Use of monolithic anion exchange chromatography and light scattering for quantification of extracellular vesicles

Abstract

The present disclosure relates to methods for detecting and purifying EV (e.g., exosomes) present in a sample using monolithic anion exchange chromatography and light scattering. The method allows for a more accurate and high throughput method of quantifying EV present in a sample. Some aspects of the methods provided herein further include analyzing one or more additional properties of EV.

Description

Technical Field

[0001] The present disclosure relates to methods for quantifying extracellular vesicles (e.g., exosomes) in a sample (e.g., a biological sample) using monolithic anion exchange chromatography and light scattering. The methods provided herein may further include analyzing one or more properties of the extracellular vesicles present in the sample. Background Art

[0002] Extracellular vesicles (e.g., exosomes) are being used in commercial processes, including as therapeutic agents, and are being produced in industrial quantities. Methods for accurately and rapidly measuring the presence, purity, concentration, and absolute number of extracellular vesicles in a complex matrix are still rare. Current methods for detecting, separating, and purifying biological extracellular vesicles from cell culture or other biological samples require laborious and time-consuming methods. For example, current ultracentrifugation protocols are not commercially reproducible because they produce a heterogeneous mixture of extracellular vesicles, other cellular vesicles, and macromolecular complexes, and may cause vesicle aggregation. Therefore, there is a need for novel methods for purifying and quantifying such extracellular vesicles efficiently, at low cost, and reliably. Summary of the invention

[0003] Provided herein is a method for determining the amount of extracellular vesicles (EVs) present in a sample, the method comprising: (i) contacting the sample with a monolithic anion exchange (AEX) chromatography column; and (ii) then measuring a light scattering emission signal from an eluent collected from the AEX chromatography column. Also provided herein is a method for preparing an extracellular vesicle (EV) fraction from a sample, the method comprising determining the amount of EVs present in the sample, wherein the determination comprises: (i) contacting the sample with a monolithic anion exchange (AEX) chromatography column; and (ii) then measuring a light scattering emission signal from an eluent collected from the AEX chromatography column.

[0004] The present disclosure further provides a method for reducing the amount of impurities present in a sample containing extracellular vesicles (EVs), the method comprising: (i) contacting the sample with a monolithic anion exchange (AEX) chromatography column; and (ii) then measuring a light scattering emission signal from an eluent collected from the AEX chromatography column.

[0005] In some aspects, the amount of impurities is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to a reference sample (e.g., a corresponding eluent collected after contacting the sample with a non-monolithic AEX chromatography column). In some aspects, if the purity of the EVs present in the eluent is lower than the purity of the reference value, the method further comprises subjecting the eluent to one or more purification steps. In some aspects, the one or more purification steps comprise filtration, centrifugation, chromatography, or a combination thereof.

[0006] In any of the methods provided herein (eg, provided above), in some aspects, the monolithic AEX chromatography column comprises a porous surface having a pore size of about 6 μm or greater. In some aspects, the monolithic column comprises a monolithic tertiary amine column.

[0007] In any of the methods described herein (e.g., provided above), in some aspects, the eluent is collected after contacting the AEX column with an elution buffer, wherein contacting the elution buffer occurs after (i) (i.e., contacting the sample with the AEX column). In some aspects, the elution buffer comprises tris, salt, or both. In some aspects, the elution buffer comprises about 50 mM tris, about 2,000 mM NaCl, and a pH of about 7.4. In some aspects, the elution buffer further comprises sodium azide.

[0008] In any of the methods described herein (e.g., provided above), in some aspects, the method further comprises contacting the AEX chromatographic column with a wash buffer, wherein contacting the wash buffer occurs after (i) (i.e., contacting the sample with the AEX chromatographic column) and before (ii) (e.g., measuring the light scattering emission signal from the eluent). In some aspects, the wash buffer comprises tris, salt, or both. In some aspects, the wash buffer comprises about 50 mM tris, about 200 mM NaCl, and a pH of about 7.4. In some aspects, the wash buffer further comprises sodium azide.

[0009] In any method described herein (e.g., provided above), in some aspects, an excitation wavelength of about 280nm to about 700nm is used to generate a light scattering emission signal. In some aspects, an excitation wavelength of about 400nm to about 500nm is used to generate a light scattering emission signal. In some aspects, an excitation wavelength of about 420nm to about 480nm is used to generate a light scattering emission signal. In some aspects, an excitation wavelength of about 460nm is used to generate a light scattering emission signal. In some aspects, the light scattering emission signal is measured at an emission wavelength of about 0nm to about 20nm longer or shorter than the excitation wavelength. In some aspects, the light scattering emission signal is measured at an emission wavelength of about 10nm longer or shorter than the excitation wavelength. In some aspects, the light scattering emission signal is measured at an emission wavelength of about 300nm to about 600nm. In some aspects, the light scattering emission signal is measured at an emission wavelength of about 400nm to about 500nm. In some aspects, the light scattering emission signal is measured at an emission wavelength of about 470nm. In some aspects, an excitation wavelength of about 460 nm is used to generate a light scattering emission signal and the light scattering emission signal is measured at an emission wavelength of about 470 nm. In some aspects, an excitation wavelength of about 280 nm is used to generate a light scattering emission signal and the light scattering emission signal is measured at an emission wavelength of about 350 nm.

[0010] In any of the methods described herein (e.g., provided above), in some aspects, the method further comprises analyzing one or more properties of the eluent. In some aspects, the one or more properties include average particle size, particle count, polydispersity index, light scattering intensity, cholesterol content, protein content, lipid content, payload, or a combination thereof. In some aspects, the one or more properties are measured using an immunoassay selected from AlphaLISA, dynamic light scattering (DLS), electron microscopy (e.g., transmission electron microscopy or cryo-electron microscopy), or both.

[0011] In some respects, compared with the average particle size of reference sample (for example, sample before contact with AEX chromatographic column), the average particle size of eluent is reduced. In some respects, compared with the average particle size of reference sample, the average particle size of eluent is reduced, and multiple is at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times or at least about 50 times or more. In some respects, the average particle size of eluent is between about 20nm to about 300nm. In some respects, the average particle size of eluent is about 180nm.

[0012] In some aspects, the polydispersity index of the eluent is reduced compared to the polydispersity index of a reference sample (e.g., a sample before contact with an AEX chromatographic column). In some aspects, the polydispersity index of the eluent is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to the polydispersity index of a reference sample. In some aspects, the polydispersity index of the eluent is between about 0 and about 0.5. In some aspects, the polydispersity index is about 0.13.

[0013] In any of the methods described herein (e.g., provided above), in some aspects, the method further comprises subjecting the eluent to one or more purification steps. In some aspects, subjecting the eluent to one or more purification steps occurs after (ii) measuring the light scattering emission signal from the eluent. In some aspects, the one or more purification steps comprise filtration, centrifugation, chromatography, or a combination thereof.

[0014] In any of the methods described herein (e.g., provided above), in some aspects, the sample is derived from a cell culture. In some aspects, the cell culture includes a perfusion cell culture, a fed-batch cell culture, or both. In some aspects, the cell culture includes a mammalian cell. In some aspects, the mammalian cell includes a human embryonic kidney cell, a mesenchymal stem cell, a neuronal cell, or a combination thereof.

[0015] In any of the methods described herein (e.g., provided above), in some aspects, the EV includes an exogenous bioactive molecule. In some aspects, the exogenous bioactive molecule comprises a payload, a targeting moiety, or both. In some aspects, the payload comprises a therapeutic molecule, an adjuvant, an immunomodulator, or a combination thereof.

[0016] In some aspects, the EV further comprises a scaffold portion. In some aspects, the scaffold portion comprises scaffold X, scaffold Y, or both. In some aspects, scaffold X comprises prostaglandin F2 receptor negative regulator (PTGFRN protein), basigin (BSG protein), immunoglobulin superfamily member 2 (IGSF2 protein), immunoglobulin superfamily member 3 (IGSF3 protein), immunoglobulin superfamily member 8 (IGSF8 protein), integrin beta-1 (ITGB1 protein), integrin alpha-4 (ITGA4 protein), 4F2 cell surface antigen heavy chain (SLC3A2 protein), a class of ATP transporters (ATP1A1 protein, ATP1A2 protein, AT P1A3 protein, ATP1A4 protein, ATP1B3 protein, ATP2B1 protein, ATP2B2 protein, ATP2B3 protein, ATP2B4 protein), aminopeptidase N (ANPEP; CD13), neprilysin (membrane metalloendopeptidase; MME), ectonucleotide pyrophosphatase / phosphodiesterase family member 1 (ENPP1), neuropilin-1 (NRP1), CD9, CD63, CD81, PDGFR, GPI anchor protein, lactadherin (MFGE8), LAMP2, LAMP2B, or any combination thereof. In some aspects, scaffold Y comprises myristoylated alanine-rich protein kinase C substrate (MARCKS protein); myristoylated alanine-rich protein kinase C substrate-like 1 (MARCKSL1 protein); brain acidic soluble protein 1 (BASP1 protein) or any combination thereof.

[0017] In some aspects, the exogenous bioactive molecule is attached to the EV via a scaffold portion. In some aspects, the exogenous bioactive molecule is attached to the scaffold portion via a linker. In some aspects, the linker comprises a polypeptide, a non-polypeptide portion, or both.

[0018] In any of the methods described herein (e.g., provided above), in some aspects, the EV comprises an exosome.

[0019] Also provided herein is a composition comprising EVs prepared by any of the methods described herein. Also provided herein is a method of treating a disease or condition in a subject in need thereof, the method comprising administering any of the compositions provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1A schematic diagram of the monolithic anion exchange quasi-light scattering (mAEX-qLS) quantification and fractionation method described herein is provided. HEK293 cell culture medium is clarified by centrifugation or filtration and injected into the mAEX column. EV particles are resolved from cell debris, proteins, and non-EV particles by brief stepwise salt elution. Light scattering detection is enabled on a fluorescence detector to selectively quantify EV particles and remove interfering co-eluting proteins. EV peaks are further automatically fractionated and their size, lipids, surface payload, etc. are determined.

[0021] Figure 2A and 2B Provided are chromatograms of harvested culture medium from an EV-producing HEK293 cell line. Figure 2A Shown are overlays of chromatograms (Ex280 / Em350 nm) for: (i) ultracentrifuged harvest medium; (ii) harvest medium; (iii) blank; and (iv) purified EV standard. Figure 2B An overlay of light scattering chromatograms (Ex460 / Em470 nm) for the same group is shown. Inset: EV and flanking peaks including front peak and extended tailing region.

[0022] Figure 3 The hydrodynamic radius (R) of purified EV standards measured using dynamic light scattering (DLS) is provided. n ) (“diameter”) and polydispersity (PDI).

[0023] Figure 4 An overlay of chromatograms (Ex460 / Em470 nm) of purified EV standards treated with and without proteinase K is provided.

[0024] Figure 5A and 5B The relationships between EV concentration and mAEX peak area and dynamic light scattering intensity are provided, respectively.

[0025] Figure 6 The relationship between the IL-12 protein concentration measured in the fractions collected after mAEX (x-axis) and the concentration of IL-12 expressing EV particles injected onto the mAEX column (y-axis) is provided. EV particle concentration was measured by NTA. IL-12 protein concentration was measured using AlphaLISA.

[0026] Fig. 7A and 7B Provided by OptiPrep ( Fig. 7A ) or through mAEX( Figure 7B) Comparison of IL-12 protein concentration in fractions collected after purification of seven different EV clones (clones 1-7). IL-12 protein concentration after OptiPrep purification was measured using Western blot. IL-12 protein concentration after mAEX purification was measured using alphaLISA. Figure 7B In the figure, the horizontal line indicates the limit of quantitation (LOQ).

[0027] Fig. 8A and 8B Data related to clonal selection of the HEK293SF cell line producing native EVs and IL-12-modified EVs are presented. Fig. 8A Relative EV particle counts from harvested culture media of naive HEK293SF cells are provided. Figure 8B EV particle counts and IL-12 expression levels of harvested culture media from IL-12 gene transfected HEK293 SF cell lines are provided. Harvested culture media from small-scale shake flask cultures were centrifuged and the supernatant analyzed for particle concentration by mAEX. For cell lines expressing IL12-EV, EV peaks were automatically fractionated and then subjected to AlphaLISA analysis for IL12 quantification. DETAILED DESCRIPTION

[0028] I. Definitions

[0029] Unless otherwise stated, the terms used in the claims and specification are defined as follows.

[0030] It should be noted that the term "a or an" entity refers to one or more / one or more of the entity; for example, "nucleotide sequence" should be understood to mean one or more nucleotide sequences. Thus, the terms "a or an", "one or more" and "at least one" are used interchangeably herein.

[0031] Furthermore, the term "and / or" as used herein is considered to be a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0032] It should be understood that whenever an aspect is described herein with the language "comprising," other similar aspects described with "consisting of" and / or "consisting essentially of" are also provided.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press, provide a general dictionary of many of the terms used in the present disclosure for those of skill in the art.

[0034] Units, prefixes and symbols are expressed in the form accepted by the International System of Units (SI). Numerical ranges include numbers that define the range. Unless otherwise indicated, nucleotide sequences are written from left to right in a 5' to 3' orientation. Amino acid sequences are written from left to right in an amino to carboxyl orientation. The headings provided herein are not limitations on the various aspects of the disclosure, which can be understood by reference to this specification as a whole. Therefore, the terms defined immediately below are more completely defined by reference to the entire specification.

[0035] The term "about" is used herein to mean approximately, roughly, around a certain region, or in a certain region. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the listed numerical values. In general, the term "about" can modify a numerical value above or below a specified value, for example, by a variance of 10% upward or downward (higher or lower).

[0036] As used herein, the term "light scattering" refers to the scattering and / or reflection of a light source from a focused beam of light. In some embodiments, light scattering can be detected at a single angle (e.g., 90 degrees) from the source, or it can be detected at multiple angles (e.g., in the case of multi-angle light scattering). In some embodiments, the light source is a laser. In some embodiments, the light source is at a wavelength in the ultraviolet spectrum, the visible spectrum, the infrared spectrum, or a combination thereof. In some embodiments, light scattering is elastic. In some embodiments, light scattering is inelastic. In some preferred embodiments, light scattering is Rayleigh (elastic) light scattering.

[0037] As used herein, the term "excitation wavelength" refers to the wavelength of the light source used to excite the sample. The wavelength of the light source is controlled by an appropriate filter to block or pass a specific wavelength. The excitation wavelength is inversely proportional to the radiant energy of the light source, that is, the longer the excitation wavelength, the lower the radiant energy, and the shorter the excitation wavelength, the higher the radiant energy. The terms "excitation wavelength" and "absorption wavelength (absorbance wavelength or absorption wavelength)" are used interchangeably.

[0038] As used herein, the term "emission wavelength" refers to the wavelength of the signal emitted, and then the wavelength of the signal is detected by the detector. After the nanoparticles are excited by the light source, due to light scattering, an emission signal can be emitted. In some aspects, light scattering is inelastic. In some aspects, light scattering is Rayleigh (elastic) light scattering. In some aspects, the light source can be fluorescence, and the emission wavelength is the wavelength of fluorescence. In some aspects, fluorescence is intrinsic fluorescence. The term "intrinsic fluorescence" refers to the fluorescence that is naturally generated after excitation. When molecules such as aromatic amino acids, neurotransmitters, porphyrins, and green fluorescent proteins are excited, such intrinsic fluorescence can occur. In contrast, exogenous fluorescence is emitted from synthetic dyes or modified biochemical molecules added to the sample. In some aspects, the detector for the fluorescent signal can be a UV / Vis detector. In some aspects, the detector can be a fluorescence detector. In some aspects, the detector can be a multi-wavelength detector.

[0039] As used herein, the term "separation" or "fractionation" refers to a process in which a certain amount of sample is divided into a plurality of smaller amounts (fractions), wherein the composition changes. In some aspects, the sample is a mixture. In some aspects, the mixture is a suspension. Fractions are collected based on the differences in the specific properties of each component. The different properties on which the separation process is based can be chemical or physical, including properties such as chemical reactivity, solubility, molecular size, charge and phase transition temperature (such as boiling point and freezing point). Separation or fractionation techniques can be roughly divided into mechanical separation processes and diffusion separation processes. Mechanical separation techniques can be based on particle size, density and electrical mobility or magnetic mobility. Diffusion separation includes chromatographic separation, extraction and fractionation. In some aspects, fractions are nanoparticles. The broad grouping of such methods and techniques includes appropriate fractionation, general separation, analytical separation and purification. The example of separation or fractionation can be but is not limited to fractionation, fractionation distillation, fractionation freezing, fractionation melting, isotope fractionation, solvent or clean fractionation, thermal diffusion, centrifugation, ultracentrifugation, gas diffusion, chromatography, fractionation guided by bioassay, geochemical fractionation and purification. In some aspects, separation or fractionation is performed in column chromatography by the difference in affinity between the stationary phase of the column and the mobile phase of the sample. In some aspects, column chromatography is size exclusion chromatography (SEC). SEC is a chromatographic method in which molecules in a solution are separated according to their size, and in some cases according to molecular weight. It is often applied to macromolecules or macromolecular complexes, such as proteins and polymers. Chromatographic columns can be filled with dextran polymers. Agarose Small porous beads composed of HPLC or polyacrylamide. The pore size of these beads is used to estimate the size of macromolecules. In some aspects, SEC includes one or more elution steps.

[0040] As used herein, the terms "batch release assay", "batch release testing" or "lot release assay" refer to the measurement methods described herein that can be used for batch release assays or lot release assays that comply with good manufacturing practices (GMP) for biopharmaceuticals or GMP for human and veterinary drugs. The purpose of testing and controlling batch release is to ensure that the batch has been manufactured and inspected in accordance with the principles and guidelines of GMP and that the relevant records are easily identifiable for future inspections. Release assays can also provide deviation measurements for manufacturing processes and / or analytical control methods. Deviations can be evaluated according to the quality risk management process using appropriate methods as specified in the GMP guidelines. The test kit (including the specifications and details of the laboratory methods (if applicable)) is agreed upon by the pharmaceutical manufacturer in consultation with the regulatory agency during the application for marketing authorization. The specific tests vary depending on the type of product, its mechanism of action and the manufacturing process. However, the laboratory will typically analyze the physical properties of the batch samples (for tablets, this may be their color, shape, solubility, etc.) and perform extensive testing of the active ingredients to ensure that their concentrations (and any degradation products) are within regulatory tolerances as well as the manufacturer's own tolerances (usually 2% to 5%). Finally, the samples will undergo microbiological and chemical surveillance to verify that the product does not contain harmful substances (e.g., residues from the manufacturing process). In some aspects, the physical properties of the manufactured extracellular vesicles (EVs) and nanoparticles can be measured during one or more release assays. In some aspects, the light scattering, intrinsic fluorescence, and UV absorbance of the EVs and nanoparticles in the batch at various excitation wavelengths and emission wavelengths are measured and recorded to detect and / or quantify the size, purity, and concentration of the EVs and nanoparticles in production. In some aspects, the light scattering signal is measured at an excitation wavelength of 460nm and an emission wavelength of 470nm. In some aspects, the intrinsic fluorescence is measured at an excitation wavelength of 280nm and an emission wavelength of 350nm. In some aspects, the UV absorbance is measured at a wavelength of 280nm. In some aspects, other measurements to assess the properties and concentration of the nanoparticles (e.g., extracellular vesicles) described herein can be included in the release assay.

[0041] As used herein, "nanoparticle" refers to a small physical entity having a size between 1 nm and 1,000 nm as measured by its longest axis (e.g., if spherical, its diameter is measured). Nanoparticles can be produced by cells or can be synthetic, or a combination or mixture thereof. Nanoparticles can be monodisperse, polydisperse, homogeneous or heterogeneous. Nanoparticles can be present in a complex mixture of various excipients, salts, biomaterials, synthetic materials, matrices, gels or other preparations known in the art (whether natural or synthetic). In a non-limiting example, a virus-like particle (VLP) is a nanoparticle.

[0042] As used herein, the term "extracellular vesicle" or "EV" refers to a vesicle of cell origin comprising a membrane surrounding an internal space. Extracellular vesicles include membrane-bound vesicles (e.g., exosomes or nanovesicles) that are smaller than all diameters of the cells from which they are derived. In some aspects, the diameter of the extracellular vesicle ranges from 20nm to 1000nm, and may be contained in the internal space (i.e., lumen), displayed on the outer surface of the extracellular vesicle, and / or across the membrane. Various macromolecular cargoes. In some aspects, the payload may include nucleic acids, proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. In some aspects, the extracellular carrier includes a scaffold portion. For example, but not limited to, extracellular vesicles include apoptotic bodies, cell fragments, vesicles derived from cells by direct or indirect manipulation (e.g., by continuous extrusion or treatment with an alkaline solution), vesicles, vesicle organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or late endoplasmic fusion with plasma membrane). Extracellular vesicles can be derived from living or dead organisms, explanted tissues or organs, prokaryotic or eukaryotic cells and / or cultured cells. In some aspects, extracellular vesicles are produced by cells expressing one or more transgenic products. Extracellular vesicles are a type of nanoparticle.

[0043] As used herein, the term "nanovesicle" refers to a small (diameter between 20nm and 250nm, for example, between 30nm and 150nm) vesicle of cell origin, the small vesicle of the cell origin comprises a membrane surrounding the inner space, and is produced by a cell (for example, a production cell) by direct or indirect operation, so that the nanovesicle is not produced by the production cell without operation. Suitable operations to the production cell include but are not limited to continuous extrusion, treatment with an alkaline solution, ultrasonic treatment or a combination thereof. Generally, the production of nanovesicles does not result in the destruction of the production cell. In some aspects, the nanovesicle colony is substantially free of a vesicle derived from the production cell by direct budding from the plasma membrane or the fusion of late endoplasmic bodies with the plasma membrane. In some aspects, the nanovesicle comprises a scaffold portion. Once derived from a production cell, the nanovesicle can be separated from the production cell based on its size, density, biochemical parameters or a combination thereof. Nanovesicles contain lipids or fatty acids and polypeptides, and optionally contain a payload (e.g., a therapeutic agent), a receptor (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA or DNA), a sugar (e.g., a monosaccharide, a polysaccharide or a polysaccharide) or other molecules. Once derived from production cells according to the operation, nanovesicles can be separated from production cells based on their size, density, biochemical parameters or a combination thereof. Nanovesicles are an extracellular vesicle.

[0044] As used herein, the term "exosome" refers to a small (diameter between 20nm and 300nm, for example, between 40nm and 200nm) vesicle of cell origin, which contains a membrane surrounding an internal space (ie, lumen), and in some aspects, can be produced from a cell (eg, a production cell) by direct plasma membrane budding or by fusion of late endosomes with the plasma membrane. In some aspects, the exosome comprises a scaffold portion. Typically, the production of exosomes does not result in the destruction of production cells. Exosomes contain lipids or fatty acids and polypeptides, and optionally contain a payload (eg, a therapeutic agent), a receptor (eg, a targeting moiety), a polynucleotide (eg, a nucleic acid, RNA or DNA), a sugar (eg, a monosaccharide, a polysaccharide or a polysaccharide) or other molecules. As described below, exosomes can be derived from production cells and separated from production cells based on their size, density, biochemical parameters or a combination thereof. Exosomes are an extracellular vesicle. In some aspects, EVs (eg, exosomes disclosed herein) are produced by cells expressing one or more transgenic products.

[0045] As used herein, the term "parent cell" or "producer cell" includes any cell from which an extracellular vesicle can be isolated. The term also encompasses cells that share the protein, lipid, sugar, or nucleic acid components of an extracellular vesicle. For example, a "parent cell" or "producer cell" can include a cell that serves as a source for an extracellular vesicle membrane.

[0046] As used herein, the term "synthetic nanoparticle" is a small (as measured by its longest axis between 1 nm and 1,000 nm) object or structure that is not produced by living cells. Synthetic nanoparticles can contain biomacromolecules, such as lipids, proteins, nucleic acids and / or carbohydrates, but cannot be produced by living cells. Liposomes, lipid nanoparticles, detergents, other polymeric structures, synthetic beads (e.g., polystyrene beads, quantum dots or metal beads) and DNA nm structures are all types of synthetic nanoparticles. In certain embodiments, synthetic nanoparticles are spherical or nearly spherical.

[0047] As used herein, the terms "purify", "purified" and "purifying" or "isolate", "isolated" or "isolating" or "enrich", "enriching" or "enriching" are used interchangeably and refer to the state (e.g., multiple known or unknown amounts and / or concentrations) of a desired extracellular vesicle population that has been subjected to one or more purification processes (e.g., selection or enrichment of a desired extracellular vesicle composition, or alternatively removal or reduction of residual biological products as described herein). In some embodiments, the purified extracellular vesicle composition has no detectable undesirable activity, or alternatively, the level or amount of undesirable activity is at or below an acceptable level or amount. In other embodiments, the purified extracellular vesicle composition has an amount and / or concentration of desired extracellular vesicles that is at or above an acceptable amount and / or concentration. In other embodiments, the purified extracellular vesicle composition is enriched compared to the starting material from which the composition is obtained (e.g., biological material collected from tissues, body fluids, or cell preparations). Compared to the starting material, this enrichment can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or greater than 99.9999%. As used herein, the term "purity" refers to the amount and / or concentration of the desired extracellular vesicles or nanoparticles relative to the total amount of the sample. As used herein, the term "contaminant" or "impurity" refers to a fraction other than the desired extracellular vesicles or nanoparticles in a sample (e.g., protein species, cell debris, ruptured extracellular vesicles or nanoparticles, microvesicles, apoptotic debris, or other impurities).

[0048] Abbreviations used in this application include the following: size exclusion chromatography (SEC), anion exchange chromatography (AEX), two-dimensional liquid chromatography (2D-LC), nanoparticle tracking analysis (NTA), resistive pulse sensing (RPS), extracellular vesicles (EV), phosphate-buffered saline (PBS), and fluorescence-activated cell sorting (FACS).

[0049] II. Methods of the Disclosure

[0050] Described herein are methods for detecting and quantifying extracellular vesicles from a complex matrix, such as a sample. In some aspects, the present disclosure provides a method for determining the amount of extracellular vesicles (EVs) present in a sample, the method comprising: (i) contacting the sample with a monolithic anion exchange (AEX) chromatographic column; and (ii) then measuring a light scattering emission signal from an eluent collected from the AEX chromatographic column. In some aspects, the present disclosure provides a method for preparing an extracellular vesicle (EV) fraction from a sample, the method comprising determining the amount of EVs present in the sample, wherein the determination comprises: (i) contacting the sample with a monolithic anion exchange (AEX) chromatographic column; and (ii) then measuring a light scattering emission signal from an eluent collected from the AEX chromatographic column. In some aspects, the present disclosure provides a method for reducing the amount of impurities present in a sample containing extracellular vesicles (EVs), the method comprising: (i) contacting the sample with a monolithic anion exchange (AEX) chromatographic column; and (ii) then measuring a light scattering emission signal from an eluent collected from the AEX chromatographic column.

[0051] In some aspects, the amount of impurities is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to a reference sample (e.g., a corresponding eluent collected after contacting the sample with a non-monolithic AEX chromatography column). In some aspects, if the purity of the EVs present in the eluent is lower than the purity of the reference value, the method further comprises subjecting the eluent to one or more purification steps.

[0052] In some aspects, the methods of the present disclosure further comprise one or more purification steps comprising filtration, centrifugation, chromatography, or a combination thereof.

[0053] In some aspects, the monolithic AEX chromatography column comprises a porous surface having a pore size of at least about 4 μm, at least about 5 μm, at least about 6 μm, at least about 7 μm, or at least about 8 μm. In some aspects, the mAEX column comprises a porous surface having a pore size of about 4 μm to about 12 μm, about 4 μm to about 11 μm, about 4 μm to about 10 μm, about 4 μm to about 9 μm, about 4 μm to about 8 μm, about 4 μm to about 7 μm, about 4 μm to about 6 μm, about 4 μm to about 5 μm, about 5 μm to about 12 μm, about 5 μm to about 11 μm, about 5 μm to about 10 μm, about 5 μm to about 9 μm, about 5 μm to about 8 μm, about A porous surface having a pore size of 5 μm to about 7 μm, about 5 μm to about 6 μm, about 6 μm to about 11 μm, about 6 μm to about 10 μm, about 6 μm to about 9 μm, about 6 μm to about 8 μm, about 6 μm to about 7 μm, about 7 μm to about 11 μm, about 7 μm to about 10 μm, about 7 μm to about 9 μm, about 7 μm to about 8 μm, about 8 μm to about 11 μm, about 8 μm to about 10 μm, or about 8 μm to about 9 μm.

[0054] In some aspects, the monolithic column comprises a monolithic tertiary amine column.

[0055] In some aspects, the elution buffer comprises tris, salt, or both. In some aspects, the elution buffer comprises about 50 mM tris, about 2,000 mM NaCl, and a pH of about 7.4. In some aspects, the elution buffer further comprises sodium azide.

[0056] In some aspects, the method further comprises contacting the AEX chromatographic column with a wash buffer, wherein contacting with the wash buffer occurs after (i) (i.e., contacting the sample with the AEX chromatographic column) and before (ii) (i.e., measuring the light scattering emission signal from the eluent). In some aspects, the wash buffer comprises tris, salt, or both. In some aspects, the wash buffer comprises about 50 mM tris, about 200 mM NaCl, and a pH of about 7.4. In some aspects, the wash buffer further comprises sodium azide.

[0057] In some aspects, an excitation wavelength of about 280 nm to about 700 nm is used to generate a light scattering emission signal. In some aspects, the light source has a wavelength in the range of about 300 nm to about 700 nm, about 320 nm to about 700 nm, about 340 nm to about 700 nm, about 360 nm to about 700 nm, about 380 nm to about 700 nm, about 400 nm to about 700 nm, about 420 nm to about 700 nm, about 440 nm to about 700 nm, about 460 nm to about 700 nm, about 300 nm to about 660 nm, about 320 nm to about 660 nm, about 340 nm to about 660 nm, about 360 nm to about 660 nm, about 380 nm to about 660 nm, about 400 nm to about 660 nm, about 420 nm to about 660 nm, about 440 nm to about 700 nm, about 460 nm to about 700 nm, In some aspects, the light source has an excitation wavelength ranging from about 400nm to about 500nm.

[0058] In some aspects, the light scattering emission signal has an emission wavelength equal to or longer than the excitation wavelength. In some aspects, the emission wavelength is less than about 20 nm, less than about 19 nm, less than about 18 nm, less than about 17 nm, less than about 16 nm, less than about 15 nm, less than about 14 nm, less than about 13 nm, less than about 12 nm, less than about 11 nm, less than about 10 nm, less than about 9 nm, less than about 8 nm, less than about 7 nm, less than about 6 nm, less than about 5 nm, less than about 4 nm, less than about 3 nm, less than about 2 nm, or less than about 1 nm longer than the excitation wavelength.

[0059] In some aspects, the emission wavelength is about 1 nm to about 20 nm, about 1 nm to about 19 nm, about 1 nm to about 18 nm, about 1 nm to about 17 nm, about 1 nm to about 16 nm, about 1 nm to about 15 nm, about 1 nm to about 14 nm, about 1 nm to about 13 nm, about 1 nm to about 12 nm, about 1 nm to about 11 nm, about 1 nm to about 10 nm, about 1 nm to about 9 nm, about 2 nm to about 20 nm, about 2 nm to about 19 nm, about 2 nm to about 18 nm, about 2 nm to about 17 nm, about 2 nm to about 16 nm, about 2 ...2 nm to about 14 nm, about 2 nm to about 13 nm, about 2 nm to about 12 nm, about 2 nm to about m to about 11 nm, about 2 nm to about 10 nm, about 2 nm to about 9 nm, about 3 nm to about 20 nm, about 3 nm to about 19 nm, about 3 nm to about 18 nm, about 3 nm to about 17 nm, about 3 nm to about 16 nm, about 3 nm to about 15 nm, about 3 nm to about 14 nm, about 3 nm to about 13 nm, about 3 nm to about 12 nm, about 3 nm to about 11 nm, about 3 nm to about 10 nm, about 3 nm to about 9 nm, about 4 nm to about 20 nm, about 4 nm to about 19 nm, about 4 nm to about 18 nm, about 4 nm to about 17 nm, about 4 nm to about 16 nm, about 4 nm to about 15 nm, about 4 nm to about 14 nm, about 4 nm to about 13 nm, about 4 nm to about 12 nm, nm to about 12nm, about 4nm to about 11nm, about 4nm to about 10nm, about 4nm to about 9nm, about 5nm to about 20nm, about 5nm to about 19nm, about 5nm to about 18nm, about 5nm to about 17nm, about 5nm to about 16nm, about 5nm to about 15nm, about 5nm to about 14nm, about 5nm to about 13nm, about 5nm to about 12nm, about 5nm to about 11nm, about 5nm to about 10nm, about 5nm to about 9nm, about 6nm to about 20nm, about 6nm to about 19nm, about 6nm to about 18nm, about 6nm to about 17nm, about 6nm to about 16nm, about 6nm to about 15nm, about 6nm to about 14nm, about 6nm to about 13nm, about 6nm to about 12nm, about 6nm to about 11nm, about 6nm to about 10nm, about 6nm to about 9nm, about 7nm to about 20nm, about 7nm to about 19nm, about 7nm to about 18nm, about 7nm to about 17nm, about 7nm to about 16nm, about 7nm to about 15nm, about 7nm to about 14nm, about 7nm to about 13nm, about 7nm to about 12nm, about 7nm to about 11nm, about 7nm to about 10nm, about 7nm to about 9nm, about 8nm to about 20nm, about 8nm to about 19nm, about 8nm to about 18nm, about 8nm to about 17nm, about 8nm to about 16nm, about 8nm to about 15nm,From about 8 nm to about 14 nm, from about 8 nm to about 13 nm, from about 8 nm to about 12 nm, from about 8 nm to about 11 nm, from about 8 nm to about 10 nm, from about 8 nm to about 9 nm, from about 9 nm to about 20 nm, from about 9 nm to about 19 nm, from about 9 nm to about 18 nm, from about 9 nm to about 17 nm, from about 9 nm to about 16 nm, from about 9 nm to about 15 nm, from about 9 nm to about 14 nm, from about 9 nm to about 13 nm, from about 9 nm to about 12 nm, from about 9 nm to about 11 nm, from about 9 nm to about 10 nm, from about 10 nm to about 20 nm, from about 10 nm to about 19 nm, from about 10 nm to about 18 nm, from about 10 nm to about 17 nm, from about 10 nm to about 16 nm, from about 10 nm to about 15 nm, from about 10 nm to about 14 nm, from about 10 nm to about 13 nm, from about 10 nm to about 12 nm, or from about 10 nm to about 11 nm. In some aspects, the difference between the emission wavelength and the excitation wavelength ranges from about 5 nm to about 20 nm. In some aspects, the difference between the emission wavelength and the excitation wavelength ranges from about 10 nm to about 20 nm.

[0060] In some aspects, the difference between the emission wavelength and the excitation wavelength is selected from the group consisting of 1 nm, 1.5 nm, 2 nm, 2.5 nm, 3 nm, 3.5 nm, 4 nm, 4.5 nm, 5 nm, 5.5 nm, 6 nm, 6.5 nm, 7 nm, 7.5 nm, 8 nm, 8.5 nm, 9 nm, 9.5 nm, 10 nm, 10.5 nm, 11 nm, 11.5 nm, 12 nm, 12.5 nm, 13 nm, 13.5 nm, 14 nm, 14.5 nm, 15 nm, 15.5 nm, 16 nm, 16.5 nm, 17 nm, 17.5 nm, 18 nm, 18.5 nm, 19 nm, 19.5 nm, and 20 nm.

[0061] In some aspects, the light source has a wavelength at about 300 nm, about 310 nm, about 320 nm, about 330 nm, about 340 nm, about 350 nm, about 360 nm, about 370 nm, about 380 nm, about 390 nm, about 400 nm, about 410 nm, about 420 nm, about 430 nm, about 440 nm, about 450 nm, about 460 nm, about 470 nm, about 480 nm, about 490 nm, An excitation wavelength of about 500 nm, about 510 nm, about 520 nm, about 530 nm, about 540 nm, about 550 nm, about 560 nm, about 570 nm, about 580 nm, about 590 nm, about 600 nm, about 610 nm, about 620 nm, about 630 nm, about 640 nm, about 650 nm, about 660 nm, about 670 nm, about 680 nm, about 690 nm, or about 700 nm. In some aspects, the emission wavelength is between 300 nm and about 320 nm, between 310 nm and about 330 nm, between 320 nm and about 340 nm, between 330 nm and about 350 nm, between 340 nm and about 360 nm, between 350 nm and about 370 nm, between 360 nm and about 380 nm, between 370 nm and about 390 nm, between 380 nm and about 400 nm, between 390 nm and about 410 nm, respectively. 0nm, between 400nm and about 420nm, between 410nm and about 430nm, between 420nm and about 440nm, between 430nm and about 450nm, between 440nm and about 460nm, between 450nm and about 470nm, between 460nm and about 480nm, between 470nm and about 490nm, between 480nm and about 500nm, between 490nm and about 510nm, between 5 between 500 nm and about 520 nm, between 510 nm and about 530 nm, between 520 nm and about 540 nm, between 530 nm and about 550 nm, between 540 nm and about 560 nm, between 550 nm and about 570 nm, between 560 nm and about 580 nm, between 570 nm and about 590 nm, between 580 nm and about 600 nm, between 590 nm and about 610 nm, between 600 nm and about 6 700 nm and about 720 nm, between 600 nm and about 610 nm, between 610 nm and about 630 nm, between 620 nm and about 640 nm, between 630 nm and about 650 nm, between 640 nm and about 660 nm, between 650 nm and about 670 nm, between 660 nm and about 680 nm, between 670 nm and about 690 nm, between 680 nm and about 700 nm, between 690 nm and about 710 nm, or between 700 nm and about 720 nm.

[0062] In some aspects, the light source has an excitation wavelength of about 450nm to about 470nm. In some aspects, the excitation wavelength is about 460nm, and the emission wavelength is about 460nm to about 480nm. In some aspects, the excitation wavelength is about 460nm, and the emission wavelength is about 460nm, about 465nm, about 470nm, about 475nm or about 480nm. In some aspects, the excitation wavelength is about 450nm, and the emission wavelength is about 450nm to about 470nm. In some aspects, the excitation wavelength is about 450nm, and the emission wavelength is about 450nm, about 455nm, about 460nm, about 465nm or about 470nm. In some aspects, the excitation wavelength is about 470nm, and the emission wavelength is about 470nm to about 490nm. In some aspects, the excitation wavelength is about 470 nm and the emission wavelength is about 470 nm, about 475 nm, about 480 nm, about 485 nm, or about 490 nm.

[0063] In some aspects, the excitation wavelength is about 550nm to about 560nm, and the emission wavelength is about 550nm to about 580nm, and wherein the excitation wavelength is identical to or shorter than the emission wavelength. In some aspects, the excitation wavelength is about 556nm, and the emission wavelength is about 556nm, about 560nm, about 565nm, about 570nm or about 573nm. In some aspects, the light scattering emission signal is measured by a UV / Vis detector or a fluorescence detector.

[0064] In some aspects, an excitation wavelength of about 460 nm is used to generate a light scattering emission signal and the light scattering emission signal is measured at an emission wavelength of about 470 nm. In some aspects, an excitation wavelength of about 280 nm is used to generate a light scattering emission signal and the light scattering emission signal is measured at an emission wavelength of about 350 nm.

[0065] In some aspects, the method of the present disclosure further includes analyzing one or more properties of the eluent. In some aspects, the one or more properties include average particle size, particle count, polydispersity index, light scattering intensity, cholesterol content, protein content, lipid content, effective load or a combination thereof. In some aspects, the one or more properties are measured using an immunoassay selected from AlphaLISA, dynamic light scattering (DLS), electron microscopy (e.g., transmission electron microscopy or cryo-electron microscopy) or both.

[0066] In some respects, compared with the average particle size of reference sample (for example, sample before contact with AEX chromatographic column), the average particle size of eluent is reduced. In some respects, compared with the average particle size of reference sample, the average particle size of eluent is reduced, and multiple is at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times or at least about 50 times or more. In some respects, the average particle size of eluent is between about 20nm to about 300nm. In some respects, the average particle size of eluent is about 180nm.

[0067] In some aspects, the polydispersity index of the eluent is reduced compared to the polydispersity index of a reference sample (e.g., a sample before contact with an AEX chromatographic column). In some aspects, the polydispersity index of the eluent is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to the polydispersity index of a reference sample. In some aspects, the polydispersity index of the eluent is between about 0 and about 0.5. In some aspects, the polydispersity index is about 0.13.

[0068] In some aspects, the methods of the present disclosure further comprise subjecting the eluent to one or more purification steps. In some aspects, subjecting the eluent to one or more purification steps occurs after (ii) measuring the light scattering emission signal from the eluent.

[0069] In some aspects, one or more purification steps of the methods of the invention comprise filtration, centrifugation, chromatography, or a combination thereof.

[0070] Samples used in the methods of the present invention can include, but are not limited to, primary cell culture harvests, clarified cell culture media, enriched extracellular vesicle preparations, partially purified extracellular vesicle preparations (e.g., by a single two-hour ultracentrifugation step), or highly purified extracellular vesicle preparations (e.g., extracellular vesicle preparations that are additionally purified using a density gradient medium (e.g., a sucrose density gradient medium or a medium containing iodixanol solution (Sigma-Aldrich))).

[0071] The parent cells can be cultured. The cultured parent cells can be scaled up from a benchtop scale to a bioreactor scale. For example, the parent cells can be cultured until they reach a saturation density, e.g., 1×10 cells per ml. 5 , 1×10 6 , 1×10 7 or greater than 1×10 7A complex. Optionally, after reaching saturation density, the parental cells can be transferred to a larger volume of fresh culture medium. The parental cells can be cultured in a bioreactor, such as, for example, a wave bioreactor, a stirred tank bioreactor. Various configurations of bioreactors are known in the art, and suitable configurations can be selected as needed. Those skilled in the art can easily determine the configuration suitable for culturing and / or amplifying the parental cell colony without too much experimentation. The bioreactor can be oxygenated. The bioreactor can optionally contain one or more impellers, a recirculating stream, a culture medium inlet stream, and a control assembly to regulate the inflow of culture medium and nutrients or the outflow of regulating culture medium, nutrients, and waste products. In some aspects, the culture method includes a perfusion culture method.

[0072] Extracellular vesicles can be made synthetically or isolated from biological systems (such as cells or organisms). EVs can contain biomacromolecules such as lipids, proteins, nucleic acids and / or carbohydrates. Methods for preparing EVs are well known in the art. For example, nanoparticles such as liposomes, lipid nanoparticles, detergents, beads and other polymer structures can be produced by extrusion, emulsification, ultrasonic treatment, mixing, self-assembly, photolithography or crystallization.

[0073] With regard to the purification or enrichment of extracellular vesicles, it is expected that all known extracellular vesicle purification methods are considered to be applicable to this article. For example, the physical properties of extracellular vesicles can be used to separate them from culture medium or other source materials, including separation based on charge (e.g., electrophoretic separation, ion exchange chromatography), size (e.g., filtration, size exclusion chromatography, molecular sieves, etc.), density (e.g., conventional centrifugation or gradient centrifugation), Svedberg constant (e.g., sedimentation with or without external force, etc.). For ion exchange chromatography, any suitable method known in the art can be used, including but not limited to anion exchange chromatography and strong anion exchange chromatography. For density gradient centrifugation, any suitable density gradient culture medium used in the art can be used, including but not limited to sucrose density gradient culture medium and culture medium containing iodixanol solution, colloidal silica, inorganic salts, polyols, polysaccharides, poly (vinyl alcohol) iohexol and non-ionic iodinated culture medium. The purification of extracellular vesicles can be carried out by manually loading a column or other device, or can be automated using a device such as an automatic sampler.

[0074] Alternatively or additionally, separation can be based on one or more biological properties, and include methods that can employ surface labeling (e.g., precipitation, reversible binding to a solid phase, FACS separation, separation using a magnetic surface, specific ligand binding, immunoprecipitation or other antibody-mediated separation techniques, non-specific ligand binding such as annexin V, etc.). In yet further contemplated methods, chemical and / or physical methods can also be used to fuse extracellular vesicles, including PEG-induced fusion and / or ultrasound fusion.

[0075] In some aspects, the enrichment of extracellular vesicles can be performed in a general and non-selective manner (e.g., methods including continuous centrifugation), and can be performed by aggregation, wherein the extracellular vesicles are interconnected with an interconnection composition (e.g., annexin V, fibrin, or antibodies or fragments thereof against at least one of tetraspanins, ICAM-1, CD86, CD63, PTGFRN, BASP1, or any combination thereof). Non-limiting examples of interconnection compositions can be found in U.S. Patent No. 10,195,290B1, issued on February 5, 2019, and U.S. Publication No. 2019 / 0151456A1, issued on May 23, 2019. Alternatively, the enrichment of extracellular vesicles can be performed in a more specific and selective manner (e.g., using tissue-specific surface markers or cell-specific surface markers). For example, specific surface markers can be used for immunoprecipitation, FACS sorting, and / or bead-bound ligands for magnetic separation, etc.

[0076] In some aspects, size exclusion chromatography can be used to enrich extracellular vesicles. Size exclusion chromatography technology is known in the art. Exemplary non-limiting techniques are provided herein. In some aspects, the void volume fraction is separated and contains the extracellular vesicle of interest. Further, in some aspects, as generally known in the art, after the chromatographic separation of (one or more chromatographic fractions) is carried out by centrifugal techniques, the extracellular vesicles can be further separated. In some aspects, for example, density gradient centrifugation can be used to further enrich extracellular vesicles. Further, in some aspects, it may be desirable to further separate the extracellular vesicles of the parental cell source from the extracellular vesicles of other sources. For example, the extracellular vesicles of the parental cell source can be separated from the extracellular vesicles of non-parental cell sources by immunoabsorption capture using antigen antibodies specific to the parental cells. For example, anti-IL-12 antibodies or anti-PTGFRN antibodies can be used.

[0077] In some aspects, isolation of extracellular vesicles may involve a combination of methods including, but not limited to, differential centrifugation, size-based membrane filtration, concentration and / or rate zonal centrifugation, and further characterization using methods including, but not limited to, electron microscopy, flow cytometry, and / or Western blotting.

[0078] Extracellular vesicles can be extracted from the supernatant of parent cells, and membranes and internal proteins, lipids and nucleic acid compositions are displayed so that they can be effectively delivered to and interact with recipient cells. Extracellular vesicles can be derived from parent cells, which may include but are not limited to reticulocytes, erythrocytes, megakaryocytes, platelets, neutrophils, tumor cells, connective tissue cells, neural cells and stem cells. Suitable sources of extracellular vesicles include but are not limited to cells isolated from subjects, which are derived from patient-derived hematopoietic cells or erythroid progenitor cells, immortalized cell lines or cells derived from induced pluripotent stem cells, optionally cultured and differentiated. Cell culture protocols can vary according to nutrients, growth factors, starting cell lines, culture cycles and morphological characteristics of the resulting cells. In some embodiments, the sample containing extracellular vesicles is derived from multiple donor cell types (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 500, 1000, 5000 or 10000 donor cell types) and combined or pooled. Pooling can be performed by mixing cell populations prior to extracellular vesicle extraction or by mixing isolated extracellular vesicle compositions from subsets of donor cell types. Prior to isolation, the parental cells can be irradiated or otherwise treated to affect the production rate and / or composition pattern of secreted extracellular vesicles.

[0079] In certain aspects, extracellular vesicles can be derived from cell lines that are differentiated, proliferated, and cultured in vitro. This enables controllable and reproducible compositions of extracellular vesicles that are not constrained by the isolation and purification of the desired parental cell type.

[0080] In some aspects, a sample comprising extracellular vesicles is obtained from a primary cell harvest and the light scattering signature is determined. In some aspects, the primary cell harvest is clarified for larger cells and cell debris prior to determining the light scattering signature. In some aspects, the sample comprising extracellular vesicles is further purified using any of the methods described above for enriching extracellular vesicles prior to determining the light scattering signature of the sample.

[0081] In some aspects, the method includes fractionating the sample prior to determining the light scattering characteristics. In some aspects, the method includes the step of loading the extracellular vesicle preparation onto a size exclusion chromatography (SEC) column (e.g., an agarose resin SEC column). In some aspects, the method includes the step of loading the extracellular vesicle preparation onto an ion exchange chromatography column. In some aspects, the method includes the step of loading the extracellular vesicle preparation onto a strong anion exchange chromatography column.

[0082] In some aspects, the cell culture comprises mammalian cells. In some aspects, the mammalian cells include human embryonic kidney cells, mesenchymal stem cells, neuronal cells, or a combination thereof.

[0083] In some aspects, the EV comprises an exogenous bioactive molecule. In some aspects, the exogenous bioactive molecule comprises a payload, a targeting moiety, or both. In some aspects, the payload comprises a therapeutic molecule, an adjuvant, an immunomodulator, or a combination thereof.

[0084] In some aspects, the EV further comprises a scaffold portion. In some aspects, the scaffold portion comprises scaffold X, scaffold Y, or both. In some aspects, scaffold X comprises prostaglandin F2 receptor negative regulator (PTGFRN protein), basigin (BSG protein), immunoglobulin superfamily member 2 (IGSF2 protein), immunoglobulin superfamily member 3 (IGSF3 protein), immunoglobulin superfamily member 8 (IGSF8 protein), integrin beta-1 (ITGB1 protein), integrin alpha-4 (ITGA4 protein), 4F2 cell surface antigen heavy chain (SLC3A2 protein), a class of ATP transporters (ATP1A1 protein, ATP1A2 protein, AT P1A3 protein, ATP1A4 protein, ATP1B3 protein, ATP2B1 protein, ATP2B2 protein, ATP2B3 protein, ATP2B4 protein), aminopeptidase N (ANPEP; CD13), neprilysin (membrane metalloendopeptidase; MME), ectonucleotide pyrophosphatase / phosphodiesterase family member 1 (ENPP1), neuropilin-1 (NRP1), CD9, CD63, CD81, PDGFR, GPI anchor protein, lactadherin (MFGE8), LAMP2, LAMP2B, or any combination thereof. In some aspects, scaffold Y comprises myristoylated alanine-rich protein kinase C substrate (MARCKS protein); myristoylated alanine-rich protein kinase C substrate-like 1 (MARCKSL1 protein); brain acidic soluble protein 1 (BASP1 protein) or any combination thereof.

[0085] In some aspects, the exogenous bioactive molecule is attached to the EV via a scaffold portion. In some aspects, the exogenous bioactive molecule is attached to the scaffold portion via a linker. In some aspects, the linker comprises a polypeptide, a non-polypeptide portion, or both.

[0086] In some aspects, the disclosure includes a composition comprising EVs prepared by the methods of the disclosure. In some aspects, the disclosure includes a method of treating a disease or condition in a subject in need thereof, the method comprising administering a composition of the disclosure to the subject.

[0087] Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are fully explained in the literature. See, e.g., Sambrook et al., eds., (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., eds., (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover, ed., (1985) DNA Cloning, Vol. I and Vol. II; Gait, ed., (1984) Oligonucleotide Synthesis; Mullis et al., U.S. Pat. No. 4,683,195; Hames and Higgins, eds., (1984) Nucleic Acid Hybridization; Hames and Higgins, eds., (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; Dissertation, Methods In Enzymology (Academic Press, Inc., NY); Miller and Calos, eds., (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vol. 154 and Vol. 155; Mayer and Walker, eds., (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Vol. I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1986); Crooke, Antisense drug Technology: Principles, Strategies and Applications, 2nd ed., CRC Press (2007) and Ausubel et al., (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.). .

[0088] All references cited above and all references cited herein are incorporated by reference in their entirety.

[0089] The following examples are offered by way of illustration and not limitation.

[0090] Examples

[0091] Example 1: Materials and Methods

[0092] The following examples use one or more of the following materials and methods: HEK293 cell culture

[0093] HEK293SF cell line was transfected with a mammalian expression vector expressing interleukin-12 (IL12) using Transporter 5PEI (Polysciences), and cells stably expressed were selected with puromycin (Invivogen). Once the transfected cells were recovered from selection, they were single-cell cloned by plating in 384-well plates at an inoculation density of 0.5 cells / well. Monoclonality was confirmed by flat plate imaging using Cellavista 4 (Synentec) at day 0. Live clones were expanded into small-scale shake flasks through a series of multi-well plates. Shake flask fed-batch cultures were harvested at day 7, and the culture medium was clarified by centrifugation before analysis. The natural HEK293SF cell line was cloned using the same culture conditions.

[0094] mAEX and fractionation

[0095] Anion exchange chromatography (AEX) was performed on a Waters Acquity UPLC system equipped with a fluorescence detector and a fraction manager. 50 μL of EV sample was injected into a custom AEX column (CIMacTM quaternary amine monolithic column 0.1 mL, 6 μm pore size) obtained from BIA Separations (Ajdovˇsˇcina, Slovenia). The column was washed with mobile phase A (50 mM Tris, 200 mM NaCl, pH 7.4) for 2 minutes and eluted with 60% mobile phase B (50 mM Tris, 2 M NaCl, pH 7.4) at a flow rate of 1 mL / min for three minutes. Tryptophan residues of the column eluent were monitored at Ex 280 nm / Em 350 nm, and particle light scattering was monitored at Ex 460 nm / Em 470 nm. The emission wavelength at 460 nm was selected to avoid interference from proteins, carbohydrates, or lipids, and a 10 nm shift was used as the emission to meet the requirements of fluorescence detection by Empower software. The particles will scatter the emitted light and the side scattered light is detected at the detector on the diagonal axis.Automatic peak fractionation is performed on the Acquity FractionManager based on predefined retention times.

[0096] AlphaLISA Immunoassay

[0097] EV samples were incubated with anti-IL-12 (p70) conjugated acceptor beads (10ug / mL) and biotinylated anti-IL-12 (p40) (1ug / mL) solution in AlphaPlate for one hour. After incubation, streptavidin donor bead solution (80ug / mL) was added and incubated in the dark for 1 hour. A microplate reader (BMG Clariostar) was used to excite the donor beads at 680nm and read the emission wavelength of the acceptor beads between 515nm and 520nm. The IL12 concentration in the EV samples was calculated based on a standard curve generated from recombinant IL12 (R&D Biosystems).

[0098] Dynamic Light Scattering (DLS)

[0099] Dynamic light scattering was performed using a Wyatt DynaPro Plate Reader III instrument. Calibration was performed using NIST polystyrene size exclusion chromatography standards (80 nm, 150 nm, and 250 nm). 200 uL of collected AEX samples were added to a UV-transparent 96-well plate and DLS data were acquired in three wells at 25°C. Raw data were fitted using Dynamics software to obtain the size distribution of the hydrodynamic radius.

[0100] Example 2: mAEX-qLS method development and characterization

[0101] The workflow for the mAEX-qLS method described in this article is as follows: Figure 1 Briefly, clarified cell culture harvest (e.g., HEK293 cells) is injected onto a mAEX column to resolve EVs from interfering matrices (e.g., contaminants), and the concentration of EVs is quantified by enabling light scattering on a conventional fluorescence detector. The EV peak is an automatically collected fraction, followed by orthogonal characterization.

[0102] Method development

[0103] In developing the above method, the suitability of different chromatographic matrices was first evaluated. Size exclusion chromatography (SEC) was found to be unable to adequately resolve EVs from other particles of similar size and to cause rapid column degradation due to repeated injections of crude samples. Anion exchange (AEX) separations were explored to exploit the negative surface charge of EVs. Strong anion exchange columns (ProPac SAX, ThermoFisher) provided effective separations but were also prone to column fouling.

[0104] To reduce the above issues, we selected a custom monolithic tertiary amine anion exchange (mAEX) column. For example, the mAEX column selected has a large pore size (6μm), which enables rapid mass transfer and extended column durability. In addition, the column is short (0.1ml column), allowing for faster resolution of EVs from other contaminants present in the sample. Short (8 minutes) step salt elution was applied to resolve the mixture, and the eluent was monitored by intrinsic protein fluorescence (Ex 280 / Em 350nm) to maximize detection sensitivity.

[0105] like Figure 2A As shown, most of the protein material flowed through the column at an initial salt concentration of 200 mM NaCl. Based on the retention time consistency with the purified EV standard, the EV material eluted in approximately 3 minutes.

[0106] Next, the presence of non-particulate material co-eluting below the EV peak was investigated by ultracentrifugation of the culture medium at 10,000 g. Figure 2A As shown, nearly half of the EV peak area still comes from the ultracentrifuged sample, indicating the significant presence of proteinaceous material, which would lead to an overestimation of the EV concentration.

[0107] To address this issue, light scattering detection was implemented to minimize interference from co-eluting species, taking advantage of the exponentially increased scattering intensity of EV particles compared to proteins. Quasi light scattering (qLS) detection (Ex460 nm / Em 470 nm) was implemented on a conventional fluorescence detector (FLR) to avoid the need for a dedicated multi-angle light scattering detection system and facilitate robust data acquisition and integration in the Water Empower software.

[0108] like Figure 2B As shown in Figure 2, the 3-minute EV peak appears more prominent in the harvest medium sample and completely disappears after ultracentrifugation, confirming the preferential response of qLS to particulate matter. A clear peak before EV (approximately 2.5 minutes) and an extended tailing region ( Figure 2B Inset). These species were not present in the EV standards, indicating that these impurities were effectively removed by the purification process described herein. The negative baseline after 3 min is an artifact associated with changes in salt concentration, as evidenced by blank injections. Based on these results, EV quantification was performed using the qLS signal and particle number was obtained by comparing the peak area to a standard curve generated with purified EV standards.

[0109] Method Qualitative

[0110] Next, to confirm that the above method can be used to quantify EV concentration, Figure 2BCharacteristics of the EV and surrounding peaks shown in the inset. Fractions of the main EV peak were collected for particle size and polydispersity measurements by dynamic light scattering (DLS). Figure 3 As shown, the hydrodynamic radius of the fractionated material is centered around 180 nm, consistent with the expected range for HEK293 cell-derived EVs. The polydispersity index is 0.13, indicating relative homogeneity of the main peak.

[0111] The structural properties of the pre-EV peak were investigated by mild proteinase K digestion under non-denaturing conditions (PBS buffer, 37 °C, 1 h). Figure 4 As shown, digestion resulted in an increase in the intensity of the pre-peak, while the main peak shifted to an earlier elution time. This result suggests that the pre-peak is a form of proteinaceous native EV material. Preliminary results showed the presence of histones as well as extracellular matrix proteins, which may form complexes with DNA and EVs, respectively.

[0112] The specificity, reproducibility, linearity, accuracy and robustness (stability) of the method described herein were further evaluated and the results are summarized in Table 1.

[0113] Table 1. Qualitative summary of methods

[0114]

[0115] Specificity was determined based on the absence of interfering peaks in blank injections and retention times consistent with EV standards. Precision was assessed by performing six injections on each of two different days and achieving less than (<) 5% RSD in peak area. Accuracy was assessed by spike recoveries of injected samples at three concentration levels. Peak area recoveries were greater than 95%. Linearity was determined to be 1.25E10 to 1E11 particles / mL based on serial dilutions of EV standards. The stability of the method was assessed by comparing the column pressure and half-width of the 3-minute EV peak between the first injection and the two hundredth injection. After 200 injections of crude culture medium, no significant increase in column pressure and a decrease in peak resolution were observed, which demonstrated the stability of the monolithic column.

[0116] Application to clonal selection

[0117] The mAEX platform was applied to clonal selection of HEK293SF cell lines that produce both native engineered EVs and surface engineered EVs. Harvested culture media from forty-nine native HEK293SF clones were analyzed for EV concentrations on day seven and the results are summarized in Fig. 8AA wide range of productivity (over sevenfold) was found in native cell lines. As described above, LC-based methods offer the advantage of peak fractionation, allowing analysis of other properties. HEK293SF cells stably transfected to express interleukin-12 (IL-12) on the EV surface were screened. The antitumor bioactivity of IL-12-modified EVs is mediated by lipid nanoparticles and IL-12, and therefore quantification of both moieties is required as criteria for clonal selection. EV peaks from twenty samples were automatically collected and IL-12 expression levels were determined by AlphaLISA, and the results are shown in Figure 8B As shown, some clones showed a three-fold and five-fold increase in IL-12 and granule production, respectively, compared to the original cell line. IL-12 is also present on non-EV material, including cell membrane fragments and protein aggregates. Without the removal of these materials by mAEX, AlphaLISA will produce erroneously high IL-12 concentrations and will not be representative of EV-associated IL-12. In addition to IL-12 quantification, other plate-based techniques have been combined with fractionation to provide insight into product properties, such as hydrodynamic radius and polydispersity by dynamic light scattering (DLS) and cholesterol content by Amplex Red assay.

[0118] In general, the above results demonstrate that the methods provided herein have sufficient throughput and sensitivity to directly measure samples collected by fractionation, and thus provide a superior and high-throughput method for quantifying EVs in samples (e.g., cell culture harvest). The current run time of less than 10 minutes can support 192 samples in 24 hours.

[0119] Incorporated by Reference

[0120] All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes.

[0121] Equivalent

[0122] Although various specific aspects have been shown and described, the above description is not restrictive. It should be understood that various changes can be made without departing from the spirit and scope of the present disclosure. For those skilled in the art, many changes will become apparent after reading this specification.

Claims

1. A method for determining the amount of extracellular vesicles (EVs) present in a sample, the method comprising: (i) contacting the sample with a monolithic anion exchange (AEX) chromatography column; and (ii) then measuring the light scattering emission signal from the eluent collected from the AEX chromatography column.

2. A method for preparing an extracellular vesicle (EV) fraction from a sample, the method comprising: (i) contacting the sample with a monolithic anion exchange (AEX) chromatography column; (ii) collecting eluent from the monolithic AEX chromatography column; and (iii) then measuring the light scattering emission signal from the eluent to prepare the EV fraction.

3. A method of reducing the amount of impurities present in a sample containing extracellular vesicles (EVs), the method comprising: (i) contacting the sample with a monolithic anion exchange (AEX) chromatography column; (ii) collecting eluent from the monolithic AEX chromatography column; and (iii) then measuring a light scattering emission signal from the eluent, wherein the amount of impurities in the sample is reduced.

4. The method of claim 3, wherein the amount of impurities is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to a reference sample (e.g., a corresponding eluent collected after contacting the sample with a non-monolithic AEX chromatography column).

5. The method according to claim 3 or 4, wherein if the purity of the EVs present in the eluent is lower than a reference value, the method further comprises subjecting the eluent to one or more purification steps.

6. The method of claim 5, wherein the one or more purification steps comprise filtration, centrifugation, chromatography, or a combination thereof.

7. The method of any one of claims 1 to 6, wherein the monolithic AEX chromatography column comprises a porous surface having a pore size of about 6 μm or greater.

8. The method according to any one of claims 1 to 7, wherein the monolithic column comprises a monolithic tertiary amine column.

9. The method according to any one of claims 1 to 8, wherein the eluent is collected after contacting the AEX chromatography column with an elution buffer, wherein the contacting with the elution buffer occurs after (i) (i.e., contacting the sample with the AEX chromatography column).

10. The method of claim 9, wherein the elution buffer comprises tris, salt, or both.

11. The method of claim 10, wherein the elution buffer comprises about 50 mM tris, about 2,000 mM NaCl, and a pH of about 7.

4.

12. The method of claim 10 or 11, wherein the elution buffer further comprises sodium azide.

13. The method according to any one of claims 1 to 12, further comprising contacting the AEX chromatography column with a wash buffer, wherein the contacting with the wash buffer occurs after (i) (i.e., contacting the sample with the AEX chromatography column) and before (ii) (i.e., measuring the light scattering emission signal from the eluent).

14. The method of claim 13, wherein the wash buffer comprises tris, salt, or both.

15. The method of claim 14, wherein the wash buffer comprises about 50 mM tris, about 200 mM NaCl, and a pH of about 7.

4.

16. The method of claim 14 or 15, wherein the wash buffer further comprises sodium azide.

17. The method of any one of claims 1 to 16, wherein the light scattering emission signal is generated using an excitation wavelength of about 280 nm to about 700 nm.

18. The method of claim 17, wherein the light scattering emission signal is generated using an excitation wavelength of about 400 nm to about 500 nm.

19. The method of claim 18, wherein the light scattering emission signal is generated using an excitation wavelength of about 420 nm to about 480 nm.

20. The method of claim 19, wherein the light scattering emission signal is generated using an excitation wavelength of about 460 nm.

21. The method of any one of claims 1 to 20, wherein the light scattering emission signal is measured at an emission wavelength that is about 0 nm to about 20 nm longer or shorter than the excitation wavelength.

22. The method of claim 21, wherein the light scattering emission signal is measured at an emission wavelength that is about 10 nm longer or shorter than the excitation wavelength.

23. The method of any one of claims 1 to 22, wherein the light scattering emission signal is measured at an emission wavelength of about 300 nm to about 600 nm.

24. The method of claim 23, wherein the light scattering emission signal is measured at an emission wavelength of about 400 nm to about 500 nm.

25. The method of claim 24, wherein the light scattering emission signal is measured at an emission wavelength of about 470 nm.

26. The method of any one of claims 1 to 16, wherein the light scattering emission signal is generated using an excitation wavelength of about 460 nm and measured at an emission wavelength of about 470 nm.

27. The method of any one of claims 1 to 16, wherein the light scattering emission signal is generated using an excitation wavelength of about 280 nm and measured at an emission wavelength of about 350 nm.

28. The method of any one of claims 1 to 27, further comprising analyzing one or more properties of the eluent.

29. The method of claim 28, wherein the one or more properties comprise average particle size, particle count, polydispersity index, light scattering intensity, cholesterol content, protein content, lipid content, payload content, or a combination thereof.

30. The method of claim 28 or 29, wherein the one or more properties are measured using an immunoassay selected from AlphaLISA, dynamic light scattering (DLS), electron microscopy (e.g., transmission electron microscopy or cryo-electron microscopy), or a combination thereof.

31. The method of claim 30, wherein the payload content comprises the level of cytokines present in the eluent.

32. The method of claim 31 , wherein the cytokine comprises IL-12.

33. The method of claim 32, wherein the level of IL-12 is measured using an IL-12 specific AlphaLISA.

34. A method according to any one of claims 29 to 33, wherein the average particle size of the eluent is reduced compared to the average particle size of a reference sample (e.g., a sample prior to the contact with the AEX chromatography column).

35. The method of claim 34, wherein the average particle size of the eluent is reduced by at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times or more compared to the average particle size of the reference sample.

36. The method of any one of claims 34 or 35, wherein the average particle size of the eluent is between about 20 nm and about 300 nm.

37. The method of claim 36, wherein the average particle size of the eluent is about 180 nm.

38. The method of any one of claims 29 to 37, wherein the polydispersity index of the eluent is reduced compared to the polydispersity index of a reference sample (e.g., a sample prior to the contact with the AEX chromatography column).

39. The method of claim 38, wherein the polydispersity index of the eluent is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to the polydispersity index of the reference sample.

40. The method of claim 38 or 39, wherein the polydispersity index of the eluent is between about 0 and about 0.

5.

41. The method of claim 40, wherein the polydispersity index is about 0.

13.

42. The method of any one of claims 1, 2, and 6 to 41, further comprising subjecting the eluent to one or more purification steps.

43. The method of claim 42, wherein subjecting the eluent to one or more purification steps occurs after (ii) measuring the light scattering emission signal from the eluent.

44. The method of claim 42 or 43, wherein the one or more purification steps comprise filtration, centrifugation, chromatography, or a combination thereof.

45. The method of any one of claims 1 to 44, wherein the sample is derived from a cell culture.

46. ​​The method of claim 45, wherein the cell culture comprises a perfusion cell culture, a fed-batch cell culture, or both.

47. The method of claim 45 or 46, wherein the cell culture comprises mammalian cells.

48. The method of claim 47, wherein the mammalian cells comprise human embryonic kidney cells, mesenchymal stem cells, neuronal cells, or a combination thereof.

49. The method of any one of claims 1 to 48, wherein the EV comprises an exogenous biologically active molecule.

50. The method of claim 49, wherein the exogenous biologically active molecule comprises a payload, a targeting moiety, or both.

51. The method of claim 50, wherein the payload comprises a therapeutic molecule, an adjuvant, an immunomodulator, or a combination thereof.

52. The method of claim 50 or 51, wherein the payload comprises a cytokine.

53. The method of claim 52, wherein the cytokine comprises IL-12.

54. A method according to any one of claims 49 to 53, wherein the EV further comprises a scaffold portion.

55. The method of claim 54, wherein the scaffold portion comprises scaffold X, scaffold Y, or both.

56. The method according to claim 55, wherein the scaffold X comprises prostaglandin F2 receptor negative regulator (PTGFRN protein), basigin (BSG protein), immunoglobulin superfamily member 2 (IGSF2 protein), immunoglobulin superfamily member 3 (IGSF3 protein), immunoglobulin superfamily member 8 (IGSF8 protein), integrin beta-1 (ITGB1 protein), integrin alpha-4 (ITGA4 protein), 4F2 cell surface antigen heavy chain (SLC3A2 protein), a class of ATP transporters (ATP1A1 protein, ATP1A2 protein). A2 protein, ATP1A3 protein, ATP1A4 protein, ATP1B3 protein, ATP2B1 protein, ATP2B2 protein, ATP2B3 protein, ATP2B4 protein), aminopeptidase N (ANPEP; CD13), neprilysin (membrane metalloendopeptidase; MME), ectonucleotide pyrophosphatase / phosphodiesterase family member 1 (ENPP1), neuropilin-1 (NRP1), CD9, CD63, CD81, PDGFR, GPI-anchored protein, lactadherin (MFGE8), LAMP2, LAMP2B, or any combination thereof.

57. The method of claim 55 or 56, wherein the scaffold Y comprises myristoylated alanine-rich protein kinase C substrate (MARCKS protein); myristoylated alanine-rich protein kinase C substrate-like 1 (MARCKSL1 protein); brain acidic soluble protein 1 (BASP1 protein) or any combination thereof.

58. The method of any one of claims 55 to 57, wherein the exogenous bioactive molecule is attached to the EV via a scaffold moiety.

59. The method of claim 58, wherein the exogenous bioactive molecule is attached to the scaffold portion via a linker.

60. The method of claim 59, wherein the linker comprises a polypeptide, a non-polypeptide moiety, or both.

61. The method of any one of claims 1 to 60, wherein the EVs comprise exosomes.

62. A composition comprising EVs prepared by the method of any one of claims 1 to 63.

63. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a composition according to claim 62.

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