Chromatography systems, their use, and methods for separating envelope or membrane-bound biological particles.

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 an anion exchange group or an affinity group having binding affinity to the biological target compound; a second chromatography device comprising a second chromatography material comprising a control 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 compound and one or more impurities through the first and second chromatography devices in series; and the first and second chromatography devices are configured to be connected in series. This disclosure also provides the use of the chromatography system and a method for separating enveloped or membrane-like biological particles from one or more impurities.

Description

[Technical Field]

[0001] This disclosure relates to the field of isolation of biological target compounds, such as enveloped virus particles. This disclosure covers chromatographic systems and their use for separating enveloped or membranous biological particles from impurities, as well as methods for separating enveloped or membranous biological particles 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.

[0004] Viral vectors commonly used in pharmaceuticals include enveloped viral particles, such as lentiviruses (LVs). Extracellular vesicles (EVs) produced and released by cells are another example of vectors with potential in cell and gene therapy.

[0005] Lentiviruses are classified as retroviruses and possess a single-stranded RNA genome with reverse transcriptase. Lentiviruses have a viral envelope containing glycosylated proteins that act as ligands with affinity to receptors on the outer cell membrane surface of host cells. Once inside a cell, the virus transcribes its genetic material. The advantage of using lentiviruses as viral vectors is that, unlike other retroviruses which only enter mitotic cells, they enter the nuclear envelope of both dividing and non-dividing cells. Many cell types in adult individuals do not divide, and lentiviruses may be the only option for transferring genetic material to such cells.

[0006] To produce lentiviruses, several plasmids are transfected into so-called packaging cell lines. One or more plasmids, commonly referred to as packaging plasmids, encode virion proteins, such as the capsid and reverse transcriptase. Another plasmid contains the genetic material delivered by the vector. This is transcribed to produce a single-stranded RNA viral genome, characterized by the presence of a ψ (psi) sequence. This sequence is used to package the genome into virions. For lentiviruses to be used in gene therapy, after transfection, the virions must be purified from host cell proteins and DNA, as well as cellular impurities such as excess plasmids. Typically, the host cells harvested to produce lentiviruses are treated with nucleases, and the lentiviruses are purified using several filtration techniques, such as conventional flow microfiltration, ultrafiltration, and dialysis, to reduce the level of impurities to an acceptable level.

[0007] However, because lentiviruses are unstable, sensitive to shear forces and buffer components such as salts, and degrade rapidly at room temperature, current downstream purification processes for lentiviruses often result in low recovery rates of infectious viruses (typically 10-20%). Time-consuming multi-step processes are also not considered beneficial. Lentiviruses have been reported to be stable in a very narrow pH range, 7.0-7.4 (Kinetic Analyses of Stability of Simple and Complex Retroviral Vectors, F. Higashikawa et al., Virology Vol. 280, pp. 124-131 (2001)), and in treatment solutions with a conductivity window of <0.2M NaCl (Process development of lentiviral vector expression, purification and formulation for gene therapy applications, Doctoral thesis, Sara Nilsson, UCL, 2016), which makes downstream purification processes difficult. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] US6,428,707 [Patent Document 2] WO2009131526 [Non-patent literature]

[0009] [Non-Patent Document 1] Kinetic Analyses of Stability of Simple and Complex Retroviral Vectors, F. Higashikawa et al., Virology Vol. 280, pp. 124-131 (2001) [Non-Patent Document 2] Process development of lentiviral vector expression, purification and formulation for gene therapy applications, Doctoral thesis, Sara Nilsson, UCL, 2016 [Overview of the project] [Problems that the invention aims to solve]

[0010] Therefore, in this field, there is a continuous need for novel chromatography systems and purification processes that offer higher capacity and faster processing, and generally result in better process economics. [Means for solving the problem]

[0011] 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 when 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 when operating conventional chromatography systems.

[0012] 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 an anion exchange group or a biological target compound, A second chromatography device comprising a second chromatography material including a chromatographic material for adjustment, Includes, The present invention relates to a chromatography system in which a feed containing a biological target compound and one or more impurities is passed through a first chromatography device and a second chromatography device in series, wherein the selective valve arrangement is configured to allow separation of the biological target compound from impurities, and the first chromatography device and the second chromatography device are connected in series.

[0013] This disclosure also relates to the use of a chromatographic system for the separation of one or more biological target compounds disclosed herein from impurities, wherein the biological target compound is selected from the group consisting of envelope or membranous biological particles, such as enveloped virus particles, extracellular vesicles, and virus-like particles. In some cases, enveloped virus particles are lentiviral particles. In some cases, extracellular vesicles are exosomes, providing a useful application.

[0014] In addition, the present disclosure relates to a method for separating envelope or membrane-like biological particles from one or more impurities, a. A first chromatography device comprising a first chromatography material comprising a support material functionalized with a ligand, the ligand comprising an anion exchange group or an affinity group having a binding affinity to an envelope or membrane-like biological particle, adding a feed comprising the envelope or membrane-like biological particle and one or more impurities to the first chromatography device; b. Eluting the envelope or membrane-like biological particle from the first chromatography device into at least one eluate fraction; c. Adding at least one eluate fraction comprising the envelope or membrane-like biological particle obtained in step b to a second chromatography device comprising a second chromatography material comprising an adjustment chromatography material; d. Obtaining the envelope or membrane-like biological particle in at least one flow-through fraction from the second chromatography device comprising; The method is directed to a feed passing continuously through the first chromatography device and the second chromatography device to enable separation, with the first chromatography device and the second chromatography device connected in series.

[0015] Preferred embodiments of the present disclosure will be described below in the detailed description and the dependent claims. Note that the present disclosure relates to all possible combinations of the features listed in the claims.

[0016] 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

[0017] [Figure 1A] It is a schematic diagram of a chromatography system according to an embodiment of the present disclosure. [Figure 1B] It is a schematic diagram of a chromatography system according to an embodiment of the present disclosure. [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 lentiviral particles from impurities according to this disclosure. [Figure 4] This graph shows chromatograms for the separation of lentiviral particles on different chromatography system settings, as described in Example 1 of this specification. [Modes for carrying out the invention]

[0018] 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.

[0019] This disclosure addresses problems related to existing chromatography systems and processes for separating biological target compounds from impurities, as illustrated in Figure 1A. 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 support material functionalized with a ligand, wherein the ligand contains an affinity group having binding affinity to an anion exchange group or a biological target compound. 72 second chromatography device containing a second chromatography material including a control chromatography material Includes, This is solved or at least mitigated by providing a chromatography system 10 in which a feed containing a biological target compound and one or more impurities is passed through a first chromatography device and a second chromatography device in succession, the selective valve arrangement is configured to allow separation of the biological target compound from impurities, and the first chromatography device and the second chromatography device are configured to be connected in series.

[0020] 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.

[0021] The pump arrangement 40 includes at least one pump, and possibly two or more pumps.

[0022] Figure 1B illustrates an embodiment in which the chromatography system 10 further includes, in addition to the components shown in Figure 1A, a third chromatography device 74 comprising a third chromatography material including porous beads having an internally porous core and an externally porous shell, wherein the core is capable of binding to molecules via hydrophobic interactions, the pore size of the shell prevents particles having a size of ≥20 nm from contacting the core, the third chromatography device 74 is configured to be connected in series with the first chromatography device 70 and the second chromatography device 72, and the selective valve arrangement 60 is configured to enable separation by passing the feed through the first, second and third chromatography devices in succession.

[0023] The selective valve arrangement 60 allows two or three chromatography devices to be connected in series, thereby enabling two or three chromatographic purification processes to be performed in a line within the chromatography system. This avoids interruptions and delays that would conventionally occur due to the manual operation of various steps between processes, saving time and effort to 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 press of a button and continued to the end without further intervention by the user. Thus, this disclosure may be said to provide a so-called "plug-and-play" chromatography system, meaning a system intended to work perfectly without being reconfigured or adjusted by the user when first used or connected.

[0024] More specifically, the chromatography system 10 of this disclosure offers the following general advantages compared to conventional chromatography systems. - 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 conductivity of the sample, enabling high-performance isolation of viral particles, such as lentiviruses: - No holding time - No freezing-thawing cycle - No sample preparation (pH and conductivity) or handling between chromatography steps.

[0025] Regarding the purification of lentiviral vectors, there is a further advantage in that lentiviral vectors sensitive to high salt concentrations are exposed to high-salt conditions for a shorter time (minutes versus hours). Direct buffer exchange to the appropriate pH using stabilizers (e.g., sucrose) is advantageous in improving infectivity yield. However, given that lentiviruses are unstable, sensitive to shear forces, buffer components, e.g., salt, and can degrade rapidly at room temperature, it is still surprising that lentiviral particles can withstand two- or three-step purification processes connected in such lines.

[0026] 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 lentiviral 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 lentiviral purification process while achieving performance similar to conventional purification settings with respect to lentiviral recovery. Given the coupled chromatography load to keep the lentiviral stable under optimal conditions without manual intervention and to maintain high chromatographic performance, it is remarkable that a complete capture and polishing process can be successfully performed using a two- or three-step purification process connected in such a line.

[0027] 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.

[0028] 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.

[0029] More specifically, the first buffer selection valve 22 may be configured to control the supply 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 supply 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. This is applicable when the biological target compound is a lentiviral particle.

[0030] 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).

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] A non-limiting example of a chromatography system that may be used is the AKTA pure chromatography system (Cytiva, Sweden). A 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. Another non-limiting example of a chromatography system that may 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.

[0036] 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).

[0037] 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.

[0038] 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.

[0039] The term "chromatographic material" is used herein to describe the type of separation matrix.

[0040] 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.”

[0041] 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.

[0042] 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.”

[0043] 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.

[0044] Of particular interest to the analytes disclosed herein are enveloped or membranous biological particles, such as enveloped virus particles, extracellular vesicles (e.g., exosomes), and virus-like particles. A particularly interesting example of enveloped virus particles is lentiviral particles.

[0045] The term “viral particle” is used herein to describe a complete infectious virus 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), and 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 nucleocapsid of an enveloped virus is 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 insert containing the desired genetic material.

[0046] 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 term “viral particle.”

[0047] The term "virus-like particle" is intended to refer to a virus-derived structure composed of one or more different molecules that possesses the ability to self-assemble, mimics the morphology and size of a virus particle, but lacks genetic material and is incapable of infecting host cells.

[0048] The term "impurity" is intended to mean any molecule or substance present in a liquid sample that is not the desired biological target compound. In the context of this invention, "impurity" primarily refers to host cell proteins (HCPs) and host cell DNA. However, the term "impurity" can also generally refer to aggregates, such as aggregates of the biological target compound, and fragments of the biological target compound.

[0049] The term "surface" in this specification means all external surfaces, including the outer surface of a porous support and the surface of a pore.

[0050] 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.

[0051] A non-limiting example of a membrane-like structure is the Mustang® membrane (Pall Corporation, USA).

[0052] 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.

[0053] A non-restrictive example of a monolith is CIMmultus® (Sartorius, Germany).

[0054] A non-limiting example of porous particles is Capto beads (Cytiva, Sweden), which are substantially spherical particles with a diameter of approximately 90 μm.

[0055] A non-exclusive example of an extended bed medium is STREAMLINE resin (Cytiva, Sweden).

[0056] As further mentioned above, the ligand of the first chromatography material may include an anion exchange group or an affinity group having binding affinity to the envelope or membrane-like biological particles.

[0057] 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.

[0058] 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

[0059] [ka]

[0060] (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:

[0061] The wavy lines represent the supporting material containing the linker. Ligands can bond to the carbon atoms of the linker.

[0062] 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.

[0063] Another non-limiting example of a chromatography material containing a quaternary amine ligand is monolithic CIMmultus® QA (Sartorius, Germany).

[0064] If the ligand of the first chromatography material contains a potent or partially potent anion exchange group, this is instead formula II

[0065] [ka]

[0066] (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 is given by the following formula III

[0067] [ka]

[0068] (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].

[0069] 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.

[0070] 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.

[0071] If the ligand of the first 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.

[0072] More specifically, if the ligand of the first chromatography material contains a weak anion exchange group, the ligand is of formula IV

[0073] [ka]

[0074] (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 Selected independently from the group, the C3 group is either linear or branched, C 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:

[0075] 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.

[0076] If the ligand of the first chromatography material contains affinity groups, it may have binding affinity to biological target compounds selected from the group consisting of envelopes or membranous biological particles, such as enveloped virus particles, extracellular vesicles (e.g., exosomes), and virus-like particles. For example, the ligand may have binding affinity to lentiviral particles.

[0077] 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 adjusts or prepares the sample feed or solution that passes through the chromatography material. Adjustment may include, for example, desalting of 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. Alternatively, adjustment may include, or may involve changing the pH of the sample feed or solution, i.e., decreasing or increasing the pH. Furthermore, the adjusting chromatography material may further achieve the removal of any low molecular weight impurities.

[0078] 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.

[0079] The third chromatography material comprises porous beads having an internally porous core and an externally porous shell. The porosity of the core and shell may be the same or different. However, the porosity of the shell is at least such that particles having a size of ≥20 nm, such as envelopes or membrane-like biological particles, do not penetrate the shell and come into contact with the core.

[0080] Enveloped viruses generally have a size ranging from 20 nm to a maximum of 300 nm, depending on the type of virus. Lentiviruses can be 80–120 nm, often in the range of 100–120 nm. Enveloped viruses can be larger than non-enveloped viruses, such as adenoviruses, which are packaged only in a capsid. Enveloped viruses are often larger than adeno-associated viruses, which are typically about 25 nm in size. Extracellular vesicles, such as exosomes, can have a size ranging from 30–180 nm.

[0081] Shells are typically hydrophilic. Therefore, the surface of porous beads that can reach larger materials, such as envelopes or membranous biological particles, is hydrophilic and does not irreversibly adsorb or denature proteins. Shells can be formed from hydrophilic materials that expose multiple polar groups, such as oxygen and / or nitrogen atoms. Examples of such polar groups are hydroxyl, amino, carboxy, sulfonate (S and SP ligands) esters and lower alkyl (e.g., (-CH2CH2O-)nH (wherein n is an integer 2, 3, 4 and greater)) ethers.

[0082] The core strongly binds to biomolecules, such as proteins and DNA, through hydrophobic interactions. In the current context, impurities, such as residual host cell proteins and DNA (preferably fragmented DNA), can enter and bind to the porous beads, while target envelopes or membranous biological particles cannot enter but are obtained in the pass-through fraction.

[0083] The core may be hydrophobic. Preferably, the hydrophobic core is hydrophilic itself and is based on a hydrophilic material, such as a hydrophilic polymer, and is functionalized with a hydrophobic interaction ligand to provide the desired hydrophobicity. However, the core may be hydrophobic itself or be based on a hydrophobic polymer. For example, styrene / ethylstyrene / DVB, vinyl ethers and acrylates containing hydrophobic substituents, as well as fluoroalkane-containing polymers, are considered.

[0084] The hydrophobic interaction ligand may include aliphatic hydrocarbons, such as C1-C30 alkyl groups, preferably C4-C16 alkyl groups, and / or aromatic hydrocarbons, such as phenyl, anthracene, and naphthalene.

[0085] The hydrophilic polymer that can form the shell and, optionally, the core, is a polysaccharide, such as agarose. For example, both the core and the shell may contain cross-linked agarose. Porous core-shell beads can be manufactured as described in WO2009131526. In particular, both the core and the shell may be made of agarose, and the core may be functionalized with a hydrocarbon interaction ligand containing 4 to 16 carbon atoms, preferably an octyl ligand. Useful chromatographic media are available under the trade names Capto® Core 400 and Capto® Core 700, respectively, from Cytiva, Sweden. Capto® Core 700 has a shell with a 700 kDa size exclusion cutoff and a core with a multimode octylamine ligand.

[0086] The disclosure further provides the use of the chromatography system 10 described above, comprising the first and second chromatographic materials described above, and optionally a third chromatographic material described above, for the separation of a biological target compound from one or more impurities, wherein the biological target compound is selected from the group consisting of envelopes or membranous biological particles, such as enveloped virus particles (e.g., lentiviruses), extracellular vesicles (e.g., exosomes), and virus-like particles.

[0087] Currently preferred non-limiting examples of first chromatography materials include: i. A support material in the form of porous particles functionalized with a diethylethanolamine ligand, wherein the ligand is optionally attached to the support material by a polymer, such as dextran. ii. Support material in the form of a convection-based membrane structure containing a nonwoven web of polymer nanofibers functionalized with diethylethanolamine ligand, iii. Support material in the form of a convection-based membrane structure containing a nonwoven web of polymer nanofibers functionalized with N,N-diethylethylenediamine ligand, iv. A support material in the form of a convection-based membrane structure comprising a nonwoven web of polymer nanofibers functionalized with ligands having affinity groups that have binding affinity to biological target compounds selected from the group consisting of envelopes or membrane-like biological particles, such as enveloped virus particles, extracellular vesicles, and virus-like particles, wherein the enveloped virus particles are lentiviral particles and the extracellular vesicles are exosomes, and v. A support material in the form of porous particles functionalized with ligands having affinity groups that have binding affinity to biological target compounds selected from the group consisting of envelopes or membrane-like biological particles, such as enveloped virus particles, extracellular vesicles, and virus-like particles, wherein the enveloped virus particles are sometimes lentiviral particles, and the extracellular vesicles are sometimes exosomes. These are some examples.

[0088] This disclosure provides a method 100 for separating envelope or membrane-like biological particles from one or more impurities, as illustrated in Figure 3. a. A step (110) of adding a feed containing the envelope or membrane biological particles and one or more impurities to a first chromatography device (70) comprising a first chromatography material containing a support material functionalized with a ligand, wherein the ligand contains an anion exchange group or an affinity group having binding affinity to an envelope or membrane biological particle. b. Eluting the envelope or membrane-like biological particle from the first chromatography device into at least one elution fraction (120), c. Adding at least one eluate fraction containing envelopes or membrane-like biological particles obtained in step b to a second chromatography device (72) containing a second chromatography material containing a chromatographic material for adjustment (130) d. A step (140) to obtain an envelope or membrane-like biological particle in at least one pass-through fraction from a second chromatography device. Includes, The present invention provides Method 100, wherein the feed material is connected in series to a first chromatographic device and a second chromatographic device, enabling separation, and the first chromatographic device and the second chromatographic device are connected in series.

[0089] 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.

[0090] Preferably, the volume of eluted liquid from step (b) is ≤15% of the total volume of the second chromatographic material.

[0091] As illustrated in Figure 3, Method 100 consists of the following steps: e. A step (150) of adding at least one pass-through fraction, which includes an envelope or membrane-like biological particle obtained in step d, to a third chromatography device (74) which includes a third chromatography material comprising porous beads having an internally porous core and an externally porous shell, wherein 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 coming into contact with the core. f. A step of obtaining envelopes or membrane-like biological particles in at least one pass-through fraction from a third chromatography device (160) It may further include, At least one pass-through fraction obtained in step d passes continuously on and through a third chromatography device connected in series with the first and second chromatography devices.

[0092] 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.

[0093] 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).

[0094] 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 isolation of lentiviral vectors, it is essential to rapidly stabilize the virus by removing salts after the anion exchange capture step, and also to adjust the pH if possible. This 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.

[0095] 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.

[0096] 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."

[0097] 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.

[0098] 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.

[0099] 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 both binding in step (a) and elution in step (b). To achieve elution of the bound biological target compound, the buffer in step (b) contains additional components compared to the buffer used in step (a). Non-limiting examples of such additional components are amino acids, e.g., arginine or proline. For anion exchange ligands, the pH of the buffer may be approximately 6.5–8.5 for both binding in step (a) and elution in step (b). To achieve elution of the bound biological target compound, the buffer in step (b) contains a salt concentration not applied to the buffer in step (a). The buffer is appropriately selected from buffers that are generally recommended for affinity chromatography or anion exchange chromatography, respectively, and are suitable for the pH ranges described above. Non-limiting examples include tris(hydroxymethyl)aminomethane (i.e., Tris), 1,3-bis(tris(hydroxymethyl)methylamino)propane (i.e., bis-trispropane), triethanolamine, N-methyldiethanolamine, diethanolamine, 1,3-diaminopropane, ethanolamine, phosphate buffer, bis-Tris, imidazole, MOPS, and HEPES.

[0100] Steps (e) and (f) of Method 100 may include the step of applying a buffer having a pH of about 6.5 to about 7.5.

[0101] As described above, the purpose of steps (c) and (d) is to adjust or prepare the biological target compound obtained in step (d) to the conditions required for further purification and / or formulation into a pharmaceutically acceptable composition. Any further purification may be performed by carrying out the subsequent steps (e) and (f) in Method 100, in which case the buffers used in steps (c) and (d) are usually the same as the buffers used in step (e). In general, buffer 34 used in step (b) of the Method for elution of envelope or membrane biological particles from the first chromatographic material is not suitable for use in steps (e) and (f). Therefore, buffer 32 used in steps (c) and (e) for feed equilibration and loading of the second and third chromatographic materials is a different buffer from buffer 34.

[0102] Those skilled in the art can select an appropriate concentration for any one of the buffers listed above.

[0103] Currently preferred, non-limiting examples of suitable buffer combinations used for lentiviral particle isolation include the following buffer systems: A1: 50 mM Tris-HCl pH 7.4 A2: 50 mM Tris-HCl pH 7.4, 130 mM NaCl, 4% sucrose B1: 50mM Tris-HCl pH 7.4, 1.3M NaCl B2:1M NaOH

[0104] Another currently preferred, non-limiting example of a suitable combination of buffers used for the isolation of lentiviral particles is the following buffer system: A1: 50 mM sodium phosphate, pH 7.0 A2: 50 mM sodium phosphate, pH 7.0, 130 mM NaCl, 4% sucrose B1: 50 mM sodium phosphate, pH 7.0, 1.3 M NaCl B2:1M NaOH

[0105] Method 100 preferably includes the step of applying a selective valve arrangement 60 configured to enable the separation of envelope or membrane-like biological particles from impurities by passing the feed through first and second chromatography devices 70, 72 and optionally a third chromatography device 74 in succession. As described in further detail elsewhere in the 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.

[0106] 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 envelope or membrane-like biological particles to be obtained in step d and in step f.

[0107] Similarly, method 100 may further include an optional step 145 prior to step e, in which a third chromatographic material is equilibrated to the conditions required for envelopes or membrane-like biological particles to be obtained in step f.

[0108] A non-limiting embodiment of the currently preferred method 100 includes a step of separating lentiviral particles from impurities.

[0109] Method 100 disclosed herein may be a preparation method (preferably) or an analysis method.

[0110] 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 processes, which would hinder the continuous flow of feeds in line within system 10. This is advantageous because it allows for the rapid neutralization of the pH and reduction of the conductivity of the viral vector sample, contributing to a reduction in the overall time required to carry out the process.

[0111] 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.

[0112] 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.

[0113] Chapter on Experiments (Example 1: Separation of lentiviral particles from impurities by in-line capture, desalting, and polishing chromatography) 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. The connected line configuration for lentiviral particle capture and polishing using a resin column was evaluated. A hands-free, separated configuration with a single desalting column between the anion-exchange capture column and the polishing column was used to stabilize lentiviral particles, prepare the sample with a low-salt buffer containing sucrose, and ensure low conductivity to maximize infectious recovery.

[0114] The performance of the three-step line configuration (Table 1) was compared with the results obtained by applying a two-step line configuration including an anion exchange capture column and a desalting column for buffer exchange to the dilution buffer (Table 2), and a one-step configuration using direct dilution including an anion exchange capture column (Table 3).

[0115] Each of the separation processes was performed at pH 7.0 and pH 7.4, respectively.

[0116] Setting up 3 steps The modified AKTA pure™ chromatography system is - In this specification, the first chromatography device selection valve is referred to as column valve 1 (CV1), - Anion exchange capture bead material, 5 mL Capto DEAE HiTrap (Cytiva, Sweden) in the first column, - 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, 3rd column including 1 mL Capto® Core 700 HiTrap (Cytiva, Sweden) It included.

[0117] The columns connected within the three lines were equilibrated separately before sample loading. 3 × 10 10 A clarified lentivirus-GFP feed material with a total particle titer of VP / mL was loaded onto the first column, following the protocol in Table 1.

[0118] [Table 1]

[0119] 2-step setup The modified AKTA pure™ chromatography system is - The first chromatography device selection valve called column valve 1 (CV1), - Anion exchange capture bead material, 5 mL Capto DEAE HiTrap (Cytiva, Sweden) in the first column, - A second chromatography device selection valve called General Purpose Valve 1 (VV1) has been added compared to standard AKTA instruments. - A second column containing desalting material, 50 mL HiPrep 26 / 10 Desalting (Cytiva, Sweden), and - A third chromatography device selection valve has been added compared to standard AKTA instruments. General-purpose valve 2 (VV2) (However, this setting does not require it and it will be turned off for the duration of all protocols) It included.

[0120] The columns connected within the two lines were equilibrated separately before sample loading. 3 × 1010 A clarified lentivirus-GFP feed material with a total particle titer of VP / mL was loaded onto the first column, following the protocol in Table 2.

[0121] [Table 2]

[0122] pH 7.4 buffer system for both 3-step and 2-step setups A1: 50 mM Tris-HCl pH 7.4 A2: 50 mM Tris-HCl pH 7.4, 130 mM NaCl, 4% sucrose B1: 50mM Tris-HCl pH 7.4, 1.3M NaCl B2:1M NaOH

[0123] pH 7.0 buffer system for both 3-step and 2-step setups A1: 50 mM sodium phosphate, pH 7.0 A2: 50 mM sodium phosphate, pH 7.0, 130 mM NaCl, 4% sucrose B1: 50 mM sodium phosphate, pH 7.0, 1.3 M NaCl B2:1M NaOH

[0124] Setting up one process The modified AKTA pure™ chromatography system is - In this specification, the first chromatography device selection valve is referred to as column valve 1 (CV1), - Anion exchange capture bead material, 5 mL Capto DEAE HiTrap (Cytiva, Sweden) in the first column, - A second chromatography device selection valve, referred to herein as General Purpose Valve 1 (VV1), is added compared to standard AKTA instruments (however, this is not required in this configuration and is turned off for the duration of the entire protocol), and - A third chromatography device selection valve, called the general-purpose valve 2 (VV2) in this specification, which is added compared to the standard AKTA machine (but not required in this setting and turned off during the entire protocol) was included.

[0125] 3×10 10 A clarified lentivirus-GFP feedstock material with a total particle titer of VP / mL was loaded onto the only column while directly diluting it in 50 mM Tris-HCl pH 7.4 and 50 mM NaPi pH 7.0, 130 mM NaCl, 5% sucrose. The protocol in Table 3 was used.

[0126]

Table 3

[0130] The results indicate that separation at pH 7 yielded a higher infectious recovery rate compared to separation at pH 7.4, and that the connected column did not negatively affect the total particle recovery rate or the infectious particle recovery rate. More specifically, a one-step chromatography consisting of a capture step using Capto DEAE followed by direct dilution yielded a physical and infectious recovery rate of 60–70% (data not shown). A two-step chromatography with a Capto DEAE capture step and a desalting step connected in-line yielded a physical recovery rate of nearly 100% and an infectious recovery rate of 70–90% for both pH 7.4 and pH 7.0 (Figure 4). A three-step chromatography setup with a Capto DEAE capture step, desalting step, and polishing Capto Core step connected in-line yielded a physical particle recovery rate of 60% and an infectious particle recovery rate of only 30% for the second and third steps at pH 7.4. Using pH 7, which is optimal for the Capto Core step, yielded a physical particle recovery rate of approximately 90% and an infectious recovery rate of nearly 100%. The third polishing step is required to remove DNA impurities (data not shown) that co-elute with lentiviral particles during the Capto DEAE capture step.

[0131] Total particle titer was measured using p24 ELISA, and infectivity titer was determined using a cell-based transduction assay (counting GFP-expressing cells by flow cytometry).

[0132] Impurity levels in the final sample after Capto Core 700 were below the detection limits of the Micro BCA total protein assay and the Picogreen total DNA assay (data not shown). Highly purified lentivirus was obtained.

[0133] (Example 2) The experimental design for separating lentivirus particles from impurities was carried out using the same apparatus and samples as in Example 1 above, with the following variations.

[0134] Regarding the first chromatography material (used in the capture step): - Different support materials, e.g., membrane or monolith, - Different Capto(trademark) core molecular weight cutoffs, e.g., CC400 (Cytiva, Sweden), - An additional capture step, for example, by applying an affinity ligand and an anion exchange ligand or a semi-affinity ligand.

[0135] Regarding biological target compounds: - Lentivirus pseudotype, - Insertions different from GFP (target gene), - Exosomes instead of lentiviruses, - Other enveloped viruses instead of lentiviruses.

[0136] (References) [Explanation of symbols]

[0137] 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 Equilibriumization process 130 Addition process 140 Steps to obtain envelopes or membrane-like biological particles 145 Equilibrium process 150 Addition process 160 Steps to obtain envelopes or membrane-like biological particles

Claims

1. A method (100) for separating envelope or membrane-like biological particles from one or more impurities, a. A step (110) of adding a feed containing the envelope or membrane-like biological particles and one or more impurities to a first chromatography device (70) comprising a first chromatography material containing a support material functionalized with a ligand, wherein the ligand contains an anion exchange group or an affinity group having binding affinity to an envelope or membrane-like biological particle. b. A step (120) of eluting the envelope or membrane-like biological particle from the first chromatography device into at least one elution fraction, c. Adding the at least one eluate fraction containing the envelope or membrane-like biological particles obtained in step b to a second chromatography device (72) containing a second chromatography material containing a chromatographic material for adjustment (130) d. A step (140) of obtaining the envelope or membrane-like biological particle in at least one pass-through fraction from a second chromatography device. Includes, Method (100), wherein the feed passes continuously through the first chromatography device and the second chromatography device to enable separation, and the first chromatography device and the second chromatography device 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 chromatography device and the second chromatography device 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. e. A step (150) of adding at least one pass-through fraction, which includes an envelope or membrane-like biological particle obtained in step d, to a third chromatography device (74) which includes a third chromatography material comprising porous beads having an internally porous core and an externally porous shell, wherein 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 coming into contact with the core. f. A step (160) of obtaining the envelope or membrane-like biological particle in at least one pass-through fraction from the third chromatography device. It further includes, The method according to any one of claims 1 to 3, wherein the at least one pass-through fraction obtained in step d passes continuously on and through the third chromatography device connected in series with the first and second chromatography devices.

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

6. The method according to any one of claims 1 to 5, further comprising, before step c, a step (125) of equilibrating the second chromatographic material to conditions required for the envelope or membrane-like biological particles to be obtained in step d and optionally in step f.

7. The method according to any one of claims 1 to 6, further comprising, before step e, a step (145) of equilibrating the third chromatographic material to the conditions required for the envelope or membrane-like biological particles to be obtained in step f.

8. The method according to any one of claims 1 to 7, wherein the envelope or membrane particle is selected from enveloped virus particles, extracellular vesicles, and virus-like particles.

9. The method according to claim 8, wherein the enveloped virus particle is a lentivirus particle.

10. The method according to claim 8, wherein the extracellular vesicle is an exosome.

11. The ligand of the first 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 any one of claims 1 to 10, comprising an anion exchange group as defined by

12. The ligand of the first 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, and R 3 and R 4 is independently selected from C1-C3 alkyl and CH2CHOHCH3, and 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 of the potent or partially potent anion exchange chromatography material 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 any one of claims 1 to 10, provided that it is defined by [the relevant definition].

13. The ligand of the first chromatography material is, 【Chemistry 4】 (In the formula, X is independently H, OH, or C for each presence) 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 any one of claims 1 to 10, comprising an anion exchange group as defined by

14. The method according to any one of claims 1 to 10, wherein the ligand of the first chromatography material comprises an affinity group having binding affinity to the envelope or membrane-like biological particles.

15. The system according to any one of claims 9 to 14, 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.

16. The system according to any one of claims 9 to 15, wherein the second chromatography material includes a size exclusion chromatography material.

17. 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 containing a support material functionalized with a ligand, wherein the ligand contains an affinity group having binding affinity to an anion exchange group or a biological target compound, A second chromatography device (72) containing a second chromatography material including a chromatography material for adjustment. Includes, A chromatography system (10) wherein the selective valve arrangement is configured to enable the separation of the biological target compound from impurities by allowing a feed containing a biological target compound and one or more impurities to pass through the first chromatography device and the second chromatography device in series, and the first chromatography device and the second chromatography device are configured to be connected in series.

18. The system according to claim 17, wherein the selection valve arrangement further includes a second chromatography device selection valve (64).

19. A third chromatography device (74) further comprises a third chromatography material comprising porous beads having an internally porous core and an externally porous shell, wherein 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. The system according to claim 17 or 18, wherein the third chromatography device is configured to be connected in series with the first and second chromatography devices, and the selective valve arrangement is configured to enable separation by passing the feed through the first, second and third chromatography devices in succession.

20. The system according to claim 19, wherein the selection valve arrangement further includes a third chromatography device selection valve (66).

21. 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 system according to any one of claims 17 to 20, comprising an anion exchange group as defined by

22. The ligand of the first chromatography material is, Formula II 【Transformation 6】 (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 of the potent or partially potent anion exchange chromatography material is the following formula III 【Transformation 7】 (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 system according to any one of claims 17 to 20, provided that it is defined by [the relevant law].

23. The ligand of the first chromatography material is, 【Transformation 8】 (In the formula, X is independently H, OH, or C for each presence) 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 system according to any one of claims 17 to 20, comprising an anion exchange group as defined by

24. The system according to any one of claims 17 to 20, wherein the ligand of the first chromatography material comprises an affinity group having binding affinity to a biological target compound selected from the group consisting of envelopes or membrane-like biological particles, such as enveloped virus particles, extracellular vesicles, and virus-like particles, wherein the enveloped virus particles are sometimes lentiviral particles, and the extracellular vesicles are sometimes exosomes.

25. The system according to any one of claims 17 to 24, 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.

26. The system according to any one of claims 17 to 25, wherein the second chromatography material includes a size exclusion chromatography material.

27. Use of the system according to any one of claims 17 to 26 for the separation of a biological target compound from one or more impurities, wherein the biological target compound is selected from the group consisting of envelopes or membranous biological particles, such as enveloped virus particles, extracellular vesicles and virus-like particles, wherein the enveloped virus particles are lentiviral particles and the extracellular vesicles are exosomes.

28. The first chromatography material described above is i. A support material in the form of porous particles functionalized with a diethylethanolamine ligand, In some cases, the ligand is connected to the support material by a polymer, such as dextran, ii. Support material in the form of a convection-based membrane structure containing a nonwoven web of polymer nanofibers functionalized with diethylethanolamine ligand, iii. Support material in the form of a convection-based membrane structure containing a nonwoven web of polymer nanofibers functionalized with N,N-diethylethylenediamine ligand, iv. A support material in the form of a convection-based membrane structure comprising a nonwoven polymer nanofiber web functionalized with ligands containing affinity groups having binding affinity to biological target compounds selected from the group consisting of envelopes or membrane-like biological particles, such as enveloped virus particles, extracellular vesicles, and virus-like particles, In some cases, the enveloped virus particle is a lentivirus particle. In some cases, the extracellular vesicle is an exosome, a supporting material, and v. A support material in the form of porous particles functionalized with ligands having affinity groups that have binding affinity to biological target compounds selected from the group consisting of envelopes or membrane-like biological particles, such as enveloped virus particles, extracellular vesicles, and virus-like particles, wherein the enveloped virus particles are lentivirus particles. In some cases, the extracellular vesicles are exosomes, supporting material The use according to claim 27, selected from the group consisting of the following.

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