Bonded phases for use in affinity chromatography

Abstract

A bonded phase for use in affinity chromatography to recover adeno-associated virus (AAV) vectors (3) from solution, the bonded phase comprising a porous base matrix (1) having a mean flow pore size of 0.1 to 2.0 μm, functionalized by binding to the matrix an AAV vector ligand (2), the ligand (2) having binding affinity for the AAV vector (3), and a density of the ligand (2) in the polymer matrix (1) of 0.1 μmol / gram to 10 mmol / gram.

Description

[Technical Field]

[0001] The present disclosure relates to bonded phases for use in affinity chromatography for recovering adeno-associated viral (AAV) vectors from solution, chromatographs containing such bonded phases, and methods for recovering adeno-associated viral (AAV) vectors from solution. [Background technology]

[0002] In gene therapy methods, delivery of a gene of interest into cells requires the use of vectors that can be derived from recombinant viruses such as adenoviruses (AdV), adeno-associated viruses (AAV), and lentiviruses (LV). The vectors deliver nucleic acid sequences (DNA or RNA) to cells, where they can be processed by the cell's biochemical machinery to alter the properties of the cell and produce the desired therapeutic effect.

[0003] Vector material is typically produced from cell lines that have been engineered to produce the vector's components, such as its coat or capsid, and the nucleic acid material that is intended to be delivered to a cell.

[0004] After host cell lysis and clarification, the "raw material" contains the vector, cell host debris, proteins, genomic DNA, serum proteins, some components of the culture medium, helper DNA, helper virus, etc. These impurities can harm cells, reduce transduction efficiency, and even induce systemic immune or inflammatory responses. The purity, potency, and safety of clinical-grade vectors are crucial. The vector must also maintain as much intact viral activity as possible throughout the purification process. A wide variety of purification strategies, including chromatographic separation methods, have been developed.

[0005] In porous bead-based chromatography systems, the binding event between the target entity and the solid phase / ligand immobilized on the solid phase relies on diffusion into the porous beads, meaning that the binding capacity decreases as the residence time decreases. At production scale, where columns are packed with several liters of bead suspension, high flow rates are also particularly unsuitable for porous beads.

[0006] Typical binding capacities of porous beads (using bovine serum albumin / monoclonal antibody, BSA / mAb) range from 35 to 120 mg / mL, depending on the functionality of the solid phase and the species being bound. However, the typical low flow rates through such systems mean that overall productivity can only be achieved in the order of 10 to 120 mg / mL / min with a single-column porous bead system.

[0007] A drawback of using porous beads is that the capacity of the material depends on the target's accessibility to the inner surface area of ​​the beads. Typical porous bead pore sizes are 15-30 nm, limiting the purification of vectors where the target vector may be much larger than the pore size.

[0008] Separations involving membranes and monoliths can be performed at much higher flow rates than porous bead-based systems, with typical residence times of around 0.2–0.5 min. However, the typical binding capacities at 10% breakthrough of the target (mAb) for monoliths (10–20 mg / mL) and membranes (7.5–29 mg / mL) in dynamic flow are lower than those for porous beads. The inferior binding capacities of monolith and membrane materials (compared to porous bead-based materials) can be offset to some extent by utilizing higher flow rates.

[0009] Furthermore, although the pore size of membranes is much larger than that of porous beads, the binding capacity of membranes decreases as the size of the adsorbed species increases, highlighting the need for both high porosity and high accessible surface area. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Patent Publication No. 2015 / 052460 Brochure [Patent Document 2] International Patent Publication No. 2015 / 052465 Brochure [Patent Document 3] International Patent Publication No. 2018 / 011599 Brochure [Non-patent literature]

[0011] [Non-Patent Document 1] O. Hardick et al., J.Mater. Sci. 46 (2011) 3890 Summary of the Invention [Problem to be solved by the invention]

[0012] There is a need for chromatographic materials that can efficiently purify these viral vectors, such as AAV vectors, and recover the therapeutic product on an industrial scale.

[0013] It is an object of the present disclosure to provide a bonded phase for use in affinity chromatography for recovering adeno-associated virus (AAV) vectors from solution, which can be used to recover AAV vectors on an industrial scale with a sufficiently short residence time. [Means for solving the problem]

[0014] According to a first aspect, there is provided a bonded phase for use in affinity chromatography for recovering adeno-associated virus (AAV) vectors from a solution, the bonded phase comprising a porous base matrix having a mean flow pore size of 0.1 to 2.0 μm, the base matrix being functionalized by binding of an AAV vector ligand to the matrix, the ligand having binding affinity for the AAV vector, and the density of the ligand in the matrix being in the range of 0.1 μmol / gram to 10 mmol / gram.

[0015] The functionalized matrix is ​​suitable for use as a binding phase in affinity capture chromatography. During operation, the binding phase, which includes the functionalized matrix, is contacted with a mobile phase solution containing AAV vectors, and the AAV vectors are retained on the matrix by the AAV vector ligand in preference to other components also present in the solution. Such other components in the mobile phase may include impurities such as cell host debris, proteins, genomic DNA, serum proteins, some components of the culture medium, helper DNA, or helper viruses.

[0016] The bonded phase may be assembled into a capsule or cartridge that allows for uniform flow distribution throughout the bonded phase.

[0017] The above-mentioned binding phase is a convection-type binding phase, and the matrix may be a convection-based matrix, including any matrix in which a hydraulic pressure difference between the inlet and outlet of the matrix is ​​applied to perfuse the matrix, thereby achieving substantially convective transport of substances into or out of the matrix. A convection-based matrix may be, for example, an adsorption membrane in which the flow through such a material is convective rather than diffusive. The binding phase of the present invention has a large surface area for high binding capacity and the macroporosity necessary for viruses to enter the matrix. When a mobile phase is added to the convection-type binding phase, convection of the mobile phase occurs within the binding phase, thereby directly contacting the AAV vector ligand within the matrix. Therefore, the AAV vector in the mobile phase does not need to rely on diffusion to reach the AAV vector ligand.

[0018] Thus, the present invention enables chromatographic materials for the purification of AAV vectors that combine the high binding capacities traditionally associated with porous bead-based materials with the higher flow rates achievable with monolith / membrane materials. The chromatographic materials can be made sufficiently porous to allow binding regions accessible to large vectors, and suitable short residence times can be achieved.

[0019] The diameters of various viruses range from 20 to 300 nm, with AAV vectors typically being around 25 nm in diameter.

[0020] The base matrix may be a non-woven polymer matrix, which may be formed from polymer fibers, such as polymer nanofibers.

[0021] Mean flow pore size (MFP) is a measure of a material's flow properties and is measured by capillary flow porometry, where a wetting liquid with a known surface tension is expelled through the pores of a sample by applying a gas at increased pressure. The larger the MFP size, the greater the flow of liquid through the material at a given pressure. The mean flow pore size is calculated from the point where 50% of the flow passes through the sample. Therefore, the mean flow pore size corresponds to the pore size calculated at the pressure where the wetting curve and the semi-dry curve intersect.

[0022] In another definition, the mean flow pore size of the bonded phase of the present invention may be known as the effective pore size, which is defined as the size of the largest sphere that can pass through the pore.

[0023] The mean flow pore size of the base matrix may be 0.1 to 1.8 μm, 0.1 to 1.6 μm, 0.1 to 1.4 μm, 0.1 to 1.2 μm, 0.1 to 1.0 μm, 0.1 to 0.8 μm, 0.1 to 0.6 μm, 0.1 to 0.4 μm, 0.1 to 0.2 μm, 0.2 to 2.0 μm, 0.4 to 2.0 μm, 0.6 to 2.0 μm, 0.8 to 2.0 μm, 1.0 to 2.0 μm, 1.2 to 2.0 μm, 1.4 to 2.0 μm, 1.6 to 2.0 μm, 1.8 to 2.0 μm, or 0.5 to 1.5 μm.

[0024] Advantageously, when the mobile phase (i.e., a solution containing the AAV vector) is added to the bonded phase, the binding rate of the AAV vector to the AAV vector ligand depends solely on the binding kinetics. The matrix of the bonded phase of the present invention has an open pore structure, and mass transfer is controlled by convection. Therefore, the use of the bonded phase of the present invention in affinity chromatography results in shorter residence times than when using conventional resin-based bonded phases. This results in cycle times of minutes, rather than the several hours required for resin-based chromatography. Using the bonded phase of the present invention, residence times can be reduced by more than half compared to when using resin-based bonded phases. Residence times as short as one second have been observed with the bonded phase of the present invention. This reduces lead times by several weeks in method development. The exact residence time may depend on the AAV serotype and mobile phase used.

[0025] The nanofiber polymer matrix may be selected from the following hydrophilic polymers: cellulose, polyethersulfone (PES), polystyrene, methyl acrylate, dextran, and agarose.

[0026] As used herein, the phrase "AAV vector ligand" or "AAV vector-binding ligand" refers to any molecule that has suitable binding affinity for vectors based on one or more adeno-associated virus (AAV)-based vectors and can bind to a chromatographic material, such as the bonded phase, of the present invention. The AAV vector ligand may be a peptide or polypeptide, including an antibody or antibody fragment, an oligonucleotide, such as DNA or RNA, an aptamer, or the like. The AAV vector ligand may also be a camelid antibody or antibody fragment.

[0027] AAV vector-binding ligands are known in the art. For example, POROS CaptureSelect AAVX resin (ThermoFischer Scientific) has shown binding reactivity to a range of AAV serotypes, including AAV1-AAV8 and AAVrh10. Other examples of resins incorporating affinity ligands include AVIPure® AAV2 affinity resin, AVIPure® AAV8 affinity resin, and AVIPure® AAV9 affinity resin (Avitide / Repligen). As another example, Capto AVB and AVB Sepharose High Performance (Cytiva) are affinity resins with significant affinity for adeno-associated viruses of subclasses 1, 2, 3, and 5. The AVB ligand is a 14 kD fragment of a single-chain camelid antibody.

[0028] The AAV vector ligand may optionally be a polypeptide that is recombinantly produced in a eukaryotic cell, for example, a yeast cell such as Saccharomyces cerevisiae.

[0029] The density of the ligand in the functionalized polymer matrix can be 0.1 μmol / gram to 1 mmol / gram, 0.1 to 100 μmol / gram, 0.1 to 10 μmol / gram, 0.2 to 10 μmol / gram, 0.5 to 5 μmol / gram, 0.1 to 1 μmol / gram, 0.5 to 2 μmol / gram, 1 μmol / gram to 10 mmol / gram, 10 μmol / gram to 10 mmol / gram, 100 μmol / gram to 10 mmol / gram, or 1 to 10 mmol / gram, where density refers to the concentration of ligand per gram (dry mass) of matrix.

[0030] The AAV vector ligand may be attached to the binding phase by means further described below. For example, the AAV vector ligand may be attached to the matrix by an amine bond. Alternatively, the AAV vector ligand may be attached by a thiol bond.

[0031] The AAV vector ligand may have binding affinity for one or more of the AAV vector serotypes AAV1 to AAV13, for example, at least one of AAV1, AAV2, AAV3, AAV5, AAV6, and AAV10, and engineered variants of any of these.

[0032] According to a second aspect, there is provided an affinity chromatography device comprising a bonded phase as described above.

[0033] According to a third aspect, there is provided a method for recovering an adeno-associated virus (AAV) vector from a solution, comprising providing a solution containing the AAV vector and one or more impurities, contacting the solution with a binding phase as described above (optionally by adding the solution to the binding phase), and eluting the AAV vector from the binding phase by contacting the binding phase with an elution buffer.

[0034] By "impurities" herein is meant that the solution may contain, in addition to the AAV vector, cellular host debris, proteins, genomic DNA, serum proteins, some components of the culture medium, helper DNA, helper viruses, etc. Such a solution may be the harvest from the culture of a cell line engineered to produce the AAV vector.

[0035] As will be appreciated by those skilled in the art, since the goal is to bind the AAV vector to the AAV vector ligand, the step of contacting the binding phase with the solution is carried out under conditions that allow for said binding.

[0036] The solution may be added to the binding phase directly from the harvest, or there may be an optional filtration step of the harvest before adding the solution to the binding phase. The eluted solution contains the AAV vectors that are collected.

[0037] Advantageously, the contact time between the binding phase and the solution containing the AAV vector during loading, also referred to as the residence time, can also be very short, for example, the binding phase can be in contact with the solution for less than 2 minutes, for example, from 1 to 120 seconds, or from 1 to 60 seconds.

[0038] The amount of solution, such as the clarified sample volume, added to the bonded phase can be up to 13 liters per mL of sorbent volume of the bonded phase, and can be a packed bonded phase. Packed bonded phase, as used herein, means that the bonded phase is under some level of compression. The sorbent volume is the volume of the porous base matrix.

[0039] It is to be noted that the present invention relates to all possible combinations of the features recited in the claims.

[0040] These and other aspects of the invention will be described in more detail with reference to the accompanying drawings, in which embodiments of the invention are shown. [Brief explanation of the drawings]

[0041] [Figure 1] FIG. 1 shows a nonwoven polymer matrix containing nanofibers. [Figure 2] FIG. 2 shows an affinity chromatography device including a bonded phase comprising the polymer matrix of FIG. 1. [Figure 3] FIG. 1 is a schematic diagram showing a method for recovering adeno-associated virus (AAV) vectors from a solution. [Figure 4a] FIG. 1 shows an exemplary reaction scheme for preparing a matrix material for immobilization of AAV vector ligands. [Figure 4b]FIG. 1 shows an example of a linker chemistry that can be used to immobilize AAV vector ligands on the surface of cellulose acetate fibers. [Figure 5] FIG. 3 shows a graph of the dynamic binding capacity for two different AAV vectors on the chromatography device of FIG. 2 at various residence times. DETAILED DESCRIPTION OF THE INVENTION

[0042] As illustrated in the figures, some features may be exaggerated for illustrative purposes and, therefore, are provided to illustrate the general structure of embodiments of the present invention. Like reference numerals refer to like elements throughout.

[0043] The following describes a bonded phase for use in affinity chromatography to recover AAV vectors from solution. The bonded phase comprises a nonwoven polymer matrix 1 containing nanofibers (see Figure 1), with a mean flow pore size of 0.1-2.0 μm. Such a pore size is useful for allowing viral particles (approximately 20-200 nm in diameter for commonly used viral vectors) to enter the matrix 1.

[0044] The nanofiber-containing nonwoven polymer matrix 1 is a mat of one or more polymer nanofibers, each fiber being essentially randomly oriented; i.e., the fiber or fibers are not manufactured to adopt a specific pattern. The nonwoven polymer matrix 1 is typically provided by known methods. In certain circumstances, the nonwoven matrix 1 is composed of a single polymer nanofiber. Alternatively, the nonwoven matrix 1 may contain two or more types of polymer nanofibers.

[0045] The polymer nanofibers may be electrospun polymer nanofibers. Such electrospun polymer nanofibers are well known to those skilled in the art. Other methods for producing polymer nanofibers, such as drawing, may also be used.

[0046] The average diameter of polymer nanofibers is typically 10 nm to 1000 nm. For some applications, polymer nanofibers with an average diameter of 200 nm to 800 nm or 200 nm to 400 nm may be appropriate.

[0047] The length of the polymer nanofibers is not particularly limited. Thus, conventional methods, such as electrospinning, can produce polymer nanofibers hundreds of meters or even kilometers long. However, typically, the length of one or more polymer nanofibers is up to 10 km, preferably 10 m to 10 km.

[0048] The surface area of ​​the nonwoven matrix 1 is typically between 1 and 40 g / m2, between 5 and 25 g / m2, between 1 and 20 or between 5 and 15 g / m2.

[0049] The thickness of the nonwoven matrix 1 is typically between 5 and 120 μm.

[0050] Suitable polymers include polyamides such as nylon, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, polystyrene, polysulfones, e.g., polyethersulfone (PES), polycaprolactone, collagen, chitosan, polyethylene oxide, agarose, agarose acetate, cellulose, cellulose acetate, dextran, and combinations thereof. Polyethylene sulfone (PES), cellulose, and cellulose acetate are preferred. In some cases, cellulose and cellulose acetate are preferred. Cellulose acetate can be readily formed into nanofibers, for example, by electrospinning and can be readily converted to cellulose after electrospinning. In some embodiments, the matrix comprises one or more nanofibers formed from various polymers. Exemplary polymers are as defined above.

[0051] Polymer matrix 1 is functionalized by attaching AAV vector ligand 2 to matrix 1, where AAV vector ligand 2 has binding affinity for AAV vector 3, see Figure 2. The density of ligand 2 in polymer matrix 1 is 0.1 μmol / gram to 10 mmol / gram.

[0052] This functionalization makes the matrix 1 containing the AAV vector ligand 2 suitable as a binding phase for use in affinity chromatography to recover AAV vectors 3 from solution, see Figure 2.

[0053] Prior to functionalization with ligands, the nanofibers may optionally be physically modified and fused together at the points where the nanofibers cross each other by thermal or chemical methods and / or by applying pressure to the nonwoven polymer matrix. This can improve the structural stability of the matrix. Pressurization and heating conditions can also be varied to change the thickness and / or porosity of the resulting matrix.

[0054] Using multiple nonwoven matrices / sheets allows for the preparation of thicker materials, potentially resulting in even higher adsorption capacities. Thus, functionalized polymer matrices are typically formed by providing two or more nonwoven matrices stacked on top of one another, each matrix containing one or more polymer nanofibers, and simultaneously heating and pressurizing the stack of matrices to fuse the contact points between the nanofibers of adjacent matrices / sheets.

[0055] In the case of a cellulose matrix, this is typically formed by providing two or more such nonwoven matrices stacked one on top of the other, each matrix containing one or more cellulose acetate nanofibers, and simultaneously heating and pressurizing the stack of sheets to fuse the contact points between the nanofibers of adjacent matrices / sheets. The polymer matrix may be composed solely of cellulose. Alternatively, the matrix may include cellulose in combination with one or more polymer nanofibers.

[0056] Preferred processing conditions for pressing and heating the polymer nanofiber / nonwoven sheet can be found, for example, in WO 2015 / 052460 and WO 2015 / 052465.

[0057] The nanofibers contain one or more functional groups. Different functional groups may be present in different polymer nanofibers. Typical functional groups include hydroxyl, amino, and carboxyl groups. Typically, the nanofibers are treated to introduce one or more functional groups, the nanofibers are treated to deprotect or activate any functional groups, or the nanofibers are treated to increase the number / density of functional groups.

[0058] For example, when the matrix comprises cellulose, cellulose acetate nanofibers are typically provided and treated to convert the cellulose acetate to cellulose before the AAV vector ligand is attached to them. This involves deprotecting acetylated hydroxyl groups to yield hydroxyl groups. Conversion of cellulose acetate to cellulose is typically accomplished using aqueous alkali, preferably NaOH in water:ethanol.

[0059] Derivatized cellulose, ie, cellulose acetate, may be used to enhance the solubility and / or other properties of the polymer, making it more suitable for electrospinning.

[0060] Methods for increasing the number / density of functional groups on a substrate are known to those skilled in the art.

[0061] The polymer used to form the nanofibers may be functionalized prior to the step of forming the nanofibers, or alternatively, and preferably, the nanofibers are functionalized after the polymer has been formed into the nanofibers.

[0062] The ligands can typically be introduced by contacting one or more nanofibers, optionally under pressure and / or heat, and optionally having one or more covalently attached polymer chains, with a reagent that functionalizes the product as a chromatographic medium.

[0063] Generally, functionalization of media / nanofibers alters their chemical and / or physical properties, which in turn affects how the functionalized chromatographic media behaves when used in chromatographic methods. The modification can, for example, change the polarity, hydrophobicity, or biological binding properties of the functionalized chromatographic media compared to its unfunctionalized form. The modification can, in certain circumstances, change more than one of the polarity, hydrophobicity, or biological binding properties of the functionalized chromatographic media compared to its unfunctionalized form. In one embodiment, the modification changes the polarity and hydrophobicity of the functionalized chromatographic media compared to its unfunctionalized form.

[0064] Typically, the chromatographic media is functionalized with diethylethanolamine (DEAE), quaternary amine (Q), sulfopropyl (SP), carboxymethyl (CM), phenyl, or mercaptoethylpyridine (MEP) groups. These ligands may be attached to the polymer nanofibers and / or to the polymer chains if they are covalently attached to the nanofibers.

[0065] As mentioned above, ligands can be attached to nanofibers / polymer chains by treatment with an appropriately selected reagent: 2-chloro-N,N-diethylamine hydrochloride (DEACH), glycidyl trimethylammonium, 1,4-butanesulfone, sodium chloroacetate, TEMPO followed by sodium perchlorate, or allyl glycidyl ether followed by sodium disulfite, styrene oxide are examples of reagents that can be used.

[0066] Ligand groups are typically introduced into functionalized chromatographic media by reacting an appropriate reagent with one or more functional groups contained on the polymer nanofiber and / or polymer chain. Typical functional groups include hydroxyl, amino, halogen, and carboxyl groups.

[0067] One or more functional groups may be activated prior to reaction with a reagent. Conventional activation methods known in the art may be used. Thus, if the functional group is a hydroxyl group, such a group may be activated by treatment with carbonyldiimidazole (CDI), bisoxirane, cyanuric acid, N-hydroxysuccinimide ester (NHS), 2-fluoro-1-methylpyridinium toluene-4 sulfonate (FMP), NaIO4, or divinyl sulfone. If the functional group is an amino group, such a group may be activated by treatment with epichlorohydrin, glutaraldehyde, or an epoxide. If the functional group is a carboxyl group, such a group may be activated by treatment with CDI or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). If the functional group is a halogen atom, such a group may be activated by treatment with divinyl sulfone.

[0068] One of skill in the art can select appropriate reagents to introduce particular groups and moieties into particular nanofiber / polymer chains based, for example, on the desired ligand groups and moieties and the functional groups contained in the desired nanofiber / polymer chain.

[0069] The AAV vector Ligand 2 may be attached to the matrix via an amine bond. When the functional group is an amino group, such a group may be activated by treatment with, for example, epichlorohydrin, glutaraldehyde, or an epoxide. The matrix may be functionalized before or after the polymer is formed into nanofibers.

[0070] The AAV vector ligand 2 may be a camelid antibody or antibody fragment known in the art. Such a camelid antibody / antibody fragment is an AAV ligand having affinity for binding to an AAV vector. Such an AAV vector ligand has binding affinity for one or more AAV vector serotypes, such as AAV1, AAV2, AAV3, and AAV5, and to a lesser extent AAV6, AAV8, and AAVrhlO.

[0071] A total of 11 naturally occurring AAV serotypes, AAV1-11, have been isolated from animal tissues. Each serotype has different tissue tropism, and many other synthetic hybrid, modified, or chimeric serotypes have been engineered to enhance or integrate desirable traits for therapeutic efficacy. The most commonly used serotypes are currently rAAV2 and rAAV5, although this may change over time, depending in part on the clinical target. These recombinant AAV vectors (rAAV) are used in all clinical trials and differ from wild-type serotypes in that two wild-type viral genes (rep and cap) have been deleted, rendering viral replication ineffective. rAAV also incorporates a transgene expression cassette inserted between two inverted terminal repeats (ITRs) to enable expression of a therapeutic gene of interest.

[0072] When a bonded phase comprising a functionalized matrix 1 is used in affinity capture chromatography, the mobile phase, i.e., a solution containing the AAV vector 3, is passed through the bonded phase comprising the functionalized matrix 1, and the AAV vector 3 is retained on the matrix 1 by the AAV vector ligand 2 in preference to other components 4 also present in the solution. Such other components 4 in the mobile phase may include impurities such as cell host debris, proteins, genomic DNA, serum proteins, some components of the culture medium, helper DNA, or helper viruses.

[0073] The bonded phase may be used in a chromatographic system or manually with a syringe. All devices may be used with peristaltic pumps, diaphragm pumps, or positive pressure gas.

[0074] The density of ligands in the matrix may be determined by titration, which determines the number of ligand moieties in the functionalized material. Those skilled in the art will be aware of appropriate methods to use to determine the amount of a particular moiety present in a given sample of functionalized material. The dynamic binding capacity (DBC) of the target entity of the functionalized matrix is ​​typically 10-210 mg / mL (10% breakthrough), preferably 20-195 mg / mL (10% breakthrough), 30-180 mg / mL (10% breakthrough), 40-165 mg / mL (10% breakthrough), or 50-150 mg / mL (10% breakthrough). In certain embodiments, the DBC may be up to 50 mg / mL (10% breakthrough), e.g., 10-50 mg / mL (10% breakthrough). The DBC at 10% breakthrough may be determined according to standard procedures, for example, using an AKTA Pure liquid chromatography system.

[0075] The aforementioned bound phases have a large surface area due to their high binding capacity and macroporosity, which is necessary for viruses to enter the matrix. The diameters of various viruses range from 20 to 300 nm. AAV vectors are typically about 25 nm in diameter.

[0076] Due to the mean flow pore size of 0.1 to 2.0 μm and the density of ligand 2 in polymer matrix 1 of 0.1 μmol / gram to 10 mmol / gram, when a mobile phase, i.e., a solution containing AAV vector 3, is added to the bonded phase, the binding rate of the AAV vector to the AAV vector ligand is determined solely by the binding kinetics. Matrix 1 of the bonded phase of the present invention has an open pore structure, and mass transfer therein is controlled by convection. Therefore, the bonded phase of the present invention can be used in affinity chromatography at shorter residence times than when conventional resin-based bonded phases are used.

[0077] Examples of residence times that can be used in the present invention may be less than 2 minutes, for example, 1 to 120 seconds, or 1 to 60 seconds, for example, 1 to 10 seconds, 1 to 5 seconds, 1 to 45 seconds, or 30 to 45 seconds.

[0078] The residence time can be tailored to the specific AAV serotype and the associated binding affinity of the AAV vector ligand. It is believed that residence times relevant to the present invention (e.g., residence times of about 1 minute or less) may take into account the binding kinetics between the AAV serotype and the AAV vector ligand in question, with the aim of streamlining the method. In contrast, for affinity interactions on diffusion-type chromatography media, such as conventional resins, binding kinetics is typically not considered. In the case of resins, the rate-limiting step is the time required for diffusion through the resin pores. Convective chromatography, as used in the present invention, has the potential to eliminate this bottleneck and significantly shorten residence times, thus potentially limiting how short residence times can be achieved by binding kinetics.

[0079] For example, in methods according to embodiments of the invention for recovering AAV5 vectors, dwell times can be less than 30 seconds, or less than 10 seconds, e.g., 1 to 5 seconds. In other embodiments, methods for recovering AAV2 vectors can use dwell times of up to 60 seconds, e.g., 30 to 60 seconds, or about 30 seconds.

[0080] 3 shows a schematic diagram of a method for recovering an AAV vector 3 from a solution. The method includes step 100 of obtaining a solution containing an AAV vector 3 and one or more impurities 4, step 200 of adding the solution to the binding phase described above, and step 300 of eluting the AAV vector 3 from the binding phase by contacting the binding phase with an elution buffer.

[0081] The solution may be added to the binding phase directly from the harvest, or there may be an optional filtration step of the harvest (e.g., tangential flow filtration (TFF)) before adding the solution to the binding phase.

[0082] Between step 200 of adding the solution to the binding phase and step 300 of eluting, the method may include a step of washing the binding phase to which the viral product and / or product-related impurities and / or non-product-related impurities have adsorbed. This washing step is performed to remove any components that are not bound to the AVV vector ligand 2. This may be performed according to conventional methods known for the washing phase of such methods. This washing step typically involves washing with a liquid phase having a low ionic concentration.

[0083] The elution solution contains the AAV vectors 3 to be collected. Typically, the method for recovering AAV vectors 3 includes a single bind-elute step or a single flow-through step. Alternatively, the method may include multiple bind-elute steps, e.g., two, three, four, five, or more bind-elute steps, in sequence. Alternatively, the method may include multiple flow-through steps, e.g., two, three, four, five, or more flow-through steps, in sequence. Alternatively, the method may include a combination of multiple bind-elute and flow-through steps, e.g., a total of two, three, four, five, or more steps, in sequence.

[0084] After elution step 300, the method may further include the step of regenerating the matrix. Typically, this is accomplished by contacting the matrix from which the viral product and / or product-associated impurities have been eluted with a buffer. This may be carried out according to conventional methods known for the regeneration phase of such methods.

[0085] Typically, the product fraction contains a greater amount of AAV vector, expressed as a percentage of the total amount of viral product and product-associated impurities, than was present in solution. Typically, the amount of AAV vector in the product fraction, expressed as a percentage of the total amount of viral product and product-associated impurities, is 10-fold or more greater than the amount in solution.

[0086] In the method shown in Figure 3, the flow rate used depends on the size of the bonded phase and the residence time selected. Achievable residence times in the method may be from 0.1 seconds to 2 minutes. The equivalent flow rate in a 0.4 ml laboratory-scale unit would be approximately 3000 to 2 cm / h. In a 2.4 liter unit, the maximum achievable flow rate in the bonded phase would be approximately 850 cm / h.

[0087] The amount of input solution added to the bonded phase of the method can be up to 13 liters per ml of sorbent volume of the packed bonded phase without causing plugging or fouling of the matrix, thereby providing the bonded phases of the present invention and chromatographic devices employing such bonded phases with a volumetric loading capacity sufficient to allow typical AAV bioprocess feeds to reach the dynamic binding capacity of the packed bonded phase.

[0088] While the present invention has been described herein with reference to exemplary embodiments, those skilled in the art will understand that the invention is not limited thereto. In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim. Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those listed. The use of the article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. [Example]

[0089] Example 1 Pore ​​size measurement method The mean flow pore size can be measured using capillary flow analysis using commercially available equipment. In one example, the equipment used is a POROLUX™ 100 porometer (IB-FT GmbH, Berlin, Germany) according to the manufacturer's manual and the method listed in Table 1.

[0090] [Table 1]

[0091] Example 2 Preparation of bonded phases for use in chromatography The following is a non-limiting example of the generation of polymer matrix 1, the immobilization of ligand 2 thereon, and the use of the binding matrix thus formed in affinity chromatography. Figure 4a shows the reaction method for preparing matrix 1 material for immobilization of AAV vector ligand 2. Figure 4b shows the linker chemistry used to immobilize AAV vector ligand 2 on the surface of cellulose acetate fibers.

[0092] Seat manufacturing The matrix material can be prepared as described in International Patent Publication No. 2018 / 011599 or to produce a laminated nonwoven sheet of fibers. A solution of cellulose acetate (CA) with a relative molecular weight of 29,000 g / mol is dissolved in a binary mixture of glacial acetic acid and ethanol in a 3:1 ratio. This is the first solution. Once mixed, polyethylene oxide (PEG) dissolved at a concentration of 5% in deionized (DI) water is then added to the first CA solution in an amount of 1.2% of the total CA volume prior to electrospinning, producing fibers with diameters in the 300-600 nm range. Optimal conditions for nanofiber production can be found, for example, in O. Hardick et al., J. Mater. Sci. 46 (2011) 3890. Sheets of approximately 20 g / m2 of material are layered and subjected to a combination of heat and pressure treatment.

[0093] The matrix material thus formed has a mean flow pore size of 0.1-2.0 μm, as measured using bubble point porometry (Porolux, Porometer NV). By varying the combination of standard electrospinning parameters and reducing the number of fiber layers, mean flow pore sizes in this range can be reliably obtained.

[0094] CA pre-cleaning A 35x sheet (100x155mm2) of CA material is sandwiched between gauze and placed in the flow reactor. The material is washed with 5L of recirculating DI water for 20 minutes. The reactor is emptied and the washing process is repeated two more times, with the material being stored overnight for the final wash if necessary.

[0095] Glycidol process In an 8 L beaker, KOH (265 g) was added to DI water (4723 mL). The solution was stirred vigorously. Once completely dissolved, glycidol (1350 mL for a 100% glycidol-based matrix, 675 mL for a 50% glycidol-based matrix) was added and stirred vigorously for 4 minutes. The reactor was emptied of rinse water, and the KOH / glycidol solution was added to the reactor. The recirculation pump was started. A reaction temperature profile of 20 °C was used, and the solution was continuously flowed through the membrane to prevent the reaction temperature from exceeding 20 °C. After 6-7 hours of recirculation, the reaction solution was removed, and the material was washed by recirculating 5 L of DI water for 20 minutes, then emptied. This washing procedure was repeated at least three times (or as many times as necessary until a final pH was neutral). The sheet was stored in DI water overnight.

[0096] saponification process The material produced, called 0% glycidol, is known as regenerated cellulose (RC) and is synthesized in a saponification reaction in which the terminal acetate groups of the cellulose acetate backbone are cleaved, leaving behind alcohol groups. This step replaces the glycidol step, followed by the divinyl sulfone (DVS) step.

[0097] In an 8 L beaker, KOH (132 g) was added to DI water (3149 mL) along with EtOH (1574 mL). The solution was stirred vigorously until completely dissolved. The flow reactor was emptied of wash water and the KOH / EtOH solution was added. The recirculation pump was started and operated at 22-24°C for 6 hours. The reaction mixture was then removed and 5 L of DI water was added to the reactor. The recirculation pump was started and operated for 20 minutes. The wash water was removed. The water wash was repeated three more times.

[0098] Divinyl sulfone (DVS) process The flow reactor was emptied of rinse water. In an 8 L beaker, Na2CO3 (316 g) was added to DI water (4211 mL). The solution was vigorously stirred until completely dissolved. Acetonitrile (1258 mL) was added with vigorous stirring. The solution was added to the flow reactor. The recirculation pump was started for 4 minutes, and then DVS (1350 mL) was carefully added in small portions to the reaction vessel.

[0099] After 6 hours of recirculation, the reaction mixture was removed. 1:1 acetone / DI water (5 L) was added to the flow reactor, and the recirculation pump was started and run for 20 minutes. The wash solution was removed. The acetone / water wash procedure was repeated three more times.

[0100] DI water (5 L) was added to the flow reactor at 22-24°C, and the recirculation pump was started and run for 20 minutes. The wash water was removed. The water wash process was repeated once.

[0101] immobilization The selected AAV vector ligand can be immobilized on a matrix. In this example, a spin-filter solution of AAV-binding ligand with a concentration of 2.5 mg / ml was used for coupling.

[0102] A coupling solution of 3.0 M (NH4)2SO4, 0.1 M NaHCO3 was prepared and adjusted to pH 9.

[0103] A sheet of DVS-treated matrix material was placed in a sealed container (155 × 10 mm), and a fixed volume of the desired concentration of ligand solution was added along with a fixed volume of coupling solution to make the desired total volume, as shown in Table 2. The container was sealed and placed on an orbital shaker at 22–24°C for 16 hours. The supernatant was then collected. All sheets were washed with DI water for 20 minutes. This was repeated three more times, with the supernatant collected each time for later quantification of immobilization efficiency.

[0104] blocking Either ethanolamine blocking or thioglycerol blocking was performed.

[0105] Ethanolamine Blocking: A blocking solution of 0.3 M ethanolamine was adjusted to pH 9, and 25 mL was dispensed onto each sheet. The container was sealed and placed on an orbital shaker at 22-24°C for 16 hours. The blocking solution was then discarded, and the sheets were washed with DI water for 20 minutes. This process was repeated once. The sheets were washed with PBS adjusted to pH 2.0 for 20 minutes, followed by PBS at pH 7.4. This two-step process was repeated once, followed by two washes with DI water for 20 minutes each.

[0106] Thioglycerol Blocking: A blocking solution of 0.288M thioglycerol, 0.1M NaHPO4.12H2O, and 0.001M EDTA was adjusted to pH 8.3, and 25mL was dispensed onto each sheet. The container was sealed and placed on an orbital shaker at 22-24°C for 16 hours. The blocking solution was then discarded, and the sheets were washed with DI water for 20 minutes. This process was repeated once. The sheets were washed with 0.5M AcOH for 20 minutes, followed by 0.1M Tris, 0.15M NaCl, pH 8.5. This two-step process was repeated once, followed by two washes with DI water for 20 minutes each.

[0107] After blocking was performed, the material was completely immersed in 1:1:3 glycerol / ethanol / water and stored in the refrigerator for a minimum of 1 hour.

[0108] Ligand density measurement Using a NanoDrop spectrophotometer, the AAV vector ligand concentration of each collected supernatant was calculated. This was used to calculate the mass of immobilized ligand. One disk was removed from each sheet, and the supernatant was collected / concentrated as described above. Using a Mitutoyo Micrometer, the thickness of each disk was measured at five points across the sheet, and the average thickness of the sheet was calculated. This allowed the total volume of the sheet to be calculated. The mass of immobilized ligand was divided by the volume of the sheet to calculate the ligand density. The average ligand density can be calculated for each batch.

[0109] [Table 2]

[0110] The above procedure yields a ligand density of 3-7 mg / mL of adsorbent, which may correspond to approximately 0.5-5 μmol / g for some ligands, e.g., 0.7-4.6 μmol / g. At such ligand densities, it is possible to achieve a static binding capacity of over 1E15 AAV-5 capsids per mL of adsorbent when using AAV-5. Ligand densities have been measured using amino acid analysis or UV spectroscopy of the pre-wash, post-wash, and wash fixative solutions.

[0111] Example 3 Use of matrices with immobilized AAV vector ligands The recovery of AAV vectors from matrix materials with immobilized AAV vector ligands represents the fraction of AAV obtained after purification with the matrix materials of the present invention (considering that the method involves loss of AAV) and is defined only for 85% capacity loads and that particular AAV serotype at the residence time tested. Lower loads or shorter residence times may result in lower recoveries. Purity is defined as the reduction in biocontaminant content, i.e., host cell DNA and protein.

[0112] Generation of AAV vectors AAV vector material is typically produced from cells engineered to produce vector components, e.g., the envelope, or capsid, and the nucleic acid material intended to be delivered to the cell. After lysis and clarification, the "raw material" contains the vector, cellular host debris, proteins, genomic DNA, serum proteins, some components of the media, helper DNA, helper virus, etc., and serves as the input feed for subsequent purification (see below). Below are examples of cell culture methods, transfection, and harvesting.

[0113] culture material: · FreeStyle 293-F cells (Thermo Fisher Scientific) Hycell TransFx-H (GE Healthcare) supplemented with 4 mM Glutamax (Thermo Fisher Scientific) and 0.1% (v / v) Pluronic F-68 DMSO (dimethyl sulfoxide)

[0114] Thawing cells: Add 9 mL of pre-warmed medium to a 15 mL test tube. · Quickly thaw a vial of cells in a 37°C water bath. · Move the vial to the BS cabinet only when only a small amount of ice is present. With a pipette, gently resuspend the contents of the vial and slowly pipette into a 15 mL test tube. Dilute the medium to a final volume of 30 mL into a T75 tissue culture flask and place in a cell incubator set at 37°C and 8% CO2. After 3-4 days, measure the cell density. When there are at least 2E7 viable cells in the culture, dilute it in a 100 mL spinner flask. Set the agitation to 130 rpm.

[0115] Cell passage: Cells need to be passaged at approximately 1-2E6 cells / mL (usually every 2-3 days). Measure the cell density and viability in the culture medium. Also, visually assess the clumping in the cell suspension using a microscope. If clumping is severe, pipette the cell suspension vigorously (approximately 20 times) or vortex for a few seconds. Dilute the cell suspension to 0.2–0.5E6 cells / mL with pre-warmed fresh medium and return it to the cell incubator.

[0116] Freezing cells: Determine cell density and viability in the culture (viability should be >95%). Centrifuge the cell suspension and dilute the cells to approximately >1E7 cells / mL with 50% conditioned medium and 50% fresh medium. Add 925 μL of cell suspension to a cryovial. Add 75 μL of DMSO. Place the vial in a cell freezing container (surrounded by isopropyl alcohol) and keep at -80°C overnight. Transfer to a liquid N2 cell bank for long-term storage.

[0117] Cell transfection material: · FreeStyle 293-F cells Hycell TransFx-H (Cytiva) supplemented with 4 mM Glutamax (Thermo Fisher Scientific) · Opti-MEM I low serum medium (Thermo Fisher Scientific) Transporter 5 transfection reagent (Polysciences) Supercoiled Rep / Cap plasmid "pRC" (1mg / mL) Supercoiled vector genome, typically a reporter or therapeutic gene flanked by AAV-2 inverted terminal sequences "pGFP" (1 mg / mL)

[0118] The night before transfection: Replace and / or add at least 60% fresh medium (without pluronics) to the cell suspension. The final cell density should be approximately 0.7-0.8E6 cells / mL. For transfections (1 L scale), the next day: Add 500 μL of pRC and 500 μL of pGFP plasmid (both at 1 mg / mL) to 47 mL of Opti-MEM at RT. Vortex briefly. Add 2 mL of Transporter 5 transfection reagent to the DNA mixture. Vortex for 5 seconds. Incubate at RT for 20 minutes. Pipette up and down three times. Add all at once to the cell suspension.

[0119] AAV harvesting material: Triton-X100 (Sigma) · 1M MgCl2 solution Benzonase (Merck) Pluronic F-68 solution (Thermo Fisher Scientific) · 4M NaCl solution

[0120] method: 72 hours after transfection: Add the following directly to each cell bag (1L scale) in 10mL of PBS:

[0121] [Table 3]

[0122] Incubate at 37°C for 3 hours. Add 115 mL of 4 M NaCl solution (to increase the NaCl concentration to 500 mM) and incubate for 30 minutes. Filter sequentially through ULTA Prime GF 5.0 μm and ULTA Prime CG 0.2 μm filters. Take a sample for titration and store at -80°C. Clarify and concentrate using a 300kDa Pellicon TFF (tangential flow filtration) membrane cassette (Merck).

[0123] dynamic binding capacity The dynamic binding capacity (QB10) test is described below. Typically, such a capacity test is performed first, followed by a separate run loading to 85% of the dynamic binding capacity to assess purity / recovery (described further below). The data in Figure 5 show the results of dynamic binding capacity tests performed over a range of residence times for various serotypes.

[0124] device AKTA Avant 150 (Cytiva) CLARIOstar Plus microplate reader (BMG Labtech) A HiTrap chromatography unit (Cytiva) equipped with a matrix containing immobilized AAV vector ligands

[0125] chemicals Running buffer: 20 mM Tris, 500 mM NaCl, 0.001% Pluronic F-68 (Sigma Aldrich), pH 8.5 Elution buffer: 100 mM NaOAc, 500 mM NaCl, 0.001% Pluronic F-68 (Sigma Aldrich), pH 2.5 Neutralization buffer: 200 mM Tris, 500 mM NaCl, 0.001% Pluronic F-68 (Sigma Aldrich), pH 10.5 Input feed: AAV feed after TFF (tangential flow filtration) of approximately 2E14 capsids followed by charged depth filtration, at a concentration of 1E12 capsids / ml in running buffer, filtered through a 0.2 μm filter immediately before use. AAV titration ELISA kit of the appropriate serotype for the input feed (e.g., Progen Biotechnik GmbH, product no. PRAAV5)

[0126] Refining, AKTA execution Fill the A1 and sample lines with running buffer, fill the B1 line with elution buffer (or equivalent settings), and flush the running buffer through the system. · Dilute the input feed with running buffer to approximately 1.3E12 capsids / mL and filter through 0.2 μm. Feed the sample line and the remaining feed supply should be equivalent to approximately 2E14 capsids (approximately 1.3E12 capsids / mL) of the diluted input feed. Connect the HiTrap unit to the column valve. Insert a 50 mL and a 15 mL test tube into the sample collection tray. Add 3 mL of neutralization buffer to the second 50 mL tube in the collection tray. · Perform the AKTA method as detailed below: Note: Parameters not detailed here should be left at their default settings.

[0127] Setting method: Spare column pressure resistance: 1.4 MPa Delta column pressure: 1MPa · Flow rate: 10mL / min Select the column position where the HiTrap unit will be connected to the AKTA. UV1: 280nm; UV2: 254nm

[0128] Equilibration 1: Volume: 10mL · Inlet B1:100

[0129] Equilibration 2: Volume: 10mL · Inlet B1: 0%

[0130] Sample addition: Use same flow rate as method setting: Check Direct sample injection to column: Check Sample inlet: S1 Inject all samples using the intake sensor: Check Use the same inlet as the method setting: Check · Inlet B:0% Fractionation: Use a fraction collection device Fraction type: Fixed volume fraction Fraction storage location: 15mL test tube Fixed fraction volume: 10 mL

[0131] Column washing: · Inlet B:0% Wash until the total volume reaches 20 mL Fractionation: Use a fraction collection device Fraction type: Fixed volume fraction Fraction storage location: 50mL test tube Fixed fraction volume: 50 mL

[0132] Elution: Isoconcentrated dissolution volume: 20 mL 100% of B Fill the system with the selected buffer: Check Fractionation: Use a fraction collection device Fraction type: Fixed volume fraction Fraction storage location: 50mL test tube Fixed fraction volume: 50 mL

[0133] Equilibration 3: Volume: 4mL · Inlet B1: 0% Collect 1 mL samples of each fraction (inlet, FT fraction, post-load wash, and elution) and store in a mapped deep-well 96-well block and cover with an adhesive lid. Store blocks at -80°C if analysis will not be performed within 24 hours, otherwise store at 4°C.

[0134] Approximate dynamic binding capacity (QB10): Perform an AAV titration ELISA on the collected samples according to the manufacturer's recommended method. Each sample should be quantified in triplicate using three independent dilutions. At least a three-fold dilution is recommended to prevent potential matrix effects. Calculate the volume (after injection) where the conductivity is equal to 50% of the difference between the running buffer and the input feed. This is the dead volume between the sample line and the conductivity monitor and must be subtracted from the volume of the input feed recorded by the AKTA to get a more accurate input volume. Use a 5-parameter curve fit generated with ELISA standards to determine the total amount of AAV particles in each fraction. NOTE: The total amount of AAV capsids in the input feed is calculated using the adjusted input volume (input volume - system dead volume). Calculate the breakthrough (% BT) for each FT fraction i using the following formula and plot the BT curve: Fit a linear (or exponential) curve to the data points.

[0135]

number

[0136] Calculate the total number of capsids at 10% BT (HiTrap capacity). Calculate the QB10 for 1 mL of membrane, taking into account that the HiTrap volume is 0.4 mL.

[0137]

number

[0138] Purity / Recovery After performing the kinetic test, a capacity test described below may be performed to assess purity / recovery, loading 85% of the dynamic binding capacity.

[0139] device AKTA Avant (Cytiva) chromatography system. CLARIOstar Plus microplate reader (BMG Labtech). A HiTrap chromatography unit (Cytiva) containing a matrix with immobilized AAV vector ligands.

[0140] chemicals Running buffer: 20 mM Tris, 500 mM NaCl, 0.001% Pluronic F-68 (Sigma Aldrich), pH 8.5 Elution buffer: 100 mM NaOAc, 500 mM NaCl, 0.001% Pluronic F-68 (Sigma Aldrich), pH 2.5 Neutralization buffer: 200 mM Tris, 500 mM NaCl, 0.001% Pluronic F-68 (Sigma Aldrich), pH 8.5 Input feed: After TFF, approximately 85% of the calculated QB10 of the AAV feed after DF, at a concentration of 1E12 capsids / ml in running buffer, filtered through a 0.2 μm filter immediately before use. AAV titration ELISA kit of the appropriate serotype for the input feed (e.g., Progen Biotechnik GmbH, product no. PRAAV5) Pierce Coomassie (Bradford) Protein Assay Kit (Thermo Fisher Scientific, product number 23200) · Quant-iT(TM) PicoGreen(TM) dsDNA Kit (Invitrogen, product number P7589) HEK293 Host Cell Protein ELISA (Cygnus Technology, product number F650S) HCP ELISA sample diluent (Cygnus Technology, product number I700)

[0141] Refining, AKTA execution: Fill the A1, Sample, and Outlet 1 lines with Running Buffer, fill the B1 line with Elution Buffer (or equivalent settings), and flush the entire system with Running Buffer. Feed the estimated amount of input feed (85% of QB10 calculated at 1E12 capsids / mL) into the sample line. The HiTrap unit containing the matrix with immobilized ligands is connected to the column valve. Insert the 96 deep well plate, 50 mL and 15 mL test tubes into the sample collection tray. Prepare and run the following procedures: 1) load the desired volume of input feed into the HiTrap unit with a residence time of 2.4 seconds and collect the flow-through in fractions; 2) wash with 20 mL of running buffer and collect the effluent as a single fraction; and 3) elute with 20 mL of elution buffer and collect as a single fraction. Within 15 minutes of completing the run, add 3 mL of neutralization buffer to the elution fraction. · 1 mL samples of the flow-through, post-load wash fractions, and input feed were collected and stored in mapped deep-well 96-well sample blocks, where the elution fractions were collected and covered with adhesive lids. If analysis is not performed within 24 hours, store at -80°C, otherwise store at 4°C.

[0142] Evaluation and quantification of recovery: Perform an AAV titration ELISA on the collected samples according to the manufacturer's recommended method. Each sample should be quantified in triplicate using three independent dilutions. At least a three-fold dilution is recommended to prevent potential matrix effects. Calculate the volume (after injection) where the conductivity is equal to 50% of the difference between the running buffer and the input feed. This is the dead volume between the sample line and the conductivity monitor and must be subtracted from the volume of the input feed recorded by the AKTA to get a more accurate input volume. Use a 5-parameter curve fit generated with ELISA standards to determine the total amount of AAV particles in each fraction. Identify the elution peak and elution peak fractions, which therefore include only fractions where the AAV concentration is ≥ 10% of the most concentrated fraction. Recovery can be determined using the following formula:

[0143]

number

[0144] The total AAV capsids in the input feed are calculated using the adjusted input volume (input volume - system dead volume).

[0145] Purity determination: 1. Determine the amount of DNA in each sample using the Quant-iT™ PicoGreen® dsDNA kit according to the manufacturer's recommendations. Each sample should be quantified in triplicate using a wide-range standard curve (linear fit). If the eluted sample is below the limit of detection, it should be measured using the low-range standard curve.

[0146]

number

[0147] 2. Determine the total protein concentration in each sample using the Pierce Coomassie (Bradford) protein assay kit according to the manufacturer's recommendations. Each sample should be quantified in triplicate using a wide-range calibration curve (5-parameter curve fit). If the eluted sample is below the detection limit, it should be measured using the low-range calibration curve. Sample and standard dilutions should be prepared in DNA- and RNA-free water. If the protein concentration is below the detection limit of the Pierce Coomassie assay, the sample should be quantified using the HEK 293 HCP ELISA according to the manufacturer's recommendations. Three independent dilutions of each sample should be prepared in Cygnus sample dilution buffer.

[0148]

number

[0149] result The dynamic binding capacity (10% breakthrough) of matrices with immobilized AAV vector ligands was calculated for cell lysate feeds containing AAV serotypes AAV-2 and AAV-5 at residence times ranging from 1.2 seconds to 60 seconds. The results, shown in Figure 5, demonstrate successful AAV vector purification at residence times ranging from a few seconds to approximately 60 seconds. Different capacities for binding these AAV serotypes were also found, likely due to different binding kinetics between the AAV ligand and each serotype. Varying the residence time also demonstrated different levels of impact on capacity among these serotypes. A four-fold increase in AAV-5 residence time resulted in a two-fold increase in dynamic binding capacity. On the other hand, for AAV-2, the dynamic binding capacity plateaued at residence times between 30 and 60 seconds, with shorter residence times resulting in a decrease in capacity (data not shown). Essentially, at residence times <60 seconds, relevant to the binding phase of the present invention, the AAV serotype may have a certain effect on the ligand-AAV binding kinetics. Notably, no serotype-dependent ligand-AAV binding kinetics were observed at residence times >60 seconds associated with conventional resin chromatography.

[0150] Example 4 Large-scale collection Recovery of AAV5 vectors from large-scale batches was tested. By scaling up the method described above, a 200 L batch of cell lysate feed was produced in a stirred tank bioreactor (Xcellerex XDR-200, Cytiva, Sweden) and then clarified by depth filtration. Approximately 100 L of cell lysate feed was then loaded into a 40 mL unit of the binding phase of the present invention, prepared as described in Example 2 above, and run with a 10 second residence time using the buffers and conditions outlined in Table 3.

[0151] [Table 4]

[0152] The obtained recovery rates of AAV5 were 90-100%. [Explanation of symbols]

[0153] 1. Matrix 2. AAV Ligand 3. AAV Vectors 4. Other ingredients 100. Obtaining a solution containing an AAV vector 3 and one or more impurities 4 200 contacting the solution with a bonded phase 300. Eluting the AAV vector 3 from the binding phase by contacting the binding phase with an elution buffer.

Claims

1. A bonded phase for use in affinity chromatography to recover an adeno-associated virus (AAV) vector (3) from a solution containing the AAV vector (3), the bonded phase comprising a porous base matrix (1) having a mean flow pore size of 0.1 to 2.0 μm and an AAV vector ligand (2) bound to the matrix (1), the AAV vector ligand (2) having binding affinity for the AAV vector (3), and the density of the ligand (2) in the matrix (1) of the bonded phase being in the range of 0.1 μmol / gram to 10 mmol / gram.

2. The bonded phase of claim 1 , wherein the porous base matrix is ​​a non-woven polymer matrix.

3. 3. The bonded phase of claim 2, wherein the nonwoven polymer matrix comprises nanofibers, and the nanofibers optionally comprise at least one hydrophilic polymer selected from cellulose, polyethersulfone, polystyrene, methyl acrylate, dextran, and agarose.

4. 4. The bonded phase of claim 1, wherein the mean flow pore size is in the range of 10 nm to 1 μm, such as 200 nm to 1 μm.

5. 5. The bonded phase according to any one of claims 1 to 4, wherein the ligand density is in the range of 0.1 to 10 μmol / g, such as 0.2 to 10 μmol / g, for example 0.5 to 5 μmol / g.

6. 6. The bonded phase according to any one of claims 1 to 5, wherein the AAV vector ligand (2) is bound to the matrix (1) by an amine or thiol bond.

7. 7. The binding phase of any one of claims 1 to 6, wherein the AAV vector ligand (2) is an antibody or an antibody fragment.

8. 8. The binding phase of any one of claims 1 to 7, wherein the AAV vector ligand (2) has binding affinity to one or more AAV vector (3) serotypes AAV1 to AAV13, such as one or more of AAV1, AAV2, AAV3, AAV5, AAV6, and AAV10, and engineered variants thereof.

9. 9. An affinity chromatography device comprising a bonded phase according to any one of claims 1 to 8.

10. 9. Use of the binding phase of any one of claims 1 to 8 for purifying an AAV vector (3) from a solution containing the AAV vector and one or more impurities, wherein the AAV vector is optionally selected from AAV1, AAV2, AAV3, AAV5, AAV6, and AAV10, and engineered variants thereof.

11. (100) providing a solution containing an adeno-associated virus (AAV) vector (3) and one or more impurities; (200) contacting the bonded phase according to any one of claims 1 to 8 with the solution, (300) A method for recovering the AAV vector (3) from the solution, comprising the step of eluting the AAV vector (3) from the binding phase by contacting the binding phase with an elution buffer.

12. 12. The method of claim 11, wherein the amount of solution added to the bonded phase is up to 13 liters per ml of sorbent volume of packed bonded phase.

13. 13. The method of claim 11 or 12, wherein the contacting step comprises adding the solution to the bonded phase.

14. 14. The method of any one of claims 11 to 13, wherein the solution forms a mobile phase and the bonded phase is in contact with the solution for a residence time in the range of 1 to 60 seconds or less than 60 seconds.

15. 15. The method of any one of claims 11 to 14, wherein the AAV vector is AAV serotype 1, 2, 3, 5, 6, or 10, or an engineered variant thereof.

Images