Chromatographic method for purifying AAV capsid

Abstract

The present disclosure provides methods of generating and purifying recombinant adeno-associated virus (AAV) vectors comprising separating whole capsid from empty capsid using anion exchange chromatography in a weak partition mode. The present disclosure also provides a population of rAAV full capsid enriched by the method, a population of rAAV full capsid isolated and enriched by the method, and a pharmaceutical composition comprising the enriched full capsid population.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of priority of U.S. Provisional Application Serial No. 63 / 379,115, filed on October 11, 2022, and U.S. Provisional Application Serial No. 63 / 489,684, filed on March 10, 2023, which are hereby incorporated by reference in their entireties. Technical Field

[0003] The present disclosure provides methods for generating and purifying recombinant adeno - associated virus (AAV) vectors, which include separating full capsids from empty capsids using anion - exchange chromatography with a weak partitioning mode. Background Art

[0004] Adeno - associated virus (AAV) is a replication - defective parvovirus. The AAV particle contains a capsid with three capsid proteins (VP1, VP2, and VP3), encapsulating a single - stranded DNA genome of approximately 4.8 kb in length, which can be either the plus - strand or the minus - strand. Particles containing either strand are infectious and replication occurs by converting the parental infecting single - strand into a duplex form and subsequent amplification, with the progeny single - strand then being displaced and packaged into capsids.

[0005] AAV depends on co - infection with other viruses (primarily adenoviruses) for replication. Its single - stranded genome contains three genes, rep (replication), cap (capsid), and aap (assembly), which generate at least nine gene products through the use of three promoters, alternative translation start sites, and differential splicing. These coding sequences are flanked by inverted terminal repeats (ITRs) required for genome replication and packaging. The rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40), which are involved in viral genome replication and packaging, while cap expression produces the viral capsid proteins (VP1, VP2, and VP3), which form the capsid shell protecting the viral genome and are actively involved in cell binding and internalization. The aap gene encodes an assembly - activating protein (AAP) in an alternative reading frame overlapping with the cap gene. This AAP protein is thought to provide a scaffolding function for capsid assembly.

[0006] AAV particles have characteristics that make them attractive as vectors for therapeutic applications, including gene therapy and genetic vaccines. AAV infects a variety of cell types, including many mammalian cells, and thus has the potential to target many different tissues in the body. AAV infects both slowly dividing and non-dividing cells. For therapeutic applications, recombinant AAV (rAAV) is used, in which the genome contains a heterologous transgene and typically retains the ITRs but lacks the viral rep, cap, and aap genes. In the absence of the Rep protein, the transgene flanked by the ITRs can form a transcriptionally active episomal extrachromosomal element that can persist essentially throughout the life of the transduced cell.

[0007] An important goal of rAAV vector production methods is to achieve a consistent, high vector production rate while minimizing the production of product-related impurities, including rAAV capsid-residual DNA impurities and empty capsids. Measured by the vector genomes (VGs) produced per cell, rAAV vector productivity can vary widely, from less than 10 3 to 2×10 5 VGs per cell. In addition to greater cost-effectiveness, an important advantage of high productivity is that purification can be more efficient when the ratio of rAAV vector product to total harvested biomass in the starting material is high.

[0008] During rAAV vector production, removal of product-related impurities is an important part of rAAV purification. Although ultracentrifugation can separate empty capsids from full capsids, scalable chromatography methods have proven more challenging. As an alternative, anion exchange chromatography in binding and elution modes is commonly investigated in an attempt to reproduce the high full capsid ratios achieved by ultracentrifugation. However, the similar properties of full capsids and empty capsids result in a sharp trade-off between product yield and full capsid proportion, making it difficult to select the optimal conditions. This is also hampered by the complex interactions between the AAV capsid and the chromatography matrix, which often results in further product loss. Thus, there is a need for an rAAV purification method that can reduce product-related impurities such as empty capsids. SUMMARY OF THE INVENTION

[0009] The present invention provides methods for producing and purifying rAAV particles, which include separating full capsids from empty capsids (enrichment of full capsids) using anion exchange chromatography in a weak partitioning mode. A method for enriching rAAV full capsids in a mixture of full capsids and empty capsids is described herein, wherein the method includes:

[0010] (i) providing a solution comprising rAAV full capsids and empty capsids;

[0011] (ii) equilibrating an anion exchange (AEX) column or membrane; and;

[0012] (iii) Subject a solution comprising rAAV full capsids and empty capsids to weak partitioning mode AEX chromatography to separate the empty capsids from the full capsids, thereby obtaining an AEX eluate enriched in full capsids.

[0013] In an embodiment of the method, the solution is an affinity chromatography eluate, and step (iii) comprises applying the affinity chromatography eluate to a balanced AEX column or membrane, washing the AEX column or membrane at least once, and eluting the rAAV full capsids from the AEX column or membrane. In these methods, the weak partitioning mode causes the full capsids to displace the bound empty capsids from the AEX column or membrane, and the empty capsids flow through the AEX column or membrane to produce an AEX flowthrough, while the full capsids remain bound to the AEX column or membrane until elution.

[0014] In some embodiments, the affinity chromatography eluate is diluted to a target salt concentration prior to being subjected to the balanced AEX column or membrane. The target salt concentration is typically in the range of about 85 mM to about 95 mM. The target salt concentration can be, for example, about 90 mM, and the salt can be, for example, NaCl.

[0015] In an embodiment of the method, prior to loading with a solution comprising rAAV full capsids and empty capsids, the AEX column or membrane is balanced with a balance buffer comprising from about 10 mM to about 1000 mM (such as about 10, 49, 50, 51, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mM) Tris, from about 85 mM to about 95 mM NaCl (such as about 84, 84.9, 85, 86, 87, 88, 89, 89.5, 90, 90.5, 91, 92, 93, 94, 95, 95.5) and greater than about 0.5% (such as 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1.0%) (w / v) polysorbate 80, wherein the pH of the balance buffer is about 9.0 (such as about 8.8, 8.9, 9.0, 9.1, 9.2). The balance buffer can comprise about 50 mM Tris, about 90 mM NaCl and about 0.75% polysorbate 80, and have a pH of about 9.0.

[0016] In an embodiment of the method, washing the AEX column or membrane at least once includes a first wash buffer that includes (i) from about 10 mM to about 1000 mM (such as about 10, 49, 50, 51, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mM) Tris; (ii) from about 85 mM to about 95 mM (such as about 84, 84.9, 85, 86, 87, 88, 89, 89.5, 90, 90.5, 91, 92, 93, 94, 95, 95.5) NaCl, and (iii) greater than about 0.5% (such as 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1.0%) (w / v) polysorbate 80, wherein the pH of the first wash buffer is about 9.0 (such as about 8.8, 8.9, 9.0, 9.1, 9.2). The first wash buffer can include, for example, (i) about 50 mM Tris; (ii) about 90 mM NaCl and (iii) about 0.75% polysorbate 80, and the pH is about 9.0.

[0017] In an embodiment, washing the AEX column or membrane at least once further includes a second wash with a second wash buffer that includes (i) from about 10 mM to about 1000 mM (such as about 10, 49, 50, 51, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mM) Tris; (ii) from about 100 mM to about 125 mM (such as about 99, 100, 101, 105, 110, 120, 124, 125, 126 mM) NaCl, and (iii) greater than about 0.5% (such as 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1.0%) (w / v) polysorbate 80, wherein the pH of the second wash buffer is about 9.0 (such as about 8.8, 8.9, 9.0, 9.1, 9.2). The second wash buffer can include, for example, (i) about 50 mM Tris; (ii) about 125 mM NaCl and (iii) about 0.75% polysorbate 80, and the pH is about 9.0.

[0018] In an embodiment, the full capsids are eluted from the AEX column or membrane with an elution buffer comprising (i) from about 10 mM to about 1000 mM (such as about 10, 49, 50, 51, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mM) tris; (ii) from about 125 mM to about 250 mM (such as about 124, 125, 126, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 251 mM) NaCl, and (iii) greater than about 0.5% (such as 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1.0%) (w / v) polysorbate 80, wherein the pH of the elution buffer is about 9.0 (such as about 8.8, 8.9, 9.0, 9.1, 9.2). The elution buffer may comprise, for example, (i) about 50 mM tris; (ii) about 250 mM NaCl, and (iii) about 0.75% polysorbate 80, wherein the pH of the elution buffer is about 9.0.

[0019] In an embodiment of the method, the AEX chromatography column or membrane comprises a high flow rate adsorption membrane, such as a Q chromatography membrane. In an embodiment, the method may further comprise the step of subjecting the AEX eluate comprising enriched full capsids to tangential flow filtration (TFF) to produce a purified preparation (e.g., drug substance) of the full capsids.

[0020] In an embodiment, polysorbate 20 and poloxamer 188 may be used in one or more of the buffers described herein.

[0021] In an embodiment of the method, the method further comprises the step of subjecting the AEX eluate to analytical ultracentrifugation to quantify full capsid enrichment.

[0022] In an embodiment of the method, the enriched full capsids may comprise AAV serotype 2 capsid protein and a polynucleotide sequence comprising a transgene (e.g., a transgene such as aquaporin 1 (AQP1)).

[0023] In an embodiment, the enriched full capsids comprise greater than 80% full capsids (such as about 80.5, 81.5, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96% full capsids), greater than 90% full capsids, and greater than 95% full capsids.

[0024] The present invention also describes a method for separating and enriching rAAV full capsids from a mixture of full capsids and empty capsids. The method includes: separating a mixture of rAAV full capsids and empty capsids from virus-producing cells by lysing the cells and clarifying the resulting lysate; subjecting the clarified lysate to affinity chromatography to obtain an affinity chromatography eluate; and subjecting the affinity chromatography eluate to weak partitioning mode AEX chromatography to separate the empty capsids from the full capsids, obtaining an AEX eluate containing enriched full capsids. In an embodiment, the cells are mammalian cells. The cells are typically in suspension, such as cultured in shake flasks, spinner flasks, cell bags, or bioreactors.

[0025] The present invention also provides an rAAV full capsid population enriched by the method, an rAAV full capsid population separated and enriched by the method, and a pharmaceutical composition comprising the enriched full capsid population. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a graph showing the charges of empty AAV capsids and full AAV capsids above and below pI(1).

[0027] Figure 2 is a graph showing the weak partitioning process in which empty capsids are displaced by full capsids due to the created environment.

[0028] Figure 3 is a graph showing the relationship between Wash 2 and Equilibration / Feed NaCl concentration (mM) and empty capsid removal rate (VP / mL).

[0029] Figure 4 is a graph showing the relationship between Wash 2 and Equilibration / Feed NaCl concentration (mM) and the full capsid ratio (%) in the eluate.

[0030] Figure 5 is a graph showing the relationship between Wash 2 and Equilibration / Feed NaCl concentration (mM) and the VG recovery rate (%) in the eluate.

[0031] Figure 6 is a graph showing the optimal region based on Wash 2 and Equilibration / Feed NaCl concentration (mM).

[0032] Figure 7 is a graph showing the relationship between the loading ratio and the breakthrough (%) of full capsids (VG) and empty capsids (VP).

[0033] Figure 8 is a chromatogram of the optimized 2-liter (L) scale AAV-AQP1 AEX step.

[0034] Figure 9It is a chart showing the summary of the display results, demonstrating the scalability of the AEX process. The 2L scale refers to the use of 10 mL in the process of Q operation, while the 80 scale refers to the use of 75 mL in the process of Q operation.

[0035] Figure 10 It is a chart showing the breakthrough experiment results, demonstrating the breakthrough degree of the vector genome and vector particles in the flow-through.

[0036] Figure 11 It is an excerpt of a chromatogram of an experiment using 10 mL of Q to confirm the process (as described in Example 2). The focus of this excerpt is on column wash 2 and the elution phase.

[0037] Figure 12 It shows the results of analytical ultracentrifugation performed on the eluate, which was generated from a confirmation run on 10 mL of Q.

[0038] Figure 13 It is the chromatogram of the initial anion exchange chromatography.

[0039] Figure 14 It shows a breakthrough curve, indicating that both VG breakthrough and empty capsid breakthrough reach the loading challenge of 4E14 VG / mL at 90 mM NaCl feed and equilibrium conductivity.

[0040] Figure 15 It is a pair of charts showing the VG concentration on the column (top chart) when the VG loading challenge is increased to 4E14 vg / ml, and the empty capsid ratio on the column (bottom chart) when the VG loading challenge is increased to 4E14 VG / mL.

[0041] Figure 16 It is a chart showing the increase in the full capsid ratio on the column when the VG loading challenge is increased to 4E14 VG / mL

[0042] Figure 17 It is a chart showing the full capsid concentration on the column when loaded to 4E14 VG / mL at 9 mS / cm and 8 mS / cm, highlighting the achievable enrichment level.

[0043] Figure 18 It is the chromatogram of the optimized AAV-AQP1 AEX step in the 20L scale confirmation run.

[0044] Figure 19 It shows the VG recovery rate and full capsid % values after each of USP, capture chromatography, ion exchange chromatography (IEX), and tangential flow filtration (TFF) in the "initial process" and "final process".

[0045] Figure 20 A graph showing the comparison between the full capsid ratio and the VG recovery rate for 20L and 80L batches, and a graph showing the results of analytical ultracentrifugation (AUC), which confirm that Figure 8 the elution peak in Detailed Description

[0046] The present disclosure provides methods for producing and purifying rAAV, which include separating full capsids from empty capsids (enrichment of full capsids) using anion exchange chromatography in a weak partitioning mode. A method for enriching rAAV full capsids in a mixture of full capsids and empty capsids includes the following steps:

[0047] (i) Providing a solution comprising rAAV full capsids and empty capsids;

[0048] (ii) Equilibrating an anion exchange (AEX) column or membrane; and

[0049] (iii) Subjecting the solution comprising rAAV full capsids and empty capsids to weak partitioning mode AEX chromatography to separate the empty capsids from the full capsids, thereby obtaining an AEX eluate enriched in full capsids.

[0050] A method for separating and enriching rAAV full capsids in a mixture of full capsids and empty capsids includes the following steps:

[0051] (i) Separating a mixture of rAAV full capsids and empty capsids from cells by lysing virus-producing cells and clarifying the resulting lysate;

[0052] (ii) Subjecting the clarified lysate to affinity chromatography, thereby obtaining an affinity chromatography eluate; and

[0053] (iii) Subjecting the affinity chromatography eluate to weak partitioning mode AEX chromatography to separate the empty capsids from the full capsids, thereby obtaining an AEX eluate comprising enriched full capsids.

[0054] The present invention also provides rAAV full capsid populations enriched by the methods, rAAV full capsid populations separated and enriched by the methods, and pharmaceutical compositions (e.g., drugs, drug substances) comprising the enriched full capsid populations. In embodiments, the methods provide increased production titers and higher full capsid to empty capsid ratios (F:E). More specifically, the present disclosure provides methods for enriching rAAV full capsids, for example, in a mixture of full capsids and empty capsids. In embodiments, the production yield is greater than 50%, greater than 50% of the empty capsids are removed, and an F:E ratio greater than 80% is achieved.

[0055] AEX in Weak Partitioning Mode for Purifying rAAV

[0056] The purification and enrichment methods described herein involve purifying rAAV full capsids (i.e., rAAV particles containing a recombinant genome) from a solution containing rAAV full capsids and empty capsids. The solution can be the result of any rAAV production / purification method. Typically, prior to undergoing AEX chromatography, the solution is conditioned by diluting it to a target salt (e.g., NaCl) concentration. For example, the solution can be diluted to an NaCl concentration of about 90 mM (e.g., about 89, 90, 91 mM), having about 10 mM to about 1000 mM (e.g., about 10, 49, 50, 51, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mM) Tris, about 0.75% (w / v) polysorbate 80, and a pH of about 9.0 (e.g., about 8.8, 8.9, 9.0, 9.1, 9.2). In an embodiment, the solution is an affinity chromatography eluate. Affinity chromatography is one of several purification steps in a typical method for producing and purifying rAAV particles (e.g., rAAV full capsids). Methods for the production / purification of rAAV are well known in the art, and examples of such methods are described below.

[0057] In the methods described herein, weak partitioning mode AEX chromatography is a purification technique for separating full capsids from empty capsids ( Figure 1 and 2 ). This form of chromatography exploits the charge difference between capsids containing the viral genome and empty capsids. A target pH value higher than the capsid pI means that full capsids containing VG carry more negative charge than empty capsids. In the purification and enrichment methods described herein, weak partitioning describes the process of separating full capsids from empty capsids by creating an environment that is more favorable for full capsid binding.

[0058] Weak partitioning mode anion exchange chromatography is different from the flow-through mode AEX and the bind-and-elute mode AEX methods used historically (Liu et al., MAbs. 2010; 2(5):480-499; Kelley et al., Biotechnology and Bioengineering Vol. 101:553-566, 2008). In the bind-and-elute mode AEX, first the product (of interest) pool is loaded onto the anion exchange column, and then the product of interest is eluted with a higher salt concentration in a stepwise or linear gradient, causing most of the impurities to bind to the column. In the cleaning or regeneration step, the impurities are eluted from the column. In the flow-through mode AEX, the working pH is typically 8 to 8.2, and the conductivity of the product load as well as the equilibration and wash buffers is as high as 10 mS / cm. The conditions are chosen such that the product does not bind to the column, while acidic impurities such as nucleic acids and host cell proteins bind to the column. The use of weak partitioning mode anion exchange chromatography enables a dual chromatography recovery process that includes affinity chromatography and anion exchange to obtain the product of interest. Similar to flow-through chromatography, the process operates in an isocratic manner, but different from the flow-through mode, the conductivity and pH are chosen such that the binding of the product and impurities is enhanced, resulting in a product partition coefficient (Kp) between 0.1 - 20, preferably between 1 and 3. This takes advantage of the fact that the impurities to be removed are more acidic than the product. Both the product and the impurities bind to the anion exchange resin, but the binding of the impurities is tighter than in the flow-through mode, which can improve the impurity removal rate. Thus, weakly bound impurities that cannot be effectively removed in the flow-through mode can be removed to a greater extent under conditions where their partition coefficient (Kp) increases. Compared to anion exchange chromatography in the flow-through mode, weak partitioning chromatography can achieve a dual-column recovery process due to increased clearance rates of viruses, host cell proteins, and product-related species. One aspect of weak partitioning chromatography is the need to optimize the pH and counterion conditions for each product. This is in contrast to some platform chromatography processes that are able to use standardized conditions for most products on anion exchange matrices (resins or membranes).

[0059] In a method for enriching rAAV full capsids in a mixture of full capsids and empty capsids, a solution comprising rAAV full capsids and empty capsids is provided. As discussed above, the solution can be the result of any rAAV production / purification method, and in an embodiment, the solution is an affinity chromatography eluate. The AEX column or membrane that will receive the solution is typically pre-equilibrated with a suitable pre-equilibration buffer. For example, a suitable pre-equilibration buffer can include about 50 mM Tris, about 1 M NaCl, and about 0.75% polysorbate 80, and have a pH of about 9.0 (e.g., about 8.8, 8.9, 9.0, 9.1, 9.2). After the pre-equilibration step, the AEX column or membrane is equilibrated with an equilibration buffer. Generally, the equilibration buffer includes from about 10 mM to about 1000 mM (e.g., about 10, 49, 50, 51, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mM) Tris, from about 85 mM to about 95 mM (e.g., about 84, 84.9, 85, 86, 87, 88, 89, 89.5, 90, 90.5, 91, 92, 93, 94, 95, 95.5) salt (e.g., NaCl), and greater than about 0.5% (e.g., 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1.0%) (w / v) polysorbate 80. In some embodiments, the equilibration buffer includes about 50 mM Tris, about 90 mM NaCl, greater than 0.5% (w / v) polysorbate 80, and about 0.75% polysorbate 80, and has a pH of about 9.0. In the method, any suitable AEX column or membrane can be used. In an embodiment, a high-flow rate adsorption membrane is used. An example of such a membrane is the Q chromatography membrane. Another example of such a membrane is the Q membrane from Pall Corporation.

[0060] After equilibration of the AEX column or membrane, the solution (referred to as "feed", "feed material", and "conditioned feed" in Examples 1 and 2) is applied to the equilibrated AEX column or membrane. The solution can be applied at any suitable loading ratio. In the experiments described in Examples 1 and 2, a loading ratio of 5×10 13 –1×10 15 VG / mL was used, and an elution % of 81.5% full capsid ratio was produced. The flow-through of the loaded solution typically consists of empty capsids (the full capsids will have a stronger interaction with the column or membrane and displace the empty capsids). In an embodiment, the flow-through or a sample thereof can be analyzed by any suitable method to determine its viral genome and viral particle concentration.

[0061] After solution application, the AEX column or membrane is washed at least once (e.g., once, twice, three times). In at least the first wash, the AEX column or membrane is washed with a first wash buffer comprising from about 10 mM to about 1000 mM (e.g., about 10, 49, 50, 51, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mM) Tris, from about 85 mM to about 95 mM (e.g., about 84.5, 84.9, 85, 85.5, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 95.5, 96 mM) NaCl, and greater than about 0.5% (e.g., about 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1.0%) (w / v) polysorbate 80. The pH of the first wash buffer is typically about 9.0 (e.g., about 8.8, 8.9, 9.0, 9.1, 9.2). For example, the first wash buffer may comprise about 50 mM tris, about 90 mM NaCl, and about 0.75% polysorbate 80, and have a pH of about 9.0. In an embodiment, a second wash is performed with a second wash buffer comprising from about 10 mM to about 1000 mM (e.g., about 10, 49, 50, 51, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mM) Tris, from about 100 mM to about 125 mM NaCl (e.g., about 99, 100, 101, 105, 110, 120, 124, 125, 126 mM) NaCl, and greater than about 0.5% (e.g., 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1.0%) (w / v) polysorbate 80. The pH of the second wash buffer is typically about 9.0 (e.g., about 8.8, 8.9, 9.0, 9.1, 9.2). For example, the second wash buffer may comprise about 50 mM tris, about 125 mM NaCl, and about 0.75% polysorbate 80, and have a pH of about 9.0. The column or membrane wash typically consists mainly of empty capsids, but may also contain full capsids. The viral genome and viral particle concentration of the wash or wash fraction (sample) can be analyzed by any suitable method.

[0062] After at least one wash (e.g., two washes), the full capsids are eluted from the column or membrane, generating an AEX eluate enriched in full capsids. The full capsids are typically eluted from the AEX column or membrane with an elution buffer that includes from about 10 mM to about 1000 mM (e.g., about 10, 49, 50, 51, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mM) Tris, from about 125 mM to about 250 mM (e.g., about 124, 125, 126, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 251 mM) NaCl, and greater than about 0.5% (e.g., 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1.0%) (w / v) polysorbate 80. The pH of the elution buffer is typically about 9.0 (e.g., about 8.8, 8.9, 9.0, 9.1, 9.2). An example of an elution buffer is a buffer that includes about 50 mM tris, about 250 mM NaCl, and about 0.75% polysorbate 80 and has a pH of about 9.0. Samples of the AEX eluate enriched in full capsids can be analyzed for viral genome and viral particle concentration, as well as their full capsid ratio and viral genome recovery. Thus, in embodiments, the method can further include performing pPCR and analytical ultracentrifugation (AUC) on the AEX eluate enriched in full capsids or a sample thereof to quantify full capsid enrichment (e.g., monitoring the quality and efficacy of vector purification, measuring the relative amount of empty capsids in a recombinant viral particle preparation). AUC is a widely applicable and informative method for studying macromolecular characteristics such as size, shape, stoichiometry, and binding properties, all in a true solution-state environment. AUC can assess both quantitative and qualitative information at moderately high concentrations. Methods for characterizing recombinant viral particle preparations using AUC are known (see, e.g., U.S. Patent Publication No. 20200225139, which is incorporated herein by reference).

[0063] For all pre-equilibration, equilibration, wash, and elution steps, any suitable membrane volume (MV) and flow rate (MV / min) can be used. Examples of suitable MV and MV / min are shown in Tables 6 and 8. Specific examples of methods for enriching rAAV full capsids in a mixture of full and empty capsids are described in Examples 1 and 2.

[0064] In embodiments, the AEX eluate enriched in full capsids is further purified by TFF (e.g., TFF diafiltration) and formulated for administration as a gene therapy (e.g., a drug, a drug substance).

[0065] In embodiments, the method provides a ratio of rAAV full particles (full capsids) to empty AAV particles (empty capsids) of at least about 30%, such as about 30%-40%, at least 65%, about 65%-95%, at least 80%, about 80%-85% (e.g., 79%, 79.5%, 80%, 80.5%, 81%, 81.5%, 82%, 83%, 85%, 85%), about 90%-95% (e.g., 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%), etc. Primers and probes designed against relevant sequences in the helper plasmid or high-copy number genomic sequences can be used to evaluate nuclease resistance and the level of AAV encapsidated DNA impurities by qPCR. The sensitivity of nuclease treatment performed prior to qPCR can distinguish nuclease-sensitive 'naked' residual DNA impurities from nuclease-insensitive encapsidated residual DNA impurities. A capsid-specific ELISA assay can be used to measure total AAV capsids, and the amount of empty capsids can be determined by comparing the capsid particle titer and the VG titer. Spectrophotometry can be used for samples from which non-AAV capsid impurities have been substantially removed.

[0066] The method can be scaled up to manufacturing scale, such as cultures of about 5 to about 10, about 10 to about 20, about 20 to about 50, about 50 to about 100 (e.g., 79, 80, 81), about 100 to about 200 liters or more, and is applicable to rAAV containing multiple AAV serotypes / capsid variants. The experiments described in Examples 1 and 2 demonstrate that rAAV full capsids can be enriched at large (manufacturing) scale.

[0067] The rAAV vectors (rAAV full capsids) generated, isolated, purified, and enriched by the methods disclosed herein can be used to express transgenes in target cells. These rAAV vectors can be used in gene therapy because they can introduce a polynucleotide containing a transgene that can be maintained and expressed in the target cell into the target cell. rAAV vectors are capable of delivering heterologous polynucleotide sequences (e.g., polynucleotide sequences encoding a therapeutic protein or a reporter protein and regulatory elements for expressing the protein) to target cells of a human patient. A non-exhaustive list of examples of transgenes includes RPGR, RPE65, GAD65, GAD67, CNGB3, and AQP1. In some embodiments, the two AAV ITRs are AAV2 ITRs. In the method, the AAV cap gene can be from an AAV serotype or AAV variant, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV 11, AAV12, AAV13, AAVrh10, AAV-PHP.5, AAV-PHP.B, AAV-PHP.eB, AAV2-retro, AAV9-retro, AAVrh74, AAVrh, and mixtures thereof.

[0068] The term "vector" refers to an agent for introducing polynucleotides into target cells. The vector can be a viral vector (e.g., rAAV vector, HSV vector) or a non-viral vector such as a plasmid, or DNA associated with a compound such as liposome, gelatin, or polyamine. An expression vector is a vector containing a polynucleotide sequence encoding a gene product (e.g., protein or RNA) that has regulatory elements for expression in a host or target cell.

[0069] "rAAV", "rAAV vector", "rAAV particle", or "rAAV virion" refers to a recombinant AAV vector genome packaged in a capsid protein (i.e., enclosed by the capsid protein) for subsequent ex vivo, in vitro, or in vivo infection of target cells. These phrases exclude empty AAV capsids and AAV capsids lacking a complete recombinant AAV genome containing the transgene to be expressed in the target cells. Thus, in addition to the capsid, the rAAV vector also contains the rAAV genome. "rAAV genome" or "rAAV vector genome" refers to a polynucleotide sequence containing the transgene of interest that is ultimately packaged or capsidated to form an rAAV particle. Typically, for rAAV, most of the AAV genome (including, for example, the rep, cap, and aap genes) has been deleted, and one or two ITR sequences are retained as part of the rAAV genome together with the transgene. As used herein, "transgene" refers to a polynucleotide sequence encoding a gene product (e.g., a therapeutic protein or a reporter protein) and regulatory elements for expressing the gene product in a target cell.

[0070] "Empty capsid" and "empty particle" refer to AAV particles that have an AAV capsid but are completely or partially lacking a recombinant AAV genome containing the transgene sequence and one or two ITRs. Such empty capsids do not function to transfer the transgene to one or more target cells. In an embodiment, the isolated rAAV particles are separated from the empty AAV particles.

[0071] The rAAV genome (including, for example, the ITR) can be based on the same strain or serotype (or subgroup or variant), or it can be different from each other. As a non-limiting example, an rAAV plasmid or vector genome or particle (capsid) based on one serotype genome can be the same as one or more capsid proteins that package the vector genome. Additionally, the rAAV genome can be derived from an AAV genome (e.g., containing one or more ITRs derived from the AAV2 genome) that is different from one or more capsid proteins that package the rAAV vector genome.

[0072] The rAAV vectors (rAAV full capsids) that can be generated, isolated, purified, and enriched by the methods disclosed herein include any rAAV vectors comprising a capsid and a genome derived from any AAV strain or serotype. As non-limiting examples, the rAAV vector capsid and / or genome can be based on AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-PHP-5, AAV-PHP-B, AAV-PHP-eB, AAV2-retro, AAV9-retro, AAVrh74, AAVrh, AAVrh.10 (i.e., AAV containing AAVrh.10 ITR and AAVrh.10 capsid protein), etc. In an embodiment, the rAAV vector comprises a genome and a capsid protein derived from the same AAV strain or serotype. For example, the rAAV vector can be an rAAV2 vector (i.e., rAAV containing AAV2 ITR and AAV2 capsid protein).

[0073] In an embodiment, the AAV vector is a pseudotyped rAAV vector that contains ITRs from one AAV serotype and capsid proteins from a different AAV serotype. In some embodiments, the pseudotyped rAAV is rAAV2 / 5 (i.e., rAAV containing AAV2 ITR and AAV5 capsid protein); rAAV2 / 8 (i.e., rAAV containing AAV2 ITR and AAV8 capsid protein); rAAV2 / 9 (i.e., AAV containing AAV2 ITR and AAV9 capsid protein); rAAV2 / 10 (i.e., rAAV containing AAV2 ITR and AAV10 capsid protein). In an embodiment, the rAAV vector comprises a capsid protein that is a variant AAV capsid, such as the AAV2 variant rAAV2-retro (SEQ ID NO:44 from WO 2017 / 218842, which is incorporated herein by reference).

[0074] Methods for generating rAAV

[0075] As described above, the rAAV purification and enrichment methods can be applied to solutions (e.g., lysates, eluates) containing rAAV full capsids and empty capsids obtained or generated by any suitable production method. Methods for generating rAAV are well known in the art. Generally, the methods include amplifying production cells; introducing an rAAV vector, AAV rep and cap, and helper gene nucleic acid sequences into the production cells; culturing the transduced production cells under conditions that allow for the production of rAAV particles; and isolating the rAAV particles. Specific embodiments of the methods for generating rAAV are described in detail below.

[0076] Cell line

[0077] Methods for generating rAAV vectors typically require certain elements, including, for example: (i) a permissive host cell for rAAV production (a producer cell); (ii) helper virus functions that can be provided, for example, by a suitable construct containing genes that provide adenovirus helper functions; (iii) a transfer packaging rep / cap construct; and (iv) a suitable production medium.

[0078] A producer cell is any cell that is a permissive host cell for rAAV production once an rAAV genome production construct, a helper function construct, and a construct providing AAV functions (such as expression of rep and cap) are present. The term can also include the progeny of an originally transfected cell. Thus, a producer cell is also a host cell that has been transfected with an exogenous DNA sequence or the progeny of a host cell in which the DNA sequence has been integrated into the host cell genome. It should be understood that due to natural, accidental, or intentional mutations, the progeny of a single parental cell need not be identical in morphology or in genomic or total DNA complementary sequences to the original parent.

[0079] In embodiments, the cells used for generating rAAV particles are mammalian cells, including HEK293 cells, BHK cells, and HeLa cells. Exemplary producer cells / host cells include human embryonic kidney (HEK) cells, such as HEK293. In a preferred embodiment, the producer cells are suitable for growth in suspension, including HEK293 cells adapted to suspension. In additional preferred embodiments, the producer cells are suitable for growth in serum-free medium. In embodiments, the producer cells are increased in at least one culture vessel, which can be, for example, one or more of a shake flask, a spinner flask, a cell bag, or a bioreactor.

[0080] Producer cell lines that can be used in the rAAV production, isolation, purification, and enrichment methods disclosed herein include mammalian or insect cell lines. The term "cell line" refers to a population of cells that are capable of growing and dividing continuously or for an extended period in vitro under appropriate culture conditions. A cell line can, but need not, be a clonal population derived from a single progenitor cell. In a cell line, karyotypic changes may occur spontaneously or be induced during storage or transfer of such clonal populations and during prolonged passage in tissue culture. Thus, the progeny cells derived from a cell line may not be identical to the ancestral cells or culture.

[0081] For rAAV production to occur, the producer cell line may require the presence of one or more of an rAAV genome production construct, an accessory function construct, and / or an AAV rep / cap construct within the producer cell. These can be introduced as three constructs (e.g., three plasmids), or the producer cell may already have one or more constructs stably integrated into the producer cell genome that provide some or all of these functions. In embodiments, one or more accessory genes may include all or part of one or more adenovirus genes, herpes simplex virus type 1 genes, or baculovirus genes. As used herein, the terms "stable" or "stably integrated" with respect to a cell mean that a nucleic acid sequence, such as a selectable marker and / or a heterologous nucleic acid sequence, or a plasmid or vector (or a portion thereof) has been inserted into a chromosome (e.g., by homologous recombination, non-homologous end joining, transfection, etc.) or is maintained extrachromosomally in a recipient cell or host organism and has been retained in the chromosome or maintained extrachromosomally for a period of time.

[0082] Amplify the producer cell line

[0083] In embodiments of the methods described herein, prior to the step of introducing an rAAV genome production construct and / or other constructs providing accessory virus functions and AAV functions, an amplification phase or amplification step is used to increase the number of producer cells. The amplification phase or amplification step can be carried out in one or more cell culture vessels. For example, the amplification phase or amplification step can be carried out in a series of cell culture vessels with increasing volumes. The cell culture medium for amplifying the producer cell line can be any medium suitable for the growth (i.e., increase in number) of the producer cells. In a preferred embodiment, the amplification phase medium is animal component-free and does not include, for example, serum or other components derived from animals. Chemically defined, animal component-free media are commercially available.

[0084] In embodiments, an anti-caking supplement (sometimes referred to herein as an anti-caking agent (ACA)) is added to the amplification medium to reduce cell aggregation. The ACA can be obtained commercially, for example, from Irvine Scientific. The anti-caking supplement can be added to the amplification phase medium at one or more time points. In embodiments, the anti-caking supplement comprises dextran sulfate, heparin, and / or other sulfated glycosaminoglycans that inhibit producer cell aggregation. In embodiments, the anti-caking supplement comprises sodium heparin, which can be added to the medium at a concentration of about 25 μg / ml to about 250 μg / ml, such as about 25 μg / ml, about 50 μg / ml, about 100 μg / ml, about 150 μg / ml, and / or about 200 μg / ml.

[0085] In an embodiment, the amplification phase medium comprises and / or is supplemented to comprise one or more of glutamine, a glutamine precursor, or an amino acid dipeptide containing glutamine at a concentration of about 2 mM to about 6 mM (such as about 2 mM, about 3 mM, about 4 mM, about 5 mM, or about 6 mM). One or more of glutamine, a glutamine precursor, or an amino acid dipeptide containing glutamine can be, for example, one or more of L-alanyl-L-glutamine, L-glutamine, glutamic acid, glycyl-L-glutamine, glutamine protein hydrolysate, L-glutamic acid, and glutamine dipeptide. A commercially available example of a glutamine supplement provided as the dipeptide L-alanyl-L-glutamine is GlutaMAX (ThermoFisher).

[0086] In an embodiment, the amplification phase medium comprises and / or is supplemented to comprise a nonionic polyol surfactant, such as poloxamer 188 (a copolymer of polyethylene and polypropylene glycol ethers). In an embodiment, the nonionic polyol surfactant is present in the amplification phase medium at about 0.05% to about 0.2% (w:v) (such as about 0.05%, about 0.1%, about 0.1%, or about 0.2%). In an embodiment, the amplification phase medium comprises about 4 mM L-alanyl-L-glutamine dipeptide and 0.1% (w:v) poloxamer 188.

[0087] In an embodiment, the pH of the amplification phase medium is maintained at a pH of about 7.1 to about 7.5 (such as about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5). In an embodiment, the pH is maintained at about 7.2 to about 7.4 by CO2 bubbling. In an embodiment, before introducing one or more polynucleotide constructs into the cells, the pH of the medium is changed to about 6.9 and CO2 bubbling is stopped.

[0088] Introduce one or more polynucleotide constructs

[0089] rAAV vector production typically requires a producer cell line that provides the basic biosynthetic machinery, and (i) a construct that provides the rAAV genome (the transgene of interest and the associated regulatory elements flanking the AAV ITRs) and (ii) one or more constructs with additional genes that provide the gene products required to direct rAAV vector production. These additional genes include AAV-derived genes (such as AAV rep and cap) required to support vector genome replication and packaging, and helper virus-derived genes (such as adenovirus E1a, E1b, E2a, E4, and VA).

[0090] "Helper virus gene" or "helper virus-derived gene" refers to a virus gene that is not AAV-derived and whose replication is dependent on the gene products of AAV for which the gene codes. The term includes proteins and / or RNAs required for AAV replication, including proteins and / or RNAs involved in AAV gene transcriptional activation, stage-specific AAV mRNA splicing, and AAV DNA replication. Helper virus genes can be derived from any known AAV helper virus, such as adenovirus, herpesvirus, and vaccinia virus. Thus, "helper virus function" refers to those functions provided by helper virus genes (such as adenovirus E1a, E1b, E2a, E4, and VA) required for AAV production. These helper virus functions can be provided on one or more vectors introduced into the producer cells, stably expressed by the producer cells, or a combination of both.

[0091] As used herein, "AAV function" or "AAV helper function" refers to AAV-derived coding sequences that can be expressed in producer cells to provide AAV gene products that act in trans for productive AAV replication and packaging. Thus, AAV functions include AAV open reading frames (ORFs), including rep and cap, and other functions, such as aap of certain AAV serotypes. Such AAV functions are provided by one or more polynucleotide constructs, which can be plasmid vectors, non-plasmid vectors, or polynucleotide constructs that have been integrated into the chromosome of the producer cells to provide AAV helper functions. Plasmids that provide AAV functions that can be used in the methods disclosed herein are commercially available.

[0092] In embodiments of the methods, one or more helper virus genes are constitutively expressed by producer cells (such as HEK293 cells), while other helper virus genes are introduced into the producer cells, for example, by transfection of one or more polynucleotide constructs encoding the remaining helper virus genes required for AAV production. AAV-derived genes (such as rep and cap) can be contained in the same polynucleotide construct containing one or more helper virus genes, or can be contained on separate polynucleotide constructs. After introduction of a polynucleotide construct that provides or encodes an rAAV genome (such as an rAAV genome production construct) into a producer cell line, rAAV particles are produced. In an embodiment, rAAV particles are produced after transient transfection of producer cells with (i) an rAAV genome production vector and (ii) one or more vectors that provide helper virus genes (such as E4, E2a, and VA) and AAV genes (such as rep and cap). In an embodiment, these vectors are plasmids.

[0093] In an embodiment of the method disclosed herein, after the amplification phase, a first polynucleotide construct comprising a transgene flanked by ITRs and a second polynucleotide construct comprising helper virus genes and AAV rep and cap genes are introduced into the amplified producer cells. When the first and second polynucleotide constructs are plasmids, this system can be referred to as a dual-plasmid system.

[0094] In an embodiment of the method disclosed herein, after the amplification phase, a first polynucleotide construct comprising a transgene flanked by ITRs, a second polynucleotide construct comprising helper virus genes, and a third polynucleotide construct comprising AAV rep and cap genes are introduced into the amplified producer cells. When the first, second, and third polynucleotide constructs are plasmids, this system can be referred to as a triple-plasmid system.

[0095] In the case of using one or more recombinant plasmids to produce rAAV vectors, an "rAAV genome production plasmid" refers to a plasmid comprising: a transgene (operably linked to regulatory sequences) and one or more ITRs intended to be packaged into rAAV, as well as non-rAAV genomic components (plasmid backbone) that are important for cloning and amplification of the plasmid but are not packaged or capsidized into the rAAV vector. As used herein, the term "construct" refers to a recombinant polynucleotide construct (i.e., a polynucleotide having elements derived from different sources), which can be a plasmid.

[0096] The terms "transduction" and "transfection" refer to the introduction of polynucleotides into a host cell or target cell. In embodiments, the host cell is a producer cell, such as a HEK293 cell. In embodiments, the rAAV genome production plasmid is introduced into the producer cell together with one or more plasmids providing helper virus functions and AAV functions by a transient transfection method. Transient transfection of producer cells to introduce a first polynucleotide construct (such as an rAAV genome production plasmid) containing a transgene and ITRs; and optionally a second and / or third polynucleotide construct providing AAV functions (rep and cap genes) and helper virus functions can be accomplished by standard transfection methods, which include, for example, calcium phosphate co-precipitation, cationic lipid-based transfection, and cationic polymer-based transfection. Cationic lipid-based transfection includes, for example, Lipofectamine (a 3:1 mixture of DOSPA (2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propaniminium trifluoroacetate) and DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine)). Cationic polymer-based transfection includes, for example, the use of linear and / or branched polyethylenimine (PEI), poly-L-lysine, poly-L-arginine, polyamidoamine dendrimers, etc. In embodiments, transient transfection of producer cells is carried out using a PEI-based transfection reagent. PEI can be a linear or branched polymer. In embodiments, PEI is 20-25kD linear PEI. For example, in embodiments, PEI is jetPEI or PEIpro (available from Polyplus). Additionally, a transfection reagent containing both a cationic lipid and a cationic polymer can be used to carry out transient transfection of producer cells.

[0097] Alternatively, rAAV can be produced in insect cells (such as sf9 cells) or in HSV-infected baby hamster kidney (BHK) cells (such as BHK21) using a baculovirus vector. In both methods, rAAV production is triggered in host cells, insect cells, or mammalian cells, respectively, when co-infected with two or more recombinant viruses carrying the rAAV genome and yet another AAV rep and cap, as well as the helper virus functions required for rAAV replication and packaging.

[0098] Generating rAAV Particles

[0099] Typically, after the production phase (also referred to as the production step), there is a step of introducing the rAAV genomic vector and / or a vector providing helper virus function and / or AAV function into the production cells. In an embodiment, rAAV particles are produced by culturing the cells for at least about 48 hours (such as 47.5, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 96.5, or 97 hours) after introducing the rAAV genomic vector. In an embodiment, the transfected production cells are cultured (i.e., the production phase is maintained) for about 72 to about 100 hours, about 90 hours to about 100 hours, about 92 hours to about 98 hours, or about 94 to about 98 hours. In an embodiment, the production phase is maintained for about 96 hours.

[0100] The production phase medium can be any cell culture medium suitable for producing rAAV in the production cells. In an embodiment, the production medium is animal product-free, such as serum-free. As used herein, "free of" means that the medium has undetectable levels of animal products, such as serum. In an embodiment, the pH of the production medium is lower compared to the pH of the amplification phase medium. In an embodiment, the pH of the production medium is maintained at about 6.8 to about 7.4 (such as about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, or about 7.14). In an embodiment, the pH is maintained at about 6.9 to about 7.3.

[0101] In an embodiment of the method, the production phase includes adding calcium ions to the production phase cell culture medium. Adding calcium ions to the production medium, also referred to herein as calcium supplementation, includes adding calcium ions in the form of a calcium salt (Ca 2+ ), such as CaCl2. Calcium ions can be added one or more times during the production phase after introducing the rAAV genomic vector (such as after transfection). For example, calcium ions can be added one or more times between about 0 hours and about 48 hours after the start of the production phase (i.e., after transfection), such as about 1 hour, about 6 hours, about 10 hours, about 12 hours, about 20 hours, about 24 hours, about 30 hours, about 36 hours, and / or about 48 hours.

[0102] Calcium ions (such as, CaCl2) can be added to the production medium to achieve a total calcium ion concentration in the medium greater than 0.3 mM and less than 10 mM. In an embodiment, calcium ions are added to a total concentration of about 1 mM to about 9 mM, about 1 mM to about 8 mM, about 1 mM to about 7 mM, about 2 mM to about 9 mM, about 2 mM to about 8 mM, about 2 mM to about 7 mM, about 2 mM to about 6 mM, about 2 mM to about 5 mM, or about 2 mM to about 4 mM.

[0103] In an embodiment, the production phase includes adding one or more of glutamine, a glutamine precursor, or an amino acid dipeptide containing glutamine to the production phase medium. One or more of glutamine, a glutamine precursor, or an amino acid dipeptide containing glutamine can be, for example, one or more of L-alanyl-L-glutamine, L-glutamine, glutamic acid, glycyl-L-glutamine, glutamine protein hydrolysate, L-glutamic acid, and glutamine dipeptide. A solution containing at least one of glutamine, a glutamine precursor, or an amino acid dipeptide containing glutamine can be added to the production phase medium at one or more times, such as at about 6 hours, about 12 hours, about 24 hours, about 48 hours, or about 72 hours after transfection.

[0104] In an embodiment, the production phase includes adding sorbitol to the production phase medium. Sorbitol can be added to the production phase medium at one or more time points during the production phase, such as at about 6 hours, about 12 hours, about 20 hours, about 24 hours, and / or about 48 hours after transfection. In an embodiment, sorbitol is added to the production medium at a concentration of about 50 mM to about 200 mM, or about 80 mM to about 120 mM. In an embodiment, sorbitol is added to the production medium at a concentration of about 100 mM.

[0105] In an embodiment, the production phase includes adding an anti-caking supplement to the production phase medium. The anti-caking supplement can be added to the production phase medium at one or more time points (such as one or more of about 6, about 10, about 12, about 20, about 24, about 48, or about 72 hours after transfection). In an embodiment, the anti-caking supplement includes dextran sulfate, heparin, and / or other sulfated glycosaminoglycans that inhibit the aggregation of production cells. In an embodiment, the anti-caking supplement includes sodium heparin, which can be added to the medium at a concentration of about 25 μg / ml to about 250 μg / ml, such as about 25 μg / ml, about 50 μg / ml, about 100 μg / ml, about 150 μg / ml, and / or about 200 μg / ml.

[0106] In an embodiment, the anti-caking supplement is not added to the production phase medium, or is added to the production phase medium only shortly before the end of the production phase, such as within about 24 hours, about 12 hours, about 6 hours, about 3 hours, about 2 hours, or about 1 hour before the end of the production phase.

[0107] Isolation and purification of rAAV

[0108] Embodiments of the methods described herein include separating and purifying rAAV particles (rAAV full capsids) at the end of the production phase. In embodiments, rAAV particles can be separated about 48 hours or more (e.g., 47.5, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 96.5, 97 hours) after introducing the rAAV vector, and / or helper functions and / or AAV rep / cap sequences (in other words, after the start of the production phase). For example, rAAV particles can be separated about 90 to about 100 hours, about 92 hours to about 98 hours, or about 94 to about 98 hours after introducing the rAAV vector, and / or helper functions and / or AAV rep / cap sequences. In embodiments, rAAV particles are separated (e.g., cells are lysed) about 96 hours after introducing the rAAV vector and / or helper functions and / or AAV rep / cap sequences. Separating rAAV can include multiple steps, including, for example, lysing the production cells to obtain a cell lysate, clarifying the lysate to obtain a clarified lysate, and subsequent purification steps.

[0109] rAAV particles can remain within the production cells after production, and methods for releasing intracellular rAAV vectors include physical and chemical disruption, such as using detergents, microfluidization, and / or homogenization. In embodiments, zwitterionic detergents, N,N-dimethyltetradecylamine N-oxide (TDAO) (commercially available from MilliporeSigma Burlington, MA in the form of C16) are used to achieve cell membrane rupture (lysis) and release of rAAV or AAV particles (recovery) from the cells. For example, when recovering rAAV5, AAV5, rAAV2, or AAV2 particles, about 0.1% to about 0.5% (e.g., 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.51%) of C16 can be used. In other embodiments, osmotic shock (increased external osmotic pressure) is used to lyse the cells and release rAAV or AAV particles. For example, when recovering rAAV8 or AAV8 particles, the production cells can be subjected to osmotic shock for a duration of about 90 to about 120 minutes (e.g., about 89, 90, 95, 100, 105, 115, 120, 121 minutes) and an NaCl concentration of about 400 mM (e.g., about 399, 400, 401 mM). During and / or subsequent to cell lysis, nucleases, such as benzonase, can be added to degrade contaminating DNA. Typically, the resulting lysate is clarified to remove cell debris and provide a clarified cell lysate. In embodiments, the clarified lysate is subjected to AEX in a weak partitioning mode to separate full capsids from empty capsids, as described herein.

[0110] Example

[0111] Example 1. Anion exchange chromatography using a weak partitioning mode to provide a high empty AAV capsid removal rate and product yield

[0112] An anion exchange step was developed to maximize the enrichment of the full capsids of AAV-AQP1 (AAV2). The similar properties of the full capsids and empty capsids result in a trade-off between product yield and the proportion of full capsids, which makes it difficult to select the optimal conditions. Different types and concentrations of surfactants were screened to maximize the product yield of the AAV2 vector. Exploring the design space led to the identification of the weak partitioning mode as an alternative to the bind-and-elute mode ( Figure 1 , 2 ), and conditions were identified that could remove >50% of the empty capsids only in the flow-through. Combining with a high NaCl wash, an eluate containing >80% full capsids was obtained. The results showed that using the new conditions described herein, a full capsid ratio of >80% and a product yield of >50% confirmed by analytical ultracentrifugation (AUC) could be robustly achieved. Understanding the design space and identifying process failure points means that the process can be reproduced on a manufacturing scale.

[0113] In the initial experimental design, further optimized parameters and ranges were identified from the experiments fitted to the initial AEX platform and are listed in Table 1:

[0114] Table 1

[0115] Parameter Range Load challenge (VG / mL) <![CDATA[5×10 13 –1×10 14 > Equilibration / feed NaCl concentration (mM) 65-90 Wash 2 NaCl concentration (mM) 100-125 Elution NaCl concentration (mM) 200-300

[0116] The relationships between the wash 2 and equilibration / feed NaCl concentrations (mM) and (i) empty capsid removal, (ii) full capsid ratio in the eluate, and (iii) VG recovery in the eluate were analyzed. The results are shown in Figures 3 - 5 . The results of the design of experiments study (DOE) ( Figure 6 ) were combined to highlight the optimal regions within the design space, in line with the expected results. The parameters and values for the optimal regions based on the wash 2 and equilibration / feed NaCl concentrations are shown in Table 2:

[0117] Table 2

[0118] Parameter Value Empty capsids in flowthrough Greater than 50% Empty capsid removal in Wash 2 Approximately 10% VG recovery in elution Approximately 50% Full capsid ratio in elution Approximately 50%

[0119] To understand how the load ratio affects weak partitioning, the relationship between the load ratio and the breakthrough of full capsids (VG) and empty capsids (VP) was analyzed. The results ( Figure 7 ) showed that the effect of weak partitioning could be maximized by increasing the load ratio. The parameters and values are shown in Table 3:

[0120] Table 3

[0121] Parameter Value Load ratio (VG / ml.) <![CDATA[1×10 15 > Equilibration / feed NaCl concentration (mM) 90

[0122] Reference Figure 7 , taking advantage of the opportunity window means that at most 80% of the empty capsids can break through, while only 10% of the full capsids are lost during circulation.

[0123] Collate the research results, Figure 3 The results shown indicate that for maximum removal of empty capsids, both Wash 2 and elution require high NaCl concentrations. Increasing Wash 2 NaCl results in up to a 30% loss of VG; 50% VG recovery can still be achieved during the elution phase. The parameters and optimization ranges are shown in Table 4:

[0124] Table 4

[0125] Parameter Optimization range Load ratio (VG / ml.) <![CDATA[1×10 14 > Equilibration / feed NaCl concentration (mM) 90 Wash 2 NaCl concentration (mM) 125 Elution NaCl concentration (mM) 250

[0126] Figure 8 Shows the chromatogram of the optimized 2L-scale AAV-AQP1 AEX step. Note that the inversion highlights an 80% full capsid ratio (inversion of the 260 nm and 280 nm UV absorbance traces usually indicates a higher full capsid ratio due to the presence of encapsidated DNA). The parameters and values are shown in Table 5:

[0127] Table 5

[0128]

[0129] As Figure 9 shown, for both VG recovery and full capsid ratio, the process can be scaled from 2L to 80L.

[0130] Example 2: AEX weak partitioning scheme for enriching full capsids and increasing yield

[0131] The experiments described below demonstrate that the rAAV purification method described herein is consistent in terms of full capsid ratio and VG recovery, indicating that the process is scalable.

[0132] Material

[0133] AAV feed material

[0134] The feed material for the experiments described in this study consisted of adeno-associated virus (AAV) capsids of serotype 2, which packaged a transgene encoding the aquaporin 1 gene.

[0135] These capsids were produced by cell culture using HEK293 cells transiently transfected with a triple plasmid complex. After the production phase of the cell culture was completed, the cells were lysed using Triton X-100 to release AAV, and the resulting lysate was then treated with benzonase. The lysate was then clarified using a 0.2μm PES filter.

[0136] The AAV capsids were initially purified using AAVX (ThermoFisher) affinity chromatography resin and an AKTA Avant (Cytiva) chromatography system. This purification was carried out as follows: the clarified lysate was loaded onto an AAVX column with a 5cm bed height at a loading rate of 200mL of clarified lysate per mL of AAVX resin, and a residence time of 1 minute. The captured AAV capsids were eluted using an elution buffer consisting of 50mM glycine, 180mM NaCl, 0.25% (w / v) polysorbate 80, pH 2.7.

[0137] Chemicals

[0138] All chemicals used in these experiments were GMP (Good Manufacturing Practice) grade reagents purchased from Merck KGaA.

[0139] Method

[0140] Anion Exchange Chromatography

[0141] All small-scale anion exchange chromatography (AEX) experiments were carried out using a 1mL Sartobind Q (Sartorius) AEX membrane and an AKTA Avant (Cytiva) chromatography system.

[0142] The buffer used for these experiments consisted of 50mM tris, 0.75% (w / v) polysorbate 80, pH 9 and various concentrations of NaCl required for the experimental phase. The feed used for these experiments was the AAVX eluate, which was adjusted by diluting to the target NaCl concentration with 50mM tris, 0.75% (w / v) polysorbate 80, pH 9.0. Table 6 describes the methods used in the AEX experiments in this study.

[0143] Table 6: Overview of the methods used in the AEX experiments

[0144]

[0145]

[0146] Experimental Design

[0147] A DoE study was conducted to evaluate the effects of NaCl concentration in the loading, washing, and elution phases, as well as the loading ratio, on the full capsid ratio and vector genome (VG) recovery. This study included 16 runs, and Table 7 describes the parameter values tested in each experiment. Samples of the flow-through, wash 2, and eluate were collected for each run and analyzed for their vector genome and vector particle concentrations.

[0148] Table 7: Design of experiments study conducted to evaluate the effects of the tested process variables on full capsid purification

[0149]

[0150] Breakthrough experiment

[0151] A breakthrough experiment was conducted to further evaluate the effect of the loading ratio on the AEX process. The AEX membrane was loaded to a loading ratio of 1×10 15 VG / mL, and the feed was adjusted to a NaCl concentration of 90 mM. The flow-through was divided into 1 mL fractions and the resulting fractions were analyzed to determine their VG and VP concentrations.

[0152] Confirmation and scale-up experiments

[0153] The optimized process was confirmed by purification on a 10 mL Sartobind Q module. Table 8 details the method used for this run. Samples of the flow-through, column wash 2, and eluate were analyzed for VG and VP concentrations. Additionally, the eluate samples were analyzed by analytical ultracentrifugation and qPCR to confirm the full capsid ratio and VG recovery.

[0154] Table 8: Method used to confirm the optimized AEX process.

[0155]

[0156] To demonstrate scalability, the process was scaled up to a 75 mL Q membrane. This run was performed using the same method detailed in Table 8, and the eluate samples were analyzed by analytical ultracentrifugation and qPCR to determine their full capsid ratio and VG recovery.

[0157] Analytical methods

[0158] The vector genome concentration in the samples generated in this study was determined by qPCR using primers and probes specific to the AQP1 transgene region. The vector particle concentration was determined using the AAVX Titer Kit (Gyros Protein Technologies).

[0159] In DoE and breakthrough experiments, the empty capsid fraction was estimated by dividing the vector genome concentration by the vector particle concentration. In confirmation and scale-up experiments, the full capsid fraction was measured by analytical ultracentrifugation using the Optima AUC (Beckman).

[0160] Result

[0161] DoE

[0162] The design space of the AEX process was investigated by conducting a design of experiments study with the goal of identifying conditions that maximized the whole capsid ratio and VG recovery. The variables explored in this study included feed NaCl concentration, wash 2 NaCl concentration, elution NaCl concentration, and load ratio. The tested ranges for these variables are detailed in Table 9. The results of these experiments are detailed in Table 10, and the trends observed in this dataset are summarized in Figure 3 , Figure 4 and Figure 5 This is explained in .

[0163] Table 9: Range of testing for each variable in the experimental design study

[0164]

[0165] Table 10: Results of the experimental design study conducted to explore the design space of the AEX process.

[0166]

[0167] The full capsid fraction of the feed for these experiments was estimated to be less than 10%. Table 10 shows that some of the conditions tested resulted in significant increases in the estimated full capsid fraction (up to 87%). Figure 3 It is shown that higher equilibrium / feed and wash 2 NaCl concentrations result in fewer empty capsids in the elution pool. This is because higher NaCl concentrations in the feed will prevent empty capsids from binding to the AEX membrane. Similarly, higher NaCl concentrations in column wash 2 will result in elution of empty capsids at this stage, thereby removing them from the later elution pool.

[0168] Figure 4 It is shown how these effects translate into a higher fraction of full capsids in the elution pool. Since empty capsids are prevented from binding or washing off the column, the resulting elution pool has a higher fraction of full capsids. Figure 5 The effect of increasing feed and column wash 2NaCl concentrations is shown. Although whole capsids have stronger interactions with the AEX membrane, some whole capsids are lost in the flow-through and wash as feed and wash 2NaCl concentrations are increased and disrupt these interactions. Figure 6Combined the trends seen in these experiments to highlight regions where high full capsid ratios and high VG recoveries can be achieved.

[0169] Breakthrough experiment

[0170] As observed from the above DoE study, at higher feed NaCl concentrations, the empty capsids flow through the membrane without binding to the Q AEX membrane. This effect was further investigated by conducting breakthrough experiments. The experiment was conducted as follows: The feed NaCl concentration was 95 mM to maximize this effect, and the membrane was loaded to 1×10 15 VG / mL to ensure that the capsids could break through into the flow-through.

[0171] The flow-through samples were analyzed to determine their VG and VP concentrations, and the results are shown in Figure 10 . These results indicate that under these feed conditions, the carrier particles started to break through almost immediately after the start of the experiment. However, since the carrier genome did not break through until the loading rate reached approximately 2.5×10 14 VG / mL, it can be concluded that the breakthrough carrier particles were empty capsids.

[0172] The study results also indicate that the carrier genome is breaking through gradually. Typically, a sharp breakthrough profile is expected, indicating that the chromatographic matrix has reached saturation. However, this gradual breakthrough suggests that weak partitioning may be occurring. Under weak partitioning, since the charged interaction between the full capsids and the membrane is stronger, the full capsids start to displace the empty capsids bound to the chromatographic membrane. The advantage of this is to further increase the full capsid ratio of the product finally eluted from the membrane.

[0173] Confirmation runs and scale-up

[0174] Using 10 mL Q and the process conditions detailed in Table 8, larger-scale runs were conducted to confirm the identified process conditions. Figure 11 An excerpt of the chromatogram for this run is shown, focusing on column wash 2 and the elution phase. The figure shows an inversion of the 260 nm and 280 nm UV absorbance traces, which typically indicates a high full capsid ratio due to the presence of capsid DNA. The eluate from this run was analyzed by analytical ultracentrifugation to confirm the full coat ratio. It was found that the full capsid ratio increased from 31% in the feed to 81.5% in the eluate. The corresponding AUC trace of the eluate is shown in Figure 12 .

[0175] Further runs were conducted on 75 mL Q to confirm the scalability of the described process. Figure 9The results of 10 mL and 75 mL runs were compared. The figure shows that the process is consistent in terms of full capsid ratio and VG recovery, indicating that the process is scalable.

[0176] Example 3. Identification of parameters and ranges from the initial AEX platform and other experiments

[0177] Platform adaptation experiments were conducted. Figure 13 The chromatogram of the initial AEX chromatographic run is shown. The conditions used in the AEX step are as follows:

[0178]

[0179] The parameters and ranges shown in Table 11 below were determined from the initial AEX platform adaptation experiment ( Figure 13 ). These initial experiments are referred to as the "initial process" in Figure 19 , which shows the VG recovery and full capsid % values after USP, capture chromatography, ion exchange chromatography (IEX), and tangential flow filtration (TFF) in the initial process. These values were compared with those obtained from the optimized process of the present invention described herein (see Figure 19 for the "final process").

[0180] Table 11: Custom DOE conditions

[0181] Parameter Range Load challenge (VG / mL) <![CDATA[5×10 13 –1×10 14 > Feed full capsid ratio (%) <10 Equilibration / feed NaCl concentration (mM) 65-90 Wash 2 NaCl concentration (mM) 100-125 Elution NaCl concentration (mM) 200-300

[0182] Regarding the following relationships of parameters with empty capsids, full capsids, and VG recovery % in elution, see Figures 3 - 5 :

[0183] · Figure 3 : The relationship between wash 2 and equilibration / feed NaCl concentration (mM) and the empty capsids (VP / mL) present in the elution

[0184] · Figure 4 : The relationship between wash 2 and equilibration / feed NaCl concentration (mM) and the full capsid ratio (%) in the elution

[0185] · Figure 5 : The relationship between wash 2 and equilibration / feed NaCl concentration (mM) and the VG recovery (%) in the elution

[0186] Figure 6 Shows the optimal region based on the wash 2 and equilibration / feed NaCl concentration (mM) in Figures 1 - 3 . Figure 1 and 2It is a diagram showing the weak partitioning process, where empty capsids are replaced by full capsids due to the created environment. The charges of empty AAV capsids and full AAV capsids above and below pI are shown in the figure. (Cytiva Life Sciences Marlborough, Massachusetts 2022. Enhanced AAV downstream processing).

[0187] Additional breakthrough experiments were conducted to identify whether weak partitioning is utilized at different rates at different feed and equilibrium conductivities when the load challenge is increased.

[0188] Methods and materials

[0189] Run 1: Feed material and equilibrium buffer with a conductivity of 90 mM NaCl.

[0190] Run 2: Feed material and equilibrium buffer with a conductivity of 80 mM NaCl.

[0191] 0.08 mL of Sartobind Q was loaded with the AAV-AQP1 neutralized AAVX eluate to 4E14 VG / mL, and the flow-through samples were fractionated and analyzed for VG titer using qPCR and for VP titer using Gyrolab.

[0192] Table 12: Load challenge of AAQ-AQP1 AAVX material

[0193] Parameter Value Load challenge (VG / mL) 4E14

[0194] Table 13: Buffers used in two breakthrough curve experiments

[0195]

[0196] Table 14: Lengths of each step in the chromatography run. Methods used for the first run and the second run.

[0197] Step Step size Pre - equilibration 10 CV Equilibration 15 CV Sample application 0.5 mL fraction Column wash 1 15 CV Column wash 2 15 CV Elution 15V

[0198] From Figure 14 It can be seen that when calculating the breakthrough curves of empty capsids and full capsids, a target conductivity of 9 mS / cm seems to favor the binding of full capsids rather than empty capsids. To examine what is happening on the column, the VG concentration on the column was examined when the VG load challenge was increased to 4E14 vg / ml ( Figure 15 , top chart), and the ratio of empty capsids on the column was examined when the VG load challenge was increased to 4E14 VG / mL ( Figure 15, bottom graph). To determine the effect on the full capsid ratio on the column, the full capsid ratio on the column was analyzed as the VG load challenge was increased. As Figure 16 shown, an increase from 38% to >55% was observed prior to any form of wash or elution step. Similar to Figure 14 shown, when looking at Figure 17 , it can be clearly seen that loading at the target 9 mS / cm allows for a higher level of enrichment than at the target 8 mS / cm, indicating that feed and equilibration feed conductivity are important when attempting to utilize weak partitioning and maximize enrichment.

[0199] For the chromatogram of the optimized AAV-AQP1 AEX step in the 20 L scale confirmation run, see Figure 18 . As shown, a full capsid ratio of 80% was obtained. The parameters and values for this confirmation run are shown in Table 15 below. Referring again to Figure 19 , the difference between the "initial process" value and the "final process" value is shown.

[0200] Table 15:

[0201] Parameter Value Empty capsids in flowthrough (%) >40％ Full capsids in flowthrough (%) <1％ Full capsids in Wash 2 (%) 33％ Empty capsids in Wash 2 (%) 20％ VG recovery in elution (%) 53％ Full capsid ratio in elution (%) 80％

[0202] For scale-up to 20 L and 80 L batches, Figure 20 shows a comparison of the full capsid ratio and VG recovery for 20 L and 80 L batches. The figure also includes AUC results, confirming that Figure 18 the elution peak in

Claims

1. A method for enriching recombinant adeno-associated virus (rAAV) full capsids in a mixture of full capsids and empty capsids, the method comprising the following steps: (i) Providing a solution comprising rAAV full capsids and empty capsids; (ii) Equilibrating an anion exchange (AEX) column or membrane; And (iii) Subjecting the solution comprising rAAV full capsids and empty capsids to weak partitioning mode AEX chromatography to separate the empty capsids from the full capsids, thereby obtaining an AEX eluate enriched in full capsids.

2. The method according to claim 1, wherein the solution is an affinity chromatography eluate, and step (iii) comprises applying the affinity chromatography eluate to the equilibrated AEX column or membrane, washing the AEX column or membrane at least once, and eluting the rAAV full capsids from the AEX column or membrane, wherein the weak partitioning mode causes the full capsids to displace the bound empty capsids on the AEX column or membrane, and the empty capsids flow through the AEX column or membrane to produce an AEX flow-through, while the full capsids remain bound to the AEX column or membrane until elution.

3. The method according to claim 2, wherein the affinity chromatography eluate is diluted to a target salt concentration before being subjected to the equilibrated AEX column or membrane.

4. The method according to claim 3, wherein the target salt concentration ranges from about 85 mM to about 95 mM.

5. The method according to claim 4, wherein the target salt concentration is about 90 mM and the salt is NaCl.

6. The method according to claim 1, wherein the AEX column or membrane is equilibrated with an equilibration buffer before being loaded with the solution comprising rAAV full capsids and empty capsids, the equilibration buffer comprising about 50 mM Tris, about 85 mM to about 95 mM NaCl, and greater than about 0.5% (w / v) polysorbate 80, wherein the pH of the equilibration buffer is about 9.

0.

7. The method according to claim 6, wherein the equilibration buffer comprises about 50 mM Tris, about 90 mM NaCl, and about 0.75% polysorbate 80, wherein the pH of the equilibration buffer is about 9.

0.

8. The method according to claim 2, wherein washing the AEX column or membrane at least once comprises a first wash buffer, the first wash buffer comprising (i) about 50 mM tris; (ii) about 85 mM to about 95 mM NaCl, and (iii) greater than about 0.5% (w / v) polysorbate 80, wherein the pH of the first wash buffer is about 9.

0.

9. The method according to claim 8, wherein the first wash buffer comprises (i) about 50 mM tris; (ii) about 90 mM NaCl, and (iii) about 0.75% polysorbate 80, wherein the pH of the first wash buffer is about 9.

0.

10. The method according to claim 2, wherein washing the AEX column or membrane at least once further comprises a second wash with a second wash buffer, the second wash buffer comprising (i) about 50 mM tris; (ii) about 100 mM to about 125 mM NaCl, and (iii) greater than about 0.5% (w / v) polysorbate 80, wherein the pH of the second wash buffer is about 9.

0.

11. The method according to claim 10, wherein the second wash buffer comprises (i) about 50 mM tris; (ii) about 125 mM NaCl, and (iii) about 0.75% polysorbate 80, wherein the pH of the second wash buffer is about 9.

0.

12. The method according to claim 2, wherein the full capsid is eluted from the AEX column or membrane with an elution buffer, the elution buffer comprising (i) about 50 mM tris; (ii) about 125 mM - 250 mM NaCl, and (iii) greater than about 0.5% (w / v) polysorbate 80, wherein the pH of the elution buffer is about 9.

0.

13. The method according to claim 12, wherein the elution buffer comprises (i) about 50 mM tris; (ii) about 250 mM NaCl, and (iii) about 0.75% polysorbate 80, wherein the pH of the elution buffer is about 9.

0.

14. The method according to claim 1, wherein the AEX chromatography column or membrane comprises a high flow rate adsorption membrane.

15. The method according to claim 14, wherein the high flow rate adsorption membrane comprises Q chromatographic membrane.

16. The method according to claim 1, the method further comprising the step of subjecting the AEX eluate or a sample thereof to analytical ultracentrifugation to quantify full capsid enrichment.

17. The method according to claim 1, wherein the enriched full capsid comprises an AAV serotype 2 capsid protein and a polynucleotide sequence comprising a transgene.

18. The method according to claim 17, wherein the transgene is aquaporin 1 (AQP1).

19. The method according to claim 1, wherein the enriched full capsid comprises more than 80% full capsids.

20. The method according to claim 19, wherein the enriched full capsid comprises about 81.5% full capsids.

21. The method according to claim 19, wherein the enriched full capsid comprises more than 90% full capsids.

22. The method according to claim 1, wherein the enriched full capsid comprises more than 95% full capsids.

23. The method according to claim 1, the method further comprising the step of subjecting the AEX eluate comprising the enriched full capsid to tangential flow filtration to obtain a purified full capsid preparation.

24. A method for separating and enriching rAAV full capsids from a mixture of full capsids and empty capsids, the method comprising the steps of: (i) separating a mixture of rAAV full capsids and empty capsids from the cells by lysing virus-producing cells and clarifying the resulting lysate; (ii) subjecting the clarified lysate to affinity chromatography to obtain an affinity chromatography eluate; and (iii) subjecting the affinity chromatography eluate to weak partitioning mode AEX chromatography to separate empty capsids from full capsids, thereby obtaining an AEX eluate comprising enriched full capsids.

25. The method according to claim 24, wherein the cell is a mammalian cell.

26. The method according to claim 25, wherein the cell is cultured in suspension.

27. The method according to claim 26, wherein the cell is cultured in a shake flask, a spinner flask, a cell bag or a bioreactor.

28. A full capsid population enriched by the method according to any one of claims 1 to 27.

29. A pharmaceutical composition comprising the full capsid population according to claim 28.

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