Resin compositions and methods of use

Abstract

The present invention relates to compositions comprising certain ion exchange chromatography resins and extracellular vesicles. These resins may be particularly suitable for use in ion exchange chromatography columns and capturing extracellular vesicles.

Description

[0001] RESIN COMPOSITIONS AND METHODS OF USE

[0002] RELATED APPLICATION

[0003] This application claims priority from Australian Provisional Applications No. 2024900609 entitled “Resin compositions and methods of use” filed on 7 March 2024, and No. 2024901931 entitled “Resin compositions and methods of use” filed on 25 June 2024, the entire contents of which are hereby incorporated by reference.

[0004] FIELD

[0005] The present invention relates to compositions comprising certain ion exchange chromatography resins and extracellular vesicles. These resins may be particularly suitable for use in ion exchange chromatography columns and capturing extracellular vesicles.

[0006] BACKGROUND

[0007] Extracellular vesicles (EVs) are lipid bound vesicles secreted by cells. The main subtypes of EVs are exosomes, microvesicles and apoptotic bodies, the exact category in which EVs fall under depends on a number of factors including their biogenesis, size, content and function. EVs are secreted by all cell types and can be found in a number of bodily fluids such as plasma, urine, semen, saliva, and cerebral spinal fluid.

[0008] In recent years, the content or cargo of EVs has become increasingly scrutinized due to the recognition that EVs play crucial roles in cell-cell communication and the identification of disease associated changes in EV signaling. Indeed, studies have shown that EVs play roles in disease pathogenesis and thus EVs could serve as carriers of biomarkers useful for diagnostic purposes. As the EV landscape evolves from a niche field in the biotechnology space to a potential global cosmetics, diagnostics and pharmaceutical market, the need for reliable and scalable isolation methods for EVs is becoming increasingly evident.

[0009] Differential centrifugation and density gradient centrifugation are commonly used for EV capture and isolation (1). However, these techniques suffer with regard to the specificity of capturing and extracting different types of EVs. Furthermore, both techniques are very time-consuming processes that does not offer any potential for scalability in various commercial applications.

[0010] Improved compositions and methods for EV capture and isolation are required. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each of the appended claims.

[0011] SUMMARY OF THE INVENTION

[0012] The present inventors have surprisingly identified structural features of anionic resins that make them suitable for effective capture and isolation of extracellular vesicles (EVs). The findings are particularly surprising as they show improved efficiency over anionic resins that are “more anionic” and thus, based on charge, are expected to have a higher affinity for EVs.

[0013] In an aspect, there is provided a composition comprising an ion exchange chromatography resin and a population of extracellular vesicles (EVs), wherein the chromatography resin comprises a support matrix functionalized with ligands for binding to the EVs, wherein one or more of the ligands comprise a hydroxylated spacer which attaches an anion exchange group to the support matrix.

[0014] In an aspect, the present disclosure relates to the use of an ion exchange chromatography resin for separating extracellular vesicles from a composition, wherein the chromatography resin comprises a support matrix functionalized with ligands for binding to the EVs, wherein one or more of the ligands comprise a hydroxylated spacer which attaches an anion exchange group to the support matrix for separating extracellular vesicles from the composition.

[0015] In an aspect, there is provided a composition when used for separating extracellular vesicles (EVs), the composition comprising an ion exchange chromatography resin, wherein the chromatography resin comprises a support matrix functionalized with ligands for binding to the EVs, wherein one or more of the ligands comprise a hydroxylated spacer which attaches an anion exchange group to the support matrix.

[0016] In one embodiment, at least some of the EVs in the composition are bound to the ligands. In one embodiment, substantially all of the EVs in the composition are bound to the ligands.

[0017] In some embodiments, the hydroxylated spacer is an aliphatic spacer which is uninterrupted or interrupted and substituted with one or more hydroxyl groups. In some embodiments, the hydroxylated spacer is an aliphatic spacer which is uninterrupted and substituted with one or more hydroxyl groups. In some embodiments, the hydroxylated spacer is an aliphatic spacer which is interrupted and substituted with one or more hydroxyl groups.

[0018] In some embodiments, the hydroxylated spacer is an alkyl spacer which is uninterrupted or interrupted and substituted with one or more hydroxyl groups. In some embodiments, the hydroxylated spacer is an alkyl spacer which is uninterrupted and substituted with one or more hydroxyl groups. In some embodiments, the hydroxylated spacer is an alkyl spacer which is interrupted and substituted with one or more hydroxyl groups.

[0019] In some embodiments, the hydroxylated spacer is an ether substituted with one or more hydroxyl groups.

[0020] In some embodiments, the hydroxylated spacer is a C2-20 alkylether substituted with one or more hydroxyl groups. In some embodiments, the hydroxylated spacer is a C2-20 alkylether substituted with one hydroxyl group. In some embodiments, the hydroxylated spacer is a C2-20 alkylether substituted with two hydroxyl groups. In some embodiments, the hydroxylated spacer is a C2-20 alkylether substituted with three hydroxyl groups. In some embodiments, the hydroxylated spacer is a C2-20 alkylether substituted with four hydroxyl groups. In some embodiments, the hydroxylated spacer is a C2-20 alkylether substituted with five hydroxyl groups.

[0021] In some embodiments, the hydroxylated spacer is a reaction product of an epoxide, glycidyl ether or diglycidyl ether. In some embodiments, the hydroxylated spacer is a reaction product of an epoxide. In some embodiments, the hydroxylated spacer is a reaction product of glycidyl ether. In some embodiments, the hydroxylated spacer is a reaction product of diglycidyl ether.

[0022] In some embodiments, the one or more ligands comprising the hydroxylated spacer is defined by the following Formula (I):

[0023] Formula (I) wherein

[0024] G is an anion exchange group; and represents an attachment point on the support matrix. In some embodiments, the anion exchange group is a strong anion exchange group or a weak anion exchange group. In one embodiment, the anion exchange group is a strong anion exchange group. In one embodiment, the anion exchange group comprises one or more amine groups. In one embodiment, the anion exchange group comprises one or more positively charged nitrogen groups. In some embodiments, the positively charged nitrogen group is a quaternary ammonium group.

[0025] In some embodiments, the one or more ligands comprising the hydroxylated spacer has the following structure: wherein represents an attachment point on the support matrix.

[0026] In some embodiments, the support matrix comprises one or more of: polysaccharides, silica, polymeric and ceramic material. In some embodiments, the support matrix comprises polysaccharides. In some embodiments, the support matrix comprises silica. In some embodiments, the support matrix comprises ceramic material. In some embodiments, the support matrix comprises polysaccharides and silica. In some embodiments, the support matrix comprises polysaccharides and polymeric material. In some embodiments, the support matrix comprises polysaccharides and ceramic material. In some embodiments, the support matrix comprises silica and polymeric material. In some embodiments, the support matrix comprises silica and ceramic material. In some embodiments, the support matrix comprises polysaccharides, silica, and polymeric material. In some embodiments, the support matrix comprises polysaccharides, silica and ceramic material. In some embodiments, the support matrix comprises polysaccharides, silica, polymeric and ceramic material.

[0027] In some embodiments, the support matrix is selected from: dextran, starch, cellulose, agarose, polyvinyl alcohols, acrylamides, polyvinyl ethers and polyglycidol. In some embodiments, the support matrix comprises dextran. In some embodiments, the support matrix comprises starch. In some embodiments, the support matrix comprises cellulose. In some embodiments, the support matrix comprises agarose. In some embodiments, the support matrix comprises polyvinyl alcohols. In some embodiments, the support matrix comprises acrylamides. In some embodiments, the support matrix comprises polyvinyl ethers. In some embodiments, the support matrix comprises polyglycidol. In some embodiments, the chromatography resin has an ionic capacity of about 0.01 to about 0.30 mmol / mL.

[0028] In some embodiments, the chromatography resin has an ionic capacity of about 0.05 to about 0.25 mmol / mL, about 0.10 to about 0.20 mmol / mL, about 0.10 to about 0.25 mmol / mL, about 0.15 to about 0.25 mmol / mL, or about 0.15 to about 0.18 mmol / mL. In some embodiments, the chromatography resin has an ionic capacity of about 0.05 to about 0.25 mmol / mL. In some embodiments, the chromatography resin has an ionic capacity of about 0.10 to about 0.20 mmol / mL. In some embodiments, the chromatography resin has an ionic capacity of about 0.10 to about 0.25 mmol / mL. In some embodiments, the chromatography resin has an ionic capacity of about 0.15 to about 0.25 mmol / mL. In some embodiments, the chromatography resin has an ionic capacity of about 0.15 to about 0.18 mmol / mL. In some embodiments, the chromatography resin has an ionic capacity of about 0.095 to about 0.125 mmol / mL.ln some embodiments, the chromatography resin has an ionic capacity of about 0.095 to about 0.18 mmol / mL.

[0029] In some embodiments, each ligand is positively charged or partially positively charged at a pH of about 6 to about 10. In some embodiments, each ligand is positively charged at a pH of about 6 to about 10. In some embodiments, each ligand is partially positively charged at a pH of about 6 to about 10.

[0030] In some embodiments, the support matrix is provided as a membrane, expanded bed media, or beads. In some embodiments, the support matrix is provided as a membrane. In some embodiments, the support matrix is provided as expanded bed media. In some embodiments, the support matrix is provided as beads.

[0031] In some embodiments, the support matrix is provided as porous beads, non- porous beads, or magnetic beads. In some embodiments, the support matrix is provided as porous beads. In some embodiments, the support matrix is provided as non-porous beads. In some embodiments, the support matrix is provided as magnetic beads.

[0032] In some embodiments, the support matrix beads have a D5o particle size of about 10 pm to about 80 pm, about 10 pm to about 50 pm, about 30 pm to about 80 pm, about 30 pm to about 50 pm, or about 20 pm to about 60 pm. In some embodiments, the support matrix beads have a D50 particle size of about 10 pm to about 80 pm. In some embodiments, the support matrix beads have a D50 particle size of about 10 pm to about 50 pm. In some embodiments, the support matrix beads have a D50 particle size of about 30 pm to about 80 pm. In some embodiments, the support matrix beads have a D50 particle size of about 30 pm to about 50 pm. In some embodiments, the support matrix beads have a D50 particle size of about 40 pm to about 75 pm. In some embodiments, the support matrix beads have a D50 particle size of about 40 pm to about 50 pm. In some embodiments, the support matrix beads have a D50 particle size of about 20 pm to about 60 pm. For example, the support matrix beads can have a D50 particle size between 45 pm to about 46 pm.

[0033] In some embodiments, the support matrix beads have a D50 particle size of about 10 pm. In some embodiments, the support matrix beads have a D50 particle size of about 20 pm. In some embodiments, the support matrix beads have a D50 particle size of about 30 pm. In some embodiments, the support matrix beads have a D50 particle size of about 40 pm. In some embodiments, the support matrix beads have a D50 particle size of about 50 pm. In some embodiments, the support matrix beads have a D50 particle size of about 60 pm. In some embodiments, the support matrix beads have a D50 particle size of about 70 pm. In some embodiments, the support matrix beads have a D50 particle size of about 75 pm. In some embodiments, the support matrix beads have a D50 particle size of about 80 pm.

[0034] In some embodiments, the ion exchange chromatography resin is a strong anionic exchange resin. In some embodiments, the ion exchange chromatography resin is Capto Q ImpRes.

[0035] In some embodiments, the extracellular vesicles comprise one or more of: microvesicles, apoptotic bodies, exomeres and exosomes. In some embodiments, the extracellular vesicles comprise microvesicles. In some embodiments, the extracellular vesicles comprise apoptotic bodies. In some embodiments, the extracellular vesicles comprise exomeres. In some embodiments, the extracellular vesicles comprise exosomes. In some embodiments, the extracellular vesicles comprise microvesicles and apoptotic bodies. In some embodiments, the extracellular vesicles comprise microvesicles and exosomes. In some embodiments, the extracellular vesicles comprise apoptotic bodies and exosomes. In some embodiments, the extracellular vesicles comprise exomeres and microvesicles. In some embodiments, the extracellular vesicles comprise exomeres and apoptotic bodies. In some embodiments, the extracellular vesicles comprise exomeres and exosomes.

[0036] In some embodiments, the extracellular vesicles are from a cell or population thereof. In some embodiments, the extracellular vesicles are from an immune cell or population thereof.

[0037] In some embodiments, the immune cell is a T cell or natural killer (NK) cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a NK cell. In another embodiment, the immune cell is a macrophage.

[0038] In some embodiments, the cell is an induced pluripotent stem cell (iPSC)-derived cell.

[0039] In some embodiments, the cell is an iPSC-derived mesenchymal stem cell.

[0040] In some embodiments, the immune cell is an induced pluripotent stem cell (iPSC)-derived immune cell.

[0041] In some embodiments, the iPSC-derived immune cell is an iPSC-derived T (iT) cell or an iPSC-derived NK (iNK) cell. In some embodiments, the iPSC-derived immune cell is an iPSC-derived T (iT) cell. In some embodiments, the iPSC-derived immune cell is an iPSC-derived NK (iNK) cell. In an embodiment, the iPSC-derived immune cell is an iPSC-derived macrophage.

[0042] In some embodiments, the immune cell is an immune cell that comprises a chimeric antigen receptor (CAR).

[0043] In some embodiments, the immune cell that comprises a CAR is a CAR iPSC- derived NK (CAR-iNK) cell or a CAR iPSC-derived T (CAR-iT) cell. In some embodiments, the immune cell that comprises a CAR is a CAR iPSC-derived NK (CAR- iNK) cell. In some embodiments, the immune cell that comprises a CAR is a CAR iPSC- derived T (CAR-iT) cell.

[0044] In some embodiments, the immune cell is an activated immune cell.

[0045] In some embodiments, the activated immune cell is an activated CAR-iNK cell or an activated CAR-iT cell. In some embodiments, the activated immune cell is an activated CAR-iNK cell. In some embodiments, the activated immune cell is an activated CAR-iT cell.

[0046] In some embodiments, the extracellular vesicles are from a cell from an immortalized human cell line, or population thereof.

[0047] In some embodiments, the immortalized cell line is a HEK293 cell line.

[0048] In some embodiments, the extracellular vesicles are from a stem cell or population thereof. In some embodiments, the stem cell is a pluripotent stem cell or a multipotent stem cell. In some embodiments, the stem cell is a pluripotent stem cell. In some embodiments, the stem cell is a multipotent stem cell.

[0049] In some embodiments, the stem cell is a mesenchymal stem cell (MSC).

[0050] In some embodiments, the mesenchymal stem cell is an adipose MSC, a human umbilical cord-derived MSC or an iPSC-derived MSC.

[0051] In some embodiments, the stem cell is a bone marrow-derived stem cell (BMSC).

[0052] In some embodiments, the stem cell is a neural stem cell (NSC).

[0053] In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC).

[0054] In an aspect, there is provided an ion exchange chromatography column comprising the composition of the present disclosure.

[0055] In an aspect, there is provided a process for separating extracellular vesicles, comprising the steps of: i) contacting a composition comprising the extracellular vesicles and one or more impurities to an ion exchange chromatography resin under conditions to enable binding of extracellular vesicles to the ion exchange chromatography resin, wherein the chromatography resin comprises a support matrix functionalized with ligands for binding to the EVs, wherein one or more of the ligands comprise a hydroxylated spacer which attaches an anion exchange group to the support matrix; and ii) separating the ion exchange chromatography resin or the ion exchange chromatography column from the composition, thereby separating the extracellular vesicles from the composition.

[0056] In some embodiments, the process of the present disclosure further comprises step (iii) eluting the extracellular vesicles from the ion exchange chromatography resin or the ion exchange chromatography column by contacting the ion exchange chromatography matrix with an elution buffer.

[0057] In some embodiments, step (i) occurs at a pH of about 6 to about 10. In some embodiments, step (i) occurs at a pH of about 6 to about 8. In some embodiments, step (i) occurs at a pH of about 8 to about 10. In some embodiments, step (i) occurs at a pH of about 7 to about 8. In some embodiments, step (i) occurs at a pH of about 6 to about 7.

[0058] In some embodiments, the elution buffer has a salt concentration of about 0.5 M to about 3 M. In some embodiments, the elution buffer has a salt concentration of about 0.5 M to about 1 M. In some embodiments, the elution buffer has a salt concentration of about 0.5 M to about 1.5 M. In some embodiments, the elution buffer has a salt concentration of about 0.5 M to about 2 M. In some embodiments, the elution buffer has a salt concentration of about 2 M to about 3 M. In some embodiments, the elution buffer has a salt concentration of about 1.5 M to about 3 M. In some embodiments, the elution buffer has a salt concentration of about 1 M to about 3 M. In some embodiments, the elution buffer has a salt concentration of about 1 M to about 2.5 M. In some embodiments, the elution buffer has a salt concentration of about 1 M to about 2 M.

[0059] In some embodiments, the process of the present disclosure further comprises step (iv) collecting the formed eluate comprising elution buffer and extracellular vesicles.

[0060] In some embodiments, the process of the present disclosure further comprises step (v) removing the elution buffer and resuspending the extracellular vesicles to form a concentrated composition or isolate containing extracellular vesicles.

[0061] In some embodiments, the process of the present disclosure further comprises step (vi) filtering the concentrated composition or isolate containing extracellular vesicles.

[0062] In some embodiments, the process of the present disclosure further comprises prior to step (i) concentrating the composition comprising the extracellular vesicles.

[0063] In some embodiments, the composition comprising the extracellular vesicles is a cell culture fluid or a purified composition derived therefrom. In some embodiments, the composition comprising the extracellular vesicles is a cell culture fluid. In some embodiments, the composition comprising the extracellular vesicles is a purified composition derived from a cell culture fluid.

[0064] In some embodiments, the anion exchange resin is an ion exchange chromatography resin as defined herein.

[0065] BRIEF DESCRIPTION OF THE FIGURES

[0066] In the following description and Examples, the ion exchange chromatography resins and the processes of the present disclosure are respectively referred to as “EXOACE resins” or the “EXO-ACE isolation process”.

[0067] Preferred embodiments of the present disclosure will be further described and illustrated, by way of example only, with reference to the accompanying drawings:

[0068] Figure 1 : Schematic of an exemplary ion exchange chromatography column according to the present invention. The column set up comprises a column with a loading capacity of 10 ml, 0.8ml of EXO-ACE resin with a top frit to keep the resin packed tight and a bottom frit to keep the resin from leaking out, and a column outlet that is capped when not in use.

[0069] Figure 2: Total protein and RNA yields from initial resin screening experiments. (A) Results from the BCA protein assay showing total protein concentration of purified EV samples from different resins (pre-buffer exchange). (B) Results from the Qubit Fluorometric Quantification system showing RNA yield from final EV samples (postbuffer exchange).

[0070] Figure 3: Gene expression of common EV markers from initial resin screening experiments. qPCR results shown for (A) GAPDH, (B) OAZ1 and (C) U6.

[0071] Figure 4: Protein and RNA comparison from second screening trials. (A) Results from the Nanodrop quantification showing protein yield from each resin at each step of the purification process. (B) Total Protein vs Total RNA yield comparison of endpoint samples from each resin. Total protein was measured using the BCA assay and total RNA measured by the Qubit Fluorometric Quantification system.

[0072] Figure 5: Gene expression of common EV markers in samples from second screening trials. qPCR results shown for (A) GAPDH with 3ng RNA load, (B) GAPDH with matched volume (12 pl), (C) miR-191 with 3ng RNA load, and (D) U6 with 3ng RNA load.

[0073] Figure 6: Western Blot analysis showing protein expression of CD9, a tetraspanin commonly found in EV membranes, in samples from second screening trials. 10 pg of protein was loaded into each well. 2 repeats were performed for each sample.

[0074] Figure 7: Particle and protein analysis of the EXO-ACE eluate fractions. (A) Total particle recovery for Cell Conditioned Media, 57.33 pL Plasma, 32.317 pL Serum, 100pL Plasma and 10OpI serum. (B) Total protein recovery for Cell Conditioned Media, 57.33uL Plasma, 32.317uL Serum, 100uL Plasma and 100ul serum.

[0075] Figure 8: Experiments for confirming mode of action of EXO-ACE resins using particle and protein analysis at each step of the EXO-ACE isolation process. Results indicate (A) particle and (B) protein recovery throughout the isolation process.

[0076] Figure 9: Particle and protein analysis for EXO-ACE resins. (A) Particle vs Protein Concentration of EXO-ACE elution fractions (200 pL each). Particle concentration is shown on the left y-axis and protein concentration is shown on the right y-axis. (B) Size distribution of particles seen at each fraction (Fraction 3 to Fraction 10) during elution phase. Figure 10: Total protein in the eluate obtained from each resin (top left), total particles obtained in the eluate of each resin (top right) and the particle to protein ratio of the eluate from each resin. One way ANOVA performed for each data set and multiple comparisons were performed using Dunnet’s Multiple Comparisons Test (p<0.05, n=2).

[0077] Figure 11 : Total RNA Quantification and exosomal marker qPCR Analysis results on the eluate (prior to Buffer Exchange) from each column. One way ANOVA performed for each data set and multiple comparisons were performed using Dunnet’s Multiple Comparisons Test (p<0.05, n=2).

[0078] Figure 12: Western Blot analysis for Flotillin-1 (49kDa) and CD9 (23-27 kDa) showing Capto Q ImpRes had a stronger banding observed than the other candidates, with Capto Q XP also showing some banding. No bands were observed for DEAE Sephacel. (n=2)

[0079] DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0080] Preferred embodiments of the present invention are described below by way of example only.

[0081] Definitions

[0082] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., molecular biology, biochemistry, and ionic based purification).

[0083] Unless otherwise indicated, the molecular and statistical techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-lnterscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0084] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0085] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0086] As used herein, the term “about”, unless stated to the contrary, refers to + / - 10%, more preferably + / - 5%, even more preferably + / - 1%, of the designated value.

[0087] As used herein, the phrase “at least one of”, when used with a list of items, means different combinations of one or more of the listed items may be used and only one of the items in the list may be needed. The item may be a particular object, thing, or category. In other words, “at least one of’ means any combination of items or number of items may be used from the list, but not all of the items in the list may be required. For example, “at least one of item A, item B, and item C” may mean item A; item A and item B; item B; item A, item B, and item C; or item B and item C. In some cases, “at least one of item A, item B, and item C” may mean, for example and without limitation, two of item A, one of item B, and ten of item C; four of item B and seven of item C; or some other suitable combination.

[0088] It is to be appreciated that certain features that are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination.

[0089] Throughout the present specification, various aspects and components of the invention can be presented in a range format. The range format is included for convenience and should not be interpreted as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range, unless specifically indicated. For example, description of a range such as from 1 to 5 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 5, from 3 to 5 etc., as well as individual and partial numbers within the recited range, for example, 1 , 2, 3, 4, 4.5, or 5, unless where integers are required or implicit from context. This applies regardless of the breadth of the disclosed range. Where specific values are required, these will be indicated in the specification.

[0090] As used herein, the term “alkyl” whether used alone, or in compound words such as haloalkyl, cycloalkyl, alkylcycloalkyl, alkylcarbocyclyl, heteroalkyl, alkylheterocyclyl, alkylheteroaryl, alkylamide, alkylphosphonate and alkylaryl, represents straight chain (i.e. linear) or branched chain hydrocarbon groups. Examples of alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, i-butyl, sec-butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl groups. In one example, the alkyl group is of 1 to 20 carbon atoms (i.e. Ci-2oalkyl). In another examples, the alkyl is a group of 1 to 10 carbon atoms (i.e. Ci-ioalkyl). In another example, the alkyl group is of 1 to 6 carbon atoms (i.e. Ci-ealkyl).

[0091] As used herein, the term “aromatic” group means a cyclic group having 4m+2 TT electrons, where m is an integer equal to or greater than 1 . As used herein, “aromatic” is used interchangeably with “aryl” to refer to an aromatic group, regardless of the valency of aromatic group. As used herein, the term “aryl” whether used alone, or in compound words such as alkylaryl, represents a monocyclic (e.g. phenyl) or polycyclic (e.g. naphthyl) aromatic carbocyclic ring system. In one example, the aryl group is of 3 to 20 carbon atoms (i.e., an aromatic 3-20 membered carbocyclyl). In another example, the aryl group is of 3 to 10 carbon atoms (i.e., an aromatic 3-10 membered carbocyclyl). Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl or fluorenyl.

[0092] As used herein, the term “aliphatic spacer” refers to a divalent moiety in which the atoms forming the spacer are connected by single, double or triple bonds to form a non-aromatic spacer (e.g. does not comprise any aromatic ring structure within the backbone of the linking moiety). The aliphatic spacer may be uninterrupted or interrupted as described herein.

[0093] As used herein, the term “ether” whether used alone, or in compound words such as alkylether, refers to a class of compounds that comprise one or more ether bonds, typically represented by “-O-“.

[0094] As used herein, the term “epoxide” refers to a cyclic ether, where the ether forms a three-atom ring: two atoms of carbon and one atom of oxygen.

[0095] As used herein, the term “glycidyl ether” refers to compound comprising at least one epoxide moiety and one ether moiety. As used herein, the term “binds” or “binding” refers to the interaction of a binding molecule with a target molecule, such as an extracellular vesicle and means that the interaction is dependent upon the presence of a particular structure charge or structure on the extracellular vesicle. For example, a binding molecule such as a ligand of the present disclosure binds to a specific target molecule, such as an exosome. This binding is typically reversible by altering the pH or ionic salt concentration, or ionic strength of buffer in the environment where the binding occurs, which results in subsequent weakening of the binding due to changes in ionic attraction and release of the target molecule from the binding molecule. In most cases, the ligand of the present disclosure binds to an exosome via an anion exchange group. The anion exchange group of the present disclosure is typically of opposite charge to the exosome (e.g. positively charged compared to the negatively charged exosome), which results in a charged interaction (e.g. an ionic interaction) between the anion exchange group and oppositely charged exosome. This of course does not mean that the exosomes and / or other molecules present in a liquid composition passing over or through the resin cannot bind at other sites on the resin.

[0096] As used herein, the term “binding efficiency” refers to a measure of the proportion of EVs bound to a ligand as opposed to the total number of EVs in the sample or composition. It can also be measured by calculating the amount of EVs remaining in the sample after the separation, isolation and / or capture process.

[0097] As used herein, the term “capture” or “capturing” refers to the binding of the ligand as disclosed herein to extracellular vesicles, in preferably a releasable manner, for example, in order to remove EVs from a sample or composition of interest and washing away the remaining unbound components. In an example, an EV is “captured” once it is bound by a resin of the disclosure.

[0098] As used herein, the term “separate” or “separating” refers to the separation of EVs from at least some components of a sample or composition.

[0099] As used herein, the term “isolate” or “isolating” or “isolation” relates to the steps of “capturing” EVs but further includes one or more steps to isolate the EVs. These terms include gross physical separation of the extracellular vesicles from their environment (e.g. removal / purification from a sample obtained from a subject; removal / purification from a population of an EV subtype such as exosomes; removal / purification from cell culture media). In an example, ionic based separation methods are used. Such methods leverage the ionic attraction of a binding molecule to isolate extracellular vesicles from a sample on the basis of their surface charge or net surface charge.

[0100] As used herein, the term “elution” refers to the removal of the EVs from a ligand.

[0101] As used herein, the term “buffer exchange” refers to a step of removing the elution buffer from EVs and re-suspending the EVs in a buffer or formulation of choice. This step also further concentrates the EVs. One or more buffer exchange steps may be conducted.

[0102] As used herein, the term “terminal filtration” refers to a filtration step that is conducted after the isolation and / or buffer exchange steps to remove one or more contaminants or other undesirable components. A terminal filtration step may be conducted to obtain a sterile sample that is suitable for clinical use.

[0103] As used herein, the term “regeneration” or “column regeneration” refers to a step whereby the columns are returned to storage conditions so that the columns can be reused. The regeneration step is performed to remove tightly bound product after elution to prevent precipitation and column fouling. A regeneration or column regeneration step can also be conducted alongside a “cleaning-in-place (CIP)” step. For example, following a column regeneration step the columns may be re-used 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 times.

[0104] As used herein, the term “extracellular vesicles” (EVs) refer to a heterogeneous group of membranous structures derived from outward budding of the plasma membrane or endosomal system of cells. EVs are delimited by a lipid bilayer and cannot replicate on their own. Extracellular vesicles can comprise exomeres, small extracellular vesicles, exosomes and microvesicles. The extracellular vesicles derived from tumour cells or populations thereof are referred to as “tumour-derived extracellular vesicles” herein. Such extracellular vesicles are generally secreted from tumour cells into their surrounding micro-environment. Extracellular vesicles comprise small extracellular vesicles (50-200 nm), microvesicles (0.2-1 pm), exomeres (<50 nm) and exosomes (50- 150 nm), migrasomes (500-3000 nm) oncosomes (1000-10000 nm) and apoptotic bodies (50 nm up to 5000 nm). Exomeres are membrane bound nanoparticles and are typically classified as having a size of <50 nm, for example, 30-50 nm. In one example, the extracellular vesicles are exomeres (<50 nm), small extracellular vesicles (50-200 nm), microvesicles (0.2-1 pm), migrasomes (500-3000 nm), oncosomes (1000-10000 nm) or exosomes (30-150 nm). In another example, the extracellular vesicles are exosomes. In another example, the extracellular vesicles are exomeres. Those of skill in the art will appreciate that EVs can be captured / isolated from various sources. In an example, the captured / isolated EVs have been secreted from a cell or population thereof. The term “population” as used herein in the context of cells refers to a group of cells. The population of cells need not be a purified population of the cells of interest but can simply comprise the cells of interest. However, the person skilled in the art will appreciate that further purification steps can be conducted on the population if required. In an example, the EVs are from a cell or population thereof, i.e. , the EVs have been secreted or released from the cell(s). In an example, the cell is a prokaryotic cell. In an example, the cell is a bacterial cell. In an example, the cell is a eukaryotic cell. In an example, the cell is a human cell. In an example, the cell is a human cell(s) in vivo and the EVs are captured / isolated from a sample obtained from a subject. In an example, the cell is an in vitro cell (e.g. a cell culture) and the EVs are isolated from the cell culture medium. In an example, the cell is an immune cell. In an example, the immune cell is a T cell. In an example, the T cell is a developing T cell. In an example, the T cell is a mature T cell. In an example, the immune cell is a Natural Killer (NK) cell. In an example, the NK cell is a developing NK cell. In an example, the NK cell is a mature NK cell. In an example, the immune cell is a macrophage.

[0105] In an example, the immune cell is a genetically modified immune cell. In an example, the cell is an immune cell that comprises a chimeric antigen receptor (CAR). The term “chimeric antigen receptor” as used herein refers to an engineered receptor that has been developed in the laboratory to bind to specific proteins or antigens on the surface of a target cell. Large numbers of immune cells may be extracted from a patient and / or expanded in the laboratory, before being engineered to express the CAR. Alternatively, cells can be grown in vitro such as by differentiated induced pluripotent stem cells (iPSCs) to the required cell type (such as an immune cell) prior to being engineered to express the CAR. These CAR carrying immune cells can then be reintroduced to the subject. CAR carrying immune cells have also been shown to secrete or release EVs. In an example, the cell is a CAR-T cell.

[0106] In an example, the cell is a CAR-NK cell. In an example, the immune cell is an activated immune cell. As used herein, the term “activated immune cell” refers to an immune cell that has been exposed to a target antigen. Activated immune cells have been shown to release cytokines and co-stimulatory factors that can improve antigen binding specificity. In an example, the cell is an activated CAR-T cell. In an example, the cell is an activated CAR-NK cell. In an example, the cell is a stem cell. In an example, the stem cell is a multipotent stem cell. In an example, the cell is a mesenchymal stem cell. In an example, the stem cell is a pluripotent stem cell. In an example, the mesenchymal stem cell is an adipose MSC, a human umbilical cord-derived MSC or an iPSC-derived MSC. In an example, the stem cell is a bone marrow-derived stem cell (BMSC). In an example, the stem cell is a neural stem cell (NSC). In an example, the stem cell is an induced pluripotent stem cell (iPSC). In an example, the cell is an iPSC- derived cell. In an example, the cell is an iPSC-derived immune cell. In an example, the cell is an iPSC-derived NK (iNK) cell. In an example, the cell is an iPSC-derived T (iT) cell. In an example, the cell is a CAR iPSC-derived NK cell (CAR-iNK). In an example, the cell is a CAR iPSC-derived T cell (CAR-iT). In an example, the cell is from an immortalized cell line. In an example, the cell is from an immortalized human cell line. In an example, the immortalized cell line is a HEK293 cell line. In an example, the iPSC- derived immune cell is an iPSC-derived macrophage. In an example, the EV is from a virus. For example, the EV can be isolated from a viral culture (e.g. a cell line is infected with a virus). In an example, the EVs are not viral EVs. For example, the isolated EVs can be exclusively cell derived EVs. In certain embodiments, the EVs in these aforementioned examples may be exosomes. In certain embodiments, the EVs in these aforementioned examples may be exomeres.

[0107] In other examples, the EVs are bacterial EVs, non-bacteria microbial EVs, EVs from animal secretions and fluids, EVs from insect secretions and fluids or EVs from plant secretions and fluids. Other examples of EVs include EV mimetic, artificial cell- derived vesicles (ACDVs), synthetic vesicles, small EVs (sEVs; <200nm in diameter), large EVs (lEVs; >200nm in diameter), exomeres (<50 nm in diameter), ectosomes and, exosome-like vesicles. Accordingly, in an example, the EV is a synthetic EV. For example, the EV may comprise synthetic lipids.

[0108] As used herein, the term “yield” refers to the number of EVs successfully separated, captured and / or isolated from a sample or composition. The term also encompasses the proportion of the total number of EVs as opposed to the known or theoretical / measured amount of EVs within a sample. The term “percentage yield” may also be used.

[0109] As used herein, the term “sample” shall be taken to mean any fluid or tissue or material including cells or populations thereof derived from a living or dead animal including humans, or prepared or isolated in the laboratory such as from a cell culture. The sample may contain extracellular vesicles (EVs), nucleic acids, carbohydrates, lipids and / or polypeptides. Samples include, but are not limited to, urine, saliva, faeces, tears, sweat, synovial fluid, vitreous humor, nasal secretions, seminal fluid, cervicovaginal fluid, cerebrospinal fluid, plasma, blood, serum, and conditioned medium of a cell culture, or a purified fraction thereof. Other exemplary samples may include mammary fluid, pleural effusions, pericardial effusions, amniotic fluid, bronchial fluid, lymph, bile, platelets. The sample may be a fluid sample, biological sample, or biological fluid sample. In an example, the sample is supernatant from a cell culture. For example, the supernatant can be obtained from a cell population under culture conditions. In an example, the supernatant is purified before contact with a composition of the disclosure. In another example, the sample is a cell lysate. In an example, the sample is obtained from ascites. In an example, the EVs are derived from ascites.

[0110] Ion exchange chromatography resins

[0111] Resins suitable for use in the present disclosure are those found by the inventors to separate, capture and / or isolate extracellular vesicles, particularly on the basis of ionic attraction to the net surface charge of extracellular vesicles. The inventors have discovered that resins comprising a support matrix functionalized with ligands for binding to the EVs, wherein one or more of the ligands comprise a hydroxylated spacer which attaches an anion exchange group to the support matrix, result in particularly high binding efficiency and yields when isolating EVs. This finding is surprising because it is conventionally accepted in the art that EVs have a net negative surface charge, and thus it would be expected that anionic resins with the highest positive charge would result in the highest binding efficiency and yields. However, the inventors have tested a range of resins and found that anion exchange resins comprising one or more of the ligands comprise a hydroxylated spacer (e.g. a spacer comprising one or more hydroxyl (-OH) groups) which attaches an anion exchange group to the support matrix results in higher binding efficiency and yields as compared to other anion exchange resins that have stronger ionic capacity and thus higher theoretical ionic attraction to EVs.

[0112] As described in the Examples, the inventors have found that the performance of resins in separating, capturing and / or isolating EVs is influenced by the ionic charge or ionic capacity of the resin, but surprisingly this performance is enhanced by the presence of hydroxyl groups in the spacer element that are presented to the net negative surface charges of EVs. While it would be expected that resins comprising anion exchange groups of similar strength (e.g. quaternary amine groups) would behave similarly, this was not the case. In particular, during rigorous testing of numerous resins, and confirmatory analysis based on an understanding of the predicted and theoretical binding of EVs to the resins, the present inventors found that the presence of one or more hydroxyl groups on the spacer of the ligand is particularly important to binding efficiency and thus EV yield.

[0113] The ligand according to the present disclosure binds to the EVs. The binding is typically achieved through the one or more anion exchange groups present on the ligand. As used herein, the term “anion exchange group” refers to ionizable basic groups attached to the support matrix via the hydroxylated spacer. They are typically positively charged groups (e.g. cationic) that are capable of exchanging negatively charged anions (e.g. exosomes) with solution into which they come in contact. Whilst the binding of the EVs typically occurs at the anion exchange group, this does not preclude the binding of EVs (or other molecules for that matter) at other sites on the ligand and / or support matrix (e.g. non-specific binding or other charged or non-charged interactions). For example, the ion exchange may be a mixed mode chromatography resin, where along with the ligand comprising one or more anion exchange groups described herein, there may be other functional groups which provide for additional interactions over and above those afforded by the anion exchange group(s) - such as hydrogen bonding, van der Waals forces, London dispersion forces, hydrophobic or hydrophilic interactions etc. Indeed, it is also possible that the ion exchange resin may comprise cationic exchange groups as well as the one or more anion exchange groups attached to the support matrix via the hydroxylated spacer described herein.

[0114] Accordingly, it will be appreciated that the overall net charge of the ion exchange chromatography resin may vary, despite the presence of the one or more anion exchange groups (and to a lesser extent the polar hydroxyl groups of the hydroxylated spacer) described herein. In most cases, the overall net charge of an ion exchange resin typically depends on the number and type of ionic exchange groups present. For example, the resin described herein may comprise additional cationic exchange groups (such as SO3-) attached to the surface of the support matrix which may lead to the ion exchange resin having an overall net negative charge (e.g. resulting in a cationic exchange chromatography resin), but nonetheless can still capture EVs owing to the presence of one or more ligands of the present disclosure functionalising the surface of the support matrix, wherein one or more of the ligands comprise a hydroxylated spacer coupled to anion exchange group(s). In one embodiment, the ion exchange chromatography resin is an anionic exchange chromatography resin (e.g. the overall number of anion exchange groups present afford the resin an overall net positive charge).

[0115] It will be appreciated that the ligand is attached to the support matrix (i.e. the support matrix is functionalised with the ligands). The attachment to the support matrix may be via any suitable means, such as grafting to one or more groups present on the support matrix. For example, the ligand may be attached to the support matrix via grafting to a hydroxyl group that is present on or within the matrix (e.g. an agarose support matrix comprises hydroxyl groups, some of which can be functionalised with the ligand). In this example, the attachment results in the formation of an ether (-O-) bond which is considered to be part of the ligand described herein.

[0116] The ligand comprises an anion exchange group which is coupled to the support matrix via a longer linker molecule, also known as a spacer group, or a spacer. Spacer groups are known in this field, and are commonly used to increase sterically the distance between the exchanger groups and support matrix. Spacers are sometimes denoted tentacles or flexible arms. In essence, the hydroxylated spacer described herein is a moiety that extends the anion exchange group away from the surface of the support matrix thus promoting exposure of the anion exchange group to target molecules, such as exosomes, suspended in composition as it passes / flows over or through the resin.

[0117] The hydroxylated spacer according to the present disclosure may be any suitable molecule, provided it comprises one or more hydroxyl groups and can attach an anion exchange group to the surface (e.g. an external and / or internal surface) of the support matrix. Provided these hydroxyl groups are present, no real limitation is placed on the structure of the spacer. That is, the hydroxylated spacer is, in essence, a divalent moiety capable of linking, joining, bonding or attaching an anion exchange group to the support matrix.

[0118] The number of hydroxyl groups present on the spacer can vary, and in some embodiments, the spacer may comprise at least one, two, three, four, five, six, seven, eight, nine, or ten or more hydroxyl groups. If two or more hydroxyl groups are present, they may be located at any position along the spacer. In one embodiment, the hydroxylated spacer comprises two or more hydroxyl groups. Despite the presence of polar hydroxyl groups along with spacer, which is believed to reduce the overall net positive charge of the ligand (noting that the anion exchange group is positively charged, and in some cases of strong positive character such as that for a quaternary ammonium compound), surprisingly and counterintuitively, the performance of the resulting resin in separating, capturing and / or isolating EVs is enhanced.

[0119] Whilst the length and nature of the hydroxylated spacer can vary, in some embodiments, the hydroxylated spacer is non-polymeric. That is, the hydroxylated spacer is a small molecule. For example, such non-polymeric spacers do not encompass polysaccharides or polymeric structures. Ligands comprising non-polymeric spacer groups have the advantage that they can assemble in a linear fashion on a given surface of the support matrix, which in turn can provide a high degree of anion exchange group functionalisation for a given area on or within the support matrix, providing higher capture efficacy for EVs. In contrast, owing to their length, polymeric spacer groups may self-assemble, twist or bend, resulting in a non-linear structure which can limit the number of anion exchange groups attached to a given area thus reducing capture efficiency. In some embodiments, the molecular weight of the hydroxylated spacer may be less than 5000, 4000, 3000, 2000, 1000, 700 or 500 Da.

[0120] In some embodiments, the hydroxylated spacer may comprise aliphatic or aromatic groups (including a combination thereof), which are uninterrupted or interrupted and substituted with one or more hydroxyl groups. In one embodiment, the hydroxylated spacer is an aliphatic spacer which is uninterrupted or interrupted.

[0121] The hydroxylated spacer may be interrupted with one or more groups. For example, the hydroxylated spacer may be interrupted with one or more heteroatoms (e.g. one or more of O, N or S). In one embodiment, the hydroxylated spacer may be interrupted with one or more groups selected from from -O-, -S-, -C(=O)-, -C(=O)NH-, - NH-, -C(=O)O-, -C(=O)S-, -S(=O)2-, -NHC(=S)NH-, and -NHC(=O)NH-. In one embodiment, the hydroxylated spacer may be interrupted with one or more -O-.

[0122] In one embodiment, the hydroxylated spacer may be an alkyl spacer which is uninterrupted or interrupted and substituted with one or more hydroxyl groups. The hydroxylated spacer may be a C2-2oalkyl which is uninterrupted or interrupted and substituted with one or more hydroxyl groups. The hydroxylated spacer may be selected from a C2-2oalkyl, C4-2oalkyl, Ce-2oalkyl or Cs-2oalkyl which is uninterrupted or interrupted and substituted with one or more hydroxyl groups. The hydroxylated alkyl spacer may be uninterrupted or interrupted with one or more heteroatoms (e.g. one or more of O, N or S). The hydroxylated alkyl spacer may be uninterrupted or interrupted with one or more groups selected from -O-, -S-, -C(=O)-, -C(=O)NH-, -NH-, -C(=O)O-, -C(=O)S-, - S(=O)2-, -NHC(=S)NH-, and -NHC(=O)NH-. In one embodiment, the hydroxylated alkyl spacer may be interrupted with one or more -O- (i.e. is an ether).

[0123] The hydroxylated spacer may be attached at a single point to the support matrix, or may be attached at two or more points to the support matrix (e.g. may form a bridge between two surfaces of the support matrix). For example, the hydroxylated spacer may be covalently bound to the support matrix at two separate points on a surface such forming a hydroxylated bridge. The anion exchange group or groups may be attached at any point along the hydroxylated spacer / bridge. This structural arrangement may be particularly suitable for highly porous support matrices where the hydroxylated spacer can form bridge attachments between surfaces forming the pores. When a liquid composition comprising EVs is passed through the porous supports, it passes across / over the hydroxylated spacer / bridge comprising anion exchange groups which can bind to EVs.

[0124] In one embodiment, the hydroxylated spacer is an ether substituted with one or more hydroxyl groups. The ether may comprise any number of -O- groups. The hydroxylated spacer may be an alkylether substituted with one or more hydroxyl groups. The hydroxylated spacer may be selected from a C2-2oalkylether, C4-2oalkylether, Ce- 2oalkylether, or Cs-2oalkylether, which is substituted with one or more hydroxyl groups.

[0125] The one or more hydroxyl groups of the hydroxylated spacer may be formed via an epoxide ring opening of an epoxide, glycidyl ether or diglycidyl ether (or indeed a number of other possible reactive compounds) upon coupling / attaching with the support matrix and / or anion exchange group. Therefore, in some embodiments, the hydroxylated spacer may comprise a reaction product of an epoxide, glycidyl ether or diglycidyl ether. In some embodiments, the hydroxylated spacer may be formed from a reaction of an epoxide, glycidyl ether or diglycidyl ether with the support matrix and anion exchange group. For example, the support matrix may comprise activated hydroxyl groups (such as agarose) which can covalently react with an epoxide, glycidyl ether or diglycidyl ether resulting in the attachment of the spacer to the solid support matrix, typically via an ether bond. The epoxide, glycidyl ether or diglycidyl ether may already have had the anion exchange group coupled thereto, or it may be subsequently reacted with another reactive group present on the attached spacer (e.g. another epoxide group that has not reactively coupled to the hydroxyl of the agarose). However, the person skilled in the art would appreciate that other reactive means of forming the hydroxylated spacer are envisaged, provided the resulting spacer attaches the anion exchange group to the solid support matrix, and comprises one or more hydroxyl groups.

[0126] The hydroxylated spacer together with the anion exchange group form a ligand defined by the following Formula (I). In other words, the one or more ligands comprising the hydroxylated spacer is defined by the following Formula (I):

[0127] Formula (I) wherein

[0128] G is an anion exchange group; and represents an attachment point on the support matrix.

[0129] Any suitable anion exchange group may be used for G or indeed elsewhere where reference is made to such group as part of the ligand, and it will be appreciated that the ligand may comprise any number of anion exchange groups. For example, the hydroxylated spacer may be coupled to 1 , 2, 3, 4, 5 or more anion exchange groups at any point on the spacer. In other words, the anion exchange group may be located anywhere on hydroxylated spacer. It will be appreciated that the skilled person is capable of coupling the anion exchange group to the hydroxylated spacer (or a precursor thereof) via suitable coupling chemistry (e.g. epoxide ring opening via reaction). For example, the anion exchange group may be any positively charged group capable of binding to oppositely charged molecules, such as exosomes. In some embodiments, the anion exchange group is a strong anion exchange group or a weak anion exchange group. In one embodiment, the anion exchange group is a strong anion exchange group. In one embodiment, the anion exchange group comprises one or more amine groups. In one embodiment, the anion exchange group comprises one or more nitrogen groups, including one or more positively charged nitrogen groups. In some embodiments, the positively charged nitrogen group is a quaternary ammonium group. In one embodiment, the ligand and / or anion exchange group comprises only one nitrogen group.

[0130] The anion exchange group may be a positively charged nitrogen, sulfur or phosphorous group. In one embodiment, the anion exchange group is a positively charged nitrogen group. The positively charged nitrogen group may be a primary, secondary, tertiary amine group, or in some cases a quaternary ammonium group (sometimes called a quaternary ammonium cation). Primary, secondary and tertiary amine groups are considered to behave like weak bases and are examples of weak anion exchange groups. In contrast, quaternary ammonium groups are considered to behave like strong bases and are examples of strong anion exchange groups. The positively charged nitrogen groups may be selected from those well known in the art, including for example diethylaminoethyl (DEAE), trimethylammonium, trimethylaminoethyl (TMAE) and the like. The positively charged nitrogen group may be permanent, or may be positively charged at a given pH of the composition.

[0131] In one embodiment, the positively charged nitrogen group is a quaternary ammonium group. In one embodiment, the one or more ligands comprise a hydroxylated spacer which attaches a quaternary ammonium group to the support matrix. Any suitable quaternary ammonium group can be used, and in some examples, an alkyl substituted quaternary ammonium group is particularly suitable, including a trimethylammonium cation group.

[0132] In one embodiment, the anion exchange group is not substituted with or does not comprise a hydroxyl group. In one embodiment, the anion exchange group is not substituted with or does not comprise an alkanol group. In one embodiment, the quaternary ammonium group is not substituted with or does not comprise an alkanol group. In one embodiment, the quaternary ammonium group is not substituted or does not comprise a hydroxyl group. It will be appreciated that such provisos do not exclude the hydroxylated spacer which is attached to the quaternary ammonium compound. That is aside from the hydroxylated spacer attached to the positively charged nitrogen, the other substituents thereon forming the quaternary ammonium group do not comprise a hydroxyl group, one embodiment, the anion exchange group does not comprise an aromatic group. In one embodiment, the quaternary ammonium group is not substituted with a benzyl group. In one embodiment, the one or more ligands does not comprise the following structure:

[0133] The hydroxylated spacer together with the anion exchange group may form a ligand defined by the following structure: In other words, the one or more ligands comprising the hydroxylated spacer has the following structure: wherein represents an attachment point on the support matrix.

[0134] The support matrix may comprise any number of the ligands described herein. No real limitation is placed on the type of support matrix. In some embodiments, the support matrix may be made from an organic or inorganic material, and may be porous or non-porous. In some embodiments, the support matrix is prepared from a native polymer, such as cross-linked carbohydrate material, e.g. agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan, alginate, pectin, starch, etc. In some embodiments, the support matrix comprises one or more of: polysaccharides, silica, polymeric and ceramic material. A polysaccharide support matrix provides some advantages in that the presence of free hydroxyl groups on or within the polysaccharide matrix can act as attachment points for the hydroxylated spacer described herein, through conventional reactive coupling chemistry known to the skilled person. Examples of polysaccharides include dextran, starch, cellulose and agarose. Agarose is a particularly suitable support matrix as it does not substantially absorb biomolecules that may be present in the liquid composition being passed over or therethrough, has good flow properties and / or can tolerate a wide range of pH’s and ionic strengths.

[0135] Examples of a polymer material include polymers comprising polyvinyl alcohols, polyacryl- and polymethacrylamides, and polyvinyl ethers.

[0136] In some embodiments, the support matrix is provided as a monolith, porous beads, non-porous beads, magnetic beads, expanded media, or membrane

[0137] In some embodiments, the support matrix is provided as particles or beads having a substantially spherical shape.

[0138] Where the support matrix is provided as beads, different sizes of beads may be used. The size of beads may be presented as a D50 particle size. The particle size average (D50) is also known as the median diameter or the medium value of the particle size distribution, it is the value of the particle diameter at 50% in the cumulative distribution. It means 50% of the particles in the sample are larger than this number, and 50% are smaller.

[0139] In some embodiments, the support matrix beads have a D50 particle size of about 10 pm to about 80 pm, about 10 pm to about 50 pm, about 30 pm to about 80 pm, about 30 pm to about 50 pm, and about 20 pm to about 60 pm. The ionic capacity of the ion exchange chromatography resin can, according to some embodiments or examples described herein, also influence the performance of the resins in separating, capturing and / or isolating EV’s. In particular, it has been found that despite having a lower ionic capacity compared to other resins (likely owing to the presence of the hydroxylated spacer), binding efficiency to EV’s is surprisingly improved. Ionic capacity is defined as that is the number of charged functional groups per mL of resin (mmol / mL). The ionic capacity may be at least about 0.01 , 0.05, 0.08, 0.1 , 0.12, 0.14, 0.15, 0.18, 0.2, 0.25, 0.3, 0.4 or 0.5 mmol / mL. The ionic capacity may be less than about 0.5, 0.4, 0.30, 0.25, 0.2, 0.18, 0.15, 0.14, 0.12, 0.1 , 0.08, 0.05 or 0.01 mmol / mL. The ionic capacity may be in a range provided by any two of these upper and / or lower values, such as between about 0.05 to about 0.025 mmol / mL, e.g. between about 0.15 to about 0.18 mmol / mL.

[0140] In some embodiments, the composition has a pH of at least about 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11 , 11.5 or 12. The composition may have a pH of less than about 12, 11.5, 11 , 10.5, 10., 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5 or 5. The pH of the composition may be in a range provided by any two of these upper and / or lower valued, for example between about 6 to about 10. According to some examples, the ligand may be positively charged or at least partially positively charged at a pH of about 6 to about 10. The pH of the composition becomes less relevant when strong anion exchange groups, such as those based on quaternary amines are used, which are always charged. However, when weaker anion exchange groups are employed, the charge can vary and as such compositions having the aforementioned pH range may provide the advantage that the groups have positive character thus improving binding efficiency. Where the anion exchange group is a strong anion exchange group, such as a quaternary ammonium compound (e.g. trialkylammonium such as trimethylammonium), the pH of the composition becomes less relevant as these groups are almost always positively charged.

[0141] In one embodiment, the resin an anion exchange chromatography resin. In one embodiment, the resin is Capto Q ImpRes. In one embodiment, there is provided a composition comprising the Capto Q ImpRes and a population of extracellular vesicles (EVs) bound to the resin. Ion exchange chromatography columns

[0142] The present disclosure also provides ion exchange chromatography column comprising the resin of the present disclosure. In an example, the chromatography resin is an anion exchange chromatography resin.

[0143] In an aspect, the chromatography column comprises: a housing comprising a side wall, opposed axially spaced first and second end units separated by said side wall, a top frit adjacent to the first end unit and a bottom frit which is adjacent to the second end unit wherein the frits together with the side wall define an enclosed bed space for containing the resin of the present disclosure.

[0144] In some embodiments, the first end unit comprises an inlet which is in fluid communication with the resin of the present disclosure for adding a composition comprising EVs to the resin of the present disclosure.

[0145] In some embodiments, the second end unit comprises an outlet which is in fluid communication with the resin of the present disclosure for removing one or more components of the composition comprising EVs.

[0146] In some embodiments, the ion exchange chromatography column is pre-packed with the resin of the present disclosure.

[0147] In some embodiments, the ion exchange chromatography column is a reuseable or a disposable column.

[0148] Figure 1 provides an exemplary design of an ion exchange chromatography column of the present disclosure. However, the person skilled in the art will appreciate that other specific constructions and modes of operation may also be appropriate depending on the type of process being applied.

[0149] In an exemplary operation of the column, a composition comprising EVs is flowed through the top first end unit and flowed or passed through the pre-packed resin, resulting in separation of EVs from the composition. The remaining components of the composition pass through the resin and exit the second end unit for collection or disposal as appropriate. The composition can be flowed or passed through the resin by gravity (i.e. , is gravity fed), subjected to pressure and / or particular flow rates or flow velocities using, for example, a pump or vacuum.

[0150] Processes and methods of separating EVs

[0151] As mentioned at the outset, while a number of techniques are established in the art for separating, capturing and / or isolating EVs from a sample or composition. These existing techniques typically function by affinity capture (i.e. , binding to certain proteins on the external surface of EVs) or by utilizing the known biophysical properties of the EVs such as size, density and surface structure. However, these techniques suffer from a number of key limitations, including scalability and operational complexities. Other methods such as polymer assisted precipitation and tangential flow filtration suffer from the requirement of additional steps post-capture / isolation that complicate workflows. Affinity capture methods such as by binding to surface EV markers suffer from binding specificity and low yields.

[0152] As described above, the inventors have discovered that resins comprising a support matrix functionalized with ligands for binding to the EVs, wherein one or more of the ligands comprise a hydroxylated spacer which attaches an anion exchange group to the support matrix, result in a high attraction of EVs and thus high efficiencies of capturing, separation and / or isolating EVs from a sample. The inventors have found that resins having ligands comprising a hydroxylated spacer result in desirable EV binding while separating from other undesirable components in the compositions or samples, such as proteins and cellular contaminants. Further, the elution profile for EVs and other undesirable components when using the resins according to the present disclosure results in a pure and concentrated sample of EVs. The use of the chromatography resins according to the present disclosure has also shown to be a purification method that can be easily scaled up for industrial production with many of the basic principles remaining the same. Costs are reduced compared to other affinity-based techniques as the production of polymer based ionic matrices are much more cost-effective than the production of antibodies. Adsorption matrices used in ionic affinity chromatography can be recycled and used several times further reducing the cost associated with industrial scale chromatography. A further advantage is that the pure and concentrated sample of EVs makes it easier to perform other optional downstream steps such as filtration, due to reduced clogging of the filters.

[0153] On the basis of this finding, the inventors have developed a process for separating EVs from a composition utilizing an ion exchange chromatography approach.

[0154] Accordingly, the present disclosure provides a process for separating extracellular vesicles, comprising the steps of: i) contacting a composition comprising the extracellular vesicles and one or more impurities to an ion exchange chromatography resin or an ion exchange chromatography column comprising the ion exchange chromatography resin, under conditions to enable binding of extracellular vesicles to the ion exchange chromatography resin, wherein the chromatography resin comprises a support matrix functionalized with ligands for binding to the EVs, wherein one or more of the ligands comprise a hydroxylated spacer which attaches an anion exchange group to the support matrix; and ii) separating the ion exchange chromatography resin or the ion exchange chromatography column from the composition, thereby separating the extracellular vesicles from the composition.

[0155] Prior to commencing the process according to the present disclosure, additional steps may be taken such as adjusting the pH and / or salt concentration of the sample or composition. Binding of the EVs to the resin is influenced by pH and / or salt concentration and so the composition may need to undergo preparation for isolation.

[0156] In some embodiments, the composition is pre-processed prior to step (i). Examples of pre-processing steps include adjusting the pH of the composition, salt adjustment of the composition, clarifying, filtering and / or removal of impurities or contaminants such as cell debris or cells. In some embodiments, the impurities or contaminants comprise one or more of: proteins, lipids, carbohydrates, nucleic acids, cell debris and cells.

[0157] In some embodiments, the composition has a pH of about 6 to about 10. In some embodiments, the composition has a pH of about 6 to about 8. In some embodiments, the composition has a pH of about 8 to about 10. In some embodiments, the composition has a pH of about 7 to about 8. In some embodiments, the composition has a pH of about 6 to about 7. In some embodiments, the composition has a pH of about 7 to about 7.6. In some embodiments, the composition has a pH of about 7 to about 7.4. In some embodiments, the composition has a pH of about 7.2 to about 7.4.

[0158] In some embodiments, the composition has a salt concentration of about 0.5 M to about 3 M. In some embodiments, the composition has a salt concentration of about 0.5 M to about 1 M. In some embodiments, the composition has a salt concentration of about 0.5 M to about 1.5 M. In some embodiments, the composition has a salt concentration of about 0.5 M to about 2 M. In some embodiments, the composition has a salt concentration of about 2 M to about 3 M. In some embodiments, the composition has a salt concentration of about 1.5 M to about 3 M. In some embodiments, the composition has a salt concentration of about 1 M to about 3 M. In some embodiments, the composition has a salt concentration of about 1 M to about 2.5 M. In some embodiments, the composition has a salt concentration of about 1 M to about 2 M. In some embodiments, the composition has a salt concentration of about 1 M to about 1 .5 M.

[0159] In some embodiments, the composition is a sample, biological sample or biological fluid sample. In some embodiments, the sample is a human sample. In some embodiments, the sample is urine, saliva, faeces, tears, cerebrospinal fluid, plasma, blood or serum. Examples of a sample or biological sample include a freshly collected sample or a sample that has been stored, e.g. frozen or refrigerated. Samples or biological samples may be prior processed before being subjected to the methods of the present disclosure, such as clarifying, filtering or debris / contaminants removal. Suitable methods include centrifugation, ultracentrifugation, filtration, diafiltration or ultrafiltration.

[0160] Suitable filters for use include those having a size in the range about 0.1 to about 1.0 pm may be employed, for example, about 0.8 pm or 0.22 pm. One or more filtration steps may be performed including with successive filtrations using filters with decreasing porosity.

[0161] The composition or starting material may be a cell culture fluid or a purified composition derived therefrom. For example, the cell culture fluid may be purified by one or more of: precipitation, centrifugation, filtration, diafiltration and ultrafiltration. This purification step may result in the removal of one or more components of the cell culture fluid to improve the binding efficiency (for example, via reduced clogging of the resin while conducting the process of the present disclosure) and / or improving EV yields by removal of undesirable components. Examples of cell culture fluid include a supernatant from a cell culture or conditioned medium of a cell culture. The supernatant may be obtained from a cell or cell population under culture conditions.

[0162] In some embodiments, the composition comprising the extracellular vesicles is cell culture fluid or a purified composition derived therefrom.

[0163] In some embodiments, the composition is substantially cell-free.

[0164] The composition comprising EVs and one or more impurities are contacted to an ion exchange chromatography resin to enable binding of EVs to the resin. As outlined above, the specific binding conditions that enable binding of EVs to the resin include alteration of the pH or salt concentration of the composition comprising the EVs. Additional binding conditions include altering the flow rate or velocity, time and / or temperature in which the contacting step (i) is conducted.

[0165] In some embodiments, the composition is subjected to a flow rate or flow velocity of about 1 mL / min to about 30 mL / min. In some embodiments, the composition is subjected to a flow rate or flow velocity of about 1 mL / min to about 20 mL / min. In some embodiments, the composition is subjected to a flow rate or flow velocity of about 4 mL / min to about 20 mL / min. In some embodiments, the composition is subjected to a flow rate or flow velocity of about 10 mL / min to about 30 mL / min. In some embodiments, the composition is subjected to a flow rate or flow velocity of about 20 mL / min to about 30 mL / min. In some embodiments, the composition is subjected to a flow rate or flow velocity of about 1 mL / min to about 5 mL / min. In some embodiments, the composition is subjected to a flow rate or flow velocity of about 1 mL / min to about 10 mL / min. In some embodiments, the composition is subjected to a flow rate or flow velocity of about 5 mL / min to about 15 mL / min.

[0166] Flow rate or flow velocity can also be expressed in linear flow velocity which standardizes flow rates for columns of different dimensions. This is typically expressed in cm per hour (cm / h) units.

[0167] In some embodiments, the composition is subjected to a linear flow rate or linear flow velocity of about 50 cm / h to about 500 cm / h. In some embodiments, the composition is subjected to a linear flow rate or linear flow velocity of about 100 cm / h to about 500 cm / h. In some embodiments, the composition is subjected to a linear flow rate or linear flow velocity of about 200 cm / h to about 400 cm / h. In some embodiments, the composition is subjected to a linear flow rate or linear flow velocity of about 200 cm / h to about 300 cm / h. In some embodiments, the composition is subjected to a linear flow rate or linear flow velocity of about 150 cm / h to about 300 cm / h. In some embodiments, the composition is subjected to a linear flow rate or linear flow velocity of about 250 cm / h to about 350 cm / h. In some embodiments, the composition is subjected to a linear flow rate or linear flow velocity of about 50 cm / h to about 200 cm / h. In some embodiments, the composition is subjected to a linear flow rate or linear flow velocity of about 100 cm / h to about 300 cm / h.

[0168] As an alternative to the expression of flow rate or velocities, compositions subjected to particular flow rate or velocities can also be said to be subjected to a particular pressure. The pressures may be expressed in units of bar, MPa or psi. Reference to one particular unit should be understood as being reference to the other units as well. For example, reference to 3 bar should be understood to also refer to 0.3 MPa or 43.5 psi. Such measurements for conversion between these units are well known to the person skilled in the art. In some embodiments, the composition is subjected to a pressure of about 0.5 bar to about 10 bar. In some embodiments, the composition is subjected to a pressure of about 1 bar to about 10 bar. In some embodiments, the composition is subjected to a pressure of about 1 bar to about 5 bar. In some embodiments, the composition is subjected to a pressure of about 1 bar to about 3 bar. In some embodiments, the composition is subjected to a pressure of about 3 bar to about 10 bar. In some embodiments, the composition is subjected to a pressure of about 3 bar to about 8 bar. In some embodiments, the composition is subjected to a pressure of about 5 bar to about 10 bar.

[0169] The compositions may also be allowed to flow through the ion exchange chromatography resin or an ion exchange chromatography column comprising the ion exchange chromatography resin using only gravity, without the need for any negative pressure or introduction of a centrifugal force.

[0170] In some embodiments, the composition is contacted to the ion exchange chromatography resin or the ion exchange chromatography column comprising the ion exchange chromatography resin by passing or flowing the composition through the resin or the column using low pressure or gravity.

[0171] An alternative way in which step (i) can be expressed is: passing or flowing a composition comprising the extracellular vesicles and one or more impurities through an ion exchange chromatography resin or an ion exchange chromatography column comprising the ion exchange chromatography resin, under conditions to enable binding of extracellular vesicles to the ion exchange chromatography resin, wherein the chromatography resin comprises a support matrix functionalized with ligands for binding to the EVs, wherein one or more of the ligands comprise a hydroxylated spacer which attaches an anion exchange group to the support matrix.

[0172] In some embodiments, the process according to the present disclosure further comprises step (iii) eluting the extracellular vesicles from the ion exchange chromatography resin or the ion exchange chromatography column by contacting the ion exchange chromatography matrix with an elution buffer.

[0173] The inventors have found that the resins according to the present disclosure allow for uniform elution of EVs from the resin by contacting with an elution buffer. In particular, the inventors found that during the sample flowthrough and wash step, the majority of protein is lost while EVs remain bound to the resin. Following the elution step and exposure to the elution buffer, the change in conductivity of the resin is altered resulting in release of the EVs into the eluate. While not wishing to be bound by any particular theory, this suggests that the attraction of EVs to the resin is not due to a size exclusion or filtration property of the resin but rather by charge. This allows for efficient separation of EVs from the composition and results in a relatively pure eluate of EVs.

[0174] The properties of the elution buffer such as pH and salt concentration can be altered depending on the properties of the composition. For example, the elution buffer may have a lower or higher pH than the composition to allow for efficient elution of EVs from the resin.

[0175] In some embodiments, the elution buffer has a higher pH than the composition. In some embodiments, the elution buffer has a lower pH than the composition.

[0176] In some embodiments, the elution buffer has a salt concentration of about 0.5 M to about 3 M. In some embodiments, the elution buffer has a salt concentration of about 0.5 M to about 1 M. In some embodiments, the elution buffer has a salt concentration of about 0.5 M to about 1.5 M. In some embodiments, the elution buffer has a salt concentration of about 0.5 M to about 2 M. In some embodiments, the elution buffer has a salt concentration of about 2 M to about 3 M. In some embodiments, the elution buffer has a salt concentration of about 1.5 M to about 3 M. In some embodiments, the elution buffer has a salt concentration of about 1 M to about 3 M. In some embodiments, the elution buffer has a salt concentration of about 1 M to about 2.5 M. In some embodiments, the elution buffer has a salt concentration of about 1 M to about 2 M.

[0177] Changes to the properties of the elution buffer, such as pH and salt concentration, can also be used to enrich the eluate for a desired subtype of EV.

[0178] The term “enriched” refers to increasing the concentration of a desired subtype of EV, for example one or more of microvesicles, exosomes, exomeres and apoptotic bodies. For example, enriched may refer to the composition having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of the desired subtype of EV, for example, exosomes. Put differently, enriched may mean that the composition is substantially free of undesired EV subtypes as opposed to the desired EV subtype. Taking the example wherein the EV subtype of interest are exosomes, a composition enriched for exosomes may refer to the composition having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% exosomes; or alternatively a composition that is substantially free of microvesicles, exomeres and / or apoptotic bodies. Accordingly, the present disclosure also relates to a composition comprising EVs, or a population of EVs enriched for microvesicles, exomeres, exosomes and / or apoptotic bodies.

[0179] In some embodiments, the composition comprising EVs or the population of EVs is enriched for microvesicles. In some embodiments, the composition comprising EVs or the population of EVs is enriched for exomeres. In some embodiments, the composition comprising EVs or the population of EVs is enriched for exosomes. In some embodiments, the composition comprising EVs or the population of EVs is enriched for apoptotic bodies. In some embodiments, the composition comprising EVs or the population of EVs is enriched for microvesicles and exosomes. In some embodiments, the composition comprising EVs or the population of EVs is enriched for microvesicles and apoptotic bodies. In some embodiments, the composition comprising EVs or the population of EVs is enriched for exosomes and apoptotic bodies. In some embodiments, the composition comprising EVs or the population of EVs is enriched for exomeres and apoptotic bodies. In some embodiments, the composition comprising EVs or the population of EVs is enriched for exomeres and microvesicles. In some embodiments, the composition comprising EVs or the population of EVs is enriched for exomeres and exosomes.

[0180] Various ways of determining if a composition or population is enriched for particular EV subtypes could be envisaged by a person skilled in the art. For example, the EVs could be subjected to further analysis to determine the magnitude or strength of expression of particular markers indicative of a particular EV subtype.

[0181] Markers for specific types of EVs may also be assessed to confirm whether the composition or population is enriched for EVs generally.

[0182] In some embodiments, the EVs are CD9+, Flotilin-1+, and / or TSG101+.

[0183] In some embodiments, the process of the present disclosure further comprises step (iv) collecting the formed eluate comprising elution buffer and extracellular vesicles.

[0184] The eluate may next be subjected to a buffer exchange step. In this step, the elution buffer is removed and the EVs resuspended in a buffer or formulation of choice. This step also further concentrates the EVs. Suitable ultrafilters for conducting the buffer exchange step include ultrafilters with a cut-off range from 10kDa to 100kDa. The method of conducting such a buffer exchange step and the resultant yield of EVs will vary based on the cut-off size and the membrane composition of the ultrafilter used. An example of a suitable ultrafilter is the HansaBiomed EV-Spinners ultrafilter. The EV- Spinners ultrafilter is a type of spin column and comprises three components: an inner tube that has a filter membrane on its bottom, an outer tube where the concentrated EV sample retentate is collected, and a removal tool such as an inverted tweezer that allows removal of the inner tube.

[0185] In some embodiments, the process of the present disclosure further comprises step (v) removing the elution buffer and resuspending the extracellular vesicles to form a concentrated composition or isolate containing extracellular vesicles.

[0186] In some embodiments, step (v) comprises loading the elution buffer into a spin column and applying centrifugal force to remove the elution buffer.

[0187] In some embodiments, step (v) comprises resuspending the extracellular vesicles in a salt buffer. In some embodiments, the salt buffer is phosphate buffered solution (PBS).

[0188] In some embodiments, step (v) comprises removing the elution buffer using an ultrafilter with a cut-off of about 10 kDa to about 100 kDa. In some embodiments, step (v) comprises removing the elution buffer using an ultrafilter with a cut-off of about 10 kDa to about 50 kDa. In some embodiments, step (v) comprises removing the elution buffer using an ultrafilter with a cut-off of about 50 kDa to about 100 kDa. In some embodiments, step (v) comprises removing the elution buffer using an ultrafilter with a cut-off of about 40 kDa to about 80 kDa. In some embodiments, step (v) comprises removing the elution buffer using an ultrafilter with a cut-off of about 30 kDa to about 60 kDa.

[0189] The resultant concentrated composition or isolate comprising EVs may next undergo a filtration step to obtain a sterile sample suitable for clinical use. This step may also be referred to as a “terminal filtration step”.

[0190] In some embodiments, the process of the present disclosure further comprises step (vi) filtering the concentrated composition or isolate containing extracellular vesicles.

[0191] In some embodiments, the filtering of step (vi) comprises subjecting the composition or concentrated isolate containing extracellular vesicles to one or more of: filtration, diafiltration and ultrafiltration.

[0192] In some embodiments, the composition is Good Manufacturing Practice (GMP) grade or complies with GMP standards. In some embodiments, the composition is suitable for clinical use. The composition comprising the extracellular vesicles may be concentrated for EVs prior to step (i) of the process of the present disclosure.

[0193] In some embodiments, the process of the present disclosure further comprises prior to step (i) concentrating the composition comprising the extracellular vesicles.

[0194] The columns may undergo a “regeneration” or “column regeneration” step in which the columns are returned to storage conditions. Preferably, the columns are returned to a state where the columns can be re-used without compromising EV yields or column integrity.

[0195] In some embodiments, the process of the present disclosure further comprises a regeneration or column regeneration step. In some embodiments, the regeneration or column regeneration step is repeated one or more times. In some embodiments, the regeneration or column regeneration step is repeated 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 times.

[0196] In some embodiments, the process of the present disclosure further comprises one or more of: a cleaning-in-place (CIP) step, a column stripping step, and a column sanitization step.

[0197] In some embodiments, the CIP step and / or column sanitization step is conducted using sodium hydroxide.

[0198] Kits

[0199] The present disclosure also relates to systems and kits for use in the processes or uses of the invention. The kit may comprise one or more containers (such as tubes, vials, bottles and the like) either containing or separately containing the reagents of the kit, including but not limited to the compositions of the present disclosure. For example, various components of the compositions of the present disclosure may be separated into individual vials or containers, or combined into a single vial or container. Examples of typical reagents suitable for use in the kits according to the invention would be known to a person skilled in the art, and include but are not limited to forward and reverse primers, probes, buffers, polymerase enzymes, digestion enzymes, reverse transcriptase enzymes, detection antibodies or fragments thereof, fluorescent labelling dyes, aptamers. The compositions subjected to the processes or uses of the invention may also be biological samples. The kit may also comprise reagents for use in suitably preparing the biological samples, for example for pre-processing blood samples or to prepare liver tissue sections for immunohistochemistry purposes. The kits may also comprise washing solutions, diluents and buffers. Suitable buffers include those suitable for biological samples such as phosphate, acetate, citrate and TRIS buffers. The buffers may additionally be pH balanced, and typically the pH of the buffer will range between 6 to 8.

[0200] Example detection antibodies contained in the kits may be antibodies capable of binding and allowing for the detection and measurement of expression of surface proteins and the biomarkers. These detection antibodies may suitably be used in binding assays such as an ELISA, and may be either directly labelled or indirectly labelled through the use of a further agent such as a fluorophore or secondary antibody.

[0201] The kit may also comprise written instructions for performing the method. The kit may also provide standards or reference points for measuring or detecting markers of EVs or EV subtypes. The standards may be for example reference proteins or peptides required to establish a standard curve that will allow more accurate determination of expression levels.

[0202] EXAMPLES

[0203] The present invention is further described below by reference to the following non-limiting Examples.

[0204] Example 1 : Materials and Methods

[0205] Resin selection

[0206] Several chromatography resins were evaluated for their suitability for EV purification. Resins were then selected for the initial screening trial with an expected EV capture rate of at least 50%.

[0207] Table 1. List of resins selected for the EXO-ACE initial screening trial

[0208] Catalogue Product Name Material Charge Manufacturer

[0209] No.

[0210] 17546601 Capto DeVirS, 25 mL Dextran Sulphate -ve Cytiva

[0211] 17051010 Capto Q Fast Flow, 25 Sephadex (Cross- +ve Cytiva mL Linked Agarose) (Strong

[0212] Anion)

[0213] 17546201 Capto Heparin, 25 mL Heparin Sulphate -ve Cytiva

[0214] 17547610 Capto Q ImpRes, 25 CH2N+(CH3)3 (High +ve Cytiva mL Flow Agarose) Packing of chromatography column

[0215] The selected resins were ordered in their slurry form and then packed into customized columns. These columns were then hand packed on site. An exemplary design of the chromatography column is shown in Figure 1.

[0216] Isolation process

[0217] Isolation occurs at several stages as follows: a) Column Prep: The columns need to be taken out of storage conditions and undergo preparation for isolation. b) Sample Prep: The samples need to be prepared for isolation. c) Isolation Step: The samples are loaded on to the column and concentrated or high purity EV eluate is obtained.

[0218] Following steps (a) - (c), the following optional steps may be conducted: d) Buffer Exchange: The eluate undergoes this process to remove the elution buffer and suspend the EVs in a buffer / formulation of choice. The EVs also undergo further concentration during this stage. e) Terminal Filtration: The final sample undergoes a filtration step to obtain a sterile sample suitable for clinical use. f) Column CIP: The columns undergo a regeneration stage, at the end of which the columns are returned to storage conditions; ready to be used again.

[0219] The columns are also suitable for re-use (the number of uses currently limited to 10).

[0220] Protein analysis

[0221] The Nanodrop One Spectrophotometer (ThermoFisher) was used to measure protein concentration at various stages of the isolation process. While not as accurate as BCA protein analysis, the Nanodrop provides a quick reading of protein concentration and valuable data in terms of relative capture rates.

[0222] The BCA colorimetric protein concentration assay was also used in the initial screening trials for measuring protein concentrations due to its higher accuracy compared to the Nanodrop, however use of the BCA assay was later considered non- viable due to the presence of phenol red in some eluate samples.

[0223] Western Blot analysis was also performed to detect and measure the presence of EV-specific markers in samples, and also provides a qualitative characterization of EVs. Gene expression analysis

[0224] Quantitative PCR (qPCR) analysis was performed to detect and measure the expression of EV specific markers in samples. The presence of EV specific markers serves as confirmation that EVs are present. qPCR analysis also aids in the quantitative characterization of EVs.

[0225] Particle analysis

[0226] The ZetaView ZNTA (Particle Metrix) was used to measure particle concentration and size distribution at various stages of the isolation process. The ZetaView ZNTA provides detailed reading of the concentration and distribution of particles within the EV size range. Although not all particles are EVs, it is a very sensitive assay and allows for measurements at very low concentrations and at high resolution where qPCR may not be viable.

[0227] Imaging of EVs

[0228] Cryogenic electron microscopy (cryo-EM) was also conducted to provide images of EV structure. The use of cryo-EM provides visual confirmation of the presence of EVs.

[0229] Statistical analysis

[0230] Various statistical analysis techniques were used depending on suitability. Where multiple groups with variable factors were compared one-way or two-way ANOVA with Tukey’s multiple comparison was used. Where technical repeats were used coefficient of variance was calculated for comparison and judge reproducibility of data. Normality of data was assumed based on sample size.

[0231] Example 2: Initial screening trials

[0232] Four columns were packed, each containing the screening trial candidates as follows: Resin A (Capto Devir S), Resin B (Capto Q), Resin C (Capto Heparin) and Resin D (Capto Q Impres). Cell conditioned media was collected after growing MCF7 cells in culture (lot id: MCF7.P14.CM62). 10ml of conditioned media was loaded onto each resin. For these initial screening trial experiments, samples consequently underwent buffer exchange using PBS (dilution factor = 100x).

[0233] A BCA assay was performed using samples post EXO-ACE-purification (before buffer exchange) as shown in Figure 2A. The results suggest that the cationic resins (- ve) had a higher capture rate for protein (29-43%) compared to the anionic (+ve) resins (16-17%).

[0234] While protein capture may be a good indicator of EV capture, cell conditioned media may also contain non-EV proteins. Therefore further analysis was performed to elucidate the results.

[0235] Comparison of RNA yield at the purification end point (post-buffer exchange) shows that there was no significant difference in RNA yield between the groups, as shown in Figure 2B. The RNA quantification was performed using matched volumes.

[0236] Analysis of genetic expression of EV markers using qPCR using matched RNA input showed no significant difference between the 4 resins as shown in Figure 3. This concurs with the RNA quantification results suggesting no significant difference in EV capture.

[0237] Together the above results elucidated that the anionic resins performed equivalent to the cationic resins in terms of EV purification. Furthermore, anionic resins also had lower protein yield, suggesting the purified EV samples obtained had lower protein impurities than their cationic counterparts.

[0238] Example 3: Screening trial repeat and comparison with competitor columns

[0239] Following the previous promising results in Example 2, the screening trials were repeated albeit testing was conducted in comparison with a competitor column, the IZON size exclusion chromatography (SEC) columns. Izon SEC columns are commercially available and are a market leader in EV isolation.

[0240] Based on the initial resin trial Capto Devir S was considered the lead cationic resin and Capto Q Impres was considered the lead anionic resins. Further data needed to be obtained to determine reproducibility. Furthermore, in depth analysis of EV marker profile needed to be performed.

[0241] This experiment contained 5 groups: Resin A (Capto Devir S), Resin B (Capto Q), Resin C (Capto Heparin), Resin D (Capto Q Impres) and Resin E (Izon SEC qEV10). The qEV10 variant was chosen for this experiment as the loading capacity for this column is the same as the EXO-ACE columns. Cell conditioned media from the same lot as trial 1 was chosen (lot id: MCF7.P14.CM62). Although buffer exchange is not required for Izon SEC Columns, it was performed for experimental consistency.

[0242] Consistent with the results of the initial screening trials, protein quantification with BCA showed no significant difference between the EXO-ACE resin groups as shown in Figure 4A. The results suggest that both cationic resins have a higher protein yield after elution, when measured with a BCA assay. The effect is neutralized after the buffer exchange step. The Izon columns resulted with a significantly higher protein yield post elution, but this drops after buffer exchange. This suggests that there is a significantly higher amount of protein impurities present post isolation than the cationic candidates, which is lost in the flow through of the Buffer Exchange filter.

[0243] As shown in Figure 4B, RNA yield comparison was performed on the end point samples (post-buffer exchange) showing that Capto Q Impres had a higher RNA yield than any of the other candidates. This data suggests that the Izon column has a similar EV capture rate to the other candidates (apart from Capto Q Impres).

[0244] For the following experiment four sets of qPCR data were obtained as following: GAPDH with 3ng load, GAPDH with matched volume (12pl), miR-191 with 3ng load and U6 with 3ng load. The results as shown in Figure 5 show that the Izon column consistently trended towards a higher Ct value than the other groups (too high to measure in the case of miR-191), while Capto Q Impres consistently trended towards having the lowest Ct value. This data suggests a lower level of expression of EV markers in resulting samples from the Izon columns as compared to the present EXO-ACE resins.

[0245] Western blot analysis was next performed to detect CD9, a tetraspanin commonly found in EV membranes. CD9 was selected because of previous studies conducted showed that EVs from MCF7 cell lines produced a strong CD9 band. For these experiments, 10pg of protein was loaded into each well. Resin EV samples were concentrated using Norgen RNA / Protein Kit to ensure target loading concentration was being met. Starting conditioned media was loaded without processing. As shown in Figure 6, all resin candidates except Capto Heparin (-ve) and Izon shows banding. This data suggests that the present resin candidates (barring Capto Heparin) and in particular the CaptoQ Impres resulted in high efficacy of isolation of EVs. In contrast, the competitor Izon SEC column was not effective in isolating EVs.

[0246] Example 4: Particle and protein analysis of EVs isolated by EXO-ACE resins

[0247] Having shown that EV markers are evident in EXO-ACE elution samples, the present inventors next sought to determine the particle recovery in eluates from three different sample sources: cell conditioned media, normal human plasma, and normal human serum. For this experiment there were 5 groups (n=2 each). The first group was cell conditioned media from MCF7 cells (Lot ID: MCF7.P9.CM689). The next two groups were pooled normal human plasma (BiolVT, Frankfurt, Germany) and pooled normal human serum (ProteoGenix, Schiltigheim, France), where each column was loaded with 100pL of sample diluted to 10ml (to match the volume of loaded CM). For the 4th and 5th group, the plasma and serum samples (as above) but diluted to match the particle concentration of the CM input sample (the dilution was calculated on previous data obtained).

[0248] The particle analysis of the EXO-ACE resin eluate fraction fractions for each sample shows a very high recovery for each group (78% to 98%) suggesting that most particles present in the input material are being bound to the EXO-ACE resin (see Figure 7A). Furthermore, there is no significant difference in particle recovery between the 100pL load and the further diluted loads for both plasma and serum suggesting that resin saturation has not been reached. This suggest that the columns have a greater maximum capacity for capture of EVs than has been presently tested.

[0249] Protein analysis (see Figure 7B) shows that in contrast to the previous particle analysis, protein recovery is very low (2.81% to 4.87%). A high particle to protein ratio suggests that most of the free protein is lost during the EXO-ACE isolation process leaving a relatively pure EV sample for each group. Furthermore, protein recovery is conserved between the 100pL plasma and serum loads and their diluted counterparts, further indicating that the protein obtained is associated with the particles and not free protein impurities.

[0250] The data obtained during this experiment suggests that the EXO-ACE resins can isolate EVs from several different sample types with a high yield and purity.

[0251] Example 5: Confirmation of mode of action of EXO-ACE resins

[0252] Having established that EVs are being isolated using EXO-ACE, particle analysis was conducted on EXO-ACE elution fractions and to the column flow through at various stages to determine the mechanism of action of the resin. It was hypothesized that there are two possible modes of action which may be occurring independently or as a combination: affinity chromatography, whereby the EVs are binding to the resin via ionic affinity and are being released upon elution; or Size Exclusion, whereby EVs are being isolated from other components based on their size due to the porous nature of the resin. The mode of action confirmation was performed using collated data from several experiments.

[0253] During the previous study (see Example 4), flow through was collected at each stage of EXO-ACE isolation as follows: The flow through when the sample is loaded onto the column, the flow through during the subsequent PBS wash step and the eluate. Comparison of the total particle and total protein from each step was compared to the input (see Figure 8). The data shows that little to no particles are present during the sample flowthrough and PBS wash step, suggesting that almost all the particles in the input material is bound to the column and not released during the PBS wash step. However, majority of the protein is lost in the sample flowthrough and PBS wash step, indicating that free protein does not bind to the resin and is simply washed off during the EXO-ACE isolation process. This data suggests that the protein and particles are not being separated due to a size exclusion / filtration property of the resin, but instead, EVs are binding to the resin and released only when the conductivity of the column bed is altered via the introduction of the elution buffer.

[0254] Looking back at the results shown in Example 4, the Capto Q Impres showed the highest efficiency of EV capture. This is despite the Capto Q Fast Flow resin having stronger anionic properties (as illustrated by the higher ionic capacity of the Capto Q Fast Flow) and both resins having similar chemical structures, being composed of a quaternary amine (e.g. a stronger anion exchange group) functionalised agarose. The core difference between the chemical structure of the two resins is the presence of a hydroxylated spacer group in the Capto Q Impres. Without wishing to be bound by any particular theory, it is possible that the spacer group is effectively extending the charged quaternary amine functional group outwards beyond the agarose matrix resulting in either better flow of sample through and around the ligand to improve binding of EVs. Another possibility is that the hydroxyl groups increases the hydrophilicity of the ion chromatography exchange resin, such that it exhibits little to no hydrophobic adsorption of water-soluble molecules / contaminants that may also be present in the liquid composition comprising the EVs, thus reducing non-specific adsorption which would otherwise perhaps compete with the binding of the EVs. Regardless, this finding is surprising since EVs are conventionally accepted as carrying a net negative surface charge and owing to their lipid surface are typically hydrophobic, so the presence of hydroxylated spacer groups would be expected to repel EVs based on its charge / polarity, and thus reduce efficacy of EV capture. Both the Capto Q Impres and Capto Q Fast Flow comprise a quaternary amine functionalised group which would be predicted to be attracting the negatively charged EVs to a similar degree, so the above suggests that the hydroxylated spacer group is important with respect to the efficacy of capturing EVs.

[0255] In a follow-up experiment, 3 technical replicates of EVs isolated from conditioned media (Lot# PC3.P11.CM56) using 3 EXO-ACE columns were collected as 200pl fractions (total 10 fractions leading to a total volume of 2ml) during the elution stage (normally collected as a single 1.5ml aliquot). Each individual fraction underwent particle and protein analysis.

[0256] It was hypothesised that if both EVs and free proteins are binding to the resin via affinity they would be released simultaneously, but if they were being separated because of unintentional size exclusion / filtration effect, then they would be eluted in different fractions.

[0257] The particle analysis (see Figure 9A) showed that little to no particles are eluted during the first two fractions (OpL to 400pL). After 400pL the particle concentration begins to rise showing a peak concentration in the 5th fraction (800pL to 1000pL), steadily declining until the last fraction (1800pL to 2000pL). The protein measurement follows a similar trend peaking at fraction 6 (1000pL to 1200pL).

[0258] The above data suggests that particles and proteins are eluted from the column at the same time upon the addition of elution buffer. This overlap of the elution profile suggests that much of the protein measured in the EXO-ACE purified EV sample is due to protein that is associated with the EVs (most likely contained within).

[0259] Size distribution analysis of the particles seen at each fraction (see Figure 9B) shows that although concentration of the particles observed varies, there is no difference in the overall size distribution curve (no significant difference in median particle size), suggesting that no distinct populations of the EVs are present in each fraction. This suggests that all bound EVs are being released simultaneously as the elution buffer permeates through the resin.

[0260] Example 6: Further comparison of EXO-ACE resins with other anionic resins

[0261] Having confirmed the mode of action for EXO-ACE resins, the inventors next sought to perform a characterisation of EVs isolated using EXO-ACE resins and compare them to those isolated using resins that are anionic but do not have a similar structure. To perform this comparison, an extensive review was conducted by the inventors to identify additional suitable anionic resins, adhering to specific criteria. This involved a comprehensive examination of scientific publications, patents, and product datasheets to ensure that the selected resins met these structural requirements. Two exemplary EXO-ACE resins having a hydroxylated spacer were selected and compared with one candidate comparative resin having similar ionic capacity but comprising a ligand that deviated from the EXO-ACE resin structure. The resins used in this study are described in Table 2 below. EV isolation was performed as described in Example 1.

[0262] Table 2: List of resins selected for further validation study.

[0263] Resin Structure Supplier Ionic capacity Particle size

[0264] Several analytical techniques were used for the evaluation of EV capture as highlighted below in Table 3.

[0265] Table 3. Analytical techniques used, the outcome of these techniques and the reasoning of why they were used.

[0266] Various statistical analysis techniques were used depending on suitability. Where multiple groups with variable factors were compared one-way or two-way ANOVA with Dunnet’s multiple comparison was used. Where technical repeats were used coefficient of variance was calculated for comparison and judge reproducibility of data. Normality of data was assumed due to small sample sizes.

[0267] Results

[0268] Particle and protein analysis revealed (Figure 10) that both Capto Q ImpRes and Capto Q XP resins (each featuring hydroxyl spacer groups terminating in an anionic active binding group) exhibited high particle counts in the eluate compared to the other candidate resin, DEAE Sephacel. Capto Q XP demonstrated a lower protein count than Capto Q ImpRes.

[0269] RNA quantification and qPCR analysis of exosomal markers (Figure 11) mirrored the trends observed in particle and protein analysis. No significant differences were noted between the two resins containing hydroxyl spacers. However, the DEAE Sephacel resin exhibited significantly reduced total RNA and increased Ct values, indicating a lower presence of EVs.

[0270] Once again, Western Blot Analysis (Figure 12) mirrored the results of all previous analytical tests. Capto Q ImpRes showed the strongest banding for both Flotillin-1 and CD-9, followed by Capto Q XP while DEAE Sephacel did not show any banding. Discussion

[0271] The results of this validation study showed that resins with a hydroxylated spacer terminating in an anionic binding site, such as Q ImpRes and Q XP are superior for EV isolation. The protein-to-particle ratio was observed to be higher in the Capto Q XP isolated EVs compared to Capto Q ImpRes. However, subsequent RNA and Western blot analyses suggested that Capto Q ImpRes generated purer EV samples. Overall, the superior performance of resins with a hydroxylated spacer terminating in an anionic binding site over another anionic resin which had similar ionic capacity supports the broad use of the EXO-ACE resins for improved isolation of EVs.

[0272] Example 7: Isolation of EVs from CAR-T and CAR-NK populations using EXO-ACE resin

[0273] Primary T and NK cells are obtained and genetically modified to express a chimeric antigen receptor. EVs are isolated from primary T, NK cells as well as CAR-T and CAR-NK cells as described in the Examples above.

[0274] For EV isolation from culture media (CM), the CM is clarified using a low and high speed centrifugation step to allow for pelleting of cells and debris. The resultant clarified CM is sterile-filtered (0.22 pm) prior to EXO-ACE isolation as described in the Examples above, followed by an optional buffer-exchange step to reduce the high-salt concentration so EVs are appropriate for in vitro functional assays.

[0275] Isolated EVs are then assessed for expression of various markers such as gene expression or protein expression.

[0276] Example 8: Summary of findings

[0277] The results of the study suggested that all selected resin candidates are viable for EV isolation with the clear leader being Capto Q ImpRes. However, all the results show that anionic resins consistently isolate more EVs than cationic resins while having a low amount of protein impurities. Therefore, taking all the results into account the Capto Q ImpRes was considered the lead candidate for EXO-ACE resins.

[0278] The data shown further demonstrates that multiple EV markers are enriched in the EV sample isolated using EXO-ACE resins. The particle and protein profile suggests that the EXO-ACE resins have a high affinity for EVs and produces a highly pure sample.

[0279] The inventors also confirmed the mode of action of EXO-ACE resins to be via ionic affinity and eliminated the possibility of any size-exclusion / filtration effects of the resin bed. The data by the inventors support the theory that it is the hydroxylated spacer group on the Capto Q ImpRes and CaptoQ XP resins that are providing for a surprising technical effect. It is highly plausible that other anionic exchange resins having a hydroxylated spacer groups would also share this technical effect.

[0280] Overall, the data within the Examples supports that the present EXO-ACE columns pose a significant advantage over the leading functional EV isolation kit available in the market (in terms of sample purity, concentration and even yield).

[0281] LIST OF REFERENCES

[0282] 1. Zhang et al., Methods and Technologies for Exosome Isolation, 2018, Small Methods.

[0283] 2. Stahl et al. , Exosomes and microvesicles in normal physiology, pathophysiology and renal diseases, 2019, Pediatr Nephrol.

[0284] 3. Doyle and Wang, Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis, 2019, Cells.

[0285] 4. Coskun, Separation Techniques: Chromatography, 2016, North Clin Istanb.

[0286] 5. Midekessa et al., Zeta Potential of Extracellular Vesicles: Toward Understanding the Attributes that Determine Colloidal Stability, 2020, ACS Omega.

[0287] 6. Ganetsos and Barker, Preparative and production scale chromatography, 1993, CRC Press.

Claims

CLAIMS:

1. A composition comprising an ion exchange chromatography resin and a population of extracellular vesicles (EVs), wherein the chromatography resin comprises a support matrix functionalized with ligands for binding to the EVs, wherein one or more of the ligands comprise a hydroxylated spacer which attaches an anion exchange group to the support matrix.

2. The composition of claim 1 , wherein the hydroxylated spacer is an aliphatic spacer which is uninterrupted or interrupted and substituted with one or more hydroxyl groups.

3. The composition of claim 1 or 2, wherein the hydroxylated spacer is an alkyl spacer which is uninterrupted or interrupted and substituted with one or more hydroxyl groups.

4. The composition of any one of claims 1 to 3, wherein the hydroxylated spacer is an ether substituted with one or more hydroxyl groups.

5. The composition of any one of claims 1 to 4, wherein the hydroxylated spacer is a C2-2oalkylether substituted with one or more hydroxyl groups.

6. The composition of any one of claims 1 to 5, wherein the hydroxylated spacer is a reaction product of an epoxide, glycidyl ether or diglycidyl ether.

7. The composition of any one of claims 1 to 6, wherein the one or more ligands comprising the hydroxylated spacer is defined by the following Formula (I):Formula (I) whereinG is an anion exchange group; and- w represents an attachment point on the support matrix.

8. The composition of any one of claims 1 to 7, wherein the anion exchange group comprises one or more positively charged nitrogen groups.

9. The composition of claim 8, where in the positively charged nitrogen group is a quaternary ammonium group.

10. The composition of any one of claims 1 to 9, wherein the one or more ligands comprising the hydroxylated spacer has the following structure:wherein -~w represents an attachment point on the support matrix.

11. The composition of any one of claims 1 to 10, wherein the support matrix comprises one or more of: polysaccharides, silica, polymeric and ceramic material.

12. The composition of claim 11 , wherein the support matrix is selected from: dextran, starch, cellulose, agarose, polyvinyl alcohols, acrylamides, polyvinyl ethers and polyglycidol.

13. The composition of any one of claims 1 to 12, wherein the chromatography resin has an ionic capacity of about 0.01 to about 0.30 mmol / mL.

14. The composition of claim 13, wherein the chromatography resin has an ionic capacity of about 0.05 to about 0.25 mmol / mL, about 0.10 to about 0.20 mmol / mL, about 0.10 to about 0.25 mmol / mL, about 0.15 to about 0.25 mmol / mL, or about 0.15 to about 0.18 mmol / mL.

15. The composition of any one of claims 1 to 14, wherein each ligand is positively charged or partially positively charged at a pH of about 6 to about 10.

16. The composition of any one of claims 1 to 15, wherein the support matrix is provided as a membrane, expanded bed media, or beads.

17. The composition of claim 16, where the support matrix is provided as porous beads, non-porous beads, or magnetic beads.

18. The composition of claim 16 or 17, wherein the support matrix beads have a D50 particle size of about 10 pm to about 80 pm, about 10 pm to about 50 pm, about 30 pm to about 80 pm, about 30 pm to about 50 pm, or about 20 pm to about 60 pm.

19. The composition of any one of claims 1 to 18, wherein the extracellular vesicles comprise one or more of microvesicles, apoptotic bodies, exomeres and exosomes.

20. An ion exchange chromatography column comprising the composition of any of claims 1-19.21 . A process for separating extracellular vesicles, comprising the steps of: i) contacting a composition comprising the extracellular vesicles and one or more impurities to an ion exchange chromatography resin or an ion exchange chromatography column comprising the ion exchange chromatography resin, under conditions to enable binding of extracellular vesicles to the ion exchange chromatography resin, wherein the chromatography resin comprises a support matrix functionalized with ligands for binding to the EVs, wherein one or more of the ligands comprise a hydroxylated spacer which attaches an anion exchange group to the support matrix; and ii) separating the ion exchange chromatography resin or the ion exchange chromatography column from the composition, thereby separating the extracellular vesicles from the composition.

22. The process of claim 21 , further comprising step (iii) eluting the extracellular vesicles from the ion exchange chromatography resin or the ion exchangechromatography column by contacting the ion exchange chromatography matrix with an elution buffer.

23. The process of claim 21 or 22, wherein step (i) occurs at a pH of about 6 to about 10.

24. The process of claim 22 or 23, wherein the elution buffer has a salt concentration of about 0.5 M to about 3 M.

25. The process of any one of claims 22 to 24, further comprising step (iv) collecting the formed eluate comprising elution buffer and extracellular vesicles.

26. The process of claims 25, further comprising step (v) removing the elution buffer and resuspending the extracellular vesicles to form a concentrated composition or isolate containing extracellular vesicles.

27. The process of claim 26, further comprising step (vi) filtering the concentrated composition or isolate containing extracellular vesicles.

28. The process of any one of claims 21 to 27, further comprising prior to step (i) concentrating the composition comprising the extracellular vesicles.

29. The process of any one of claims 22 to 28, wherein the elution buffer has a salt concentration of about 1 M to about 2 M.

30. The process of any one of claims 21 to 29, wherein the composition comprising the extracellular vesicles is a cell culture fluid or a purified composition derived therefrom.31 . The process of any one of claims 21 to 29, wherein the anion exchange resin is an ion exchange chromatography resin defined by any one of claims 1 to 18.

32. Use of an ion exchange chromatography resin for separating extracellular vesicles from a composition, wherein the chromatography resin comprises asupport matrix functionalized with ligands for binding to the EVs, wherein one or more of the ligands comprise a hydroxylated spacer which attaches an anion exchange group to the support matrix for separating extracellular vesicles from the composition.

33. The process of any one of claims 21 to 31 , or the use of claim 32, wherein the extracellular vesicles comprise one or more of: microvesicles, apoptotic bodies, exomeres and exosomes.

34. The composition of any one of claims 1 to 19, the process of any one of claims 21 to 31 , or the use of claim 32, wherein the extracellular vesicles are from an immune cell or population thereof.

35. The composition, process or use of claim 34, wherein the immune cell is a T cell, NK cell or a macrophage.

36. The composition, process or use of claim 34, wherein the immune cell is an induced pluripotent stem cell (iPSC)-derived immune cell.

37. The composition, process or use of claim 36, wherein the iPSC-derived immune cell is an iPSC-derived T (iT) cell, an iPSC-derived NK (iNK) cell or an iPSC- derived macrophage.

38. The composition, process or use of claim 34, wherein the immune cell is an immune cell that comprises a chimeric antigen receptor (CAR).

39. The composition, process or use of claim 38, wherein the immune cell that comprises a CAR is a CAR iPSC-derived NK (CAR-iNK) cell or a CAR iPSC- derived T (CAR-iT) cell.

40. The composition, process or use of claim 34, wherein the immune cell is an activated immune cell.41 . The composition, process or use of claim 40, wherein the activated immune cell is an activated CAR-iNK cell or an activated CAR-iT cell.

42. The composition of any one of claims 1 to 19, the process of any one of claims 21 to 31 , or the use of claim 32, wherein the extracellular vesicles are from a cell from an immortalized human cell line, or population thereof.

43. The composition, process or use of claim 42, wherein the immortalized cell line is a HEK293 cell line.

44. The composition of any one of claims 1 to 19, the process of any one of claims 21 to 31, or the use of claim 32, wherein the extracellular vesicles are from a stem cell or population thereof.

45. The composition, process or use of claim 44, wherein the stem cell is a pluripotent stem cell or a multipotent stem cell.

46. The composition, process or use of claim 44 or 45, wherein the stem cell is a mesenchymal stem cell.

47. The composition, process or use of claim 44 or 45, wherein the stem cell is an induced pluripotent stem cell (iPSC).