Chromatographic methods for the purification of AAV capsids

Abstract

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

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application Serial No. 63 / 379,115, filed October 11, 2022, and U.S. Provisional Application Serial No. 63 / 489,684, filed March 10, 2023, which are incorporated by reference in their entireties.

[0002] The present disclosure provides methods for producing and purifying recombinant adeno-associated virus (AAV) vectors, comprising separating full capsids from empty capsids using anion exchange chromatography in weak partitioning mode. [Background technology]

[0003] Adeno-associated virus (AAV) is a replication-deficient parvovirus. AAV particles contain a capsid with three capsid proteins—VP1, VP2, and VP3—that encloses a single-stranded DNA genome approximately 4.8 kb in length, which can be either plus- or minus-stranded. Particles containing either strand are infectious and replicate by converting the parent infectious single strand into a double-stranded form, which is then amplified, from which the progeny single strand is displaced and packaged into the capsid.

[0004] AAV depends on co-infection with other viruses, primarily adenovirus, 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 initiation sites, and alternative 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) involved in viral genome replication and packaging, whereas cap expression generates viral capsid proteins (VP1, VP2, and VP3) that form the outer capsid shell protecting the viral genome and are actively involved in cell binding and internalization. The aap gene, in an alternative reading frame overlapping with the cap gene, encodes an assembly-activating protein (AAP). This AAP protein is thought to provide a scaffolding function for capsid assembly.

[0005] AAV particles have characteristics that make them attractive as vectors for therapeutic applications, including gene therapy and gene vaccines. AAV can infect a wide range of cell types, including many mammalian cells, offering the potential for targeting many different tissues in vivo. AAV infects 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 Rep proteins, the ITR-flanked transgene can form a transcriptionally active extranuclear chromosomal element or episome that can persist essentially for the lifespan of the transduced cell.

[0006] A key goal of rAAV vector production methods is to achieve stable, high vector productivity while minimizing the generation of product-associated impurities, including residual AAV-encapsidated DNA impurities and empty capsids. Measured as vector genomes (VG) produced per cell, rAAV vector productivity can reach 10 VG per cell. 3 Less than 2 x 10 5 The VG range can be highly variable. In addition to being cost-effective, a key advantage of high productivity is that purification can be more efficient when the starting material has a higher ratio of rAAV vector product to total harvested biomass.

[0007] During rAAV vector production, removal of product-related impurities is a critical part of rAAV purification. While it is possible to separate empty capsids from full capsids using ultracentrifugation, scalable chromatography methods have proven more difficult. As an alternative, anion exchange chromatography in bind-and-elute mode is commonly investigated in an attempt to reproduce the high full capsid ratio achieved by ultracentrifugation. However, the similar properties of full and empty capsids result in a sharp trade-off between product yield and full capsid ratio, making it difficult to select optimal conditions. This is also hindered by complex interactions between AAV capsids and the chromatography matrix, which often results in additional product loss. Therefore, there is a need for rAAV purification methods that reduce product-related impurities, such as empty capsids. Summary of the Invention

[0008] The present invention provides methods for producing and purifying rAAV particles, which involve separating full capsids from empty capsids (enriching full capsids) using anion exchange chromatography in a weak partitioning mode. Described herein is a method for enriching rAAV full capsids in a mixture of full and empty capsids, which method comprises: (i) providing a solution containing full and empty rAAV capsids; (ii) equilibrating an anion exchange (AEX) column or membrane; and (iii) subjecting the solution containing rAAV complete capsids and empty capsids to weak partitioning mode AEX chromatography to separate the empty capsids from the complete capsids and obtain an AEX eluate enriched in complete capsids.

[0009] In embodiments of the method, the solution is an affinity chromatography eluent, and step (iii) comprises applying the affinity chromatography eluent to an 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. In these methods, a weak partitioning mode displaces bound empty capsids from the AEX column or membrane with full capsids, causing the empty capsids to flow through the AEX column or membrane, resulting in AEX flow-through, and the full capsids remaining bound to the AEX or membrane column until elution.

[0010] In some embodiments, the affinity chromatography eluate is diluted to a target salt concentration before being applied to the equilibrated 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.

[0011] In an embodiment of this method, the AEX column or membrane is buffered with 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 85 mM to about 95 mM NaCl (e.g., 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% (e.g., 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1.0%) (w / v) polysorbate 80, wherein the equilibration buffer has a pH of about 9.0 (e.g., about 8.8, 8.9, 9.0, 9.1, 9.2). The equilibration buffer may comprise about 50 mM Tris, about 90 mM NaCl, and about 0.75% polysorbate 80 and may have a pH of about 9.0.

[0012] In embodiments of the method, washing the AEX column or membrane at least once comprises washing the AEX column or membrane with (i) 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, (ii) about 85 mM to about 95 mM (e.g., about 84, 84.9, 85, 86, 87, 88, 89, 8 and (iii) a first wash buffer comprising (i) about 50 mM Tris, (ii) about 90 mM NaCl, and (iii) about 0.75% (e.g., 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1.0%) (w / v) polysorbate 80, wherein the first wash buffer has a pH of about 9.0 (e.g., about 8.8, 8.9, 9.0, 9.1, 9.2). The first wash buffer may, for example, comprise (i) about 50 mM Tris, (ii) about 90 mM NaCl, and (iii) about 0.75% polysorbate 80, and may have a pH of about 9.0.

[0013] In embodiments, washing the AEX column or membrane at least once comprises washing with (i) 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, (ii) about 100 mM to about 125 mM (e.g., about 99, 100, 101, 105, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 and a second wash with a second wash buffer comprising (i) about 50 mM Tris, (ii) about 125 mM NaCl, and (iii) about 0.75% (e.g., 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1.0%) (w / v) polysorbate 80, where the second wash buffer has a pH of about 9.0 (e.g., about 8.8, 8.9, 9.0, 9.1, 9.2). The second wash buffer can, for example, comprise (i) about 50 mM Tris, (ii) about 125 mM NaCl, and (iii) about 0.75% polysorbate 80, and can have a pH of about 9.0.

[0014] In embodiments, the intact capsids are purified from the AEX column or membrane by elution with (i) 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, (ii) about 125 mM to about 250 mM (e.g., about 124, 125, 126, 130, 140, 150, 160, 170, 180, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, The elution buffer may comprise, for example, (i) about 50 mM Tris, (ii) about 250 mM NaCl, and (iii) about 0.75% (e.g., 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1.0%) (w / v) polysorbate 80, wherein the elution buffer has a pH of about 9.0 (e.g., 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 elution buffer has a pH of about 9.0.

[0015] In embodiments of the method, the AEX chromatography column or membrane comprises a high-flux adsorptive membrane, e.g., a Sartobind® Q chromatography membrane. In embodiments, the method can further comprise subjecting the AEX eluate containing the enriched intact capsids to tangential flow filtration (TFF) to obtain a purified preparation of intact capsids (e.g., drug substance).

[0016] In embodiments, polysorbate 20 and poloxamer 188 may be used in one or more of the buffers described herein.

[0017] In an embodiment of this method, the method further comprises subjecting the AEX eluate to analytical ultracentrifugation to quantify full capsid enrichment.

[0018] In embodiments of this method, the enriched complete capsids may comprise AAV serotype 2 capsid proteins and a polynucleotide sequence comprising a transgene, for example, a transgene such as aquaporin 1 (AQP1).

[0019] In embodiments, enriched full capsids include greater than 80% full capsids (e.g., 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.

[0020] Also described herein is a method for isolating and enriching rAAV complete capsids from a mixture of complete and empty capsids. The method includes: isolating a mixture of rAAV complete 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 empty capsids from complete capsids to obtain an AEX eluate containing enriched complete capsids. In embodiments, the cells are mammalian cells. The cells are typically cultured in suspension, for example, in shaker flasks, spinner flasks, cell bags, or bioreactors.

[0021] The present invention also provides populations of rAAV complete capsids enriched by the method, populations of rAAV complete capsids isolated and enriched by the method, and pharmaceutical compositions comprising the enriched populations of complete capsids. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 shows the charges of empty and full AAV capsids above and below pI(1). [Figure 2] FIG. 1 illustrates the weak partitioning process, where empty capsids are replaced by full capsids depending on the environment in which they are formed. [Figure 3] 1 is a graph showing the relationship between NaCl concentration (mM) in Wash 2 and equivalents / feed and empty capsid removal (VP / mL). [Figure 4] 1 is a graph showing the relationship between NaCl concentration (mM) of Wash 2 and Equivalents / Feed and the percentage of intact capsids in the eluate. [Figure 5] 1 is a graph showing the relationship between NaCl concentration (mM) of Wash 2 and Eq / Feed and VG recovery (%) in the eluent. [Figure 6] 1 is a plot showing the optimum region based on NaCl concentration (mM) for Wash 2 and equivalents / feed. [Figure 7] Graph showing the relationship between load ratio and breakthrough (%) of full capsids (VG) and empty capsids (VP). [Figure 8] Chromatogram of the optimized AAV-AQP1 AEX step at a 2 liter (L) scale. [Figure 9] 1 is a graph showing a summary of results demonstrating the scalability of the AEX process, where 2 L scale refers to a process operated using 10 mL of Sartobind® Q and 80 refers to a process operated using 75 mL of Sartobind® Q. [Figure 10] 1 is a graph showing the results of a breakthrough experiment showing the extent of breakthrough of vector genomes and vector particles in the flow-through. [Figure 11] 1 is an excerpt of a chromatogram from an experiment (described in Example 2) conducted to validate the process using 10 mL of Sartobind® Q. This excerpt focuses on the column wash 2 and elution steps. [Figure 12] 1 shows the results of analytical ultracentrifugation performed on eluent produced from a validation run performed on 10 mL of Sartobind® Q. [Figure 13] Chromatogram of initial anion exchange chromatography run-through. [Figure 14] Breakthrough curves showing both VG breakthrough and empty capsid breakthrough up to a load of 4E14 VG / mL at 90 mM NaCl feed and equilibrium conductivity are shown. [Figure 15] 1 is a pair of graphs showing the VG concentration of the column as the VG load is increased to 4E14 vg / ml (top graph) and the empty capsid ratio of the column as the VG load is increased to 4E14 VG / mL (bottom graph). [Figure 16] 1 is a graph showing the increase in the ratio of intact capsids on the column as the VG load increases to 4E14 VG / mL. [Figure 17]To highlight the level of enrichment achievable, a graph shows the concentration of intact capsids on the column when loaded to 4E14VG / mL at both 9mS / cm and 8mS / cm. [Figure 18] Chromatogram of the optimized AAV-AQP1 AEX step from a 20 L scale validation run. [Figure 19] The VG recovery and % intact capsid values ​​after USP, capture chromatography, ion exchange chromatography (IEX), and tangential flow filtration (TFF) are shown for the "initial process" and "final process," respectively. [Figure 20] 9 is a graph showing a comparison of intact capsid ratio and VG recovery between the 20L and 80L batches, as well as a plot showing analytical ultracentrifugation (AUC) results confirming that the elution peak in FIG. 8 contains 82% intact capsids. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present disclosure provides a method for producing and purifying rAAV, which involves separating full capsids from empty capsids (enriching full capsids) using anion exchange chromatography in a weak partitioning mode. The method for enriching rAAV full capsids in a mixture of full and empty capsids comprises the following steps: (i) providing a solution containing full and empty rAAV capsids; (ii) equilibrating an anion exchange (AEX) column or membrane; and (iii) subjecting the solution containing rAAV complete capsids and empty capsids to weak partitioning mode AEX chromatography to separate the empty capsids from the complete capsids and obtain an AEX eluate enriched in complete capsids.

[0024] The method for isolating and enriching rAAV full capsids in a mixture of full and empty capsids comprises the following steps: (i) isolating a mixture of full and empty rAAV capsids from virus-producing cells by lysing the cells and clarifying the resulting lysate; (ii) subjecting the clarified lysate to affinity chromatography to obtain an affinity chromatography eluate; (iii) subjecting the affinity chromatography eluate to weak partitioning mode AEX chromatography to separate empty capsids from full capsids to obtain an AEX eluate containing enriched full capsids.

[0025] The present invention also provides populations of rAAV full capsids enriched by the present methods, populations of rAAV full capsids isolated and enriched by the present methods, and pharmaceutical compositions (e.g., drugs, drug substances) comprising the enriched populations of full capsids. In embodiments, the present methods result in increased production titers and a higher ratio of full capsids to empty capsids (F:E). More specifically, the present disclosure provides methods for enriching rAAV full capsids in a mixture of full and empty capsids, for example. In embodiments, production yields are greater than 50%, greater than 50% of empty capsids are removed, and an F:E ratio of greater than 80% is achieved.

[0026] AEX in weak partitioning mode for purifying rAAV The purification and concentration methods described herein involve purifying rAAV full capsids (i.e., rAAV particles containing a recombinant genome) from a solution containing full and empty rAAV capsids. The solution may be the result of any rAAV production / purification method. Typically, the solution is conditioned by dilution to a target salt (e.g., NaCl) concentration before being subjected to AEX chromatography. For example, the solution is diluted to a NaCl concentration of about 90 mM (e.g., about 89, 90, 91 mM) with 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 about 9.0 (e.g., about 8.8, 8.9, 9.0, 9.1, 9.2). In embodiments, the solution is an affinity chromatography eluent. Affinity chromatography is one of several purification steps in a typical method for producing and purifying rAAV particles (e.g., rAAV complete capsids). Methods for rAAV production / purification are well known in the art, and examples of such methods are described below.

[0027] In the methods described herein, weak partitioning mode AEX chromatography is a purification technique used to separate complete capsids from empty capsids (Figures 1 and 2). This form of chromatography exploits the charge difference between capsids containing the viral genome and empty capsids. Targeting a pH value above the capsid pI means that VG-containing complete capsids carry a greater negative charge than empty capsids. In the purification and concentration methods described herein, weak partitioning describes the process of separating complete capsids from empty capsids by creating an environment more favorable for complete capsid binding.

[0028] Anion exchange chromatography in weak partitioning mode differs from the historically used flow-through and bind-elute AEX methods (Liu et al., MAbs. 2010;2(5):480-499; Kelley et al., Biotechnology and Bioengineering vol. 101:553-566, 2008). In bind-and-elute AEX, the (desired) product pool is first loaded onto an anion exchange column, and then the desired product is eluted with a step or linear gradient at a higher salt concentration, leaving the majority of impurities bound to the column. Impurities are eluted from the column during a wash or regeneration step. In flow-through AEX, the operating pH is typically 8-8.2, and the conductivity in the product load and equilibration and wash buffers is a maximum of 10 mS / cm. Conditions are selected 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 anion exchange chromatography in weak partitioning mode can enable a two-chromatographic recovery process for a desired product, including affinity chromatography and anion exchange. Similar to flow-through chromatography, the process is run isocratically; however, in contrast to flow-through mode, the conductivity and pH are selected to enhance binding of both product and impurities, achieving a product partition coefficient (Kp) of 0.1 to 20, preferably 1 to 3. This takes advantage of the fact that the impurities to be removed are more acidic than the product. While both product and impurities bind to the anion exchange resin, impurities bind much more tightly than in flow-through mode, which can result in increased impurity removal. Thus, weakly bound impurities that are not efficiently removed in flow-through mode can be removed to a greater extent under conditions where their partition coefficient (Kp) is increased. Compared to anion exchange chromatography in flow-through mode, weak partitioning chromatography can enable a two-column recovery process due to the increased clearance of viruses, host cell proteins, and product-related species. One aspect of weak partitioning chromatography is that pH and counterion conditions must be optimized for each product.This is in contrast to some platform chromatography processes, where standardized conditions can be used with an anion exchange matrix (resin or membrane) for most products.

[0029] In a method for enriching rAAV full capsids in a mixture of full and empty capsids, a solution containing rAAV full capsids and empty capsids is provided. As described above, the solution can be the result of any rAAV production / purification method, and in embodiments, the solution is an affinity chromatography eluate. The AEX column or membrane that receives the solution is typically pre-equilibrated with an appropriate pre-equilibration buffer. For example, a suitable pre-equilibration buffer contains about 50 mM Tris, about 1 M NaCl, and about 0.75% polysorbate 80, and has 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 the equilibration buffer. Typically, the equilibration buffer comprises 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 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 comprises about 50 mM Tris, about 90 mM NaCl, and greater than 0.5% (w / v) polysorbate 80 and greater than about 0.75% polysorbate 80, with a pH of about 9.0. Any suitable AEX column(s) or membrane(s) can be used in this method. In embodiments, a high-flux adsorptive membrane is used. An example of such a membrane is the Sartobind® Q chromatography membrane. Another example of such a membrane is Pall Corporation's Mustang® Q membrane.

[0030] After equilibration of the AEX column or membrane, a solution (referred to as "feed," "feed material," and "conditioned feed" in Examples 1 and 2) is added to the equilibrated AEX column or membrane. The solution can be applied at any suitable load ratio. In the experiments described in Examples 1 and 2, 5×10 13 ~1×10 15 A load ratio of VG / mL was used, resulting in an elution percentage of 81.5% full capsids. The flow-through of the load solution typically consists of empty capsids (full capsids have a stronger interaction with the column or membrane and displace empty capsids). In embodiments, the flow-through or a sample thereof can be analyzed to determine its viral genome and viral particle concentration by any suitable method.

[0031] Following application of the solution, the AEX column or membrane is washed at least once (eg, once, twice, three times). In at least the first wash, the AEX column or membrane is washed with a first wash buffer comprising 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 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 first wash buffer typically has a pH of about 9.0 (e.g., about 8.8, 8.9, 9.0, 9.1, 9.2). For example, the first wash buffer may contain about 50 mM Tris, about 90 mM NaCl, and about 0.75% polysorbate 80, and may have a pH of about 9.0. In embodiments, there are two washes with a second wash buffer comprising 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 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 second wash buffer is typically at a pH of about 9.0 (e.g., about 8.8, 8.9, 9.0, 9.1, 9.2). For example, the second wash buffer can contain about 50 mM Tris, about 125 mM NaCl, and about 0.75% polysorbate 80, with the second wash buffer having a pH of about 9.0. The column or membrane wash typically consists mostly of empty capsids but may contain full capsids. The wash, or a fraction (sample) of the wash, can be analyzed for their viral genome and viral particle concentrations by any suitable method.

[0032] Following at least one wash (eg, two washes), the intact capsids are eluted from the column or membrane to provide an AEX eluate enriched for intact capsids. Intact capsids are generally eluted from the AEX column or membrane with an eluent comprising 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 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., about 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1.0%) (w / v) polysorbate 80. The eluate typically has a pH of 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 containing about 50 mM Tris, about 250 mM NaCl, and about 0.75% polysorbate 80, and having a pH of about 9.0. A sample of the AEX eluate enriched for complete capsids can be analyzed for its viral genome and viral particle concentrations, as well as its complete capsid ratio and viral genome recovery rate. Thus, in embodiments, the method further includes subjecting the AEX eluate enriched for complete capsids, or a sample thereof, to PCR and analytical ultracentrifugation (AUC) to quantify the complete capsid enrichment (e.g., to monitor the quality and effectiveness of vector purification, to measure the relative amount of empty capsids in a preparation of recombinant viral particles). AUC is a broadly applicable and information-rich method for investigating macromolecular properties such as size, shape, stoichiometry, and binding characteristics, all in a true solution-state environment. AUC can assess quantitative and qualitative information at moderately high concentrations. Methods for using AUC to characterize preparations of recombinant viral particles are known (see, e.g., U.S. Patent Publication No. 20200225139, incorporated herein by reference).

[0033] Any suitable membrane volume (MV) and flow rate (MV / min) can be used for all pre-equilibration, equilibration, washing, and elution steps. Examples of suitable MV and MV / min are provided 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.

[0034] In embodiments, the AEX eluate enriched for intact capsids is subjected to TFF (e.g., TFF dialysis) for further purification and preparation of a pharmaceutical product (e.g., a drug, drug substance) that can be administered as a gene therapy drug.

[0035] In embodiments, the method provides a ratio of rAAV complete particles (complete capsids) to empty AAV particles (empty capsids) of at least about 30%, e.g., about 30% to 40%, at least 65%, about 65% to 95%, at least 80%, about 80% to 85% (e.g., 79%, 79.5%, 80%, 80.5%, 81%, 81.5%, 82%, 83%, 85%, 85%), about 90% to 95% (e.g., 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%), etc. The level of nuclease-resistant AAV-encapsulated DNA impurities may be assessed by qPCR using primers and probes designed for relevant sequences in the helper plasmid or against high-copy-number genomic sequences. Susceptibility to nuclease treatment prior to qPCR allows for differentiation of nuclease-sensitive "naked" residual DNA impurities from nuclease-insensitive encapsulated residual DNA impurities. Intact AAV capsids may be measured using a capsid-specific ELISA assay and the amount of empty capsid determined by comparing the capsid particle titer to the VG titer. Spectrophotometry may be used on samples from which non-AAV capsid impurities have been substantially removed.

[0036] This method is scalable to production scales, e.g., about 5 to about 10 liters, about 10 to about 20 liters, about 20 to about 50 liters, about 50 to about 100 liters (e.g., 79, 80, 81), about 100 to about 200 liters, or even larger cultures, and is applicable to rAAVs, including various AAV serotypes / capsid variants. The experiments described in Examples 1 and 2 demonstrate that rAAV complete capsids can be concentrated at large (production) scales.

[0037] The rAAV vectors (rAAV complete capsids) produced by the methods disclosed herein are useful for expressing transgenes in target cells. These rAAV vectors can be used for gene therapy because they can introduce into target cells a polynucleotide containing a transgene that can be maintained and expressed in the target cells. The rAAV vectors can deliver heterologous polynucleotide sequences (e.g., polynucleotide sequences encoding therapeutic or reporter proteins and regulatory elements for protein expression) to target cells in human patients. A non-exclusive list of example transgenes includes RPGR, RPE65, GAD65, GAD67, CNGB3, and AQP1. In some embodiments, the two AAV ITRs are AAV2 ITRs. In this method, the AAV cap gene may be derived from an AAV serotype or AAV variant, such as, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, AAV-PHP.5, AAV-PHP.B, AAV-PHP.eB, AAV2-retro, AAV9-retro, AAVrh74, AAVrh, and hybrids thereof.

[0038] The term "vector" refers to a vehicle for introducing a polynucleotide into a target cell. A vector may be a viral vector (e.g., rAAV vector, HSV vector) or a non-viral vector such as a plasmid, or DNA combined with a compound such as liposome, gelatin, or polyamine. An expression vector is a vector that contains a polynucleotide sequence that encodes a gene product (e.g., protein or RNA) with regulatory elements for expression in a host cell or target cell.

[0039] "rAAV," "rAAV vector," "rAAV particle," or "rAAV virion" refers to a recombinant AAV vector genome that is packaged into (i.e., encapsidated by) capsid proteins for subsequent infection of target cells ex vivo, in vitro, or in vivo. These terms exclude empty AAV capsids and AAV capsids that lack a complete recombinant AAV genome, including a transgene to be expressed in a target cell. Thus, in addition to the capsid, an rAAV vector contains an rAAV genome. "rAAV genome" or "rAAV vector genome" refers to a polynucleotide sequence containing a transgene of interest that is ultimately packaged or encapsidated to form an rAAV particle. Typically, for rAAVs, most of the AAV genome (e.g., including the rep, cap, and aap genes) has been deleted, with one or both ITR sequences remaining as part of the rAAV genome along with the transgene. As used herein, "transgene" refers to a polynucleotide sequence that encodes a gene product (e.g., a therapeutic protein or a reporter protein) and regulatory elements for the expression of the gene product in a target cell.

[0040] "Empty capsid" and "empty particle" refer to an AAV particle that has an AAV capsid shell but lacks all or part of the recombinant AAV genome, including the transgene sequence and one or two ITRs. Such empty capsids do not function to introduce a transgene into one or more target cells. In embodiments, isolated rAAV particles are separated from empty AAV particles.

[0041] rAAV genomes (e.g., including ITRs) may be based on the same strain or serotype (or subgroup or variant), or they may be different from each other. As a non-limiting example, rAAV plasmids, or vector genomes or particles (capsids) based on a single serotype genome may be identical in one or more capsid proteins that package the vector genome. Furthermore, the rAAV genome may be derived from an AAV genome (e.g., including one or more ITRs derived from the AAV2 genome) that is different from the one or more capsid proteins that package the rAAV vector genome.

[0042] rAAV vectors that may be produced, isolated, purified, and concentrated by the methods disclosed herein include any rAAV vector comprising a capsid and genome derived from any AAV strain or serotype. By way of non-limiting example, the rAAV vector capsid and / or genome may 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., an AAV comprising AAVrh.10 ITRs and AAVrh.10 capsid proteins), and the like. In embodiments, the rAAV vector comprises a genome and capsid proteins derived from the same AAV strain or serotype. For example, the rAAV vector may be an rAAV2 vector (i.e., an rAAV comprising AAV2 ITRs and AAV2 capsid proteins).

[0043] In embodiments, the AAV vector is a pseudotyped rAAV vector comprising ITRs from one AAV serotype and capsid proteins from a different AAV serotype. In some embodiments, the pseudotyped rAAV is rAAV2 / 5 (i.e., an rAAV comprising AAV2 ITRs and AAV5 capsid proteins), rAAV2 / 8 (i.e., an rAAV comprising AAV2 ITRs and AAV8 capsid proteins), rAAV2 / 9 (i.e., an AAV comprising AAV2 ITRs and AAV9 capsid proteins), or rAAV2 / 10 (i.e., an rAAV comprising AAV2 ITRs and AAV10 capsid proteins). In embodiments, the rAAV vector comprises capsid proteins that are variant AAV capsids, such as the AAV2 variant rAAV2-retro (SEQ ID NO: 44 from WO2017 / 218842, incorporated herein by reference).

[0044] Methods for generating rAAV As explained above, rAAV purification and concentration methods can be applied to solutions (e.g., lysates, eluates) containing rAAV full and empty capsids obtained or produced by any suitable production method. Methods for producing rAAV are well known in the art. In general, the methods include expanding producer cells, introducing nucleic acid sequences for an rAAV vector, AAV rep and cap, and helper genes into the producer cells, culturing the transduced producer cells under conditions such that rAAV particles are produced, and isolating the rAAV particles. Specific embodiments of methods for producing rAAV are described in detail below.

[0045] cell line rAAV vector production methods generally require certain elements, including, for example, (i) a permissive host cell (producer cell) for rAAV production, (ii) helper virus functions, which may be supplied by an appropriate construct containing, for example, genes providing adenovirus helper functions, (iii) a trans-packaging rep / cap construct, and (iv) a suitable production medium.

[0046] A producer cell is any cell that is a permissive host cell for producing rAAV when an rAAV genome production construct, a helper function construct, and a construct providing AAV functions (e.g., expression of rep and cap) are present. The term may also include the progeny of the original transfected cell. Thus, a producer cell is also the progeny of a host cell that has been transfected with an exogenous DNA sequence or that has integrated that DNA sequence into the host cell genome. It is understood that the progeny of a single parent cell may not necessarily be completely identical in morphology or genomic or total DNA complement to the original parent due to natural, accidental, or deliberate mutation.

[0047] In embodiments, cells used to produce rAAV particles are mammalian cells, including HEK293 cells, BHK cells, and HeLa cells. Exemplary producer / host cells include human embryonic kidney (HEK) cells, such as HEK293. In preferred embodiments, producer cells are adapted for growth in suspension, including HEK293 cells adapted to suspension culture. In further preferred embodiments, producer cells are adapted for growth in serum-free medium. In embodiments, producer cells are grown in at least one culture vessel, which may be, for example, one or more of a shaker flask, spinner flask, cell bag, or bioreactor.

[0048] Producer cell lines that may be used in the methods of rAAV production, isolation, purification, and enrichment disclosed herein include mammalian or insect cell lines. The term "cell line" refers to a cell population capable of continuous or long-term growth and division in vitro under appropriate culture conditions. A cell line may be, but is not necessarily, a clonal population derived from a single progenitor cell. Spontaneous or induced changes in karyotype may occur in cell lines during storage or transfer of such clonal populations, as well as during long-term passage in tissue culture. Thus, progeny cells derived from a cell line may not be exactly identical to the ancestral cells or cultures.

[0049] To achieve rAAV production, producer cell lines may require the presence within the producer cells of one or more of the following: an rAAV genome production construct, a helper function construct, and / or an AAV rep / cap construct. These may be introduced as three constructs (e.g., three plasmids), or the producer cell may already harbor one or more constructs that stably integrate some or all of these functions into the producer cell genome. In embodiments, the one or more helper genes may include all or part of one or more adenovirus, herpes simplex virus type 1, or baculovirus genes. As used herein, the terms "stable" or "stably integrated" with respect to a cell means that a nucleic acid sequence, such as a selectable marker and / or a heterologous nucleic acid sequence, or a plasmid or vector (or 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 remained intrachromosomally or has been maintained extrachromosomally for some time.

[0050] Expansion of producer cell lines In embodiments of the methods described herein, an expansion step is used to increase the number of producer cells prior to the step of introducing the rAAV genome production construct (including the rAAV genome) and / or other constructs providing helper virus and AAV functions. The expansion step may be performed in one or more cell culture vessels. For example, the expansion step may be performed in a series of cell culture vessels of increasing volume. The cell culture medium used to expand the producer cell line may be any medium suitable for growing (i.e., increasing in number) the producer cells. In preferred embodiments, the expansion step culture medium is animal-component-free, e.g., does not contain serum or other components derived from animals. Chemically defined, animal-component-free media are commercially available.

[0051] In embodiments, an anti-aggregation supplement, sometimes referred to herein as an anti-aggregation agent (ACA), is added to the expansion medium to reduce cell clumping. ACA is commercially available, for example, from Irvine Scientific. The anti-aggregation supplement may be added to the expansion stage culture medium at one or more time points. In embodiments, the anti-aggregation supplement includes dextran sulfate, heparin, and / or other sulfated glycosaminoglycans that inhibit clumping of producer cells. In embodiments, the anti-aggregation supplement includes heparin sodium, which may be added to the medium to a concentration of about 25 μg / ml to about 250 μg / ml, e.g., about 25 μg / ml, about 50 μg / ml, about 100 μg / ml, about 150 μg / ml, and / or about 200 μg / ml.

[0052] In embodiments, the expansion stage culture medium contains and / or is supplemented to contain one or more glutamine, glutamine precursors, or glutamine-containing amino acid dipeptides at a concentration of about 2 mM to about 6 mM (e.g., about 2 mM, about 3 mM, about 4 mM, about 5 mM, or about 6 mM). The one or more glutamine, glutamine precursors, or glutamine-containing amino acid dipeptides may be, for example, one or more of L-alanyl-L-glutamine, L-glutamine, glutamate, 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).

[0053] In embodiments, the expansion stage culture medium includes and / or is supplemented to include a non-ionic polyol surfactant, such as poloxamer 188 (a copolymer of polyethylene and polypropylene ether glycol). In embodiments, the non-ionic polyol surfactant is present in the expansion stage culture medium at about 0.05% to about 0.2% (w:v) (e.g., about 0.05%, about 0.1%, about 0.1%, or about 0.2%). In embodiments, the expansion stage culture medium includes about 4 mM L-alanyl-L-glutamine dipeptide and 0.1% (w:v) poloxamer 188.

[0054] In embodiments, the pH of the expansion stage culture medium is maintained at a pH of about 7.1 to about 7.5 (e.g., about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5). In embodiments, the pH is maintained at about 7.2 to about 7.4 by CO sparging. In embodiments, prior to introducing one or more polynucleotide constructs into the cells, the pH of the culture medium is changed to about 6.9 and CO sparging is stopped.

[0055] Introduction of one or more polynucleotide constructs Production of rAAV vectors typically requires a producer cell line that provides the basic biosynthetic machinery, as well as (i) a construct that provides the rAAV genome (the transgene of interest and associated regulatory elements flanking the AAV ITRs), and (ii) one or more constructs containing additional genes that provide gene products necessary to direct rAAV vector production. These additional genes include genes from AAV (e.g., AAV rep and cap) and helper virus (e.g., adenovirus E1a, E1b, E2a, E4, and VA) necessary to support replication and packaging of the vector genome.

[0056] "Helper virus genes" or "helper virus-derived genes" refer to non-AAV-derived viral genes whose gene products AAV depends on for replication. This term includes proteins and / or RNAs required for AAV replication, including those involved in activating AAV gene transcription, stage-specific AAV mRNA splicing, and AAV DNA replication. Helper virus genes may be derived from any of the known AAV helper viruses, such as adenovirus, herpesvirus, and vaccinia virus. Thus, "helper virus functions" refer to those functions provided by helper virus genes (e.g., adenovirus E1a, E1b, E2a, E4, and VA) required for AAV production. These helper virus functions may be provided on one or more vectors introduced into the producer cell, stably expressed by the producer cell, or a combination of both.

[0057] As used herein, "AAV functions" or "AAV accessory functions" refer to coding sequences from AAV that can be expressed in producer cells to provide AAV gene products that function in trans for productive AAV replication and packaging. Thus, AAV functions include AAV open reading frames (ORFs), including rep and cap, as well as others, such as aap, of certain AAV serotypes. Such AAV functions are provided by one or more polynucleotide constructs, which may be plasmid vectors, non-plasmid vectors, or polynucleotide constructs integrated into the chromosome of the producer cell that provide AAV helper functions. Plasmids providing AAV functions that may be used in the methods disclosed herein are commercially available.

[0058] In embodiments of this method, one or more helper virus genes are constitutively expressed by producer cells (e.g., 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. Genes from AAV (e.g., rep and cap) may be included in the same polynucleotide construct containing one or more helper virus genes, or may be included on separate polynucleotide constructs. rAAV particles are generated after a polynucleotide construct providing or encoding the rAAV genome (e.g., an rAAV genome production vector) is introduced into a producer cell line. In embodiments, rAAV particles are produced after transient transfection of producer cells with (i) an rAAV genome production vector and (ii) one or more vectors providing helper virus genes (e.g., E4, E2a, and VA) and AAV genes (e.g., rep and cap). In embodiments, these vectors are plasmids.

[0059] In embodiments of the methods disclosed herein, following the expansion step, a first polynucleotide construct comprising the transgene flanked by ITRs and a second polynucleotide construct comprising helper virus genes and the AAV rep and cap genes are introduced into the expanded producer cells. When the first and second polynucleotide constructs are plasmids, the system may be referred to as a two-plasmid system.

[0060] In embodiments of the methods disclosed herein, following the expansion step, a first polynucleotide construct comprising a transgene flanked by ITRs, a second polynucleotide construct comprising helper virus genes, and a third polynucleotide construct comprising the AAV rep and cap genes are introduced into the expanded producer cells. When the first polynucleotide construct, second polynucleotide construct, and third polynucleotide construct are plasmids, the system may be referred to as a three-plasmid system.

[0061] When one or more recombinant plasmids are used to produce an rAAV vector, a "rAAV genome-producing plasmid" refers to a plasmid that contains a transgene (operably linked to regulatory sequences) and one or more ITRs intended for packaging into an rAAV, as well as non-rAAV genome components (plasmid backbone) that are important for cloning and amplification of the plasmid but are not packaged or encapsidated 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 may be a plasmid.

[0062] The terms "introduction" and "transfect" refer to the introduction of a polynucleotide into a host cell or target cell. In embodiments, the host cell is a producer cell, e.g., HEK293 cell. In embodiments, an rAAV genome production plasmid is introduced into the producer cell by transient transfection along with one or more plasmids providing helper virus and AAV functions. Transient transfection of producer cells to introduce a first polynucleotide construct (e.g., an rAAV genome production vector) comprising a transgene and ITR(s), and optionally a second polynucleotide construct and / or a third polynucleotide construct providing AAV functions (rep and cap genes) and helper virus functions, may be achieved by standard transfection methods, including, for example, calcium phosphate co-precipitation, cationic lipid-based transfection, and cationic polymer-based transfection. Examples of cationic lipid-based transfection include lipofectamine (a 3:1 mixture of DOSPA (2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propandinium trifluoroacetate) and DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine)). Examples of cationic polymer-based transfection include linear and / or branched polyethyleneimine (PEI), poly-L-lysine, poly-L-arginine, and polyamidoamine dendrimers. In embodiments, transient transfection of producer cells is performed using a PEI-based transfection reagent. The PEI may be a linear or branched polymer. In embodiments, the PEI is a linear PEI of 20 to 25 kD. For example, in embodiments, the PEI is jetPEI or PEIpro (available from Polyplus). In addition, transient transfection of producer cells may be performed using a transfection reagent that includes both a cationic lipid and a cationic polymer.

[0063] rAAV may alternatively be produced in insect cells (e.g., sf9 cells) using baculovirus vectors, or in HSV-infected baby hamster kidney (BHK) cells (e.g., BHK21). In either method, rAAV production is induced in host cells, insect cells, or mammalian cells, respectively, by coinfection with two or more recombinant viruses carrying the rAAV genome, one or more AAVrep and cap, and helper virus functions necessary for rAAV replication and packaging.

[0064] Production of rAAV particles Generally, the production phase (also referred to as the production step) follows the step of introducing the rAAV genome vector and / or a vector providing helper virus and / or AAV functions into producer cells. In embodiments, rAAV particles are produced by culturing the cells for at least about 48 hours (e.g., 47.5, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 96.5, or 97 hours) after introduction of the rAAV genome vector. In embodiments, the transfected producer cells are cultured (i.e., the production phase is maintained) for about 72 to about 100 hours, about 90 to about 100 hours, about 92 to about 98 hours, or about 94 to about 98 hours. In embodiments, the production phase is maintained for about 96 hours.

[0065] The production stage medium may be any cell culture medium suitable for producing rAAV in producer cells. In embodiments, the production medium is free of animal products, such as serum. "Free" in this context means that the medium has undetectable levels of animal products, such as serum. In embodiments, the pH of the production medium is reduced compared to the pH of the expansion stage medium. In embodiments, the production medium is maintained at a pH of about 6.8 to about 7.4 (e.g., about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, or about 7.14). In embodiments, the pH is maintained at about 6.9 to about 7.3.

[0066] In embodiments of the method, the production stage includes adding calcium ions to the production stage cell culture medium. Adding calcium ions to the production medium, also referred to herein as calcium supplementation, refers to the addition of calcium ions (Ca 2+ ) in the form of a calcium salt, e.g., CaCl. Calcium ions may be added one or more times during the production stage after introduction of the rAAV genome vector (e.g., after transfection). For example, calcium ions may be added one or more times between about 0 hours and about 48 hours, e.g., 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, from the start of the production stage (i.e., after transfection).

[0067] Calcium ions (e.g., CaCl) may be added to the production medium so that the total concentration of calcium ions in the culture medium is greater than 0.3 mM and less than 10 mM. In embodiments, 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.

[0068] In embodiments, the production stage includes adding one or more glutamine, glutamine precursors, or glutamine-containing amino acid dipeptides to the production stage medium. The one or more glutamine, glutamine precursors, or glutamine-containing amino acid dipeptides may be, for example, one or more of L-alanyl-L-glutamine, L-glutamine, glutamate, glycyl-L-glutamine, glutamine protein hydrolysate, L-glutamic acid, and glutamine dipeptide. A solution containing at least one glutamine, glutamine precursor, or glutamine-containing amino acid dipeptide may be added to the production stage medium, for example, at one or more of about 6 hours, about 12 hours, about 24 hours, about 48 hours, or about 72 hours after transfection.

[0069] In embodiments, the production stage includes adding sorbitol to the production stage medium. Sorbitol may be added to the production stage medium at one or more time points during the production stage, for example, about 6 hours, about 12 hours, about 20 hours, about 24 hours, and / or about 48 hours after transfection. In embodiments, 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 embodiments, sorbitol is added to the production medium to a concentration of about 100 mM.

[0070] In embodiments, the production stage includes adding an anti-coagulation supplement to the production stage medium. The anti-coagulation supplement may be added to the production stage culture medium at one or more time points (e.g., at one or more of about 6 hours, about 10 hours, about 12 hours, about 20 hours, about 24 hours, about 48 hours, or about 72 hours after transfection). In embodiments, the anti-coagulation supplement includes dextran sulfate, heparin, and / or other sulfated glycosaminoglycans that inhibit aggregation of producer cells. In embodiments, the anti-coagulation supplement includes heparin sodium, which may be added to the medium to a concentration of about 25 μg / ml to about 250 μg / ml, e.g., about 25 μg / ml, about 50 μg / ml, about 100 μg / ml, about 150 μg / ml, and / or about 200 μg / ml.

[0071] In embodiments, anti-aggregation supplements are not added to the production stage medium or are added to the production stage medium only shortly before the end of the production stage, e.g., within about 24 hours, within about 12 hours, within about 6 hours, within about 3 hours, within about 2 hours, or within about 1 hour of the end of the production stage.

[0072] Isolation and purification of rAAV Embodiments of the methods described herein include isolating and purifying rAAV particles (rAAV complete capsids) at the end of the production phase. In embodiments, rAAV particles may be isolated 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 introduction of the rAAV vector, and / or helper functions, and / or AAV rep / cap sequences (i.e., after the start of the production phase). For example, rAAV particles may be isolated about 90 to about 100 hours, about 92 to about 98 hours, or about 94 to about 98 hours after introduction of the rAAV vector, and / or helper functions, and / or AAV rep / cap sequences. In embodiments, rAAV particles are isolated (e.g., cells are lysed) approximately 96 hours after introduction of the rAAV vector, and / or helper functions and / or AAV rep / cap sequences. Isolating the rAAV may involve multiple steps, including, for example, lysing the producer cells to obtain a cell lysate, clarifying the lysate to obtain a clarified lysate, and subsequent purification steps.

[0073] rAAV particles may be retained within producer cells after production, and methods for releasing intracellular rAAV vectors include physical and chemical disruption, e.g., the use of detergents, microfluidization, and / or homogenization. In embodiments, cell membrane disruption (lysis) and release (recovery) of rAAV or AAV particles from cells is achieved using the zwitterionic detergent N,N-dimethyltetradecylamine N-oxide (TDAO) (commercially available as Deviron® C16 from MilliporeSigma, Burlington, MA). 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 Deviron® C16 can be used. In other embodiments, hyperosmotic shock (increased external molarity) is used to lyse cells and release rAAV or AAV particles. For example, to recover rAAV8 or AAV8 particles, producer cells can be subjected to a hyperosmotic shock at a NaCl concentration of about 400 mM (e.g., about 399, 400, 401 mM) for an incubation duration of about 90 to about 120 minutes (e.g., about 89, 90, 95, 100, 105, 115, 120, 121 minutes). Concurrently with and / or following cell lysis, a nuclease such as benzonase may be added to degrade contaminating DNA. Typically, the resulting lysate is clarified to remove cellular 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. [Example]

[0074] Example 1. Use of anion exchange chromatography in weak partitioning mode to provide high empty AAV capsid removal and product yield An anion exchange step was developed to maximize full capsid enrichment of AAV-AQP1 (AAV2). The similar properties of full and empty capsids posed a trade-off between product yield and full capsid ratio, making it difficult to select optimal conditions. Different detergent types and concentrations were screened to maximize AAV2 vector product yield. By exploring the design space, a weak partitioning mode was identified as an alternative to the bind-and-elute mode (Figures 1 and 2), and conditions were identified that achieved greater than 50% removal of empty capsids in the flow-through alone. In combination with a high NaCl wash, an eluate with greater than 80% full capsids was achieved. These results demonstrate that greater than 80% full capsid ratios, as confirmed by analytical ultracentrifugation (AUC), and greater than 50% product yields can be robustly achieved using the novel conditions described herein. Understanding the design space and identifying process failure points meant that the process was reproducible at manufacturing scale.

[0075] In the initial experimental design, parameters and ranges for further optimization were identified from the initial AEX platform adaptation experiments and are listed in Table 1.

[0076] [Table 1]

[0077] The relationship between the NaCl concentration (mM) in Wash 2 and equiv / feed and (i) empty capsid removal, (ii) the proportion of full capsids in the eluate, and (iii) VG recovery in the eluate was analyzed. The results are shown in Figures 3-5. Design of Experiments (DOE) results were combined to highlight the optimal region within the design space in line with the desired outcome (Figure 6). The parameters and values ​​from the optimal region based on the NaCl concentration in Wash 2 and equiv / feed are shown in Table 2:

[0078] [Table 2]

[0079] To understand how the loading ratio affects weak partitioning, we analyzed the relationship between the loading ratio and the breakthrough of full capsids (VG) and empty capsids (VP). The results (Figure 7) showed that the effect of weak partitioning was maximized by increasing the loading ratio. The parameters and values ​​are shown in Table 3:

[0080] [Table 3]

[0081] Referring to Figure 7, exploiting the window of opportunity allows up to 80% of the empty capsids to pass through, while only 10% of the full capsids are lost in the flow-through.

[0082] Collating the findings, the results shown in Figure 3 indicate that high NaCl concentrations are required in both wash 2 and elution to maximize empty capsid removal. Increasing NaCl in wash 2 resulted in up to 30% VG loss. 50% VG recovery was still recovered in the elution step. The parameters and optimized ranges are shown in Table 4:

[0083] [Table 4]

[0084] The chromatogram of the optimized AAV-AQP1 AEX step at 2L scale is shown in Figure 8; note the 80% full capsid ratio highlighted by the inversion (inversion of the UV absorbance trace at 260 nm and 280 nm, which typically indicates high full capsids due to the presence of encapsidated DNA). Parameters and values ​​are shown in Table 5:

[0085] [Table 5]

[0086] As shown in Figure 9, this process is scalable from 2 L to 80 L in terms of both VG recovery and full capsid ratio.

[0087] Example 2: AEX Weak Partitioning Protocol for Enrichment and Increased Yield of Intact Capsids The experiments described below demonstrate that the rAAV purification method described herein is consistent with respect to intact capsid ratio and VG recovery, and demonstrate that the process is scalable.

[0088] material AAV supply materials The feed material for the experiments described in this study consisted of serotype 2 adeno-associated virus (AAV) capsids packaged with a transgene encoding the aquaporin 1 gene.

[0089] These capsids were produced by cell culture using HEK293 cells transiently transfected with the triple-plasmid complex. After completion of the cell culture production phase, the cells were lysed using Triton X-100 to release the AAV, and the resulting lysate was treated with benzonase. The lysate was then clarified using a 0.2 μm PES filter.

[0090] Initial purification of AAV capsids was performed using AAVX (ThermoFisher) affinity chromatography resin and an AKTA Avant (Cytiva) chromatography system. This was accomplished by loading the clarified lysate onto a 5 cm bed height AAVX column at a load ratio of 200 mL of clarified lysate per mL of AAVX resin and a residence time of 1 minute. Captured AAV capsids were eluted using an elution buffer consisting of 50 mM glycine, 180 mM NaCl, and 0.25% (w / v) polysorbate 80 (pH 2.7).

[0091] chemicals All chemicals used in these experiments were GMP (Good Manufacturing Practice) grade reagents purchased from Merck KGaA.

[0092] method Anion exchange chromatography All small-scale anion exchange chromatography (AEX) experiments were performed using 1 mL Sartobind Q (Sartorius) AEX membranes and an AKTA Avant (Cytiva) chromatography system.

[0093] The buffer for these experiments consisted of 50 mM Tris, 0.75% (w / v) polysorbate 80, pH 9, and various concentrations of NaCl as required for the experimental steps. The feed for these experiments was AAVX eluent conditioned by dilution to the target NaCl concentration with 50 mM Tris, 0.75% (w / v) polysorbate 80, pH 9.0. Table 6 describes the method used for the AEX experiments in this study.

[0094] [Table 6]

[0095] Experimental design A DoE study was performed to evaluate the effect of NaCl concentration in the load, wash, and elution steps, as well as the load ratio, on the complete capsid ratio and vector genome (VG) recovery. The study consisted of 16 experiments, and the parameter values ​​tested in each experiment are listed in Table 7. Flow-through, wash 2, and eluate samples were collected from each run and analyzed for their vector genome and vector particle concentrations.

[0096] [Table 7]

[0097] Breakthrough Experiment To further evaluate the effect of loading ratio on the AEX process, a breakthrough experiment was performed. The AEX membrane was loaded with 1 × 10 ethanol at a feed conditioned to 90 mM NaCl. 15The flow-through was fractionated into 1 mL fractions and the resulting fractions were analyzed to determine their VG and VP concentrations.

[0098] Validation and scale-up experiments The optimized process was confirmed by performing the purification on a 10 mL Sartobind Q module. The method used for this run is detailed in Table 8. Samples of the flow-through, column wash 2, and eluate were analyzed for their VG and VP concentrations. Additionally, samples of the eluate were analyzed by analytical ultracentrifugation and qPCR to confirm the intact capsid ratio and VG recovery.

[0099] [Table 8]

[0100] To demonstrate scalability, the process was scaled up to 75 mL of Sartobind® Q membranes. This run was performed using the same method detailed in Table 8, and samples of the eluate were analyzed by analytical ultracentrifugation and qPCR to determine their intact capsid ratio and VG recovery.

[0101] Analysis method 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, and the vector particle concentration was determined using the Gyrolab® AAVX Titer kit (Gyrus Protein Technologies).

[0102] During DoE and breakthrough experiments, the empty capsid ratio was estimated by dividing the vector genome concentration by the vector particle concentration. In validation and scale-up experiments, the full capsid ratio was measured by analytical ultracentrifugation using an Optima AUC (Beckman).

[0103] result DoE The design space of the AEX process was explored by performing an experimental design aimed at identifying conditions that maximized intact capsid ratio and VG recovery. The variables investigated in this study were feed NaCl concentration, wash 2 NaCl concentration, elution NaCl concentration, and load ratio. The ranges tested for these variables are detailed in Table 9. The results of these experiments are detailed in Table 10, while the trends observed in this dataset are shown in Figures 3, 4, and 5.

[0104] [Table 9]

[0105] [Table 10-1]

[0106] [Table 10-2]

[0107] The feed for these experiments had an estimated full capsid fraction of less than 10%. Table 10 shows that the estimated total capsid fraction increased significantly (up to 87%) for some of the conditions tested. Figure 3 shows that higher equivalents / feed and wash 2 NaCl concentrations resulted in a reduction of empty capsids in the eluate pool. This is because higher NaCl concentrations in the feed prevent empty capsids from binding to the AEX membrane. Similarly, higher NaCl concentrations in column wash 2 elute empty capsids at this stage, removing them from the subsequent eluate pool.

[0108] Figure 4 shows how these effects translate into a higher full capsid ratio in the eluate pool. When empty capsids are prevented from binding or washed from the column, the resulting eluate pool has a high full capsid ratio. The effect of increasing the NaCl concentration in the feed and column wash 2 is shown in Figure 5. Despite the stronger interactions of full capsids with the AEX membrane, some of these full capsids are lost to the flow-through and wash water as the NaCl concentrations in the feed and wash 2 increase, disrupting these interactions. Figure 6 combines the trends seen in these experiments and highlights areas where high full capsid ratios and high VG recoveries can be achieved.

[0109] Breakthrough Experiment In the DoE study described above, it was observed that empty capsids passed through the Sartobind® Q AEX membrane without binding to it at high feed NaCl concentrations. This effect was further investigated by conducting breakthrough experiments. This experiment was performed at a feed NaCl concentration of 95 mM to maximize this effect, and the membrane was loaded with 1 x 10 capsids to ensure capsids broke through to the flow-through. 15 Loaded VG / mL.

[0110] Flow-through samples were analyzed to determine their VG and VP concentrations. The results are shown in Figure 10. These results indicate that under these feeding conditions, vector particles begin to break through almost immediately after the start of the experiment. However, vector genomes were still present at approximately 2.5 x 10 14 Since no breakthrough occurs up to a load ratio of 1000 VG / mL, it can be concluded that the vector particles that do breakthrough are empty capsids.

[0111] These results also indicate that the vector genome undergoes gradual breakthrough. Typically, a sharp breakthrough profile is expected, indicating that the chromatography matrix has reached saturation. However, this gradual breakthrough indicates that weak partitioning may be occurring. Under weak partitioning, full capsids begin to displace empty capsids bound to the chromatography membrane due to the stronger charge interaction that full capsids have with the membrane. This has the advantage of further increasing the full capsid ratio of the product ultimately eluted from the membrane.

[0112] Confirmation run and scale-up The identified process conditions were confirmed by a larger-scale run using 10 mL of Sartobind® Q; the process conditions are detailed in Table 8. An excerpt of the chromatogram from this run, focusing on the column wash 2 and elution steps, is shown in Figure 11. This figure shows an inversion of the UV absorbance trace at 260 nm and 280 nm, which typically indicates a high full capsid ratio due to the presence of encapsidated DNA. The eluate from this run was analyzed by analytical ultracentrifugation to confirm the full capsid ratio. The full capsid ratio was found to increase from 31% in the feed to 81.5% in the eluate. The corresponding AUC trace of the eluate is shown in Figure 12.

[0113] To confirm the scalability of the process, an additional run was performed on 75 mL of Sartobind® Q. The results of the 10 mL and 75 mL runs are compared in Figure 9. This figure shows that the process is consistent in terms of intact capsid ratio and VG recovery, indicating that the process is scalable.

[0114] Example 3. Identification of parameters and ranges from initial AEX platform and additional experiments A platform adaptation experiment was performed. Figure 13 shows the chromatogram of the initial AEX chromatography run-through. The conditions used for the AEX step were as follows:

[0115] [Table 11]

[0116] The parameters and ranges shown in Table 11 below were identified from initial AEX platform adaptation experiments (Figure 13). These initial experiments are referred to as "Initial Process" in Figure 19, which shows the VG recovery and % intact capsid values ​​after USP, capture chromatography, ion exchange chromatography (IEX), and tangential flow filtration (TFF), respectively, in the initial process. These values ​​are compared to those obtained from the inventive optimization process described herein (see "Final Process" in Figure 19).

[0117] [Table 12]

[0118] See Figures 3-5 for the following relationships between parameters and % recovery of empty capsids, full capsids, and VG in the eluent: Figure 3: NaCl concentration (mM) in Wash 2 and Eq / Feed vs. empty capsids (VP / mL) present in the eluate Figure 4: NaCl concentration (mM) in Wash 2 and Eq / Feed vs. intact capsid percentage in the eluate Figure 5: Relationship between NaCl concentration (mM) in Wash 2 and Eq / Feed and VG recovery (%) in the eluent

[0119] Figure 6: Optimal regions based on NaCl concentration (mM) for wash 2 and equivalents / feed for Figures 1-3. Figures 1 and 2 illustrate the weak partitioning process, where empty capsids are replaced by full capsids depending on the environment in which they are formed. The charges of empty and full AAV capsids above and below pI are shown. (Cytiva Life Sciences Marlborough, Massachusetts 2022. Enhanced AAV downstream processing).

[0120] Additional breakthrough experiments were performed to identify whether weak partitioning is utilized at different rates at different feed and equilibrium conductivities as the load is increased.

[0121] Methods and Materials Run 1: Feed material and equilibration buffer at conductivity 90 mM NaCl. Run 2: Feed material and equilibration buffer at conductivity 80 mM NaCl.

[0122] A 0.08 mL Sartobind Q was loaded with AAV-AQP1 neutralized AAVX eluate to 4E14VG / mL, and the flow-through sample was fractionated and analyzed by qPCR for VG titer and Gyrolab for VP titer.

[0123] [Table 13]

[0124] [Table 14]

[0125] [Table 15]

[0126] Figure 14 shows that when calculating breakthrough curves for both empty and full capsids, a target conductivity of 9 mS / cm appears to favor binding of full capsids over empty capsids. To examine what is happening on the column, we examined the VG concentration on the column as the VG load was increased to 4E14 vg / ml (Figure 15, top graph). We also examined the empty capsid ratio on the column as the VG load was increased to 4E14 VG / mL (Figure 15, bottom graph). To determine the impact on the full capsid ratio on the column, we analyzed the full capsid ratio on the column as the VG load was increased. As shown in Figure 16, an increase from 38% to >55% was observed before any form of wash or elution step. Similar to what was shown in Figure 14, Figure 17 clearly shows that a target of 9 mS / cm allows for higher levels of concentration than a target of 8 mS / cm, suggesting that the feed and equilibrium feed conductivities are important when attempting to maximize concentration using weak partitioning.

[0127] See Figure 18 for a chromatogram of the optimized AAV-AQP1 AEX step from the 20 L scale validation run. As shown in this figure, an 80% full capsid ratio was obtained. The parameters and values ​​from this validation run are shown in Table 15 below. Referring again to Figure 19, the difference between the "initial process" and "final process" values ​​is shown.

[0128] [Table 16]

[0129] For scale-up to 20 L and 80 L batches, a comparison of the intact capsid ratio and VG recovery for the 20 L and 80 L batches is shown in Figure 20. This figure also includes AUC results confirming that the elution peak in Figure 18 contains 80% intact capsids.

Claims

1. 1. A method for concentrating recombinant adeno-associated virus (rAAV) complete capsids in a mixture of complete and empty capsids, the method comprising: (i) providing a solution containing rAAV full capsids and empty capsids; (ii) equilibrating an anion exchange (AEX) column or membrane; (iii) subjecting the solution containing the rAAV complete capsids and empty capsids to weak partitioning mode AEX chromatography to separate the empty capsids from the complete capsids, thereby obtaining an AEX eluate enriched in complete capsids.

2. 2. The method of claim 1, wherein the solution is an affinity chromatography eluent, and step (iii) comprises applying the affinity chromatography eluent to the equilibrated AEX column or membrane, washing the AEX column or membrane at least once, and eluting rAAV complete capsids from the AEX column or membrane, wherein the weak partitioning mode causes complete capsids to displace bound empty capsids from the AEX column or membrane, the empty capsids flowing through the AEX column or membrane to generate an AEX flow-through, and the complete capsids remaining bound to the AEX column or membrane until elution.

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

4. 4. The method of claim 3, wherein the target salt concentration is in the range of about 85 mM to about 95 mM.

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

6. 2. The method of claim 1, wherein the AEX column or membrane is equilibrated with an 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 prior to loading with a solution containing the rAAV full and empty capsids, wherein the equilibration buffer has a pH of about 9.

0.

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

0.

8. 3. The method of claim 2, wherein washing the AEX column or membrane at least once comprises a 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 first wash buffer is at about pH 9.

0.

9. 9. The method of 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, and wherein the first wash buffer has a pH of about 9.

0.

10. 3. The method of claim 2, wherein washing the AEX column or membrane at least once further comprises a second wash with a 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 second wash buffer has a pH of about 9.

0.

11. 11. The method of 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 second wash buffer has a pH of about 9.

0.

12. 3. The method of claim 2, wherein the intact capsids are eluted from the AEX column or membrane with an elution buffer comprising: (i) about 50 mM tris; (ii) about 125 mM to 250 mM NaCl; and (iii) greater than about 0.5% (w / v) polysorbate 80, wherein the elution buffer has a pH of about 9.

0.

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

0.

14. 10. The method of claim 1, wherein the AEX chromatography column or membrane comprises a high flux adsorptive membrane.

15. 15. The method of claim 14, wherein the high flux adsorptive membrane comprises a Sartobind® Q chromatography membrane.

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

17. 2. The method of claim 1, wherein the enriched complete capsids comprise AAV serotype 2 capsid proteins and a polynucleotide sequence comprising a transgene.

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

19. 2. The method of claim 1, wherein the enriched complete capsids comprise greater than 80% complete capsids.

20. 20. The method of claim 19, wherein the enriched complete capsids comprise about 81.5% complete capsids.

21. 20. The method of claim 19, wherein the enriched whole capsids comprise greater than 90% whole capsids.

22. 2. The method of claim 1, wherein the enriched complete capsids comprise greater than 95% complete capsids.

23. 10. The method of claim 1, further comprising subjecting the AEX eluate containing enriched whole capsids to tangential flow filtration to obtain a purified preparation of whole capsids.

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

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

26. 26. The method of claim 25, wherein the cells are cultured in suspension.

27. 27. The method of claim 26, wherein the cells are cultured in a shaker flask, a spinner flask, a cell bag, or a bioreactor.

28. A population of complete capsids enriched by the method of any one of claims 1 to 27.

29. 30. A pharmaceutical composition comprising a population of complete capsids according to claim 28.

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