Chromatographic system, its use, and method for separating adeno-associated capsids

Abstract

This disclosure relates to a chromatography system comprising a buffer valve configuration configured to allow independent control of a first buffer feed and a second buffer feed; a pump configuration configured to supply a feed containing the first buffer feed, the second buffer feed, and a biological target compound and one or more impurities; a selection valve configuration including a first chromatography device selection valve; a first chromatography device comprising a first chromatography material comprising a ligand-functionalized support material, wherein the ligand comprises affinity groups having binding affinity to the biological target compound, an ion exchange group, or a multimode group; a second chromatography device comprising a second chromatography material comprising a modulating chromatography material; and a third chromatography device comprising a third chromatography material, wherein the selection valve configuration is configured to allow separation of the biological target compound from impurities by passing a feed containing the biological target molecule and one or more impurities through the first, second, and third chromatography devices in series. Further provided are the use of the chromatography system and methods for separating adeno-associated virus capsids from one or more impurities.

Description

[Technical Field]

[0001] This disclosure relates to the field of isolation of biological target compounds, such as adeno-associated capsids. This disclosure covers chromatographic systems and their use for isolating adeno-associated virus capsids from impurities, as well as methods for isolating adeno-associated virus capsids from impurities. [Background technology]

[0002] Liquid chromatography is a separation technique used to separate and analyze complex liquid mixtures of compounds. The process involves a stationary phase and a liquid mobile phase, where the liquid mixture to be separated is introduced into the mobile phase and passes through it. Different components of the mixture interact with the stationary phase to varying degrees, and these are separated and collected at different times. Different types of liquid chromatography techniques exist based on the stationary and mobile phases used, such as ion exchange chromatography, size exclusion chromatography, and affinity chromatography. Different types of chromatography require different sample preparation methods, process conditions, and buffers.

[0003] Various biological target compounds, such as antibodies and viral vectors, are produced by fermentation of host cells. These biological target compounds must then be separated from the host cell material and other impurities contained in the fermentation broth before they can be used, for example, for medical and analytical purposes. To achieve high recovery rates with high levels of purity of the biological target molecules, a combination of several steps using different types of liquid chromatography is often applied. However, the process for separating biological target compounds often requires manual switching between different chromatographic devices and sample preparation between chromatographic steps to adjust the sample for the next step, which makes the overall process time-consuming. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] WO2023285011 A1 [Patent Document 2] US6,428,707 [Patent Document 3] WO2018 / 011599 [Non-patent literature]

[0005] [Non-Patent Document 1] Xiaotong Fu et al., *Analytical Strategies for Quantification of Adeno-Associated Virus Empty Capsids to Support Process Development*, *Human Gene Therapy Methods*, 2019, Vol. 30 (No. 4): pp. 144-152. [Non-Patent Document 2] O. Hardick et al., J. Mater. Sci., Vol. 46 (2011), p. 3890. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Adeno-associated viruses (AAVs) are non-enveloped viruses that possess a linear single-stranded DNA (ssDNA) genome and can be manipulated to deliver DNA to target cells. Recombinant adeno-associated virus (rAAV) vectors have emerged as one of the most versatile and successful gene therapy delivery vehicles. For AAV particles to be used as vectors in therapy, the viral particles must be purified from cellular impurities such as DNA after transfection. Furthermore, since the therapeutic efficacy of AAV vectors depends on a high percentage of viral particles that are fully encapsulated with the genetic material of interest, it is important to separate such fully encapsulated AAVs from empty or partially encapsulated AAV particles. WO2023285011 A1 describes a method for separating fully encapsulated AAV particles from unencapsulated AAV particles. However, in this art, there is a continuous need for novel chromatographic systems that offer higher volume and faster processing, and generally better process economics. [Means for solving the problem]

[0007] One object of this disclosure is to provide a chromatography system that enables faster and simpler separation of biological target compounds from impurities, while having performance similar to previously known chromatography systems. The chromatography system is configured so that a feed containing the biological target compound and impurities can be subjected to several purification steps without interruption by passing through the system continuously. Several chromatography devices are connected in line within the chromatography system. This achieves a significantly reduced time compared to using conventional chromatography systems. Conventionally, the purification process flow is typically interrupted and prolonged because buffers and / or chromatography devices are manually switched within the system, and because several steps, such as preparation of the chromatography medium and / or the performance of certain filtration / purification steps, are performed in separate containers or devices outside the chromatography system. Using the chromatography system of this disclosure requires less human intervention compared to operating conventional chromatography systems.

[0008] More specifically, this disclosure is, A buffer valve arrangement configured to allow independent control of a first buffer supply and a second buffer supply, A pump configuration configured to supply a first buffer supply, a second buffer supply, and a supply containing a biological target compound and one or more impurities, A selection valve arrangement including a first chromatography device selection valve, A first chromatography device comprising a first chromatography material containing a support material functionalized with a ligand, wherein the ligand contains an affinity group having binding affinity to a biological target compound, an ion exchange group, or a multimodal group, A second chromatography device comprising a second chromatography material including a chromatographic material for adjustment, Third chromatography device containing a third chromatography material Includes, The present invention relates to a chromatography system in which a feed containing a biological target molecule and one or more impurities is passed through first, second, and third chromatography devices in series, wherein a selective valve arrangement is configured to allow separation of the biological target compound from impurities, and the first, second, and third chromatography devices are connected in series.

[0009] This disclosure also relates to the use of a chromatographic system for the separation of one or more impurities of a biological target compound disclosed herein, wherein the biological target compound is an adeno-associated virus capsid, e.g., adeno-associated virus serotype 1 (AAV1), adeno-associated virus serotype 2 (AAV2), adeno-associated virus serotype 3 (AAV3), adeno-associated virus serotype 4 (AAV4), adeno-associated virus serotype 5 (AAV5), and The invention provides a use selected from adeno-associated virus serotype 6 (AAV6), adeno-associated virus serotype 7 (AAV7), adeno-associated virus serotype 8 (AAV8), adeno-associated virus serotype 9 (AAV9), adeno-associated virus serotype 10 (AAV10), adeno-associated virus serotype 11 (AAV11), adeno-associated virus serotype 12 (AAV12), or adeno-associated virus serotype 13 (AAV13), or the capsids of their variants.

[0010] In addition, the present disclosure relates to a method for isolating an adeno-associated virus capsid from one or more impurities, wherein the adeno-associated virus capsid is completely encapsulated in genetic material. a. Adding a feed containing an adeno-associated virus capsid completely encapsulated in genetic material and one or more impurities to a first chromatography device comprising a first chromatography material comprising a ligand-functionalized support material, wherein the ligand comprises affinity groups having binding affinity to the adeno-associated virus capsid, an ion exchange group, or a multimodal group. b. eluting the adeno-associated virus capsid into at least one eluate fraction from a first chromatography device; c. adding at least one eluate fraction containing the adeno-associated virus capsid obtained in step b to a second chromatography device containing a second chromatography material including an adjusting chromatography material; d. obtaining the adeno-associated virus capsid in at least one flow-through fraction from the second chromatography device; e. adding at least one flow-through fraction containing the adeno-associated virus capsid obtained in step d to a third chromatography device containing a third chromatography material; f. obtaining the adeno-associated virus capsid in at least one eluate fraction from the third chromatography device comprising a method wherein a feed continuously passes through the first, second, and third chromatography devices to enable separation of one or more impurities of the adeno-associated virus capsid, and the first, second, and third chromatography devices are connected in series.

[0011] Preferred embodiments of the present disclosure are described below in the detailed description and the dependent claims. It should be noted that the present disclosure relates to all possible combinations of the features listed in the claims.

[0012] These and other aspects of the present disclosure will be described in more detail with reference to the accompanying drawings showing embodiments of the invention.

Brief Description of the Drawings

[0013] [Figure 1] It is a schematic diagram of a chromatography system according to an embodiment. [Figure 2] It is a schematic diagram of a chromatography system according to a further embodiment. [Figure 3]This flowchart outlines the steps of the method for separating adeno-associated virus capsids from impurities as disclosed herein. [Figure 4] This graph shows chromatograms for the separation of AAV5 full capsid and empty capsid under different chromatography system settings, as described in Example 1 of this specification. [Modes for carrying out the invention]

[0014] As illustrated in the drawings, some features may be emphasized for illustrative purposes and are therefore provided to illustrate the general structure of embodiments of the present disclosure.

[0015] This disclosure addresses problems related to existing chromatography systems and processes for separating biological target compounds from impurities, as illustrated in Figure 1. A buffer valve arrangement 20 configured to allow independent control of the first buffer supply and the second buffer supply, A pump configuration 40 configured to supply a first buffer supply, a second buffer supply, and a supply containing a biological target compound and one or more impurities, A selection valve arrangement 60 including a first chromatography device selection valve (62), A first chromatography device 70 comprises a first chromatography material containing a ligand-functionalized support material, wherein the ligand contains an affinity group having binding affinity to a biological target compound, an ion exchange group, or a multimode group. A second chromatography device 72 containing a second chromatography material including a control chromatography material, Third chromatography device 74 containing a third chromatography material Includes, This is solved or at least mitigated by providing a chromatography system 10 in which a feed containing a biological target molecule and one or more impurities is passed through first, second and third chromatography devices in series, the selective valve arrangement is configured to allow separation of the biological target compound from impurities, and the first, second and third chromatography devices are configured to be connected in series.

[0016] The buffer valve arrangement 20 has at least one inlet (indicated by the arrow in Figure 1) and a corresponding outlet for the buffer supply, and typically two or more inlets and corresponding outlets for independent control of two or more buffer supplies.

[0017] The pump arrangement 40 includes at least one pump, and possibly two or more pumps.

[0018] The selective valve arrangement 60 connects three chromatography devices in series, thereby enabling a three-step chromatographic purification process within a single line of chromatography systems. This avoids interruptions and delays that would conventionally occur due to the manual operation of various steps between processes, saving time and effort for the user operating the equipment. The chromatography system can be fully set up and programmed before starting the purification process, and the process can then be started with a single button press and continued to completion without further user intervention. Thus, this disclosure may be said to provide a so-called "plug-and-play" chromatography system, meaning a system intended to function perfectly without being reconfigured or adjusted by the user when first used or connected.

[0019] More specifically, the chromatography system 10 of this disclosure offers the following general advantages compared to conventional chromatography systems.

[0020] - A coupled desalting and pH adjustment step after affinity capture. This eliminates the need for dilution to reduce the conductivity of the sample and adjust the pH before polishing, resulting in a lower sample feed volume and thus shorter loading time, potentially saving time. Furthermore, this provides complete control of the sample's conductivity, enabling high-performance packing and empty AAV capsid separation. - No holding time, - No freezing-thawing cycle, - No sample preparation (pH and conductivity) or handling between chromatography steps.

[0021] For the purification of adeno-associated virus (AAV) vectors, there is an additional advantage in that AAV vectors, which are sensitive to low pH for extended periods, are exposed to acidic conditions for a shorter period (minutes versus hours). In addition, it has been shown that when purifying AAV vectors, a step of tangent flow filtration can be avoided by using the chromatography system 10 disclosed herein, compared to when using a conventional chromatography system for a conventional purification process.

[0022] As will be described in more detail elsewhere in this specification, the chromatography system 10 can be applied with various formats of chromatography devices and chromatography materials. Non-limiting examples of chromatography materials suitable for in-line connected purification of AAV vectors include combinations of membrane adsorbents with convection-based membrane structures consisting of nanofibers, monoliths, and resins (beads). The chromatography systems disclosed herein provide a significantly time-reduced (up to 70%) and simplified AAV purification process while achieving performance similar to conventional purification settings in terms of recovery and percentage of AAV capsids fully encapsulated in genetic material. Given the combined chromatographic load and conductivity precision required for the polishing step, it is remarkable that a complete capture and polishing process can be successfully performed using such an in-line connected three-step purification process.

[0023] Figure 2 illustrates a currently preferred non-limiting embodiment of the chromatography system 10, where the buffer valve arrangement consists of a first buffer selection valve 22 and a second buffer selection valve 24.

[0024] The first buffer selection valve 22 may be configured to control a first buffer supply used to equilibrate the chromatography device before loading a sample supply containing a biological target compound, which is referred to herein instead as Buffer A, and the second buffer selection valve 24 may be configured to control a second buffer supply used to elute the biological target compound from the chromatography system 10, which is referred to herein instead as Buffer B.

[0025] More specifically, the first buffer selection valve 22 may be configured to control the feed of the first buffer 30 for equilibrating the first chromatography device 70, which is referred to herein instead as buffer A1, and the first buffer selection valve 22 may further be configured to control the feed of the first buffer 32 for equilibrating the second chromatography device 72, which is referred to herein instead as buffer A2. Buffer 32 may also be used for equilibrating the third chromatography device 74. If the biological target compound is not bound to the second chromatography device and the second chromatography device is instead in a pass-through state, buffer 32 may also be used to pass the biological target compound from the second chromatography device to the third chromatography device.

[0026] Furthermore, the second buffer selection valve 24 may be configured to control the supply of a second buffer 34 for the elution of a biological target compound from a first chromatography device 70, which is referred to herein instead as buffer B1, and to control the supply of a second buffer 36 for the elution of a biological target compound from a second chromatography device 72, which is referred to herein instead as buffer B2. Buffer B2 may also be used for the elution of a biological target compound from a third chromatography device 74, if applicable. Alternatively, separation buffer supply B3 may be used for the elution of a biological target compound from a third chromatography device 74 (not shown).

[0027] The system 10 shown in Figure 2 further includes a pump arrangement comprising a system pump 42 and a sample pump 44. The system pump 42 is configured to supply feeds of buffer 30, buffer 32, buffer 34, and buffer 36. The sample pump 44 is configured to supply a feed of sample 46 containing a biological target compound and one or more impurities.

[0028] The system 10 shown in Figure 2 also includes an injection valve 50. It should be understood that any injection valve conventionally used in the art may be used.

[0029] According to the embodiment shown in Figure 2, the selection valve arrangement consists of a first chromatography device selection valve 62, a second chromatography device selection valve 64, and a third chromatography device selection valve 66. The chromatography device selection valve 62 is connected to the first chromatography device 70, the second chromatography device selection valve 64 is connected to the second chromatography device 72, and the third chromatography device selection valve 66 is connected to the third chromatography device 74, where the three chromatography devices are connected in series in a line within the system 10.

[0030] However, the selection valve configuration 60 is intended to consist of a single chromatography device selection valve 62 connected in series with the first, second, and third chromatography devices 70, 72, and 74 (as shown in Figure 1). Alternatively, the selection valve configuration 60 may consist of a combination of a first chromatography device selection valve 62 and a second chromatography device selection valve 64 (not shown), which is connected in series with the first, second, and third chromatography devices 70, 72, and 74. In embodiments where the number of chromatography device selection valves is less than the number of chromatography devices, it should be understood that at least one of the selection valves can be operated in a different flow path, and that it is possible to perform multiple steps of chromatographic purification on a chromatography system containing fewer selection valves than the number of chromatography devices.

[0031] A non-limiting example of a chromatography system that may be used is the AKTA pure chromatography system (Cytiva, Sweden). The standard AKTA pure system has only one column valve (corresponding to the chromatography device selection valve in system 10). The AKTA pure system used in the experimental chapter of this specification is modified by adding two additional chromatography device selection valves to obtain the system according to the embodiment shown in Figure 2.

[0032] Another non-limiting example of a chromatography system that can be used is the NGC chromatography system (BioRad, USA). This may be modified by adding a versatile valve to obtain the system according to the embodiment shown in Figure 2.

[0033] Non-limiting examples of chromatography device selection valves that may be part of System 10 of this disclosure include so-called general-purpose valves, such as the general-purpose valve V9-V (Cytiva, Sweden).

[0034] The chromatography system 10 shown in Figure 2 further includes a UV detector 80, a conductivity detector 82, an outlet valve 84, and a fraction collector 86, all of which are standard components of a chromatography system.

[0035] The chromatography system 10 disclosed herein is primarily intended for use in preparation applications for feed materials in volumes ranging from a few milliliters to several hundred liters. The system may also be used for analytical applications. A distinguishing feature of system 10 is that it offers greater advantages when applied in large-scale processes than in small-scale processes.

[0036] The term "chromatographic material" is used herein to describe the type of separation matrix.

[0037] The term “separation matrix” is used herein to describe a material comprising a support material to which one or more ligands containing functional groups are coupled. The functional groups of the ligands are bound to the compound herein, also called the analyte, and are separated from a liquid sample and / or from other compounds present in the liquid sample. The separation matrix may further include compounds that couple the ligands to the support material. The terms “linker,” “bulker,” and “surface bulker” may be used to describe compounds such as those further described below. In this specification, the term “support material” may be used interchangeably with the term “support.”

[0038] Chromatographic materials referred to herein may include linkers that connect ligands to support structures; i.e., coupling of ligands to support structures is provided by introducing a linker between the support structure and the ligand. Coupling can be performed according to any conventional covalent coupling methodology, for example, by using epichlorohydrins; epibromohydrins; allyl glycidyl ethers; bis-epoxides, e.g., butanediol diglycidyl ether; halogen-substituted aliphatic substances, e.g., dichloropropanol; and divinyl sulfones. Non-limiting examples of suitable linkers include vinyl sulfones, vinyl sulfones combined with glycidol, polyethylene glycol (PEG) having 2 to 6 carbon atoms, carbohydrates having 3 to 6 carbon atoms, or polyhydric alcohols having 3 to 6 carbon atoms. Alternatively, the ligand may be coupled to the support structure via longer linker molecules also known as "surface extenders" or simply "extenders." Extenders are well known in this art and are commonly used to sterically increase the distance between the ligand and the support structure. Extenders may be described as tentacles or flexible arms. For a more detailed description of possible chemical structures, see, for example, US6,428,707, which is included herein by reference. In short, fillers can be in the form of polymers, such as homopolymers or copolymers. Hydrophilic polymer fillers can be of synthetic origin, i.e., have a synthetic skeleton, or of biological origin, i.e., biopolymers with a naturally occurring skeleton. Typical synthetic polymers include polyvinyl alcohol, polyacrylamide and polymethacrylamide, polyvinyl ethers, etc. Typical biopolymers are polysaccharides, such as starch, cellulose, dextran, and agarose.

[0039] In this context, “ligand” is a molecule having known or unknown affinity to a given analyte, and including any functional groups or scavengers immobilized on its surface, while “analyte” includes any specific binding partner to the ligand. The term “ligand” may be used interchangeably herein with the terms “specific binding molecule,” “specific binding partner,” “scavenger molecule,” and “scavenger.”

[0040] In this specification, molecules in a liquid sample that interact with a ligand are referred to as “biological target compounds” or “analytes.” The term “biological target compounds” encompasses a wide range of biomolecules and compounds. Non-limiting examples include plasmids, exosomes, mRNA, viral particles and proteins, and, for example, monoclonal antibodies.

[0041] Of particular interest in this disclosure are adeno-associated virus capsids, and more specifically, adeno-associated virus capsids completely encapsulated in genetic material.

[0042] The term "viral particle" is used herein to describe a complete infectious viral particle. This particle contains a core containing the viral genome (i.e., viral genome) in either the form of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), where the core is surrounded by a morphologically defined shell. The shell is called a capsid. The capsid and the encapsulated viral genome together constitute a so-called nucleocapsid. The nucleocapsids of some viruses are surrounded by a lipoprotein bilayer envelope. In the field of bioprocessing, for the purpose of producing viral vectors for various applications such as therapeutics, the genome of a viral particle is modified to include a gene insertion containing the desired genetic material. The modified viral particle infects host cells in a cell culture, the viral particle proliferates in the host cells, and the viral particle is then purified from the cell culture by any means of isolation and purification. Hereinafter, viral particles isolated from a cell culture by the methods of this disclosure may instead be referred to as "biological target compounds," "target molecules," or "targets." The term "viral particle" is intended to refer to a type of viral particle, and it should be understood that the singular form of the term may encompass a number of individual viral particles. In this specification, the term "viral particle" may be used interchangeably with the terms "vector" and "capsid," respectively, as further defined below.

[0043] The term “vector” is used herein to describe viral particles, typically recombinant viral particles intended for use to achieve gene transfer for the modification of a particular cell type or tissue. Viral particles can be manipulated to provide, for example, a vector that expresses a therapeutic gene. Several viral species are currently being investigated for use in delivering genetic material (e.g., genes) to cells to provide either transient or persistent transgene expression. These include adenoviruses, retroviruses (gamma-retroviruses and lentiviruses), poxviruses, adeno-associated viruses (AAVs), baculoviruses, and herpes simplex viruses. In this specification, the term “vector” may be used interchangeably with the terms “viral particle” and “capsid,” respectively.

[0044] The term "capsid" refers to the shell of a viral particle. The capsid surrounds the core of the viral particle and typically contains the viral genome. Modified (recombinant) capsids, such as those produced in the upstream processes of synthesis, will contain the complete viral genome, which contains targeted genetic material for one or more uses, for example, for various therapeutic applications. However, due to low inclusion efficiency, assembled capsids do not always contain any genetic material, or they only form capsids from cleaved gene fragments, resulting in so-called empty capsids and partially filled capsids, respectively. These capsids lack therapeutic function and further compete for inter-receptor binding in cell-mediated processes. This can reduce overall therapeutic efficacy and potentially trigger unwanted immune responses. As a result, tracking these capsids throughout the entire manufacturing process is essential to ensure consistent product quality and appropriate drug response (Xiaotong Fu et al., Analytical Strategies for Quantification of Adeno-Associated Virus Empty Capsids to Support Process Development, Human gene therapy methods, 2019, Vol. 30 (No. 4): pp. 144-152). In up to 20-30% of artificially produced virus particle populations in cell cultures, the capsid is only partially filled with genetic material. Furthermore, in up to 98% of artificially produced virus particles, the capsid contains no part of the viral genome at all, i.e., it is empty. However, while generally 80-90% of artificially produced virus particles have empty capsids, in the best cases, only as low as 50% empty capsids are currently achieved.

[0045] In this specification, the term "capsid" may be used interchangeably with the terms "vector" and "viral particle," respectively. In the context of this disclosure, a capsid may or may not contain genetic material.

[0046] The term "genetic material of interest" is intended to mean genetic material that is considered relevant and valuable in the field of bioprocessing because it is produced by viral replication and purified for use in various applications, including but not limited to therapeutic applications. In non-limiting examples, genetic material of interest may include therapeutically relevant genetic material, such as therapeutically relevant nucleotide sequences.

[0047] The term "capsid completely encapsulated with genetic material" refers to a capsid that has been properly manufactured (by the host cell), or in other words, A capsid containing the complete viral genome, or in other words, A capsid containing 100% of the viral genome, or in other words, Capsid containing functional viral genome This is used herein to describe [the subject].

[0048] The viral genome includes gene insertions containing the genetic material of interest, as defined elsewhere in this specification.

[0049] A capsid containing a complete viral genome may be referred to herein instead as a “complete capsid” or “fully enclosed capsid.” The terms “complete capsid,” “fully enclosed capsid,” and “capsid fully enclosed with genetic material” may be used interchangeably throughout this context.

[0050] The term "capsid not completely enclosed in genetic material" refers to a capsid that is not properly manufactured (by the host cell), or in other words, A capsid that does not contain the complete viral genome, or in other words, Capsid containing less than 100% of the viral genome This is used herein to describe [the subject].

[0051] Capsids that are not completely encapsulated with genetic material are either partially filled with genetic material or not filled with any genetic material at all.

[0052] The term "capsid not completely enclosed in genetic material" encompasses the terms "partially filled capsid" and "empty capsid," as defined below.

[0053] A "partially filled capsid" is a capsid that contains a portion of the viral genome, for example, a missing portion of the viral genome, or in other words, A capsid containing a partial viral genome, or in other words, A capsid containing an incomplete viral genome, or in other words, A capsid containing a missing viral genome, or in other words, Capsids containing more than 0% and less than 100% of the complete viral genome, for example, about 1% to about 99%, for example, about 5% to about 95%, for example, about 10% to about 90%, or for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% of the complete viral genome. Partially filled capsids are improperly manufactured capsids, and therefore it is desirable to separate and remove as many partially filled capsids as possible from the capsid population before using the population for its intended purpose, such as therapeutic use. Partially filled capsids may instead be referred to as “intermediate capsids.”

[0054] An "empty capsid" is a capsid that does not contain any part of the viral genome, that is, a capsid containing 0% of the viral genome, or in other words, Capsids that are not filled with any genetic material whatsoever As defined herein, empty capsids do not contain any of the target genetic material. Consequently, it is desirable to concentrate the complete capsids by isolating and removing as many empty capsids as possible from the capsid population before using the capsid population for its intended purpose, e.g., therapeutic use, i.e., to increase the percentage of complete capsids instead of the percentage of partially filled and empty capsids (and may be required by clinical regulations, for example).

[0055] The percentage of complete capsids and empty capsids in a capsid population can be estimated or analyzed using several methods known in the art. Some of these methods are briefly described below.

[0056] 1: In the chromatogram, the A260:280 ratio provides an estimate of the percentage of complete capsid present in the peak (a ratio of 1–1.5 indicates a high concentration of complete capsid, while a ratio of 0.5–0.7 indicates a predominantly empty capsid).

[0057] 2. qPCR:ELISA ratio. qPCR quantifies the viral genome, while ELISA quantifies the total viral particles. The ratio of two assays with variability is not very accurate and is unstable. Orthogonal analysis is required for confirmation (see 3, 4, or 5 below).

[0058] 3. Analytical anion exchange to separate complete and empty capsids (ratio A260:280 and peak region for calculating percentages). Accuracy depends on the peak definition.

[0059] 4. Ultracentrifugation (AUC). This method detects and quantifies particles of different densities (corresponding to complete capsids, partially filled capsids, and empty capsids). This is currently known as the "golden standard" in the art. However, ultracentrifugation is not scalable and is therefore not suitable for the analysis of large batches of capsids.

[0060] 5. Transmission electron microscopy (TEM). Image analysis for counting particles (complete capsids, partially filled capsids, and empty capsids). Allows for the introduction of artificial materials from sample preparation.

[0061] Several methods for estimating or analyzing the percentage of complete and empty capsids in a capsid population are described in detail by Xiaotong Fu et al., *Analytical Strategies for Quantification of Adeno-Associated Virus Empty Capsids to Support Process Development*, *Human gene therapy methods*, 2019, Vol. 30 (No. 4): pp. 144-152, their disclosure of which is incorporated herein by reference.

[0062] In this specification, the term “impurities” includes cell host fragments, proteins, genomic DNA, serum proteins, several elements of the medium, helper DNA, helper viruses, etc., which may be present in the liquid sample in addition to the purified, fully encapsulated adeno-associated virus capsid. The term “impurities” further includes adeno-associated virus capsid that is not fully encapsulated.

[0063] The term "surface" in this specification means all external surfaces, including the outer surface of a porous support and the surface of a pore.

[0064] The support material for the first chromatography material may include a film structure, nanofibers, monoliths, porous particles, non-porous particles, or an expanded bed media.

[0065] A non-limiting example of a membrane-like structure is the Mustang® membrane (Pall Corporation, USA).

[0066] Another non-limiting example of a membrane structure is Fibro® (Cytiva, Sweden), which is also a non-limiting example of a support material containing nanofibers. Fibro® is a convection-based membrane structure containing nanofibers made from cellulose or a cellulose derivative.

[0067] A non-restrictive example of a monolith is CIMmultus® (Sartorius, Germany).

[0068] A non-limiting example of porous particles is Capto beads (Cytiva, Sweden), which are substantially spherical particles with a diameter of approximately 90 μm.

[0069] A non-exclusive example of an extended bed medium is STREAMLINE resin (Cytiva, Sweden).

[0070] As further mentioned above, the ligand for the first chromatography material may include a biological target compound, an ion exchange group (more particularly, an anion exchange group or a cation exchange group), or an affinity group having binding affinity to a multimode group.

[0071] If the ligand of the first chromatography material contains an affinity group, it may have binding affinity to the adeno-associated virus capsid. In this specification, such ligands are referred to instead as AAV vector ligands. AAV vector ligands may be peptides or polypeptides containing antibodies or antibody fragments, oligonucleotides, e.g., DNA or RNA, e.g., aptamers. AAV vector ligands may be camel antibodies or antibody fragments. AAV vector-binding ligands are known in the art. For example, POROS CaptureSelect AAVX chromatography material (ThermoFisher Scientific, USA) has demonstrated binding reactivity to a set of AAV serotypes including AAV1-AAV8 and AAVrh10. Other examples of chromatography materials incorporating affinity ligands include AVIPure® AAV2 affinity resin, AVIPure® AAV8 affinity resin, and AVIPure® AAV9 affinity resin (Avitide / Repligen, USA). As another example, Capto AVB and AVB Sepharose High Performance (Cytiva, Sweden) are affinity chromatography materials that have been shown to have affinity for adeno-associated viruses from subclasses 1, 2, 3, and 5.

[0072] If the ligand of the first chromatographic material contains an anion exchange group, this may be a strong or partially strong anion exchange group, more particularly a quaternized amine group. Quaternary amine groups are strong anion exchange groups and are always positively charged regardless of the pH to which they are supplied. For DEAE-based types of chromatographic materials, the degree of quaternization of amine groups can vary among the amine groups contained in the chromatographic material. A degree of quaternization of approximately 12% to 100% of the amine groups in the entire chromatographic material is generally considered to result in a chromatographic material that behaves like a strong or at least partially strong anion exchange chromatographic material, since at least 12% of all these amines are always charged.

[0073] More specifically, if the ligand of the first chromatography material contains a potent or partially potent anion exchange group, the ligand is of formula I

[0074] [ka]

[0075] (In the formula, R1 is selected from H and C1-C3 alkyl groups, and R2 and R3 are independently selected from H, C1-C3 alkyl groups, CH2OH, and CH2CHOHCH3, preferably each of R1, R2, and R3 is CH3.) It can be defined by:

[0076] The wavy lines represent the supporting material containing the linker. Ligands can bond to the carbon atoms of the linker.

[0077] There are currently available chromatographic materials containing ligands defined by formula I (wherein R1, R2, and R3 are each CH3), such as the chromatographic material available under the name Capto Q (Cytiva, Sweden). Capto Q further contains dextran as a surface extender. Capto Q is a non-limiting example of a potent anion-exchange chromatographic material with approximately 100% quaternized amine groups.

[0078] If the ligand of the first chromatography material contains a cation exchange group, this could be, for example, a sulfonic acid group. A non-limiting example is Capto S (Cytiva, Sweden). Another non-limiting example is CIMmultus® SO3 (Sartorius, Germany).

[0079] When the ligand of the first chromatography material contains a multimodal group, the multimodal group may be, for example, a multimodal weak cation exchanger or a multimodal weak anion exchanger. A non-limiting example is Capto MMC (Cytiva, Sweden), which is a multimodal weak cation exchanger.

[0080] The second type of chromatography material is the adjusting chromatography material, and the term “adjusting chromatography material” is intended to mean that it is loaded onto the chromatography material for a later purification step and / or final formulation of the product, and that it adjusts or prepares the sample feed or solution that passes through the chromatography material. Adjustment may include, for example, desalting the sample feed or solution, i.e., reducing the salt concentration. Alternatively, adjustment may include, or may involve increasing the salt concentration of the sample feed or solution. Or, further, adjustment may include, or may involve changing the pH of the sample feed or solution, i.e., decreasing or increasing the pH.

[0081] If the adjustment consists of desalting, the adjustment chromatography material may appropriately include size exclusion chromatography material. The salt is delayed by the size exclusion material, while the biological target compound, which is much larger than the salt molecule, passes through the size exclusion material without delay and is therefore obtained in the pass-through fraction from the second chromatography material.

[0082] The support material for the third chromatography material may include a membrane structure, nanofibers, monoliths, porous particles, non-porous particles, or an extended bed medium. Such support materials are described in more detail elsewhere in this specification.

[0083] The ligand for the third chromatography material may contain an anion exchange group or a multimode group.

[0084] If the ligand of the third chromatographic material contains an anion exchange group, this may include a potent or partially potent anion exchange group, as defined and described in more detail above with respect to the first chromatographic material.

[0085] More specifically, if the ligand of the third chromatography material contains a potent or partially potent anion exchange group, the ligand is of formula I

[0086] [ka]

[0087] (In the formula, R1 is selected from H and C1-C3 alkyl groups, and R2 and R3 are independently selected from H, C1-C3 alkyl groups, CH2OH, and CH2CHOHCH3, preferably each of R1, R2, and R3 is CH3.) It can be defined by:

[0088] The wavy lines represent the supporting material containing the linker. Ligands can bond to the carbon atoms of the linker.

[0089] A non-limiting example of a chromatography material containing a ligand defined by formula I (wherein R1, R2, and R3 are each CH3) is Capto Q (Cytiva, Sweden).

[0090] Another non-limiting example of a chromatography material containing a quaternary amine ligand is monolithic CIMmultus® QA (Sartorius, Germany).

[0091] If the ligand of the third chromatography material contains a potent or partially potent anion exchange group, this is instead formula II

[0092] [ka]

[0093] (In the formula, m is an integer between 1 and 3. R1 and R2 may be independently selected from C1-C3 alkyl groups, R3 and R4 may be independently selected from C1-C3 alkyl groups and CH2CHOHCH3 groups, and R5 may be defined by hydrogen, C1-C3 alkyl groups and CH2CHOHCH3 groups. However, if m is 1, the ligand for the potent or partially potent anion exchange chromatography material is given by the following formula III

[0094] [ka]

[0095] (In the formula, n is an integer between 0 and 3, However, if n is 0, R3 and R4 are independently selected from C1-C3 alkyl groups, and R5 is hydrogen or CH2CHOHCH3. This is subject to the condition that it is defined by [the specified method].

[0096] As a non-restrictive example, a ligand is defined by formula III and includes two or more combinations of the following structures (i) to (iv). (i)n is 0, R3 and R4 are ethyl, and R5 is hydrogen or CH2CHOHCH3. (ii)n is 1, R1, R2, R3, R4 are ethyl, and R5 is hydrogen or CH2CHOHCH3. (iii)n is 2, R1 and R2 are ethyl, R3 and R4 are ethyl, and R5 is hydrogen or CH2CHOHCH3. (iv)n is 3, R1 and R2 are ethyl, R3 and R4 are ethyl, and R5 is hydrogen or CH2CHOHCH3.

[0097] One currently available chromatographic material containing ligands defined by formula III and combinations of structures (i) to (iv) described above is a chromatographic resin called Capto DEAE (Cytiva, Sweden). Capto DEAE further contains dextran as a surface extender. Capto DEAE is a non-limiting example of a potent or partially potent anion exchange chromatographic material with a degree of quaternization of approximately 15% of amine groups.

[0098] If the ligand for the third chromatography material contains an anion exchange group, it may instead contain a weak anion exchange group. In contrast to the quaternized amine groups defined above, almost all other ionic exchange groups are weak; that is, their charge varies from fully charged to uncharged within a reasonable pH range used (e.g., pH 2-11), and they have a neutral charge (equal amounts of + and - charge) at pI.

[0099] More specifically, if the ligand of the third chromatography material contains a weak anion exchange group, the ligand is of formula IV

[0100] [ka]

[0101] (In the formula, X is independently H, OH and C for each entity) 1~3 Selected from the base, R1, R2, R3, and R4 are H and C 1~3 The group is independently selected, and the C3 group is either linear or branched. 1~3 The base is HR, OC 1~2 SC 1~2 It includes a group independently selected from NH, NHR, and NR2, where R is H and C 1~3 (Selected from the base) It can be defined by:

[0102] Non-limiting examples of ligands defined by formula IV include N,N,N'-triethylethylenediamine, diethylenetriamine, N,N'-dimethylethylenediamine, N-methylethylenediamine, 1,3-diaminopropane, 1,3-diamino-2-hydroxypropane, 2-methyl-1,3-propanediamine, and N,N-diethylethylenediamine. The currently preferred ligand containing a weak anion exchange group is N,N-diethylethylenediamine.

[0103] When the ligand of a third chromatography material contains a multimodal group, the multimodal group may be, for example, a multimodal weak cation exchanger or a multimodal weak anion exchanger. A non-limiting example is Capto MMC (Cytiva, Sweden), which is a multimodal weak cation exchanger.

[0104] The disclosure further relates to the use of the chromatography system 10 described above, comprising the first, second, and third chromatographic materials described above, for the separation of a biological target compound from one or more impurities, wherein the biological target compound is an adeno-associated virus capsid, e.g., adeno-associated virus serotype 1 (AAV1), adeno-associated virus serotype 2 (AAV2), adeno-associated virus serotype 3 (AAV3), adeno-associated virus serotype 4 (AAV4), adeno-associated virus The invention provides a use selected from serotype 5 (AAV5), adeno-associated virus serotype 6 (AAV6), adeno-associated virus serotype 7 (AAV7), adeno-associated virus serotype 8 (AAV8), adeno-associated virus serotype 9 (AAV9), adeno-associated virus serotype 10 (AAV10), adeno-associated virus serotype 11 (AAV11), adeno-associated virus serotype 12 (AAV12), or adeno-associated virus serotype 13 (AAV13), or capsids of their variants.

[0105] The term “variant” in relation to adeno-associated virus (AAV) serotypes as listed above is intended to mean a modified or manipulated AAV in which the capsid structure has been modified, for example, to improve clinical performance against a specific target organ. As a non-limiting example, an AAV8 variant may include the capsid portion of AAV8, in addition to the capsid portion of another AAV serotype, such as AAV5. However, an AAV8 variant as referred to herein must retain significant structural similarity to an unmodified AAV8 capsid, for example, retaining at least 50%, e.g., 60%, 70%, 80%, or 90% of the external surface structure of an unmodified AAV8 capsid. This applies equally to variants of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, and 13, respectively, compared to unmodified AAV serotypes 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, and 13. Furthermore, as a non-limiting example, in the context of the purification or isolation of AAV8 variants, “variant” is defined herein as an adeno-associated virus having a binding ability to a ligand of a particular chromatographic material that is performatively equivalent to the binding ability of the original AAV8 to the said chromatographic material. This applies equally to variants of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, and 13, respectively, compared to the original AAV serotypes 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, and 13. The particular chromatographic material may be, for example, a chromatographic material as described in more detail elsewhere in this specification. Adeno-associated virus variants can be obtained, for example, through spontaneous mutations or manipulated modifications of one or more nucleotides in the adeno-associated virus genome (i.e., through human-to-human interactions).

[0106] In particular, the adeno-associated virus capsid may be selected from the group consisting of serotypes AAV2, AAV5, AAV8, and AAV9, or from any one variant of the said serotypes.

[0107] Currently preferred non-limiting examples of first chromatography materials include: (i) A support material in the form of porous particles functionalized with ligands containing affinity groups having binding affinity to adeno-associated virus capsids, (ii) Support material in the form of a convection-based membrane structure, including a non-woven web of polymer nanofibers functionalized with a ligand containing affinity groups having binding affinity to adeno-associated virus capsids, (iii) Support material in the form of porous particles functionalized with ligands containing a multimode weak cation exchanger, and (iv) Support material in the form of porous particles functionalized with ligands containing sulfonic acid groups These are some examples.

[0108] Currently preferred third chromatography materials, non-limiting examples include: i. A support material in the form of porous particles functionalized with a ligand defined by formula I (wherein R1, R2, and R3 are each CH3), wherein the ligand optionally binds to the support material via dextran, and optionally the adeno-associated virus capsid that is separated is the capsid of AAV2, AAV5, AAV8, or AAV9. ii. A support material in the form of a convection-based membrane structure comprising a nonwoven polymer nanofiber web functionalized with a ligand defined by formula I (wherein R1, R2, and R3 are each CH3), wherein the isolated adeno-associated virus capsid is the capsid of AAV2, AAV5, or AAV8. iii. A support material in the form of a convection-based membrane structure comprising a nonwoven web of porous particles or polymer nanofibers functionalized with a diethylethanolamine ligand, wherein the adeno-associated virus capsid that is optionally isolated is the capsid of AAV2, AAV5, or AAV8, and iv. A support material in the form of a convection-based membrane structure comprising a nonwoven web of porous particles or polymer nanofibers functionalized with an N,N-diethylethylenediamine ligand, wherein the adeno-associated virus capsid optionally isolated is the capsid of AAV2, AAV5, or AAV8. These are some examples.

[0109] Furthermore, when using the chromatography system 10 for the separation of AAV capsids, it is currently preferable to select a first chromatography material from the above-listed options (i) to (iv), and a third chromatography material from the above-listed options i to iv. Therefore, the system 10 appropriately includes any combination of the above-listed options for the first chromatography material and the above-listed options for the third chromatography material. In addition, the second chromatography material, which is a conditioning chromatography material, is currently preferred to be a desalting chromatography material.

[0110] The disclosure also includes, as illustrated in Figure 3, a method 100 for isolating an adeno-associated virus capsid from one or more impurities, wherein the adeno-associated virus capsid is completely encapsulated in genetic material. a. Step 110, adding a feed containing an adeno-associated virus capsid completely encapsulated in genetic material and one or more impurities to a first chromatography device 70, which comprises a first chromatography material containing a ligand-functionalized support material, wherein the ligand contains affinity groups having binding affinity to the adeno-associated virus capsid, an ion exchange group, or a multimodal group. b. Step 120 of eluting the adeno-associated virus capsid from the first chromatography device into at least one elution fraction. c. Step 130, which involves adding at least one eluate fraction containing the adeno-associated virus capsid obtained in step b to a second chromatography device 72 containing a second chromatography material including a chromatographic material for adjustment. d. Step 140 to obtain the adeno-associated virus capsid in at least one pass-through fraction from a second chromatography device. e. Step 150, which involves adding at least one pass-through fraction containing the adeno-associated virus capsid obtained in step d to a third chromatography device 74 containing a third chromatography material. f. Step 160 to obtain the adeno-associated virus capsid in at least one eluate fraction from a third chromatography device. Includes, The present invention provides a method in which a feed material passes sequentially through first, second, and third chromatography devices to enable the separation of the adeno-associated virus capsid from one or more impurities, wherein the first, second, and third chromatography devices are connected in series.

[0111] The term "eluate" is used in its conventional sense in this field, which refers to the portion of the liquid sample that elutes from the chromatography column after the liquid sample has been loaded onto the chromatography column.

[0112] It should be understood that the first, second, and third chromatographic materials and corresponding chromatographic devices 70, 72, and 74 mentioned in Method 100 are as defined and illustrated in further detail above in connection with the description of the chromatographic system 10.

[0113] In Method 100, the chromatographic materials referred to in steps (a) and (b) may be referred to as capture chromatographic materials, meaning that the chromatographic materials are applied in the capture step, which in the context of liquid chromatography refers to the first step of the separation procedure. In this specification, the capture step performed in steps (a) and (b) of Method 100 achieves significant purification of the biological target compound from soluble impurities. Additional steps, such as clarification and filtration (e.g., tangent flow filtration), may be performed prior to steps (a) and (b).

[0114] Furthermore, the objective of steps (c) and (d) of Method 100 is to adjust or prepare the biological target compound for the conditions required to carry out the subsequent purification steps (e) and (f). More particularly, for the separation of AAV vectors, it is essential to reduce the conductivity of the sample to achieve good separation of complete and empty AAV capsids in the subsequent purification steps. The in-line connected desalting carried out in steps (c) and (d) is an important part of this disclosure and helps to achieve good separation results while reducing process time. Conventionally, the desalting step would be carried out in a separate device that is not in line with other chromatographic devices.

[0115] In Method 100, the chromatography material referred to in steps (e) and (f) may be referred to as polishing chromatography material, meaning that the chromatography material is applied in the polishing step.

[0116] The term "polishing step" in the context of liquid chromatography refers to the final purification step where trace amounts of impurities are removed to leave an active and safe product. The impurities removed during the polishing step are often conformational isomers of the target molecule, i.e., forms of the target molecule with a specific molecular configuration, or suspected leaked products. The polishing step may instead be called the "second purification step."

[0117] In method 100 illustrated in Figure 3, the flow rate used depends on the type of chromatography material used, the dimensions of the chromatography material or the chromatography device containing the chromatography material, and the selected residence time. For example, very high flow rates can be applied to convection-based membrane support materials, such as Fibro®, rather than to porous particles, such as Capto beads.

[0118] Furthermore, as will be understood by those skilled in the art, the different buffers used in Method 100 are selected depending on which chromatographic material is applied and which biological target compound is to be purified.

[0119] In steps (a) and (b) of the method, the pH of the buffer used may vary depending on the type of ligand used. For affinity ligands, the buffer may have a pH of approximately 7–8 for binding (step (a)) and a pH of 1.5–4 for elution (step (b)). For cation exchange ligands and multimode cation exchange ligands, the pH of the buffer may be approximately 4.5–6.5 for both binding (step (a)) and elution (step (b)).

[0120] Steps (e) and (f) of Method 100 may include the step of applying a buffer having a pH of about 6.0 to about 10.5, for example, about 7.0 to about 10.0, for example, about 7.5 to about 9.5. According to a non-limiting example, a pH of about 9.5 may be applied to chromatographic material containing a ligand defined by formula I. Furthermore, a pH of about 7.5 may be applied to chromatographic material containing a ligand defined by formula II or formula III. The buffer may be appropriately selected from buffers commonly recommended for anion exchange chromatography, and may include, for example, tris(hydroxymethyl)aminomethane (i.e., Tris), 1,3-bis(tris(hydroxymethyl)methylamino)propane (i.e., bis-trispropane), triethanolamine, N-methyldiethanolamine, diethanolamine, 1,3-diaminopropane, or ethanolamine.

[0121] The buffer applied in step (f) also includes a compound that may help to elute the capsid bound to the chromatographic material. This compound is not present in the buffer applied in step (e). Non-limiting examples of such compounds are salts, such as salts of monovalent metal ions. Non-limiting examples include NaCl, LiCl, KCl, or other equivalent metal salts known in the art that are suitable for use in salt elution. Furthermore, step (f) may include, for example, applying a gradient of such compounds to improve the elution of adeno-associated virus capsids fully encapsulated in genetic material from the chromatographic material. Such a gradient may be a linear gradient, a step gradient, or a combination thereof. In the current preferred embodiment, step (f) includes applying a step gradient of increased salt concentration, eluting a capsid that is not fully encapsulated at a first lower salt concentration, and eluting a fully encapsulated capsid at a second higher salt concentration.

[0122] The buffer applied in step (f) may also include a compound that improves the separation between capsids completely encapsulated in the genetic material and capsids not completely encapsulated in the genetic material. This compound may or may not be present in the buffer applied in step (e). The compound that improves the separation may be selected from, for example, carbohydrates, divalent metal ions, and detergents.

[0123] As described above, the purpose of steps (c) and (d) is to prepare or modify the biological target compound for the conditions required to carry out the subsequent steps (e) and (f). Therefore, the buffers used in steps (c) and (d) are usually the same as the buffers used in step (e). Generally, buffer 34 used in step (b) of the method for eluting the capsid from the first chromatographic material is not suitable for binding the capsid to the third chromatographic material. Accordingly, buffer 32 used in steps (c) and (e) for equilibrating the feed and loading it onto the second and third chromatographic materials is a different buffer from buffer 34.

[0124] Those skilled in the art can select an appropriate concentration for any one of the buffers listed above.

[0125] Currently preferred, non-limiting examples of suitable buffer combinations used for AAV capsid separation include: Buffer 30 (Buffer A1): 20mM Tris-HCl pH 7.5, 0.5M NaCl Buffer 32 (Buffer A2): 20mM bis-tris-propane (BTP) pH 9.0, 2mM MgCl2 Buffer 34 (Buffer B1): 100 mM Glycine pH 2.5 Buffer 36 (Buffer B2): 20mM Bis-Tris-Propane (BTP) That is the case.

[0126] Method 100 appropriately includes the step of applying a selective valve arrangement 60 configured to enable the separation of the AAV capsid from impurities by passing the feed through first, second, and third chromatography devices 70, 72, and 74 in succession. As will be described in more detail elsewhere in this specification, the selective valve arrangement includes a first chromatography device selective valve 62, optionally further including a second chromatography device selective valve 64, and optionally also including a third chromatography device selective valve 66.

[0127] As can be seen from Figure 3, method 100 may further include an optional step 125 prior to step c, in which the second chromatographic material is equilibrated to the conditions required for the adeno-associated virus capsid to be obtained in step d and in step f.

[0128] Similarly, method 100 may further include an optional step 145 prior to step e, in which the third chromatographic material is equilibrated to the conditions required for the adeno-associated virus capsid to be obtained in step f.

[0129] Method 100 disclosed herein may be a preparation method (preferably) or an analytical method.

[0130] A distinguishing feature of the chromatography system 10 of this disclosure is that it allows for the equilibration of the second and / or third chromatography material in line while performing method 100, in contrast to equilibrating them separately as isolated steps, which would hinder the continuous flow of feeds in line within system 10. This is advantageous because it allows for rapid neutralization of the pH and reduction of the conductivity of the AAV vector sample, contributing to a reduction in the overall time required to carry out the process.

[0131] Since the loading of different chromatography devices within system 10 can be performed partially simultaneously, further time reductions can be achieved. For example, it is not necessary to wait for the sample feed to pass through the second or third device before loading more sample into the first device.

[0132] The capsids isolated from impurities using Method 100 may be capsids of adeno-associated virus serotype 1 (AAV1), adeno-associated virus serotype 2 (AAV2), adeno-associated virus serotype 3 (AAV3), adeno-associated virus serotype 4 (AAV4), adeno-associated virus serotype 5 (AAV5), adeno-associated virus serotype 6 (AAV6), adeno-associated virus serotype 7 (AAV7), adeno-associated virus serotype 8 (AAV8), adeno-associated virus serotype 9 (AAV9), adeno-associated virus serotype 10 (AAV10), adeno-associated virus serotype 11 (AAV11), adeno-associated virus serotype 12 (AAV12), or adeno-associated virus serotype 13 (AAV13), or variants thereof.

[0133] In particular, the adeno-associated virus capsid may be selected from the group consisting of serotypes AAV2, AAV5, AAV8, and AAV9, or from any one variant of the said serotypes.

[0134] The term "variant" is defined elsewhere in this specification.

[0135] This disclosure is not bound by the exemplary embodiments described below, and it should be understood that some conceivable modifications of this disclosure are possible within the following claims. Any reference numerals placed in parentheses within the claims should not be construed as limiting the claims. Where the verb “including” and its conjugations are used, this does not preclude the existence of elements or processes other than those described. Where an element is preceded by the article “a” or “an,” this does not preclude the existence of multiple such elements.

[0136] Chapter on Experiments [Examples]

[0137] (Example 1) Separation of AAV5 complete capsids and empty capsids by affinity capture, desalting, and polishing chromatography connected in a single line. The three-step chromatography process was performed in a single run using an AKTA pure™ chromatography system (Cytiva, Sweden) modified with the addition of two general-purpose valves. Two alternative line-connected configurations for AAV5 capture and polishing were evaluated, using either a bead resin column or a nanofiber membrane adsorbent unit. A hands-free, separated configuration with a single desalting column between the affinity capture device and the polishing column was used to prepare the sample for high-performance complete capsid and empty capsid separation and to ensure low conductivity loading.

[0138] Setting 1 The modified AKTA pure™ chromatography system is - In this specification, the first chromatography device selection valve is referred to as column valve 1 (CV1), - Affinity capture bead material, first column containing 1 mL Capto AVB HiTrap (Cytiva, Sweden), - A second chromatography device selection valve, referred to herein as General Purpose Valve 1 (VV1), is added compared to standard AKTA instruments. - Second column containing desalting material, 50 mL HiPrep 26 / 10 Desalting (Cytiva, Sweden), - A third chromatography device selection valve, referred to herein as the general-purpose valve 2 (VV2), has been added compared to standard AKTA instruments, and - Polishing bead material containing anion exchange ligand, and a third column containing 1 mL Capto Q HiTrap (Cytiva, Sweden) It included.

[0139] The columns connected within the three lines were equilibrated separately before sample loading. In total, approximately 1 × 10⁻⁶ 12 It has a titer of 2.6 × 10⁶ virus particles (VP) / mL and contains the viral genome (VG). 11 AAV5-GFP, clarified, concentrated (by tangent flow filtration), and buffer-exchanged material (tangent flow filtration (TFF), 10× ultrafiltration, and 5× diafiltration) containing 100% virus particles / mL (i.e., approximately 26% complete capsid), was loaded onto the first column using the protocols shown in Table 1 below.

[0140] [Table 1]

[0141] Second setting The modified AKTA chromatography system is - In this specification, the first chromatography device selection valve is referred to as column valve 1 (CV1), - A first chromatography device, more specifically, an affinity-capturing nanofiber material, a membrane adsorbent unit including a 0.4 mL Fibro AVB prototype, - A second chromatography device selection valve, referred to herein as General Purpose Valve 1 (VV1), is added compared to standard AKTA instruments. - A second chromatography device, more specifically, a column containing desalting material, 50 mL HiPrep 26 / 10 Desalting (Cytiva, Sweden), - A third chromatography device selection valve, referred to herein as the general-purpose valve 2 (VV2), has been added compared to standard AKTA instruments, and - A third chromatography device, more specifically, a membrane adsorbent unit containing polished nanofiber material and a 0.4 mL Fibro anion exchange prototype (Fibro-NG-DAX IC 162 μmol / mL). It included.

[0142] The chromatographic devices connected within three lines were separately equilibrated before sample loading. Approximately 1.3×10 11 VP / mL and 3.0×10 10 VG / mL (approximately 23% of full capsid) titer clarified AAV5-GFP material was loaded onto the first chromatographic device and the following protocol in Table 2 was used.

[0143]

Table 2

[0144] Buffer systems used for both the first and second settings: A1: 20 mM Tris-HCl pH 7.5, 0.5 M NaCl<000(0607>A2: 20 mM bis-Tris-propane (BTP) pH 9.0, 2 mM MgCl2 B1: 100 mM glycine pH 2.5 B2: 20 mM BTP

[0145] For the second setting, an affinity capture Fibro prototype and a polishing Fibro prototype were prepared by manufacturing a support material containing a non-woven web of cellulose acetate nanofibers and functionalizing it with a ligand as follows.

[0146] Preparation of the affinity capture fiber prototype: Sheet manufacturing: The support material can be manufactured as described in WO2018 / 011599, or to produce a nonwoven sheet of laminated fibers. A solution of cellulose acetate (CA) with a relative molecular weight of 29,000 g / mol was dissolved in a two-component mixture of glacial acetic acid and ethanol in a 3:1 ratio (the so-called first CA solution). Prior to electrospinning to produce fibers with diameters ranging from 300 to 600 nm, polyethylene oxide dissolved at a concentration of 5% in deionized (DI) water was then added to the first CA solution in an amount of 1.2% of the total volume of CA. Optimized conditions for nanofiber production can be found, for example, in O. Hardick et al., J. Mater. Sci. Vol. 46 (2011), p. 3890. Sheets of material at approximately 20 g / m2 were stacked and subjected to a combination of heating and pressure treatments.

[0147] The resulting support material has an average flow pore diameter of 0.1–2.0 μm, which can be measured using bubble point porometry, for example, with a Porolux 100 porometer (IB-FT GmbH, Germany). Despite variations in standard electrospinning parameter combinations and a decrease in the number of fiber layers, this average flow pore diameter can be easily obtained.

[0148] CA prewash: CA material (100×155mm 2 A 35x sheet of ) was sandwiched between gauze and loaded into the flow reactor. The material was washed for 20 minutes by recirculating 5L of DI water. The reactor was emptied, the washing process was repeated two more times, and the material was stored overnight in the final washed material if necessary.

[0149] Glycidol process: In an 8L beaker, 265g of KOH was added to 4723mL of DI water. The solution was vigorously stirred. Once completely dissolved, glycidol (1350mL or 675mL, depending on the 100% or 50% glycidol-based matrix, respectively) was added and vigorously stirred for 4 minutes. The reactor was emptied of washing water and the KOH / glycidol solution was added to the reactor. The recirculation pump was started. Using a reaction temperature profile of 20°C, 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 with 5L of DI water by recirculation for 20 minutes, then emptied. The washing process was repeated at least 3 times (or as many times as necessary until the final pH was neutral). The sheets were stored overnight in DI water.

[0150] Saponification process: The resulting material, referred to as 0% glycidol, is known as regenerated cellulose (RC) and is synthesized in a saponification reaction in which the terminal acetate groups on the cellulose acetate skeleton are cleaved, leaving behind alcohol groups. This step occurs as a substitution in the glycidol step, followed by the divinyl sulfone (DVS) step (see below). In an 8 L beaker, KOH (132 g) was added to DI water (3149 mL) along with EtOH (1574 mL). The solution was vigorously stirred until completely dissolved. The washing water was emptied from the flow reactor, and the KOH / EtOH solution was added. The recirculation pump was started and run for 6 hours at 22-24°C. The reaction mixture was then removed, and 5 L of DI water was added to the reactor. The recirculation pump was started and run for 20 minutes. The washing water was removed, and the water washing step was repeated three more times.

[0151] Divinyl sulfone (DVS) process: The wash water in the flow reactor was emptied. In an 8 L beaker, Na2CO3 (316 g) was added to DI water (4211 mL). The solution was stirred vigorously until completely dissolved. Acetonitrile (1258 mL) was added while stirring vigorously. The solution was added to the flow reactor. The recirculation pump was started for 4 minutes, and then DVS (1350 mL) was carefully added to the reaction vessel all at once. 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 washing process was repeated three more times. DI water (5 L) at 22-24°C was added to the flow reactor, and the recirculation pump was started and run for 20 minutes. The wash water was removed, and the water washing process was repeated once.

[0152] Fixation of affinity ligands: The selected AAV vector ligand can be immobilized on the support material. In this example, a spin-filtered solution of AAV-conjugated ligand at a concentration of 2.5 mg / ml was used for coupling. A coupling solution of 3.0 M (NH4)2SO4 and 0.1 M NaHCO3 was prepared and adjusted to pH 9. The DVS-treated support material sheet was placed in a sealable container (155 × 105 mm). 2 A certain amount of ligand solution of a desired concentration (0.5–6.0 mg / mL) was placed in a container and added to a certain volume of coupling solution, either 17 or 34 mL, to form a desired total volume of either 25 mL or 50 mL. The container was sealed and placed in an orbital shaker at 22–24°C for 16 hours. After this, the supernatant was collected. All sheets were washed with DI water for 20 minutes. This was repeated 3 times, and the washing supernatant was collected each time for subsequent quantification of fixation efficiency.

[0153] blocking: Blocking with ethanolamine or thioglycerol was performed.

[0154] Ethanolamine blocking: A 0.3 M ethanolamine blocking solution was adjusted to pH 9, and 25 mL was dispensed into each sheet. The containers were sealed and placed in an orbital shaker at 22-24°C for 16 hours. After this, the blocking solution was discarded, and the sheets were washed with DI water for 20 minutes. This was repeated once. The sheets were then 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 2 × DI water washing for 20 minutes on each side.

[0155] Thioglycerol blocking: A blocking solution of 0.288 M thioglycerol, 0.1 M Na2HPO4.12H2O, and 0.001 M EDTA was prepared to pH 8.3, and 25 mL was dispensed into each sheet. The containers were sealed and placed in an orbital shaker at 22-24°C for 16 hours. After this, the blocking solution was discarded, and the sheets were washed with DI water for 20 minutes. This was repeated once. The sheets were then washed with 0.5 M AcOH for 20 minutes, followed by 0.1 M Tris at pH 8.5 and 0.15 M NaCl. This two-step process was repeated once, followed by 2 × DI water washing for 20 minutes each.

[0156] After blocking, the materials were completely immersed in a 1:1:3 glycerol / ethanol / water solution and stored in the refrigerator for at least one hour.

[0157] Ligand density measurement: The AAV vector ligand concentration was calculated using a NanoDrop spectrophotometer. This was used to calculate the mass of the immobilized ligand. One disk was taken from each sheet, and the supernatant was collected / concentrated as described above. The thickness of each disk was measured at five points across the entire sheet using a Mitutoyo micrometer to calculate the average thickness of the sheet. From this, the total volume of the sheet was calculated. The ligand density was calculated by dividing the mass of the immobilized ligand by the volume of the sheet. The average ligand density can be calculated for each batch.

[0158] Using the procedure described above, ligand densities of 3–7 mg / mL of adsorbent were obtained, which can correspond to approximately 0.5–5 μmol / g for some ligands, e.g., 0.7–4.6 μmol / g. At such ligand densities, when using AAV5, it is possible to achieve a static binding capacity of AAV5 capsid exceeding 1E15 per mL of adsorbent. Ligand densities were measured using amino acid analysis or UV spectroscopy of the pre-fixation solution, post-fixation solution, and wash-fixation solution.

[0159] Preparation of polished Fibro prototypes and Fibro-VS-DAX(NG): This prototype includes an anionic ligand containing an N,N-diethylethylenediamine group (hereinafter referred to as "DAX") by using an activated linker containing vinyl sulfone (VS) and not glycidol (hereinafter referred to as "non-glycidol" or "NG" instead).

[0160] Fibro-VS(NG) Preparation: The Fibro cellulose acetate (CA) sheet, prepared as described above for the affinity-capturing Fibro prototype, was inserted into the reactor with gauze in between. The sheet was washed with MQ water (3 × 5 L, 15 minutes per wash). The reactor was drained, the temperature was raised to 30°C, and KOH reaction solution (309 g in 33% EtOH in 5500 mL) was added. The solution was circulated for 2 hours. The reactor was drained, and the sheet was washed with MQ water (4 × 5 L) and acetone (2 × 5 L). The reactor was drained, and the sheet was left to dry overnight. The reactor was then filled with 3 L of sodium carbonate buffer (306.6 g of Na2CO3 in 2960 mL of MQ water) and acetonitrile (1224 mL). The solution was cooled to 15°C and then pumped into the reaction chamber. Divinyl sulfone (DVS) (945 mL) was added directly to the reaction chamber. The circulation was restarted, and the reaction was allowed to proceed at room temperature for 6 hours. The reaction mixture was drained, and the sheet was washed with 1:1 acetone / MQ water (4 x 5 L), followed by MQ water (4 x 5 L).

[0161] DAX coupling: Sheets were placed in food boxes and washed with MQ water (4 × 100 mL, 20 minutes) on a vibrating table (84 rpm). 25 mL of MQ water and 2 mL of DAX were added to each box. The boxes were placed on a heated vibrating table (45°C, 75 rpm) for 23 hours. The reaction solution was decanted, and the sheets were washed with MQ water (6 × 100 mL, 20 minutes).

[0162] Deactivation: Thioglycerol (15.2 mL) was added to Tris buffer (600 mL, Tris 0.1 M, EDTA 0.001 M, pH 10) and the pH was adjusted to 8.6. This solution (153 mL) was added to a food container, the reaction mixture was sealed, and the container was placed on a shaking table (84 rpm) at room temperature for 16 hours. The reaction solution was decanted, and the sheet was washed with 20% EtOH (3 × 100 mL, 20 min) and MQ water (3 × 100 mL, 20 min). Titration showed an ionic capacity of 162 μmol / mL for the membrane.

[0163] Preparation of chromatography devices: Each of the Fibro chromatography material prototypes was assembled in a HiTrap Fibro® (Cytiva, Sweden) 0.4 mL type chromatography device, which is referred to herein as a Fibro unit.

[0164] result Complete and empty separations were similar to the in-line connected workflows and the corresponding step-by-step workflows of comparisons involving manual neutralization, dilution, or buffer exchange (not shown).

[0165] As shown in Figures 4A and 4B, the anion exchange polishing chromatograms for the first and second settings showed similar separation results with the predicted UV260:280 ratio, i.e., peak 2, for the complete capsid.

[0166] For the first setting, empty capsids bound to and eluted the resin anion exchange polishing material at 33% of buffer B (peak 1), while complete capsids bound and eluted at 100% of buffer B (peak 2). See Figure 4A for details.

[0167] In the second setting, most of the empty capsids did not bind to the Fibro anion-exchanged abrasive material, but passed through the abrasive material unimpeded, and therefore the profile of Peak 1 is broader and flatter than in the first setting. Only a small amount of empty capsids bound to the abrasive material and eluted at 2% of Buffer B. Complete capsids bound and eluted at 100% of Buffer B (Peak 2), see Figure 4B.

[0168] (Example 2) The experimental design for separating fully sealed AAV5 capsids from empty AAV5 capsids was carried out using the same apparatus and samples as in Example 1 above, with the following variations.

[0169] Regarding the first chromatography material (used in the capture step): 1) Ligands containing cation exchange groups 2) Ligands containing multimodal groups

[0170] For both of the chromatographic materials listed above, one or more pass-through steps may be added between the capture and conditioning steps. For any of such additional pass-through steps, a suitable chromatographic material may include porous beads having an internally porous core and an externally porous shell, where the core is capable of binding to molecules via hydrophobic interactions, and the pore size of the shell prevents particles having a size of ≥20 nm from contacting the core.

[0171] (Example 3) Isolation of capsids from different adeno-associated virus serotypes The experimental design for the isolation of complete capsids from empty capsids of adeno-associated virus serotypes AAV1, AAV2, AAV4, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, and AAV13 is carried out according to the variable conditions of Examples 1 and 2 above.

[0172] (References) [Explanation of Symbols]

[0173] 10 Chromatography Systems 20 Buffer valve arrangement 22 First buffer selection valve 24. Second buffer selection valve 30 buffers 32 buffers 34 buffers 36 buffers 40 Pump placement 42 System Pumps 44 Sample pump 46 samples 50 Injection valve 60 Selectable Valve Arrangement 62 First chromatography device selection valve 64 Second chromatography device selection valve 66 Selection valve for the third chromatography device 70 Chromatography devices 72 Second Chromatography Device 74. The third chromatography device 80 UV detector 82 Conductivity detector 84 Outlet valve 86 Fraction Collector 100 ways 110 Addition process 120 Elution process 125 Equilibrium process 130 Addition process 140 Process for obtaining adeno-associated virus capsid 145 Equilibriumization process 150 Addition process 160 Process for obtaining adeno-associated virus capsid

Claims

1. A method (100) for isolating an adeno-associated virus capsid from one or more impurities, wherein the adeno-associated virus capsid is completely encapsulated in genetic material. a. A step (110) of adding a feed containing an adeno-associated virus capsid completely encapsulated in genetic material and one or more impurities to a first chromatography device (70) comprising a first chromatography material comprising a ligand-functionalized support material, wherein the ligand comprises affinity groups having binding affinity to the adeno-associated virus capsid, an ion exchange group, or a multimode group. b. A step (120) of eluting the adeno-associated virus capsid from the first chromatography device into at least one elution fraction, c. Adding the at least one eluate fraction containing the adeno-associated virus capsid obtained in step b to a second chromatography device (72) containing a second chromatography material including a chromatographic material for adjustment (130) d. A step (140) of obtaining the adeno-associated virus capsid in at least one pass-through fraction from the second chromatography device, e. A step (150) of adding the at least one pass-through fraction containing the adeno-associated virus capsid obtained in step d to a third chromatography device (74) containing a third chromatography material. f. A step of obtaining the adeno-associated virus capsid in at least one eluate fraction from the third chromatography device (160) Includes, A method wherein the feed passes sequentially through the first, second, and third chromatography devices to enable the separation of the adeno-associated virus capsid from one or more impurities, and the first, second, and third chromatography devices are connected in series.

2. The method according to claim 1, comprising the step of applying a selective valve arrangement (60) configured to enable the separation by passing the feed through the first, second, and third chromatography devices in succession, wherein the selective valve arrangement includes a first chromatography device selective valve (62).

3. The method according to claim 2, wherein the selection valve arrangement further includes a second chromatography device selection valve (64).

4. The method according to claim 3, wherein the selection valve arrangement further includes a third chromatography device selection valve (66).

5. The method according to any one of claims 1 to 4, further comprising, before step c, a step (125) of equilibrating the second chromatographic material to the conditions required for the adeno-associated virus capsid to be obtained in step d and in step f.

6. The method according to any one of claims 1 to 5, further comprising, before step e, a step (145) of equilibrating the third chromatographic material to the conditions required for the adeno-associated virus capsid to be obtained in step f.

7. The adeno-associated virus capsid is adeno-associated virus serotype 1 (AAV1), adeno-associated virus serotype 2 (AAV2), adeno-associated virus serotype 3 (AAV3), adeno-associated virus serotype 4 (AAV4), adeno-associated virus serotype 5 (AAV5), adeno-associated virus serotype 6 (AAV6), adeno-associated virus serotype 7 (AAV7), adeno-associated virus serotype 8 (AAV8), The method according to any one of claims 1 to 6, wherein the adeno-associated virus serotype 9 (AAV9), adeno-associated virus serotype 10 (AAV10), adeno-associated virus serotype 11 (AAV11), adeno-associated virus serotype 12 (AAV12), or adeno-associated virus serotype 13 (AAV13), or a variant thereof, optionally a capsid of AAV2, AAV5, AAV8, or AAV9.

8. The method according to any one of claims 1 to 7, wherein the one or more impurities include an adeno-associated virus capsid that is not completely encapsulated in the genetic material.

9. The method according to any one of claims 1 to 8, wherein the third chromatography material comprises a support material functionalized with a ligand, and the ligand comprises an anion exchange group or a multimode group.

10. The ligand of the third chromatography material is of formula I 【Chemistry 1】 (In the formula, R 1 R is selected from H and C1-C3 alkyl groups. 2 and R 3 The is independently selected from H, C1-C3 alkyl, CH2OH, and CH2CHOHCH3, preferably R 1 , R 2 and R 3 Each of them is CH3. The method according to claim 9, comprising an anion exchange group as defined by

11. The ligand of the third chromatography material is, Formula II 【Chemistry 2】 (In the formula, m is an integer between 1 and 3. R 1 and R 2 is independently selected from C1-C3 alkyl, R 3 and R 4 is independently selected from C1-C3 alkyl and CH2CHOHCH3, R 5 is selected from hydrogen, C1-C3 alkyl and CH2CHOHCH3) It includes an anion exchange group as defined by, However, if m is 1, the ligand is the following formula III 【Transformation 3】 (In the formula, n is an integer from 0 to 3, However, if n is 0, R 3 and R 4 R is independently selected from C1-C3 alkyl groups. 5 (This is conditional on the substance being hydrogen or CH2CHOHCH3.) The method according to claim 9, provided that it is defined by [the specified method].

12. The ligand of the third chromatography material is, 【Chemistry 4】 (In the formula, X is independently H, OH and C for each entity) 1～3 Selected from the base, R 1 , R 2 , R 3 and R 4 H and C 1～3 Selected independently from the original, C 3 The base is either a straight chain or a branched chain. C 1～3 The base is HR, OC 1～2 SC 1～2 NH, NHR and NR 2 Includes a base that is independently selected from, R is H and C 1～3 (Selected from the base) The method according to claim 9, comprising an anion exchange group as defined by

13. The method according to any one of claims 1 to 12, wherein the ligand of the first chromatography material comprises an affinity group having binding affinity to an adeno-associated virus capsid.

14. The ligand of the first chromatography material is of formula I 【Transformation 5】 (In the formula, R 1 R is selected from H and C1-C3 alkyl groups. 2 and R 3 The is independently selected from H, C1-C3 alkyl, CH2OH, and CH2CHOHCH3, preferably R 1 , R 2 and R 3 Each of them is CH3. The method according to any one of claims 1 to 12, comprising an anion exchange group as defined by

15. The chromatography system according to any one of claims 1 to 12, wherein the ligand of the first chromatography material comprises a cation exchange group, and optionally the cation exchange group is a sulfonic acid group.

16. The method according to any one of claims 1 to 12, wherein the ligand of the first chromatography material comprises a multimode group.

17. The method according to claim 16, wherein the multimode group is a multimode weak cation exchange group.

18. The method according to claim 16, wherein the multimode group is a multimode weak anion exchange group.

19. The method according to any one of claims 1 to 18, wherein the support material of the first chromatography material comprises a film structure, nanofibers, monoliths, porous particles, non-porous particles, or an expanded bed medium.

20. The method according to any one of claims 1 to 19, wherein the second chromatography material includes a size exclusion chromatography material.

21. The method according to any one of claims 1 to 20, wherein the support material for the third chromatography material comprises a film structure, nanofibers, monoliths, porous particles, non-porous particles, or an expanded bed medium.

22. A buffer valve arrangement (20) configured to allow independent control of a first buffer supply and a second buffer supply, A pump configuration (40) configured to supply a first buffer supply, a second buffer supply, and a supply containing a biological target compound and one or more impurities, A selection valve arrangement (60) including a first chromatography device selection valve (62), A first chromatography device (70) comprising a first chromatography material comprising a support material functionalized with a ligand, wherein the ligand comprises an affinity group having binding affinity to a biological target compound, an ion exchange group, or a multimode group, A second chromatography device (72) comprising a second chromatography material including a chromatographic material for adjustment, A third chromatography device containing a third chromatography material (74) Includes, A chromatography system (10) wherein a feed containing a biological target molecule and one or more impurities is passed through the first, second, and third chromatography devices in series, the selective valve arrangement is configured to enable the separation of the biological target compound from impurities, and the first, second, and third chromatography devices are configured to be connected in series.

23. The chromatography system according to claim 22, wherein the selection valve arrangement further includes a second chromatography device selection valve (64).

24. The chromatography system according to claim 23, wherein the selection valve arrangement further includes a third chromatography device selection valve (66).

25. The chromatography system according to any one of claims 22 to 24, wherein the third chromatography material comprises a support material functionalized with a ligand, and the ligand comprises an anion exchange group or a multimode group.

26. The ligand of the third chromatography material is of formula I 【Transformation 6】 (In the formula, R 1 R is selected from H and C1-C3 alkyl groups. 2 and R 3 The is independently selected from H, C1-C3 alkyl, CH2OH, and CH2CHOHCH3, preferably R 1 , R 2 and R 3 Each of them is CH3. The chromatography system according to claim 25, comprising an anion exchange group defined by...

27. The ligand of the third chromatography material is, Formula II 【Transformation 7】 (In the formula, m is an integer between 1 and 3. R 1 and R 2 R is independently selected from C1-C3 alkyl groups. 3 and R 4 R is independently selected from C1-C3 alkyl and CH2CHOHCH3. 5 (Selected from hydrogen, C1-C3 alkyl, and CH2CHOHCH3) It includes an anion exchange group as defined by, However, if m is 1, the ligand is the following formula III 【Transformation 8】 (In the formula, n is an integer from 0 to 3, However, if n is 0, R 3 and R 4 R is independently selected from C1-C3 alkyl groups. 5 (This is conditional on the substance being hydrogen or CH2CHOHCH3.) The chromatography system according to claim 25, provided that it is defined by

28. The ligand of the third chromatography material is, 【Chemistry 9】 (In the formula, X is independently H, OH and C for each entity) 1～3 Selected from the base, R 1 , R 2 , R 3 and R 4 H and C 1～3 Selected independently from the original, C 3 The base is either a straight chain or a branched chain. C 1～3 The base is HR, OC 1～2 SC 1～2 NH, NHR and NR 2 Includes a base that is independently selected from, R is H and C 1～3 (Selected from the base) The chromatography system according to claim 25, comprising an anion exchange group defined by...

29. The chromatography system according to any one of claims 22 to 28, wherein the ligand of the first chromatography material comprises an affinity group having binding affinity to an adeno-associated virus capsid.

30. The ligand of the first chromatography material is of formula I 【Chemistry 10】 (In the formula, R 1 R is selected from H and C1-C3 alkyl groups. 2 and R 3 The is independently selected from H, C1-C3 alkyl, CH2OH, and CH2CHOHCH3, preferably R 1 , R 2 and R 3 Each of them is CH3. A chromatography system according to any one of claims 22 to 28, comprising an anion exchange group as defined by:

31. The chromatography system according to any one of claims 22 to 28, wherein the ligand of the first chromatography material comprises a cation exchange group, and optionally the cation exchange group is a sulfonic acid group.

32. The chromatography system according to any one of claims 22 to 28, wherein the ligand of the first chromatography material comprises a multimode group, and optionally the multimode group is a multimode weak cation exchange group or a multimode weak anion exchange group.

33. The chromatography system according to any one of claims 22 to 32, wherein the support material of the first chromatography material comprises a film structure, nanofibers, monoliths, porous particles, non-porous particles, or an expanded bed medium.

34. The chromatography system according to any one of claims 22 to 33, wherein the second chromatography material includes a size exclusion chromatography material.

35. The chromatography system according to any one of claims 22 to 34, wherein the support material for the third chromatography material comprises a film structure, nanofibers, monoliths, porous particles, non-porous particles, or an expanded bed medium.

36. The aforementioned biological target compound is an adeno-associated virus capsid, for example, adeno-associated virus serotype 1 (AAV1), adeno-associated virus serotype 2 (AAV2), adeno-associated virus serotype 3 (AAV3), adeno-associated virus serotype 4 (AAV4), adeno-associated virus serotype 5 (AAV5), adeno-associated virus serotype 6 (AAV6), adeno-associated virus serotype 7 (AAV7), adeno-associated virus serotype 8 (AAV8), adeno-associated virus serotype 9 (AAV9). Use of the chromatography system according to any one of claims 22 to 35 for the separation of one or more impurities of a biological target compound, selected from adeno-associated virus serotype 10 (AAV10), adeno-associated virus serotype 11 (AAV11), adeno-associated virus serotype 12 (AAV12) or adeno-associated virus serotype 13 (AAV13), or their variants, optionally selected from capsids of AAV2, AAV5, AAV8 or AAV9.

37. The first chromatography material described above is i. Support material in the form of porous particles functionalized with ligands containing affinity groups having binding affinity to adeno-associated virus capsids. ii. Support material in the form of a convection-based membrane structure, including a nonwoven polymer nanofiber web functionalized with ligands containing affinity groups having binding affinity to adeno-associated virus capsids. iii. Support materials in the form of porous particles functionalized with ligands containing multimode weak cation exchangers, and iv. Support material in the form of porous particles functionalized with ligands containing sulfonic acid groups The use according to claim 36, selected from the group consisting of the following.

38. The third chromatography material is i.Formula I (wherein, R 1 , R 2 and R 3 A support material in the form of porous particles functionalized with ligands defined by (each of which is CH3), wherein the ligands are optionally bound to the support material by dextran, and optionally the adeno-associated virus capsid is the capsid of AAV2, AAV5, AAV8, or AAV9. ii.Formula I (wherein, R 1 , R 2 and R 3 A support material in the form of a convection-based membrane structure comprising a nonwoven web of polymer nanofibers functionalized with ligands defined by (each of which is CH3), wherein the adeno-associated virus capsid is the capsid of AAV2, AAV5, or AAV8, iii. A support material in the form of a convection-based membrane structure comprising a nonwoven web of porous particles or polymer nanofibers functionalized with a diethylethanolamine ligand, wherein the adeno-associated virus capsid is the capsid of AAV2, AAV5, or AAV8, and iv. A support material in the form of a convection-based membrane structure comprising a nonwoven web of porous particles or polymer nanofibers functionalized with an N,N-diethylethylenediamine ligand, wherein the adeno-associated virus capsid is the capsid of AAV2, AAV5, or AAV8. The use according to claim 36 or 37, selected from the group consisting of the following.

Images