Chromatography system, use thereof and method for separating collected or film-like

Abstract

The present disclosure relates to a chromatography system comprising a buffer valve arrangement configured to allow independent control of a first buffer feed and a second buffer feed; a pump arrangement configured to supply the first buffer feed, the second buffer feed, and a feed comprising a biological target compound and one or more impurities; a selector valve arrangement comprising a first chromatography device selector valve; a first chromatography device comprising a first chromatography material comprising a support material functionalized with a ligand wherein the ligand comprises an anion exchange group or an affinity group having a binding affinity for a biological target compound; a second chromatography device comprising a second chromatography material, the second chromatography material comprising a regulatory chromatography material; wherein the selector valve arrangement is configured to be able to separate a biological target compound from one or more impurities by allowing a feed comprising the biological target compound and the one or more impurities to continuously pass through the first chromatography device and the second chromatography device, and wherein the first chromatography device and the second chromatography device are configured to be connected in series. The present disclosure also provides uses of the chromatography system and a method for separating enveloped or membranous bio-particles from one or more impurities.

Description

Technical Field

[0001] This disclosure relates to the field of separating biological target compounds, such as enveloped viral particles. This disclosure relates to chromatographic systems and their use in separating enveloped or membrane-bound biological particles from impurities, as well as methods for separating enveloped or membrane-bound biological particles from impurities. Background Technology

[0002] Liquid chromatography (LC) is a separation technique used to separate and analyze complex liquid mixtures of compounds. This method involves a stationary phase and a liquid mobile phase, in which the liquid mixture to be separated is introduced into the mobile phase and passes through the stationary phase. Different components of the mixture will interact with the stationary phase to varying degrees, resulting in their separation and collection at different times. Various types of LC techniques exist, differing 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, different process conditions, and different buffer solutions.

[0003] Various biotarget compounds, such as antibodies and viral vectors, are produced through the fermentation of host cells. These biotarget compounds must then be separated from the host cell material and other impurities contained in the fermentation broth before they can be used for applications such as medicine and analysis. A combination of several steps using different types of liquid chromatography is typically employed, with the goal of achieving high recoveries of biotarget molecules at high purity levels. However, the process for separating biotarget compounds often requires manual switching between different chromatographic devices and sample preparation between chromatographic steps to condition the sample for the next step, making the process time-consuming overall.

[0004] Commonly used viral vectors in medical products 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 therapies.

[0005] Lentivirals are classified as retroviruses and possess a single-stranded RNA genome carrying reverse transcriptase. Lentivirals have a viral envelope in which glycosylated proteins act as ligands with affinity for receptors on the outer cell membrane surface of the host cell. Transcription of the viral genetic material occurs upon entry into the cell. The advantage of using LV as a viral vector is that it can penetrate the nuclear envelope in both dividing and non-dividing cells, unlike other retroviruses that only penetrate cells undergoing mitosis. Many cell types in adult individuals do not divide, and LV may be the only option for transferring genetic material into such cells.

[0006] To generate lentiviruses, several plasmids are transfected into so-called packaging cell lines. One or more plasmids, commonly referred to as packaging plasmids, encode viral particle proteins, such as the capsid and reverse transcriptase. Another plasmid contains the genetic material to be delivered by the vector. It is transcribed to produce a single-stranded RNA viral genome and is labeled by the presence of a ψ (psi) sequence. This sequence is used to package the genome into viral particles. For lentiviruses to be used in gene therapy, it is necessary to purify the viral particles from cellular impurities, such as host cell proteins and DNA, as well as excess plasmids after transfection. Typically, the harvested host cells that produce lentiviruses are treated with nucleases, and the lentiviruses are purified using several filtration techniques, such as normalflow microfiltration, ultrafiltration, and osmosis, to reduce impurity levels to approved levels.

[0007] However, current downstream purification processes for lentiviruses typically yield low recoveries of infectious viruses (usually 10-20%) because lentiviruses are unstable and sensitive to shear forces, buffer components such as salts, and rapidly degrade at room temperature. Time-consuming, multi-step processes are also not considered advantageous. Lentiviral viruses have been reported to be processed within a very narrow pH range of 7.0-7.4 (Kinetic Analyses of Stability of Simple and Complex Retroviral Vectors, F. Higashikawa et al., Virology The lentiviral vector expression (280, 124-131 (2001)) and the conductivity window <0.2M NaCl (Process development of lentiviral vector expression, purification and formulation for gene therapy applications, Doctoral thesis, Sara Nilsson, UCL, 2016) are stable, which makes downstream purification processes challenging.

[0008] Therefore, there is a continued need in the field for new chromatographic systems and purification methods that offer higher capacity and faster processing, resulting in better overall process economy. Summary of the Invention

[0009] One object of this disclosure is to provide a chromatographic system capable of separating biological target compounds from impurities more quickly and easily, with performance similar to previously known chromatographic systems. The chromatographic system is constructed such that a feed containing both the biological target compound and impurities can undergo several purification steps uninterruptedly as it continuously passes through the system. Several chromatographic devices are connected online within the chromatographic system. This results in a significant reduction in time compared to using conventional chromatographic systems. Traditionally, the purification process flow is often interrupted and prolonged, for example, due to manual changes to buffer solutions and / or chromatographic devices within the system, and because some steps are performed in separate containers or devices outside the chromatographic system, such as adjusting the chromatographic media and / or performing certain filtration / purification steps. Using the chromatographic system of this disclosure requires less human intervention compared to operating previously known chromatographic systems.

[0010] More specifically, this disclosure relates to a chromatography system comprising: A buffer valve arrangement is configured to allow independent control of the first and second buffer feeds; The pump arrangement is configured to supply a first buffer feed, a second buffer feed, and a feed containing a biological target compound and one or more impurities; The selection valve arrangement includes a first chromatographic device selection valve; A first chromatographic apparatus comprising a first chromatographic material comprising a support material functionalized with a ligand, wherein the ligand comprises anion exchange groups or affinity groups having binding affinity for biological target compounds; A second chromatographic apparatus comprising a second chromatographic material, the second chromatographic material comprising a regulating chromatographic material; The selection valve arrangement is configured to separate the biological target compound from the impurities by allowing a feed containing a biological target compound and one or more impurities to continuously pass through the first chromatographic unit and the second chromatographic unit, wherein the first chromatographic unit and the second chromatographic unit are configured to be connected in series.

[0011] This disclosure also provides the use of the chromatographic system disclosed herein for separating biological target compounds from one or more impurities, wherein the biological target compounds are selected from enveloped or membrane-bound biological particles, such as enveloped viral particles, extracellular vesicles, and virus-like particles. Optionally, the enveloped viral particles are lentiviral particles. Optionally, the extracellular vesicles mentioned therein are exosomes.

[0012] Additionally, this disclosure relates to a method for separating coated or membrane-bound bioparticles from one or more impurities, the method comprising: a. Adding a feed comprising coated or membrane-bound bioparticles and one or more impurities to a first chromatographic apparatus, the first chromatographic apparatus comprising a first chromatographic material comprising a support material functionalized with ligands, wherein the ligands comprise anion exchange groups or affinity groups having binding affinity to the coated or membrane-bound bioparticles; b. Elute the membrane-bound or membrane-bound biological particles from the first chromatographic apparatus in at least one eluent fraction; c. Add the at least one eluent fraction containing a membrane or membrane-bound bioparticle obtained in step b to a second chromatographic apparatus, the second chromatographic apparatus containing a second chromatographic material, the second chromatographic material containing a conditioning chromatographic material; d. Obtain the coated or membrane-bound bioparticles from a second chromatographic apparatus in at least one flow fraction; The feed continuously passes through the first and second chromatographic units to enable separation, wherein the first and second chromatographic units are connected in series.

[0013] Preferred aspects of this disclosure are described in the detailed description and dependent claims below. It should be noted that this disclosure relates to all possible combinations of features recited in the claims. Attached Figure Description

[0014] These and other aspects of this disclosure will now be described in more detail with reference to the accompanying drawings, which illustrate embodiments of the invention, in which: Figure 1 AB is a schematic outline of a chromatographic system according to an embodiment of the present disclosure.

[0015] Figure 2 This is a schematic diagram of a chromatographic system according to a further implementation scheme.

[0016] Figure 3 This is a flowchart outlining the steps of a method for separating lentiviral particles from impurities according to this disclosure.

[0017] Figure 4 The chromatograms shown are those of lentivirus particles separated on different chromatographic system settings as described in Example 1 of this document.

[0018] As illustrated in the figure, some features may be exaggerated for illustrative purposes, and are therefore provided to illustrate the general structure of embodiments of this disclosure. Detailed Implementation

[0019] like Figure 1AAs can be seen, this disclosure addresses or at least mitigates problems associated with existing chromatographic systems and methods for separating biological target compounds from impurities by providing a chromatographic system 10, said chromatographic system 10 comprising: Buffer valve arrangement 20 is configured to allow independent control of the first buffer feed and the second buffer feed; Pump arrangement 40 is configured to supply a first buffer feed, a second buffer feed, and a feed containing a biological target compound and one or more impurities; Selector valve arrangement 60 includes a first chromatographic apparatus selector valve 62; A first chromatographic apparatus 70 includes a first chromatographic material comprising a support material functionalized with a ligand, wherein the ligand comprises anion exchange groups or affinity groups having binding affinity to biological target compounds. The second chromatographic apparatus 72 includes a second chromatographic material, the second chromatographic material including a regulating chromatographic material; The selection valve arrangement is configured to separate the biological target compound from the impurities by allowing a feed containing a biological target compound and one or more impurities to continuously pass through the first chromatographic unit and the second chromatographic unit, wherein the first chromatographic unit and the second chromatographic unit are configured to be connected in series.

[0020] The buffer valve arrangement 20 has at least one inlet (indicated by arrows in Figure 1) and a corresponding outlet for buffer feed, typically two or more inlets and corresponding outlets, for independently controlling the feed of two or more buffer solutions.

[0021] Pump arrangement 40 includes at least one pump, optionally two or more pumps.

[0022] Figure 1B An implementation plan was described, in which, besides Figure 1A In addition to the components shown, the chromatography system 10 also includes a third chromatography device 74, which contains a third chromatography material comprising porous beads having an internal porous core and an external porous shell, wherein the core is capable of binding molecules through hydrophobic interactions, and wherein the pore size of the shell prevents particles with a size ≥20 nm from contacting the core, wherein 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 wherein the selection valve arrangement 60 is configured to enable separation by allowing the feed to continuously pass through the first, second and third chromatography devices.

[0023] The selector valve arrangement 60 allows two or three chromatographic units to be connected in series, enabling online two- or three-step chromatographic purification processes within the chromatographic system. This avoids interruptions and delays traditionally encountered due to manual operation of individual steps during the process, saving users time and effort in operating the equipment. The chromatographic system can be fully set up and programmed before starting the purification process, which can then be initiated with the push of a button and allowed to continue until completion without further user intervention. This disclosure can therefore be considered to provide a so-called "plug and play" chromatographic system, meaning a system designed to work perfectly upon first use or connection, requiring no user reconfiguration or adjustment.

[0024] More specifically, the chromatographic system 10 of this disclosure offers the following general advantages compared to conventional chromatographic systems: - Desalting and pH adjustment steps are linked after affinity capture. This potentially saves time because dilution to reduce conductivity and adjust sample pH are not required prior to purification, resulting in lower sample feed volumes and thus shorter loading times. Furthermore, it provides complete control over sample conductivity to allow for high-performance separation of viral particles such as lentiviruses; - No holding time; - No freeze-thaw cycles; - No sample conditioning (pH and conductivity) or sample manipulation between chromatographic steps.

[0025] For the purification of lentiviral vectors, an additional advantage is the shorter exposure time (minutes vs. hours) to high-salt conditions for lentiviral vectors sensitive to high salt concentrations. Direct buffer exchange with a stabilizer (e.g., sucrose) to the appropriate pH is beneficial for increasing infectious yield. However, given that lentiviruses are unstable and sensitive to shear forces, buffer components such as salt, and readily degrade at room temperature, it is still surprising that lentiviral particles can tolerate such online-linked two- or three-step purification processes.

[0026] The chromatographic system 10 can be applied with a variety of chromatographic devices and materials, as described in more detail elsewhere herein. Non-limiting examples of chromatographic materials suitable for online-connected lentiviral vector purification include membrane adsorbers, convection-based membrane structures incorporating nanofibers, bulk materials, and combinations of resins (beads). The chromatographic system disclosed herein offers significant time reductions (up to 70%) and a simplified lentiviral purification process, while achieving performance similar to conventional purification settings in terms of lentiviral recovery. Considering the combined chromatographic challenges of maintaining lentiviral stability under optimal conditions without manual intervention and maintaining high chromatographic performance, the successful implementation of a complete capture and purification process using such an online-connected two- or three-step purification procedure is remarkable.

[0027] Figure 2 A currently preferred, non-limiting embodiment of the chromatography system 10 is described, wherein the buffer valve arrangement comprises 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 the first buffer feed, also referred to herein as buffer A, which is used when equilibrating the chromatographic apparatus before loading a sample containing a biological target compound onto the chromatographic apparatus, and the second buffer selection valve 24 may be configured to control the second buffer feed, also referred herein as buffer B, which is used when eluting the biological target compound from the chromatographic system 10.

[0029] More specifically, the first buffer selection valve 22 may be configured to control the feed of a first buffer solution 30 for equilibrating the first chromatographic apparatus 70, also referred to herein as buffer solution A1, and the first buffer selection valve 22 may be further configured to control the feed of a first buffer solution 32 for equilibrating the second chromatographic apparatus 72, also referred to herein as buffer solution A2. Buffer solution 32 may also be used to equilibrate the third chromatographic apparatus 74. Buffer solution 32 may also be used to transfer the biological target compound from the second chromatographic apparatus to the third chromatographic apparatus, provided that the biological target compound does not bind to the second chromatographic apparatus but leaves the second chromatographic apparatus in a flow-through. This is applicable, for example, in cases where the biological target compound is a lentiviral particle.

[0030] Furthermore, the second buffer selection valve 24 can be configured to control the feed of a second buffer 34, also referred to herein as buffer B1, for eluting the biological target compound from the first chromatographic unit 70, and the feed of a second buffer 36, also referred herein as buffer B2, for eluting the biological target compound from the second chromatographic unit 72. Buffer B2 can also optionally be used for eluting the biological target compound from the third chromatographic unit 74. Alternatively, a separate buffer feed B3 can be used for eluting the biological target compound (not shown) from the third chromatographic unit 74.

[0031] according to Figure 2 The system 10 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 feeds of a sample 46 containing a biological target compound and one or more impurities.

[0032] according to Figure 2 The system 10 also includes an injection valve 50. It should be understood that any injection valve conventionally used in the art can be used.

[0033] according to Figure 2 The illustrated implementation shows that the selection valve arrangement consists of a first chromatographic device selection valve 62, a second chromatographic device selection valve 64, and a third chromatographic device selection valve 66. Chromatographic device selection valve 62 is connected to the first chromatographic device 70, the second chromatographic device selection valve 64 is connected to the second chromatographic device 72, and the third chromatographic device selection valve 66 is connected to the third chromatographic device 74, wherein the three chromatographic devices are connected online in series within the system 10.

[0034] However, the selector valve arrangement 60 is contemplated to consist of a single chromatographic device selector valve 62 connected in series with the first, second, and third chromatographic devices 70, 72, and 74 (as shown in Figure 1). Alternatively, the selector valve arrangement 60 may consist of a combination (not shown) of the first chromatographic device selector valve 62 and the second chromatographic device selector valve 64 connected in series with the first, second, and third chromatographic devices 70, 72, and 74. It should be understood that in embodiments where the number of chromatographic device selector valves is less than the number of chromatographic devices, at least one selector valve can operate a different flow path, which makes multi-step chromatographic purification possible on a chromatographic system comprising fewer selector valves than chromatographic devices.

[0035] A non-limiting example of a usable chromatographic system is the ÄKTA pure chromatographic system (Cytiva, Sweden). The ÄKTA pure system, in its standard configuration, has only one column valve (corresponding to the chromatographic device selection valve of system 10). The ÄKTA pure system used in the experimental section of this paper has been modified by adding two additional chromatographic device selection valves to achieve the desired effect. Figure 2 The system shown is an embodiment of the present invention. Another non-limiting example of a usable chromatography system is the NGC chromatography system (BioRad, USA). It can be modified by adding a multi-functional valve to achieve the desired effect. Figure 2 The system shown is the implementation scheme.

[0036] Non-limiting examples of chromatographic apparatus selection valves that may be part of system 10 of this disclosure are so-called multi-function valves, such as multi-function valve V9-V (Cytiva, Sweden).

[0037] according to Figure 2 The chromatography system 10 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 the chromatography system.

[0038] The chromatographic system 10 disclosed herein is primarily intended for preparative applications involving feed materials with volumes ranging from a few mL to several hundred liters. The system can also be used for analytical applications. When applied in large-scale processes, the distinctive features of system 10 offer more significant advantages than those for small-scale processes.

[0039] The term "chromatographic material" is used in this document to refer to a type of separation matrix.

[0040] The term "separation matrix" is used herein to refer to a material comprising a support material coupled with one or more ligands containing functional groups. The functional groups of the ligands bind to compounds (also referred to herein as analytes) that will separate from the liquid sample and / or from other compounds present in the liquid sample. The separation matrix may further comprise compounds that couple the ligands to the support material. The terms "connector," "stretcher," and "surface stretcher" may be used to describe such compounds, as further described below. In this document, the term "support material" is used interchangeably with the term "support."

[0041] The chromatographic materials mentioned herein may include a linker connecting the ligand and the support; that is, coupling of the ligand and the support is provided by introducing a linker between the support and the ligand. Coupling can be performed according to any conventional covalent coupling method, for example by using epichlorohydrin; epibromohydrin; allyl-glycidyl ether; biepoxides, such as butanediol diglycidyl ether; halogen-substituted aliphatic substances, such as dichloropropanol; and divinyl sulfone. Non-limiting examples of suitable linkers include vinyl sulfone, combinations of vinyl sulfone and glycidyl, polyethylene glycol (PEG) having 2-6 carbon atoms, carbohydrates having 3-6 carbon atoms, or polyols having 3-6 carbon atoms. Alternatively, the ligand may be coupled to the support via a longer linker molecule, also known as a “surface extender” or simply “extender.” Extenders are well known in the art and are commonly used to spatially increase the distance between the ligand and the support. Extenders are sometimes referred to as tentacles or flexible arms. For a more detailed description of possible chemical structures, see, for example, US 6,428,707, which is incorporated herein by reference. In short, the stretching agent can be in the form of a polymer, such as a homopolymer or copolymer. Hydrophilic polymeric stretching agents can be of synthetic origin, i.e., having a synthetic backbone, or of biological origin, i.e., biopolymers with a naturally occurring backbone. Typical synthetic polymers are polyvinyl alcohol, polyacrylamide and polymethacrylamide, polyvinyl ether, etc. Typical biopolymers are polysaccharides, such as starch, cellulose, dextran, agarose.

[0042] In this context, a "ligand" is a molecule that has a known or unknown affinity for a given analyte and includes any functional groups or trapping agents immobilized on its surface, while an "analyte" includes any specific binding partner of the ligand. The term "ligand" may be used interchangeably herein with the terms "specifically binding molecule," "specifically binding partner," "trapping molecule," and "trapping agent."

[0043] In this paper, molecules that interact with ligands in a liquid sample are referred to as “biotarget compounds” or “analytes.” The term “biotarget compounds” encompasses a wide range of types of biomolecules and compounds. Non-limiting examples include plasmids, exosomes, mRNA, viral particles, and proteins such as monoclonal antibodies.

[0044] Of particular interest as analytes according to this disclosure are enveloped or membrane-bound biological particles, such as enveloped viral particles, extracellular vesicles (e.g., exosomes), and virus-like particles. A particularly interesting example of enveloped viral particles is lentiviral particles.

[0045] The term "viral particle" is used herein to refer to a complete infectious viral particle. It comprises a core containing the virus's genome (i.e., the viral genome) in the form of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and is surrounded by a morphologically defined outer shell. This outer shell is called the capsid. The capsid and the encapsulated viral genome together constitute a so-called nucleocapsid. The nucleocapsid of enveloped viruses is surrounded by a lipoprotein bilayer. In the field of bioprocessing, to produce viral vectors for various applications such as therapeutic purposes, the genome of the viral particle is modified to include genetic inserts containing the target genetic material.

[0046] The term "vector" is used herein to refer to viral particles, typically recombinant viral particles, designed to facilitate gene transfer to modify specific cell types or tissues. Viral particles may, for example, be modified to provide vectors for expressing therapeutic genes. Several types of viruses are currently being investigated for delivering genetic material (e.g., genes) into cells to provide transient or permanent transgenic expression. These include adenoviruses, retroviruses (gamma retroviruses and lentiviruses), poxviruses, adeno-associated viruses (AAVs), baculoviruses, and herpes simplex viruses. In this document, the term "vector" is 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 has the ability to self-assemble, mimicking the shape and size of a virus particle but lacking genetic material, and therefore cannot infect host cells.

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

[0049] The term "surface" in this document refers to all external surfaces, and in the case of porous supports, includes both the external surface and the pore surface.

[0050] The supporting material for the first chromatographic material may include membrane structures, nanofibers, monolithic materials, porous particles, non-porous particles, or expanded bed media.

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

[0052] Another non-limiting example of a membrane structure is Fibro. TM (Cytiva, Sweden), which is also a non-limiting example of a support material containing nanofibers. Fibro TM It is a convection-based membrane structure that contains nanofibers made of cellulose or cellulose derivatives.

[0053] A non-limiting example of a monolithic material is CIMmultus® (Sartorius, Germany).

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

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

[0056] As further mentioned above, the ligands of the first chromatographic material may contain anion exchange groups or affinity groups that have binding affinity to the coating or membrane-bound biological particles.

[0057] In the case where the ligands of the first chromatographic material contain anion-exchange groups, these can be strong or partially strong anion-exchange groups, more specifically quaternized amine groups. A quaternary ammonium group is a strong anion-exchange group that is always positively charged regardless of the pH it experiences. For DEAE-based chromatographic materials, the degree of quaternization of the amine groups can vary among the amine groups included in the chromatographic material. A total quaternization degree of approximately 12% to approximately 100% of the amine groups in the chromatographic material is generally considered to result in chromatographic materials behaving similarly to strong or at least partially strong anion-exchange chromatographic materials, because these at least 12% of all amine groups are always charged.

[0058] More specifically, when the ligand of the first chromatographic material contains a strong or partially strong anion exchange group, the ligand can be defined by Formula I: (I) 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 wherein R1, R2 and R3 are each CH3.

[0059] The wavy portion represents the supporting material, including the connector. The ligand can attach to the carbon atoms of the connector.

[0060] Currently available chromatographic materials contain ligands defined by Formula I, wherein R1, R2, and R3 are each CH3; for example, chromatographic materials are available under the name Capto Q (Cytiva, Sweden). Capto Q further contains dextran as a surface-extending agent. Capto Q is a non-limiting example of a strong anion exchange chromatographic material having approximately 100% quaternized amine groups.

[0061] Another non-limiting example of chromatographic materials containing quaternary ammonium ligands is the bulk CIMmultus® QA (Sartorius, Germany).

[0062] When the ligands of the first chromatographic material contain strong or partially strong anion exchange groups, alternatively, it can be defined by Formula II: (II) in: m is an integer from 1 to 3; R1 and R2 are independently selected from C1-C3 alkyl groups; R3 and R4 are independently selected from C1-C3 alkyl groups and CH2CHOHCH3; and R5 is selected from hydrogen, C1-C3 alkyl groups and CH2CHOHCH3; The condition is that if m is 1, then the ligand is defined by equation III: (III) Where n is an integer between 0 and 3; The condition is that if n is 0, then R3 and R4 are independently selected from C1-C3 alkyl groups, and R5 is hydrogen or CH2CHOHCH3.

[0063] As a non-restrictive example, the ligand is defined by Equation III and includes a combination of two or more of the following structures (i)-(iv): (i) n is 0; R3 and R4 are ethyl groups; and R5 is hydrogen or CH2CHOHCH3; (ii) n is 1; R1, R2, R3, and R4 are ethyl groups; and R5 is hydrogen or CH2CHOHCH3; (iii) n is 2; R1 and R2 are each ethyl; R3 and R4 are ethyl; and R5 is hydrogen or CH2CHOHCH3; (iv) n is 3; R1 and R2 are each ethyl; R3 and R4 are ethyl; and R5 is hydrogen or CH2CHOHCH3.

[0064] One currently available chromatographic material comprising ligands defined by Formula III and including combinations of the structures (i)-(iv) above is a chromatographic resin called Capto DEAE (Cytiva, Sweden). Capto DEAE further comprises dextran as a surface-extending agent. Capto DEAE is a non-limiting example of a strong or partially strong anion exchange chromatographic material having a degree of quaternization of about 15% of amine groups.

[0065] In cases where the ligand of the first chromatographic material contains anion exchange groups, it may, alternatively, contain weak anion exchange groups. In contrast to the quaternized amine groups defined above, almost all other ion exchange groups are weak, i.e., their charge changes from fully charged to uncharged within a reasonable pH range (e.g., pH 2–11), and they have a neutral charge (equal amounts of + and - charges) at pI.

[0066] More specifically, when the ligand of the first chromatographic material contains a weak anion exchange group, the ligand can be defined by Formula IV: (IV) For each occurrence, X is independently selected from H, OH, and C. 1-3 The groups, and R1, R2, R3, and R4 are independently selected from H and C. 1-3 Groups, wherein the C3 group is straight-chain or branched, wherein C 1-3 The functional groups include those independently selected from OH and OC. 1-2 SC 1-2 The groups NH, NHR and NR2, wherein R is selected from H and C. 1-3 Group.

[0067] Non-limiting examples of ligands defined by Formula IV are 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. Currently preferred ligands containing a weak anion exchange group are N,N-diethylethylenediamine.

[0068] When the ligand of the first chromatographic material contains an affinity group, it can have binding affinity for a biological target compound selected from enveloped or membrane-bound biological particles, such as enveloped viral particles, extracellular vesicles (e.g., exosomes), and virus-like particles. For example, the ligand can have binding affinity for lentiviral particles.

[0069] The second chromatographic material is a conditioning chromatographic material, where the term "conditioning chromatographic material" is intended to refer to its conditioning or preparation of a sample feed or solution that is loaded onto and passed through the chromatographic material for subsequent purification steps and / or final product formulation. Conditioning may, for example, include desalting, i.e., reducing the salt concentration of, or being composed of, the sample feed or solution. Alternatively, conditioning may include increasing the salt concentration of, or being composed of, the sample feed or solution. Or, additionally, conditioning may include changing the pH of, i.e., decreasing or increasing, the pH of, or being composed of, the sample feed or solution. Furthermore, conditioning chromatographic materials may additionally achieve the removal of any low molecular weight impurities.

[0070] When the conditioning consists of desalting, the conditioning chromatographic material may appropriately include size-limiting chromatographic material. Salts are blocked by the size-limiting material, while biological target compounds much larger than salt molecules pass through the size-limiting material without obstruction and are thus obtained in the flow fraction from the second chromatographic material.

[0071] The third chromatographic material comprises porous beads having an internal porous core and an external porous shell. The porosity of the core and shell may be the same or different. However, at least the porosity of the shell prevents particles with a size ≥20 nm, such as membranes or membrane-bound bioparticles, from permeating through the shell to contact the core.

[0072] Enveloped viruses typically range in size from 20 nm to 300 nm, depending on the virus type. Lentivirals can range in size from 80–120 nm, typically 100–120 nm. Enveloped viruses can be larger than non-enveloped viruses (e.g., adenoviruses), which are only encapsulated in a capsid. Enveloped viruses are generally larger than adeno-associated viruses, which typically have a size of about 25 nm. Extracellular vesicles, such as exosomes, can range in size from 30–180 nm.

[0073] The shell is typically hydrophilic. Therefore, the surface of porous beads accessible to large entities such as membranes or membrane-bound bioparticles is hydrophilic and does not irreversibly adsorb or denature proteins. The shell can be formed from a hydrophilic material that exposes multiple polar groups, such as those containing oxygen and / or nitrogen atoms. Examples of such polar groups are hydroxyl, amino, carboxyl, sulfonates (S and SP ligands), and lower alkyl ethers (e.g., (-CH2CH2O-)nH, where n is an integer of 2, 3, 4, or greater).

[0074] The core binds strongly to biomolecules such as proteins and DNA through hydrophobic interactions. In this 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 membrane-bound bioparticles cannot enter and are instead acquired in the flow fraction.

[0075] The core can be hydrophobic. Preferably, the hydrophobic core is itself hydrophilic and is based on a hydrophilic material, such as a hydrophilic polymer, and functionalized with hydrophobic interacting ligands to provide the desired hydrophobicity. However, alternatively, the core can be based on a hydrophobic polymer and be hydrophobic itself. For example, styrene / ethylstyrene / DVB, vinyl ethers, and acrylates containing hydrophobic substituents, as well as polymers containing fluoroalkanes, are considered.

[0076] The hydrophobic interaction ligand may contain aliphatic hydrocarbons, such as C1-C30 alkyl, preferably C4-C16 alkyl, and / or aromatic hydrocarbons, such as phenyl, anthracene, and naphthalene.

[0077] The hydrophilic polymer upon which the shell and optionally the core can be based is a polysaccharide, such as agarose. For example, both the core and shell can comprise cross-linked agarose. Porous core-shell beads can be produced as described in WO2009131526. Specifically, the core and shell can be made of agarose, and the core can be functionalized with a hydrocarbon-interacting ligand comprising 4-16 carbons, preferably an octyl ligand. Useful chromatographic media can be produced under the trade name Capto, available from Cytiva, Sweden. TM Core 400 and Capto TM Core 700 obtained. Capto TM The Core 700 bead features a 700 kDa size resistive cutoff shell and a core containing a multimode octylamine ligand.

[0078] This disclosure further provides the above-described chromatographic system 10, including the first and second chromatographic materials and optionally the third chromatographic material, for use in separating a biological target compound from one or more impurities, wherein the biological target compound is selected from enveloped or membrane-bound biological particles, such as enveloped viral particles (e.g., lentiviruses), extracellular vesicles (e.g., exosomes), and virus-like particles.

[0079] Currently preferred, non-limiting examples of the first chromatographic material include: i. A support material in the form of porous particles functionalized with a diethylethanolamine ligand; optionally, said ligand is connected to the support material via a polymer, such as dextran; ii. A support material in the form of a convection-based membrane structure comprising a nonwoven web of polymer nanofibers functionalized with diethylethanolamine ligands; iii. A support material in the form of a convection-based membrane structure comprising a nonwoven web of polymer nanofibers functionalized with N,N-diethylethylenediamine ligands; iv. A support material in the form of a convection-based membrane structure comprising a nonwoven web of polymer nanofibers functionalized with ligands, said ligands comprising affinity groups having binding affinity for a biological target compound selected from enveloped or membrane-bound biological particles, such as enveloped viral particles, extracellular vesicles, and virus-like particles; optionally wherein the enveloped viral particles are lentiviral particles, and optionally wherein the extracellular vesicles are exosomes; and v. A support material in the form of porous particles functionalized with ligands, said ligands comprising affinity groups having binding affinity for biological target compounds selected from enveloped or membrane-bound biological particles, such as enveloped viral particles, extracellular vesicles, and virus-like particles; optionally wherein the enveloped viral particles are lentiviral particles, and optionally wherein the extracellular vesicles are exosomes.

[0080] like Figure 3 As described herein, this disclosure further provides a method 100 for separating coated or membrane-bound biological particles from one or more impurities, the method comprising: a. Add (110) a feed comprising coated or membrane-bound bioparticles and one or more impurities to a first chromatographic apparatus (70), the first chromatographic apparatus (70) comprising a first chromatographic material comprising a support material functionalized with a ligand, wherein the ligand comprises anion exchange groups or affinity groups having binding affinity to the coated or membrane-bound bioparticles; b. Elute the membrane-coated or membrane-bound biological particles (120) from the first chromatographic apparatus in at least one eluent fraction; c. Add (130) the at least one eluent fraction containing a membrane or membrane-bound biological particle obtained in step b to a second chromatographic apparatus (72), the second chromatographic apparatus (72) containing a second chromatographic material, the second chromatographic material containing a conditioning chromatographic material; d. Obtain the (140) membrane-coated or membrane-bound bioparticles from a second chromatographic apparatus in at least one flow fraction; The feed continuously passes through the first and second chromatographic units to enable separation, wherein the first and second chromatographic units are connected in series.

[0081] The term "elution" is used in its conventional sense in the field, referring to the portion of a liquid sample eluted from a chromatographic column after the liquid sample has been loaded onto the column.

[0082] Preferably, the volume of eluent from step (b) is ≤15% of the total volume of the second chromatographic material.

[0083] like Figure 3 As shown, method 100 may further include the following steps: e. Add (150) the at least one flow fraction containing membrane or membrane-bound bioparticles obtained in step d to a third chromatographic device (74), the third chromatographic device (74) comprising a third chromatographic material comprising porous beads having an internal porous core and an external porous shell, wherein the core is capable of binding molecules through hydrophobic interactions, and wherein the pore size of the shell prevents particles having a size ≥20 nm from contacting the core; f. Obtain the (160) membrane-coated or membrane-bound bioparticles from a third chromatographic apparatus in at least one flow fraction; The at least one flow fraction obtained in step d is continuously transferred to and through a third chromatographic apparatus, which is connected in series with the first and second chromatographic apparatuses.

[0084] It should be understood that the first, second, and third chromatographic materials and the corresponding chromatographic devices 70, 72, and 74 mentioned in method 100 are further defined and illustrated in detail above in conjunction with the description of chromatographic system 10.

[0085] In method 100, the chromatographic material mentioned in steps (a) and (b) may be referred to as a capture chromatographic material, meaning that the chromatographic material is applied in the capture step, which is referred to as the initial step of the separation procedure in the context of liquid chromatography. In this document, the capture steps performed in steps (a) and (b) of method 100 achieve efficient purification of the biological target compound from soluble impurities. Additional steps, such as clarification and filtration (e.g., tangential flow filtration), may be performed prior to steps (a) and (b).

[0086] Furthermore, steps (c) and (d) of method 100 aim to adjust or prepare the biotarget compound for the conditions required for subsequent purification steps (e) and (f). More specifically, for the separation of lentiviral vectors, it is crucial to rapidly stabilize the virus by removing salt after the anion exchange capture step, and possibly adjust the pH. This online desalting performed in steps (c) and (d) is an important part of this disclosure, contributing to good separation results while reducing process time. Conventionally, the desalting step is performed in a separate apparatus, rather than being online connected to other chromatographic apparatus.

[0087] In method 100, the chromatographic material mentioned in steps (e) and (f) may be referred to as purified chromatographic material, meaning that the chromatographic material is applied in the purification step.

[0088] In the context of liquid chromatography, the term "purification step" refers to the final purification step in which trace impurities are removed to leave the active, safe product. The impurities removed during the purification step are typically conformational isomers of the target molecule—that is, the target molecule form with a specific molecular conformation—or suspected leaked products. Alternatively, the purification step may be referred to as a "secondary purification step."

[0089] exist Figure 3 In method 100, the flow rate used depends on the type of chromatographic material used and the size of the chromatographic material or the chromatographic apparatus containing said chromatographic material, as well as the selected residence time. For example, much higher flow rates can be applied to convection-based membrane support materials, such as Fibro, relative to porous particles like Capto beads. TM .

[0090] Furthermore, as those skilled in the art will understand, the different buffers used in method 100 are selected based on the chromatographic material being applied and the biological target compound to be purified.

[0091] In steps (a) and (b) of the method, the pH of the buffer solution used may vary depending on the type of ligand used. For binding in step (a) and elution in step (b), the buffer solution may, for example, have a pH of approximately 7-8 for affinity ligands. To achieve elution of the bound biotarget compound, the buffer solution in step (b) contains additional components compared to the buffer solution used in step (a). Non-limiting examples of such additional components are amino acids, such as arginine or proline. For binding in step (a) and elution in step (b), the buffer solution pH may be approximately 6.5-8.5 for anion exchange ligands. To achieve elution of the bound biotarget compound, the buffer solution in step (b) contains a salt concentration not applied in the buffer solution of step (a). The buffer solution is suitably selected from buffers generally recommended for affinity chromatography or anion exchange chromatography, respectively, and is 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.

[0092] Steps (e) and (f) of method 100 may include applying a buffer solution having a pH of about 6.5 to about 7.5.

[0093] As described above, the goal of steps (c) and (d) is to adjust or prepare the biotarget compound under the conditions required for further purification of the biotarget compound obtained in step (d) and / or for formulation into a pharmaceutically acceptable composition. Any further purification may be performed by performing subsequent steps (e) and (f) of method 100, in which case the buffer used in steps (c) and (d) is generally the same as the buffer used in step (e). Generally, buffer 34 used in step (b) of the method, which is used to elute coated or membrane-bound bioparticles from the first chromatographic material, is not suitable for use in steps (e) and (f). Therefore, buffer 32 used in steps (c) and (e), which is used to equilibrate and load the feed onto the second and third chromatographic materials, is a different buffer from buffer 34.

[0094] For any of the buffer solutions listed above, those skilled in the art can select an appropriate concentration.

[0095] Currently preferred, non-limiting examples of suitable buffer combinations for isolating lentiviral particles are 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: 50 mM Tris-HCl pH 7.4, 1.3 M NaCl B2: 1 M NaOH Another currently preferred, non-limiting example of a suitable buffer combination for separating lentivirus 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: 1 M NaOH Method 100 suitably includes the application of a selection valve arrangement 60, configured to separate coated or membrane-bound bioparticles from impurities by allowing feed to continuously pass through first and second chromatographic units 70, 72 and optionally a third chromatographic unit 74. The selection valve arrangement includes a first chromatographic unit selection valve 62 and may optionally further include a second chromatographic unit selection valve 64 and also optionally a third chromatographic unit selection valve 66, as described in more detail elsewhere herein.

[0096] like Figure 3As shown, method 100 may further include the optional step of equilibrating the second chromatographic material 125 to the conditions required to obtain the coated or membrane-bound bioparticles in step d and in step f prior to step c.

[0097] Similarly, method 100 may further include an optional step of equilibrating the third chromatographic material 145 to the conditions required in step f to obtain the coated or membrane-bound bioparticles prior to step e.

[0098] A currently preferred non-limiting embodiment of method 100 includes separating lentiviral particles from impurities.

[0099] The method 100 disclosed herein can be a preparative method (preferably) or an analytical method.

[0100] The distinguishing feature of the chromatographic system 10 of this disclosure is that it is possible to equilibrate the second and / or third chromatographic materials online while the method 100 is running, in contrast to equilibrating them separately as an isolated step within the system 10, thereby interrupting the continuous flow of online feed. This is advantageous because it enables rapid neutralization of pH and reduction of the conductivity of the viral vector sample, and helps to reduce the overall time required to perform the process.

[0101] Further time reductions are possible because loading of different chromatographic devices can occur partially simultaneously within system 10. For example, it is not necessary to wait until the sample feed has passed through the second or third device before loading more sample into the first device, and so on.

[0102] It should be understood that this disclosure is not limited to its exemplary embodiments described below, and several conceivable modifications of this disclosure are possible within the scope of the appended claims. Any reference signs placed between parentheses in the claims should not be construed as limiting the claims. The use of the verb "comprising" and its morphological variations does not exclude the presence of elements or steps other than those stated. The article "an" or "a" preceding an element does not exclude the presence of a plurality of such elements.

[0103] Experimental Section Example 1: Separation of lentiviral particles from impurities by online-connected capture, desalting, and purification chromatography In one run, using ÄKTA pure TMA three-step chromatographic process was performed using a chromatographic system (Cytiva, Sweden), which had been modified by adding two multi-functional valves. The setup for the online connection used to capture and purify lentiviral particles using a resin column was evaluated. A hands-free walk-away setup with a desalting column between the anion-exchange capture column and the purification column was used to condition the sample and ensure low conductivity, while a low-salt buffer containing sucrose was used to stabilize the lentiviral particles to maximize infectious recovery.

[0104] The performance of the three-step online connection setup (Table 1) was compared with the results obtained by applying a two-step online connection setup (Table 2) that includes an anion exchange trap column and a desalting column for buffer exchange into dilution buffer, and a one-step setup (Table 3) that includes an anion exchange trap column and direct dilution.

[0105] The separation processes were carried out at pH 7.0 and pH 7.4, respectively.

[0106] Three-step setup Modified ÄKTA pure TM The chromatography system includes: - The first chromatographic apparatus selection valve, referred to herein as column valve 1 (CV1), - The first column contains anion exchange trapping beads, 5 mL Capto DEAE HiTrap (Cytiva, Sweden). - A second chromatographic apparatus selection valve, referred to herein as multifunctional valve 1 (VV1), has been added compared to the standard ÄKTA instrument. - The second column contains desalting material, 50 mL HiPrep 26 / 10 Desalting (Cytiva, Sweden). - A third chromatographic device selection valve, referred to herein as multifunctional valve 2 (VV2), has been added compared to the standard ÄKTA instrument, and - The third column contains refined bead material, 1 mL Capto TM Core 700 HiTrap (Cytiva, Sweden).

[0107] Equilibrate the three online-connected columns separately before sample loading. The columns will have a 3 x 10... 10 Clarified lentivirus-GFP feed material with a total particle titer of VP / mL was loaded onto the first column and followed the protocol in Table 1.

[0108] Table 1: Scheme for three-step setup (Unicorn™ software). stage Flow rate Entrance %B Volume (ml) Hierarchical separation CV1 VV1 VV2 balance 5 ml / min A1 / B1 10 5 - open close close Sample loading 5 ml / min A1 / B1 - 15 50 ml open close close washing 5 ml / min A1 / B1 10 25 - open close close Washed into Desalt 5 ml / min A1 / B1 50 15 - open open close Desalting into CC700 0.3 ml / min A2 / B1 0 25 25 ml close open open peeling 5 ml / min A1 / B1 100 15 - open close close CIP 5 ml / min A1 / B2 100 15 - open close close washing 5 ml / min A1 / B1 100 15 - open close close Rebalancing 5 ml / min A1 / B1 10 5 - open close close

[0109] Two-step setup Modified ÄKTA pure TM The chromatography system includes: - First chromatographic apparatus selection valve, column valve 1 (CV1), - The first column contains anion exchange trapping beads, 5 mL Capto DEAE HiTrap (Cytiva, Sweden). - A second chromatographic apparatus selection valve, multi-function valve 1 (VV1), added compared to the standard ÄKTA instrument. - The second column contains desalting material, 50 mL HiPrep 26 / 10 Desalting (Cytiva, Sweden), and - A third chromatographic apparatus selection valve, multi-function valve 2 (VV2), is added compared to the standard ÄKTA instrument (however, it is not required in this setup; it is closed throughout the protocol).

[0110] Equilibrate the two online-connected columns separately before sample loading. The columns will have a diameter of 3 x 10⁻⁶. 10 Clarified lentivirus-GFP feed material with a total particle titer of VP / mL was loaded onto the first column and followed the protocol in Table 2.

[0111] Table 2: Scheme for two-step setup (Unicorn™ software). Link Flow rate Entrance %B Volume (ml) Hierarchical separation CV1 VV1 VV2 balance 5 ml / min A1 / B1 10 5 - open close close Sample loading 5 ml / min A1 / B1 - 15 50 ml open close close washing 5 ml / min A1 / B1 10 25 - open close close Washed into Desalt 5 ml / min A1 / B1 50 15 - open open close Desalination 5 ml / min A2 / B1 0 25 25 ml close open close peeling 5 ml / min A1 / B1 100 15 - open close close CIP 5 ml / min A1 / B2 100 15 - open close close washing 5 ml / min A1 / B1 100 15 - open close close Rebalancing 5 ml / min A1 / B1 10 5 - open close close

[0112] pH 7.4 buffer systems for three-step and two-step setups: A1: 50 mM Tris-HCl, pH 7.4 A2: 50 mM Tris-HCl pH 7.4, 130 mM NaCl, 4% sucrose B1: 50 mM Tris-HCl pH 7.4, 1.3 M NaCl B2: 1 M NaOH pH 7.0 buffer systems for three-step and two-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: 1 M NaOH.

[0113] One-step setup Modified ÄKTA pure TM The chromatography system includes: - The first chromatographic apparatus selection valve, referred to herein as column valve 1 (CV1), - The first column contains anion exchange trapping beads, 5 mL Capto DEAE HiTrap (Cytiva, Sweden). - A second chromatographic apparatus selection valve, referred to herein as multi-function valve 1 (VV1), has been added compared to the standard ÄKTA instrument (however, it is not required in this setup; it is closed throughout the protocol), and - A third chromatographic apparatus selection valve, referred to herein as multifunctional valve 2 (VV2), has been added compared to the standard ÄKTA instrument (however, it is not required in this setup; it is closed throughout the protocol).

[0114] It will have 3 x 10 10 Clarified lentiviral-GFP feed material with a total particle titer of VP / mL was loaded onto a single column and directly diluted in 50 mM Tris-HCl pH 7.4 and 50 mM NaPi pH 7.0, 130 mM NaCl, and 5% sucrose. The protocol in Table 3 was used.

[0115] Table 3: Scheme for one-step setup (Unicorn™ software). Link Flow rate Entrance %B Volume (ml) Hierarchical separation CV1 VV1 VV2 balance 5 ml / min A1 / B1 10 5 - open close close Sample loading 5 ml / min A1 / B1 - 15 50 ml open close close washing 5 ml / min A1 / B1 10 25 - open close close Washout 5 ml / min A1 / B1 50 15 5 ml open close close peeling 5 ml / min A1 / B1 100 15 - open close close CIP 5 ml / min A1 / B2 100 15 - open close close washing 5 ml / min A1 / B1 100 15 - open close close Rebalancing 5 ml / min A1 / B1 10 5 - open close close

[0116] pH 7.4 buffer system for one-step setup: A1: 50 mM Tris-HCl, pH 7.4 B1: 50 mM Tris-HCl pH 7.4, 1.3 M NaCl B2: 1 M NaOH pH 7.0 buffer system for one-step setup: A1: 50 mM sodium phosphate, pH 7.0 B1: 50 mM sodium phosphate, pH 7.0, 1.3 M NaCl B2: 1 M NaOH.

[0117] result Figure 4 The results show the separation of lentiviral particles from impurities at pH 7.4 and pH 7.0 using three different processes (numbered 1-3): 1: Capto DEAE capture column, then directly diluted. 2: Online-connected Capto DEAE capture and desalination columns. 3: Online-connected Capto DEAE capture column, desalting column, and refining column.

[0118] The results showed that separation at pH 7 provided higher infectious recovery compared to separation at pH 7.4, and the connected column did not negatively impact the total or infectious particle recovery. More specifically, one-step chromatography consisting of a capture step via Capto DEAE followed by direct dilution resulted in 60–70% physical and infectious recoveries (data not shown). Two-step chromatography with a Capto DEAE capture step and an online-connected desalting step resulted in near 100% physical recovery and 70–90% infectious recovery for both pH 7.4 and pH 7.0. Figure 4 A three-step chromatographic setup with an online-connected Capto DEAE capture step, desalting step, and Capto Core purification step resulted in 60% physical particle recovery and only 30% infectious particle recovery for the second and third steps at pH 7.4. Using pH 7, which is better for the Capto Core step, resulted in approximately 90% physical particle recovery and close to 100% infectious particle recovery. A third purification step is required to remove DNA impurities that were co-eluted with lentiviral particles in the Capto DEAE capture step (data not shown).

[0119] p24 ELISA was used to determine total particle titer, and cell-based transduction assays (calculated by flow cytometry for GFP-expressing cells) were used to determine infectious titer.

[0120] Following Capto Core 700, impurity levels in the final sample were below the detection limits for Micro BCA total protein assay and Picogreen total DNA assay (data not shown). Highly purified lentivirus was obtained.

[0121] Example 2 The experimental design for separating lentivirus particles from impurities is carried out using the same equipment and samples as in Example 1 above, with the following modifications: Regarding the first chromatographic material (for the capture step): - Different support materials, such as membranes or solid materials; - Different Capto TM Core molecular weight cutoff, for example, CC400 (Cytiva, Sweden); - Additional capture steps, such as by applying affinity ligands and anion exchange ligands or hemi-affinity ligands.

[0122] Regarding biological target compounds: - Lentiviral pseudotypes; - Inserts (target gene) that are different from GFP; - Exosomes, not lentiviruses; - Other enveloped viruses, not lentiviruses.

[0123] References F. Higashikawa et al., Virology 280, 124-131 (2001), Kinetic Analyses of Stability of Simple and Complex Retroviral Vectors Sara Nilsson, Doctoral thesis, UCL (2016), Process development oflentiviral vector expression, purification and formulation for genetherapy applications WO2009131526.

Claims

1. A method (100) for separating coated or membrane-bound bioparticles from one or more impurities, the method comprising: a. Add (110) a feed comprising coated or membrane-bound bioparticles and one or more impurities to a first chromatographic apparatus (70), the first chromatographic apparatus (70) comprising a first chromatographic material comprising a support material functionalized with a ligand, wherein the ligand comprises anion exchange groups or affinity groups having binding affinity to the coated or membrane-bound bioparticles; b. Elute the membrane-coated or membrane-bound biological particles (120) from the first chromatographic apparatus in at least one eluent fraction; c. Add (130) the at least one eluent fraction containing a membrane or membrane-bound biological particle obtained in step b to a second chromatographic apparatus (72), the second chromatographic apparatus (72) containing a second chromatographic material, the second chromatographic material containing a conditioning chromatographic material; d. Obtain the (140) membrane-coated or membrane-bound bioparticles from the second chromatographic apparatus in at least one flow fraction; The feed continuously passes through the first chromatographic apparatus and the second chromatographic apparatus to enable separation, wherein the first chromatographic apparatus and the second chromatographic apparatus are connected in series.

2. The method of claim 1, wherein the method includes applying a selection valve arrangement (60) configured to enable separation by allowing the feed to continuously pass through the first chromatographic apparatus and the second chromatographic apparatus; wherein the selection valve arrangement includes a first chromatographic apparatus selection valve (62).

3. The method of claim 2, wherein the selection valve arrangement further comprises a second chromatographic device selection valve (64).

4. The method according to any one of claims 1-3, further comprising: e. Add (150) the at least one flow fraction containing a membrane or membrane-bound bioparticle obtained in step d to a third chromatographic device (74), the third chromatographic device comprising a third chromatographic material comprising porous beads having an internal porous core and an external porous shell, wherein the core is capable of binding molecules through hydrophobic interactions, and wherein the pore size of the shell prevents particles having a size ≥20 nm from contacting the core; f. Obtain the (160) membrane-coated or membrane-bound bioparticles from the third chromatographic apparatus in at least one flow fraction; The at least one flow fraction obtained in step d is continuously transferred to and through the third chromatographic apparatus, which is connected in series with the first and second chromatographic apparatuses.

5. The method of claim 4, wherein the selection valve arrangement further comprises a third chromatographic device selection valve (66).

6. The method according to any one of claims 1-5, further comprising balancing (125) the second chromatographic material to the conditions required for the coating or membrane-bound bioparticles to be obtained in step d and optionally in step f prior to step c.

7. The method according to any one of claims 1-6, further comprising balancing (145) the third chromatographic material to the conditions required for the coating or membrane-bound bioparticles to be obtained in step f prior to step e.

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

9. The method of claim 8, wherein the enveloped viral particle is a lentiviral particle.

10. The method of claim 8, wherein the extracellular vesicles are exosomes.

11. The method according to any one of claims 1-10, wherein the ligand of the first chromatographic material comprises an anion exchange group defined by formula I: (I) 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 wherein R1, R2 and R3 are each CH3.

12. The method according to any one of claims 1-10, wherein the ligand of the first chromatographic material comprises an anion exchange group defined by formula II: (II) in: m is an integer from 1 to 3; R1 and R2 are independently selected from C1-C3 alkyl groups; R3 and R4 are independently selected from C1-C3 alkyl groups and CH2CHOHCH3; and R5 is selected from hydrogen, C1-C3 alkyl groups and CH2CHOHCH3. The condition is that if m is 1, then the ligands of strong or partially strong anion exchange chromatographic materials are defined by the following equation III: (III) Where n is an integer between 0 and 3; The condition is that if n is 0, then R3 and R4 are independently selected from C1-C3 alkyl groups, and R5 is hydrogen or CH2CHOHCH3.

13. The method according to any one of claims 1-10, wherein the ligand of the first chromatographic material comprises an anion exchange group defined by formula IV: (IV) For each occurrence, X is independently selected from H, OH, or C. 1-3 Groups, and R1, R2, R3, and R4 are independently selected from H and C. 1-3 Group, The C3 group can be either straight-chain or branched. Where C 1-3 The functional groups include those independently selected from OH and OC. 1-2 SC 1-2 The groups NH, NHR and NR2, R is selected from H and C. 1-3 Group.

14. The method according to any one of claims 1-10, wherein the ligand of the first chromatographic material comprises an affinity group having binding affinity for the coating or membrane-bound bioparticle.

15. The system according to any one of claims 9-14, wherein the support material of the first chromatographic material comprises a membrane structure, nanofibers, monolithic material, porous particles, non-porous particles, or an expanded bed medium.

16. The system according to any one of claims 9-15, wherein the second chromatographic material comprises size exclusion chromatographic material.

17. A chromatographic system (10), said system comprising: A buffer valve arrangement (20) is configured to allow independent control of the first buffer feed and the second buffer feed; Pump arrangement (40) is configured to supply a first buffer feed, a second buffer feed, and a feed containing a biological target compound and one or more impurities; The selection valve arrangement (60) includes a first chromatographic device selection valve (62); A first chromatographic apparatus (70) includes a first chromatographic material comprising a support material functionalized with a ligand, wherein the ligand comprises anion exchange groups or affinity groups having binding affinity to biological target compounds. A second chromatographic apparatus (72) comprising a second chromatographic material, the second chromatographic material comprising a regulating chromatographic material; The selection valve arrangement is configured to separate the biological target compound from the impurities by allowing a feed containing a biological target compound and one or more impurities to continuously pass through the first chromatographic unit and the second chromatographic unit, wherein the first chromatographic unit and the second chromatographic unit are configured to be connected in series.

18. The system of claim 17, wherein the selection valve arrangement further comprises a second chromatographic device selection valve (64).

19. The system of claim 17 or 18, further comprising a third chromatographic device (74) comprising a third chromatographic material comprising porous beads having an internal porous core and an external porous shell, wherein the core is capable of binding molecules through hydrophobic interactions, and wherein the pore size of the shell prevents particles having a size ≥20 nm from contacting the core. The third chromatographic device is configured to be connected in series with the first and second chromatographic devices, and the selection valve arrangement is configured to enable separation by allowing the feed to continuously pass through the first, second, and third chromatographic devices.

20. The system of claim 19, wherein the selection valve arrangement further comprises a third chromatographic device selection valve (66).

21. The system according to any one of claims 17-20, wherein the ligand of the first chromatographic material comprises an anion exchange group defined by formula I: (I) 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 wherein R1, R2 and R3 are each CH3.

22. The system according to any one of claims 17-20, wherein the ligand of the first chromatographic material comprises an anion exchange group defined by formula II: (II) in: m is an integer from 1 to 3; R1 and R2 are independently selected from C1-C3 alkyl groups; R3 and R4 are independently selected from C1-C3 alkyl groups and CH2CHOHCH3; and R5 is selected from hydrogen, C1-C3 alkyl groups and CH2CHOHCH3. The condition is that if m is 1, then the ligands of strong or partially strong anion exchange chromatographic materials are defined by the following equation III: (III) Where n is an integer between 0 and 3; The condition is that if n is 0, then R3 and R4 are independently selected from C1-C3 alkyl groups, and R5 is hydrogen or CH2CHOHCH3.

23. The system according to any one of claims 17-20, wherein the ligand of the first chromatographic material comprises an anion exchange group defined by formula IV: (IV) For each occurrence, X is independently selected from H, OH, or C. 1-3 Groups, and R1, R2, R3, and R4 are independently selected from H and C. 1-3 Group, The C3 group can be either straight-chain or branched. Where C 1-3 The functional groups include those independently selected from OH and OC. 1-2 SC 1-2 The groups NH, NHR and NR2, R is selected from H and C. 1-3 Group.

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

25. The system according to any one of claims 17-24, wherein the support material of the first chromatographic material comprises a membrane structure, nanofibers, monolithic material, porous particles, non-porous particles, or an expanded bed medium.

26. The system according to any one of claims 17-25, wherein the second chromatographic material comprises size exclusion chromatographic material.

27. Use of the system according to any one of claims 17-26 for separating a biological target compound from one or more impurities, wherein the biological target compound is selected from enveloped or membrane-bound biological particles, such as enveloped viral particles, extracellular vesicles, and virus-like particles; optionally, wherein the enveloped viral particles are lentiviral particles; optionally, wherein the extracellular vesicles are exosomes.

28. The use according to claim 27, wherein the first chromatographic material is selected from: i. A porous particulate support material functionalized with diethylethanolamine ligand; Optionally, the ligand is connected to the support material via a polymer, such as dextran; ii. A support material in the form of a convection-based membrane structure comprising a nonwoven web of polymer nanofibers functionalized with diethylethanolamine ligands; iii. A support material in the form of a convection-based membrane structure comprising a nonwoven web of polymer nanofibers functionalized with N,N-diethylethylenediamine ligands; iv. A support material in the form of a convection-based membrane structure comprising a nonwoven web of polymer nanofibers functionalized with ligands, the ligands comprising affinity groups having binding affinity for biological target compounds selected from enveloped or membrane-bound biological particles, such as enveloped viral particles, extracellular vesicles, and virus-like particles. Optionally, the enveloped viral particles are lentiviral particles. Optionally, the extracellular vesicles mentioned above are exosomes; and v. A support material in the form of porous particles functionalized with ligands, said ligands comprising affinity groups having binding affinity for biological target compounds selected from enveloped or membrane-bound biological particles, such as enveloped viral particles, extracellular vesicles, and virus-like particles; optionally said enveloped viral particles are lentiviral particles; optionally said extracellular vesicles are exosomes.

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