames (ORFs) comprising: rep and cap, and others, such as Assembly Activator Proteins (AAPs) of certain AAV serotypes. Rep expression products have been shown to have many functions, including, inter alia: recognizing, binding and cutting an AAV DNA replication origin; DNA helicase activity; and modulating transcription from AAV (or other heterologous) promoters. Cap expression products (capsids) provide the necessary packaging functions. AAV helper functions are used to supplement AAV functions deleted in the AAV vector genome in trans.

The term "AAV helper construct" generally refers to a nucleic acid sequence comprising a nucleotide sequence that provides AAV function deleted from an AAV vector used to generate a transduced AVV vector for delivery of the nucleic acid sequence of interest to a subject, e.g., by gene therapy. AAV helper constructs are typically used to provide transient expression of AAV rep and/or cap genes to complement the deleted AAV functions necessary for AAV vector replication. Helper constructs typically lack AAV ITRs and are neither capable of replication nor packaging themselves. AAV helper constructs may be in the form of plasmids, phages, transposons, cosmids, viruses or virions. Numerous AAV helper constructs have been described, such as plasmids pAAV/Ad and pIM29+45 encoding 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). Many other vectors encoding Rep and/or Cap expression products have been described (see, e.g., U.S. Pat. nos. 5,139,941 and 6,376,237).

The term "accessory function" refers to viral and/or cellular functions that are not AAV-derived, upon which AAV replicates. The term includes proteins and RNAs required in AAV replication, including those involved in activating AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products, and AAV capsid packaging. The viral-based accessory function may be derived from any known helper virus, such as adenovirus, herpes virus (other than herpes simplex virus type 1), and vaccinia virus.

An "accessory function vector" generally refers to a nucleic acid molecule comprising a polynucleotide sequence that provides an accessory function. Such sequences may be on accessory functional vectors and transfected into suitable host cells. Accessory functional vectors are capable of supporting rAAV virion production in host cells. The accessory functional vector may be in the form of a plasmid, phage, transposon or cosmid. In addition, the accessory function does not require complete supplementation of the adenovirus gene. For example, adenovirus mutants that are incapable of DNA replication and late gene synthesis have been reported to permit AAV replication (Ito et al, (1970) J.Gen.Virol.9:243; ishibashi et al, (1971) Virology 45:317). Similarly, mutants within the E2B and E3 regions have been shown to support AAV replication, suggesting that the E2B and E3 regions may not be involved in providing ancillary functions (Carter et al, (1983) Virology 126:505). Adenovirus defective in the El region or adenovirus with a deleted E4 region cannot support AAV replication. Thus, the El A and E4 regions appear to be necessary 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 (P.Tijssen eds., 1990)); e3 (Carter et al (1983), supra); and E4 (Carter et al (1983), supra; carter (1995)). The investigation of the accessory functions provided by adenoviruses with mutations in the EIB coding region produced conflicting results, but ElB k may be required for AAV virion production, while EIB 19k is not (Samulski et al, (1988) J.Virol.62:206-210). In addition, international publication WO 97/17458 and Matshashita et al, (1998) Gene Therapy 5:938-945 describe an accessory functional vector encoding various adenovirus genes. Exemplary accessory functional vectors include an adenovirus VA RNA coding region, an adenovirus E4 ORF6 coding region, an adenovirus E2a 72kD coding region, an adenovirus E1A coding region, and an adenovirus EIB region lacking the intact ElB k coding region. Such an accessory function vector is described, for example, in International publication No. WO 01/83797.

As used herein, the term "serotype" is used to refer to the differentiation of AAV having a capsid that is serologically distinct from other AAV serotypes. Serological uniqueness is determined based on the lack of cross-reactivity between antibodies against one AAV and antibodies against another AAV. The cross-reactivity differences are typically due to differences in capsid protein sequences/antigenic determinants (e.g., due to VP1, VP2, and/or VP3 sequence differences in AAV serotypes).

Under conventional definition, a serotype means that the neutralizing activity of serum specific for all existing and characterized serotypes has been tested for the virus of interest and antibodies have not been found to neutralize the virus of interest. Serological differences from any presently existing serotype may or may not exist as more naturally occurring viral isolates are discovered and/or capsid mutants are generated. Thus, in the case where there is no serological difference in a new virus (e.g., AAV), the new virus (e.g., AAV) will be a subgroup or variant of the corresponding serotype. In many cases, mutant viruses with capsid sequence modifications have not been subjected to serological testing for neutralization activity to determine whether they belong to another serotype based on the traditional definition of serotypes. Thus, for convenience and to avoid duplication, the term "serotype" broadly refers to both serologically distinct viruses (e.g., AAV) and viruses that may not be serologically distinct (e.g., AAV) within a given serotype subgroup or variant.

rAAV vectors include any viral strain or serotype. As non-limiting examples, the rAAV plasmid or vector genome or particle (capsid) can be based on any AAV serotype, including, for example, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9 (hu 14), AAV10, AAV11, AAV12, rh8, rh10, rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ8, NP59, anc-80, and variants thereof, including AAV capsid variants set forth in the following: pulcherla et al mol. Ter., 19 (6) 1070-1078 (2011) (describing AAV9 variants, including in particular AAV 9.47), U.S. patent No. 7,906,111 (describing in particular AAV9 (hu 14)), 10,532,111 (describing in particular NP 59), 10,738,087 (describing in particular Anc-80), 9,169,299 (describing "LK 03"), 9,840,719 (describing "RHM 4-1"), 7,749,492, 7,588,772 (describing "DJ" and "DJ 8"), 9,587,282, and patent applications WO 2012/145601, WO 2013/158879, WO 2015/01353, WO 2018/156654, US2013/0059732, all of which are incorporated herein by reference in their entirety. All of the foregoing descriptions of AAV (including sequence information thereof) are incorporated by reference in their entirety. Such vectors may be based on the same strain or serotype (or subgroup or variant) or different from each other. As a non-limiting example, a rAAV plasmid or vector genome or particle (capsid) based on one serotype genome may be identical to one or more capsid proteins of the packaging vector. In addition, the rAAV plasmid or vector genome may be based on an AAV (e.g., AAV 2) serotype genome that differs from one or more capsid proteins of the packaging vector genome, in which case at least one of the three capsid proteins may be, but is not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9 (hu 14), AAV10, AAV11, AAV12, rh8, rh10, rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ8, NP59, anc-80, and variants thereof, including AAV capsid variants set forth in: pulcherla et al mol. Ter., 19 (6) 1070-1078 (2011) (describing AAV9 variants, including in particular AAV 9.47), U.S. patent No. 7,906,111 (describing in particular AAV9 (hu 14)), 10,532,111 (describing in particular NP 59), 10,738,087 (describing in particular Anc-80), 9,169,299 (describing "LK 03"), 9,840,719 (describing "RHM 4-1"), 7,749,492, 7,588,772 (describing "DJ" and "DJ 8"), 9,587,282, and patent applications WO 2012/145601, WO 2013/158879, WO 2015/01353, WO 2018/156654, US2013/0059732, all of which are incorporated herein by reference in their entirety. Thus, rAAV vectors include gene/protein sequences that are identical to gene/protein sequences that are unique to a particular serotype and mixed serotypes. Various embodiments are applicable to any rAAV or AAV capsid from any source, provided that the capsid is capable of binding to an ion exchange chromatography column.

In various exemplary embodiments, a rAAV vector includes or consists 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: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9 (hu 14), AAV10, AAV11, AAV12, rh8, rh10, rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ8, NP59, anc-80 and variants thereof, including AAV capsid variants set forth in: pulcherla et al mol. Ter., 19 (6) 1070-1078 (2011) (describing AAV9 variants, including in particular AAV 9.47), U.S. patent No. 7,906,111 (describing in particular AAV9 (hu 14)), 10,532,111 (describing in particular NP 59), 10,738,087 (describing in particular Anc-80), 9,169,299 (describing "LK 03"), 9,840,719 (describing "RHM 4-1"), 7,749,492, 7,588,772 (describing "DJ" and "DJ 8"), 9,587,282, and patent applications WO 2012/145601, WO 2013/158879, WO 2015/01353, WO 2018/156654, US2013/0059732, all of which are incorporated herein by reference in their entirety. In various exemplary embodiments, the rAAV vector comprises or consists 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: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9 (hu 14), AAV10, AAV11, AAV12, rh8, rh10, rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ8, NP59, anc-80 and variants thereof, including AAV capsid variants set forth in: pulcherla et al mol. Ter., 19 (6) 1070-1078 (2011) (describing AAV9 variants, including in particular AAV 9.47), U.S. patent No. 7,906,111 (describing in particular AAV9 (hu 14)), 10,532,111 (describing in particular NP 59), 10,738,087 (describing in particular Anc-80), 9,169,299 (describing "LK 03"), 9,840,719 (describing "RHM 4-1"), 7,749,492, 7,588,772 (describing "DJ" and "DJ 8"), 9,587,282, and patent applications WO 2012/145601, WO 2013/158879, WO 2015/01353, WO 2018/156654, US2013/0059732, all of which are incorporated herein by reference in their entirety.

rAAV (including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9 (hu 14), AAV10, AAV11, AAV12, rh8, rh10, rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ8, NP59, anc-80 and variants thereof, including AAV capsid variants as set forth in pulcherla et al, mol. Ther.,19 (6) 1070-1078 (2011) (including AAV 9.47), US patent No. 7,906,111 (particularly AAV9 (hu14), 10,532,111 (particularly NP 59), 10,738,087 (particularly including Anc-80), 9,169,299 (including "LK 03"), 9,840,719 (including "m 4-1"), 7,749,492, 7,588,772 (including "DJ" and "DJ 8"), 9,587,282 and WO 2012/WO 20135, WO/20135, 201973 and 20135, or a plurality of variants thereof may be constructed by a variety of techniques including by crossing the sequence of known techniques, and by a variety of such techniques, and by a variety of the present inventors. Such vectors have one or more wild-type AAV genes deleted in whole or in part, but retain at least one functional flanking ITR sequence that is necessary for rescue, replication and packaging of the recombinant vector into rAAV vector particles. Thus, the rAAV vector genome will include cis sequences (e.g., functional ITR sequences) required for replication and packaging.

The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to all forms of nucleic acid, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids include genomic DNA, cDNA, and antisense DNA, as well as spliced or non-spliced 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-spliced RNA, or antisense RNA). Nucleic acids include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. The nucleic acid may be single, double or triplex, linear or circular, and may have any length. In discussing nucleic acids, the sequence or structure of a particular polynucleotide may be described herein according to the convention of providing sequences in the 5 'to 3' direction.

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

"polypeptide", "protein" and "peptide" encoded by a "nucleic acid sequence" include full-length native sequences (as naturally occurring proteins), as well as functional subsequences, modified forms or sequence variants, so long as the subsequences, modified forms or variants retain some degree of function of the native full-length protein. Such polypeptides, proteins and peptides encoded by the nucleic acid sequences may be, but need not be, identical to endogenous proteins that are defective in the mammal being treated or that are under-expressed or absent.

"transgene" is used herein to conveniently refer to a nucleic acid (e.g., heterologous) that is intended to or has been introduced into a cell or organism. Transgenes include any nucleic acid, such as a heterologous nucleic acid or heterologous polynucleotide sequence encoding a therapeutic protein.

In cells with transgenes, the transgene has been introduced/transferred by way of a plasmid or AAV vector, "transduction" or "transfection" of the cell. The terms "transduction" and "transfection" refer to the introduction of a molecule (e.g., a nucleic acid) into a host cell (e.g., HEK 293) or a cell of an organism. The transgene may or may not be integrated into the genomic nucleic acid of the recipient cell. If the introduced nucleic acid is integrated into the nucleic acid (genomic DNA) of the recipient cell or organism, it can be stably maintained in the cell or organism and further transferred or inherited to the progeny cell or organism of the recipient cell or organism's cell.

"host cell" means, for example, microorganisms, yeast cells, insect cells, and mammalian cells that can or have been used as AAV vector plasmids, AAV helper constructs, accessory functional vectors, or other recipients of transfer DNA. The term includes progeny of the original cell that has been transfected. Thus, a "host cell" generally refers to a cell that has been transfected with an exogenous DNA sequence. It will be appreciated that the morphology or genomic or total DNA complement of the offspring of a single parent cell may not necessarily be exactly the same as the original parent due to natural, accidental or deliberate mutation. Exemplary host cells include Human Embryonic Kidney (HEK) cells (e.g., HEK 293).

As used herein, a "therapeutic protein" is a peptide or protein that can reduce or decrease symptoms caused by insufficient, absent, or defective amounts of the protein in a cell or subject. The "therapeutic" protein encoded by the transgene may confer a benefit to the subject, e.g., correcting genetic defects, correcting gene (expression or function) defects, and the like.

Non-limiting examples of heterologous nucleic acids encoding gene products (e.g., therapeutic proteins) useful according to the invention include those useful in the treatment of diseases or disorders including, but not limited to, "hemostasis" or blood clotting disorders (e.g., hemophilia a), hemophilia a patients with inhibitory antibodies, hemophilia B, clotting factors (VII, VIII, IX and X, XI, V, XII, II, von willebrand factor) deficiency, FV/FVIII combination 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); over anticoagulation associated with heparin, low molecular weight heparin, pentose, warfarin, small molecule antithrombotics (i.e., FXa inhibitors); and platelet disorders such as giant platelet syndrome (Bernard Soulier syndrome), glanzman platelet insufficiency, and reservoir defects (storage pool deficiency).

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 of the nervous system. In certain embodiments, the CNS or nervous system degenerative disease is alzheimer's disease, huntington's disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, kennedy disease, polyglutamine repeat disease (polyglutamine repeat disease), or parkinson's disease. In certain embodiments, the CNS or nervous system degenerative disease is polyglutamine repeat disease. In certain embodiments, the polyglutamine repeat disease is spinocerebellar ataxia (SCA 1, SCA2, SCA3, SCA6, SCA7, or SCA 17).

In certain embodiments, the heterologous nucleic acid encodes a protein selected from the group consisting of: GAA (acid alpha-glucosidase) for treating pompe disease; ATP7B (copper transport atpase 2) for the treatment of wilson's disease; alpha-galactosidase for use in the treatment of brix disease; ASS1 (argininosuccinate synthase) for the treatment of citrullinemia type 1; beta-glucocerebrosidase for the treatment of gaucher type 1 disease; beta-hexosaminidase a for the treatment of tassel disease (Tay Sachs disease); SERPING1 (C1 protease inhibitor or C1 esterase inhibitor) for use in the treatment of Hereditary Angioedema (HAE) (also known as type I and type II C1 inhibitor deficiency); and glucose-6-phosphatase for use in the treatment of type I Glycogen Storage Disease (GSDI).

In certain embodiments, the heterologous nucleic acid encodes CFTR (cystic fibrosis transmembrane regulator), blood coagulation (factor XIII, factor IX, factor VIII, factor X, factor VII, factor VIIa, protein C, etc.), functionally acquired clotting factors, antibodies, retinal pigment epithelium-specific 65kDa protein (RPE 65), erythropoietin, LDL receptors, lipoprotein lipase, ornithine transcarbamylase, beta-globin, alpha-globin, shadow protein, alpha-antitrypsin, adenosine Deaminase (ADA), metal transporter (ATP 7A or ATP 7), sulfonamide enzymes involved in lysosomal storage diseases (ARSA), hypoxanthine guanine phosphoribosyl transferase, beta-25 glucocerebrosidase, sphingomyelinase, lysosomal aminohexosidases, branched-chain ketoacid dehydrogenases, hormones, growth factors, insulin-like growth factors 1 or 2, platelet-derived growth factors, epidermal growth factors, nerve growth factors, neurotrophic factors-3 and-4, brain-derived neurotrophic factors, glial cell-derived growth factors, transforming growth factors alpha and beta, cytokines, alpha-interferon, beta-interferon, interferon-gamma, interleukin-2, interleukin-4, interleukin 12, granulocyte-macrophage colony stimulating factor, lymphotoxins, suicide gene products, herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor necrosis factor, drug-resistant proteins, tumor suppressor proteins (e.g., P53, rb, wt-1), NF1, hippel-lindau (VHL), adenomatous Polyposis Coli (APC)), peptides with immunomodulatory properties, tolerogenic or immunogenic peptides or proteins Tregitope or hCDR1, insulin, glucokinase, guanylate cyclase 2D (LCA-GUCY 2D), rab guard 1 (Rab escort protein 1) (choroid-free), LCA 5 (LCA-lebericlin), ornithine ketoacid aminotransferase (gyrate atrophy), retinolytic element 1 (X-linked retinal split), USH1C (irschel syndrome 1C), X-linked retinitis pigmentosa gtpase (XLRP), MERTK (AR form of RP: retinal pigment degeneration), DFNB1 (connexin 26 deafness), ACHM 2, 3 and 4 (total color blindness), PKD-1 or PKD-2 (polycystic kidney disease), TPP1, CLN2, sulfatase, N-acetylglucosamine-1-phosphotransferase, cathepsin a, GM2-AP, NPC1, VPC2, sphingolipid activator protein, one or more zinc finger nucleases for genome editing, or one or more donor sequences that serve as repair templates for genome editing.

Nucleic acid molecules, vectors (e.g., cloning vectors, expression vectors (e.g., vector genomes), and plasmids) can be prepared using recombinant DNA techniques. The availability of nucleotide sequence information enables the preparation of nucleic acid molecules in a variety of ways. For example, heterologous nucleic acids encoding Factor IX (FIX) constituting a vector or plasmid can be prepared via cellular expression or in vitro translation and chemical synthesis techniques using a variety of standard cloning, recombinant DNA techniques. The purity of the polynucleotide may be determined by sequencing, gel electrophoresis, and the like. For example, nucleic acids may be isolated using hybridization or computer-based database screening techniques. Such techniques include, but are not limited to: (1) Hybridization of genomic DNA or cDNA library with probes to detect homologous nucleotide sequences; (2) Antibody screening to detect polypeptides having shared structural features, e.g., using an expression library; (3) Performing Polymerase Chain Reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to a nucleic acid sequence of interest; (4) computer retrieving the relevant sequences in the sequence database; and (5) differential screening of the subtractive nucleic acid library.

Methods known in the art for producing rAAV virions: for example, the use of an AAV vector in combination with AAV helper sequence transfection is co-infected with an AAV helper virus (e.g., adenovirus, herpes virus, or vaccinia virus) or transfected with a recombinant AAV vector, AAV helper vector, and accessory functional vector. Non-limiting methods for producing rAAV virions are described, for example, in U.S. Pat. Nos. 6,001,650 and 6,004,797, international application PCT/US16/64414 (published as WO 2017/096039), and U.S. provisional application Nos. 62/516,432 and 62/531,626. Following 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.

Method for purifying complete rAAV particles

and

(c) Eluting the intact rAAV particles bound to the chromatographic medium with an elution buffer to obtain a purified preparation having an increased ratio of intact rAAV particles to non-intact particles.

According to embodiments of the present application, a rAAV formulation may be any mixture comprising intact rAAV particles and non-intact particles (including empty particles and partial particles) and variants thereof. The rAAV formulation may also include a mixture of various non-intact particles (e.g., empty particles and partial particles) for further separation. For example, the rAAV formulation can be a cell lysate, a treated cell lysate, a supernatant, or a previously purified formulation. In view of the present disclosure, rAAV formulations useful in the methods of the present application may be obtained using any method of collecting rAAV known in the art. For example, cell lysates 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 it may be further processed or stored prior to use in the methods of the present application.

Typically, the cell lysate is clarified to remove cellular debris (e.g., filtration and centrifugation) to provide a clarified cell lysate. The lysate (optionally clarified) contains intact rAAV particles, non-intact particles, and other rAAV vector production/process-related impurities, such as soluble cellular components (which may include, inter alia, cellular proteins, lipids, and/or nucleic acids) from the host cell, and cell culture medium components. The optionally clarified lysate may then be subjected to additional purification steps to remove other process-related impurities by any method known in the art. The resulting treated lysate may be diluted or concentrated with an appropriate buffer before use in the methods of the present application.

In some embodiments, the non-intact particles comprise empty particles.

In some embodiments, the non-intact particles comprise a portion of particles.

In some embodiments, the chromatographic medium is an ion exchange column chromatographic medium, preferably an anion exchange chromatographic medium. The anion exchange chromatography medium used in the process may be a strong anion exchange resin or a weak anion exchange resin. Preferably, the anion exchange chromatography medium comprises an anion exchange ligand such as a proprietary quaternary amine, quaternized polyethylenimine, polyethylenimine or dimethylaminopropyl group. More preferably, the anion exchange chromatography medium is selected from weak anion exchange resins (e.g., poros 50D, poros50 PI) or strong anion exchange resins (e.g., poros XQ, poros50 HQ). Other examples of anion exchange chromatography media include, but are not limited to, DEAE sepharose FF, Q-sepharose (HP and FF), Q sepharose FF (low and high substitution), capto Q, Q XP, source 30Q and 15Q, fractogel DEAE and MPHQ. Other examples of anion exchange chromatography media include, for example, CIMmultus ^TM Integral QA monolith.

In some embodiments, the chromatographic medium is an ion exchange chromatographic medium, preferably an anion exchange chromatographic medium.

In some embodiments, the chromatographic medium is, for example, CIMmultus ^TM Integral QA monolith.

In some embodiments, a variety of chromatographic media are used.

In some embodiments, where multiple chromatographic media are used, the media are the same.

In some embodiments, the ion exchange chromatography medium is bound (meaning either before or after) to an affinity chromatography medium (e.g., AVB Sepharose ^TM High Performance (GE Healthcare, markylor, mass.) or size exclusion chromatography (e.g.Superdex200 (GE Healthcare)).

In some embodiments, when the rAAV formulation is loaded into the column comprising chromatographic medium in step (b) in a loading buffer, only the intact rAAV particles bind to the chromatographic medium, while the non-intact particles do not bind to the chromatographic medium and flow through the column. In some embodiments, the flow-through from the column comprises empty particles. In some embodiments, the flow-through from the column comprises a portion of the particles. In some embodiments, the flow-through from the column comprises both intact rAAV particles and empty particles. In some embodiments, the flow-through from the column comprises both whole rAAV particles and partial particles. In some embodiments, the flow-through from the column comprises both intact rAAV particles and non-intact particles (including empty particles and partial particles).

In some embodiments, upon application of the rAAV formulation in a loading buffer to the column comprising chromatographic medium in step (b), both intact rAAV particles and non-intact particles (including variants like portions) bind to the chromatographic medium, but the binding affinity of the intact rAAV particles to the chromatographic medium is higher than the non-intact particles; and the amount of intact particles and non-intact particles applied to the column exceeds the binding capacity of the chromatographic medium such that the non-intact particles bound to the chromatographic medium are displaced by the intact rAAV particles into the loading flow-through from the column. The loaded flow-through from the column comprises both intact rAAV particles and non-intact particles. In certain embodiments, some non-intact particles (e.g., empty particles and/or partial particles) are bound to the chromatographic medium, which may be eluted by a wash buffer, followed by elution of the intact rAAV particles with an elution buffer.

While not wanting to be bound by theory, it is believed that separation of intact AAV particles and non-intact AAV particles occurs during loading due to the displacement phenomenon. When a rAAV formulation comprising a mixture of particles is first applied to a column, all particles bind to available sites on the column. When the available sites are occupied towards the inlet of the column, intact rAAV particles with higher affinity for the chromatographic medium displace bound non-intact particles with lower affinity for the chromatographic medium, and the intact particles are enriched in the top of the column. In this process, the non-intact particles bind to the downstream chromatographic medium. As more mixture is applied to the column, more intact rAAV particles will bind to the upstream chromatographic medium, displace the bound non-intact particles and push the non-intact particles further downstream. In certain embodiments, after sample loading is complete, no or a reduced amount of non-intact particles are bound to the chromatographic medium as compared to the intact rAAV. Because the replacement of non-intact particles by intact particles is a result of their different affinities for chromatographic media, enhancing the contrast of their affinities can improve separation. The affinity with which the particles bind to the chromatographic medium is affected by factors such as those associated with the rAAV particle, the resin, and the environment, including buffer conditions (salt and pH). Similarly, when the incomplete rAAV particle includes a partial particle, the reasoning applies to the partial particle. As a variant of the population of non-intact particles, part of the particles may bind to the chromatographic medium with higher affinity than other non-intact particles (in particular empty particles) but with lower affinity than intact particles, and thus both intact particles and part of the particles may displace other non-intact particles. Enhancing the contrast of the affinity of the whole particles and part of the particles to the chromatographic medium may further improve the separation of part of the particles from the whole particles, among other non-whole particles. Also similarly, when non-intact rAAV particles include empty particles, the disclosed methods can be used to improve separation of empty particles from intact particles and other non-intact particles.

Once penetration occurs in the column (beyond the point of binding capacity of the column chromatography media), the benefit of displacement is realized. When the chromatographic medium resin begins to partially cross its saturation level, competition between intact particles and non-intact particles ensues, resulting in replacement of the bound non-intact particles by the incoming intact particles.

Based on their different affinities for chromatographic media, the benefits of substitution can be similarly applicable to the isolation of other rAAV impurities and product variants, such as partially filled capsids with truncated transgenes, empty, partial or complete capsid variants with post-translational modifications, or fragments or aggregates.

In some embodiments, when the non-intact particles include both empty particles and partial particles, all of the non-intact particles bind to available sites on the column. When the available sites are occupied, part of the particles (which have a relatively higher affinity for the chromatographic medium than the empty particles) displace the bound empty particles.

Chromatographic media (e.g., anion exchange resins) can be equilibrated, washed, and eluted with various buffers under various conditions (e.g., pH and buffer volumes). The following is intended to describe specific non-limiting examples, but is not intended to limit the invention.

Standard buffers can be used and anion exchange chromatography balanced according to manufacturer's instructions. After equilibration, the sample is then loaded. The chromatographic medium is then washed at least one or more times, for example 2-10 column volumes. Elution from the chromatographic medium was performed by 2-20 column volumes of high salt buffer.

According to embodiments of the present application, equilibration buffers for anion exchange chromatography and solutions for washing and elution are suitable at a pH of about pH 6.0 to pH 12. In addition, suitable equilibration buffers and solutions for washing and eluting of anion exchange columns are generally cationic or zwitterionic in nature. Such buffers include, but are not limited to, buffers with the following buffers: n-methylpiperazine; piperazine; bis-Tris; bis-Tris propane; triethanolamine; tris; tris acetate; n-methyldiethanolamine; 1, 3-diaminopropane; ethanolamine; acetic acid, and the like. For eluting the sample, salts (e.g. NaCl, KCl, mgCl ₂ 、CaCl ₂ Sulfate, formate, or acetate) to increase the ionic strength of the starting buffer.

In some embodiments, the loading buffer comprises at least one buffer selected from the group consisting of: tris, bis-Tris propane, tris acetate, ethanolamine and phosphate. In certain embodiments, the loading buffer comprises the selected buffer at a concentration of about 20-50mM, such as 20mM, 30mM, 40mM, 50mM, 100mM, or any concentration in between. In a preferred embodiment, the loading buffer comprises 20-50mM Tris.

In some embodiments, the loading buffer comprises 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 ⁺ . For example, the loading buffer may comprise a buffer selected from NaCl, mgCl ₂ And CaCl ₂ Is a salt of a metal. In a preferred embodiment, the loading buffer comprises NaCl, mgCl ₂ 、CuCl ₂ LiCl and CaCl ₂ . The anionic component of the salt is not critical and no particular anion is preferred.

In some embodiments, 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 ⁺ 。

In some embodiments, when the loading buffer comprises 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 ⁺ Only the intact rAAV particles bind to the chromatographic medium, while the non-intact particles do not bind to the chromatographic medium and flow through the column. In a preferred embodiment, the loading buffer comprises at least CaCl ₂ 。

In some embodiments, the loading buffer comprises sodium salt at about 10-100mM, such as 10, 20, 30, 40, 50, 60, 70, 80, 90, 100mM or any concentration in between. In preferred embodiments, the concentration of sodium salt is between about 20-60mM, such as 20, 25, 30, 35, 40, 45, 50, 55, 60mM or any concentration in between.

In some embodiments, the loading buffer comprises magnesium salt at any concentration between about 0-20mM, such as 0, 5, 10, 15, 20mM, or in between. In preferred embodiments, the concentration of magnesium salt is between about 1-10mM, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10mM or any concentration in between.

In some embodiments, the loading buffer comprises lithium salt at a concentration of about 0-100mM, such as 0, 20, 30, 40, 50, 60, 70, 80, 90, 100mM or any concentration in between. In preferred embodiments, the concentration of lithium salt is between about 0-75mM, such as 0, 15, 25, 35, 45, 55, 65, 75mM or any concentration in between.

In some embodiments, the loading buffer comprises calcium salt at a concentration of about 0-10mM, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10mM, or any concentration in between. In preferred embodiments, the concentration of the calcium salt is between about 0.1 and 2.5mM, such as 0.1, 0.5, 1.0, 1.5, 2.0, 2.5mM, or any concentration in between.

In some embodiments, the loading buffer comprises copper salt at a concentration of about 0-5mM, such as 0, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5mM, or any concentration in between. In preferred embodiments, the concentration of copper salt is between about 0.1 and 2.5mM, such as 0.1, 0.5, 1.0, 1.5, 2.0, 2.5mM, or any concentration in between.

In some embodiments, the loading buffer comprises ammonium salt at a concentration of about 5-100mM, such as 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100mM, or any concentration in between. In preferred embodiments, the concentration of ammonium is between about 10-50mM, such as 10, 15, 20, 25, 30, 35, 40, 45, 50mM or any concentration in between.

In some embodiments, 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 。

In some embodiments, the pH of the loading buffer is about 7-10, such as 7, 7.5, 8, 8.5, 9, 9.5, 10 or any pH in between, preferably about 8-9.

In certain embodiments, the loading buffer comprises about 20-60mM NaCl, 1-5mM MgCl ₂ 、0.1-2.5mM CaCl ₂ Preferably in Tris, more preferably in 20-50mM Tris.

In certain embodiments, the loading buffer comprises about 20-60mM NaCl, 10-30mM (NH) ₄ ) ₂ SO ₄ 、1-5mM MgCl ₂ 、0.1-2.5mM CuCl ₂ Preferably in Tris, more preferably in 20-100mM Tris.

In some embodiments, the loading buffer comprises at least one surfactant.

After loading step (b), loading the rAAV formulation in a loading buffer into a column comprising a chromatographic medium, wherein the intact rAAV particles have a higher binding affinity to the chromatographic medium than the non-intact particles, and the intact rAAV particles bind to the chromatographic medium. The column may optionally be washed with a wash buffer prior to elution. For example, the wash buffer may have an increased salt concentration and/or an increased pH for elution compared to loading conditions in order to remove non-intact particles bound to the chromatographic medium.

The intact particles bound to the chromatographic medium are then eluted in a purer form, e.g., using increased salt concentration and/or an adjusted pH for elution as compared to loading conditions. Adjusting the pH may enhance the separation of intact particles from non-intact particles. Preferably, only salt gradients are employed without changing the pH to achieve elution of intact particles.

In some embodiments, the elution buffer comprises at least one buffer selected from the group consisting of: tris, bis-Tris propane, tris acetate, ethanolamine and phosphate. In certain embodiments, the elution buffer comprises the selected buffer at a concentration of about 20-70mM, such as 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, or any concentration in between. In a preferred embodiment, the elution buffer comprises 40-60mM Tris.

In some embodiments, the elution buffer comprises 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 ⁺ . For example, the elution buffer may comprise one or more compounds preferably selected from NaCl, mgCl ₂ 、LiCl、CuCl ₂ And CaCl ₂ Is a salt of (a). The anionic component of the salt is not critical.

In some embodiments, 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 ⁺ 。

In some embodiments, the elution buffer comprises about 0-1000mM, such as 0, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000mM or any concentration in between NaCl. In preferred embodiments, the concentration of NaCl is between about 20-300mM, such as 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300mM or any concentration in between.

In some embodiments, the elution buffer comprises MgCl at any concentration between about 0 and 30mM, such as 0, 5, 10, 15, 20, 25, 30mM, or in between ₂ . In a preferred embodiment, mgCl ₂ Such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15mM or any concentration in between.

In some embodiments, the elution buffer comprises LiCl at a concentration of about 0-200mM, such as 0, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200mM or any concentration in between. In preferred embodiments, the concentration of LiCl is between about 0-150mM, such as 0, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150mM or any concentration in between.

In some embodiments, the elution buffer comprises about 0.1-20mM, such as 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20mM or any concentration in between CaCl ₂ . In a preferred embodiment, caCl ₂ At a concentration of about 5-10mMSuch as 5, 6, 7, 8, 9, 10mM or any concentration in between.

In some embodiments, the elution buffer comprises about 0-10mM, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10mM or any concentration in between CuCl ₂ . In a preferred embodiment, cuCl ₂ Between about 0-3mM, such as 0, 1, 2, 3mM or any concentration in between.

In some embodiments, the elution buffer comprises about 5-100mM, such as 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100mM, or any concentration in between (NH) ₄ ) ₂ SO ₄ . In a preferred embodiment, (NH) ₄ ) ₂ SO ₄ Between about 10-50mM, such as 10, 15, 20, 25, 30, 35, 40, 45, 50mM or any concentration in between.

In some embodiments, the pH of the elution buffer is about 6-10, such as 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 or any pH in between, preferably about 7-9.

In certain embodiments, the elution buffer comprises about 20-150mM NaCl, preferably in Tris, more preferably in 40-60mM Tris.

In some embodiments, the elution buffer comprises at least one surfactant.

(c) Loading the first loaded carrier into a second column comprising a second chromatographic medium to obtain a loaded or partially loaded second column and a first loaded carrier from the second column, wherein the second chromatographic medium is the same or the same type as the first chromatographic medium;

(e) Bypassing the second column after the step (c) or after the washing step of (d) if washing step (d) is performed, and eluting the intact rAAV particles bound to the first chromatographic medium with an elution buffer to obtain a first eluate from the first column and an eluted first column, wherein the first eluate has an increased ratio of intact rAAV particles to non-intact rAAV particles;

(f) Optionally, if the second column is not saturated (i.e., loaded) after the first column is run through, a second batch of the rAAV formulation in a loading buffer may be applied to a partially loaded second column, wherein the amount of intact rAAV particles and non-intact particles applied to the second column exceeds the binding capacity of the second chromatographic medium such that non-intact particles bound to the second chromatographic medium are displaced by the intact rAAV particles into a second loading run through from the second column;

(g) If step (f) is performed, loading a second loaded permeate from the second column into the washed first column between steps (d) and (e) to obtain a second loaded first column, and optionally washing the second loaded first column, followed by continuing the elution in step (e);

(i) Bypassing the first column after step (g) or after the washing step (h) if washing step (h) is performed, and eluting the intact rAAV particles bound to the second chromatographic medium with an elution buffer to obtain a second eluate and an eluted second column, wherein the second eluate has an increased ratio of intact rAAV particles to non-intact rAAV particles; and

(j) Combining the first eluate and the second eluate to produce a purified preparation of intact rAAV particles.

In some embodiments, the non-intact particles comprise empty particles.

In some embodiments, the non-intact particles comprise a portion of particles.

In some embodiments, a dual column purification process is set up and operated according to the implementation scheme as shown in fig. 12. Steps (a) and (b) of the dual column method are similar to the first two steps of the single column displacement chromatography method. After these two steps, the intact rAAV particles are bound to a first chromatographic medium and the first loading vehicle from the first column comprises both intact rAAV particles and non-intact particles.

In step (f), a second batch of rAAV formulation in loading buffer is applied to the second column partially loaded in step (c). The amount of the second batch of rAAV formulation may be the same as the first batch or different from the first batch. Preferably, the second batch is the same as the first batch.

According to embodiments of the present application, the amounts of intact rAAV particles and non-intact particles applied to the second column in step (f) include the amount from the first loading carrier and the amount from the second batch of rAAV formulation, and exceed the binding capacity of the second chromatographic medium. During loading of the second batch into the partially loaded second column, non-intact particles bound to the second chromatographic medium are replaced by intact rAAV particles in a second loading flow-through from the second column.

In a subsequent step (g), the second loading permeate from the second column is loaded onto the first column which was previously washed or eluted. The first column is then partially loaded after step (g), and the amount of intact rAAV particles and non-intact particles in the second loaded flow-through from the second column is now no more than the binding capacity of the first chromatographic medium.

Depending on the amount of rAAV formulation, steps (b) through (i) may be performed in one cycle or multiple cycles. The term "one cycle" as used herein refers to steps from step (b) to step (i) which are performed sequentially once. The term "multiple cycles" as used herein refers to steps from step (b) to step (i) which are performed more than once in sequence.

When steps (b) through (i) are performed for multiple cycles, the "first batch" in step (b) is repeated to refer to a new "first batch" (first batch of the number of cycles) of rAAV formulation, and the "second batch" in step (f) is repeated to refer to a new "second batch" of rAAV formulation in the same cycle.

In some embodiments, when the non-intact particles include both empty particles and partial particles, the second eluate in step (i) has increased ratios of intact rAAV particles and partial rAAV particles to empty rAAV particles.

In some embodiments, where multiple cycles are performed, the eluted column may undergo subsequent steps that are beneficial or necessary to maintain consistent column binding capacity throughout the cycle, prior to the next cycle of loading. For example, such steps include, but are not limited to, stripping the column, cleaning and/or sterilizing the column, and/or rebalancing the column.

In some embodiments, the loading buffer comprises at least one buffer selected from the group consisting of: tris, bis-Tris propane, tris acetate, ethanolamine and phosphate. In certain embodiments, the loading buffer comprises the selected buffer at a concentration of about 20-50mM, such as 20mM, 30mM, 40mM, 50mM, or any concentration in between. In a preferred embodiment, the loading buffer comprises 20-50mM Tris.

In some embodiments, the loading buffer comprises in the range of 0-200mM 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 anionic component of the salt is not critical and no particular anion is preferred. For example, the loading buffer may comprise one or more compounds preferably selected from NaCl, mgCl ₂ 、LiCl、CuCl ₂ And CaCl ₂ Is a salt of (a).

In some embodiments, the loading buffer comprises ammonium salt at about 5-100mM, such as 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100mM or any concentration in between. In preferred embodiments, the concentration of ammonium is between about 10-50mM, such as 10, 15, 20, 25, 30, 35, 40, 45, 50mM or any concentration in between.

In some embodiments, the loading buffer comprises at least one surfactant.

In some embodiments, the pH of the loading buffer is about 6-10, such as 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 or any pH in between, preferably about 8-9.

In certain embodiments, the loading buffer comprises about 20-60mM NaCl, 5-30mM (NH) ₄ ) ₂ SO ₄ 、1-5mM MgCl ₂ 、0.1-3mM CuCl ₂ Preferably in Tris, more preferably in 20-50mM Tris, with 0.00005% to 0.01% poloxamer 188, more preferably with 0.0002% to 0.001% poloxamer 188.

After the loading step, the intact particles bind to the chromatographic medium and the non-intact particles leave the column in the flow-through. The column may optionally 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 pH for elution compared to loading conditions in order to remove non-intact particles bound to the chromatographic medium.

The intact particles bound to the chromatographic medium are then subsequently eluted in a purer form, e.g., using increased salt concentration and/or adjusted pH for elution as compared to loading conditions. Adjusting the pH may allow for enhanced separation. Preferably, only salt gradients are employed without increasing the pH to achieve elution of intact particles.

In some embodiments, the elution buffer comprises at least one buffer selected from the group consisting of: tris, bis-Tris propane, tris acetate, ethanolamine and phosphate. In certain embodiments, the loading buffer comprises the selected buffer at a concentration of about 20-70mM, such as 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, or any concentration in between. In a preferred embodiment, the loading buffer comprises 40-60mM Tris.

In some embodiments, the elution buffer comprises 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 ⁺ . For example, the elution buffer may comprise one or more compounds preferably selected from NaCl, mgCl ₂ 、LiCl、CuCl ₂ And CaCl ₂ Is a salt of (a). The anionic component of the salt is not critical.

In some embodiments, 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 ⁺ . For example, the loading buffer may comprise one or more compounds preferably selected from NaCl, mgCl ₂ 、LiCl、CuCl ₂ And CaCl ₂ Is a salt of (a).

In some embodiments, the elution buffer comprises sodium salt at a concentration of about 0-1000mM, such as 0, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000mM or any concentration in between. In preferred embodiments, the concentration of sodium salt is between about 20-300mM, such as 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300mM or any concentration in between.

In some embodiments, the elution buffer comprises magnesium salt at any concentration between about 0-30mM, such as 0, 5, 10, 15, 20, 25, 30mM, or in between. In preferred embodiments, the concentration of magnesium salt is between about 2-15mM, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15mM or any concentration in between.

In some embodiments, the elution buffer comprises about 0-200mM, such as 0, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200mM or any concentration in between of lithium salt. In preferred embodiments, the concentration of lithium salt is between about 0-150mM, such as 0, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150mM or any concentration in between.

In some embodiments, the elution buffer comprises calcium salt at about 0.1-20mM, such as 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20mM, or any concentration in between. In preferred embodiments, the concentration of the calcium salt is between about 5-10mM, such as 5, 6, 7, 8, 9, 10mM or any concentration in between.

In some embodiments, the elution buffer comprises copper salt at a concentration of about 0-5mM, such as 0, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5mM, or any concentration in between. In preferred embodiments, the concentration of copper salt is between about 0.1 and 2.5mM, such as 0.1, 0.5, 1.0, 1.5, 2.0, 2.5mM, or any concentration in between.

In some embodiments, the elution buffer comprises ammonium salt at a concentration of about 5-100mM, such as 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100mM or any concentration in between. In preferred embodiments, the concentration of ammonium is between about 10-50mM, such as 10, 15, 20, 25, 30, 35, 40, 45, 50mM or any concentration in between.

In some embodiments, the pH of the elution buffer is about 6-10, such as 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 or any pH in between, preferably about 8-9.

In some embodiments, the elution buffer comprises at least one surfactant.

In some embodiments, the methods use no less than two columns (e.g., three columns) to purify the whole rAAV particle. Specifically, the method does not include steps (g) to (j), but further includes:

(k) Loading a second loading fluid from the second column into a third column comprising a third chromatographic medium after step (f), to obtain a loaded or partially loaded third column, preferably the third chromatographic medium is 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) bypassing the first column after step (k) or after washing step (l) if washing step (l) is performed, and eluting the intact rAAV particles bound to the second chromatographic medium with an elution buffer to obtain a second eluate and an eluted second column, wherein the second eluate has an increased ratio of the intact rAAV particles to the non-intact rAAV particles;

(n) optionally, if the third column is not saturated (i.e., loaded) after the second flow-through, a third batch of the rAAV formulation in a loading buffer can be applied to a partially loaded third column, wherein the amount of the intact rAAV particles and the non-intact particles applied to the third column exceeds the binding capacity of the third chromatography medium such that the non-intact particles bound to the third chromatography medium are displaced by the intact rAAV particles into a third loaded flow-through from the third column;

if step (n) is performed, loading a third loaded permeate from the third column into the washed first column between steps (d) and (e) to obtain a second loaded first column, and optionally washing the second loaded first column, followed by continuing the elution in step (e);

optionally washing the third column with a washing buffer to obtain a washed third column;

(q) bypassing the first column and the second column, and eluting the intact rAAV particles bound to the third chromatographic medium with an elution buffer to obtain a third eluate and an eluted third column, wherein the third eluate has an increased ratio of the intact rAAV particles to the non-intact rAAV particles; and

(r) combining the first eluate, the second eluate, and the third eluate to produce a purified preparation of intact rAAV particles.

In some embodiments, when the non-intact particles include empty particles and a portion of the particles, the intact particles are enriched in the first column, the portion of the particles are enriched in the second column, and the empty particles are enriched in the third column.

To the best of the inventors' knowledge, there is no displacement chromatography method for purifying intact rAAV particles from non-intact rAAV particles. The purity of the whole particles is significantly increased after the process of the present application compared to existing purification methods for purifying whole particles, such as conventional anion exchange chromatography. In some embodiments, the purified formulation is substantially free of non-intact particles, more specifically, substantially free of empty particles and/or partial particles. In other embodiments, the purified preparation has an increased ratio of the intact rAAV particles to the non-intact particles compared to the rAAV preparation. Preferably, the ratio of intact rAAV particles to non-intact particles in the purified formulation is no less than 9:1, such as no less than 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, or 50:1, or any ratio in between, more preferably no less than 49:1.

According to embodiments of the present application, the ratio of intact rAAV particles to non-intact particles in the purified formulation may be calculated by the number of intact rAAV particles and non-intact particles. In some embodiments, the ratio is derived from a calibration curve based on the molar concentrations of intact rAAV particles and non-intact particles. In some embodiments, the ratio is calculated based on the number of intact rAAV particles and non-intact particles.

In some embodiments, the ratio of intact particles to non-intact particles in the purified intact rAAV particles is not less than 9:1, preferably not less than 49:1.

The displacement chromatography method of the present application can also achieve high yields/recovery of purified whole particles. In some embodiments, the yield of the purified whole rAAV particle is no less than 70%, preferably no less than 80%, more preferably no less than 90%, and most preferably no less than 95%. The term "yield" as used herein refers to the percentage or proportion of intact rAAV particles in the purified preparation relative to intact rAAV particles in the initial rAAV preparation. There are various methods of calculating the yield. One way to calculate the yield is to multiply 100 by the percentage (%) = (amount of intact rAAV particles in the purified preparation)/(amount of intact rAAV particles in the initial rAAV preparation). Another way to calculate the percent yield is to divide the copy number of the transgene in the purified preparation by the copy number of the transgene in the initial preparation and multiply by 100.

(b) Loading the rAAV formulation in a loading buffer into a column comprising chromatographic medium, wherein the loading buffer comprises CaCl ₂ And the intact rAAV particle is bound to the chromatographic medium;

In some embodiments, the column chromatography medium is selected from the group consisting of Poros HQ, poros PD, polyethylenimine (PI), capto ImpRes Q, and Poros XQ, preferably Poros XQ.

In some embodiments, the amount of intact rAAV particles and non-intact particles applied to the column does not exceed the binding capacity of the chromatographic medium.

In some embodiments, the amount of intact rAAV particles and non-intact particles applied to the column exceeds the binding capacity of the chromatographic medium such that non-intact particles bound to the first chromatographic medium are displaced by intact rAAV particles into the first loading carrier from the first column.

In some embodiments, the non-intact particles comprise empty particles.

In some embodiments, the non-intact particles comprise a portion of particles.

According to embodiments of the present application, the addition of a 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 ⁺ So that more of the intact rAAV particles bind to the chromatographic medium and less of the non-intact particles bind to the chromatographic medium. Thus, the loaded flow-through from the column contains less intact particles, thus increasing the recovery of intact particles. The anionic component of a given salt is not critical.

In some embodiments, the loading buffer further comprises a sodium salt and/or a magnesium salt. In a preferred embodiment, the loading buffer further comprises both sodium and magnesium salts.

In some embodiments, the loading buffer comprises at least one surfactant.

After the loading step, the intact particles bind to the chromatographic medium and the non-intact particles leave the column in the flow-through. The column may optionally 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 loading conditions in order to remove non-intact particles bound to the chromatographic medium.

The intact particles bound to the chromatographic medium are then subsequently eluted in a purer form, e.g., using increased salt concentration and/or adjusted pH for elution as compared to loading conditions. Adjusting the pH may increase separation of intact rAAV particles from non-intact rAAV particles. Preferably, only salt gradients are employed without increasing the pH to achieve elution of intact particles.

In some embodiments, 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 ⁺ . For example, load bufferingThe flushing liquid may comprise one or more substances preferably selected from NaCl, mgCl ₂ 、LiCl、CuCl ₂ And CaCl ₂ Is a salt of (a).

In some embodiments, the elution buffer comprises at least one buffer selected from the group consisting of: tris, bis-Tris propane, tris acetate and phosphate. In certain embodiments, the loading buffer comprises the selected buffer at a concentration of about 20-70mM, such as 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, or any concentration in between. In a preferred embodiment, the loading buffer comprises 40-60mM Tris.

In some embodiments, the elution buffer comprises at least one surfactant.

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 sodium and magnesium salts. The anionic component of the salt is not critical.

In some embodiments, the elution buffer comprises sodium salt at a concentration of about 0-1000mM, such as 0, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000mM or any concentration in between. In preferred embodiments, the concentration of sodium salt is between about 20-150mM, such as 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150mM or any concentration in between.

In some embodiments, the yield of purified whole rAAV particles is no less than 70%, preferably no less than 80%, more preferably no less than 90%, and most preferably no less than 95%. As used herein, the term "yield" refers to the percentage of intact rAAV particles in the purified preparation relative to intact rAAV particles in the initial rAAV preparation. For example, yield (%) = (amount of intact rAAV particles in purified preparation)/(amount of intact rAAV particles in initial rAAV preparation) is expressed as a percentage.

The following embodiments are applicable to each of the general aspects disclosed herein, including the general aspects described above.

The 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 can be from any source (e.g., human, avian, bovine, canine, equine, primate, non-primate, ovine, or any derivative class thereof).

In some embodiments, the rAAV particle is derived from one or more AAV selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9 (hu 14), AAV10, AAV11, AAV12, rh8, rh10, rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ8, NP59, anc-80 and variants thereof, including AAV capsid variants set forth in: pulcherla et al mol. Ter., 19 (6) 1070-1078 (2011) (describing AAV9 variants, including in particular AAV 9.47), U.S. patent No. 7,906,111 (describing in particular AAV9 (hu 14)), 10,532,111 (describing in particular NP 59), 10,738,087 (describing in particular Anc-80), 9,169,299 (describing "LK 03"), 9,840,719 (describing "RHM 4-1"), 7,749,492, 7,588,772 (describing "DJ" and "DJ 8"), 9,587,282, and patent applications WO 2012/145601, WO 2013/158879, WO 2015/013313, WO 2018/156654, US2013/0059732, all of which are incorporated herein by reference in their entirety, including AAV capsids with peptide modifications (such as cell-targeting peptides).

In some embodiments, the intact 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 a (TGFa), platelet-derived growth factor (PDGF), insulin growth factors I and II (IGF-I and IGF-II), TGFp, activin, inhibin, bone Morphogenic Protein (BMP), nerve Growth Factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin NT-3 and NT4/5, ciliary nerve factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, axon factor (neurturin), axon-1 and neurite factor (HGF), liver factor-1 and Hepatocyte Growth Factor (HGF), hedgehog-1 and hedgehog, and hedgehog-2.

In some embodiments, the intact rAAV particle comprises a transgene encoding a gene product selected from the group consisting of: thrombopoietin (TPO), interleukins (IL 1 through IL-17), monocyte chemotactic proteins, leukemia inhibitory factors, 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.

In some embodiments, the intact rAAV particle comprises a transgene encoding a protein useful for correcting an inborn error of metabolism, the protein selected from the group consisting of: carbamoyl synthase I, ornithine transcarbamylase, argininosuccinate synthase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor V, factor VIII, factor IX, cystathionine 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, liver phosphorylase, phosphorylase kinase, glycine decarboxylase, RPE65, protein H, protein T, cystic Fibrosis Transmembrane Regulator (CFTR) sequence, and dystrophin cDNA sequence.

In some embodiments, the intact rAAV particle comprises a transgene encoding factor VIII and factor IX.

The whole rAAV particle of interest purified by the methods according to embodiments of the present application can be produced by a host cell. As an initial step, host cells producing rAAV virions can generally be harvested, optionally in combination with harvesting cell culture supernatant (medium), wherein the host cells producing rAAV virions have been cultured (suspended or adherent). In the methods herein, the harvested cells and optionally the cell culture supernatant may be used as such or concentrated as appropriate. In addition, if infection is used to express accessory functions, residual helper virus may be inactivated. For example, adenovirus may be inactivated by heating to a temperature of about 60 ℃ for, e.g., 20 minutes or more, which only inactivates helper virus, as AAV is thermostable and helper adenovirus is thermolabile.

The rAAV particles are released by disrupting the cells, e.g., by chemical or physical means (e.g., detergent, microfluidization, and/or homogenization), to lyse the cells and/or supernatant of the harvest. In parallel during or subsequent to cell lysis, nucleases (e.g., benzonase) may be added to degrade contaminating DNA. Typically, the resulting lysate is clarified to remove cellular debris (e.g., filtered, centrifuged) to provide a clarified cell lysate. In one specific example, the lysate is filtered with a micron diameter pore size filter (e.g., a 0.1-10.0 μ iota η pore size filter, e.g., a 0.45 μ iota η and/or pore size 0.2 μ η iota filter) to produce a clarified lysate.

The lysate (optionally clarified) contains AAV particles (intact rAAV particles and AAV non-intact particles) and AAV vector production/process related impurities, such as soluble cellular components (which may include, inter alia, cellular proteins, lipids, and/or nucleic acids) from the host cells, and cell culture medium components. The optionally clarified lysate is then subjected to additional purification steps to purify the intact AAV particles from the impurities using chromatography. The clarified lysate may be diluted or concentrated in an appropriate buffer prior to performing the chromatographic methods of the present application.

Examples

Example 1 resin Screen for separation of intact rAAV particles and non-intact particles

In this example, different resins were screened to identify the best resin for separating intact rAAV particles from non-intact particles.

Methods and materials

The sample contained non-intact particles and intact RHM4-1rAAV particles at a ratio of 2.4:1 (i.e., 70% impurity/30% product). The column size was 0.353mL 3mm x 50mm. Resin screening was performed using pulse loading (10% of binding capacity) and a residence time of 1 minute. During the screening process, 3 pH values (8.0, 8.6 and 9.25) and three binding strengths with salt (6.8, 5 and 1.6 mS/cm) were tested. The loading buffer was 50mM Tris or 25mM Tris. The flow-through effluent from the column was analyzed by High Performance Liquid Chromatography (HPLC) to determine the separation of intact particles and non-intact particles.

Results

^TM Poros50 HQ resin Thermo Scientific; POROS- (Walsh Semer, mass): the resin is based on quaternized polyethylenimine functionality some weak Anion Exchange (AEX) functionality. As indicated in fig. 1A-1C, the separation of intact and non-intact particles on Poros50 HQ resin at all three pH values at 50mM Tris and 30mM NaCl was not significantly incompleteAlso, the separation at pH 8.6 is slightly better than the other two pH values.

Poros 50D resin: the resin is based on dimethylaminopropyl functional groups. As indicated in fig. 2A-2D, rAAV particles bound poorly to Poros 50D resin under the screening conditions and decreased with increasing pH. Various loading buffers are used in the figure: pH 8.6 and 50mM Tris (FIG. 2A), pH 8.6 and 25mM Tris (FIG. 2B), pH 8.0 and 25mM Tris (FIG. 2C), and pH 9.25 and 25mM Tris (FIG. 2D).

Poros50 PI resin: the resin is based on polyethyleneimine functions. As indicated in fig. 3A-3E, the binding of rAAV particles to Poros50 PI resin decreased with increasing pH. It was also noted that the addition of NaCl only slightly improved the degree of separation. The figures evaluate Poros50 PI resins at different pH and salt concentrations: pH 8.0 NaCl free (FIG. 3A), pH 8.6 NaCl free (FIG. 3B), pH 8.6 and 30mM NaCl (FIG. 3C), pH 9.2 NaCl free (FIG. 4C) and pH 9.2 and 30mM NaCl (FIG. 3E).

Capto ImpResQ resin: the resin uses Capto with ligand of ionic group ^TM (Cytiva Life Sciences-Markhler, massachusetts) basal agarose matrix. As indicated in fig. 4A-4C, the binding of rAAV particles to Capto ImpRes Q resin increased with increasing pH. However, the Capto ImpRes Q resin separated poorly from intact particles and non-intact particles under the screening conditions. These figures evaluate Capto ImpRes Q resins at different buffers and different pH values as follows: pH 8.0 and 50nm Tris-NaCl free (FIG. 4A), pH 8.6 and 25mM Tris-NaCl free (FIG. 4B), and pH 9.0 and 30mM Tris-NaCl free (FIG. 4C).

PosoXQ resin: the resin is based on specific quaternary amine functionalities. Screening demonstrated that Poros XQ resin was optimal for separation of intact particles and non-intact particles. As indicated in fig. 5, separation at pH 8.75 was optimal compared to other pH values (pH 8 and 9.25). The bound salts also affect separation, as indicated in fig. 6. In addition, when performed at pH 8.75, the separations at different flow rates (84 cm/h, 150cm/h, and 300 cm/h) were similar, as indicated in FIG. 7.

Example 2 optimization of Displacement chromatography with Poros XQ resin

In this example, different conditions were screened to identify optimal conditions for displacement chromatographic separation of intact RHM4-1 rAAV particles and non-intact particles on a Poros XQ resin.

FIG. 8A shows that when the sample is loaded in 30mM Tris, pH 8.6, and the loading is low, there are two product peaks in the HPLC analysis of the effluent. Of these two peaks, the ratio of non-intact particles to intact particles (E/F) in peak P1 was 9.9, while the E/F ratio in peak P2 was 0.8. This indicates that the affinity of the non-intact particles to the Poros XQ resin is weaker than the affinity of the intact particles to the resin, and therefore the non-intact particles elute earlier than the intact particles.

In contrast, when the sample was loaded in an amount exceeding the binding capacity, the E/F ratio in the flow-through (FT) in the penetration was 2.4, as indicated in fig. 8B. FIG. 8B shows that the addition of 30mM NaCl to the loading material was insufficient to promote displacement. Thus, the E/F ratio of the flow-through is the same as the E/F ratio of the loading substance.

Next, different binding strengths at different salt concentrations were screened to identify the optimal salt concentration for better separation of non-intact particles and intact particles under displacement column chromatography. As indicated in fig. 9A and 9B, the NaCl concentration in the loading buffer was correspondingly increased from 45mM to 60mM and the E/F ratio in the flow-through was increased from 3.4 to 229, indicating more displacement of intact particles to non-intact particles at higher salt concentrations. Meanwhile, the purity of the product peak P2 was improved from E/f=1 to E/f=0.7.

However, increasing the salt concentration further to 75mM and 90mM did not further enhance displacement, as the overall affinity of the sample for the resin was reduced, as shown in fig. 9C and 9D, respectively. Thus, fig. 9A-9D demonstrate that optimal displacement occurs at moderate binding strengths, where the difference in binding affinity between non-intact particles and intact particles is most pronounced. More generally, the results indicate that binding strength that can be adjusted with salt concentration can be used as a means to facilitate displacement of non-intact particles, which can lead to improved separation.

In addition, step elution conditions were also tested to determine the salt range in the elution buffer. The loading buffer used for the test contained 50mM Tris pH 8.5 and 30mM NaCl. As shown in fig. 10A-10D, step elution was suitable for all tested elution buffers, where the concentration of NaCl was respectively: 60mM, 75mM, 90mM and 120mM.

Further substitutions were also investigated in this example. Further displacement occurs when loading more samples beyond breakthrough loading, as shown in fig. 11A-11B, where the non-intact particles bound to the bottom of the column are also displaced by intact particles and exit in the flow-through. Fig. 11B shows that the purity of rAAV in the purified preparation increases to nearly 100% due to further displacement. In addition, further displacement chromatography also provided purified whole particles in high yields (greater than 90%).

EXAMPLE 3 use of CaCl in sample Loading and washing buffer ₂ As an additive

In studies with Poros XQ resin to separate intact RHM4-1 rAAV particles from non-intact particles, product loss was noted in displacement chromatography purification. For example, in a breakthrough run, when the loading buffer was 50mM Tris/60mM NaCl at pH 8.5, the recovery of the single column method was 89.81% and the recovery of the proposed double column method was 74.92%. The loss is manifested primarily by the presence of intact particles in the flow-through. The addition of additives to the sample loading buffer or wash buffer to increase the degree of separation and reduce the amount of intact rAAV particles in the flow-through was investigated, thereby increasing recovery.

As shown in FIG. 12, the sample load was first tested for MgCl ₂ Is added to the system. MgCl ₂ Can be used as an additive to improve the degree of separation. However, in the subsequent washing step, mgCl needs to be added ₂ To wash away non-intact particles bound to the column. It was found that MgCl was added to the sample load even under unbound conditions ₂ Nor is it robust because the conductivity difference between the loading buffer and the elution buffer is only about 1mS/cm.

Other additives were also tested. CaCl (CaCl) ₂ Identified as the additive that resulted in the best flow-through for purification on Poros XQ resin. As shown in FIG. 13, caCl was added ₂ Improved incomplete rAAV particles and complete rAAV particlesIs separated from the other components. In addition, caCl is used ₂ The conductivity difference between the loading buffer and the elution buffer is robust, e.g. about 4mS/cm.

It was also found that if no CaCl was added to the sample load ₂ Both yield and product purity are compromised when purifying intact rAAV particles from non-intact particles. As shown in FIG. 15, when no additive other than NaCl was used in the sample load and the wash buffer contained 1mM CaCl ₂ And 2.5mM MgCl ₂ In this case, it is difficult to remove all the non-intact particles bound to the column via washing. In addition, increased CaCl is used later ₂ During concentration elution (fig. 15), the bound non-intact particles appear unstable on the resin and create new impurities which in turn affect the product purity.

Thus, the inventors have unexpectedly found that the addition of CaCl to sample loads (and preferably also wash buffer and elution buffer) ₂ Resulting in optimal isolation and improved yields.

EXAMPLE 4 use of LiCl as an additive in sample Loading and washing buffer

Lithium chloride is another additive tested in Poros XQ resin purification. As shown in FIG. 17, addition of LiCl improved the separation of incomplete RHM4-1 rAAV particles from complete RHM4-1 rAAV particles. In addition, in the case of LiCl, the conductivity difference between the loading buffer and the elution buffer was about 0.2mS/cm.

Example 5 when CaCl is to be added ₂ Enhancement of separation and yield in displacement chromatography with addition to sample loading and wash buffer

Due to the use of additives (like CaCl) in the loading buffer and optionally in the wash buffer and elution buffer as shown in examples 3 and 4 ₂ ) The resulting improved separation of non-intact RHM4-1 rAAV particles and intact RHM4-1 rAAV particles can also be used in combination with the displacement chromatography method shown in example 2. As shown in fig. 18, combining these methods will yield optimal isolation and yields of intact rAAV particles over non-intact particles.

Example 6. Enhancement of separation with additives and lowering pH.

As shown in fig. 19, due to the use of additives (like CaCl in the loading buffer and optionally in the wash buffer and elution buffer as shown in examples 3 and 4 ₂ ) The resulting improved separation of non-intact RHM4-1 rAAV particles and intact RHM4-1 rAAV particles can be enhanced by lowering the pH using a gradient or step elution.

EXAMPLE 7 use of additive (NH) ₄ ) ₂ SO ₄ Enhancement of separation.

As shown in fig. 20A-20B, improved separation of particles from intact RHM4-1 rAAV particles over PXQ resin can be achieved by using (NH ₄ ) ₂ SO ₄ As an additive in the loading buffer.

EXAMPLE 8 use of additive (NH) ₄ ) ₂ SO ₄ Enhancement of separation.

As shown in FIG. 21, improved separation of particles from intact RHM4-1 rAAV particles over BIA monolithic resin can be accomplished by using (NH ₄ ) ₂ SO ₄ As an additive in the loading buffer.

EXAMPLE 9 use of additive CuCl ₂ Enhancement of separation.

As shown in FIGS. 22A-22E, improved separation of particles from intact RHM4-1 rAAV particles over PXQ resin can be accomplished by using CuCl ₂ As an additive in the loading buffer.

EXAMPLE 10 use of CuCl ₂ And the improvement in separation of part of the particles is enhanced with lower pH and increasing salt elution. As shown in fig. 23A-23E, due to the use of additives in the loading buffer (like CuCl ₂ ) The resulting improved separation of empty particles, partial LK03 rAAV particles, and complete LK03 rAAV particles may be enhanced by eluting at a lower pH, with a gradient of increasing salts, or by stepwise elution.

Example 11. Enhancement of separation of a portion of particles with multiple columns. As shown in fig. 24-26, the improved separation of empty particles, partial LK03 rAAV particles, and complete LK03 rAAV particles observed using 2 columns may be enhanced by using at least 3 columns. As shown in fig. 25, the method can be used to remove impurities and aggregates that have a stronger affinity for the column than the intact particles. In this two column arrangement, the second column is enriched for intact particles, as aggregates and impurities having a stronger affinity for the column than the intact particles bind to the first column. In a multi-column setup, the first column is enriched for the strongest binding particles (highest retention time) and the subsequent column is enriched for the next strongest binding particles. After determining the column for which the target rAAV particle or impurity has the greatest affinity, the disclosed methods can be employed to purify the desired rAAV particle or impurity. This example further demonstrates the use of the disclosed methods for enriching different particle variants in a multi-column set-up.

It is to be understood that the examples and embodiments described herein are for illustrative purposes only and that changes may be made to the embodiments described above without departing from the general inventive concept thereof. For example, the inventive concept includes separating a part of particles from empty particles, or separating impurities having stronger affinity to a resin than the whole particles, as shown in fig. 25, so that the whole particles are enriched in the second column instead of the first column. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.

Claims

1. A method for purifying intact recombinant adeno-associated virus (rAAV) particles, the method comprising:

(a) Providing a rAAV formulation comprising intact rAAV particles and non-intact rAAV particles;

(b) Loading a rAAV formulation in a loading buffer to a column comprising a chromatographic medium, wherein the intact rAAV particles have a higher binding affinity to the chromatographic medium than the non-intact particles and the amount of the intact rAAV particles and the non-intact particles applied to the column exceeds the binding capacity of the chromatographic medium such that the non-intact particles bound to the chromatographic medium are displaced by the intact rAAV particles into a flow-through from the column; and

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 method of claim 2, wherein the column chromatography medium is selected from the group consisting of Poros 50HQ, poros 50D, poros PI, capto ImpRes Q, CIMmultus ^TM QA monolithic column and Poros XQ, preferably Poros XQ.

4. A method for purifying intact recombinant adeno-associated virus (rAAV) particles, the method comprising:

(c) Loading the first loaded carrier into a second column comprising a second chromatographic medium to obtain a loaded or partially loaded second column, wherein the second chromatographic medium is of the same type as the first chromatographic medium;

(e) Eluting the intact rAAV particles bound to the first chromatographic medium with an elution buffer to obtain a first eluate from the first column and an eluted first column, wherein the first eluate has an increased ratio of the intact rAAV particles to the non-intact rAAV particles;

(f) Optionally, applying a second batch of the rAAV formulation in a loading buffer to the at least partially loaded second column, wherein the amount of the intact rAAV particles and the non-intact particles applied to the second column exceeds the binding capacity of the second chromatographic medium such that non-intact particles bound to the second chromatographic medium are displaced by the intact rAAV particles into a second loading flow-through from the second column;

(g) Optionally loading a second loaded permeate from the second column into the eluted first column after step (e) to obtain a second loaded first column, and optionally washing the second loaded first column, followed by continuing the elution in step (e);

(i) Eluting the intact rAAV particles bound to the second chromatographic medium with an elution buffer to obtain a second eluate and an eluted second column, wherein the second eluate has an increased ratio of the intact rAAV particles to the non-intact rAAV particles; and

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 chromatography medium.

6. The method of claim 4 or 5, wherein steps (b) through (i) are performed for one or more cycles.

7. The method of any one of claims 4-6, wherein after step (c), the second column is partially loaded.

8. The method of any one of claims 4-7, wherein the first column chromatography medium is selected from the group consisting of Poros 50HQ, poros 50D, poros PI, capto ImpRes Q, CIMmultus ^TM QA monolithic column and Poros XQ, preferably Poros XQ.

9. A method for purifying intact recombinant adeno-associated virus (rAAV) particles, the method comprising:

(g) Loading a second loading fluid from the second column into a third column comprising a third chromatographic medium to obtain a loaded or partially loaded third column, preferably the third chromatographic medium is of the same type as the first chromatographic medium;

(i) Bypassing the first column after step (k) or after washing step (l) if washing step (l) is performed, and eluting the intact rAAV particles bound to the second chromatographic medium with an elution buffer to obtain a second eluate and an eluted second column, wherein the second eluate has an increased ratio of the intact rAAV particles to the non-intact rAAV particles;

(j) Optionally, if the third column is not saturated (i.e., loaded) after the second flow-through, a third batch of the rAAV formulation in a loading buffer can be applied to a partially loaded third column, wherein the amount of the intact rAAV particles and the non-intact particles applied to the third column exceeds the binding capacity of the third chromatographic medium such that the non-intact particles bound to the third chromatographic medium are replaced by the intact rAAV particles into a third loaded flow-through from the third column;

(k) If step (n) is performed, loading a third loaded permeate from the third column into the washed first column between steps (d) and (e) to obtain a second loaded first column, and optionally washing the second loaded first column, followed by continuing the elution in step (e);

(l) Optionally washing the third column with a washing buffer to obtain a washed third column;

(m) bypassing the first column and the second column, and eluting the intact rAAV particles bound to the third chromatographic medium with an elution buffer to obtain a third eluate and an eluted third column, wherein the third eluate has an increased ratio of the intact rAAV particles to the non-intact rAAV particles; and

(n) combining the first eluate, the second eluate, and the third eluate to produce a purified preparation of intact rAAV particles.

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 chromatography medium.

11. The method of claim 9 or 10, wherein steps (b) to (m) are performed for one or more cycles.

12. The method of any one of claims 9-11, wherein after step (c), the second column is partially loaded.

13. The method of any one of claims 9-12, wherein after step (g), the third column is partially loaded.

14. The method of any one of claims 9-13, wherein the first column chromatography medium is selected from the group consisting of Poros 50HQ, poros 50D, poros 50 PI, capto ImpRes Q, CIMmultus ^TM QA monolithic column and Poros XQ, preferably Poros XQ.

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

16. The method of any one of claims 1-15, wherein the loading buffer comprises at least one buffer selected from the group consisting of: tris, bis-Tris propane, tris acetate, ethanolamine and phosphate.

17. The method of any one of claims 1-16, wherein the loading buffer comprises 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 ⁺ 。

18. The method according to any one of claims 1-17, wherein the loading buffer comprises about 10-100mM sodium salt, preferably 20-60mM NaCl.

19. The method of any one of claims 1-17, wherein the loading buffer comprises about 0-20mM magnesium salt, preferably 1-10mM MgCl ₂ 。

20. The method according to any one of claims 1-17, wherein the loading buffer comprises about 0-10mM calcium salt, preferably 0.1-2.5mM CaCl ₂ 。

21. The method according to any one of claims 1-17, wherein the loading buffer comprises about 0-100mM lithium salt, preferably 0-75mM LiCl.

22. The method of any one of claims 1-17, wherein the loading buffer comprises about 0-5mM copper salt, preferably 0.1-2.5mM CuCl ₂ 。

23. The method of any one of claims 1-17, wherein the loading buffer comprises about 5-100mM ammonium salt, preferably 10-50mM (NH ₄ ) ₂ SO ₄ 。

24. The method of any one of claims 1-23, wherein the loading buffer comprises about 20-60mM NaCl, 20-50mM Tris, 1-5mM MgCl ₂ 、0-75mM LiCl、0-10mM CuCl ₂ And/or 0.1-2.5mM CaCl ₂ 。

25. The method of any one of claims 1-23, wherein the loading buffer comprises about 20-60mM NaCl, 10-30mM (NH ₄ ) ₂ SO ₄ 、1-5mM MgCl ₂ 、0.1-2.5mM CuCl ₂ Preferably in Tris, more preferably in 20-100mM Tris.

26. The method of any one of claims 1-25, wherein the pH of the loading buffer is about 6-10.

27. The method of any one of claims 1-26, wherein the loading buffer comprises 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.

28. The method of claim 27, wherein the concentration of the surfactant in the loading buffer is between 0.0001% and 0.1%.

29. The method of any one of claims 1-28, wherein the elution buffer comprises 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 ⁺ 。

30. The method according to any one of claims 1-29, wherein the elution buffer comprises about 0-1000mM NaCl, preferably 20-300mM NaCl.

31. The method of any one of claims 1-29, wherein the elution buffer comprises about 0-30mM MgCl ₂ Preferably 2-15mM MgCl ₂ 。

32. The method according to any one of claims 1-29, wherein the elution buffer comprises about 0-200mM LiCl, preferably 0-150mM LiCl.

33. The method of any one of claims 1-29, wherein the elution buffer comprises about 0.1-20mM CaCl ₂ Preferably 5-10mM CaCl ₂ 。

34. The method of any one of claims 1-29, wherein the elution buffer comprises about 0-10mM CuCl ₂ Preferably 0-3mM CuCl ₂ 。

35. The method of any one of claims 1-29, wherein the elution buffer comprises about 5-100mM ammonium salt, preferably 10-50mM (NH ₄ ) ₂ SO ₄ 。

36. The method of any one of claims 1-35, wherein the elution buffer comprises at least one buffer selected from the group consisting of: tris, bis-Tris propane, tris acetate, ethanolamine and phosphate.

37. The method of any one of claims 1-36, wherein the elution buffer comprises about 20-200mM NaCl, preferably in Tris buffer.

38. The method of any one of claims 1-37, wherein the elution buffer comprises 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.

39. The method of claim 38, wherein the concentration of the surfactant in the elution buffer is between 0.0001% and 0.1%.

40. The method of any one of claims 1-39, wherein the pH of the elution buffer is about 6-10, preferably 8-9.

41. The method of claim 9 or 10, wherein an impurity in the rAAV formulation binds the first column with greater affinity than the intact rAAV particle.

42. A method for purifying intact recombinant adeno-associated virus (rAAV) particles, the method comprising:

(b) Loading the rAAV formulation in a loading buffer into a column comprising chromatographic medium, wherein the loading buffer comprises CaCl ₂ And the intact rAAV particle is bound to the chromatographic medium; and

(c) Eluting the intact rAAV particles bound to the chromatographic medium with an elution buffer to obtain a purified preparation, wherein the elution buffer comprises CaCl ₂ 。

43. The method of claim 42, wherein the chromatographic medium is an ion exchange column chromatographic medium, preferably an anion exchange chromatographic medium.

44. The method of claim 43, wherein the column chromatography medium is selected from the group consisting of Poros 50HQ, poros 50D, poros PI, capto ImpRes Q, CIMmultus ^TM QA monolithic column and Poros XQ, preferably Poros XQ.

45. The method of any one of claims 42-44, wherein the loading buffer comprises 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 ⁺ 。

46. The method of any one of claims 42-45, wherein the loading buffer comprises about 0-10mM CaCl ₂ Preferably 0.1-2.5mM CaCl ₂ 。

47. The method of any one of claims 42-45, wherein the loading buffer comprises about 10-100mM NaCl, preferably 20-60mM NaCl.

48. The method of any one of claims 42-45, wherein the loading buffer comprises about 0-20mM MgCl ₂ Preferably 1-10mM MgCl ₂ 。

49. The method of any one of claims 42-45, wherein the loading buffer comprises about 0-100mM LiCl, preferably 0-75mM LiCl.

50. The method of any one of claims 42-45, wherein the loading buffer comprises about 0-5mM copper salt, preferably 0.1-2.5mM CuCl ₂ 。

51. The method of any one of claims 42-45, wherein the loading buffer comprises about 5-100mM ammonium salt, preferably 10-50mM (NH ₄ ) ₂ SO ₄ 。

52. The method of any one of claims 42-51, wherein the loading buffer comprises at least one buffer selected from the group consisting of: tris, bis-Tris propane, tris acetate, ethanolamine and phosphate.

53. The method of any one of claims 42-52, wherein the loading buffer comprises about 20-60mM NaCl, 1-5mM MgCl ₂ 、0-75mM LiCl、0.1-2.5mM CaCl ₂ Preferably in 20-50mM Tris.

54. The method of any one of claims 42-52, wherein the loading buffer comprises about 20-60mM NaCl, 10-30mM (NH ₄ ) ₂ SO ₄ 、1-5mM MgCl ₂ 、0.1-2.5mM CuCl ₂ Preferably in Tris, more preferably in 20-100mM Tris.

55. The method of any one of claims 42-54, wherein the loading buffer comprises 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.

56. The method of claim 55, wherein the concentration of the surfactant in the loading buffer is between 0.0001% and 0.1%.

57. The method of any one of claims 42-56, wherein the pH of the loading buffer is about 6-10, preferably 8-9.

58. The method of any one of claims 42-57, wherein the elution buffer comprises 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 ⁺ 。

59. The method of any one of claims 42-58, wherein the elution buffer comprises about 0.1-20mM CaCl ₂ Preferably 5-10mM CaCl ₂ 。

60. The method according to any one of claims 42-58, wherein the elution buffer comprises about 0-1000mM NaCl, preferably 20-300mM NaCl.

61. The method of any one of claims 42-58, wherein the elution buffer comprises about 0-30mM MgCl ₂ Preferably 2-15mM MgCl ₂ 。

62. The method of any one of claims 42-58, wherein the elution buffer comprises about 0-200mM LiCl, preferably 0-150mM LiCl.

63. The method of any one of claims 42-62, wherein the elution buffer comprises at least one buffer selected from the group consisting of: tris, bis-Tris propane, tris acetate, ethanolamine and phosphate.

64. The method of any one of claims 42-63, wherein the elution buffer comprises about 20-200mM NaCl, preferably in 40-60mM Tris buffer.

65. The method of any one of claims 42-64, wherein the elution buffer comprises 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.

66. The method of claim 65, wherein the concentration of the surfactant in the elution buffer is between 0.0001% and 0.1%.

67. The method of any one of claims 42-66, wherein the pH of the elution buffer is about 6-10, preferably 8-9.

68. The method of any one of claims 1-67, wherein the yield of purified whole rAAV particles is not less than 70%, preferably not less than 80%, more preferably not less than 90%, most preferably not less than 95%.

69. The method of any one of claims 1-68, wherein the ratio of the intact rAAV particles to the non-intact particles in the purified preparation is not less than 9:1, preferably not less than 49:1.

70. The method of any one of claims 1-69, wherein the non-intact particles comprise empty particles and/or partial particles, preferably both empty particles and partial particles.

71. A method for purifying a portion of a rAAV particle, the method comprising:

(d) Non-intact rAAV formulations comprising empty particles and partial particles are provided;

(e) Loading the incomplete rAAV preparation in a loading buffer into a column comprising a chromatographic medium, wherein the partial rAAV particles have a higher binding affinity to the chromatographic medium than the empty particles; and

(f) Eluting a portion of the rAAV particles bound to the chromatographic medium with an elution buffer to obtain a purified preparation.

72. The method of any one of claims 1-71, wherein the intact rAAV particle comprises a transgene encoding a polypeptide or a nucleic acid selected from the group consisting of: siRNA, antisense molecules, miRNA, ribozymes, and shRNA.

73. A method for purifying an empty recombinant adeno-associated virus (rAAV) particle, the method comprising:

(b) Loading the rAAV formulation in a loading buffer into a column comprising a chromatographic medium, wherein the empty rAAV particle has a higher binding affinity to the chromatographic medium than the whole particle or a fraction of particles and the amount of at least one of the whole particle and the fraction of particles applied to the column and the empty rAAV particle exceeds the binding capacity of the chromatographic medium such that at least one of the whole particle and the fraction of particles bound to the chromatographic medium is displaced by the empty rAAV particle into a flow-through from the column; and

74. The method of any one of claims 1-73, wherein the rAAV particle comprises a capsid derived from one or more AAV selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9 (hu 14), AAV10, AAV11, AAV12, rh8, rh10, rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ8, NP59, anc-80, and variants thereof.