A polymer for improved purification of extracellular vesicles

Abstract

The present disclosure relates generally to methods for reducing the lipoprotein content of a lipoprotein sample to provide lipoprotein reduced compositions, uses of copolymers to reduce the lipoprotein content of a lipoprotein sample, lipoprotein-reduced compositions, and resins comprising a copolymer for reducing the lipoprotein content of a lipoprotein sample.

Description

A POLYMER FOR IMPROVED PURIFICATION OF EXTRACELLULAR VESICLES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority from Australian Provisional Patent Application No 2023903967 filed on 7 December 2023, the contents of which are incorporated by reference herein in their entirety.TECHNICAL FIELD

[0002] The present disclosure generally relates to methods for reducing the lipoprotein content of a lipoprotein sample to provide lipoprotein reduced compositions, uses of copolymers to reduce the lipoprotein content of a lipoprotein sample, lipoprotein-reduced compositions, and resins comprising a copolymer for reducing the lipoprotein content of a lipoprotein sample.BACKGROUND

[0003] Lipoproteins are biochemical assemblies of lipids and proteins. They include species such as chylomicrons, high-density lipoprotein, intermediate-density lipoprotein, low-density lipoprotein, very low-density lipoprotein, and other apolipoprotein and lipid-like complexes. The presence of lipoproteins can represent a significant challenge to the purification of complex biological samples.

[0004] One context in which the difficulty of lipoprotein separation frequently arises, is in the isolation of extracellular vesicles (EVs) from biological samples. Compared to lipoproteins, EVs are a minority nanoparticle population in plasma, being approximately six orders of magnitude lower in concentration. Using existing methods for the isolation of EVs, such isolated samples are estimated to have a lipoproteins concentration up to 20 to 100-fold higher than that of the extracellular vesicles. In particular, low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) are known to co-isolate with EVs, making lipoprotein contamination a prevalent issue in the field. Although attempts have been made to combine two or more isolation methods to decrease lipoprotein abundance, the output of such methods still retain a significant amount of lipoprotein content.

[0005] The establishment of a method for the reduction of lipoprotein content from samples, is critical for the obtaining of biological entities of improved purity for research, therapy, and / or diagnostics, which in consequence, many enable the development of a range of new applications.SUMMARY

[0006] The present inventors have surprisingly found that contacting a sample containing lipoprotein with a copolymer as described herein, may facilitate removal of the lipoprotein from the sample. Without intending to be limited by theory, it is believed that this surprising result isdue to the described copolymers preferentially interacting with lipoproteins (which have phospholipid monolayers), over extracellular vesicles (which have phospholipid bilayers).

[0007] By implementing one or more of the methods and / or uses as described herein, the reduction in at least a portion of lipoprotein present in a sample, may provide at least one of the following advantages for one or more applications:- increased EV labelling; and / or- reduced macrophages interactions; and / or- enhanced classification efficiency; and / or- sensitivity to detect EV biomarkers; and / or- specificity, to detect EV biomarkers.

[0008] In addition, the methods and / or uses disclosed herein may provide for, inter alia, a pretreatment step that provides one or more benefits (for example a quantifiable measurement such as an improved reduction in ApoB levels per particle), in comparison to known methods in the art, such as, for example, the acidification of plasma to facilitate the separation of EVs from lipoproteins. This acidification method may be as exemplified by Mladenovic, D. et al., J Thromb Haemost, 21, 1032-1042, (2023), the contents of which are incorporated by reference herein in its entirety.

[0009] In one aspect, the present disclosure provides a method of reducing the lipoprotein content of a lipoprotein sample to provide a lipoprotein-reduced composition, the method comprising:(a) contacting the lipoprotein sample for a period of time with a copolymer derived from: at least one hydrophobic monomer; and at least one hydrophilic monomer, wherein the molar ratio of the at least one hydrophobic monomer and the at least one hydrophilic monomer is in the range of about 1 : 1 to about 50: 1.

[0010] In some embodiments, the contacting the lipoprotein sample for a period of time with a copolymer provides a treated composition, and the method further comprises:(b) subjecting the treated composition to a separation step.

[0011] In another aspect, the present disclosure provides a method of reducing the lipoprotein content of a lipoprotein sample to provide a lipoprotein-reduced composition, the method comprising:(a) contacting the lipoprotein sample for a period of time with an amphiphilic copolymer derived from: at least one hydrophobic monomer selected from styrene, alkyl monoalkene, diisobutylene or 7V-alkylacrylamide; and at least one hydrophilic monomer selected from maleic acid, maleic anhydride, maleimide, an acrylate or the salt or conjugate base thereof,wherein the molar ratio of the at least one hydrophobic monomer and the at least one hydrophilic monomer is in the range of about 1 : 1 to about 10: 1, and wherein the lipoprotein sample and the lipoprotein-reduced composition comprises one or more extracellular vesicles.

[0012] In some embodiments, the copolymer is an amphiphilic copolymer.

[0013] In some embodiments, the separation step comprises at least one of: tangential flow filtration, size-exclusion chromatography, precipitation, dead-end filtration, differential ultracentrifugation, density gradient ultracentrifugation, microfluidics, flow field fractionation, affinity-based methods, chromatography-based methods, and acoustic trapping, or any method suitable for separating particles less than about 10 to about 30 nm from particles greater than about 10 to about 30 nm.

[0014] In some embodiments, the copolymer is disposed on at least a portion of a solid support.

[0015] In some embodiments, the separation step comprises chromatography; the copolymer is disposed on at least a portion of a solid support; and the solid support is a porous monolith.

[0016] In some embodiments, the lipoprotein sample is a biological fluid.

[0017] In some embodiments, the method is used to isolate and / or purify extracellular vesicles from the lipoprotein sample.

[0018] In another aspect, the present disclosure provides for a method of reducing the lipoprotein content of a lipoprotein sample to provide a lipoprotein-reduced composition, the method comprising:(a) contacting the lipoprotein sample for a period of time with an amphiphilic copolymer derived from: at least one hydrophobic monomer selected from styrene, alkyl monoalkene, diisobutylene or V-alkylacrylamide; and at least one hydrophilic monomer selected from maleic acid, maleic anhydride, maleimide, an acrylate or the salt or conjugate base thereof, wherein the molar ratio of the at least one hydrophobic monomer and the at least one hydrophilic monomer is in the range of about 1 : 1 to about 10: 1, and wherein the lipoprotein sample and the lipoprotein-reduced composition comprise one or more extracellular vesicles.

[0019] In another aspect, the present disclosure provides a lipoprotein-reduced composition obtainable or obtained by a method according to any aspect, embodiment or example described herein.

[0020] In another aspect, the present disclosure provides a lipoprotein-reduced composition obtainable or obtained by a method according to any aspect, embodiment or example described herein, when used for isolating EV particles, optionally: with an increased fluorescent labelling capacity; and / orreduced macrophage interactions, compared to the lipoprotein sample before contact with the copolymer.

[0021] In another aspect, the present disclosure provides a lipoprotein-reduced composition obtainable or obtained by the method according to any aspect, embodiment or example described herein, wherein the EVs comprised in the lipoprotein-reduced composition have an increased fluorescent labelling capacity; and / or have reduced macrophage interactions, compared to the lipoprotein sample before contact with the copolymer.

[0022] In another aspect, the present disclosure provides for a lipoprotein-reduced composition obtainable or obtained by the method according to any aspect, embodiment or example described herein, wherein the EVs comprised in the lipoprotein-reduced composition have an increased fluorescent labelling capacity; and / or have reduced macrophage interactions, compared to the lipoprotein sample before contact with the copolymer.

[0023] In another aspect, the present disclosure provides use of a copolymer to reduce the lipoprotein content of a lipoprotein sample to provide a lipoprotein-reduced composition, wherein the copolymer contacts the lipoprotein sample for a period of time, and wherein the copolymer is derived from: at least one hydrophobic monomer; and at least one hydrophilic monomer, and wherein the molar ratio of the at least one hydrophobic monomer and the at least one hydrophilic monomer is in the range of about 1 : 1 to about 50: 1.

[0024] In another aspect, the present disclosure provides a separation matrix comprising a solid support and a copolymer derived from a hydrophobic monomer and a hydrophilic monomer, wherein the combination of hydrophobic monomer and hydrophilic monomer is selected from styrene and maleic acid or the salt or conjugate base thereof, or styrene and maleic anhydride.

[0025] In another aspect, the present disclosure provides use of the separation matrix according to any aspect, embodiment, or example described herein to reduce the lipoprotein content of a lipoprotein sample to provide a lipoprotein-reduced composition, wherein the use comprises:(a) contacting the lipoprotein sample for a period of time with the separation matrix.

[0026] Other aspects and embodiments relating to the present disclosure are described herein. It will be appreciated that each example, aspect and embodiment of the present disclosure described herein is to be applied mutatis mutandis to each and every other example, aspect or embodiment unless specifically stated otherwise. The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent substituents, compositions, methods and processes are clearly within the scope of the disclosure as described herein.BRIEF DESCRIPTION OF DRAWINGS

[0027] Whilst it will be appreciated that a variety of embodiments disclosed herein may be used, described herein are a number of examples with reference to the following drawings:

[0028] Figure 1 describes the effects of styrene-maleic acid (SMA) on particle concentrations in plasma and plasma-derived extracellular vesicle (EV) samples. Particle concentrations obtained by nanoparticle tracking analysis (NTA) upon the exposure of various samples to SMA: plasma (A), plasma processed by tangential flow filtration (TFF) (B), and plasma processed by TFF followed by size-exclusion chromatography (SEC) (C).

[0029] Figure 2 describes the effects of SMA pre-treatment on protein / lipoprotein contaminant levels in EV samples. Comparison of contaminant levels in plasma processed by TFF, TFF combined with SEC, and SMA pre-treatment combined with TFF. (A) Protein concentration. (B) Western blot of apolipoprotein (Apo)E normalized for protein concentration. (C, D) Enzyme linked immunosorbent assays (ELISAs) for ApoAl and ApoB levels normalized by number of particles.

[0030] Figure 3 describes the effects of SMA pre-treatment on EV markers. Comparison of size (A) and particle concentration (B) normalized by input plasma. The CD63 ELISA (C) and Western blot of CD63, Alix, and CD9 (D) were normalized for number of particles and by protein concentration, respectively.

[0031] Figure 4 describes the effects of SMA pre-treatment on the morphology and abundance of biological nanoparticles. Images were obtained by cryogenic transmission electron microscopy (cryo-TEM). (A) Representative Cryo-TEM images of very low-density lipoprotein (VLDL; left), low-density lipoprotein (LDL; centre) and TFF-processed plasma (right) with or without pretreatment with SMA. Scale bar, 50 nm. Black arrows indicate LDL and VLDL, white arrows indicate EVs, and asterisks indicate the supporting film. (B) Quantification of the number of biological nanoparticles. Data represent mean ± SD of at least eight images with a 126.87 pm2each. (C) Size distribution of VLDL and LDL particles assessed by cryo-TEM. Numbers were normalized to the non-SMA samples. (D) Particle size distribution (nanoparticle tracking analysis) of an EV sample of high purity (combined TFF and SEC isolation; gold standard) exposed to SMA post-isolation.

[0032] Figure 5 describes the effect of directly diluting SMA and other copolymers (powder) with plasma and incubating for a shorter pre-treatment period (one hour at 4 °C). Contaminant lipoprotein markers, ApoAl (A) and ApoB (B), were measured after plasma pre-treatment with copolymers (SMA, DIBMA 10, AASTY 11-45, AMPHIPOL-18, and SMALP 200) followed by SEC.

[0033] Figure 6 describes the impact of SMA pre-treatment on fluorescent particle labelling and macrophage interactions. (A) Fluorescence intensity of plasma-derived particles (TFF isolation) after Dil (left) / DiO (right) labelling and dialysis. The same number of particles were labelled in each group. Quantitative (B) particle association with macrophages (Raw 264.7) normalized based on particle concentration and input fluorescence. Scale bar represents 50 pm.

[0034] Figure 7 describes the impact of SMA pre-treatment on detection of EV-associated biomolecular correlates of disease using the accelerated SMA pre-treatment protocol (one hour at 4 °C) followed by SEC. (A) ELISAs of ApoAl and ApoB levels normalized by number of particles for samples processed by SEC. (B) Particle concentration normalized by input plasma for samples processed by SEC. Posterior predictive probability to identify a case (that is, gestational diabetes mellitus) in EV samples without (-) SMA (C) and with (+) SMA (D). (E) Biomarker analysis presented as receiver operating characteristic (ROC) with sensitivity without (blue circle) and with (red circle) SMA at 98% specificity.

[0035] Figure 8 describes (A) pre-treatment of plasma with SMA reduced level of ApoB 100 in monolithic chromatography (MC). (B) Fluorescence intensity of the flow-through from plasma samples spiked with fluorescently labelled human embryonic kidney (HEK293) EVs, albumin, or very low-density lipoproteins (VLDL). (C) ApoB 100 levels measured before and after the purification process. Data represent results from three independent runs. (D) Fluorescence intensity of the flow-through from plasma spiked with fluorescently labelled VLDL before and after simple dead-end ultrafiltration. (E) Levels of EV markers CD63 and CD9 were evaluated by Western blot and normalized to total protein concentration. Data in subfigures B and D represent mean + / - standard deviation (SD) of triplicates. Statistics analysis of variance (ANOVA) followed by Tukey’s correction. ****, p < 0.0001.DESCRIPTION OF EMBODIMENTS

[0036] The present disclosure describes the following various non-limiting embodiments, which relate to methods for reducing lipoprotein content, uses of copolymers, separation matrices comprising copolymers, their uses, and methods for the preparation thereof, and lipoprotein- reduced compositions.General Terms

[0037] In the following description, reference is made to the accompanying drawings which form a part hereof, and which is shown, by way of illustration, several embodiments. It is understood that other embodiments may be used, and structural changes may be made without departing from the scope of the present disclosure.

[0038] With regards to the definitions provided herein, unless stated otherwise, or implicit from context, the defined terms and phrases include the provided meanings. Unless explicitly statedotherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired by a person skilled in the relevant art. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0039] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present disclosure. It 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 claim of this application.

[0040] Throughout this disclosure, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter. Thus, as used herein, the singular forms “a”, “an” and “the” include plural aspects unless the context clearly dictates otherwise. For example, reference to “a” includes a single as well as two or more; reference to “an” includes a single as well as two or more; reference to “the” includes a single as well as two or more and so forth.

[0041] Those skilled in the art will appreciate that the disclosure herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the examples, steps, features, methods, processes, and compositions, referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0042] 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.

[0043] Unless otherwise indicated, the terms “first,” “second,” etc., are used herein merely as labels, and are not intended to impose ordinal, positional, or hierarchical requirements on the items to which these terms refer. Moreover, reference to a “second” item does not require or preclude the existence of lower-numbered item (e.g., a “first” item) and / or a higher-numbered item (e.g., a “third” item).

[0044] 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 listmay 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.

[0045] As used herein, the term “about”, unless stated to the contrary, typically refers to a range of up to + / - 10% of the designated value, and includes smaller ranges therein, for example + / - 5% or + / - 1% of the designated value.

[0046] 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.

[0047] 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 subranges 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, 4.75, and 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.

[0048] 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.

[0049] Throughout this specification, the term "consisting essentially of' is intended to exclude elements which would materially affect the properties of the claimed composition, method or process.

[0050] The terms "comprising", "comprise" and "comprises" herein are intended to be optionally substitutable with the terms "consisting essentially of, "consist essentially of, "consists essentially of, "consisting of, "consist of and "consists of, respectively, in every instance.

[0051] Herein “weight %” may be abbreviated to as “wt%” or “wt.%”. The weight % may be w / w or w / v, unless specifically indicated or clear from context.Specific Terms

[0052] The term “halo” or “halogen” whether employed alone or in compound words such as haloalkyl, represents fluorine, chlorine, bromine or iodine. Further, when used in compound words such as haloalkyl, the alkyl may be partially halogenated or fully substituted with halogen atoms which may be independently the same or different. Examples of haloalkyl groups include fluoromethyl, chloromethyl, bromomethyl, iodomethyl, fluoropropyl, fluorobutyl, difluoromethyl difluoroethyl, trifluoromethyl and trifluoroethyl groups. Further examples of haloalkyl groups include -CF3, -CCh, and -CH2CF3, -CF2CF3 and -CH2CHFCI.

[0053] 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, but are not limited to: 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).

[0054] As used herein, the term “heteroalkyl” represents straight chain (i.e., linear), or branched chain hydrocarbon groups which are analogous to an alkyl group, but in which one or more carbon atoms is / are replaced by one or more heteroatoms selected from nitrogen, sulfur, and oxygen.

[0055] As used herein, the term “alkenyl” represents straight (i.e., linear) or branched chain unsaturated hydrocarbon groups containing at least one carbon-carbon double bond. Examples of alkenyl groups include, but are not limited to: ethylene, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl and decenyl groups. In one example, the alkenyl group is of 2 to 20 carbon atoms (i.e., C2-2oalkenyl). In another example, the alkenyl is a group 2 to 10 carbon atoms (i.e., C2-ioalkenyl). In another example, the alkenyl group is of 2 to 6 carbon atoms (i.e., C2- ealkenyl)

[0056] As used herein, the term “alkynyl” represents straight (i.e., linear), or branched chain unsaturated hydrocarbon groups containing at least one carbon-carbon triple bond. Examples of alkenyl groups include, but are not limited to: ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl and decynyl groups. In one example, the alkynyl group is of 2 to 20 carbon atoms (i.e., C2-2oalkynyl). In one example, the alkynyl group is of 2 to 10 carbon atoms (i.e., C2-ioalkynyl). In another examples, the alkynyl group is of 2 to 6 carbon atoms (i.e., C2- ealkynyl).

[0057] As used herein, the term “haloalkyl” represents to an alkyl group having at least one halogen substituent, where “alkyl” and “halogen” are as described above. For example, the haloalkyl group may have at least one, two or three halogen substituents. Examples of haloalkyl groups include, but are not limited to: fluoromethyl, chloromethyl, bromomethyl, iodomethyl, fluoropropyl, fluorobutyl, difluoromethyl difluoroethyl, trifluoromethyl and trifluoroethyl groups. Further examples of haloalkyl groups include -CF3, -CCI3, and -CH2CF3, -CF2CF3 and -CH2CHFCI. In one example, the haloalkyl group is of 1 to 20 carbon atoms (i.e., Ci- 2ohaloalkyl). In one example, the haloalkyl group is of 1 to 10 carbon atoms (i.e., Ci-iohaloalkyl). In another example, the haloalkyl group is of 1 to 6 carbon atoms (i.e., Ci-ehaloalkyl).

[0058] As used herein, the terms “carbocyclyl” and “carbocycle” whether used alone, or in compound words such as alkylcarbocyclyl, represents a monocyclic or polycyclic ring system wherein the ring atoms are all carbon atoms, e.g., of about 3 to about 20 carbon atoms, and which may be aromatic, non-aromatic, saturated, or unsaturated, and may be substituted and / or contain fused rings. In one example, the carbocyclyl group is of 3 to 20 carbon atoms (i.e., C3-2o-membered carbocyclyl). In another example, the carbocyclyl group is of 3 to 10 carbon atoms (i.e., C3-10- membered carbocyclyl). Examples of such groups include, but are not limited to: aryl groups such as phenyl, naphthyl, anthracenyl or fluorenyl, saturated groups such as cycloalkyl and cycloalkenyl groups e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl groups, or fully or partially hydrogenated phenyl, naphthyl and fluorenyl. It will be appreciated that the polycyclic ring system includes bicyclic and tricyclic ring systems.

[0059] As used herein, the term “cycloalkyl” whether used alone, or in compound words such as alkylcycloalkyl, refers to a monocyclic or polycyclic carbocyclic ring system of varying sizes, e.g., from about 3 to about 20 carbon atoms, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl. It will be appreciated that the polycyclic ring system includes bicyclic and tricyclic ring systems.

[0060] As used herein, the term “heterocyclyl” whether used alone or in compound words such as alkylheterocyclyl, refers to a monocyclic or polycyclic ring system wherein the ring atoms are provided by at least two different elements, typically a combination of carbon and one or more of nitrogen, sulfur, and oxygen, and wherein the ring system may be aromatic such as a “heteroaryl” group, non-aromatic, saturated, or unsaturated, and may be substituted and / or contain fused rings. Heterocyclyl groups containing a suitable nitrogen atom include, but are not limited to: the corresponding N-oxides. In one example, the heterocyclyl group is of 3 to 20 atoms (i.e., 3-20- membered heterocyclyl). In another example, the heterocyclyl group is of 3 to 10 atoms (i.e., 3- 10-membered heterocyclyl). The heteroatom may be N, O or S. Examples of monocyclic non-aromatic heterocyclyl groups include, but are not limited to: aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, tetrahydrofuranyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl and azepanyl. Examples of bicyclic heterocyclyl groups in which one of the rings is non-aromatic include, but are not limited to: dihydrobenzofuranyl, indanyl, indolinyl, isoindolinyl, tetrahydroisoquinolinyl, tetrahydroquinolyl, and benzoazepanyl. Examples of monocyclic aromatic heterocyclyl groups (also referred to as monocyclic heteroaryl groups) include, but are not limited to: furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl (e.g., the radical derived from pyridine), triazolyl, triazinyl, pyridazyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrazolyl, and pyrimidinyl. Examples of bicyclic aromatic heterocyclyl groups (also referred to as bicyclic heteroaryl groups) include, but are not limited to: quinoxalinyl, quinazolinul, pyridopyrazinyl, benzoxazolyl, benzothiophenyl, benzimidazolyl, naphthyridinyl, quinolinyl, benzofuranyl, indolyl, benzothiazolyl, oxazolyl[4,5- b]pyridyl, pyridopyrimidinyl, isoquinolinyl, and benzohydroxazole. It will be appreciated that the polycyclic ring system includes bicyclic and tricyclic ring systems.

[0061] As will be understood, an “aromatic” group means a cyclic group having 4m+27i 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.

[0062] 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 and / or fluorenyl. It will be appreciated that the polycyclic ring system includes bicyclic and tricyclic ring systems. Related to the term aryl, the term “aralkyl” as used herein refers to an alkyl group wherein a hydrogen atom is replaced by an aryl group as a substituent. Examples of alkylaryl groups include, but are not limited to, an optionally substituted benzyl (e.g., -CEE-phenyl).

[0063] As used herein, the term “heteroaryl” whether used alone, or in compound words such as alkylheteroaryl, represents a monocyclic or polycyclic aromatic ring system wherein the ring atoms are provided by at least two different elements, typically a combination of carbon and one or more of nitrogen, sulfur, and oxygen, and may be substituted and / or contain fused rings. Heteroaryl groups containing a suitable nitrogen atom include the corresponding N-oxides. In one example, the heteroaryl group is of 3 to 20 atoms (i.e., 3-20-membered heteroaryl). In another example, the heteroaryl group is of 3 to 10 atoms (i.e., 3-10-membered heteroaryl). Examples ofmonocyclic heteroaryl groups include, but are not limited to: furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl, triazolyl, triazinyl, pyridazyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrazolyl, and pyrimidinyl. Examples of bicyclic heteroaryl groups include, but are not limited to: quinoxalinyl, quinazolinul, pyridopyrazinyl, benzoxazolyl, benzothiophenyl, benzimidazolyl, naphthyridinyl, quinolinyl, benzofuranyl, indolyl, benzothiazolyl, oxazolyl[4,5-b]pyridyl, pyridopyrimidinyl, isoquinolinyl, and benzohydroxazole. All regioisomers are contemplated, e.g., 2-pyridyl, 3-pyridyl and 4-pyridyl. It will be appreciated that the polycyclic ring system includes bicyclic and tricyclic ring systems.

[0064] As used herein, the term “saturated” refers to a group where all available valence bonds of the backbone atoms are attached to other atoms Representative examples of saturated groups include, but are not limited to: butyl, cyclohexyl, piperidine, and the like.

[0065] As used herein, the term “unsaturated” refers to a group where at least one valence bond of two adjacent backbone atoms is not attached to other atoms. Representative examples include, but are not limited to: alkenes (e.g., -CH2-CH2CH=CH), phenyl, pyrrole, and the like.

[0066] As used herein, the term “optionally substituted” means that a functional group is either substituted or unsubstituted, at any available position.

[0067] As used herein, the term “substituted” refers to a group having one or more hydrogens or other atoms removed from a carbon or suitable heteroatom and replaced with a further group (i.e., substituent).

[0068] As used herein, the term “unsubstituted” refers to a group that does not have any further groups attached thereto or substituted therefore.

[0069] The terms "optionally substituted”, “comprises one or more substituents” or “substituted” means that a corresponding radical, atom, group or moiety on a compound may have one or more substituents present. Where a plurality of substituents, or a selection of various substituents is specified, the substituents are selected independently of one another and do not need to be identical. In some cases, at least one hydrogen atom on the radical, group or moiety is replaced with a substituent. In the case of an oxo substituent (=0) two hydrogen atoms may be replaced. In this regard, substituents may include, but are not limited to: alkyl, alkenyl, alkynyl, carbocyclyl, halogen, nitro, cyano, hydroxy, sulfonic, thiol, ether, amino, alkylamino, dialkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, heteroaryloxy, aralkyloxy, alkylthio, carboxamido, sulfonamido, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, carboxy, carboxyalkyl, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclo, alkoxyalkyl, (amino)alkyl, hydroxyalkylamino, (alkylamino)alkyl, (dialkylamino)alkyl, (cyano)alkyl, (carboxamido)alkyl, mercaptoalkyl, (heterocyclo)alkyl, (cycloalkylamino)alkyl, (C1-C4 haloalkoxy)alkyl, (heteroaryl)alkyl, or perylene, oxo, heterocycle, -ORX, -NRXRY, -NRxC(=O)Ry-NRxSO2Ry, -C(=O)RX, -C(=O)ORX, -C(=O)NRxRy, -SOqRxand -SOqNRxRy, wherein q is 0, 1 or 2, Rxand Ryare the same or different and independently selected from hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents may be further substituted with one or more of oxo, halogen, -OH, -CN, alkyl, -ORX, heterocycle, -NRxRy, -NRxC(=O)Ry-NRxSO2Ry,-C(=O)Rx, - C(=O)ORX, -C(=O)NRxRy, -SORXand -SONRxRy.

[0070] As used herein, “hydrophilic” shall refer to a substance or portion thereof that more readily associates with water than with lipids.

[0071] As used herein, “hydrophobic” shall refer to a substance or portion thereof that more readily associates with lipids than with water.

[0072] As used herein, “lipoprotein content” collectively refers to the “lipoprotein” or “lipoproteins” that are present in a lipoprotein sample (or lipoprotein-reduced sample). Nonlimiting examples of “lipoproteins” that may be present in a sample include, but are not limited to: chylomicrons, low-density lipoprotein, high-density lipoprotein, very low-density lipoprotein, intermediate-density lipoprotein and other entities containing complexes of apolipoproteins and lipids produced naturally or synthetically. “Lipoprotein content” may be quantified e.g., in terms of absolute particle count, or particle concentration (viz. lipoprotein particles per unit volume of sample).

[0073] As used herein, “lipoprotein-reduced composition” shall refer to a second composition that has a lower amount of lipoprotein(s) in comparison to a first composition from which the second composition is derived, e.g., by any means, method or process of destroying, degrading, removing, and / or reducing the lipoprotein content in the first composition.

[0074] As used herein, “monomer” shall refer to a compound that can be polymerised chemically, actinically or thermally.

[0075] As used herein, “polymer” shall refer to a material formed by polymerisation and / or crosslinking or one or more monomers and / or crosslinkers.

[0076] As used herein, “copolymer” shall refer to a polymer obtained via polymerisation or two, three, four or more different monomers. The copolymer may be in the form selected from, but not limited to: a block copolymer, an alternating copolymer, a periodic copolymer, a statistical copolymer, a stereoblock copolymer, a gradient copolymer, a graft copolymer, a star copolymer, or a mixture thereof. The copolymer, or a portion thereof, may be in a form selected from: linear, branched, hyperbranched, dendritic or dendrimer, brush or comb, and mixtures thereof.

[0077] As used herein, “lipoprotein sample” shall refer to any composition comprising lipoprotein. Examples of such compositions include, but are not limited to: natural, biological, non-biological, or synthetic compositions, or mixtures thereof.

[0078] As used herein, “separation matrix / matrices” refers to a material useful for the separation of any molecule, compound, or complex from one or more other distinct molecules, compounds or complexes, and the separation matrix comprises a porous or nonporous solid support, and a porous surface layer. In the field of chromatography, matrices are often represented by resins or media.

[0079] As used herein, “porous surface layer” refers to a polymeric surface comprising a plurality of pores of similar size or various sizes. The porous surface layer may be associated with a polymeric support.

[0080] As used herein, “surface of a separation matrix” refers to surfaces that comprise both the external surface of the support and the pore surfaces and also the inner surface of the porous layer, which is associated with the bulk porous support.Methods of Reducing Lipoprotein Content

[0081] Disclosed herein is a method of reducing the lipoprotein content of a lipoprotein sample to provide a lipoprotein-reduced composition, the method comprising:(a) contacting the lipoprotein sample for a period of time with a copolymer derived from: at least one hydrophobic monomer; and at least one hydrophilic monomer, wherein the molar ratio of the at least one hydrophobic monomer and the at least one hydrophilic monomer is in the range of about 1 : 1 to about 50: 1.

[0082] It will be understood that reducing the lipoprotein content of a lipoprotein sample encompasses a reduction, removal, and / or degradation of at least a portion of the lipoprotein particles that are present in the lipoprotein sample.

[0083] There is not intended to be any limitation on the means or manner by which the lipoprotein sample is contacted for a period of time with the copolymer. For example, the lipoprotein sample and copolymer are comprised in a composition of any form e.g., but without limitation, a solution, suspension, mixture, gel, paste. Thus, in some embodiments, the contacting the lipoprotein sample for a period of time with a copolymer provides a treated composition. In some embodiments, the copolymer is disposed on a solid support, and the lipoprotein sample is a liquid. In some embodiments, the copolymer is disposed on a solid support, and the lipoprotein sample is in the form of a solution-phase or suspended composition.

[0084] In some embodiments, the contacting the lipoprotein sample for a period of time with a copolymer provides a treated composition, and the method further comprises:(b) subjecting the treated composition to a separation step.

[0085] In some embodiments, step (a) and step (b) may occur simultaneously. In some embodiments, the contacting the lipoprotein sample for a period of time with a copolymer occurs at the same time as a separation step. That is to say, in some embodiments the method is a methodof reducing the lipoprotein content of a lipoprotein sample to provide a lipoprotein-reduced composition, the method comprising:(a) contacting the lipoprotein sample for a period of time with a copolymer derived from: at least one hydrophobic monomer; and at least one hydrophilic monomer, wherein the molar ratio of the at least one hydrophobic monomer and the at least one hydrophilic monomer is in the range of about 1 : 1 to about 50: 1, and wherein step (a) further comprises a separation step, which occurs simultaneously with the contacting of the lipoprotein sample by the copolymer. In some embodiments, separation step comprises separating the lipoprotein content of the lipoprotein sample, from the remainder of lipoprotein sample.Copolymers

[0086] The identity of the copolymer derived from at least one hydrophobic monomer; and at least one hydrophilic monomer is not particularly limited. It will be understood that “copolymer derived from” refers to a copolymer formed via at least one polymerisation reaction involving the hydrophobic monomer and / or the hydrophilic monomer. Any appropriate polymerisation reaction known in the art may be suitable for deriving the copolymer. It will be appreciated that copolymers derived from at least one hydrophobic monomer, and at least one hydrophilic monomer may optionally be end-capped with an end-cap group. An “end-cap group” refers to a group attached to or replacing the end group of the polymer backbone. End-cap groups can be introduced into the polymer by end-capping processing, which is known in the art. The end-capper can be added after the polymerisation reaction. Alternatively, the end-cap reagent can be added in situ before or during the polymerisation reaction. Further, the addition of the end-cap reagent may be used to terminate the polymerisation reaction and thereby control the molecular weight of the formed polymer. Non-limiting examples of end-cap groups include, but are not limited to: H, alkylaryl, aryl (e.g., phenyl) and lower alkyl, and mixtures thereof.

[0087] In some embodiments, the copolymer is an amphiphilic copolymer. In some embodiments, the amphiphilic copolymer is an amphipol. Amphipols broadly encompass amphiphilic polymers capable of solubilising a membrane protein in its native / natural conformation. Non-limiting examples of amphipols include styrene-maleic acid copolymers, diisobutylene-maleic acid copolymers, acrylic acid-styrene copolymers, (partially) A-alkylated polyacrylates, ‘CyclAPols’, and those disclosed in Zoonens M, Popot JL. ‘Amphipols for each season’, J. Membr. Biol. 2014 Oct; 247(9-10):759-96, the contents of which is hereby incorporated by reference herein in its entirety, in particular Tables 1 and 2.Hydrophobic Monomer

[0088] The identity of the at least one hydrophobic monomer is not particularly limited. The hydrophobic monomer may be any monomeric unit having at least one hydrophobic group and at least one polymerisable double bond. Non-limiting examples of polymerisable double bonds include, but are not limited to: acrylic, methacrylic, acrylamido, methacrylamido, fumaric, maleic, styryl, isopropenylphenyl, (9-vinylcarbonate, (9-vinylcarbamate, allylic, (9-vinylacetyl and N- vinyllactam and -vinylamido double bonds, and mixtures thereof.

[0089] Non-limiting examples of suitable hydrophobic groups include linear, branched and cyclic hydrocarbons (including alkanes, alkenes and alkynes), aromatic compounds, and substituted or interrupted derivatives thereof, so long as the hydrophobic character of the group is retained. Hydrophobic groups may also include non-hydrocarbon groups and / or may contain one or more heteroatoms (for example, nitrogen, oxygen, sulfur, or phosphorus). In some embodiments, the hydrophobic group is an aliphatic group. In certain embodiments, the hydrophobic group comprises a C4-C12 alkyl, alkenyl, cycloalkyl, or cycloalkenyl group. In some embodiments, the hydrophobic group comprises fewer than 10 carbons or fewer than 8 carbons. For example, the hydrophobic group can comprise a C2-C10, C2-C8, or C2-C6 (e.g., C3-C8 or C3- Ce), alkyl group. The alkyl or alkenyl groups can be linear, branched, or cyclic. In some embodiments, the hydrophobic group is attached to the polymer backbone directly or via a linkage comprising, for instance, an ester, an amide, or an ether group, optionally further comprising a hydrocarbon linker, e.g., an alkylene linker (e.g., a methylene or ethylene linker).

[0090] The hydrophobic group can be linked to the hydrophobic monomer via any form of covalent attachment e.g., an amide, ester, or glycosidic bond. In some cases, the hydrophobic group includes an amide reaction product of a carboxylic group of the hydrophobic monomer and an amine compound, or vice versa.

[0091] The hydrophobic monomer can further include an alkyl group, for example as a spacer or linker between the hydrophobic group and the polymer backbone. The length of the alkyl group is not particularly limiting. Thus, the hydrophobic group can be separated from the copolymer backbone by an alkyl group larger than a C10 alkyl group, a C1-C10 alkyl group, or a Ci-6 alkyl group, for example. In some cases, the hydrophobic group can include a Ci-Ce alkyl group including a terminal hydrophobic group.

[0092] Non-limiting examples of suitable hydrophobic monomers include C2-C24 olefins such as ethylene, propylene, n-butene, isobutene, n-hexene, n-octene, isooctene, n-decene, isotridecene, Cs-Cs cycloolefins such as cyclopentene, cyclopentadiene, cyclooctene, fluoroolefms and fluorochloroolefins such as vinylidene fluoride, chlorotrifluoroethylene and tetrafluoroethylene, vinyl aromatics such as styrene and alpha-methylstyrene, diolefins such as butadiene, isoprene andchlorobutadiene, monoethylenically unsaturated monomers comprising at least one C2-C36 alkyl group, vinyl esters of linear or branched alkane carboxylic acids having 2 to 36 carbon atoms such as vinyl acetate, vinyl propionate, vinyl n-butyrate, vinyl isobutyrate, vinyl hexanoate, vinyl octanoate, vinyl laurate and vinyl stearate, esters of acrylic acid or methacrylic acid with linear or branched C1-C36 alkanols, such as ethyl (meth)acrylate, n-butyl (meth)acrylate, tert-butyl (meth)acrylate, n-hexyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, 2-propylheptyl (meth)acrylate, lauryl (meth)acrylate and stearyl (meth)acrylate and also vinyl ethers (polyvinyl ethers) and allyl ethers (polyallylethers) of C2-C36 alkanols, such as n-butyl vinyl ether and octadecyl vinyl ether. Further examples include, but are not limited to: a C4-C40 alkylacrylate, a C4-C40 alkylmethacrylate, a C4-C40 alkenylacrylate, a C4-C40 alkenylmethacrylate, or a mixture of any two or more such hydrophobic monomers. In some embodiments, the monomer is a C8-C34 alkylacrylate or C8-C40 alkenylacrylate. In some embodiments, the monomer is a C8-C34 alkylacrylate or C8-C34 alkylmethacrylate. Suitable hydrophobic monomers include, but are not limited to: capryl acrylate, pelargonyl acrylate, undecyl acrylate, lauryl acrylate, tridecyl acrylate, myristyl acrylate, pentadecyl acrylate, cetyl acrylate, palmitoleyl acrylate, heptadecyl acrylate, stearyl acrylate, isostearyl acrylate, elaidyl acrylate, linoleyl acrylate, elaidolinolenyl acrylate, nondecyl acrylate, arachidyl acrylate, heneiscosyl acrylate, behenyl acrylate, erycyl acrylate, lignoceryl acrylate, ceryl acrylate, heptacosanyl acrylate, montanyl acrylate, docosanyl acrylate, myricyl acrylate, dotriacontanyl acrylate, geddyl acrylate, capryl methacrylate, pelargonyl methacrylate, undecyl methacrylate, lauryl methacrylate, tridecyl methacrylate, myristyl methacrylate, pentadecyl methacrylate, cetyl methacrylate, palmitoleyl methacrylate, heptadecyl methacrylate, stearyl methacrylate, isostearyl methacrylate, elaidyl methacrylate, linoleyl methacrylate, elaidolinolenyl methacrylate, nondecyl methacrylate, arachidyl methacrylate, heneiscosyl methacrylate, behenyl methacrylate, erycyl methacrylate, lignoceryl methacrylate, ceryl methacrylate, heptacosanyl methacrylate, montanyl methacrylate, nancosanyl methacrylate, myricyl methacrylate, dotriacontanyl methacrylate, and geddyl methacrylate. Further non-limiting examples include, but are not limited to: benzyl methacrylate, butyl methacrylate, methyl methacrylate, ethyl methacrylate, propyl methacrylate, hexyl methacrylate, 2-ethylhexyl methacrylate, octyl methacrylate, lauryl ethacrylate, stearyl methacrylate, phenyl methacrylate, phenoxyethyl methacrylate, methacrylonitrile, glycidyl methacrylate, p-tolyl methacrylate, sorbyl methacrylate, methyl acrylate, ethyl acrylate, propyl acrylate, butyl acrylate, hexyl acrylate, 2- ethylhexyl acrylate, octyl acrylate, lauryl acrylate, stearyl acrylate, phenyl acrylate, phenoxyethyl acrylate, acrylonitrile, glycidyl acrylate, p-tolyl acrylate, sorbyl acrylate, styrene, alpha-methyl styrene, substituted styrenes, 7V-alkyl acrylamides (including linear, branched and cyclic A -alkyl derivatives of acrylamide), 7V-alkyl methacrylamides, vinyl acetate, vinyl butyrate and vinylbenzoate. Such groups may be substituted e.g., with one or more alkyl groups (e.g., Ci-Cs alkyl groups), and / or substituted with one or more heteroatoms (e.g., N, O, S heteroatoms), whilst retaining overall hydrophobic character / properties.Hydrophilic Monomers

[0093] The identity of the at least one hydrophilic monomer is not particularly limited. The hydrophilic monomer may be any monomeric unit having at least one hydrophilic group and at least one polymerisable double bond. Examples of polymerizable double bonds include, but are not limited to: acrylic, methacrylic, acrylamido, methacrylamido, fumaric, maleic, styryl, isopropenylphenyl, (9-vinylcarbonate, (9-vinylcarbamate, allylic, (9-vinylacetyl and / V-vinyllactam and 7V-vinylamido double bonds, and mixtures thereof.

[0094] The hydrophilic group can include any hydrophilic group. For example, the pendant hydrophilic group can include at least one of: hydroxyl, amino, carboxylic acid, carboxylate, phosphate, phosphonate, sulfonic acid, sulfuric acid, thiosulfates, carboxylic ether, carboxylic ester, phosphoric acid, phosphate ester, amide, phosphonamide, alkylamines, alkyl substituted ammonium salts, quatemized pyridine salts, quatemized imidazole salts, derivatives thereof, salts thereof, or combinations thereof. In some embodiments, the hydrophilic group may include at least one of: a carboxylic acid group, carboxylate group, or salt thereof.

[0095] The hydrophilic monomer may contain a masked or protected hydrophilic group, e.g., in the case of carboxylic acid, the ester, anhydride, or alkali metal salt thereof. Methods to convert the masked or protected hydrophilic group are known in the art, e.g., hydrolysis. In some embodiments, a hydrophilic group is installed via modification of an acid or anhydride.

[0096] The hydrophilic group can be linked to the hydrophilic monomer via any form of covalent attachment e.g., an amide, ester, or glycosidic bond. The hydrophilic group can be linked to the hydrophilic monomer by the reaction of a carboxylic group or corresponding anhydride of the hydrophilic monomer and a nucleophilic group provided on the hydrophilic group, for example, an amino or hydroxyl group. In some cases, the hydrophilic group includes an amide reaction product of a carboxylic group of the hydrophilic monomer and an amine compound. In some cases, the pendant hydrophilic group includes an amide reaction product of maleic anhydride and an amine compound. In some cases, the pendant hydrophilic group includes an amide reaction product of maleimide and a carboxylic group compound.

[0097] The monomer can further include an alkyl group, for example as a spacer or linker between the hydrophilic group and the polymer backbone. The length of the alkyl group is not particularly limiting. Thus, the hydrophilic group can be separated from the copolymer backbone by an alkyl group larger than a Cio alkyl group, a Ci-Cio alkyl group, or a Ci-6 alkyl group, for example. In some cases, the hydrophilic group can include a Ci-Ce alkyl group including a terminalhydrophilic group. In some cases, the hydrophilic group can include a Ci-6 alkyl group including a terminal hydrophilic group selected from hydroxyl, amino, carboxylic acid, carboxylate, phosphate, phosphonate, carboxylic ether, carboxylic ester, phosphate ester, amide, phosphonamide, ammonium, or salts of the foregoing.

[0098] Non-limiting examples of suitable hydrophilic monomer units include, but are not limited to: vinyl acetic acid, maleic acid, monoalkyl maleate, dialkyl maleate, maleic anhydride, fumaric acid, monoalkyl fumarate dialkyl fumarate, itaconic acid, monoalkyl itaconate, dialkyl itaconate, itaconic anhydride, citraconic acid, monoalkyl citraconate, dialkyl citraconate, citraconic anhydride, mesaconic acid, monoalkyl mesaconate, dialkyl mesaconate, glutaconic acid, monoalkyl glutaconate, dialkyl glutaconate, glutaconic anhydride, alkyl acrylates, alkyl alkacrylates, alkali metal salts thereof, esters thereof, and combinations thereof.Further Embodiments

[0099] In some embodiments, the at least one hydrophobic monomer is selected from: vinyl aromatics, vinyl esters of linear or branched C2-C36 alkane carboxylic acids, esters of acrylic acid or methacrylic acid with linear or branched C1-C36 alkanols, vinyl ethers of linear of branched Ci- C36 alkanols, C2-C36 alkene, acrylamide or N-functionalised acrylamide, or a mixture thereof.

[0100] In some embodiments, the vinyl aromatic is styrene or C1-C36 alkylstyrene. In some embodiments, the alkyl substitution of the styrene occurs at the ortho, meta, or para position(s). In some embodiments, the alkylstyrene is substituted one, two, three, four or five times. In some embodiments, the vinyl aromatic is a Ci-Cs alkylstyrene. In some embodiments, the vinyl aromatic is a methylstyrene. In some embodiments, the vinyl aromatic is styrene.

[0101] In some embodiments the alkene is an alkyl mono-alkene. In some embodiments, alkyl mono-alkene refers to any linear, branched or cyclic C2-C20 alkene that contains one double bond. In some embodiments, alkyl mono-alkene refers to any linear, branched or cyclic C2-C15 alkene that contains one double bond. In some embodiments, alkyl mono-alkene refers to any linear, branched or cyclic C2-C10 alkene that contains one double bond. In some embodiments, alkyl mono-alkene refers to any linear, branched or cyclic C2-C8 alkene that contains one double bond. In some embodiments, the alkyl mono-alkene is a compound of Formula (1):(Formula 1) wherein: R1, R2and R3are each independently H, CH3 or C2H5, andR4is H or a linear, branched or cyclic radical having from 1 to 6 carbon atoms or an aromatic radical having from 6 to 12 carbon atoms.

[0102] In some embodiments, the alkyl mono-alkene is butylene. In some embodiments, the alkyl mono-alkene is isobutylene or diisobutylene. In some embodiments, the alkyl mono-alkene is diisobutylene.

[0103] In some embodiments, the A-functionalised acrylamide is 7V-R5acrylamide, wherein R5is selected from linear, branched or cyclic C1-C36 alkyl, linear, branched or cyclic C1-C20 alkyl, linear, branched or cyclic C1-C15 alkyl, linear, branched or cyclic C1-C12 alkyl, linear, branched or cyclic C1-C10 alkyl, linear, branched or cyclic Ci-Cs alkyl, linear, branched or cyclic Ci-Ce alkyl, linear, branched or cyclic C1-C4 alkyl, linear, branched or cyclic C1-C3 alkyl, C1-C2 alkyl, Ci alkyl, linear, branched or cyclic C2-C36 alkenyl, linear, branched or cyclic C2-C20 alkenyl, linear, branched or cyclic C2-C15 alkenyl, linear, branched or cyclic C2-C12 alkenyl, linear, branched or cyclic C2-C10 alkenyl, linear, branched or cyclic C2-C8 alkenyl, linear, branched or cyclic C2-C6 alkenyl, linear, branched or cyclic C2-C4 alkenyl, linear, branched or cyclic C2-C3 alkenyl, C2 alkenyl, linear, branched or cyclic C2-C36 alkynyl, linear, branched or cyclic C2-C20 alkynyl, linear, branched or cyclic C2-C15 alkynyl, linear, branched or cyclic C2-C12 alkynyl, linear, branched or cyclic C2-C10 alkynyl, linear, branched or cyclic C2-C8 alkynyl, linear, branched or cyclic C2-C6 alkynyl, linear, branched or cyclic C2-C4 alkynyl, linear, branched or cyclic C2-C3 alkynyl, C2 alkynyl, C3-C12 heterocycloalkyl, C3-C10 heterocycloalkyl, optionally substituted with linear, branched, or cyclic Ci-Cs alkyls, linear, branched or cyclic C2-C8 alkenyls, or linear, branched or cyclic C2-C8 alkynyls.

[0104] In some embodiments, the A-functionalised acrylamide is 7V-R5acrylamide, wherein R5is selected from linear, branched or cyclic C1-C36 alkenyl, branched or cyclic C1-C4 alkyl, linear, branched or cyclic C1-C3 alkyl, C1-C2 alkyl, Ci alkyl, C3-C12 heterocycloalkyl, C3-C10 heterocycloalkyl, optionally substituted with linear, branched, or cyclic Ci-Cs alkyls, linear, branched or cyclic C2-C8 alkenyls, or linear, branched or cyclic C2-C8 alkynyls.

[0105] In some embodiments, the A-functionalised acrylamide is 7V-R5acrylamide, wherein R5is selected from linear, branched or cyclic C1-C10 alkyl or C3-C10 heterocycloalkyl, optionally substituted with linear, branched, or cyclic Ci-Cs alkyls, linear, branched or cyclic C2-C8 alkenyls, or linear, branched or cyclic C2-C8 alkynyls.

[0106] In some embodiments, the A-functionalised acrylamide is 7V-R5acrylamide, wherein R5is selected from linear, branched or cyclic C1-C10 alkyl or C3-C10 heterocycloalkyl.

[0107] In some embodiments, the at least one hydrophobic monomer is selected from styrene, alkyl mono-alkene, or 7V-alkylacrylamide.

[0108] In some embodiments, the at least one hydrophobic monomer is selected from styrene, linear, branched or cyclic C2-C8 alkene, or 7V-(R5)acrylamide, wherein R5is H, Ci-Cioalkyl, or C3- C10 heterocycloalkyl.

[0109] In some embodiments, the at least one hydrophobic monomer is selected from styrene, linear, branched or cyclic C2-C8 alkene, or 7V-(R5)acrylamide, wherein R5is Ci-Cioalkyl, or C3-C10 heterocycloalkyl.

[0110] In some embodiments, the at least one hydrophobic monomer is selected from styrene, isobutylene or diisobutylene, or 7V-(R5)acrylamide, wherein R5is Ci-Cioalkyl, or C3-C10 heterocycloalkyl. In some embodiments, the at least one hydrophobic monomer is selected from styrene, isobutylene or diisobutylene, or 7V-(R5)acrylamide, wherein R5is Ci-Cioalkyl. In some embodiments, the at least one hydrophobic monomer is selected from styrene, isobutylene or diisobutylene, or 7V-(R5)acrylamide, wherein R5is C3-C10 heterocycloalkyl.

[0111] In some embodiments, the at least one hydrophilic monomer is selected from maleic acid, maleic anhydride, acrylates, or the salt or conjugate base thereof.

[0112] In some embodiments the acrylate is acrylic acid or the salt or conjugate base thereof, or an acrylic ester. In some embodiments the acrylate is acrylic acid or the salt or conjugate base thereof.

[0113] In some embodiments, the acrylic ester is a compound of Formula (2): H2CCHC(=O)O-R5(Formula 2) wherein R5is according to any embodiment described above.

[0114] In some embodiments, the at least one hydrophobic monomer is selected from styrene, isobutylene or diisobutylene, or 7V-(R5)acrylamide, wherein R5is Ci-Cioalkyl, or C3-C10 heterocycloalkyl, and the at least one hydrophilic monomer is selected from maleic acid, maleic anhydride, acrylates, or the salt or conjugate base thereof.

[0115] In some embodiments, the at least one hydrophobic monomer is selected from styrene, isobutylene or diisobutylene, or 7V-(R5)acrylamide, wherein R5is Ci-Cioalkyl, or C3-C10 heterocycloalkyl, and the at least one hydrophilic monomer is selected from maleic acid, maleic anhydride, acrylic acid, or the salt or conjugate base thereof.

[0116] In some embodiments, the at least one hydrophobic monomer and the at least one hydrophilic monomer are selected from a combination selected from: styrene and maleic acid or the salt or conjugate base thereof, styrene and maleic anhydride, diisobutylene and maleic acid or the salt or conjugate base thereof, diisobutylene and maleic anhydride, styrene and acrylic acid or the salt or conjugate base thereof, styrene and an acrylic ester, acrylamide and acrylic acid or the salt or conjugate base thereof, acrylamide and an acrylic ester.

[0117] In some embodiments, the at least one hydrophobic monomer is styrene, and the at least one hydrophilic monomer is selected from maleic acid or the salt or conjugate base thereof, or maleic anhydride.

[0118] In some embodiments, the at least one hydrophobic monomer is styrene, and the at least one hydrophilic monomer is selected from maleic acid or the salt or conjugate base thereof.

[0119] In some embodiments, the at least one hydrophobic monomer is styrene, and the at least one hydrophilic monomer is selected from maleic acid or the salt or conjugate base thereof.Copolymer - Functionalisation / Hydrolysis

[0120] It will be apparent to persons skilled in the art that certain copolymers, including anhydride copolymers, may be hydrolysed for use according to the present method, and such hydrolysed polymers may optionally be used in the form of a salt, or the conjugate base. In some embodiments, the copolymer is in the form of an alkali salt (for example, Li+, Na+and K+), or conjugate base. In some embodiments, the at least one hydrophilic monomer from which the copolymer is derived is in the form of an alkali salt (for example, Li+, Na+and K+), or conjugate base. Partially hydrolysed copolymers may also be of use in the present method, however, in aqueous solution these are likely to hydrolyse further.

[0121] Copolymers comprising an acid, such as a carboxylic acid, may be functionalised prior to, during or after polymerisation by any method known in the art e.g., esterification with an alcohol containing hydrocarbon, or an amide-bond formation between a carboxylic acid and an amine. Pre-polymerisation functionalisation of e.g., an acrylic acid, will yield an acrylic ester, which may be used to derive a copolymer that is suitable in the present method.

[0122] More generally, copolymers may comprise an acid, or another reactive groups that may be functionalised, prior to, during or after polymerisation by any method known in the art. Functionalisation may be monovalent. Functionalisation may be multivalent, in the sense that the functionalisation forms cross-links between two or more reactive groups.

[0123] In some embodiments, less than, or less than about: 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1%, of the residues in the copolymer derived from the hydrophobic monomer and hydrophilic monomer are further functionalised. In some embodiments, none of residues in the copolymer derived from the hydrophobic monomer and hydrophilic monomer are further functionalised. By ‘further functionalised’, it is meant that, although the hydrophobic monomer and the hydrophilic monomer may inherently be considered functionalised following copolymerisation and / or hydrolysis, there has been no additional functionalisation step performed after polymerisation (e.g. no amide or ester formation). For example, in some embodiments wherein the copolymer is derived from styrene and maleic acid or the salt or conjugate base thereof, or styrene and maleic anhydride (with subsequent hydrolysis), less than, or less than about: 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1%, of the carboxylic acid residues are functionalised. In some embodiments, where the copolymer is derived from styrene and maleic acid or the salt or conjugate base thereof,or styrene and maleic anhydride (with subsequent hydrolysis), the percentage of maleic anhydride and / or maleic acid derived residues that comprise a carboxylate or carboxylic acid group is about or greater than, or greater than about: 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.5%. In some embodiments, where the copolymer is derived from styrene and maleic acid or the salt or conjugate base thereof, or styrene and maleic anhydride (with subsequent hydrolysis), the percentage of maleic anhydride and / or maleic acid derived residues that comprise a carboxylate or carboxylic acid group is about 100%.

[0124] In some embodiments, less than, or less than about: 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1%, of the residues in the copolymer derived from the hydrophobic monomer and hydrophilic monomer are crosslinked. In some embodiments, none of residues in the copolymer derived from the hydrophobic monomer and hydrophilic monomer are crosslinked.

[0125] Equally, anhydrides and / or esters may be hydrolysed after polymerisation to unmask one or more hydrophilic groups, such as a carboxylic acid.Copolymer - Synthesis and Commercial Availability

[0126] Styrene / maleic anhydride copolymers are conveniently prepared by a precipitation process, typically in an aromatic hydrocarbon solvent, for example toluene or di chlorobenzene.

[0127] Polymerisation may be initiated using free-radical initiators, for example azobisisobutyronitrile (AIBN), and the molecular weight may be controlled by the use of endcapping agents such as highly alkylated aromatic hydrocarbons, for example p-cymene. The ratio of monomers in the polymer may be controlled by variation of the feed composition, and may be determined by means known to those skilled in the art, for example by titration to determine maleic acid content of the hydrolysed polymer.

[0128] A number of suitable styrene / maleic anhydride, styrene / maleic acid, styrene / mal eimide copolymers are commercially available, e.g., from Sartomer Inc., Anatrace, Orbiscope or Cube Biotech, and include copolymers sold under the tradenames SMA2000, SMA3000, SMA4000, SMA-EA, SMA-ED, SMA-QA, SMAd-A, SMALP 300, SMALP 200, SMALP 140, SMALP 520- E, and SMALP 140-1.

[0129] Certain salts of hydrolysed styrene / maleic anhydride copolymers are available commercially, for example, SMA3000HNa is a sodium salt of hydrolysed SMA3000, SMA3000HK is a potassium salt of hydrolysed SMA3000, and SMA4000HNa is a sodium salt of hydrolysed SMA4000.

[0130] Other salt forms are also available commercially, such as the ammonium salt. A number of styrene / maleic anhydride copolymer esters are commercially available. These esters may behydrolysed for use in the present invention. Such esters include the Sartomer Inc. products known as SMA1440, SMA17352, SMA2625, SMA3840 and SMA3190.

[0131] Commercial grades of the styrene / maleic anhydride copolymers, as supplied for industrial uses, may contain monomer, end-capping agent residuals and initiator residuals (e.g., maleic anhydride, styrene, cumene and acetophenone), which residuals are generally undesirable in compositions for use in personal care, cosmetic, pharmaceutical or biomedical products. Residual impurities may be removed or reduced in quantity by means known to those skilled in the art, such techniques include but are not limited to the selective solvation of the residual components into alcohols (for example methanol, ethanol or isopropanol) or into chlorinated solvents (for example chloroform or dichloromethane).Copolymer - Molar Ratio

[0132] In some embodiments, the molar ratio of the hydrophobic monomer to the hydrophilic monomer is about, or greater than about: 1:1, 1.8:1, 2:1, 2.2:13:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1 or 50:1. In some embodiments, the molar ratio of the hydrophobic monomer to the hydrophilic monomer is less than about: 1:1, 1.8:1, 2:1, 2.2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1 or 50:1. In some embodiments, the molar ratio of the hydrophobic monomer to the hydrophilic monomer is in a range provided by any two of the previously described upper and / or lower amounts. For example, the molar ratio of the hydrophobic monomer to the hydrophilic monomer Is between about 1 : 1 and about 50:1, about 1:1 and about 10:1, or about 1.8:1 and about 2.2:1.

[0133] In some embodiments, the molar ratio of the hydrophilic monomer to the hydrophobic monomer is about, or greater than about: 1:1, 1.8:1, 2:1, 2.2:13:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1 or 50:1. In some embodiments, the molar ratio of the hydrophilic monomer to the hydrophobic monomer is less than about: 1:1, 1.8:1, 2:1, 2.2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1 or 50:1. In some embodiments, the molar ratio of the hydrophilic monomer to the hydrophobic monomer is in a range provided by any two of the previously described upper and / or lower amounts.

[0134] In some embodiments, the molar ratio of the hydrophobic monomer to the hydrophilic monomer is about 2:1. In some embodiments, the copolymer comprises a hydrophobic monomer content (in wt. %) of at least about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 98%. In some embodiments, the copolymer comprises a hydrophobic monomer content (in wt. %) of at most about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 98%. In some embodiments, the copolymer comprises a hydrophobic monomer content (in wt. %) in a range provided by any two of the previously described upper and / or lower amounts, e.g., the copolymer comprises a hydrophobic monomer content (in wt. %) between about 1 and 98, 5 and 95, 10 and 90, or 40 and 80.

[0135] In some embodiments, the molar ratio of the styrene to the maleic acid or maleic anhydride is about, or greater than about: 1:1, 1.8:1, 2:1, 2.2:13:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1 or 50:1. In some embodiments, the molar ratio of the styrene to the maleic acid or maleic anhydride is less than about: 1:1, 1.8:1, 2:1, 2.2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1 or 50:1. In some embodiments, the molar ratio of the styrene to the maleic acid or maleic anhydride is in a range provided by any two of the previously described upper and / or lower amounts, e.g., the molar ratio of the styrene to the maleic acid or maleic anhydride is between about 1:1 and about 50:1, about 1:1 and about 10:1, or about 1.8:1 and about 2.2:1. In some embodiments, the molar ratio of the styrene to the maleic acid or maleic anhydride is about 2:1. In some embodiments, the molar ratio of the styrene to the maleic acid is about, or greater than about: 1:1, 1.8:1, 2:1, 2.2:13: 1,4: 1,5: 1,6: 1,7: 1,8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1 or 50:1. In some embodiments, the molar ratio of the styrene to the maleic acid is less than about 1:1, 1.8:1, 2:1, 2.2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1 or 50:1. In some embodiments, the molar ratio of the styrene to the maleic acid is in a range provided by any two of the previously described upper and / or lower amounts, e.g., the molar ratio of the styrene to the maleic acid is between about 1 : 1 and about 50:1, about 1:1 and about 10:1, or about 1.8:1 and about 2.2:1. In some embodiments, the molar ratio of the styrene to the maleic acid is about 2:1.Copolymer - Molecular Weight

[0136] In some embodiments, the copolymer has an average molecular weight (in Da) greater than about: 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000 or 20000. In some embodiments, the copolymer has an average molecular weight (in Da) less than about: 20000, 19000, 18000, 17000, 16000, 15000, 14000, 13000, 12000, 11000, 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, or 1000. In some embodiments, the copolymer has an average molecular weight (in Da) in a range provided by any two of the previously described upper and / or lower amounts, e.g., the copolymer has an average molecular weight (in Da) between about 1000 and about 20000, about 3000 and about 10000, or about 5000 and about 12000. The molecular weight may be calculated or measured using an appropriate method known in the art.Copolymer - Amount

[0137] It will be appreciated by the person skilled in the art that the amount of copolymer contacting the lipoprotein sample for a period of time may be expressed in a variety of ways, e.g., the ratio of particles to moles of copolymer, or the ratio of the mass of copolymer per lipoprotein sample volume (viz. effective copolymer density).

[0138] In some embodiments, the ratio of particles to moles of copolymer (expressed as the ratio of IO10particles / 10 nmol of copolymer) is about, or greater than about: 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10. In some embodiments, the ratio of particles to moles of copolymer (expressed as the ratio of IO10particles / 10 nmol of copolymer) is less than about: 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1 : 10. In some embodiments, the ratio of particles to moles of copolymer (expressed as the ratio of 1010particles / 10 nmol of copolymer) is in a range provided by any two the previously described upper and / or lower amounts, e.g., the ratio of copolymer to lipoprotein sample volume is between about 10:1 and about 1:10, about 1:1 to about 1:10, or about 1:4 and about 1:6. In some embodiments, the ratio of particles to moles of copolymer (expressed as the ratio of 1010particles / 10 nmol of copolymer) is about: 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0139] In some embodiments, the ratio of copolymer to lipoprotein sample volume (in ug / uL) is about, or greater than about: 1000:1, 100:1, 50:1, 40:1, 36:1, 32:1, 28:1, 24:1, 20:1, 16:1, 12:1, 8:1, 6:1, 5:1, 4:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:40. In some embodiments, the ratio of copolymer to lipoprotein sample volume (in ug / uL) is less than about: 1000:1, 100:1, 50:1, 40:1, 36:1, 32:1, 28:1, 24:1, 20:1, 16:1, 12:1, 8:1, 6:1, 5:1, 4:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:40. In some embodiments, the ratio of copolymer to lipoprotein sample volume (in ug / uL) is in a range provided by any two the previously described upper and / or lower amounts, e.g., the ratio of copolymer to lipoprotein sample volume is between about 1000:1 and about 1:40, about 40:1 and about 1 :40, about 40: 1 and about 1:1, about 10:1 and about 1 : 1, or about 6:1 and about 4: 1. In some embodiments, the ratio of copolymer to lipoprotein sample volume (in ug / uL) is about 1000:1, 100:1, 50:1, 40:1, 36:1, 32:1, 28:1, 24:1, 20:1, 16:1, 12:1, 8:1, 6:1, 5:1, 4:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:40.Copolymer - Activation

[0140] It will be appreciated by persons skilled in the art that copolymers may exist in an inactive, latent, or protected form. Such a protected form may offer practical benefits, such as longer shelflife of the copolymer, or simplify and / or facilitate a particular synthetic methodology towards the synthesis of that copolymer. It will be understood that such protected copolymers are intended to be suitable for use in any one of the methods described herein. It will also be understood that such protected copolymers may be activated, deprotected prior to, or during, any one of the methods described herein. For example, the anhydride residues in a copolymer of maleic anhydride may be hydrolysed by any method known in the art (e.g., by contacting with water, and optional heating). Accordingly, in some embodiments, the method further comprises:(aOl) activating the copolymer.

[0141] In some embodiments step (aOl) is performed before step (a). In some embodiments step (aOl) is performed simultaneously with step (a). In some embodiments, activating the copolymer comprises hydrolysis.

[0142] Thus, in some embodiments, contacting the lipoprotein sample with the copolymer comprises either providing a copolymer of maleic anhydride, and hydrolysing the maleic anhydride to maleic acid, or providing a copolymer of an acrylic ester, and hydrolysing the acrylic ester to acrylic acid. In some embodiments, contacting the lipoprotein sample for a period of time with the copolymer comprises providing a copolymer of maleic anhydride, and hydrolysing the maleic anhydride to maleic acid. In some embodiments, contacting the lipoprotein sample for a period of time with the copolymer comprises providing a copolymer of an acrylic ester, and hydrolysing the acrylic ester to acrylic acid.Contacting - ConditionsAgitation

[0143] In some embodiments, contacting the lipoprotein sample for a period of time with a copolymer comprises agitating a composition comprising the lipoprotein sample and copolymer for at least a portion of the period of time. In some embodiments, the agitating is for more than one portion of the period of time. In some embodiments, the portions of the period time occur at regular intervals. In some embodiments, the agitating comprises stirring. The person skilled in the art will appreciate that various methods and / or apparatus will be suitable for agitation. Without intending to limit the means by which agitation may be affected, suitable apparatus may include, e.g., a rotary tube mixer, or a vortex mixer. pH

[0144] In some embodiments, the pH during one or more steps of a method as described herein is about or greater than about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. In some embodiments, the pH during one or more steps of a method as described herein, is less than about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. In some embodiments, the pH during one or more steps as described herein, is in a range provided by any two of the previously described upper and / or lower amounts, e.g., the pH during one or more steps is between about 1 and about 14, about 3 and about 11, or about 6 and about 8. In some embodiments, the pH during step (a) is greater than about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. In some embodiments, the pH during step (a) is less than about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. In some embodiments, the pH during step (a) is in a range provided by any two of the previously described upper and / or lower amounts, e.g., the pH during step (a) is between about 1 and about 14, about 3 and about 11, or about 6 and about 8. In some embodiments, the pH during step (b) is greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. In some embodiments, the pH during step (b) is less than about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13 or 14. In some embodiments, the pH during step (b) is in a range provided by any two of the previously described upper and / or lower amounts, e.g., the pH during step (b) is between 1 and 14, 3 and 11, or 6 and 8. In some embodiments, the pH during step (a) and / or step (b) is greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. In some embodiments, the pH during step (a) and / or step (b) is less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. In some embodiments, the pH during step (a) and / or step (b) is in a range provided by any two of the previously described upper and / or lower amounts, e.g., the pH during step (a) and / or step (b) is between 1 and 14, 3 and 11, or 6 and 8. In some embodiments, the pH is biological pH. As used herein, “biological pH” refers to the pH of blood, plasma or serum in the body, and which does not interfere with normal degradation of materials present therein. The person skilled in the art will appreciate that the pH of blood, plasma or serum may range from between about 7.0 and about 7.6. Typically, the normal pH of blood, plasma or serum is between about 7.35 and about 7.45. In some embodiments, the normal pH of blood, plasma or serum is between about 7.39 and about 7.41.Period of Time

[0145] In some embodiments, the period of time (in hours), is about or greater than about: 0.33, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24. In some embodiments, the period of time (in hours) is less than about: 0.33, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24. In some embodiments, the period of time is in a range provided by any of the previously described upper and / or lower amounts, e.g., the period of time (in hours) is between about 0.33 and about 24, or about 0.33 and about 2.Temperature

[0146] In some embodiments, the contacting the lipoprotein sample with the copolymer is performed at a temperature (in °C) of about: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40. In some embodiments, the contacting the lipoprotein sample with the copolymer is performed at a temperature (in °C) greater than about: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40. In some embodiments, the contacting the lipoprotein sample with the copolymer is performed at a temperature (in °C) less than about: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40. In some embodiments, the contacting the lipoprotein sample with the copolymer is performed at a temperature (in °C) in a range provided by any two of the previously described upper and / or lower amounts, e.g., the contacting the lipoprotein sample with the copolymer is performed at a temperature (in °C) between about 1 and about 40, about 1 and about 20, about 2 and about 10, orabout 3 and about 5. In some embodiments, the contacting the lipoprotein sample with the copolymer is performed at room temperature. In some embodiments, the contacting the lipoprotein sample with the copolymer is performed at ambient temperature. In some embodiments, the contacting the lipoprotein sample with the copolymer is performed at body temperature.Separation Step

[0147] The person skilled in the art will appreciate that the separation step may comprise any one or more of a variety of separation methods that are known in the art for the separation of proteinaceous and / or biological material or particles, including those separation methods separate based on molecular weight, hydrodynamic radius, hydrophobicity, density, affinity, and mixtures thereof. It will be appreciated that the use of more than one separation method in the separation step may provide improved separation efficiency and / or purity.

[0148] In some embodiments, the separation step is any method or technique suitable for separating particles from a complex mixture. In some embodiments, the separation step comprises any method suitable for separating lipoprotein from a complex mixture. In some embodiments, the separation step comprises any method suitable for separating particles less than about 10 nm, from a complex mixture. Non-limiting examples of suitable methods for separation include, but are not limited to: precipitation, acid extraction, electrophoresis, immunoadsorption, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, immunoaffinity chromatography, size exclusion chromatography, liquid chromatography (LC), high performance liquid chromatography (HPLC), fast protein liquid chromatography (FPLC), hydroxyl apatite chromatography, lectin chromatography, tangential flow filtration (TFF), dead-end filtration, differential ultracentrifugation, density gradient ultracentrifugation, microfluidics, flow field fractionation, other chromatography-based methods, acoustic trapping, and mixtures thereof. Such methods of separation are known to persons skilled in the art.

[0149] In some embodiments, the separation step comprises at least one of tangential flow filtration, size-exclusion chromatography, precipitation, differential ultracentrifugation, dead-end filtration, density gradient ultracentrifugation, microfluidics, flow field fractionation, affinitybased methods, chromatography-based methods, and / or acoustic trapping.

[0150] In a particular embodiment, the separation step comprises chromatography (e.g. HPLC or FPLC), comprising use of a monolithic column. In a particular embodiment, the separation step comprises a chromatography step (e.g. HPLC or FPLC), followed by a filtration step. In a particular embodiment, the separation step comprises chromatography (e.g. HPLC or FPLC), comprising use of a monolithic column followed by ultrafiltration. In a particular embodiment, theseparation step comprises chromatography (e.g. HPLC or FPLC), comprising use of a monolithic column followed by dead-end filtration.

[0151] In some embodiments, the separation step comprises filtration. In some embodiments, the separation step comprises ultrafiltration. In some embodiments, the separation step comprises dead-end filtration. In some embodiments, filtration, ultrafiltration or dead-end filtration comprises use of a filter with a molecular weight cut-off (in kDa) cut-off of between about 0.65 pm and about 1000 kDa. In some embodiments, the filter has a molecular weight cut off (in kDa) of about, or greater than about: 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000. In some embodiments, the filter has a molecular weight cut off of about, or greater than about 1000 kDa. In some embodiments, at least one filter has a size cut off (in pm) less than about: 10, 5, 2, 1, 0.65, 0.5, 0.4, 0.3, 0.2, or 0.1.

[0152] In some embodiments, the separation step comprises tangential flow filtration and / or sizeexclusion chromatography. In some embodiments, the separation step comprises tangential flow filtration. In some embodiments, the separation step comprises size-exclusion chromatography.

[0153] In some embodiments, the tangential flow filtration comprises passing the treated composition through one filter. In some embodiments, the tangential flow filtration comprises passing the treated composition through one or more filters. In some embodiments, the tangential flow filtration comprises passing the treated composition through a plurality of filters, for example two filters. In some embodiments, the tangential flow filtration comprises passing the treated composition through two or more filters. In some embodiments, passing the treated composition through the one or more filters or two or more filters comprise at least one filter for removing large contaminants (such as debris), and at least one filter for removing small contaminants (such as non-extracellular vesicle-associated proteins). In some embodiments, passing the treated composition through the one or more filters or two or more filters comprise at least one filter with a size cutoff of about 0.65 pm; and at least one filter with a molecular weight cut off greater than about 500 kDa. In some embodiments, at least one filter has a molecular weight cut off (in kDa) of about, or greater than about: 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000. In some embodiments, at least one filter has a size cut off (in pm) less than about: 10, 5, 2, 1, 0.65, 0.5, 0.4, 0.3, 0.2, or 0.1.Lipoprotein Sample

[0154] In some embodiments, the lipoprotein sample is any composition with a lipoprotein content. In some embodiments, the lipoprotein sample is a composition comprising lipoprotein. In some embodiments, the lipoprotein sample is a biological fluid.

[0155] In some embodiments, the lipoprotein sample comprises, substantially comprises, consists essentially of, or consists of: blood, serum plasma, lymph, interstitial fluid, cerebrospinalfluid, synovial fluid, amniotic fluid, milk, seminal fluid, bile, tears, pericardial fluid, peritoneal fluid, pleural fluid, lipoaspirate, saliva, sweat, conditioned cell culture medium, or mixtures thereof.

[0156] In some embodiments, the lipoprotein sample comprises, substantially comprises, consists essentially of, or consists of: plasma, serum, lipoaspirate, urine, or mixtures thereof. In some embodiments, the lipoprotein sample comprises, substantially comprises, consists essentially of, or consists of serum.

[0157] In some embodiments, the lipoprotein sample comprises, substantially comprises, consists essentially of, or consists of: plasma, lipoaspirate, urine, or mixtures thereof.

[0158] In some embodiments, the lipoprotein sample comprises, substantially comprises, consists essentially of, or consists of lipoaspirate. In some embodiments, the lipoprotein sample comprises, substantially comprises, consists essentially of, or consists of urine.

[0159] In some embodiments, the lipoprotein sample comprises, substantially comprises, consists essentially of, or consists of plasma. In some embodiments, the plasma is mammalian plasma. In some embodiments, the plasma is human plasma.Pre-treatment of Lipoprotein Sample

[0160] It will be appreciated by persons skilled in the art that the lipoprotein sample may be pretreated by any method or methods known to persons skilled in the art. Non-limiting examples of pre-treatment methods include, but are not limited to at least one of: centrifugation, decanting, filtration, concentration and / or dilution. Where a sample contains multiple components, centrifugation may be used to assist in separating those different components. For example, in the case of blood, centrifugation may be used to separate e.g., plasma, lipids, red blood cells, and white blood cells, so as to remove components that are unwanted from the desired final lipoprotein- reduced composition. Decanting may be used e.g., to isolate particular phases obtained via centrifugation. Filtration (non-limiting examples of which include gravity, vacuum, pressure, centrifugal, microfiltration, ultrafiltration, nanofiltration, or crossflow filtration), may be used e.g., to remove large particulate matter or debris from a sample, or to remove small particles, prior to further processing of the sample according to the rest of the method. The sample may be concentrated and / or diluted according to any technique known in the art, and may be done so e.g., in order to adjust the protein concentration and / or particle concentration of the lipoprotein sample.

[0161] In some embodiments, the method further comprises pre-treating the lipoprotein sample with centrifugation. In some embodiments, the method further comprises pre-treating the lipoprotein sample with decantation. In some embodiments, the method further comprises pretreating the lipoprotein sample with filtration. In some embodiments, the method further comprises pre-treating the lipoprotein sample with dilution.

[0162] In some embodiments, one or more of the methods described herein comprises a step of: (aOO) pre-treating the lipoprotein sample with one or more of the following: centrifugation, decantation, filtration, and / or dilution, wherein, in one or more embodiments, step (aOO) is performed before step (a).Lipoprotein content

[0163] It will be understood that the lipoprotein sample comprises lipoprotein, and that the lipoprotein-reduced composition comprises less lipoprotein than the lipoprotein sample.

[0164] It will be understood by a person skilled in the art that there are various accepted methods or techniques by which lipoprotein content and / or a reduction in lipoprotein content, may be quantified and / or inferred. Any such method or technique may be suitable for determining the lipoprotein content and / or reduction of lipoprotein content, for example, by determining the number and / or concentration of particles in the lipoprotein sample or lipoprotein-reduced composition, e.g., by nanoparticle tracking analysis (NTA). Other examples include, but are not limited to, quantifying the amount of, or change in the amount of, protein(s) which are characteristic of lipoprotein, e.g., at least one of: apolipoprotein (such as ApoA-1, ApoA-II, ApoA- V, ApoE, ApoB, ApoB-48, ApoB-100, ApoC-II, and / or ApoC-III), paraoxonase, lecithin- cholesterol acyltransferase, cholesteryl ester transfer protein. The person skilled in the art will appreciate that such proteins can be quantified via a variety of methods, e.g., enzyme-linked immunosorbent assay (ELISA) with one or more specific antibodies. Other suitable methods include, but are not limited to: ultracentrifugation, electrophoresis, gel filtration, western blot, or HPLC, all of which are known in the art.

[0165] In some embodiments, the method provides a reduction in lipoprotein content (in IO10particles per mL), of about or greater than about: 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0. In some embodiments, the method provides a reduction in lipoprotein content (in 1010particles per mL), of less than about: 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0. In some embodiments, the method provides a reduction in lipoprotein content (in 1010particles per mL, in terms of the lipoprotein sample relative to the lipoprotein-reduced sample), in a range provided by any two of the previously described upper and / or lower amounts, e.g., the method provides a reduction in lipoprotein content (in 1010particles per mL), between about 0.1 and about 10.0.

[0166] In some embodiments, the method provides a reduction in lipoprotein content of the lipoprotein sample (as a %) of about, or greater than about: 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 98. In some embodiments, the method provides a reduction in lipoprotein content of the lipoprotein sample (as a %), of less than about: 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 98. In some embodiments, the method provides a reduction in lipoprotein content of the lipoproteinsample (as a %), in a range provided by any two of the previously described upper and / or lower amounts, e.g., the method provides a reduction in lipoprotein content of the lipoprotein sample (as a %) between about 5 and about 95, about 10 and about 95, or about 20 and about 80. In some embodiments, the lipoprotein-reduced lipoprotein sample does not comprise lipoprotein, or substantially no lipoprotein.

[0167] In some embodiments, the method provides a reduction in the concentration of particles comprising a lipid monolayer (as a %), of about, or greater than about: 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 98. In some embodiments, the method provides a reduction in the concentration of particles comprising a lipid monolayer (as a %), less than about: 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 98. In some embodiments, the method provides a reduction in the concentration of particles comprising a lipid monolayer (as a %) in a range provided by any two of the previously described upper and / or lower amounts, e.g., the method provides a reduction in the concentration of particles comprising a lipid monolayer (as a %), between about: 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 98.

[0168] In some embodiments, the method provides a reduction in apolipoprotein. In some embodiments, the method provides a reduction in ApoAl, or ApoB. In some embodiments, the method provides a reduction in ApoAl and ApoB. It will be understood that this reduction may be quantified in terms of mass of apolipoprotein per particles, or as a percentage reduction.

[0169] In some embodiments, the method provides a reduction in apolipoprotein, ApoAl, or ApoB (as a %), of about, or greater than about: 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 98. In some embodiments, the method provides a reduction in apolipoprotein, ApoAl, or ApoB (as a %), less than about: 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 98.

[0170] In some embodiments, the method provides a reduction in apolipoprotein, ApoAl, or ApoB (in ug of apolipoprotein, ApoAl or ApoB per 1010particles), of about, or greater than about: 3, 2.8, 2.6, 2.4, 2.2, 2, 1.8, 1.6, 1.4, 1.2, 1.0, 0.8, 0.6, 0.4, 0.2, or 0.1. In some embodiments, the method provides a reduction in apolipoprotein, ApoAl, or ApoB (in ug of apolipoprotein, ApoAl or ApoB per 1010particles), less than about: 3, 2.8, 2.6, 2.4, 2.2, 2, 1.8, 1.6, 1.4, 1.2, 1.0, 0.8, 0.6, 0.4, 0.2, or 0.1 In some embodiments, the method provides a reduction in apolipoprotein, ApoAl, or ApoB (in ug of apolipoprotein, ApoAl or ApoB per 1010particles), in a range provided by any two of the previously described upper and / or lower amounts, e.g., provides a reduction in apolipoprotein, ApoAl, or ApoB (in ug of apolipoprotein, ApoAl or ApoB per 1010particles), between about 3 and about 0.1, about 0.8 and about 2.4, or about 1.6 and about 2.0.

[0171] In some embodiments, the method provides a reduction in lipoprotein content (in 1010particles per mL), of about, or greater than about: 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0. In some embodiments, the method providesa reduction in lipoprotein content (in IO10particles per mL), less than about: 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0. In some embodiments, the method provides a reduction in lipoprotein content (in 1010particles per mL, in terms of the lipoprotein sample relative to the lipoprotein-reduced sample), in a range provided by any two of the previously described upper and / or lower amounts, e.g., the method provides a reduction in lipoprotein content (in 1010particles per mL), between about 0.1 and about 10.0. Lipoprotein-reduced Composition

[0172] It will be understood that the method can be used to improve the purity of a lipoprotein sample that comprises extracellular vesicles, by reducing the lipoprotein-content of the lipoprotein sample. In some embodiments, the method is used to isolate and / or purify extracellular vesicles from the sample. Therefore, in some embodiments, the lipoprotein sample and / or lipoprotein- reduced composition comprise extracellular vesicles. In some embodiments, the lipoprotein- reduced composition comprises extracellular vesicles. In some embodiments, the lipoprotein- reduced composition comprises one or more extracellular vesicles. In some embodiments, the lipoprotein sample comprises extracellular vesicles. In some embodiments, the lipoprotein sample comprises one or more extracellular vesicles. In some embodiments, the lipoprotein-reduced composition comprises extracellular vesicles in a higher concentration than the lipoprotein sample.

[0173] It will be appreciated by persons skilled in the art that there are various methods for assessing extracellular vesicle purity, e.g., protein to particle ratio and the decrease thereof from the lipoprotein sample to the lipoprotein-reduced composition, or an increase in particle concentration per input volume of lipoprotein sample. Other suitable methods include, but are not limited to, assessment of any extracellular vesicle marker, including, but not limited to: CD9, CD63, CD81, LAPM1, TSG101, Alix and combinations thereof.

[0174] In some embodiments, the method provides a lipoprotein-reduced composition comprising extracellular vesicles (EV) with an EV purity that is (in basis points with respect to the purity of the lipoprotein sample), of about, or greater than about: 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98.

[0175] In some embodiments, the protein to particle ratio of the lipoprotein-reduced composition is at least about: 200, 150, 120, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 or 5 times less than that of the lipoprotein sample. In some embodiments, the protein to particle ratio of the lipoprotein- reduced composition is between about 200 and about 5, between about 150 and about 10, between about 100 and about 20, or between about 30 and about 60 times less than that of the lipoprotein sample.

[0176] In some embodiments, the method provides a lipoprotein-reduced composition with a particle concentration (in 107'13particles per mL of lipoprotein sample), of about, or greater thanabout: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the method provides a lipoprotein-reduced composition with a particle concentration (in 107'13particles per mL of lipoprotein sample), less than about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the method provides a lipoprotein-reduced composition with a particle concentration (in 107'13particles per mL of lipoprotein sample), in a range provided by any two of the previously described upper and / or lower amounts, e.g., a lipoprotein-reduced composition with a particle concentration (in 107'13particles per mL of lipoprotein sample), between about 1 and about 15, about 2 and about 14, about 3 and about 13, about 4 and about 12, about 5 and about 11, about 6 and about 10, or about 7 and about 9.

[0177] In some embodiments, the method as described herein may produce a lipoprotein-reduced composition, which provides at least one of: enhanced classification efficiency; and / or sensitivity and / or specificity to detect EV biomarkers, compared to the lipoprotein sample before contact with the copolymer. In one embodiment the lipoprotein-reduced composition provides an enhanced classification efficiency, compared to the lipoprotein sample before contact with the copolymer. In another embodiment, the lipoprotein-reduced composition provides enhanced sensitivity to detect EV biomarkers, compared to the lipoprotein sample before contact with the copolymer. In yet another embodiment, the lipoprotein-reduced composition provides enhanced specificity to detect EV biomarkers, compared to the lipoprotein sample before contact with the copolymer.

[0178] The present disclosure also provides for a lipoprotein-reduced composition obtainable according to any aspect, embodiment, or example herein. In some embodiments, the lipoprotein- reduced composition is obtained according to any aspect, embodiment, or example herein.

[0179] The present disclosure also provides for a lipoprotein-reduced composition obtainable or obtained according to any aspect, embodiment, or example herein, when used for isolating EV particles, optionally: with an increased fluorescent labelling capacity; and / or reduced macrophage interactions, compared to the lipoprotein sample before contact with the copolymer.Separation Matrix Comprising Copolymer

[0180] The present disclosure also provides for a separation matrix comprising a solid support and a copolymer derived from at least one hydrophobic monomer and at least one hydrophilic monomer. The copolymer, hydrophobic monomer, and hydrophilic monomer may be according to any aspect, embodiment or example described herein. It will be appreciated that use of a separation matrix comprising a solid support and a copolymer may be advantageous at least in that it allowsfor the mutual contacting of the copolymer and lipoprotein sample to occur simultaneously with the separation step, which may improve efficiency of the purification / i solation process.

[0181] In some embodiments, the separation matrix is a separation matrix comprising a solid support and a copolymer derived from at least one hydrophobic monomer and at least one hydrophilic monomer, wherein the at least one hydrophobic monomer is selected from styrene, alkyl monoalkene, or 7V-alkylacrylamide; and wherein the at least one hydrophilic monomer is selected from maleic acid, maleic anhydride, acrylates, or the salt or conjugate base thereof.

[0182] The at least one hydrophobic monomer and at least one hydrophilic monomer may be selected from any such hydrophobic monomer and hydrophilic monomer described according to any aspect, method or embodiment herein.

[0183] In some embodiments, the at least one hydrophobic monomer and the at least one hydrophilic monomer are selected from a combination selected from: styrene and maleic acid or the salt or conjugate base thereof, styrene and maleic anhydride, diisobutylene and maleic acid or the salt or conjugate base thereof, diisobutylene and maleic anhydride, styrene and acrylic acid or the salt or conjugate base thereof, styrene and an acrylic ester, acrylamide and acrylic acid or the salt or conjugate base thereof, acrylamide and an acrylic ester.

[0184] In some embodiments, the at least one hydrophobic monomer is styrene, and the at least one hydrophilic monomer is selected from maleic acid or the salt or conjugate base thereof, or maleic anhydride.

[0185] In some embodiments, the at least one hydrophobic monomer is styrene, and the at least one hydrophilic monomer is selected from maleic acid or the salt or conjugate base thereof.

[0186] In some embodiments, the at least one hydrophobic monomer is styrene, and the at least one hydrophilic monomer is selected from maleic acid or the salt or conjugate base thereof.

[0187] In some embodiments, the separation matrix is suitable for separation. In some embodiments, the separation matrix is suitable for chromatography. In some embodiments, the separation matrix is suitable for size-exclusion chromatography.Solid Support

[0188] In some embodiments, the solid support is selected from: chromatographic resins, membranes, porous monoliths, woven fabrics or non-woven fabrics. In some embodiments, the solid support is selected from: chromatographic resins, membranes, or porous monoliths. In some embodiments, the solid support is a non-woven fabric. In some embodiments, the solid support is: a size-exclusion chromatographic resin, ion-exchange chromatographic resin, affinity- chromatographic resin, or porous monolith. In some embodiments, the solid support is selectedfrom chromatographic resins or porous monoliths. In some embodiments, the solid support is a chromatographic resin. In some embodiments, the solid support is a membrane. In some embodiments, the solid support is a porous monolith. In some embodiments, the solid support is a woven fabric.

[0189] In some embodiments, the solid support comprises, substantially comprises, consists essentially of, or consists of at least one of: sepharose, sephadex, poly(acrylate), methacrylate, polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose, silica, or combinations thereof. In some embodiments, the solid support comprises, substantially comprises, consists essentially of, or consists of sepharose. In some embodiments, the solid support comprises, substantially comprises, consists essentially of, or consists of sephadex. In some embodiments, the solid support comprises, substantially comprises, consists essentially of, or consists of poly(acrylate). In some embodiments, the solid support comprises, substantially comprises, consists essentially of, or consists of polymethacrylate. In some embodiments, the solid support comprises, substantially comprises, consists essentially of, or consists of methacrylate. In some embodiments, the solid support comprises, substantially comprises, consists essentially of, or consists of polystyrene. In some embodiments, the solid support comprises, substantially comprises, consists essentially of, or consists of poly(acrylamide). In some embodiments, the solid support comprises, substantially comprises, consists essentially of, or consists of polyol. In some embodiments, the solid support comprises, substantially comprises, consists essentially of, or consists of agarose. In some embodiments, the solid support comprises, substantially comprises, consists essentially of, or consists of agar. In some embodiments, the solid support comprises, substantially comprises, consists essentially of, or consists of cellulose. In some embodiments, the solid support comprises, substantially comprises, consists essentially of, or consists of dextran. In some embodiments, the solid support comprises, substantially comprises, consists essentially of, or consists of starch. In some embodiments, the solid support comprises, substantially comprises, consists essentially of, or consists of heparin. In some embodiments, the solid support comprises, substantially comprises, consists essentially of, or consists of glycogen. In some embodiments, the solid support comprises, substantially comprises, consists essentially of, or consists of amylopectin. In some embodiments, the solid support comprises, substantially comprises, consists essentially of, or consists of mannan. In some embodiments, the solid support comprises, substantially comprises, consists essentially of, or consists of inulin. In some embodiments, the solid support comprises, substantially comprises, consists essentially of, or consists of nitrocellulose. In some embodiments, the solid support comprises, substantially comprises, consists essentially of, or consists of diazocellulose.In some embodiments, the solid support comprises, substantially comprises, consists essentially of, or consists of silica.

[0190] In some embodiments, the solid support comprises, substantially comprises, consists essentially of, or consists of: sepharose, sephadex, agarose, dextran, silica, poly(acrylate), or combinations thereof.

[0191] In some embodiments, the solid support comprises, substantially comprises, consists essentially of, or consists of: agarose, dextran, poly (acrylate), or combinations thereof.

[0192] In some embodiments the solid support is porous. In one or more embodiments, the pore size is important in partially determining the capacity and / or the selectivity of the solid support. In some embodiments, the solid support is in the form of porous beads or a porous membrane. In some embodiments, the solid support has an average pore size (in A), of about, or greater than about: 1, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000. In some embodiments, the solid support has an average pore size (in A), of less than about: 1, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000. In some embodiments, the solid support has an average pore size (in A), in range provided by any two of the previously described amounts, e.g., the solid support has an average pore size (in A) of about 50 to about 1000, about 50 to about 800, about 75 to about 500, or about 50 to about 200.

[0193] In some embodiments, the solid support has an average pore size (in pm), of about, or greater than about: 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50. In some embodiments, the solid support has an average pore size (in pm), of less than about: 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1 or 0.5. In some embodiments, the solid support has an average pore size (in pm), in range provided by any two of the previously described amounts, e.g., the solid support has an average pore size (in pm) of about 1 to about 50, about 1 to about 20, about 1 to about 10, or about 4 to about 8.

[0194] In some embodiments, the solid support has an average particle diameter (in microns), of about, or greater than about: 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50. In some embodiments, the solid support has an average particle diameter (in microns), less than about: 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50. In some embodiments, the solid support has an average particle diameter (in microns), in a range provided by any two of the previously described amounts, e.g., the solid support has an average particle diameter (in microns) of about 1 to about 50, or about 5 to about 30. In some embodiments, the solid support has an average particle diameter (in microns), of about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0195] In a particular example, the solid support is a porous monolith and the copolymer is derived from at least one hydrophobic monomer and at least one hydrophilic monomer, wherein the at least one hydrophobic monomer is selected from styrene, alkyl monoalkene, or 7V-alkylacrylamide; and wherein the at least one hydrophilic monomer is selected from maleic acid, maleic anhydride, acrylates, or the salt or conjugate base thereof.Copolymers

[0196] Herein, the copolymer may comprise two or more monomers. In one embodiment, the copolymer or a potion thereof is a block copolymer. In one embodiment, the copolymer or a potion thereof is an alternating copolymer. In one embodiment, the copolymer or a potion thereof is a periodic copolymer. In one embodiment, the copolymer or a potion thereof is a statistical copolymer. In one embodiment, the copolymer or a potion thereof is a stereoblock copolymer. In one embodiment, the copolymer or a potion thereof is a gradient copolymer. In one embodiment, the copolymer or a potion thereof is a graft copolymer. In one embodiment, the copolymer or a potion thereof is a star copolymer. In one embodiment, the copolymer, or a portion thereof is linear or substantially linear. In one embodiment, the copolymer, or a portion thereof is branched. In one embodiment, the copolymer, or a portion thereof is hyperbranched. In one embodiment, the copolymer, or a portion thereof is a dendrimer or dendritic. In one embodiment, the copolymer, or a portion thereof is a brush or a comb.

[0197] In some embodiments, the copolymer is disposed on at least a portion of the solid support. In some embodiments, the copolymer is disposed on the solid support with a loading (in mmol of copolymer per gram of resin), of about, or greater than about: 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 5.0, 10.0, 100.0 or 500.0. In some embodiments, the copolymer is disposed on the solid support with a loading (in ug of copolymer per gram of resin), that is about, or greater than about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 200, 500 or 1000.

[0198] In some embodiments, the molar ratio of the hydrophobic monomer to the hydrophilic monomer is about or greater than about: 1 : 1, 1.8:1, 2: 1, 2.2: 1 3: 1, 4: 1, 5: 1, 6:1, 7: 1, 8: 1, 9:1, 10: 1, 15: 1, 20: 1, 25: 1, 30: 1, 35:1, 40: 1, 45: 1 or 50: 1. In some embodiments, the molar ratio of the hydrophobic monomer to the hydrophilic monomer is less than about: 1 : 1, 1.8: 1, 2: 1, 2.2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 15:1, 20: 1, 25: 1, 30:1, 35: 1, 40: 1, 45: 1 or 50: 1. In some embodiments, the molar ratio of the hydrophobic monomer to the hydrophilic monomer is in a range provided by any two of the previously described upper and / or lower amounts, e.g., the molar ratio of the hydrophobic monomer to the hydrophilic monomer is between about 1 : 1 and about 50: 1, about 1 : 1 and about 10: 1, or about 1.8:1 and about 2.2:1. In some embodiments, the molarratio of the hydrophobic monomer to the hydrophilic monomer is about 2: 1. In some embodiments, the copolymer comprises a hydrophobic monomer content (in wt. %), of at least about: 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 98. In some embodiments, the copolymer comprises a hydrophobic monomer content (in wt. %) of at most about: 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 9%. In some embodiments, the copolymer comprises a hydrophobic monomer content (in wt. %) in a range provided by any two of the previously described upper and / or lower amounts, e.g., the copolymer comprises a hydrophobic monomer content (in wt. %) between about 1 and about 98, about 5 and about 95, about 10 and about 90, or about 40 and about 80.

[0199] In some embodiments, the molar ratio of the styrene to the maleic acid or maleic anhydride is about, or greater than about: 1:1, 1.8:1, 2:1, 2.2:13:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1 or 50:1. In some embodiments, the molar ratio of the styrene to the maleic acid or maleic anhydride is less than about: 1:1, 1.8:1, 2:1, 2.2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1 or 50:1. In some embodiments, the molar ratio of the styrene to the maleic acid or maleic anhydride is in a range provided by any two of the previously described upper and / or lower amounts, e.g., the molar ratio of the styrene to the maleic acid or maleic anhydride is between about 1:1 and about 50:1, about 1:1 and about 10:1, or about 1.8:1 and about 2.2:1. In some embodiments, the molar ratio of the styrene to the maleic acid or maleic anhydride is about 2:1. In some embodiments, the molar ratio of the styrene to the maleic acid is about, or greater than about: 1:1, 1.8:1, 2:1, 2.2:13: 1,4: 1,5: 1,6: 1,7: 1,8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1 or 50:1. In some embodiments, the molar ratio of the styrene to the maleic acid is less than about: 1:1, 1.8:1, 2:1, 2.2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1 or 50:1. In some embodiments, the molar ratio of the styrene to the maleic acid is in a range provided by any two of the previously described upper and / or lower amounts, e.g., the molar ratio of the styrene to the maleic acid is between about 1 : 1 and about 50:1, about 1:1 and about 10:1, or about 1.8:1 and about 2.2:1. In some embodiments, the molar ratio of the styrene to the maleic acid is about 2:1.Method for the Preparation of Separation Matrix

[0200] The present disclosure also provides for a method of preparing a separation matrix according to any aspect, embodiment or example provided herein.

[0201] In some embodiments, the method comprises a conjugation step between a solid support and a copolymer.

[0202] Any method of forming a covalent bond between the solid support and the copolymer or derivative or analogue thereof, can be used in accordance with the present disclosure. Each covalent bond can be formed by direct conjugation of any of these or derivatives or analogues thereof.

[0203] Without intending to limit the scope of the disclosure, covalent bonding can occur through the formation of e.g., amide, ester, ether, thioether, disulfide, imino, sulfonamide groups between e.g., acid, acid chloride, aldehyde, hydroxy, amino, alcohol, alkyl halide, sulfhydryl, or hydrazo groups. Other suitable reactions include, but are not limited to, O-alkylation (etherification); N- alkylation; C-alkylation; chiral alkylation; S-alkylation; esterification; transesterification; displacement (e.g., with cyanide, hydroxide, fluoride, thiocyanate, cyanate, iodide, sulfide, sulfite, azide, nitrite, or nitrate); other nucleophilic aliphatic and aromatic substitutions; oxidation; hydrolysis; epoxidation and chiral epoxidation; Michael addition; aldol condensation; Wittig condensation; Darzens condensation; carbene reactions; thiophosphorylation; reduction; carbonylation; transition metal co-catalysis; HCl / HBr / HOCl / EESCU reactions. The person skilled in the art will appreciate that the suitability of a reaction for forming a covalent bond will depend upon the selection of the solid support and copolymer, as well the functional groups present in any synthetic intermediate. The person skilled in the art will appreciate that a number of synthetic methods are available for the syntheses of the solid support, the copolymer and analogous or derivatives thereof, or that in many cases, such molecules may be available commercially. Suitable examples may include, but not limited to: amino acids, peptides, amino alcohols, polyethylylene glycols, alkanes, alkenes, alkynes, azide aromatic compounds, carbohydrates, carboxylic acids, esters, organophosphorus compounds, and sulfonates.

[0204] The person skilled in the art will appreciate that the overall synthetic method, and any particular reaction or step therein, can be chosen depending upon the selection of the solid support and copolymer which may include optional substitution as described herein. Non-limiting examples of suitable bond forming reactions are described in Richard C. Larock (ed), ‘Comprehensive Organic Transformations: A Guide to Functional Group Preparations,’ 4th edition, John Wiley & Sons (2018), and in Theodora E. Greene and Peter G.M. Wuts, ‘Protective Groups in Organic Synthesis,’ 2nd edition, John Wiley & Sons, Inc. New York (1991), the contents of which are incorporated herein in their entirety.

[0205] In some embodiments, the solid support is functionalised with a reactive chemical moiety. In some embodiments, the solid support is functionalised with at least one of: a carboxy imidazole, carbonyl, aldehyde, hydrazide, amine, carboxylic acid, thiol, alkyne, or Michael acceptor. In some embodiments, the copolymer is functionalised with at least one of a: carboxy imidazole, carbonyl, aldehyde, hydrazide, amine, carboxylic acid, thiol, alkyne, or Michael acceptor. In some embodiments, the conjugation step is a 1,2 or 1,4-conjugate addition. In some embodiments, the conjugation step is a 1,4-conjugate addition. In some embodiments, the conjugation step forms an amide, amine, disulfide, or hydrazone group between the solid support and the copolymer.

[0206] The person skilled in the art will appreciate that various solid supports, amenable to functionalisation (e.g. by virtue of the solid support containing activated / activable groups such as carboxyimidazole, aldehyde, hydrazides, etc.), are commercially available, e.g. CIMmic® (Sartorius). It will be appreciated that such solid supports may be modified in accordance with the manufacturer’s instructions, or other methods known to relevant persons skilled in the art.Uses of Copolymers or Separation Matrices Comprising CopolymersCopolymers

[0207] The present disclosure also provides for use of a copolymer to reduce the lipoprotein content of a lipoprotein sample to provide a lipoprotein-reduced composition, wherein the use comprises:(a) contacting the lipoprotein sample for a period of time with a copolymer derived from: at least one hydrophobic monomer; and at least one hydrophilic monomer, and wherein the molar ratio of the at least one hydrophobic monomer and the at least one hydrophilic monomer is in the range of about 1 : 1 to about 50: 1.

[0208] There is not intended to be any limitation on the means or manner by which the lipoprotein sample is contacted for a period of time with the copolymer. For example, the lipoprotein sample and copolymer are comprised in a composition of any form e.g., but without limitation: a solution, suspension, mixture, gel, or a paste. Thus, in some embodiments, the contacting the lipoprotein sample for a period of time with a copolymer provides a treated composition. In some embodiments, the copolymer is disposed on at least a portion of a solid support. In some embodiments, the copolymer is disposed on at least a portion of a solid support, and the lipoprotein sample is in the form of a solution-phase or suspended composition.

[0209] In some embodiments, the contacting the lipoprotein sample for a period of time with a copolymer provides a treated composition, and the method further comprises:(b) subjecting the treated composition to a separation step.

[0210] In some embodiments, step (a) and step (b) may occur simultaneously. In some embodiments, the contacting the lipoprotein sample for a period of time with a copolymer occurs at the same time as a separation step. That is to say, in some embodiments the use provides a lipoprotein-reduced composition, the use comprising:(a) contacting the lipoprotein sample for a period of time with a copolymer derived from: at least one hydrophobic monomer; and at least one hydrophilic monomer, wherein the molar ratio of the at least one hydrophobic monomer and the at least one hydrophilic monomer is in the range of about 1 : 1 to about 50: 1, and wherein step (a) further comprises a separation step, which occurs simultaneously with the contacting of the lipoprotein sample by the copolymer. In some embodiments, separation stepcomprises separating the lipoprotein content of the lipoprotein sample, from the remainder of lipoprotein sample.Separation Matrices

[0211] The present disclosure also provides for use of the separation matrix described according to any aspect, embodiment, or example provide herein.

[0212] In some embodiments, the use is of a separation matrix to reduce the lipoprotein content of a lipoprotein sample to provide a lipoprotein-reduced composition, wherein the use comprises:(a) contacting the lipoprotein sample for a period of time with the separation matrix.

[0213] It will be appreciated that each aspect, embodiment, and example of the present disclosure described herein is to be applied mutatis mutandis to each and every other aspect, embodiment, and example pertaining to use of a separation matrix to reduce the lipoprotein content of a lipoprotein sample, e.g., and without limitation, the separation matrix may be any separation matrix described herein, the copolymer may be any copolymer described herein, the lipoprotein may be any lipoprotein described herein, the hydrophobic monomer may be any hydrophobic monomer described herein, the hydrophilic monomer may be any hydrophilic monomer described herein, the molar ratio of the at least one hydrophobic monomer and the at least one hydrophilic monomer may be any molar ratio as described herein, the period of time may be any period of time described herein.EXAMPLE EMBODIMENTS

[0214] The present disclosure may be described by one or more of the following example embodiments.

[0215] Example Embodiment 1. A method of reducing the lipoprotein content of a lipoprotein sample to provide a lipoprotein-reduced composition, the method comprising:(a) contacting the lipoprotein sample for a period of time with a copolymer (optionally an amphiphilic copolymer) derived from: at least one hydrophobic monomer (optionally selected from styrene, alkyl mono-alkene, diisobutylene or 7V-alkylacrylamide); and at least one hydrophilic monomer (optionally selected from maleic acid, maleic anhydride, maleimide, an acrylate or the salt or conjugate base thereof), wherein the molar ratio of the at least one hydrophobic monomer and the at least one hydrophilic monomer is in the range of about 1 : 1 to about 50:1, or about 1 : 1 to about 10: 1, and optionally wherein the lipoprotein sample and the lipoprotein-reduced composition comprises one or more extracellular vesicles.

[0216] Example Embodiment 2. The method according to Example Embodiment 1, wherein the contacting the lipoprotein sample for a period of time with a copolymer provides a treated composition, and wherein the method further comprises:(b) subjecting the treated composition to a separation step.

[0217] Example Embodiment 3. The method according to Example Embodiment 1 or Example Embodiment 2, wherein the copolymer is an amphiphilic copolymer.

[0218] Example Embodiment 4. The method according to any one Example Embodiments 1 to 3, wherein at least one hydrophobic monomer is selected from styrene, alkyl mono-alkene, or N- alkylacrylamide; and wherein at least one hydrophilic monomer is selected from maleic acid, maleic anhydride, maleimide, acrylates or the salt or conjugate base thereof.

[0219] Example Embodiment 5. The method according to any one of Example Embodiments 1 to 4, wherein the at least one hydrophobic monomer and the at least one hydrophilic monomer are selected from a combination selected from: styrene and maleic acid or the salt or conjugate base thereof, styrene and maleic anhydride, diisobutylene and maleic acid or the salt or conjugate base thereof, diisobutylene and maleic anhydride, styrene and acrylic acid or the salt or conjugate base thereof, styrene and an acrylic ester, acrylamide and acrylic acid or the salt or conjugate base thereof, acrylamide and an acrylic ester.

[0220] Example Embodiment 6. The method according to any one of Example Embodiments 1 to 5, wherein at least one hydrophobic monomer and at least one hydrophilic monomer are selected from a combination selected from a combination selected from: styrene and maleic acid or the salt or conjugate base thereof, or styrene and maleic anhydride.

[0221] Example Embodiment 7. The method according to any one of Example Embodiments 1 to 6, wherein at least one hydrophobic monomer and at least one hydrophilic monomer are selected from a combination selected from: styrene and maleic acid or the salt or conjugate base thereof, or styrene and maleic anhydride, and wherein the molar ratio of the styrene to the maleic acid or the salt or conjugate base thereof, or the styrene to the maleic anhydride is in a range of about 1 : 1 and about 10: 1.

[0222] Example Embodiment 8. The method according to any one of Example Embodiments 1 to 7, wherein the molar ratio of the styrene to the maleic acid or the salt or conjugate base thereof, or the styrene to the maleic anhydride is in a range of about 1.8: 1 and about 2.2: 1, or about 2: 1.

[0223] Example Embodiment 9. The method according to any one of Example Embodiments 1 to 8, wherein the ratio of copolymer to lipoprotein sample volume (in ug / uL) is between about 10: 1 and about 1 : 10.

[0224] Example Embodiment 10. The method according to any one of Example Embodiments 1 to 9, wherein the copolymer has a molecular weight of between about 3000 Da and about 20000 Da, or between about 5000 Da and about 12000 Da.

[0225] Example Embodiment 11. The method according to any one Example Embodiments 1 to 10, wherein contacting the lipoprotein sample comprises agitating a composition comprising the lipoprotein sample and copolymer for at least a portion of the period of time.

[0226] Example Embodiment 12. The method according to Example Embodiment 11, wherein the agitating comprises stirring.

[0227] Example Embodiment 13. The method according to any one of Example Embodiments 1 to 12, wherein the pH during step (a) and / or step (b) if present is between about 6 and about 8 for at least a portion of the period of time.

[0228] Example Embodiment 14. The method according to any one of Example Embodiments 1 to 13, wherein the period of time is about 0.5 to about 24 hours.

[0229] Example Embodiment 15. The method according to any one of Example Embodiments 1 to 14, wherein contacting the lipoprotein sample with the copolymer is performed at a temperature between about 1 °C and about 20 °C, or between about 2 °C and about 10 °C, or about 4 °C.

[0230] Example Embodiment 16. The method according to any one of Example Embodiments 2 to 15, wherein the separation step comprises at least one of: tangential flow filtration, sizeexclusion chromatography, precipitation, dead-end filtration, differential ultracentrifugation, density gradient ultracentrifugation, microfluidics, flow field fractionation, affinity-based methods, chromatography-based methods, and acoustic trapping, or any method suitable for separating particles less than about 10 to about 30 nm from particles greater than about 10 to about 30 nm.

[0231] Example Embodiment 17. The method according to any one of Example Embodiments 2 to 16, wherein the separation step comprises tangential flow filtration.

[0232] Example Embodiment 18. The method according to Example Embodiment 16 or Example Embodiment 17, wherein the tangential flow filtration comprises passing the treated composition through two or more filters.

[0233] Example Embodiment 19. The method according to Example Embodiment 18, wherein the two or more filters comprise at least one filter with a molecule weight cutoff of about 0.65 uM; and at least one filter with a molecular weight cut off greater than about 500 kDa.

[0234] Example Embodiment 20. The method according to any one of Example Embodiments 2 to 16, wherein the separation step comprises size-exclusion chromatography.

[0235] Example Embodiment 21. The method according to any one of Example Embodiments 1 to 20, wherein the copolymer is disposed on at least a portion of a solid support.

[0236] Example Embodiment 22. The method according to any one of Example Embodiments 1 to 21, wherein the solid support is a size-exclusion chromatographic resin, ion-exchange chromatographic resin, affinity- chromatographic resin, or porous monolith.

[0237] Example Embodiment 23. The method according to any one of Example Embodiments 1 to 22, wherein the solid support is a size-exclusion chromatography resin.

[0238] Example Embodiment 24. The method according to any one of Example Embodiments 1 to 23, wherein the method further comprises:(aOl) activating and / or hydrolysing the copolymer, wherein step (aOl) is performed before step (a).

[0239] Example Embodiment 25. The method according to any one of Example Embodiments 1 to 24, wherein the method further comprises:(aOO) pre-treating the lipoprotein sample with one or more of the following: centrifugation, decantation, filtration, and / or dilution, wherein step (aOO) is performed before step (a).

[0240] Example Embodiment 26. The method according to any one of Example Embodiments 1 to 25, wherein contacting the lipoprotein sample with the copolymer comprises either providing a copolymer of maleic anhydride, and hydrolysing the maleic anhydride to maleic acid, or providing a copolymer of an acrylic ester, and hydrolysing the acrylic ester to acrylic acid.

[0241] Example Embodiment 27. The method according to any one of Example Embodiments 1 to 26, wherein the lipoprotein sample is a biological fluid.

[0242] Example Embodiment 28. The method according to any one of Example Embodiments 1 to 27, wherein the lipoprotein sample comprises, substantially comprises, consists essentially of, or consists of: blood, plasma, lymph, interstitial fluid, cerebrospinal fluid, synovial fluid, amniotic fluid, milk, seminal fluid, bile, tears, pericardial fluid, peritoneal fluid, pleural fluid, lipoaspirate, saliva, sweat, conditioned cell culture medium, or mixtures thereof.

[0243] Example Embodiment 29. The method according to any one of Example Embodiments 1 to 28, wherein the lipoprotein sample comprises, substantially comprises, consists essentially of, or consists of: plasma, lipoaspirate, urine or mixtures thereof.

[0244] Example Embodiment 30. The method according to any one of Example Embodiments 1 to 29, wherein the lipoprotein sample comprises, substantially comprises, consists essentially of, or consists of plasma.

[0245] Example Embodiment 31. The method according to Example Embodiment 30, wherein the plasma is human plasma.

[0246] Example Embodiment 32. The method according to any one Example Embodiments 1 to 31, wherein the lipoprotein sample and the lipoprotein-reduced composition comprises one or more extracellular vesicles, and the lipoprotein-reduced composition comprises extracellular vesicles in a higher concentration than the lipoprotein sample.

[0247] Example Embodiment 33. The method according to any one of Example Embodiments 1 to 32, when used to isolate and / or purify the extracellular vesicles from the lipoprotein sample.

[0248] Example Embodiment 34. The method according to any one of Example Embodiments 1 to 33, wherein the protein to particle ratio of the lipoprotein-reduced composition is at least about 200, 150, 120, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 or 5 times less than that of the lipoprotein sample.

[0249] Example Embodiment 35. The method according to any one of Example Embodiments 1 to 34, wherein the lipoprotein-reduced composition provides at least one of: enhanced classification efficiency; and / or sensitivity and / or specificity to detect EV biomarkers, compared to the lipoprotein sample before contact with the copolymer.

[0250] Example Embodiment 36. A lipoprotein-reduced composition obtainable or obtained by the method according to any one of Example Embodiments 1 to 35.

[0251] Example Embodiment 37. A lipoprotein-reduced composition obtainable or obtained by the method according to any one of claims 1 to 35, optionally when used for isolating EV particles, and optionally wherein the EVs comprised in the lipoprotein-reduced composition have an increased fluorescent labelling capacity and / or have reduced macrophage interactions, compared to the lipoprotein sample before contact with the copolymer.

[0252] Example Embodiment 38. Use of a copolymer to reduce the lipoprotein content of a lipoprotein sample to provide a lipoprotein-reduced composition, wherein the copolymer contacts the lipoprotein sample for a period of time, and wherein the copolymer is derived from: at least one hydrophobic monomer; and at least one hydrophilic monomer, and wherein the molar ratio of the at least one hydrophobic monomer and the at least one hydrophilic monomer is in the range of about 1 : 1 to about 50: 1.

[0253] Example Embodiment 39. The use according to Example Embodiment 38, wherein the lipoprotein-reduced composition provides at least one of: enhanced classification efficiency; and / or sensitivity and / or specificity to detect EV biomarkers, compared to the lipoprotein sample before contact with the copolymer.

[0254] Example Embodiment 40. A separation matrix comprising a solid support and a copolymer derived from a hydrophobic monomer and a hydrophilic monomer, wherein the combination of hydrophobic monomer and hydrophilic monomer is selected from styrene and maleic acid or the salt or conjugate base thereof, or styrene and maleic anhydride.

[0255] Example Embodiment 41. The separation matrix of Example Embodiment 40, wherein the molar ratio of the styrene to the maleic acid or the salt or conjugate base thereof or maleic anhydride is in a range of about 1 :1 and about 10: 1, about 1.8: 1 and about 2.2: 1, or about 2: 1.

[0256] Example Embodiment 42. The separation matrix of Example Embodiment 40 or Example Embodiment 41, wherein the solid support comprises or consists essentially of agarose, dextran, polymethacrylate, or combinations thereof.

[0257] Example Embodiment 43. The separation matrix of any one of Example Embodiments 40 to 42, wherein the resin is suitable for chromatography, optionally fast protein liquid chromatography (FPLC).

[0258] Example Embodiment 44. The separation matrix of any one of Example Embodiments 40 to 43, wherein the resin has an average pore size of between about 1 to 50 pm.

[0259] Example Embodiment 45. The separation matrix of any one of Example Embodiments 40 to 44, when used to remove at least a portion of one or more lipoproteins from a lipoprotein sample.

[0260] Example Embodiment 46. The separation matrix of any one any one of Example Embodiments 40 to 45, when used to isolate and / or purify extracellular vesicles from a lipoprotein sample.

[0261] Example Embodiment 47. Use of the separation matrix according to any one of Example Embodiments 40 to 46 to reduce the lipoprotein content of a lipoprotein sample to provide a lipoprotein-reduced composition, wherein the use comprises:(a) contacting the lipoprotein sample for a period of time with the separation matrix.

[0262] Example Embodiment 48. The method according to any one of claims 1 to 35, wherein the copolymer is an amphiphilic copolymer, optionally an amphipol.EXAMPLES

[0263] The present disclosure may be described by one or more of the following examples.Materials

[0264] 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid (HEPES), sodium chloride (NaCl), sucrose, magnesium chloride (MgCl), styrene-maleic anhydride (SMA) copolymer 2: 1 pre-hydrolyzed (Lipodisq 2: 1), Pur-A-Lyzer™ Midi Dialysis Kit, fetal bovine serum (FBS), distilled ultrapure sterile water, tris hydrochloride, iodoacetamide (IAA), ammonium bicarbonate (ABC) and sodium hydroxide (NaOH) were purchased from Merk Life Science (Darmstadt, DE). PierceTM micro-BCA protein assay kit, cell-grade phosphate buffered saline (PBS - pH 7.4), NuPAGE bis-tris gels, NuPAGE MOPS SDS Running Buffer (20X), NuPAGE Transfer Buffer (20X), SuperSignal™ West Femto kit, CD9, apolipoprotein E (ApoE) monoclonal antibodies, FluoroBrite Dulbecco’s Modified Eagle Medium (DMEM) media, Roswell Park Memorial Institute medium (RPMI), penicillin / streptomycin antibiotics, glutamine, Hoechst 33342 solution (20 mM), l,r-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil), 3,3'- dioctadecyloxacarbocyanine perchlorate (DiO), dithiothreitol (DTT), mass spectrometry-gradetrypsin protease, urea were purchased from Thermo Scientific (Whatman, MA, USA). ALG-2- interacting protein X (Alix) monoclonal antibody was purchased from Cell Signaling Technology (Danvers, Massachusetts, USA). Apolipoprotein Al (ApoAl) and Apolipoprotein (ApoB) ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). CD63 ELISA kit was purchased from System Bioscience (Palo Alto, CA, USA). Full-Range Rainbow Molecular Weight Markers were purchased from GE Healthcare Life Sciences (Chicago, Illinois, USA). D02-S500- 05-S 500 kDa and D02-E750-05-S 750 kDa cut-off membranes for TFF were purchased from Repligen (San Francisco, CA, USA).SMA preparation

[0265] SMA was hydrated (5% w / v) with HEPES 20 mM pH 7.4, NaCl 100 mM buffer. The resulting polymeric solution was sonicated by ultrasonic bath (Grant Instrument, UK) for one minute to promote complete dissolution of the polymer.EV isolation and concentration by tangential flow filtration

[0266] Human plasma from healthy donors was obtained from the Australian Red Cross Lifeblood under Human Research Ethics Approval number 2022 / HE000725. Plasma was collected with anticoagulant (citrate phosphate dextrose-adenine-; CPCD-1) and frozen at -80 °C for up to one year. To purify the plasma from large debris, a small volume (1-5 mL) was centrifuged (800 x g; 30 minutes) with an Eppendorf 5810 R (Eppendorf, Hamburg, Germany). The resulting lipid top layer was discarded, while 400 pL of centrifuged plasma was diluted with PBS (1 :18 v / v). Plasma-derived EVs were isolated by a KrosFlo Research 2i Tangential Flow Filtration System (Repligen, Waltham, Massachusetts, US). Briefly, plasma was processed twice through two different filters. The first filter, a sterile hollow fiber modified- polyethersulfone membrane with 0.65 pm cut-off pores, was used to remove large contaminants (debris), while the second filter, a sterile hollow fiber polysulfone membrane with molecular cut-off 750 kDa, was used to remove small contaminants, such as non-EV associated proteins. Filters were washed with PBS before the runs and with 5 mM NaOH and PBS between runs. Plasma treated with SMA (ratio 1 :5) was used to isolate EVs, while plasma without copolymer represented the control. The plasma input flow rate was set to 50 mL / min in order not to exceed 2000 s'1for the feed stream shear rate. The obtained EVs were concentrated to a volume of 50 mL and diafiltered six times with sucrose buffer (5% (w / v) sucrose, 50 mM Tris and 2 mM MgCl,), which has cryoprotective properties. The EV sample was concentrated up to 9.6 mL and stored in aliquots at -80 °C.EV isolation by SEC

[0267] EVs were isolated via SEC according to the following. Briefly, qEV Gen 2 (Izon) columns were washed and equilibrated with 30 mL of sucrose solution. Each sample (400 pL) wasprocessed following the manufacturer’s instructions. Fractions 7-11 (0.5 mL each fraction), enriched with EVs, were collected, and further assessed.Protein quantification, bicinchoninic acid assay (BCA)

[0268] Proteins from all samples were quantified using the Pierce method following the manufacturer’s guidelines.Enzyme linked immunosorbent assay (ELISA)

[0269] The manufacturer's guidelines were followed to determine the levels of ApoAl, ApoB, and CD63.Western blotting

[0270] Briefly, after protein quantification by the BCA assay, the samples were normalized to obtain the same protein concentration. Samples were boiled for five minutes at 90 °C, loaded in a 4-12% Bis-Tris gel in MOPS buffer (1.73 pg / well) and electrophoretically separated for 1.5-2 hours at 120 V. Sample were transferred to nitrocellulose membranes at 200 mA for 1.5 hours. Membranes were blocked at room temperature in 5% milk (w / v) dissolved in 1 x Tris-buffered saline (TBS) with 0.1% Tween (TBST). Membranes were incubated overnight with CD9, Alix, and ApoE primary antibodies (1 :500 in 1% milk in TBST w / v) at 4 °C. Membranes were then washed in TBST and incubated 1 :3000 with the secondary antibodies for two hours at room temperature. After washing the membranes, they were developed using the SuperSignal™ West Femto kit with ChemiDoc MP Imager (Bio-Rad, Hercules, California, US).Physicochemical characterisation

[0271] Nanoparticle tracking analysis (NT A) was used to determine the average nanoparticle size and particle concentration (particles / mL). Samples were diluted 1 : 10 - 1 : 1000 (v / v) in ultrapure sterile water and analysed by NanoSight NS300 (Malvern Panalytical, Malvern, Westborough, MA, USA) using NanoSight NTA 3.4 software. Each sample was subject to three capture replicates (camera level 13) and a 60-second measurement with a 40 pL / min flow rate. Isotonic conditions were maintained before and after dilution of samples for NTA analysis. Prior to assessing the effects of SMA co-incubation on particle concentration in different samples, samples were adjusted (through dilution) to have the same particle concentration, so that the SMA to volume ratio and particle to volume ratio were consistent between samples.Cryogenic transmission electron microscopy (cryo-TEM) analysis

[0272] Samples were imaged by cryo-TEM. Specimen preparation was conducted in a robotic vitrification system, Leica EM GP2 at controlled temperature and humidity of 22 °C and 95% respectively. The samples (3 pL) were placed on a carbon coated perforated formvar film supported on a 200 mesh copper TEM grid. The excess solution was automatically blotted for 3- 3.5 seconds and rapidly plunged into liquid ethane close to its freezing point (-182.8 °C). Gridswere then quickly transferred into liquid nitrogen for storage. Samples were imaged in a frozen hydrated state at -176 °C using a Jeol Cryo ARM 300 (JEM-Z300FSC) TEM, equipped with a cold field emission gun (FEG), and an in-column Omega energy filter. Zero energy loss images were acquired at an acceleration voltage of 300 kV and a filter setting of 20 eV. Images were recorded by a Gatan K3 direct detector camera under low-dose exposure conditions using SerialEM* software. Biological nanoparticles were counted in at least eight micrographs with a surface area of 126.87 pm2each.Fluorescent labelling and measuring fluorescence intensity

[0273] Fluorescent labelling was performed by incubating samples with Dil or DiO (5 pM) for two hours at 37 °C with gentle mixing after the first hour. Free dye was then removed using Pur- A-Lyzer™ Midi Dialysis Kit (3.5 kDa membranes) in a sucrose buffer (200-fold volume) for three hours on a stirrer. Fluorescent intensity was assessed using the EnSight Multimode Plate Reader (PerkinElmer, Waltham, MA, USA). The excitation and emission wavelengths were 462 nm and 588 nm, respectively, for DiO. The excitation and emission wavelengths were 528 nm and 583 nm, respectively, for Dil. The wavelengths were determined based on the highest emission wavelength.Macrophage association studies

[0274] RAW 264.7 macrophages were cultured in Roswell Park Memorial Institute medium (RPMI) supplemented with FBS (10%), penicillin / streptomycin (1%) and glutamine (1%). The cells were maintained at 37 °C in 5% CO2 and not used above passage 20. For EV interaction studies, cells were seeded in 96-well plates at a density of 10,000 cells per well and incubated for 24 hours at 37 °C in 5% CO2. The Raw 264.7 cells were then exposed to fluorescently labelled EV samples. For the quantitative interaction assay (cell uptake / association), the same number of particles from each sample was added to the cells to take into account potential concentrationdependent saturation effects (membrane binding / endocytosis) on cellular uptake / association. The cells were washed three times with PBS, and their fluorescence intensity was assessed using the EnSight Multimode Plate Reader (PerkinElmer, Waltham, MA, USA) after a four-hour incubation period with fluorescent EV samples. The emission wavelengths of 582 nm and 586 nm were used, corresponding to the highest peaks observed in the fluorescence intensity studies. The fluorescence intensity was then normalised to the fluorescence intensity of the added sample.Clinical samples

[0275] The Research Ethics Committee of Mercy Health and The University of Queensland (2021 / HE001826) approved this study. Written, informed consent was obtained from all participating women. Plasma was collected with anticoagulant (ethylenediaminetetraacetic acid ZEDTA) and frozen at -80 °C. Plasma samples were obtained from two groups of women whodelivered singleton infants at term (> 37 weeks of gestation) with normal glucose tolerance test (n = 5), and women diagnosed with gestational diabetes mellitus (n = 5). Gestational diabetes mellitus was diagnosed by testing with a three sample 75 g oral glucose tolerance test at 24-28 weeks, with cut-offs set according to recommendations of the Australasian Diabetes in Pregnancy Society and the World Health Organization. Demographic data of all participants (discovery and validation cohort) involved in this study are summarized in Table 1.Table 1. Demographic data of all participants (discovery and validation cohort).NGT1(n = 5) GDM2(n = 5) p- valueMaternal characteristics.z, 34.6 ± 5.413 38.2 ± 3.493Age (years) (30.5, 37 5) (35.5, 42)0 25„ . ,+ z, 166.2 ± 7.563 155.8 ± 2.775 „Height (cm) (160, 173) (153.5, 158.5)0 02. 9.4 ± 0.5477 9.2 ± 0.4472ApgarS (9, 10)(9.9 5)0 541- NGT, normal glucose tolerance;2- GDM, gestational diabetes mellitus;;3- OGTT, oral glucose tolerance test.Quantitative mass spectrometry

[0276] Previous studies have identified dysregulation in the levels of extracellular vesicle- associated pappalysin-1 (PAPP -A), in gestational diabetes mellitus. As a proof of concept, the levels of PAPP-A within EVs isolated both in the absence and presence of SMA using quantitative mass spectrometry. Tryptic digestion of the EV samples and sequential window acquisition of all theoretical fragment ion spectra (SWATH) mass spectrometry were performed. Equal volumes of EV samples from individually isolated clinical plasma (100 pL), were reduced, alkylated, and digested using the filter-aided sample preparation (FASP), method. To generate the spectral library required for SWATH analysis, a peptide pool was created from all the samples and injected for data-dependent acquisition (DDA) on a ZenoTOF 7600 mass spectrometer (AB SCIEX, Framingham, MA, USA). The resulting data were searched using ProteinPilot 5.0.2 (AB SCIEX) with the UniProt databases (human: UP000005640). SWATH acquisition and non-normalized peak area extraction was performed.Classification efficiency

[0277] The classification efficiency (that is, the proportion of cases correctly identified) by measuring PAPP-A in circulating EV samples isolated in the absence or presence of SMA, in patients with normal glucose tolerance or gestational diabetes mellitus was assessed by receiver operating characteristic (ROC) curve analysis and summarised as the area under the curve (AUC). The ROC curve was based on the predicted posterior probability of membership of the disease group. PAPP-A classifier for inclusion in a multivariate index assay were selected based on their individual predictive and degree of redundancy, as implemented in the WEKA data mining package. An additive logistic regression model (LogitBoost, WEKA), with leave-on-out cross- validation.Statistical analysis

[0278] Statistical analysis was performed in Prism 7.0a software (GraphPad) using the analysis of variance (ANOVA) test or the / -test.Example 1 - Effects of styrene-maleic acid (SMA) on particle concentrations in plasma and plasma-derived extracellular vesicle (EV) samples

[0279] Potential preferential interactions of SMA with lipoproteins as opposed to EVs was assessed by incubating the polymer with samples of varying levels of lipoprotein content: crude plasma (high content), TFF-processed plasma (medium content) and TFF+SEC-processed plasma (low content) (Figure 1).

[0280] Figure 1 describes the effects of styrene-maleic acid (SMA) on particle concentrations in plasma and plasma-derived extracellular vesicle (EV) samples. A greater SMA-induced reduction in particle concentration is correlated to the levels of lipoprotein contaminants in the samples. Particle concentrations obtained by nanoparticle tracking analysis (NTA) upon the exposure ofvarious samples to SMA: plasma (A), plasma processed by tangential flow filtration (TFF) (B), and plasma processed by TFF followed by size-exclusion chromatography (SEC) (C). Data represent mean ± standard deviation (SD) of three replicates. Statistics by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison post-hoc analysis. **, / ? < 0.01; ***, p < 0.001; **** p < 0.0001. ns, non-significant.

[0281] Varying amounts of SMA were added to an independent sample from each aforementioned sample category of varying lipoprotein content, and the resulting suspension was equilibrated under gentle and continuous stirring for 12 hours at 4 °C unless otherwise indicated. The particle to SMA ratios analysed were 1 : 1, 1 :2, 1 :5, 1 : 10, 1 :20 and 1 :50, along with a control (no SMA). A ratio of 1010particles / 10 nmol of SMA is arbitrarily referred to as a 1 : 1 ratio. The results are presented in Figure 1.

[0282] The exposure of crude plasma to SMA caused a two to five-fold reduction in particle concentration, with lower particle to SMA ratios (< 1 :5) displaying a more pronounced effect (Figure 1A). Specifically, the 1 :5 particle to SMA ratio demonstrated a greater reduction in the particle concentration in plasma compared to the 1 : 1 and 1 :2 ratios, and the particle concentration was not further reduced at the 1 : 10, 1 :20, and 1 :50 ratios (Figure 1A). The inability of higher concentrations of SMA to further reduce the particle concentration may indicate that the remaining particles are not susceptible to SMA-induced alterations. Similarly, SMA caused a reduction in the particle concentration of TFF-processed plasma (Figure IB), although to a lesser extent than that seen with crude plasma. When crude plasma was processed through both TFF and SEC, SMA had no significant effects on particle concentration (Figure 1C).

[0283] In conclusion, a greater SMA-induced reduction in particle concentration was correlated to the levels of lipoprotein contaminants in the samples, suggesting that SMA may display a preferential interaction towards lipoprotein, compared to EVs. A 1 :5 particle to SMA ratio was used in subsequent studies as higher SMA concentrations failed to further reduce particle concentration in lipoprotein-rich samples (crude plasma).Example 2 - Tangential flow filtration (TFF) parameters for plasma and plasma pre-treated with styrene-maleic acid 2:1 (SMA)

[0284] To assess whether the SMA-induced reduction in particle concentration in lipoprotein- enriched samples could be exploited to improve e.g., the isolation of EVs, plasma was treated with SMA prior to TFF. TFF was selected as an exemplary isolation method, as it displays highly efficient size-based separation of components and characteristics suitable for clinical-grade manufacturing. The results revealed that treatment with SMA reduced the processing time for TFF from five hours to three hours (Table 2). Additionally, during TFF the flow rate was increased, and the shear rate was decreased for pre-treated plasma (Table 2). Increased flow ratesaccompanied by decreased shear rates are likely to indicate reduced pore clogging due to a reduction in larger particles in the samples.

[0285] Table 2 - Tangential flow filtration (TFF) parameters for plasma and plasma pre-treated with styrene-maleic acid 2: 1 (SMA).Plasma + SMAPlasma (TFF) (TFF)Flow rate, filter with 650115 nm pores (mL / min)Shear rate, filter with 650— 1200 nm poresFlow rate, filter with 75090 kDa pores (mL / min)Shear rate, filter with 750— 1500- 1900 kDa poresTime (hours)Example 3 - Effects of SMA pre-treatment on contaminant levels in EV samples

[0286] To gain an improved understanding of the effects of SMA pre-treatment upon contaminant and EV markers, a series of analyses were performed (protein quantification, ELISA, and Western blotting). Plasma (without any SMA or TFF / SEC treatment), plasma processed byTFF (without SMA treatment), and plasma processed by TFF followed by SEC (without SMA treatment), were used as controls for comparison.

[0287] The protein to particle ratio is an accepted method of assessment of EV purity (see e.g., Thery, C. et al., ‘Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines’, J. Extracell Vesicles Vol. 7 (2008), Article: 1535750). Protein to particle ratio was assessed across the controls, and for plasma treated with SMA (SMA pre-treatment) and then subjected to TFF purification to isolate the EVs.

[0288] Figure 2 describes the effects of SMA pre-treatment on contaminant levels in EV samples: (A) comparison of protein contaminant levels in plasma processed by TFF, TFF combined with SEC, and SMA pre-treatment combined with TFF; (B) western blot of apolipoprotein (Apo)E normalized for protein concentration; (C, D) enzyme linked immunosorbent assays (ELISAs) for ApoAl and ApoB levels normalized by number of particles. Data represent mean ± SD of three replicates. Statistics by one-way ANOVA with Sidak’s multiple comparison post-hoc analysis. ****, p < 0.0001 compared to the TFF processed plasma. ####, p < 0.0001 compared to plasma processed by TFF and SEC. LP, lipoprotein.

[0289] Exposure to SMA prior to plasma processing by TFF resulted in a substantial reduction in protein contaminants, indicating higher EV purity (Figure 2A). The SMA-mediated reductionin protein contaminants is likely to be due to enhanced filtration performance (Table 2) due to reduced pore clogging. Notably, SMA pre-treatment with TFF was superior to TFF combined with SEC for the reduction of protein contaminants (Figure 2A).

[0290] To assess the ability of SMA pre-treatment to remove specific contaminants, the various protein components (ApoE, ApoAl, and ApoB) of lipoproteins were assessed. SMA pre-treatment followed by TFF resulted in a substantial reduction in ApoE (Figure 2B), which is a protein component of several types of lipoproteins. Plasma processing with TFF followed by SEC (without SMA pre-treatment) showed a greater reduction in ApoE compared to TFF alone, however, the magnitude of this reduction was less than the SMA pre-treated example (Figure 2B).

[0291] Enzyme linked immunosorbent assays (ELISAs) for ApoAl and ApoB levels, normalized by the number of particles, also indicated that SMA pre-treatment is superior to TFF alone in terms of reducing lipoprotein contaminants, such as ApoAl / high-density lipoprotein (HDL) (Figure 2C) and ApoB / low density lipoprotein (LDL) and very low-density lipoprotein (VLDL) (Figure 2D). There was a trend indicating improved removal of lipoprotein contaminants with SMA pretreatment and TFF, compared to TFF combined with SEC (Figures 2C and 2D). In summary, the simple addition of SMA prior to TFF outperformed the current gold standard for obtaining high purity EV samples on the basis of a combination of multiple isolation methods (e.g., TFF and SEC).Example 4 - Effects of SMA pre-treatment on EV markers

[0292] Figure 3 describes the effects of SMA pre-treatment on EV markers: comparison of size (A) and particle concentration normalized by input plasma (B); evaluation of EV markers (CD63, Alix, and CD9) by ELISA (C) or Western blot (D). The CD63 ELISA and Western blot were normalized for number of particles and by protein concentration, respectively. Data represent mean ± SD of three replicates. Statistics by one-way ANOVA with Tukey's (a, b) or Sidak’s (d) multiple comparison post-hoc analysis. ****, p < 0.0001 compared to TFF-processed plasma. ####, / ? < 0.0001 compared to plasma processed by TFF followed by SEC.

[0293] The effect of SMA pre-treatment on particle size was assessed using NT A, revealing a lack of significant size variations compared to particles isolated by TFF alone or TFF combined with SEC (Figure 3 A). On the contrary, treatment with SMA led to a substantial increase in particle concentration when normalized to the volume of input plasma (Figure 3B), an effect that is likely to be caused by an SMA-induced improvement in TFF performance (reduced filter clogging), as observed in Table 2. The SMA-induced reduction in protein content (Figure 2B) and ApoAl / ApoB levels (Figures 2D and 2E) per particle indicates that the population contributing to the increase in particle concentration is unlikely to be lipoproteins or protein aggregates. Western blotting and ELISA were performed to evaluate whether SMA pre-treatment caused an increase in EV markers.The addition of SMA to plasma prior to TFF resulted in superior levels of CD63 compared to TFF alone or TFF combined with SEC (Figure 3C). Additionally, Western blot results indicate that the use of SMA induces an increase in levels of the EV marker, CD9, compared to TFF alone and increased levels of Alix compared to TFF combined with SEC (Figure 3D).

[0294] Overall, SMA pre-treatment reduces contaminant markers (e.g., those of lipoprotein), while increasing EV-enriched markers, suggesting that the polymer shows selectivity for lipoprotein removal from samples.Example 5 - Effects of SMA pre-treatment on the morphology and abundance of biological nanoparticles

[0295] Figure 4 shows the effects of SMA pre-treatment on the morphology and abundance of biological nanoparticles. Images were obtained by cryogenic transmission electron microscopy (cryo-TEM). (A) Representative Cryo-TEM images of VLDL (left), LDL (centre) and TFF- processed plasma (right) with or without pre-treatment with SMA. Scale bar, 50 nm. Black arrows indicate LDL and VLDL, white arrows indicate EVs, and asterisks indicate the supporting film. (B) Quantification of the number of biological nanoparticles. (C) Size distribution of VLDL and LDL particles assessed by cryo-TEM. Numbers were normalized to the non-SMA samples. (D) Particle size distribution (nanoparticle tracking analysis) of an EV sample of high purity (combined TFF and SEC isolation; gold standard) exposed to SMA post-isolation. Data represent mean ± SD of at least eight images with a 126.87 pm2each. Statistics by Ltest analysis or oneway ANOVA with Sidak’s multiple comparison post-hoc analysis. *,p < 0.05; ****, p < 0.0001 compared to the non-pre-treated sample.

[0296] To gain an understanding of the mechanism by which SMA pre-treatment selectively removes lipoproteins from samples, SMA was incubated with VLDL, LDL, or plasma, and cryo- TEM was performed to assess morphological changes. VLDL and LDL are identified by a lipid monolayer enclosed by a hydrophobic lipid core. VLDL has an approximate size range of 25-200 nm with a polygonal faceted shape due to crystallisation. LDL has a smaller size of approximately 18-23 nm with a less evident spherical shape and stripes due to pseudo-random arrangement of lipids within the core. EVs are approximately 30-10,000 nm in size and are surrounded by a distinct lipid bilayer that encloses a less electrodense core. The results revealed that the characteristic morphology and size of VLDL and LDL were mostly absent following exposure to SMA (Figure 4A). However, when TFF-processed plasma was pre-treated with SMA, EVs remained intact (Figure 4A). Additionally, it is worth noting that in TFF-processed plasma without SMA pretreatment, VLDL and LDL-like structures were observed, while they were no longer present upon polymer pre-incubation (Figure 4A). Quantification of biological nanoparticles based on cryo- TEM images revealed a significant reduction in VLDL and LDL upon exposure to SMA (Figure4B). A similar trend in terms of lipoprotein reduction was observed in SMA pre-treated plasma samples, while the number of EVs remained unchanged (Figure 4B). Furthermore, the sizes of the remaining lipoproteins in the SMA treated LDL / VLDL samples were substantially smaller compared to the ones in the untreated samples (Figure 4C). It is likely that these remaining smaller structures are removed during the 750 kDa filtration step in TFF. To assess whether the EV size distribution changed upon exposure to SMA, an EV sample of high purity (combined TFF and SEC isolation; gold standard) was assessed. The results revealed that the EV size distribution profiles with and without SMA treatment were similar (Figure 4D). These results indicate that SMA causes a selective breakdown of lipoproteins, enabling enrichment of other components from the sample, e.g., EVs, through subsequent size-based isolation.Example 6 - Effects of SMA and other copolymer pre-treatment on contaminant levels

[0297] Figure 5 describes the effect of directly diluting SMA and other copolymers (powder) with plasma and incubating for a shorter pre-treatment period (one hour at 4 °C) was assessed. A 1 :5 particle to copolymer ratio was used in each case. Specifically, contaminant lipoprotein markers, ApoAl (Figure 5A) and ApoB (Figure 5B), were measured after plasma pre-treatment with copolymers (SMA, DIB MA 10, AASTY 11-45, AMPHIPOL-18, and SMALP 200) followed by SEC. Data are presented as the mean ± SD of three replicates. Statistics are based on one-way ANOVA test. ****, p < 0.0001 samples versus plasma; ####, p < 0.0001 copolymer samples versus SEC alone.Example 7

[0298] Figure 6 describes the impact of SMA pre-treatment on fluorescent particle labelling and macrophage interactions. (A) Fluorescence intensity of plasma-derived particles (TFF isolation) after Dil (left) / DiO (right) labelling and dialysis. The same number of particles were labelled in each group. Quantitative (B) particle association with macrophages (Raw 264.7) normalized based on particle concentration and input fluorescence. The scale bar represents 50 pm.

[0299] The SMA pre-treatment step combined with TFF effectively improved the purity and plasma-derived EVs. Next, the benefits of these improvements on downstream applications were assessed. Fluorescent labelling of EVs to assess cellular uptake, trafficking, and biodistribution is frequently performed in the field. The fluorescent labelling capacity of plasma EV samples isolated with and without SMA pre-treatment were compared. The results revealed 14 to 15-fold improvement in Dil and DiO fluorescent intensity / particle in the pre-treated samples (Figure 6A), which would have substantial benefits for downstream applications. One such application is the use of EVs as drug carriers, where various approaches can be used for drug encapsulation postisolation, including, sonication, electroporation, extrusion, and fusion with synthetic nanoparticles. One of the major goals in drug delivery is to avoid rapid clearance, which prevents drug deliverysystems from reaching target cells / tissues. Therefore, the extent of interactions between plasma EV samples and macrophages, which play a key role in the clearance of drug delivery systems, was assessed. The results indicate that SMA pre-treatment substantially reduces particle interactions with macrophages (Figure 6B), which is likely to be beneficial for drug delivery applications. Macrophages express various lipoprotein receptors and internalise high amounts of lipoproteins, which could also contribute to the untreated samples displaying increased interactions with these cells.Example 8

[0300] Figure 7 describes the impact of SMA pre-treatment on detection of EV-associated biomolecular correlates of disease using the accelerated SMA pre-treatment protocol (one hour at 4 °C) followed by SEC. (A) ELISAs of ApoAl and ApoB levels normalized by number of particles for samples processed by SEC. (B) Particle concentration normalized by input plasma for samples processed by SEC. Posterior predictive probability to identify a case (that is, gestational diabetes mellitus) in EV samples without (-) SMA (C) and with (+) SMA (D). (E) Biomarker analysis presented as receiver operating characteristic (ROC) with sensitivity without (blue circle) and with (red circle) SMA at 98% specificity.

[0301] Next, downstream applications based of EV-associated biomolecules as correlates of disease were assessed. TFF is an isolation technique that is ideal for mechanistic and therapeutic EV studies as large volumes of samples can be processed to obtain EVs in high yields. However, for EV biomarker studies, SEC is widely used as this method accommodate low sample volumes. To expand the potential applicability of SMA pre-treatment for biomarker studies, SEC alone was assessed as a potential strategy for low volume samples. As biomarker studies usually require analysis of multiple samples, the throughput of the method was increased by reducing the SMA and plasma incubation time from 12 hours to one hour. Notably, the one-hour SMA incubation time followed by subsequent SEC isolation resulted in a 10-fold reduction in Apo- Al (HDL) and an 11 -fold reduction in ApoB (VLDL / LDL) (Figure 7A). The addition of the SMA pre-treatment step prior to SEC did not alter the particle concentration (Figure 7B), which is in contrast to the TFF isolation method where the particle yields substantially increased (TFF; Figure 3B). These results suggest that the performance of TFF in terms of particle concentration is substantially affected by the presence of lipoproteins, most likely due to filter clogging, which reduces efficient separation of particles, while the performance (yield) of SEC may be less impacted by lipoprotein contaminants.

[0302] Next, the accelerated (one hour incubation) pre-treatment SEC protocol was applied to assess the impact of SMA pre-treatment on the detection of previously identified EV-associated molecular correlates of health versus disease. PAPP-A in circulating EVs has previously beenlinked to normal glucose tolerance in pregnancy. In this study, the effects of SMA pre-treatment on the ability to detect EV-associated PAPP-A to distinguish normal pregnancy from gestational diabetes mellitus was assessed.

[0303] The results reveal that the concentration of PAPP-A isolated with SMA pre-treatment prior to SEC improved the ability to identify pregnant women with gestational diabetes mellitus from those with normal glucose tolerance (Figure 7C to 7E). Changes in the distribution of the posterior predictive probability to identify cases in the absence (Figure 7C) compared to the presence (Figure 7D) of SMA were observed with p values of 0.4206 and 0.0754, respectively. The area under the ROC curve in the absence or in the presence of SMA was 0.56 and 0.80, respectively. The overall classification accuracy (that is, the ability of the model to correctly identify women with gestational diabetes mellitus and normal glucose tolerance) was 40% and 70% in the absence and presence of SMA, respectively. Notably, at 98% specificity (that is, the ability of the model to correctly identify the true negatives among all the negatives in the cohort), the sensitivity was significantly better (> 20%) for the PAPP-A analysis with SMA pre-treatment (Sensitivity = 62% (95% CI, 23%-92%)) compared to without (Sensitivity = 40% (95% CI, 7%- 76%)) (Figure 7E).Example 9 - Covalent attachment of SMA to chromatography columns improves extracellular vesicle isolation from biofluids

[0304] SMA was covalently attached to a chromatography column to assess to what extent lipoproteins could be removed from extracellular vesicle (EV) samples through binding to SMA. Specifically, SMA was attached to a monolithic chromatography column. It was hypothesised that biofluids, such as plasma, could be loaded onto a SMA-modified monolith column, resulting in lipoprotein binding to the column, while EVs would elute in the flow-through. By subsequently increasing the salt concentration, the interaction between SMA and lipoproteins would be disrupted, allowing lipoproteins to elute in subsequent fractions. The column can then be regenerated with a cleaning protocol to fully remove lipoproteins, enabling reuse.Materials and MethodsIsolation of human embryonic kidney 293 (HEK293) cell-derived EVs

[0305] HEK-293 cells were cultured and maintained in high-glucose Dulbecco’s modified Eagle medium (DMEM; Life Technologies) supplemented with 10% fetal bovine serum (FBS; Sigma) and 1% penicillin / streptomycin (Gemini Bioproducts) at 37 °C in a 5% CO2 atmosphere. Cells were used between passages 5 and 20. Before EV isolation, cells were subjected to a 24-hour starvation period once they reached 90% confluence and over 95% viability, as confirmed by Trypan blue exclusion. Conditioned medium was collected and centrifuged at 800 x g for 30 minutes at 4 °C (Eppendorf, Hamburg, Germany) to remove cell debris. The resulting supernatantwas then filtered twice using tangential flow filtration (TFF) KrosFlo KR2i (Repligen, Massachusetts, US) to further purify EVs. The first filtration step used a sterile hollow fiber modified-polyethersulfone membrane with 0.65 pm pores (Repligen, Massachusetts, US) to eliminate large contaminants at a flow rate of 130 mL / min. The second filtration step employed a 750 kDa cut-off hollow fiber membrane (Repligen, Massachusetts, US) to remove smaller contaminants, including non-EV-associated proteins, at a controlled flow rate of 50 mL / min to prevent shear forces exceeding 2000 s '. Prior to filtration, filters were washed with phosphate buffered saline (PBS), and they were cleaned between runs with 5 mM NaOH followed by PBS. The EVs were concentrated to 50 mL and diafiltered with a 750 kDa filter in six cycles using a cryoprotective sucrose buffer (5% w / v sucrose, 50 mM Tris, and 2 mM magnesium chloride). The final EV concentrate was reduced to 10 mL, aliquoted, and stored at -80 °C.Fluorescent labelling of HEK293 EVs and VLDL

[0306] HEK-293 cell-derived EVs and VLDL (Merck, Darmstadt, DE) were separately fluorescently labelled with a lipophilic dye. Briefly, 5 pM of l,l'-Dioctadecyl-3,3,3',3'- tetram ethylindocarbocyanine perchlorate (Dil) (Merck, Darmstadt, DE) was added to EVs or VLDL, protected from light, and incubated at +37 °C for three hours, with gentle mixing by inversion every 30 minutes. Excess unlabelled Dil was removed through dialysis using 3.5 kDa Pur-A-Lyzer (Merck, Darmstadt, DE) for 4 hours at room temperature in sucrose buffer (5% w / v sucrose, 50 mM Tris, and 2 mM magnesium chloride), with a 200- to 800-fold excess of buffer volume, under constant stirring. Labelled EVs and VLDL were stored at -80 °C and +4 °C, respectively, protected from light.Monolithic chromatography

[0307] Plasma purification was performed by monolithic chromatography using AKTA pure (Cytiva, Massachusetts, US). Human plasma from healthy donors was collected with an anticoagulant (citrate phosphate dextrose-adenine, CPCD-1) and stored at -80 °C for up to one year. To remove large debris, a small plasma volume (1-5 mL) was centrifuged at 800 x g for 30 minutes using an Eppendorf 5810 R (Eppendorf, Hamburg, Germany). The resulting lipid top layer was discarded, and 200 pL of the centrifuged plasma was diluted with ultrapure water (1 :5 v / v; Merck, Darmstadt, DE).

[0308] To trace the main plasma components, that is, EVs, lipoproteins, and proteins, separate runs were performed using spiked samples of fluorescently labelled HEK293 EVs, fluorescently labelled VLDL, and FITC-albumin (Merck, Darmstadt, DE). Control runs were performed without spiking.

[0309] Monolithic chromatography columns (CIMmic®, Sartorius - Gottingen, Germany) with a 6 pm channel size, modified through covalent diisopropylcarbodiimide (DIC) / A-hydroxysuccinimide (NHS) conjugation (according to the manufacturer’s instructions) of styrenemaleic acid (SMA), were used. Washing and equipment equilibration were performed at a flow rate of 1 mL / min using 20 mM HEPES, 100 mM NaCl, pH 7.4 buffer. Equilibration was achieved with 0.5 mL of 20 mM HEPES, 100 mMNaCl, pH 7.4, at a flow rate of 1 mL / min. Plasma samples (200 pL) were diluted 1 :5 with ultrapure water and loaded manually onto the column using a 1 mL loop at a setting of 0.05 mL / min to achieve a final volume of 1.2 mL, ensuring complete sample loading. The column was then washed with 2 mL of 20 mM HEPES, 100 mM NaCl, pH 7.4, and the flow-through was collected. Lipoprotein elution was performed in 22 fractions of 0.5 mL each, using a linear salt gradient with 20 mM HEPES, 100 mM NaCl, pH 7.4, and 20 mM HEPES, 2 M NaCl, pH 7.4, as buffers at a flow rate of 1 mL / min. The column was regenerated using a 0.1% sodium dodecyl sulfate (SDS) buffer at a flow rate of 10 mL / min.Fluorometric analysis

[0310] The fluorescence intensity of flow-through samples were immediately analysed using an EnSight plate reader (PerkinElmer, Massachusetts, US) with distinct settings for each fluorescently labelled component. For DiLlabelled HEK293 EVs, the excitation (Ex) was set to 528 nm and the emission (Em) to 596 nm, with a measurement height of 9.5 mm and 100 flashes. For DiLlabelled VLDL, Ex was set to 528 nm and Em to 568 nm, with a height of 8.0 mm and 100 flashes. For FITC-albumin, Ex was set to 490 nm and Em to 520 nm, with a height of 9.5 mm and 100 flashes.Dead-end ultrafiltration

[0311] To enhance lipoprotein removal, Vivaspin™ 500 filters with a 1000 kDa molecular weight cutoff (Sartorius, Gottingen, Germany) were tested. Briefly, flow-through obtained from plasma spiked with DiLlabelled VLDL was loaded into the Vivaspin™ 500 unit and centrifuged at 4000 x g for 30 minutes at +4°C. The permeate was immediately analysed for fluorescence intensity as previously described.Apolipoprotein Al (ApoAl) enzyme-Linked immunosorbent Assay (ELISA)

[0312] ApoAl levels were determined using an ELISA kit (R&D Systems, Minnesota, US) according to the manufacturer’s guidelines.Western blot

[0313] After protein quantification with the BCA assay (Thermo Fisher Scientific, Massachusetts, US), samples were normalized to achieve equal protein concentrations. Each sample (22.17 pg per well) was boiled for five minutes at 90°C, loaded onto a 4-12% Bis-Tris gel in NuPage™ MOPS SDS running buffer (Thermo Fisher Scientific, Massachusetts, US), and separated by electrophoresis at 120 V for 1.5-2 hours. Proteins were then transferred to a nitrocellulose membrane at 200 mA for 1.5 hours. Membranes were blocked at room temperaturein 5% milk (w / v) prepared in l x Tris-buffered saline with 0.1% Tween (TBST). They were then incubated overnight at 4°C with primary antibodies against CD63 and CD9 (1 :250 in 1% milk in TBST, w / v). After washing in TBST, membranes were incubated with secondary antibodies (1 :3000 dilution) for 2 hours at room temperature. Following additional washes, the membranes were developed using the SuperSignal West Femto kit and imaged with the ChemiDoc MP Imager (Bio-Rad, Hercules, California, US).Results and Discussion

[0314] Monolithic chromatography in combination with SMA was assessed as a method for isolation of plasma-derived EVs. Pretreatment of plasma with SMA prior to monolithic chromatography, resulted in reduced apolipoprotein (Apo)B100 levels in the EV fraction (Figure 8A). Results demonstrated an approximately 61% reduction in ApoBlOO levels in samples pretreated with SMA (Figure 8A) further supporting the effectiveness of SMA as a strategy for lipoprotein removal. Next, SMA was covalently attached to monolithic chromatography columns to assess lipoprotein removal. . To evaluate whether fluorescently-labelled lipoproteins spiked into plasma samples bind to the monolith column, fluorescence analysis was conducted on the flow- through, representing unbound components (Figure 8B). Results indicated that lipoproteins had the lowest recovery in the flow-through, suggesting substantial binding to the SMA-modified monolith column, while 100% of the proteins and approximately 72% of the EVs were recovered in the flow-through (Figure 8B). To further confirm these findings, ApoBlOO levels (the primary protein marker for VLDL) were analysed, showing a 64% reduction following monolith chromatography (Figure 8C). The ELISA results indicated a slightly higher degree of lipoprotein binding to the column compared to the fluorescence results, which could be due to the lipophilic fluorophore detaching from the lipoproteins after labelling. Plasma contains various macromolecules that may exhibit affinity for the lipophilic fluorophore used to label VLDL, potentially causing fluorophore transfer to other macromolecules in the plasma. To further decrease lipoprotein levels, we performed a simple and rapid dead-end ultrafiltration (1000 kDa), which resulted in an additional 56% reduction of lipoproteins in the flow-through (Figure 8D). By combining monolith column chromatography with dead-end filtration, approximately 80% of lipoproteins were successfully removed.

[0315] To assess whether EVs were effectively recovered in the flow-through, we analysed EV markers (Figure 8E). All flow-through samples showed enrichment in EV markers CD63 and CD9 (Figure 8E). As expected, samples spiked with HEK293 EVs exhibited higher CD63 levels, corresponding to the increased EV content (Figure 8E). These results confirm that EVs are efficiently eluted and enriched in the flow-through and display low binding to the column.

[0316] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

CLAIMS:

1. A method of reducing the lipoprotein content of a lipoprotein sample to provide a lipoprotein-reduced composition, the method comprising:(a) contacting the lipoprotein sample for a period of time with an amphiphilic copolymer derived from: at least one hydrophobic monomer selected from styrene, alkyl mono-alkene, diisobutylene or 7V-alkylacrylamide; and at least one hydrophilic monomer selected from maleic acid, maleic anhydride, maleimide, an acrylate or the salt or conjugate base thereof, wherein the molar ratio of the at least one hydrophobic monomer and the at least one hydrophilic monomer is in the range of about 1 : 1 to about 10:1, and wherein the lipoprotein sample and the lipoprotein-reduced composition comprise one or more extracellular vesicles.

2. The method according to claim 1, when used to isolate and / or purify the extracellular vesicles from the lipoprotein sample.

3. The method according to claim 1 or claim 2, wherein the contacting the lipoprotein sample for a period of time with a copolymer provides a treated composition, and wherein the method further comprises:(b) subjecting the treated composition to a separation step comprising at least one of: tangential flow filtration, size-exclusion chromatography, precipitation, dead-end filtration, differential ultracentrifugation, density gradient ultracentrifugation, microfluidics, flow field fractionation, affinity-based methods, chromatography-based methods, and acoustic trapping.

4. The method according to any one of claims 1 to 3, wherein the amphiphilic copolymer is an amphipol.

5. The method according to any one of claims 1 to 4, wherein the copolymer comprises: styrene and maleic acid or the salt or conjugate base thereof; or styrene and maleic anhydride, wherein the molar ratio of the styrene to the maleic acid or the salt or conjugate base thereof, or the styrene to the maleic anhydride is in a range of about 1.8:1 to about 2.2: 1.

6. The method according to any one of claims 1 to 5, wherein the ratio of copolymer to lipoprotein sample volume (in ug / uL) is between about 10: 1 and about 1 : 10.

7. The method according to any one of claims 1 to 6, wherein the copolymer has a molecular weight of between about 5000 Da and about 12000 Da.

8. The method according to any one of claims 1 to 7, meeting at least one of the following:the pH during step (a) and / or step (b) if present is between about 6 and about 8 for at least a portion of the period of time; and / or the period of time is about 0.5 to about 24 hours; and / or contacting the lipoprotein sample with the copolymer is performed at a temperature between about 1 °C and about 20 °C.

9. The method according to any one of claims 3 to 8, wherein the separation step comprises tangential flow filtration, wherein the tangential flow filtration comprises passing the treated composition through two or more filters, wherein the two or more filters comprise at least one filter with a molecule weight cutoff of about 0.65 uM; and at least one filter with a molecular weight cut off greater than about 500 kDa.

10. The method according to any one of claims 3 to 9, wherein: the separation step comprises size-exclusion chromatography.

11. The method according to any one of claims 3 to 8, wherein: the separation step comprises chromatography; the copolymer is disposed on at least a portion of a solid support; and the solid support is a porous monolith.

12. The method according to any one of claims 1 to 11, wherein the lipoprotein sample is a biological fluid which comprises, substantially comprises, consists essentially of, or consists of human plasma.

13. The method according to any one claims 1 to 12, wherein the lipoprotein-reduced composition comprises extracellular vesicles in a higher concentration than the lipoprotein sample.

14. A lipoprotein-reduced composition obtainable or obtained by the method according to any one of claims 1 to 13.

15. A lipoprotein-reduced composition obtainable or obtained by the method according to any one of claims 1 to 13, wherein the EVs comprised in the lipoprotein-reduced composition have an increased fluorescent labelling capacity; and / or have reduced macrophage interactions, compared to the lipoprotein sample before contact with the copolymer.

16. A separation matrix comprising a solid support and a copolymer derived from a hydrophobic monomer and a hydrophilic monomer, wherein the combination of hydrophobic monomer and hydrophilic monomer is selected from styrene and maleic acid or the salt or conjugate base thereof, or styrene and maleic anhydride, wherein: the molar ratio of the styrene to the maleic acid or the salt or conjugate base thereof or maleic anhydride is in a range of about 1 : 1 and about 10: 1, andthe solid support comprises or consists essentially of agarose, dextran, polymethacrylate, or combinations thereof.

17. The separation matrix of claim 16, wherein: the resin is suitable for fast protein liquid chromatography (FPLC); and the resin has an average pore size of between about 1 to 50 pm.

18. The separation matrix of claims 16 or claim 17, when used to: remove at least a portion of one or more lipoproteins from a lipoprotein sample; or isolate and / or purify extracellular vesicles from a lipoprotein sample.

19. Use of the separation matrix according to any one of claims 16 to 18 to reduce the lipoprotein content of a lipoprotein sample to provide a lipoprotein-reduced composition, wherein the use comprises:(a) contacting the lipoprotein sample for a period of time with the separation matrix.

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