Methods and compositions for purifying small extracellular vesicles

Abstract

The present invention relates to a method for purifying small extracellular vesicles by cation exchange mixed-mode chromatography.

Description

[Technical field]

[0001] The present invention relates to a method for purifying small extracellular vesicles by cation exchange mixed-mode chromatography. [Background technology]

[0002] Small extracellular vesicles are small lipid bilayer vesicles. They form within the intracellular space and are secreted by cells. They typically range between 50 and 150 nm in diameter. Their spherical lipid bilayer is rich in various proteins such as tetraspanins. They display complex glycosylation patterns with specific glycoconjugates on their surface. An exemplary small extracellular vesicle is the exosome.

[0003] Small extracellular vesicles can carry small biomolecules, such as proteins, lipids, and nucleic acids. The composition and cargo of small extracellular vesicles varies depending on the parent cell and biological state. In recent years, there has been an increasing interest in exosomes for diagnostic and therapeutic applications. In clinical diagnostics, exosomes can prove their value in the field of liquid biopsies for the investigation of biomarkers to detect diseases in body fluids. However, they have also attracted attention for therapeutic purposes. Due to their advantages compared to artificial nanoparticles, they are ideally suited for drug delivery.

[0004] Exosomes are of natural origin, originate from the endosomal system, and appear to have low toxicity and low immunogenicity. Their surface and loading can be specifically adapted to cells or targets. Native exosomes are inherently bioactive and therefore can be envisaged for use in cancer immunotherapy or regenerative medicine. In ongoing preclinical research of exosomes, bulk isolates are deployed. These isolates are highly heterogeneous in their composition and cargo, so that the therapeutic potential of exosomes is unfortunately not fully exploited at present.

[0005] Separation of subpopulations can currently be achieved by density gradients, immunoadsorption, and affinity beads. These methods are not scalable or have limitations in terms of purity and yield. The heterogeneity of exosomal contents may necessitate the separation of subpopulations to enhance therapeutic effects and reduce undesirable side effects. As a result, there is a need to develop large-scale purification methods for exosomal subpopulations.

[0006] WO2019 / 241836 discloses the use of certain surfaces bearing polymers containing anionic or electron-rich groups for binding of exosomes. WO2020 / 191369 discloses the use of a combined ion exchange purification step for the purification of extracellular vesicles. Mixed mode chromatography is also mentioned, but always in combination with several other ion exchange chromatography steps. Summary of the Invention

[0007] It has now been found that mixed-mode cation exchange chromatography is particularly effective in purifying exosomes from process-related impurities such as host cell proteins, as well as for the separation of exosome subpopulations. No additional ion exchange chromatography step is necessary. Particularly preferred is the use of a pH gradient.

[0008] The present invention is therefore directed to a method for purifying small extracellular vesicles, such as exosomes, comprising the steps of: a) contacting a sample containing small extracellular vesicles with a mixed-mode cation exchange chromatography matrix, the chromatography matrix comprising cation exchange groups and hydrophobic groups. b) optionally washing the chromatography matrix; c) eluting the small extracellular vesicles bound to the chromatography matrix in step a) with an elution buffer, whereby the pH of the elution buffer is increased stepwise or as a gradient from a pH between 4 and 7 to a pH of 8.5 or higher.

[0009] In a preferred embodiment, the pH is increased as a linear gradient. In another embodiment, the method includes collecting small extracellular vesicles that flow through the chromatography matrix without being bound to it.

[0010] In a preferred embodiment, the chromatography matrix is ​​a membrane or a monolith. In a highly preferred embodiment, the chromatography matrix is ​​a membrane, in particular a hydrogel membrane.

[0011] In another preferred embodiment, the elution in step c) is performed with a linear pH gradient from a pH between 4 and 7 to a pH between 8.5 and 10, while keeping the conductivity at a constant level, and two or more fractions containing different subpopulations of small extracellular vesicles are eluted at different pH ranges. For example, one fraction containing a first subpopulation is eluted at a pH between 7 and 8, and another subpopulation is eluted at a pH between 8 and 9.

[0012] In another preferred embodiment, the small extracellular vesicles are exosomes. The present invention is further directed to a method for purifying small extracellular vesicles from cell culture supernatant by isolating and / or purifying the small extracellular vesicles by contacting them with a mixed-mode cation exchange chromatography matrix according to the method of the present invention.

[0013] In one embodiment, the method of the invention comprises one or more of the following steps: - Clarification - Filtration - Dialysis / Diafiltration - Tangential flow filtration - Treatment with nucleases, e.g. RNase and / or DNase - Treatment with chloroform - Ion exchange chromatography - Affinity Chromatography - Hydrophobic Interaction Chromatography - Centrifugation - PEG precipitation DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Before describing the invention in detail, it is to be understood that the invention is not limited to particular compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "ligand" includes a plurality of ligands, and reference to an "antibody" includes a plurality of antibodies, and so forth.

[0015] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For purposes of the present invention as described herein, the following terms are defined.

[0016] Small extracellular vesicles (EVs) are nano-sized cell-derived particles released by almost all types of cells. They are small lipid bilayer vesicles and have a diameter between 20-200 nm, preferably between 50-150 nm. Depending on their biogenesis and size, EVs can be generally divided into three subgroups: exosomes, microvesicles and apoptotic bodies. Microvesicles, in contrast to exosomes, are formed by outward blebbing of the cell membrane. However, exosomes and microvesicles overlap in size and are not always easily and clearly distinguishable. Therefore, exosomes and microvesicles have often been referred to as small EVs (sEVs) and used interchangeably.

[0017] Typically, the sample contains two or more different subpopulations of small extracellular vesicles. Such subpopulations differ in their composition, for example, in their glycosylation patterns and their surface protein patterns, and / or in their cargo. Small EVs can be native or engineered.

[0018] As used herein, the term "cell" or "cell line" refers to a single cell or a cell population. The cell may be capable of continuous or sustained growth and division in vitro. In some embodiments, by way of example, the terms "HEK293 cells", "293 cells" or their grammatical equivalents are used interchangeably herein and refer to the host / packaging cell line used in the methods disclosed herein. The cell may also originate from an organism, such as an animal, a human subject, and may include tissue cells, blood cells, or others.

[0019] Suitable cells and cell lines are described for use in producing exosomes. The cells themselves may be selected from any biological organism, including prokaryotic (e.g., bacterial) cells and eukaryotic cells, including insect cells, yeast cells and mammalian cells. Particularly preferred host cells are selected from any mammalian species, including but not limited to mesenchymal stem cells (MSCs), keratinocytes, primary fibroblasts, hepatocytes and myoblasts from mammals, including humans, monkeys, mice, rats, rabbits and hamsters, or cell lines such as A549, WEHI, 3T3, 10T1 / 2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, HEK 293 cells, Saos, C2C12, L cells, HT1080 and HepG2.

[0020] Purification refers to increasing the purity of a target molecule, in this case an exosome, for example by removing one or more impurities. The term "impurity" or "contaminant" as used herein refers to any foreign or deleterious molecule or species, including DNA, RNA, one or more host cell proteins, nucleic acids, endotoxins, biological macromolecules such as lipids, impurities of synthetic origin such as detergents, and one or more additives that may be present in a sample containing exosomes to be purified and thereby separated from one or more of the impurities.

[0021] As used herein, and unless otherwise stated, the term "sample" refers to any composition or mixture containing exosomes. The sample is preferably a liquid sample containing exosomes. The sample may be derived from biological or other sources. Biological sources include eukaryotic and prokaryotic sources such as plant and animal cells, tissues and organs. The sample may also include diluents, buffers, detergents, and contaminating species, debris, etc. that are found mixed with the target molecule, i.e., small extracellular vesicles. The sample may be "partially purified" (i.e., subjected to one or more purification steps, such as a filtration step) or may be obtained directly from an organism or cell that produces small extracellular vesicles; for example, the sample may include harvested cell culture fluid.

[0022] The terms "purifying," "separating," or "isolating," as used interchangeably herein, refer to increasing the purity of targeted exosomes from a composition or sample that contains targeted exosomes and one or more impurities and / or non-targeted exosomes.

[0023] The term "chromatography" refers to any type of technique that separates an analyte of interest (e.g., a target exosome) from other molecules present in a sample. Often, the target exosome is separated from other molecules as a result of differences in the rate at which individual molecules of a mixture bind to and / or migrate through a chromatographic matrix under the influence of a mobile phase.

[0024] The terms "matrix" or "chromatographic matrix" are used interchangeably herein and refer to a solid phase through which a sample passes during chromatographic separation. A matrix typically comprises a substrate and a ligand covalently bound to the substrate. The matrix of the present invention preferably comprises or consists of a particle, a membrane or a monolith, preferably the substrate is a membrane or monolith, most preferably a membrane.

[0025] A "ligand" is a functional group that is part of a chromatography matrix, typically attached to the matrix substrate, and that determines the binding and interaction properties of the matrix. Examples of "ligands" include, but are not limited to, ion exchange groups, hydrophobic interaction groups, hydrophilic interaction groups, thiophilic interaction groups, metal affinity groups, affinity groups, bioaffinity groups, and mixed mode groups (combinations of the foregoing). It is also possible for one ligand to have more than one binding / interaction property.

[0026] The matrix of the present invention comprises at least cation exchange groups and hydrophobic interaction groups. It is a mixed mode cation exchange chromatography matrix. The cation exchange groups may be strong cation exchange groups, such as, for example, sulfonic acid groups. They may also be weak cation exchange groups, such as carboxymethyl or carboxylic acid. Examples of hydrophobic interaction groups are hydrophobic groups, such as phenyl, butyl, propyl, hexyl. The matrix may comprise two or more different cation exchange groups, for example, weak and strong cation exchange groups. It may also comprise two or more different hydrophobic interaction groups. It may additionally comprise further other types of ligands. The groups may be part of the substrate or they may also be part of the ligand. One ligand may comprise one or more different cation exchange groups and / or hydrophobic groups.

[0027] The ligand can be attached to the substrate of the matrix by any type of covalent attachment. Covalent attachment can be performed by directly binding a functional group to a suitable residue on the substrate, such as, for example, OH, NH2, carboxyl, phenol, anhydride, aldehyde, epoxide, or thiol. It is also possible to attach the ligand via a suitable linker. It is also possible to produce the matrix by polymerizing a monomer that contains the ligand and a polymerizable moiety. Examples of matrices produced by polymerization of suitable monomers are polystyrene, polymethacrylamide or polyacrylamide-based matrices produced by polymerizing suitable styrene or acryloyl monomers.

[0028] In another embodiment, the chromatography matrix can be produced by grafting ligands onto or from the substrate. Grafting is suitable from processes involving controlled free radical polymerization, such as the method of atom transfer free radical polymerization (ATRP). A highly preferred one-step grafting, for example from the polymerization reaction of acrylamides, methacrylates, acrylates, methacrylates, etc. functionalized with ionic, hydrophilic or hydrophobic groups, can be initiated by cerium (IV) on hydroxyl-containing supports without the need to activate the support.

[0029] When a chromatography matrix is ​​used in a chromatographic separation, it is typically used within a separation device, also called a housing, as a means for holding the matrix.

[0030] In one embodiment, the device comprises a housing with an inlet and an outlet and a fluid path between the inlet and the outlet. In a preferred embodiment, the device is a chromatography column. Chromatography columns are known to those skilled in the art. They usually comprise a cylindrical tube or cartridge packed with a stationary phase, and a filter and / or a means for fixing the stationary phase in the tube or cartridge, and optionally a connection for solvent delivery to and from the tube or cartridge. The size of the chromatography column varies depending on the application, for example analytical or preparative. In one embodiment, the column, or generally the separation device, is a single-use device.

[0031] A "buffer" is a solution that resists changes in pH by the action of its acid-base conjugate components. For example, various buffers that can be employed depending on the desired pH of the buffer are described in Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., ed. Calbiochem Corporation (1975). Non-limiting examples of buffers include MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, and ammonium buffers, and combinations thereof.

[0032] According to the present invention, the term "buffer" or "solvent" is used for any liquid composition used to load, wash, elute, re-equilibrate, strip and / or sanitize a chromatography matrix.

[0033] When "loading" a chromatography column in binding and elution mode, a sample or composition containing target molecules and one or more impurities is loaded onto the chromatography column. In preparative chromatography, the sample is preferably loaded directly without adding a loading buffer. If a loading buffer is used, the buffer has a composition, conductivity, and / or pH such that the target exosomes bind to the stationary phase, while ideally all impurities such as host cell proteins flow through the column without being bound. Typically, if used, the loading buffer has the same or similar composition as the equilibration buffer used to prepare the column for loading. The final composition of the sample loaded onto the column is called the feed. The feed may include the sample and the loading buffer, but preferably it is only the sample.

[0034] "Washing" or "washing" a chromatography matrix means passing a suitable liquid, e.g., a buffer, through or over the matrix. Typically, washing is used to remove weakly bound impurities from the matrix in bind / elute mode before eluting the target molecule. In addition, washing steps can be used to reduce residual detergent levels, enhance virus clearance, and / or modify conductivity carryover during elution.

[0035] "Eluting" a molecule (e.g., a target exosome) from a matrix means that the molecule is removed from it. Elution can occur by changing solution conditions such that a buffer different from the loading buffer and / or wash buffer competes with the molecule of interest for ligand sites on the matrix, or by altering the equilibrium of the target molecule between the stationary and mobile phases such that the target molecule is preferentially present in the elution buffer.

[0036] A non-limiting example is to elute molecules from an ion exchange resin by changing the ionic strength of the buffer surrounding the ion exchange material, such that the buffer competes with the molecules for the charged sites on the ion exchange material. Alternatively, a pH change is also suitable.

[0037] The terms "flow-through process", "flow-through mode", and "flow-through operation", as used interchangeably herein, refer to a chromatographic process in which at least one target molecule (e.g., an exosome) contained in a sample along with one or more impurities is intended to flow through a chromatographic matrix that generally binds the one or more impurities, where the target molecule generally does not bind (i.e., flows through) and elutes from the chromatographic matrix along with the loading buffer.

[0038] The terms "binding and elution mode" and "binding and elution process" as used herein refer to a separation technique in which at least one target molecule (e.g., an exosome) contained in a sample is bound to a suitable chromatographic matrix and then eluted with a buffer different from the loading buffer.

[0039] The term "ion density" as used herein refers to the number of ions per unit volume or mass of a given separation material, more specifically, the number of ions of a given type (e.g., positive or negative ions) per unit volume or mass of the separation material. Most often, the number of ions is estimated by titrating a given separation material. Moreover, the amount of ions is given in equivalents (eq) per unit of mass or volume for the separation material.

[0040] The term "conductivity" as used herein refers to an intrinsic property of most materials that quantifies how strongly it resists or conducts electric current. In an aqueous solution, such as a buffer solution, electric current is carried by charged ions. Conductivity is determined by the number of charged ions, the amount of charge they carry, and how fast they move. Thus, in most aqueous solutions, the higher the concentration of dissolved salts, the higher the conductivity. Increasing the temperature allows the ions to move faster, thus increasing the conductivity. Typically, unless otherwise specified, conductivity is defined at room temperature. The base unit of conductance is the Siemens (S). It is defined as the reciprocal of the resistance in Ohms measured between the opposing faces of a 1 cm cube of liquid. Thus, values ​​are estimated in S / cm.

[0041] Log reduction is a measure of how thoroughly a purification process reduces the concentration of a contaminant. It is defined as the common logarithm of the ratio of the level of contamination before and after the process, with an increase of 1 corresponding to a reduction in concentration by a factor of 10. In general, a log reduction of n means that the remaining concentration of the contaminant is only 10-n times lower than the original. Thus, for example, a log reduction of 0 is no reduction at all, while a log reduction of 1 corresponds to a 90 percent reduction from the original concentration, and a log reduction of 2 corresponds to a 99 percent reduction from the original concentration.

[0042] Membranes as chromatographic matrices can be distinguished from particle-based chromatography by the fact that the interaction between solutes, e.g., target exosomes or contaminants, and the matrix does not occur in the dead-end pores of the particle, but mainly occurs in the through-pores of the membrane. Exemplary membrane types are flat sheet systems, stacks of membranes, microporous polymer sheets with integrated cellulose, polystyrene or silica-based membranes, as well as radial flow cartridges, hollow fiber modules and hydrogel membranes. Preferred are hydrogel membranes. Such membranes include a membrane support and a hydrogel formed in the pores of the support. The membrane support provides mechanical strength to the hydrogel. The hydrogel determines the properties of the final product, such as pore size and binding chemistry.

[0043] The membrane support can consist of any porous membrane, such as a polymeric membrane, a ceramic-based membrane, a woven or non-woven fibrous material, etc. Suitable polymeric materials for the membrane support are cellulose or cellulose derivatives, as well as other, preferably inert, polymers, such as polyethylene, polypropylene, polybutylene terephthalate or polyvinylidene difluoride.

[0044] Hydrogels can be formed through the in situ reaction of one or more polymerizable monomers with one or more crosslinkers and / or one or more crosslinkable polymers to form crosslinked gels, preferably with macropores. Suitable polymerizable monomers include monomers containing vinyl or acrylic groups. Preferred are monomers containing additional functional groups that either directly form the ligands of the matrix or are suitable for attaching ligands. Suitable crosslinkers are compounds that contain at least two vinyl or acrylic groups.

[0045] Further details on suitable membrane supports, monomers, crosslinkers, etc., as well as suitable production conditions can be found in WO04073843 and WO2010 / 027955. Particularly preferred are membranes made of inert flexible fibrous reticular supports, such as Natrix® type membranes, Merck KGaA, Germany, which contain an assembly of porous polyacrylamide hydrogels with cation exchange groups and hydrophobic groups inside and around the fibrous reticular support. Further details on suitable hydrogel mixed-mode membranes can be found in WO2014018635.

[0046] Depending on the membrane device used, the respective process is carried out with different operating principles such as dead-end operation, cross-flow operation and radial flow operating systems. Dead-end operation is preferred.

[0047] Examples of membranes that are suitable for use in the method of the present invention are: - Mustang® type membranes, such as Pall, which have a polyethersulfone (PES) based support and a cross-linked polymer coating functionalized with suitable ligands. - Sartobind® type membranes, such as Sartorius, made of stabilized reinforced cellulose functionalized with suitable ligands. - Membranes made of stabilized reinforced cellulose containing a hydrogel with suitable ligands, such as Sartobind® Jumbo Membranes, Sartorius, made of stabilized reinforced cellulose. - 3M™ Emphaze™ Hybrid Purifier type membranes, such as 3M, made of a fine fiber nonwoven scaffold containing a hydrogel with suitable ligands. - Membranes made of inert flexible fibrous reticulated supports, such as Natrix® type chromatography membranes, Merck KGaA, Germany, which contain a porous polyacrylamide hydrogel with suitable cation exchange and hydrophobic ligands within and around the fibrous reticulated support.

[0048] Monoliths or monolithic adsorbents, like membranes, have through-holes, such as interconnected channels, that allow liquid to flow from one side of the monolith through the monolith to the other side of the monolith. As the mobile phase flows through these through-holes, the molecules to be separated are transported by convection rather than by diffusion. Due to their structure, monolithic adsorbents exhibit flow-rate independent separation efficiency and dynamic capacity.

[0049] The monolith is typically formed in situ from the reactant solution and can have any shape or constrained geometry, typically a frit-free structure, which ensures convenience of operation. Preferably, the monolithic material has a dual porous structure, mesopores and macropores. The micron-sized macropores are through-holes and ensure fast dynamic transport and low back pressure in applications, while the mesopores contribute to sufficient surface area and thus high loading capacity.

[0050] The monoliths can be made of organic, inorganic or organic / inorganic hybrid materials, with organic polymer-based monoliths being preferred. The synthesis of organic polymer monoliths is typically carried out by one-step polymerization, which provides tunable porous structures with tailored functional groups. Generally, a prepolymerization mixture consisting of monomers, crosslinkers, porogenic solvents, and initiators in appropriate ratios is polymerized in a suitable container, also called a mold, which determines the form of the monolith. Polymerization is typically initiated by the use of heat, UV radiation, microwaves, or gamma radiation in the presence of an initiator. After reacting at the appropriate temperature for a specified time, the resulting material is typically washed with a solvent to remove unreacted components and the porogenic solvent.

[0051] Suitable organic polymers are polymethacrylates, polyacrylamides, polystyrenes, polyurethanes, and the like, such as poly(methacrylic acid-ethylene dimethacrylate), poly(glycidyl methacrylate-ethylene dimethacrylate) or poly(acrylamide-vinylpyridine-N,N'-methylenebisacrylamide).

[0052] Inorganic monoliths can be made of silica or other inorganic oxides. Preferably, they are made of silica. Silica monoliths are usually prepared via a sol-gel process with phase separation. This mainly involves hydrolysis, condensation, and polycondensation of silica precursors. Typically, tetraethoxysilane (TEOS) or tetramethyl orthosilicate (TMOS) is dispersed in a suitable solvent in the presence of a porogen (e.g., poly(ethylene glycol) (PEG)), followed by the addition of a catalyst, acid or base, or a dual catalyst, acid and base, in sequence. After reacting for a given time, the resulting gel-like product is washed with a solvent to remove unreacted precursor, porogen, and catalyst, followed by a suitable post-treatment, typically a thermal treatment.

[0053] The monolith can be modified with suitable functional groups, in this case cation exchange groups and hydrophobic interaction groups, to allow targeted interaction with samples containing target molecules and thus targeted separation. Typically, the monolith is contained within a housing, such as a column. Membranes and monoliths can also be produced by 3D printing processes.

[0054] The particulate substrate can be prepared, for example, from organic polymers. This type of organic polymer can be polysaccharides, such as agarose, dextran, starch, cellulose, etc., or synthetic polymers, such as poly(acrylamide), poly(methacrylamide), poly(acrylate), poly(methacrylate), hydrophilically substituted poly(alkylaryl ether), hydrophilically substituted poly(alkylvinyl ether), poly(vinyl alcohol), poly(styrene), and copolymers of the corresponding monomers. These organic polymers can also be preferably employed in the form of crosslinked hydrophilic networks. This also includes polymers made of styrene and divinylbenzene, which, like other hydrophobic polymers, can preferably be employed in hydrophilized form.

[0055] Alternatively, inorganic materials such as silica, zirconium oxide, titanium dioxide, aluminum oxide, etc. can be employed as particulate substrates. It is also possible to employ composite materials, i.e. particles that can themselves be magnetized, for example by copolymerization of magnetic particles or magnetic cores. It is also possible to use shells, i.e. core-shell materials, where at least the surface or coating bears OH groups.

[0056] However, since the materials according to the invention should preferably withstand long periods of use, e.g., alkaline cleaning or regeneration at basic pH, the use of hydrophilic substrates that are hydrolytically stable or difficult to hydrolyze is preferred.

[0057] The base matrix may consist of irregularly shaped or spherical particles, the particle size of which may be between 2 and 1000 μm. An average particle size between 3 and 300 μm is preferred, and in the most preferred embodiment the average particle size is between 20 and 63 μm.

[0058] The particulate substrate may be in the form of, inter alia, non-porous or preferably porous particles. The average pore size may be between 2 and 300 nm. Pore sizes between 5 and 200 nm are preferred, and the most preferred average pore size is between 40 and 110 nm.

[0059] In a highly preferred embodiment, the particulate substrate is formed by copolymerization of a hydrophilic substituted alkyl vinyl ether selected from the group of 1,4-butanediol monovinyl ether, 1,5-pentanediol monovinyl ether, diethylene glycol monovinyl ether or cyclohexanedimethanol monovinyl ether and divinylethyleneurea (1,3-divinylimidazolin-2-one) as a crosslinker. An example of a suitable commercially available vinyl ether-based substrate is Eshmuno®, Merck KGaA, Germany.

[0060] Preferred particle matrices are those with weak cation exchange, strong cation exchange and hydrophobic groups, e.g. matrices with sulfonic acid, carboxylic acid and phenyl groups, such as Eshmuno® HCX, Merck KGaA, Germany.

[0061] Also preferred is a particulate chromatography matrix comprising a hydroxyl-containing substrate, preferably a vinyl ether-based substrate, having polymer chains grafted to its surface by covalent bonds, characterized in that: a) the polymer chains are covalently attached to the base matrix via hydroxyl groups; b) the polymer chain comprises an end group -N(Y)-R3; Y's are independently H or CH 3 is preferably H, R3 is -CHCOOMR4, R4 is C1-C4 alkyl, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, preferably isopropyl and isobutyl, very preferably isobutyl, or C1-C4 perfluoroalkyl; and M is, independently of each other, H, Na, K, or NH 4 + It is.

[0062] One example is Eshmuno® CMX, Merck KGaA. The substrate may also be in the form of a fiber, hollow fiber, or coating.

[0063] The present invention provides a method for separating or purifying small extracellular vesicles.This means that one or more subpopulations of small extracellular vesicles can be separated from one or more other subpopulations of small extracellular vesicles and / or from other impurities in a sample.Preferably, at least one small extracellular vesicle is separated from at least one impurity, in particular DNA, and from at least one other subpopulation of small extracellular vesicles.

[0064] It has been found that chromatographic separation on a mixed-mode cation exchange chromatography matrix containing at least one type of cation exchange group and one type of hydrophobic group using a pH change for elution is suitable for separating even subpopulations of small extracellular vesicles. It is hypothesized that different glycosylation patterns, different surface protein patterns, and / or different cargoes within the vesicles result in slight differences in the charge of the vesicles. Unexpectedly, such minimal differences are sufficient to result in fractionated elution when a pH gradient is applied to the mixed-mode cation chromatography matrix.

[0065] The method of the present invention can separate, enrich and / or purify small extracellular vesicles, preferably exosomes, and allows efficient separation.In a particular aspect of the present invention, targeted exosomes can be separated from impurities and other non-targeted exosomes in one chromatographic step.High resolution separation can be achieved, and subpopulations of different exosome species can be isolated.

[0066] The method of the present invention has been found to be particularly suitable for separating exosomes from process-related impurities such as DNA and host cell proteins.In addition, cation exchange mixed-mode chromatography has been found to be suitable for separating exosomes, preferably based on their glycosylation rate.The higher the glycosylation rate of the surface of exosomes, the higher the interaction of exosomes with the chromatography matrix, which leads to a higher retention on the chromatography matrix.Particularly good results are obtained with membrane-based chromatography matrices.

[0067] The production of cells containing small extracellular vesicles is known to those skilled in the art. Typically, selected cells are propagated in a suitable culture medium in a bioreactor under suitable conditions. The cells may be grown as an adherent or suspension culture.

[0068] In one aspect, the present disclosure provides a method for producing exosomes by the steps of: (i) culturing cells in a bioreactor; (ii) iv) isolating and / or purifying exosomes, where step iv) comprises chromatographic purification on a mixed-mode cation exchange chromatography matrix. The exosomes can be native or engineered exosomes.

[0069] Optionally, after cell harvesting, the resulting mixture is first filtered and / or centrifuged. In one embodiment, the mixture is filtered through a filter that removes large molecular contaminants and cellular debris but allows small extracellular vesicles to pass therethrough.

[0070] In one embodiment, the released small extracellular vesicles can be separated and purified from cell culture medium using clarification.Clarification can be a microfiltration process in which relatively larger components such as lysed cells and / or impurities are removed from the solution.Clarification filters include depth filtration, charged depth filtration, and similar microfiltration techniques.

[0071] Tangential flow filtration can be used to concentrate the mixture of purified small extracellular vesicles and to remove salts and proteins.Tangential flow filtration (TFF) refers to a generally rapid and efficient method for filtration or purification of solutions containing target products and / or impurities, during which the solution or liquid stream flows parallel to the filtration membrane.

[0072] Centrifugation can be a low speed centrifugation to remove larger particles such as cell debris. This can be done, for example, at 10,000-12,000 g for 10-30 minutes. The released small extracellular vesicles can be found in the supernatant.

[0073] Isolation and / or purification of small extracellular vesicles typically involves one or more of the following process steps: - Clarification - Filtration - Dialysis / Diafiltration - Tangential flow filtration - Treatment with nucleases, e.g. RNase and / or DNase - Treatment with chloroform - Ion exchange chromatography - Affinity Chromatography - Hydrophobic Interaction Chromatography - Centrifugation - PEG precipitation

[0074] In some embodiments, a nuclease, typically an endonuclease, is added, for example to reduce the amount of host cell DNA. It can be added directly to the mixture in the bioreactor. The nuclease may degrade both DNA and RNA. In one embodiment, the endonuclease is a genetically modified endonuclease from Serratia marcescens sold under the name Benzonase® (EMD Millipore, US).

[0075] In the cation exchange mixed chromatography step, the target small extracellular vesicles are separated from at least one impurity in the sample by contacting the sample containing the small extracellular vesicles with a chromatography matrix. The contact time usually ranges from 30 seconds to 24 hours. It is advantageous to work according to the principle of liquid chromatography by passing a liquid through a chromatography column containing a mixed mode cation exchange chromatography matrix. The liquid can simply pass through the column by its gravity or be pumped by means of a pump.

[0076] An alternative method is batch chromatography, in which the separation material is mixed with the liquid by stirring or shaking for as long as necessary to allow the exosomes to bind to the chromatography matrix.It is also possible to work according to the principle of chromatographic fluidized bed, for example by introducing the liquid to be separated into a suspension containing a chromatography matrix, where the chromatography matrix is ​​selected so that it is suitable for the desired separation due to its high density and / or magnetic core.

[0077] The chromatographic process is carried out in a binding and elution mode, and the target small extracellular vesicles are bound to the chromatographic matrix. The chromatographic matrix can then be washed with one or more washing buffers, which preferably have the same ionic strength and the same pH as the liquid in which the sample contacts the chromatographic matrix. The washing buffer removes substances that do not bind to the chromatographic matrix. Further washing steps with other suitable buffers may follow this without detaching the target small extracellular vesicles.

[0078] The release of the bound small extracellular vesicles is carried out by at least changing the pH in the elution solution. In addition, the ionic strength of the elution buffer and / or the solvent contained in the elution buffer may be changed compared to the loading conditions. Thus, the target small extracellular vesicles can be obtained in a purified and concentrated form in the elution solution. The target small extracellular vesicles usually have a purity of 70% to 99%, preferably 85% to 99%, particularly preferably 90% to 99% after release.

[0079] Cation exchange mixed mode materials can be used for the purification of small extracellular vesicles, allowing for efficient removal of impurities such as host cell proteins. Moreover, preferably, the same chromatography step also provides for the removal of non-target small extracellular vesicles. In a preferred embodiment, the chromatography matrix is ​​a membrane, in particular a hydrogel membrane.

[0080] The nature of the chromatography matrix used (i.e., strong and / or weak cation exchanger, type of hydrophobic group), as well as the salt concentration, buffer used, and pH conditions will vary depending on the variant of small extracellular vesicles selected as the target small extracellular vesicles. While all known variants of small extracellular vesicles share features such as size and shape, they differ in the details of molecular topology and surface charge distribution.

[0081] Thus, while it is expected that all small extracellular vesicle variants are suitable for purification by mixed-mode cation exchange chromatography, the optimal method can be determined systematically using screening experiments of chromatography resins and buffers, and different conditions will be required for each small extracellular vesicle variant to achieve efficient purification. The determination of such conditions will be readily apparent to one skilled in the art.

[0082] Preferably, chromatographic purification is performed by using a pH change from 4-7 to a pH above 8.5, preferably between 8.5-10, most preferably between pH 8.5-9.5, in gradient or step mode while keeping the conductivity at a constant level. The sample applied to the chromatographic matrix is ​​adjusted to the pH at which the pH gradient begins.

[0083] Suitable buffer solutions for use are, for example: 0.00796M citric acid, 0.009068M sodium dihydrogen phosphate, 0.021543M glycine, 0.010668M TRIS, 0.007666M 4 succinic acid, 0.0111M NaOH, and 0.2889M NaCl, with a pH of 4.5 and a conductivity of approximately 24mS / cm, and B buffer solution (for example, 0.00796M citric acid, 0.009068M sodium dihydrogen phosphate, 0.021543M glycine, 0.010668M TRIS, 0.007666M succinic acid, 0.046M NaOH, and 0.254M NaCl, with a pH of 8.5 and a conductivity of approximately 24mS / cm). Suitable alternatives are phosphate buffer or Tris buffer.

[0084] It is also possible to perform elution from the chromatographic matrix by combining a linear or step mode pH gradient with a linear or step mode salt gradient. The salt may be selected from the group consisting of NaCl, KCl, sulfate, formate and acetate, preferably NaCl. Typically, the gradient starts with a low salt concentration, for example between 10-100 mM salt, which is then increased until the target small extracellular vesicles are eluted, for example to 250-500 mM salt.

[0085] The term "column volume" refers to the volume inside a packed column that is not occupied by the chromatography matrix. This volume includes both the interstitial volume (the volume outside the matrix) and the porosity (pore volume) within the matrix itself.

[0086] In a preferred embodiment, the applied linear gradient lasts for approximately 60-120 column volumes (CV), plus optionally an additional retention step with the target elution buffer for at least 20 CV. The recovery of the target product is greater than 60%, preferably greater than 80%.

[0087] Furthermore, the present invention provides a chromatography-based small extracellular vesicle purification step that is regenerative and applicable in a wide operating window, e.g., pH 3-10, conductivity 1 mS / cm-50 mS / cm.

[0088] In a preferred embodiment, the method of the present invention comprises the steps of: 9 ~10 12 Used to purify samples containing small extracellular vesicles / ml sample. The window span of the conductivity is between 1 mS / cm and 50 mS / cm, and in a more preferred embodiment, the conductivity range is between 2 and 30 mS / cm. Preferably, the ion density of the chromatography matrix is ​​between 10 and 1200 μeq / g.

[0089] The method of the present invention can be applied to all small extracellular vesicles without the need for a previous process step. Preferably, crude cell culture supernatant (treated and clarified with nuclease) can be directly applied to the chromatography matrix.

[0090] In a highly preferred embodiment, the sample, e.g. in the form of crude cell culture supernatant treated and clarified with a nuclease such as Benzonase® (Merck GaA, Germany), is applied to a membrane carrying cation exchange and hydrophobic groups at a pH between 4.5 and 5.5 and a conductivity of approximately 20 mS to 30 mS. Elution was performed at a constant conductivity, for example with 150 mM NaCl, at a pH between 4.5 and 5.5, and at a pH between 8.5 and 10.5, depending on the sample pH. + This is done by applying a linear pH gradient.

[0091] Elution preferably results in at least two fractions. Fraction 1 contains the majority of process-related impurities. Fraction 2 contains a portion highly enriched in small extracellular vesicles and a log reduction in impurities of 2-3 logs. Highly preferred elution results in at least two fractions containing different subpopulations of small extracellular vesicles.

[0092] Preferred buffer compositions are those with a broad pH range of 4-11, examples of which include, but are not limited to, the Good buffer system containing Tris, phosphate and acetate, as well as amino acids.

[0093] The method of the present invention can efficiently purify small extracellular vesicles. Typically, contaminants, even DNA, can be reduced by more than 2 log logarithmic reduction. Preferably, the method achieves a host cell protein and DNA reduction of more than 2 log. In addition, different small extracellular vesicle populations can be separated based on their glycosylation rate, glycosylation pattern, surface protein pattern and / or cargo, preferably based on their glycosylation rate and glycosylation pattern. Thus, process and product-related impurities can be removed in one single step.

[0094] The invention is further illustrated by the following figures and examples, without, however, being limited thereto. The entire disclosures of all applications, patents, and publications cited above and below, as well as the corresponding EP application EP 22163007.2, filed March 18, 2022, are hereby incorporated by reference.

[0095] example HEK293T cells were grown in a total of 20 ml of DMEM prepared with L-glutamine and without FBS in a T-75 culture flask for 24 hours. The cell supernatant was used to isolate EVs. The medium was first centrifuged at 300xg for 10 minutes to remove any cells. The supernatant was then transferred to a clean tube and centrifuged again at 2,000xg for 15 minutes to remove other debris. At this point, the medium was concentrated to 3 ml by centrifugation at 1000xg for 10 minutes using a 100 kDa MWCO ultrafiltration device (Amicon® Ultra-15, Millipore). Buffer exchange with Buffer A (Table 1) was performed by centrifugation again three times at 1000xg for 10 minutes using a 100 kDa MWCO ultrafiltration device. Prior to chromatographic separation, the conditioned medium was filtered through a 0.22 μm filter (Millipore).

[0096] Chromatographic binding and elution separations on the Eshmuno® CMX used the buffers shown in Tables 1 and 2. Table 3 summarizes the methodology used on the Aekta™ system.

[0097] [Table 1]

[0098] [Table 2]

[0099] [Table 3]

[0100] The column packed with Eshmuno® CMX was equilibrated with Buffer A for at least 10 CV. 15 CV of conditioned cell supernatant was applied onto the column. A 10 CV wash step with Buffer A was performed to elute any weakly bound impurities prior to gradient elution. Exosomes were then eluted in a linear pH gradient for 60 CV. To elute tightly bound proteins, vesicles or other impurities, the column was washed with 6 CV of 1 M sodium hydroxide solution, followed by re-equilibration with Buffer A. The elution was fractionated into 0.5 mL fractions.

Claims

1. A method for purifying small extracellular vesicles, comprising the following steps: a) Contacting a sample containing small extracellular vesicles with a mixed-mode cation exchange chromatography matrix. b) Optionally, wash the chromatography matrix. c) Eluting the small extracellular vesicles bound to the chromatography matrix in step a) with an elution buffer, which is done by gradually or in a gradient increasing the pH of the elution buffer from a pH between 4 and 7 to a pH of 8.5 or higher.

2. The method according to claim 1, wherein the pH of the elution buffer increases as a linear gradient.

3. The method according to claim 1, comprising recovering small extracellular vesicles that flow through a chromatography matrix without binding to it.

4. The method according to claim 1, wherein the chromatography matrix is ​​a membrane or a monolith.

5. The method according to claim 1, wherein the chromatography matrix is ​​a hydrogel membrane.

6. The method according to claim 1, wherein the conductivity of the elution buffer is maintained at a constant level.

7. The method according to claim 1, wherein the elution in step c) is carried out over a linear pH gradient from a pH between 4 and 7 to a pH above 8.5, and two or more fractions containing different subpopulations of small extracellular vesicles are eluted in different pH ranges.

8. The method according to claim 1, wherein the small extracellular vesicle is an exosome.

9. A method for purifying small extracellular vesicles from cell culture supernatant by purifying small extracellular vesicles according to the method described in claim 1.

10. A method according to claim 7 or 9, comprising one or more of the following steps: - Clarification - Filtration - Dialysis / Dialysis Filtration - Tangential flow filtration - Treatment using nucleases, e.g., RNase and / or DNase - Treatment using chloroform - Ion exchange chromatography - Affinity chromatography - Hydrophobic interaction chromatography - Centrifugal separation - PEG precipitation.