Method for separating extracellular vesicles
Patent Information
- Authority / Receiving Office
- EP · EP
- Patent Type
- Applications
- Current Assignee / Owner
- EXIT071 BV
- Filing Date
- 2024-07-25
- Publication Date
- 2026-06-10
Smart Images
Figure EP2024071131_06022025_PF_FP_ABST
Abstract
Description
[0001] METHOD FOR SEPARATING EXTRACELLULAR VESICLES
[0002] TECHNICAL FIELD
[0003] The invention relates to a method for separating extracellular vesicles, preferably exosomes, for use in extracellular vesicle-based diagnostics and therapeutics.
[0004] BACKGROUND OF THE INVENTION
[0005] Exosomes are a type of extracellular vesicles (EVs), which can be found in most body fluids, including blood, urine, saliva, sweat and tears. Whereas exosomes were initially considered waste products, it is now known that exosomes are functional vehicles, not only carrying biomolecular cargo such as proteins, lipids, and nucleic acids, but also being able to deliver this cargo to target cells. This type of cell-cell communication via exosomes has shown to be involved in various physiological and pathological processes, for example, embryonic development, immune responses, tissue regeneration and the development of cancer (Liu, Shang-Long, et al. Translational Cancer Research 8.1 (2019): 298).
[0006] For this reason, there is a growing interest in the use of exosomes and other EVs in non- invasive diagnostic methods, both for the early detection and prognosis of disease. In addition, their biocompatibility, low immunogenicity, particularly when exosomes are isolated from a patient’s own material, and ability to cross biological barriers, make exosomes promising candidates for drug delivery vehicles.
[0007] In order to benefit from their diagnostic and therapeutic potential, efforts have been made to isolate exosomes from e.g. body fluids. Unfortunately, the currently most commonly used methods, namely affinity-based methods (Scientific Reports volume 6, Article number: 33935 (2016), or methods based on differential centrifugation and size exclusion chromatography, are not yet able to deliver the desired level of purity (Chen, Jiaci, et al. "Review on strategies and technologies for exosome isolation and purification." Frontiers in Bioengineering and Biotechnology 9 (2022): 811971). This can in part be explained by the complexity of exosome-containing samples, which may comprise a variety of components, such as other extracellular vesicles, lipoproteins, protein aggregates and cellular debris, which may have overlapping size and density and can show non-specific binding properties. Other challenges that are associated with these methods are the requirement of high sample amount, risk of altering the original sample composition and the need for lengthy procedures. It is an objective of the current invention to overcome one or more of the above-mentioned challenges and in particular to develop an exosome separation method that allows improved separation of exosomes, resulting in a more pure exosome-comprising sample compared to when using the currently used methods.
[0008] SUMMARY OF THE INVENTION
[0009] The current invention relates to a method for separating extracellular vesicles, particularly exosomes by e.g. using an analytical electrophoresis system.
[0010] The current inventors have not only shown that using depletion zone isotachophoresis, enables preconcentration of exosomes, but also that the method is compatible with a variety of relevant exosome-comprising samples, preferably cell-line derived exosomes, any body fluid derived exosomes (such as from plasma, urine or interstitial fluid, tear fluid, CSF), or organ-on-a-chip derived exosomes. The use of depletion zone isotachophoresis for separating exosomes tackles several of the challenges encountered with currently used methods e.g. differential centrifugation and size exclusion chromatography, for example separation of confounding analytes of a similar size as (the) exosomes(of interest). Other advantages are that depletion zone isotachophoresis is compatible with a low amount of sample and is gentle on the exosomes, avoiding damage.
[0011] Moreover, the current inventors have demonstrated that depletion zone isotachophoresis separation of exosomes can be dramatically enhanced by using separator booster(s). The term ‘separator boosters’ as used herein refers to or may be replaced by the term ‘compounds’ and is preferably a compound with intermediate electrophoretic mobility compared to the electrophoretic mobility of the exosomes (of interest) in the exosome- containing sample. In addition or alternatively, the term may refer to amino acids and / or peptides and / or acids and / or other (co-)polymers with a group that can be charged by protonation or deprotonation, or otherwise. Preferably, the acids are selected from a group comprising acids with a molecular weight between 30 and 400 Da, more preferably between 70 and 350 Da, even more preferably between 120 and 300 Da, even more preferably between 160 and 250 Da.
[0012] The current inventors have shown that these separator boosters do not only allow the separation of exosomes from other impurities / confounding factors in the sample of interest, but also of sub-populations of different exosomes. Whereas attempts have been made to separate subpopulations of exosomes using the prior art methods, the method as per the disclosure allows improved efficiency, specificity and sensitivity. Accordingly, in one aspect, the current disclosure relates to a method for separating extracellular vesicles, preferably exosomes.
[0013] In another aspect, the current disclosure relates to the use of separator booster(s) in depletion zone isotachophoresis separation of extracellular vesicles, preferably exosomes.
[0014] In another aspect, the current disclosure relates to separator booster(s), wherein at least one separator booster is a compound having an electrophoretic mobility between -20 and -30*1 O'9m2s'1V’1. Preferably, the current disclosure relates to a combination of separator boosters, wherein a first separator booster is a compound having an electrophoretic mobility between -2 and -10*1 O'9m2s-1V’1, wherein a second separator booster is a compound having an electrophoretic mobility between -10 and -20*1 O'9m2s-1V’1, wherein a third separator booster is a compound having an electrophoretic mobility between -20 and -30*1 O'9m2s-1V'1and preferably wherein the combination of separator boosters comprises glycine, glycinephenylalanine, and / or tricine, more preferably glycine, serine, 3-methyl-L-histidine, alanineglycine, glycine-phenylalanine, tricine, MES (2-(N-morpholino)ethanesulfonic acid) and MOPSO (3-(N-morpholino)propanesulfonic acid). The combination may comprise at most 20 separator boosters.
[0015] DETAILED DESCRIPTION OF THE INVENTION
[0016] The present disclosure relates to a method for separating, preferably for separating analytes, more preferably for separating extracellular vesicles (EVs), preferably for separating exosomes, said method comprising the steps of: providing a sample comprising analytes (preferably extracellular vesicles or exosomes) and preferably comprising separator booster(s), wherein the separators boosters preferably comprise at least 1 (preferably at least 3, preferably at most 20)separator booster(s), wherein at pH 7-14 e.g. 7, 7.3, 7.5, 7.7, 8, 8.1 , 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.3, 9.5, 9.7, 10, 10.3, 10.5, 10.7, 11 , 11.3, 11.5, 11.7, 12, 12.3, 12.5, 12.7, 13, 13.3, 13.5, 13.7, 14, at least one separator booster is a compound having an electrophoretic mobility between -20 and -3000*1 O'8m2s-1V’1, preferably between -20 and -300*1 O'8m2s-1V’1, more preferably between -20 and - 30*1 O'9m2s'1V’1. More preferably a first separator booster is a compound having an electrophoretic mobility between -2 and -10*1 O'9m2s-1V’1, a second separator booster is a compound having an electrophoretic mobility between -10 and -20*1 O'9m2s'1V'1and a third separator booster is a compound having an electrophoretic mobility between -20 and -30*1 O'9m2s-1V’1; introducing said sample into an apparatus comprising at least a separation channel
[0017] (C), wherein the separation channel (C) comprises a downstream end (D) and an upstream end (II), and a depletion zone formation means (N), placed in or connected to an intermediate region between the upstream end (II) and downstream end (D); applying an electric field between 1-1000 volt per centimeter, more preferably the electrical field may be from from 1-500 volt per centimeter, even more preferably the electric field may be from 1-250, 1-100, 1-75, 1-50 volt per centimeter, yet even more preferably the electric field may be from 1 and 30 volt per centimeter, e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 24, 25, 26, 27, 28, 29, 30 volt per centimeter, yet even more preferably the electric field may be from 1 and 20 volt per centimeter, e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 volt per centimeter, yet even more preferably the electric field may be from 1 and 15 volt per centimeter e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 volt per centimeter, most preferably the electric field may be from 3 and 12 volt per centimeter e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 volt per centimeter, between the downstream end
[0018] (D) and the upstream end (II), thereby separating exosomes.
[0019] The method for separating as presented herein may advantageously be used in a variety of fields, among which environmental sciences and life sciences as well as research applications. In one application, the method may be used to separate exosomes with enhanced resolution, which can allow to better benefit from their diagnostic and therapeutic potential, for example, as non-invasive diagnostic methods, to understand biological processes and as drug delivery vehicles.
[0020] The sample provided may be obtained from a variety of sources e.g. human, animal, plant, algae, including various cells and (solid) tissue types, such as brain and tumour sample. Exosomes can also be obtained from biological fluids, including but not limited to blood, urine, saliva, mucus, sweat, tears, breast milk, semen, bronchial lavage and cerebrospinal fluid. It may also be possible to obtain exosomes from fluids with lower complexity, such as tissue culture supernatant. In view thereof, the sample as used in the method may be or comprise blood urine, saliva, mucus, sweat, tears, breast milk, semen, bronchial lavage, cerebrospinal fluid and tissue culture supernatant and / or may comprise exosomes derived or obtained from blood, urine, saliva, mucus, sweat, tears, breast milk, semen, bronchial lavage, cerebrospinal fluid and tissue culture supernatant. In addition and / or alternatively, the sample provided may be obtained from in vitro culture systems, such as organ-on-chip models, 3D printed constructs and / or organoids. In addition and / or alternatively, the sample provided may be obtained from fungi, bacteria, and / or viruses. Electrophoretic mobility as described herein refers to a measure of the rate at which a charged analyte moves in response to an applied electric field and is an important parameter used in many analytical techniques, including gel electrophoresis, capillary electrophoresis and electrophoretic deposition. The general steps involved in measuring electrophoretic mobility of a compound comprise:
[0021] Sample preparation: the sample is prepared by dissolving the compound in a suitable buffer solution to ensure the right pH and ionic strength, and remove disturbing components, if needed.
[0022] Setting up the electrophoresis apparatus: the sample is then placed in a suitable container, such as a capillary tube, which is immersed in a buffer solution. Two electrodes are placed at either end of the container, and a voltage is applied across them to create an electric field.
[0023] Measuring the movement of the compound: when the voltage is applied, the charged analyte in the sample will move towards the electrode of the opposite charge, depending on the pH and the electroosmotic flow. The movement of the compound is tracked using a detector, which can be optical, such as a fluorescence detector, electrical, such as a conductivity detector or mass spectrometry
[0024] Calculating electrophoretic mobility: the electrophoretic mobility of the compound is calculated by dividing the velocity of the particle by the strength of the applied electric field and subtracting the mobility of the electroosmotic flow.
[0025] In the current method for separating, preferably for separating extracellular vesicles, preferably exosomes, the sample preferably has a viscosity that allows the migration of the analytes (e.g. extracellular vesicles, preferably exosomes, and / or other constituents, among which lipoproteins, protein aggregates and cellular debris). Whereas the sample as used in the method of the disclosure is preferably an aqueous solution, the sample may also comprise non-aqueous solvents, surfactant solutions, or ionic liquids. The (aqueous) solution preferably has a viscosity (at 25°C) between 0.8 and 9 millipascal seconds. The aqueous solution may comprise viscosity enhancing agents, such as cellulose, gums, dextran, polyethylene glycol. This viscosity value of the sample comprising exosomes can vary, depending on the temperature and pressure of the solution. Whereas the pH of the sample may vary, the pH is preferably chosen based on the pKa values of the analytes (e.g. exosomes and / or other constituents, among which lipoproteins, protein aggregates and cellular debris), this to ensure their appropriate ionization state for separation. More specifically, the pH of the solution can affect the ionization state of the analytes (e.g. exosomes and / or other constituents, among which lipoproteins, protein aggregates and cellular debris) in the sample, and hence their electrophoretic mobility. For example, if the pH of the sample is such that a particular molecule in the sample is mostly ionized, it will have a higher electrophoretic mobility than if it were mostly non-ionized. Conversely, if the pH of the sample is such that the same molecule is mostly non-ionized, it will have a lower electrophoretic mobility. In the current method for separating exosomes, the pH of the sample preferably ranges between 7-14, for example 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1 , 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.2, 9.4, 9.6, 9.8, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13, 13.5, 14. More preferably, the pH of the sample ranges between 8-13. Even more preferably, the pH of the sample ranges between 9-12. Most preferably, the pH ranges between 10-11 e.g. 10.0, 10.1 , 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0.
[0026] As used herein, ‘separating extracellular vesicles, preferably exosomes’ particularly refers to the separation of extracellular vesicles, preferably exosomes, from other constituents in a sample. The other constituents may be any constituent comprising a different electrophoretic mobility than the exosomes of interest and can for example be lipoproteins, protein aggregates and cellular debris. As exosomes can be heterogenous vesicles, with potentially varying electrophoretic mobility, ‘separating exosomes’ as used herein, may further refer to the separation of subpopulations of exosomes. It may for example be possible to separate different types of exosomes, e.g. exosomes of different cell and / or tissue and / or organ origin, different size, different cargo and different impact on the recipient cell.
[0027] In the method of the disclosure, the sample comprising extracellular vesicles, particularly exosomes, preferably further comprises separator booster(s). These separator booster(s) may enable enhanced separation of analytes (e.g. extracellular vesicles, exosomes and / or other constituents, among which lipoproteins, protein aggregates and cellular debris) into discrete zones and / or selective analyte elution. Preferably, the ‘separator booster(s)’ do(es) not bind the analytes in such a way that the separator booster(s) can drag the analyte through the medium wherein they are present. More preferably, the ‘separator booster(s)’ preferably separate analytes by positioning between analytes (e.g. extracellular vesicles) having a different electrophoretic mobility, e.g. a lower electrophoretic mobility on one side of the analyte and higher electrophoretic mobility on another side of the analyte, preferably without requiring and / or undergoing binding interactions. Even more preferably the separator booster(s) do(es) not stain or immune-capture the analyte, nor function as a drag-tag for the analytes. As the skilled person will be aware, a drag-tag refers to a perturbing entity, that can be attached to e.g. DNA fragments to enable their separation in free-solution electrophoresis. Yet even more preferably, said ‘separator booster(s)’ do not modify (e.g. increase or decrease) the electrophoretic mobility of the analytes. Yet even more preferably, said ‘separator booster(s)’ separate(s) the analytes based on their electrophoretic mobility, do(es) not bind the analytes and / or do(es) not modify the electrophoretic mobility of the analytes. Most preferably, said ‘separator booster(s)’ separate(s) the subpopulations of exosomes based on their electrophoretic mobility, do(es) not bind the exosomes and / or do(es) not modify the electrophoretic mobility of the exosomes. The term ‘separator booster(s)’ as used herein refers to or may be replaced by the term ‘compounds’ and is preferably a compound with intermediate electrophoretic mobility compared to the analytes in the exosome- containing sample. Preferably the ‘separator booster(s)’ as used herein, have an electrophoretic mobility that is higher and / or lower than the electrophoretic mobility of one or more of the analyte(s). In addition or alternatively, the term may refer to (modified) amino acids, peptides, acids or a combination thereof. Preferably, the acids are selected from a group comprising acids with a molecular weight between 30 and 400 Da, more preferably between 70 and 350 Da, even more preferably between 120 and 300 Da, even more preferably between 160 and 250 Da. In the current disclosure the electrophoretic mobility of the separator booster(s) may for example be determined at pH 8.8, preferably in a suitable buffer, which is known by the person skilled in the art, preferably TAPS, more preferably borate buffer. Alternatively, the suitable buffer may comprise between 2-10 mmol / L NaCI, 0.05-0.5 mmol / L KCI, 0.1-2 mmol / L Na2HPO4, 0.03-2 mmol / L KH2PO4. More preferably, the suitable buffer comprises 6.8 mmol / L NaCI, 0.13 mmol / L KCI, 0.5 mmol / L Na2HPC>4 and 0.09 mmol / L KH2PO4. Even more preferably the buffer comprises phosphate buffered saline (PBS). A person skilled in the art will know that the electrophoretic mobility of separator boosters may vary upon changing the pH to lower pH and / or to higher pH such as 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 6.5, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8, 10.0, 11.0, 12.0, 13.0, 14.0 and / or buffer. Preferably, the sample comprising exosomes and separator booster(s) comprises separator boosters having, at a pH of 8.8, an electrophoretic mobility between -1 and -50*1 O'9m2s-1V’1, even more preferably between -2 and -40*1 O'9m2s-1V’1, yet even more preferably between -3 and -30*1 O'9m2s-1V’1, most preferably between -3 and -25*10'9m2s'1V’1e.g. -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*1 O’9m2s’1V’1.
[0028] The term ‘separator booster’ as used herein may refer to any alpha-amino acid, for example alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, selenocysteine, serine, threonine, tryptophan, tyrosine, valine, but preferably refer to one or more compounds selected from the list of cysteine, glutamine, glycine, histidine, methionine, phenylalanine, serine, and / or valine. The amino acids may be modified, for example by phosphorylation, methylation, acetylation, amidation, sulfation and nitrolisation. Examples of (modified) amino acids are 1-methyl-L-histidine, 3-methyl-L-histidine. The peptides may be dipeptides such as alanine-glycine, alanine-serine, glycine-serine, glycine-alanine, glycine-asparagine, glycine- threonine, glycine-histidine, glycine-phenylalanine, leucine-glycine, glycine-valine, alaninealanine, glycine-isoleucine, glycine-leucine, alanine-asparagine, glycine-tryptophan, alaninevaline, alanine-methionine, leucine-valine, alanine-phenylalanine, alanine-leucine, glycineproline, leucine-phenylalanine, leucine-leucine. Preferably, the peptides are selected from the list of alanine-alanine, alanine-glycine, glycine-phenylalanine, glycine-histidine. Separator boosters may also be acids, including sulfonic acids. Preferably, the acids are selected from a group comprising acids with a molecular weight between 30 and 400 Da, more preferably between 70 and 350 Da, even more preferably between 120 and 300 Da, even more preferably between 160 and 250 Da. Several examples of acids include d-glucuronic acid, MES (2-(N-morpholino)ethanesulfonic acid), MOPSO (3-(N-morpholino)propanesulfonic acid), TAPSO (N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropane sulfonic acid), Tricine (N- [Tris(hydroxymethyl)methyl]glycine), and TAPS (N-Tris(hydroxymethyl)methyl-3- aminopropanesulfonic acid). Preferably, the sample as used in the method for separating exosomes comprises separator boosters, wherein separator boosters are compounds with a molecular weight between 25-500 Da and / or with an electrophoretic mobility between -2 and - 40*1 O'9m2s'1V’1. More preferably the sample as used in the method for separating exosomes comprises separator boosters with a molecular weight between 50-400 Da and / or with an electrophoretic mobility between -3 and -30*1 O'9m2s-1V’1. Most preferably the sample as used in the method for separating exosomes comprises separator boosters with a molecular weight between 75-300 Da and / or with an electrophoretic mobility between -3 and - 25*10'9m2s'1V’1. Such as -3, -4, -5, -6, -7, -8, -9, -10, -11 , -12, -13, -14, -15, -16, -17, -18, - 19, -20, -21 , -22, -23, -24, -25*1 O'9m2s'1V’1. In addition or alternatively, the term ‘separator booster’ may refer to other co-polymers comprising a group that can be charged by protonation or deprotonation, or otherwise.
[0029] In a preferred embodiment, the separator booster is a compound with a charge and electrophoretic mobility, preferably an intermediate electrophoretic mobility compared to the analytes in the exosome-containing sample.
[0030] In an embodiment, the sample as used in the method comprises separator booster(s), more preferably at least 1 (preferably at least 3, preferably at most 20) separator boosters, wherein at least one separator booster is a compound having an electrophoretic mobility between -20 and -30*10-9m2s-1V'1(e.g. tricine). More preferably the sample comprises at least 3 (preferably at most 20) separator boosters, wherein the electrophoretic mobility of a first separator booster preferably ranges between -2 and -10*1 O'9m2s-1V'1(e.g. glycine), the electrophoretic mobility of a second separator booster preferably ranges between -10 and - 20*1 O'9m2s'1V'1(e.g. glycine-phenylalanine) and the electrophoretic mobility of a third separator booster preferably ranges from -20 and -30*1 O'9m2s-1V'1(e.g. tricine). Preferably, the method comprises at least three separator boosters, of which the electrophoretic mobility of the first separator booster ranges between -2 and -5*10-9m2s-1V’1, the electrophoretic mobility of the second separator booster ranges between -16 and -19*10-9m2s-1V'1and the electrophoretic mobility of the third separator booster ranges between -21 and -24*10-9m2s-1V’1. Most preferably, the method comprises at least three separator boosters, of which the electrophoretic mobility of the first separator booster ranges between -3 and -4*1 O'9m2s-1V’1, the electrophoretic mobility of the second separator booster ranges between -17 and -18*1 O'9m2s'1V'1and the electrophoretic mobility of the third separator booster ranges between -22 and -23*1 O'9m2s-1V’1. Most preferably, the method comprises at least three separator boosters, of which the electrophoretic mobility of the first separator booster ranges between - 3 and -4*10’9m2s’1V'1(e.g. -3.0, -3.1 , -3.2, -3.3, -3.4, -3.5, -3.6, -3.7, -3.8, -3.9, -4.0*10-9m2s'1V'1), the electrophoretic mobility of the second separator booster ranges between -17 and - 19*10'9m2s'1V’1(e.g. -17.0, -17.1 , -17.2, -17.3, -17.4, -17.5, -17.6, -17.7, -17.8, -17.9, - 18.0*1 O'9m2s'1V'1) and the electrophoretic mobility of the third separator booster ranges between -22 and -23*1 O’9m2s’1V'1(e.g. -22.0, -22.1 , -22.2, -22.3, -22.4, -22.5, -22.6, -22.7, - 22.8, -22.9, -23.0*1 O'9m2s’1V’1).
[0031] In addition or alternatively, the sample comprises at most 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 separator boosters. Preferably, the sample does not comprise separator boosters chosen from the group consisting of octanesulfonic acid, N-(2-acetamido)-2-aminoethane sulfonic acid and glutamic acid.
[0032] In the method for separating, particularly in the step of applying an electric field, any electrophoretic technique may be applied, such as electro-driven membrane-based filtration, dielectrophoresis, (micro)capillary electrophoresis and isotachophoresis (ITP). Preferably, the electrophoretic technique comprises ITP, which is a powerful electrokinetic technique for the concentration, separation, purification, and quantification of ionic analytes (e.g. exosomes and / or other constituents, among which lipoproteins, protein aggregates and cellular debris), especially when downscaled to microfluidic devices. In the method for separating, preferably for separating exosomes, the sample is preferably sandwiched between two buffers with different electrophoretic mobilities i.e. the sample may be between a leading and terminating electrolyte, or mixed with either the leading or terminating electrolyte. The leading electrolyte is defined by (co-)ions, which are ions with the same charge as the analyte of interest (e.g. exosomes and / or other constituents, among which lipoproteins, protein aggregates and cellular debris), with high ionic mobility (i.e. higher than the ionic mobility of the analyte of interest). The terminating electrolyte is defined by co-ions with low ionic mobility (i.e. lower than the analyte of interest). As described herein, the sample can then be subjected to an electric field. Since the particles located in the leading electrolyte will migrate faster than the analyte of interest and the particles located in the terminating electrolyte will migrate slower than the analyte of interest, the sample particles are driven towards the interface of the leading and terminating electrolyte, ordered in adjacent zones according to their electrophoretic mobility.
[0033] More preferably, the current method, particularly in the step of applying an electrical field, comprises depletion zone isotachophoresis. In the current method, the depletion zone may act as a terminating electrolyte. An ion depletion zone is an area that repels positively or negatively charged particles depending on the material surface charge. Such an ion depletion zone can be generated through ion concentration polarization, which refers to asymmetric distribution of cations and anions and may occur upon applying a voltage across an ion depletion zone formation means. In the current method, particularly in the step in which a sample is introduced into an apparatus comprising among others an ion depletion zone formation means, the ion depletion zone formation means may be any means that allows ion concentration polarization, but is preferably a nanochannel (i.e. a channel with a diameter in the nanometer range i.e. between 1-1000 nanometer, preferably between 1-500 nanometer, more preferably between 1-250 nanometer, most preferably between 1-100 nanometer), nanojunction or an ion permeative-selective membrane (e.g. a nation selective membrane). In addition and / or alternatively, the ion depletion zone formation means is a nanochannel. Within nanochannels, the ion distribution of counter- and co-ions, as defined relative to the surface charge, may be affected significantly by wall surface charge. When an electric field is applied over such a nanochannel, the current carries electrolyte counter- and co-ions asymmetrically, resulting in concentration polarization.
[0034] In the current method for separating, preferably for separating extracellular vesicles, preferably exosomes, the ion depletion formation means can be part of a fluidic apparatus. The fluidic apparatus may comprise at least one separation channel (C), wherein the separation channel (C) comprises a downstream end (D) and an upstream end (II), and a depletion zone formation means (N), placed in or connected to an intermediate region between the upstream end (II) and downstream end (D), in which the depletion zone (N) may enable ion concentration polarization. Preferably, the fluidic apparatus of the current method has at least two parallel microchannels, which are preferably connected by a nanochannel, over which an electrical field may be applied. In an embodiment, an electrical field is applied between the downstream and upstream end of the separation channel. Preferably, the electrical field varies from 1-1000 volt per centimeter, more preferably the electric field may be from 1-500 volt per centimeter, even more preferably the electric field may be from 1-250, 1-100, 1-75, 1-50, 1-30, 1-20 or 1-15 volt per centimeter, most preferably, the electric field may be from 3-12 volt per centimeter. The asymmetric distribution of anions and cations can make the nanochannel perm-selective, which is a term used to describe the ability of a membrane or material to selectively allow the passage of certain molecules or ions while preventing the passage of others, leading to concentration polarization, and in turn the formation of a depletion zone in the separation channel. In a preferred embodiment, the / a other microchannel (i.e. not the separation channel) as per the preferred fluidic apparatus, is connected to the ground, using a suitable ground voltage, as will be clear to the skilled person. In an embodiment, the sample comprises a combination of at least 5 separator boosters, wherein the first separator booster preferably has, at pH 7-14, an electrophoretic mobility between -2 and -5*1 O'9m2s-1V'1(e.g. glycine), wherein the second separator booster preferably has an electrophoretic mobility between -6 and -9*1 O'9m2s-1V'1(e.g. serine), wherein the third separator booster preferably has an electrophoretic mobility between -16 and -19*10-9m2s-1V'1(e.g. glycine-phenylalanine), wherein the fourth separator booster preferably has an electrophoretic mobility between -21 and -24*1 O'9m2s-1V'1(e.g. tricine) and wherein the fifth separator booster preferably has an electrophoretic mobility between -24 and -27*1 O'9m2s-1V'1(e.g. MOPSO). More preferably, preferably the first separator booster, at pH 7-14, has an electrophoretic mobility between -3 and -4*1 O'9m2s-1V’1(e.g. -3.0, -3.1 , -3.2, -3.3, -3.4, -3.5, -3.6, -3.7, -3.8, -3.9, -4.0*10-9m2s’1V’1), the second separator booster preferably has an electrophoretic mobility between -7 and -8*1 O'9m2s-1V’1, the third separator booster has an electrophoretic mobility between -17 and -18*1 O'9m2s-1V’1(e.g. -17.0, -17.1 , -17.2, -17.3, -17.4, -17.5, -17.6, -17.7, -17.8, -17.9, -18.0*10-9m2s’1V’1), the fourth separator booster has an electrophoretic mobility between -22 and -23*1 O'9m2s-1V'1and the fifth separator booster has an electrophoretic mobility between 24 and 26*1 O'9m2s'1V’1. More preferably the first separator booster has an electrophoretic mobility between -3 and -4*1 O'9m2s-1V’1, the second separator booster preferably has an electrophoretic mobility between -7 and -8*10-9m2s’1V'1(e.g. -7.0, -7.1 , -7.2, -7.3, -7.4, -7.5, -7.6, -7.7, -7.8, -7.9, -8.0*1 O'9m2s'1V'1), the third separator booster has an electrophoretic mobility between - 17 and -18*1 O'9m2s-1V’1, the fourth separator booster has an electrophoretic mobility between -22 and -23*1 O’9m2s’1V'1(e.g. -22.0, -22.1 , -22.2, -22.3, -22.4, -22.5, -22.6, -22.7, - 22.8, -22.9, -23.0*1 O'9m2s'1V'1) and the fifth separator booster has an electrophoretic mobility between 24 and 26*1 O’9m2s’1V'1(e.g. -24.0, -24.1 , -24.2, -24.3, -24.4, -24.5, -24.6, - 24.7, -24.8, -24.9, -25.0, 25.1 , -25.2, -25.3, -25.4, -25.5, -25.6, -25.7, -25.8, -25.9, -26.0*1 O’9m2s'1V'1). Preferably, the sample comprising separator boosters (preferably at most 20), comprises a combination of cysteine, glutamine, glycine, histidine, methionine, phenylalanine, serine, valine, alanine-alanine, alanine-glycine, glycine-histidine, glycine-phenylalanine, 1-methyl-L- histidine, 3-methyl-L-Histidine, MES, MOPSO.TAPSO, TAPS and / or tricine (preferably comprising at most 20 separator boosters).
[0035] In an embodiment, the sample comprising exosomes and at least 3 separator boosters (preferably comprising at most 20 separator boosters), comprises a combination of at least glycine, glycine-phenylalanine and tricine. The sample comprising exosomes and at least 3 and at most 20 separator boosters, comprises preferably at least 50 pg / mL, more preferably at least 40 pg / mL, even more preferably at least 30 mg / mL, yet even more preferably at least 20 pg / mL, yet even more preferably at least 10 pg / mL, yet even more preferably at least 5 pg / mL, yet even more preferably at least 1 pg / ml, yet even more preferably at least 1 pg / ml, most preferably at least 0.1 pg / mL of each separator booster. More preferably, the sample comprising exosomes and at least 3 and at most 20 separator boosters comprises between 0.1-1000 pg / ml of each separator booster, more preferably between 0.5 and 75 pg / ml, even more preferably between 0.5-50 pg / ml, yet even more preferably between 0.5-25 pg / ml, yet even more preferably between 0.5-10 pg / ml, most preferably between 1-10 pg / ml of each separator booster.
[0036] In an embodiment, said separators boosters may be present in the sample at equal concentration i.e. concentration of glycine:glycine-phenylalanine:tricine may be 1 :1 :1. Alternatively, it may be possible to vary the concentration of the individual separator boosters.
[0037] In another embodiment, the sample comprising exosomes and at least 3 and (preferably comprising at most 20) separator boosters, comprises a combination of at least glycine, serine, glycine-phenylalanine, tricine and MES. Said separators boosters may be present at equal concentration i.e. concentration of glycine:serine:glycine-phenylalanine:tricine:MES may be 1 : 1 : 1 : 1 : 1. Alternatively, it may be possible to vary the concentration of the individual separator boosters.
[0038] In yet another embodiment, the sample comprising exosomes and at least 3 (preferably comprising at most 20) separator boosters, comprises a combination of at least Glycine, Serine, 3-methyl-L-Histidine, alanine-glycine, glycine-phenylalanine, tricine, MES, and MOPSO. Said separators boosters may be present at equal concentration i.e. concentration of 1 :1 :1:1:1 :1 :1. Alternatively, it may be possible to vary the concentration of the individual separator boosters. In an embodiment, the sample comprising exosomes and at least 3 (preferably comprising at most 20) separator boosters may for example comprise at least 1 particle / ml, more preferably at least 1*102particles / ml, even more preferably at least 1*104particles / ml, yet even more preferably at least 1*106particles / ml, yet even more preferably at least 1*108particles / ml, yet even more preferable 1*101° particles / ml, yet even more preferably 1*1015particles / ml most preferably at least 1*102° particles / ml . Preferably, in the samples comprising exosomes and at least 3 (preferably comprising at most 20 separator booters), the concentration of each separator booster is that it assures the position and distance in the microchannel of preferably at least 50 pm, more preferably at least 30 pm, even more preferably at least 10 pm, yet even more preferably at least 5 pm, within at least 1 min of the experiment between two exosome zones or between one exosome zone and the next fraction comprising different components, preferably lipoproteins, protein aggregates, cellular debris.
[0039] In an embodiment, the method comprises, after applying an electrical field between the downstream end (D) and the upstream end (II), detecting the position and / or composition of the separate fractions comprising exosomes with varying electrophoretic mobility. In order to assess the position of the (separate) fractions, preferably a sensing element is employed, such as a laser, electrodes, other (temperature), CMOS sensor etc. However, the fraction of interest may be to be within reach of the sensor, or alternatively, the analyte zone of interest may be related to a second zone that is within reach of the sensor. Apart from assessing the separate fractions comprising exosomes with varying electrophoretic mobility, above- mentioned approach may be used to assess the position of the separator boosters.
[0040] In order to evaluate the analyte of interest (e.g. exosomes and / or other constituents, among which lipoproteins, protein aggregates and / or cellular debris), it is beneficial if its position in the separation channel could be precisely assessed. In the method of the disclosure, particularly after applying an electric field between downstream end (D) and the upstream end (II) of the separation channel, the position of the analyte can be determined by a process comprising the steps of: (I) creating an ion depleted zone, (II) focussing of analytes at the border of that zone; (III) separating analytes in multiple zones according to isotachophoretic principles, and (IV) tuning the balance of the flow through the focusing channel and the ion depletion flux such that the analyte zone or its related zones are positioned in the sensing area.
[0041] In an embodiment, the method comprises, after applying an electrical field between the downstream end (D) and the upstream end (II), adjusting the fluid flow (F) in the separation channel (C) and / or ion depletion rate (R) to move at least one focussed exosome zone in an upstream and / or downstream direction in the separation channel (C). The analytes ((e.g. exosomes and other constituents, among which lipoproteins, protein aggregates, and cellular debris) are preferably separated at a distance from each other and focused in the separation channel. The thus preconcentrated and self-focused analyte zones travel downstream, and once they arrive at their predetermined site as being continuously controlled, they are all subsequently transferred into an extraction channel by an applied fluid flow perpendicular to the main separation channel. The analytes may then be dispensed into reservoirs, and / or subjected to detailed analysis, or they may isolated further to achieve increased extraction purities.
[0042] The current disclosure further relates to the use of separator booster(s) in depletion zone isotachophoresis separation of extracellular vesicles, preferably exosomes. In other words, the current disclosure further relates to the use of separator boosters for separation of extracellular vesicles in depletion zone isotachophoresis, wherein the extracellular vesicles preferably are exosomes. The ‘separator booster(s)’ preferably separate analytes by positioning between analytes (e.g. extracellular vesicles) having a different electrophoretic mobility, e.g. a lower electrophoretic mobility on one side of the analyte and higher electrophoretic mobility on another side of the analyte. In addition or alternatively, the ‘separator booster(s)’ preferably separate the extracellular vesicles, preferably exosomes, without requiring and / or undergoing binding interactions, such as those interactions typically present during staining or immune-capture of the analytes. Preferably the separator booster(s) do(es) not stain or immune-capture the extracellular vesicles and / or exosomes, nor function as a drag-tag for the analytes. More preferably, said ‘separator booster(s)’ separate(s) the extracellular vesicles and / or exosomes based on their electrophoretic mobility, do(es) not bind the analytes and / or do(es) not modify the electrophoretic mobility of the extracellular vesicles and / or exosomes. Most preferably, said ‘separator booster(s)’ separate(s) the subpopulations of exosomes based on their electrophoretic mobility, do(es) not bind the exosomes and / or do(es) not modify the electrophoretic mobility of the exosomes.
[0043] The current disclosure further relates to the use, e.g. in depletion zone isotachophoresis separation of exosomes, of at least 1 (preferably at least 3, preferably at most 20) separator boosters, wherein at least one separator booster is a compound having an electrophoretic mobility between -20 and -30*10-9m2s-1V'1(e.g. tricine). More preferably the sample comprises at least 3 (preferably at most 20 separator boosters), wherein the first compound has an electrophoretic mobility between -2 and -10*1 O'9m2s-1V’1, wherein the second compound has an electrophoretic mobility between -10 and -20*1 O'9m2s-1V'1and wherein the third compound has an electrophoretic mobility between -20 and -30*1 O'9m2s-1V’1. Preferably, the use comprises at least three separator boosters, of which the electrophoretic mobility of the first separator booster ranges between -2 and -5*1 O'9m2s-1V’1, the electrophoretic mobility of the second separator booster ranges between -16 and -19*10-9m2s'1V'1and the electrophoretic mobility of the third separator booster ranges between -21 and - 24*1 O'9m2s'1V’1. Most preferably, the use comprises at least three separator boosters, of which the electrophoretic mobility of the first separator booster ranges between -3 and -4*1 O'9m2s'1V’1(e.g. -3.0, -3.1 , -3.2, -3.3, -3.4, -3.5, -3.6, -3.7, -3.8, -3.9, -4.0*10-9m2s’1V’1), the electrophoretic mobility of the second separator booster ranges between -17 and -18*10-9m2s'1V’1(e.g. -17.0, -17.1 , -17.2, -17.3, -17.4, -17.5, -17.6, -17.7, -17.8, -17.9, -18.0*10-9m2s’1V'1) and the electrophoretic mobility of the third separator booster ranges between -22 and - 23*1 O’9m2s'1V’1(e.g. -22.0, -22.1 , -22.2, -22.3, -22.4, -22.5, -22.6, -22.7, -22.8, -22.9, - 23.0*1 O’9m2s'1V’1).
[0044] The current disclosure further relates to the use of at least Glycine, Serine, 3-methyl-L- Histidine, Alanine-Glycine, Glycine-Phenylalanine, MES, and / or MOPSO in depletion zone isotachophoresis separation of exosomes.
[0045] DESCRIPTION OF FIGURES
[0046] Figure 1
[0047] Figure 1 discloses depletion zone isotachophoresis preconcentration efficiency of exosome samples obtained from plasma and cell lines. The x-axis shows the position in the microchannel and the and y-axis shows the fluorescence intensity.
[0048] Figure 2
[0049] Figure 2 discloses an electropherogram showing the separation of HEK293 cell-derived exosomes when exposed to a combination of separator boosters. As a control, the samples was exposed to deionized water. The x-axis shows the time and y-axis shows the fluorescence intensity.
[0050] Figure 3
[0051] Figure 3 discloses an electropherogram showing the separation of exosomes from protein and (lipo)protein-based impurities when exposed to a combination of separator boosters. The x-axis shows the time and y-axis shows the fluorescence intensity. Figure 4
[0052] Figure 4 discloses an electropherogram showing the separation of EVs samples spiked with lipoproteins when exposed to a combination of separator boosters. The x-axis shows the time and y-axis shows the fluorescence intensity.
[0053] Figure 5
[0054] Figure 5 discloses an electropherogram showing the separation of subpopulations of exosomes when exposed to a combination of separator boosters. The x-axis shows the time and y-axis shows the fluorescence intensity.
[0055] Figure 6
[0056] Figure 6 discloses an electropherogram showing the separation of an exosome comprising samples using dzITP, without tricine.
[0057] Figure 7
[0058] Figure 7 discloses an electropherogram showing the separation of an exosome comprising samples using dzITP, with tricine.
[0059] EXAMPLES
[0060] Example 1 : depletion zone isotachophoresis preconcentration of plasma-derived and cell-line derived exosomes
[0061] Figure 1 shows a comparison of plasma-derived (i.e. EXO plasma) concentration 1 ,35*10A9particles per mL and cell line-derived (i.e. EXO cell; SK-N-SH neuroblastoma-derived) concentration 1,8*10A8particles per mL exosome samples concentrated under the same depletion zone isotachophoresis conditions for 10 min (Channel dimensions 1.8cm length, 50 urn width, 2 urn hight. Voltage over separation channel 40 / 80, buffer 20 times diluted PBS). The exosomes were stained with CFDA-SE (5(6)-carboxyfluorescein diacetate succinimidyl ester) fluorescent staining. As can be seen, depletion zone isotachophoresis allows preconcentration of both sample types. In addition, depletion zone isotachophoresis is able to shown the difference in the different exosome samples, with cell-line derived exosomes being more homogeneous and containing a well-defined zone of vesicles, followed by protein impurities and excess fluorescent dye that was left over during the labelling procedure.
[0062] Plasma exosomes contain very heterogeneous exosomes particles and co-isolated impurities of lipoproteins, protein complexes and other analytes, that are present in the sample. Example 2: use of separator boosters in isotachophoresis separation of exosomes In this example, the separation of HEK293 cell-derived exosomes was evaluated. The exosomes were stained with CFDA-SE (5(6)-carboxyfluorescein diacetate succinimidyl ester) fluorescent staining and subsequently exposed to a combination of separator boosters (hereafter booster 1 or booster 2) or deionized water. Booster 1 comprised: 3uL of each of the following 10mg / L aqueous stock solutions: AlaAla, AlaGly, GlyPhe, 3-methyl-L-His, Ser, Gly, TAPS, MOPSO and d-glucuronic acid. Finally, 5uL MES was added to the mixture. Booster mix 2 comprised: 13.4uL of 10mg / L aqueous stock solutions of AlaAla, AlaGly, GlyPhe, GlyHis, GlyGly, Cys, Glu, His, Phe, Vai, 3-methyl-His, 1-methyl-his, Tricine, MOPSO and MES. In addition, 50uL Gly, 50uL Guluronic acid, 50uL octanesulfonic acid, 60uL Ser, 80uL Gin, 80uL Met, 80uL TAPSO 100uL ACES, 120uL TAPS. The separator boosters were mixed in a concentration of stained exosome-comprising sample:booster / deionized water 2:1.
[0063] As demonstrated in the electropherogram in Figure 2, last graph, without separator boosters, all exosome-associated analytes were contained within one singular peak at the end of the electropherogram. Addition of booster 1 did not only allow separation of the exosome- associated peaks from the dye-associated peaks, but also further divided the exosome- associated peaks into multiple separated peaks containing analytes with different electrophoretic mobilities (Figure 2, middle graph). Surprisingly, compared to when using booster 2 (24 boosters), using booster 1 (MIX 2) resulted in an electropherogram with a cleaner look by trimming unnecessary spacers that overlap with the unbound CFDA-SE peaks, while also achieving greater separation of the exosome-associated peaks (Figure 2, first and middle graph).
[0064] Example 3: use of separator boosters to separate exosomes from (lipo)protein-based impurities.
[0065] In this example, the ability of separator boosters to separate exosomes from other constituents was evaluated i.e. Evs, (lipo)proteins, excess dye. For this purpose, .. Lyophilised plasma-derived exosomes (HBM-PEP) CFDA-SE (5(6)-carboxyfluorescein diacetate succinimidyl ester) fluorescent staining was used in order to stain the exosomes. The leading electrolyte (LE) was prepared with 10 mM HCI and 0.35% hydroxypropylmethylcellulose (HPMC) and the terminating electrolyte (TE) consisted of 20 mM L-alanine (L-Ala), both adjusted with 2-amino-2-methyl-1 ,3-propanediol (ammediol) to, respectively, pH 8.8 and pH 9.4. An Agilent 7100 CE system was connected with a Zetalif 480nm LED induced fluorescence detector for cITP-LIF analysis. OV1701-OH coated fused silica capillaries with an internal diameter of 100um, total length of 32cm and effective length of 20cm were used. The samples were injected by applying -50mbar of pressure in the sample vial for 42 seconds. A voltage of 9 kV was applied across the system for 25 minutes. Separator booster mix was prepared by adding together 15uL of each of the following: MES, MOPSO, Tricine and Ser, to 5uL of each of the following: D-glucuronic acid, AlaAla, AlaGly, TAPSO, GlyPhe, GlyHis, 3-methyl-L-his, Gin, MET, Phe, 1-methyl-L-his, Gly, Vai and His.
[0066] As can be seen in Figure 3, carefully tuning the mixture of separator boosters enabled the current inventors to separate exosomes particles from protein and lipoprotein based impurities, that are not usually separated by ultracentrifugation and size-based method. The current inventors were also able to separate sub-populations of exosomes in the sample into peaks with discrete mobilities. As can be seen in Figure 4, when separating an exosome- comprising sample spiked with different types of lipoproteins i.e. HDL and LDL, the exosomes are located in the middle, indicating, that exosomes can be separated from (lipo)proteins.
[0067] Example 4: use of separator boosters to separate subpopulations of exosomes
[0068] In this example, the ability of separator boosters to separate subpopulations of exosomes was evaluated. For this purpose, a sample comprising HEK293 cell-derived, SK-N-SH neuroblastoma-derived and human plasma-derived exosomes was prepared. The sample was stained as described in example 1 and subsequently exposed to combination of separator boosters (hereafter booster 3). Booster 3 comprised AlaAla, AlaGly, GlyPhe, GlyHis, Gin, Gly, Met, His, Phe, Ser, Vai, 3-methyl-His, 1-methyl-his, Tricine, MES, MOPSO, d-glucuronic acid, TAPSO, TAPS.
[0069] As can be seen in Figure 4, significant differences were observed between the three subpopulations. The CFDA-SE-associated peaks are largely identical, but the exosome- associated peaks feature clear differences in distribution. Plasma-derived exosomes feature more analytes with heterogeneous mobilities such as EVs (possibly from different organs and tissues) and other impurities such as lipoproteins that were separated in this experiment. HEK293 cell-derived and SK-N-SH neuroblastoma-derived exosomes instead contain a larger share of EVs and they show their unique electrophoretic signatures of mobility distribution. Overall, the example demonstrates that different subpopulations of exosomes can be separated using separator compounds in isotachophoresis.
[0070] Example 5: the effect of using one separator booster on the separation efficiency using dzITP.
[0071] Brain cell-derived exosomes were prepared in PBS buffer and diluted to reach a suitable ionic strength to perform dzITP, either in the presence of absence of one separator booster i .e. tricine (1.5. mM). The analytes (e.g. exosomes and possible protein residues) were stained with CFDA-SE fluorescent dye. As can be seen by comparing Figure 6 (without tricine) and Figure 7 (with tricine), the exosomes (those closest to the nanochannel) were separated from the rest of the fluorescent analytes in the sample and in the case where Tricine was also present in the sample, the peaks were completely separated (i.e., with a resolution greater than 1.5, equal to 1.52) while in the case were Tricine is not present in the sample, the two peaks did not turn out to be completely separated and the resolution achieved was only 1 .09. Noteworthy, the plotted data was obtained after subtracting the baseline (corresponding to the background light signal, given by the experimental conditions, the optics of the experimental setup, and the camera used for the fluorescence microscope). For this, they start from a brightness level that is approximately equal to 160 arbitrary units. The formula used to calculate the resolution was taken from Desai and Armstrong, Microbiology and molecular biology reviews: MMBR 67(1):38-51 , where it was used to analyze electropherograms, and where minimum threshold values for the resolution in electropherograms are discussed. In the formula, instead of the migration time of analytes, the relative positions of the maximum of the two peaks with respect to the position of the nanochannel were used, which is considered position 0 in the reference system.
[0072] In this example, the effect of using varying amounts of separator boosters on the efficiency to separate exosomes (i.e., the capacity to isolate exosome-associated peaks without the interference of overlapping neighboring peaks that do not contribute to separating exosomes) is evaluated. A plasma-derived exosome sample is exposed to a number (3, 5, 10, 18, 24, 30) of different spacers and subsequently separated using conventional isotachophoresis or depletion zone isotachopohoresis. Noteworthy, each of the separator booster combinations comprises at least one separator booster with electrophoretic mobility ranging from -2 and - 10*1 O'9m2s'1V'1and / orone separator booster with electrophoretic mobility between -10 and - 20*1 O'9m2s'1V'1and / or one separator booster with electrophoretic mobility between -20 and -30*10-9m2s'1V’1. At least 2 sets of each spacer booster was evaluated. In this qualitative assessment (scale 1-10), a higher number indicates more efficient the separation of exosomes.
[0073] Table 1. Separation of exosomes using varying amounts of separator boosters (assessed separation resolution on scale 1-10 based on repeated experiments).
[0074] As can be seen from Table 1 , the use of separator boosters in general enhances the separation of exosomes, compared to when not using any separator booster (i.e. the higher the number, the better the separation of exosomes). Whereas the best results can be achieved with a combination of 18 separator boosters, using between 3 and 8 separator boosters results in sufficient exosome separation as well. Using more than 20 separator boosters i.e., 24, may, but does not necessarily further enhance the separation of exosomes. Moreover, Table 1 also demonstrates that compared to conventional isotachophoresis, depletion zone isotachophoresis results in superior separation of exosomes.
Claims
Claims1. A method for separating extracellular vesicles, preferably exosomes, said method comprising the steps of:- providing a sample comprising extracellular vesicles, preferably exosomes, and at least 1 separator booster, wherein at least 1 separator booster is a compound having electrophoretic mobility between -20 and -30*10-9 m2 s-1 V-1 at pH 7-14;- introducing said sample into an apparatus comprising at least a separation channel (C), wherein the separation channel (C) comprises a downstream end (D) and an upstream end (II), and an ion depletion zone formation means (N), placed in or connected to an intermediate region between the upstream end (II) and downstream end (D);- applying an electric field between 1 and 1000 volt per centimeter between the downstream end (D) and the upstream end (II), thereby separating extracellular vesicles, preferably exosomes.
2. Method according to claim 1 , further providing separate fractions comprising different components, preferably lipoproteins, protein aggregates, cellular debris, extracellular vesicles, preferably exosomes, and / or providing separate fractions comprising different extracellular vesicles, preferably exosomes.
3. Method according to any one of the preceding claims, wherein a sample is provided comprising at least 3 separator boosters, wherein a first separator booster is a compound having an electrophoretic mobility between -2 and -10*10-9 m2 s-1 V-1 at pH 7-14, wherein a second separator booster has an electrophoretic mobility between -10 and -20*10-9 m2 s-1 V-1 at pH 7-14, wherein a third separator booster has an electrophoretic mobility between -20 and -30*10-9 m2 s-1 V-1 at pH 7-14.
4. Method according to any one of the preceding claims, wherein the sample comprises at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5 separator boosters.
5. Method according to the preceding claims, wherein the separator boosters at least comprise glycine, glycine-phenylalanine and / or tricine.
6. Method according to any one of the preceding claims, wherein the separator boosters at least comprise a combination of glycine, serine, glycine-phenylalanine and tricine and MES.
7. Method according to any one of the preceding claims, wherein the separator boosters at least comprise a combination of glycine, serine, 3-methyl-L-histidine, alanine-glycine, glycinephenylalanine, tricine, MES and MOPSO.
8. Method according to any one of the preceding claims, comprising, after applying an electrical field between downstream end (D) and the upstream end (II), detecting the position and / or composition of separate fractions with varying electrophoretic mobility.
9. Method according to any one of the preceding claims, comprising, after applying an electrical field between downstream end (D) and the upstream end (II), adjusting the fluid flow (F) in separation channel (C) and / or ion depletion rate (R) to move at least one fraction in a upstream and / or downstream direction in the separation channel (C).
10. Use of separator boosters for separation of extracellular vesicles based on the electrophoretic mobility of the extracellular vesicles in depletion zone isotachophoresis, wherein the extracellular vesicles preferably are exosomes.
11. Use according to claim 10, wherein the separator boosters do not bind the extracellular vesicles, preferably exosomes.
12. Use according to any one of claims 10-11 , wherein at least 1 separator booster is used, wherein at least 1 separator booster is a compound having an electrophoretic mobility between -20 and -30*10-9 m2 s-1 V-1 at pH 7-14.
13. Use according to any one of claims 10-12, wherein at least 3 separator boosters are used comprising at least glycine, glycine-phenylalanine, and tricine.
14. Combination of at least 3 and at most 20 separator boosters, wherein a first separator booster is a compound having an electrophoretic mobility between -2 and -10*10-9 m2 s-1 V- 1 at pH 7-14, wherein a second separator booster is a compound having an electrophoretic mobility between -10 and -20*10-9 m2 s-1 V-1 at pH 7-14 and wherein a third separator booster is a compound having an electrophoretic mobility between -20 and -30*10-9 m2 s-1 V-1 at pH 7-14, further in combination with extracellular vesicles, preferably exosomes.
15. Combination of separator boosters comprising glycine, glycine-phenylalanine, and tricine, and comprising at most 20 separator boosters, further in combination with extracellular vesicles, preferably exosomes..