WO2022261716A1

WO2022261716A1 - Aqueous formulations for preservation of extracellular vesicles

Info

Publication number: WO2022261716A1
Application number: PCT/AU2022/050603
Authority: WO
Inventors: Patrick F JAMES; Karmen Jia Wen KONG
Original assignee: Exopharm Limited
Priority date: 2021-06-16
Filing date: 2022-06-16
Publication date: 2022-12-22

Abstract

The present invention relates to aqueous formulations comprising a polyol, a sugar or combination thereof, a non-ionic surfactant, a salt and a buffering agent. The aqueous formulation of the present invention further comprises extracellular vesicles (EVs). The present invention also relates to using said formulations for the preservation, in particular through freezing, and/or the administration of the EVs. Particular exemplified embodiments comprise 5% w/v sucrose or 12.5% w/v trehalose, 20 mM histidine, 150 mM NaCl and 0.01-0.2% w/v Polysorbate 20 with a pH of 6.0. These formulations can further comprise Plexaris or Cevaris which are EVs or exosomes, respectively.

Description

AQUEOUS FORMULATIONS FOR PRESERVATION OF EXTRACELLULAR

VESICLES

Field of the invention

[0001] The present invention relates to aqueous formulations, which may be suitable for use as buffer solutions for extracellular vesicles (EVs). The present invention also relates to aqueous formulations comprising an EV. The present invention further relates to methods of preserving an EV and methods of administering an EV formulation to a subject.

Background of the invention

[0002] Extracellular vesicles (EVs), such as exosomes, are a natural mechanism by which cells communicate and share material. EVs are a heterogeneous collection of biological structures that differ in size and are bound by membranes. These structures have a lipid bilayer and they may reside within a cell or in an extracellular environment. EVs range in size from about 20 nm to 1000 nm. EVs efficiently exchange information between cells and transfer biologically active proteins, lipids, and various nucleic acids including mRNA, miRNA, rRNA, IncRNA and DNA. Endogenous EVs naturally occur in vivo. EVs may also be produced ex vivo from a variety of sources - exogenous EVs.

[0003] There is interest in the use of EVs as therapeutics, which is partly due to the simplified cold chain procedures of EVs as compared to those required by cell-based treatments. However, in the absence of freezing EVs and being able to maintain their potency, the use of EVs is limited to immediately after collection only. Likewise, a thawed storage buffer would optimally be capable of maintaining EVs over a duration of time practical for timely administration to a patient, for example at least up to 48 hours post thaw. Accordingly, formulations for long term storage of EVs isolated from source cells which preserve the activity of EVs are of interest.

[0004] EV storage buffers developed to date have typically been based on phosphate buffered saline (PBS) or a cell media, such as Ringers solution. However, problems have been associated with storage of EVs in these buffers, including EV aggregation, flocculation and degradation. A common form of EV degradation is protein modification, with such modifications commonly occurring at a pH higher than 7.0. However, existing EV storage buffers based on PBS or a cell media typically acidify when frozen or during other periods during the cold chain handling process, which can lead to EV degradation. [0005] EV storage buffers have been developed which contain a cryoprotectant. Ideally, a cryoprotectant functions to protect EVs during freezing and thawing. In some cases, the protectant must also not be toxic to the cells to which the EVs are applied. However, not all cryoprotectants are necessarily capable of achieving these desired outcomes.

[0006] Accordingly, there is a need for new or alternative formulations that allow for long term storage of EVs, including formulations that maintain the integrity of EVs during the cold chain handling processes used between EV isolation and eventual patient delivery.

[0007] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art.

Summary of the invention

[0008] The present inventors have identified aqueous formulations comprising a specific combination of components which are capable of preserving EVs over an extended period of time, while maintaining the integrity of the EVs.

[0009] Accordingly, in one aspect the present invention provides an aqueous formulation comprising: a polyol, a sugar, or a combination thereof, in an amount of about 5 % (w/v) to about 15% (w/v), based on the total volume of the aqueous formulation; a surfactant; a salt; and a buffering agent having a buffering capacity at a pH equal to or less than about 7.0; wherein the aqueous formulation has a pH of equal to or less than about 7.0. [0010] In some embodiments, the aqueous formulation is for use, or when used as, a buffer solution for an EV.

[0011] In some embodiments, the aqueous formulation further comprises an EV.

[0012] In another aspect the present invention provides a method of preserving an EV comprising: combining an EV and the aqueous formulation described herein to provide an EV formulation; and freezing the EV formulation to provide a preserved EV formulation.

[0013] In another aspect the present invention provides a preserved EV formulation prepared by the method described herein.

[0014] In another aspect the present invention provides a method of administering an EV to a subject comprising: providing an EV formulation comprising an EV and the aqueous formulation described herein; and administering the EV formulation to the subject.

[0015] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

Brief description of the drawings

[0016] Figure 1. Size exclusion chromatography spectrum (214 nm) of Formulation 3 of Table 1 containing platelet EVs (PLX). Peaks A-D, Tail and Buffer peaks of the chromatography trace are denoted.

[0017] Figure 2. Plot illustrating normalised cell index over time of 1 :10 Formulation 3 of Table 1 containing PLX in Fibroblast Growth Media with 0.2% fetal calf serum (FCS) (empty triangles), compared to Formulation 3 alone in Fibroblast Growth Media with 0.2% FCS (filled triangles) and Fibroblast Growth Media with 2% FCS alone (empty circles) or 0.2% FCS alone (filled circles). [0018] Figure 3. Graphs illustrating normalised cell index of Formulation 5 containing 15 pg mL^-1 or 7.5 pg mL^-1 PLX after storage at -80°C for 0 months (Figure 3A), 6 months (Figure 3B) and 12 months (Figure 3C), compared to basal media, growth media and placebo.

[0019] Figure 4. Graphs illustrating normalised cell index of Formulation 5 containing 15 pg mL¹ or 7.5 pg mL¹ PLX after storage at -80°C for 0 months (Figure 4A) or 6 months (Figure 4B), compared to basal media and placebo. The symbol (^*) denotes significant difference from basal media (p < 0.05) and the symbol (^#) denotes significant difference from corresponding placebo control (p < 0.05).

Detailed description of the embodiments

[0020] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

[0001] Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

[0002] All of the patents and publications referred to herein are incorporated by reference in their entirety.

[0003] For the purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0004] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materials are described. For the purposes of the present disclosure, the following terms are defined below. [0005] The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “a sugar” means one sugar or more than one sugar.

[0006] As used herein, the term “and/or”, e.g., “X and/or Y” will be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0007] As used herein, the term “about” refers to a quantity, value, dimension, size, or amount that varies by as much as 10%, 5%, 1% or 0.1 % to a reference quantity, value, dimension, size, or amount.

[0008] Throughout the present disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0009] As used herein, unless the context requires otherwise, the term “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0010] A “subject” herein is preferably a human subject. It will be understood that the terms “subject” and “individual” are interchangeable in relation to an individual requiring administration of the aqueous formulation of the present disclosure.

[0011] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text. All of these different combinations constitute various alternative aspects of the invention. Formulation

[0012] In one aspect, there is provided an aqueous formulation comprising: a polyol, a sugar, or a combination thereof, in an amount of about 5 % (w/v) to about 15% (w/v), based on the total volume of the aqueous formulation; a surfactant; a salt; and a buffering agent having a buffering capacity at a pH equal to or less than about 7.0; wherein the aqueous formulation has a pH of equal to or less than about 7.0.

[0013] The buffering agent may be any suitable buffering agent having a buffering capacity at a pH equal to or less than about 7.0. The term “buffering capacity” will be understood to mean that the buffering agent is capable of substantially minimising a change in the pH of the aqueous solution. Accordingly, a suitable buffering agent may be capable of buffering an aqueous solution within the pH range of equal to or less than about 7.0. In some embodiments, the buffering agent has a buffering capacity at a pH from about 5.0 to about 7.0. In some embodiments, the aqueous formulation has a pH of less than about 7.0; especially a pH of from about 5.0 to less than about 7.0; more especially a pH of from about 5.5 to about 6.5; even more especially a pH of about 6.0.

[0014] It is known that the pH of an aqueous solution may shift upon freezing. In some embodiments, the buffering agent is capable of substantially minimising a change in the pH of the aqueous solution upon freezing. This may advantageously allow the aqueous formulation of the invention to be suitable for preserving biological material, which may include storing the biological material contained in the aqueous formulation at a freezing temperature over a period of time and subsequently thawing the aqueous formulation, wherein the structural integrity of the biological material in the frozen and thawed formulation is maintained. The biological material may be a liposome or EV, preferably an EV. In some embodiments, the buffering agent is capable of a minimising the change in pH by less than about 0.025 pH unit/°C, about 0.024 pH unit/°C, about 0.023 pH unit/°C, about 0.022 pH unit/°C, about 0.021 pH unit/°C, about 0.020 pH unit/°C, about 0.019 pH unit/°C, about 0.018 pH unit/°C, about 0.017 pH unit/°C, 0.016 pH unit/°C change, or about 0.015 pH unit/°C at a temperature below 0°C.

[0015] The buffering agent may be selected from one or more of histidine, citric acid, acetic acid, succinic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 2-(N-morpholino)ethanesulfonic acid (MES). In some embodiments, the buffering agent is histidine. Advantageously, as shown in the Examples, histidine is capable of acting as a buffering agent in the presence of the other components in the aqueous formulation of the invention, that is, histidine is shown to be functional as a buffering agent in combination with the other components of the aqueous formulation. In some embodiments, the histidine is L-histidine.

[0016] The buffering agent may be present in a concentration of from about 10 mM to about 30 mM. In some embodiments, the buffering agent is present in an amount of about 20 mM.

[0017] The aqueous formulation comprises a polyol, a sugar, or a combination thereof. The present inventors have found that the polyol, sugar or combination thereof can act as a cryoprotectant in the formulation described herein, which may preserve the structural integrity of an EV that may be contained in the formulation during freezing and thawing. Advantageously, the polyol, sugar or combination thereof is capable of acting as a cryoprotectant in the presence of the other components in the aqueous formulation of the invention. As shown in the Examples, aqueous formulations of the present invention are capable of storing EVs at freezing temperatures over an extended period of time. In contrast, it has been found that using other known cryoprotectants (eg DMSO, glycerine) can led to partial or complete lysing of EVs, consequently rendering the EVs ineffective.

[0018] The polyol, sugar or combination thereof is present in an amount of about 5 % (w/v) to about 15% (w/v), based on the total volume of the aqueous formulation. The present inventors have determined that this concentration may provide aqueous formulations suitable for storing or preserving EVs. Advantageously, as shown in the Examples, these concentrations were shown to not be toxic to cells from which EVs may be derived. Further, these concentrations may advantageously provide the aqueous formulation with osmolality suitable for use as an injectable formulation by a particular route of administration, such as intradermal injection. [0019] The polyol, sugar, or combination thereof may be selected from mannitol, sorbitol, sucrose, trehalose, mannose, dextran, and combinations thereof. In some embodiments, the polyol, sugar or combination thereof is a sugar, especially a sugar selected from sucrose, trehalose, and combinations thereof.

[0020] In some embodiments, the polyol, sugar, or combination thereof is present in an amount of about 5 % (w/v) to about 12.5% (w/v), especially about 7.5 % (w/v) to about 12.5% (w/v), based on the total volume of the aqueous formulation.

[0021] The aqueous formulation comprises a surfactant. The surfactant may be a non-ionic surfactant. In some embodiments, the non-ionic surfactant is selected from a polysorbate, a poloxamer, and combinations thereof. Examples of suitable polysorbates include polysorbate 20 and polysorbate 80. Examples of suitable poloxamers include poloxamer 188. In some embodiments, the surfactant is polysorbate 20. Advantageously, the surfactant may act to avoid or reduce the formation of protein aggregates on an EV that may be contained in the aqueous formulation.

[0022] The surfactant may be present in an amount of from about 0.001 % (w/v) to about 0.02 % (w/v), based on the total volume of the aqueous formulation. In some embodiments, the surfactant is present in an amount of from about 0.005 % (w/v) to about 0.0075 % (w/v), based on the total volume of the aqueous formulation. Advantageously, as shown in the examples, surfactant present in these concentrations were not toxic to cells.

[0023] The aqueous formulation comprises a salt. The salt may be selected from sodium chloride, potassium chloride, and combinations thereof. In some embodiments, the salt is sodium chloride.

[0024] The salt may be present in a concentration of from about 120 mM to about 180 mM. In some embodiments, the salt is present in a concentration of from about 130 mM to about 160 mM, especially a concentration of about 150 mM.

[0025] In some embodiments, the aqueous formulation comprises, or consists of: a sugar selected from sucrose, trehalose, and a combination thereof; polysorbate 20; sodium chloride; and histidine.

[0026] In some embodiments, the aqueous formulation comprises, or consists of: a sugar selected from sucrose, trehalose, and a combination thereof, in an amount of about 5 % (w/v) to about 15% (w/v), based on the total volume of the aqueous formulation; polysorbate 20 in an amount of from about 0.001 % (w/v) to about 0.02 % (w/v), based on the total volume of the aqueous formulation; sodium chloride in a concentration of from about 120 mM to about 180 mM; and histidine in a concentration of from about 10 mM to about 30 mM.

[0027] It will be understood that the aqueous formulations described herein, being aqueous, necessarily comprise water.

[0028] The term “consists of” with respect to an aqueous formulation described herein will be understood to imply that the aqueous formulations only include the components specified in the formulation (and water). That is, the aqueous formulations, other than water, do not include any further components in the aqueous formulations.

[0029] The aqueous formulation has a pH of equal to or less than about 7.0. Advantageously, providing a pH of equal to or less than about 7.0 may avoid or reduce the occurrence of protein modification to an EV that may be contained in the aqueous formulation. Such protein modifications may include deamination of amino acid side chains (eg conversion of asparagine to aspartate and/or glutamine to glutamate) and scrambling of disulphide bonds between cysteine side chains. In some embodiments, the aqueous formulation has a pH of from about 5.0 to about 7.0. In some embodiments, the aqueous formulation has a pH of less than about 7.0; especially a pH of from about 5.0 to less than about 7.0; more especially a pH of from about 5.5 to about 6.5; even more especially a pH of about 6.0.

[0030] Advantageously, the formulation of the invention may be suitable for use as a buffer solution for an extracellular vesicle (EV). The term “extracellular vesicle” as used herein is intended to encompass a vesicle within or outside a cell and which comprises a liquid or cytoplasm enclosed by a lipid bilayer. EVs typically contain cargo, which may be a therapeutic or drug cargo, for example one or more membrane proteins, cytosolic and nuclear proteins, extracellular matrix proteins, lipids, metabolites, and nucleic acids including DNA and RNA such as mRNA and non-coding RNA species. Examples of suitable types of cargo are described in Kalluri R and LeBleu V S ( Science . 2020 February 07; 367(6478). The EV may be a naive EV or an engineered EV. The term “naive EV” as used herein will be understood to mean an unmodified EV that is naturally produced by cells. Examples of suitable cells from which naive EVs can be derived include stem cells such as mesenchymal cells (MSCs), platelets and human induced pluripotent stem cells (hiPSCs), e.g., hiPSC derived neural stem cells. A naive EV may have a lipid bilayer comprising one or more phospholipids which are substantially similar to the lipid bilayer of the cell from which the EV is derived. The term “engineered EV” as used herein will be understood to mean vesicles that have been modified to express a targeting molecule on their surface and/or to carry a specific drug cargo. EVs may be obtained by methods known in the art, for example methods by described in WO201 8/112557 and WO2019/241836.

[0031] Accordingly, the present invention provides the aqueous formulation described herein when used as a buffer solution for an EV. The present invention also provides the use of the formulation described herein as a buffer solution for an EV. The present invention further provides the formulation described herein for use as a buffer solution for an EV.

[0032] The aqueous formulation of the invention may further comprise an EV. In some embodiments, the EV is selected from an exosome, a microvesicle, an oncosome and an apoptotic body, especially an exosome. The term “exosome” as used herein is intended to encompass EVs produced in the endosomal compartment of a eukaryotic cell. Exosomes typically have a diameter size of from about 40 nm to about 120 nm and contain protein and nucleic acid cargo. The term “microvesicle” as used herein is intended to encompass EVs released (shedded) from a cell plasma membrane. Microvesicles typically have a diameter size of from about 150 nm to about 1000 nm and contain protein and nucleic acid cargo. The term “oncosome” as used herein is intended to encompass EVs with pro-tumourigenic properties that are typically generated from the shedding of membrane blebs from cancer cells in more advanced stages of disease. Oncosomes (also called “large oncosomes”, LOs) typically have a diameter size of from about 1 pm to about 10 pm and contain oncogenic cargo including proteins. The term “apoptotic body” as used herein is intended to encompass EVs released from a cell plasma membrane during apoptosis. Apoptotic bodies typically have a diameter size of from about 500 nm to about 2000 nm and contain cargo including nuclear fractions and cell organelles.

[0033] The aqueous formulation of the invention may also be suitable for use as a buffer solution for a liposome. The term “liposome” as used herein is intended to encompass a vesicle having at least one lipid bilayer. A liposome may contain cargo, which may be a therapeutic or drug cargo, for example one or more membrane proteins, cytosolic and nuclear proteins, extracellular matrix proteins, metabolites, and nucleic acids including DNA and RNA such as mRNA and non-coding RNA species.

[0034] Accordingly, the present invention provides the formulation described herein when used as a buffer solution for a liposome. The present invention also provides the use of the formulation described herein as a buffer solution for a liposome. The present invention further provides the formulation described herein for use as a buffer solution for a liposome.

[0035] The aqueous formulation of the invention may further comprise a liposome. [0036] In some embodiments, the aqueous formulation comprises, or consists of: sucrose, trehalose, or a combination thereof; polysorbate 20; sodium chloride; histidine; and an EV.

[0037] In some embodiments, the aqueous formulation comprises, or consists of: sucrose, trehalose, or a combination thereof, in an amount of about 5 % (w/v) to about 15% (w/v), based on the total volume of the aqueous formulation; polysorbate 20 in an amount of from about 0.001 % (w/v) to about 0.02 % (w/v), based on the total volume of the aqueous formulation; sodium chloride in a concentration of from about 120 mM to about 180 mM; histidine in a concentration of from about 10 mM to about 30 mM; and an EV.

[0038] The aqueous formulation of the invention may be suitable for use as an injectable formulation. Advantageously, this may allow a biological material contained in the aqueous formulation described herein to be administered by injection. Accordingly, in some embodiments, the aqueous formulation is an injectable formulation. In some embodiments, the aqueous formulation is for use as an injectable formulation.

[0039] In some embodiments, the aqueous formulation has a calculated osmolality suitable for use as an injectable formulation by a particular route of administration, such as intradermal injection. In some embodiments, the aqueous formulation has a calculated osmolality of equal to or less than about 800 mOsm, equal to or less than about 700 mOsm, or equal to or less than about 600 mOsm. Osmolality may be calculated using the following equation:

Osm = x[Buffer] + y[Salt]+ [Surfactant] + [Sugar] where x is an integer denoting the number of ions present in the Buffer if the Buffer is provided as a salt, or is 1 if the Buffer is not provided as a salt; and where y is an integer denoting the number of ions present in the Salt. For example, when the Buffer is His (provided by His.HCI), xis 2; when the Buffer is citrate (provided by sodium citrate), is 2; when the Buffer is acetate (provided by sodium acetate), x is 2; when the Buffer is succinic acid (provided by disodium succinate), x is 3; when the Buffer is HEPES, x is 1 ; when the Buffer is MES (MES hydrate), x is 1 ; when the Salt is sodium chloride, y is 2; and when the Salt is potassium chloride, y is 2.

Methods of preparation

[0040] The aqueous formulation of the invention may be prepared by methods known in the art. [0041] In one aspect, the present invention provides a method of preparing an aqueous formulation comprising: dissolving in water: a polyol, a sugar, or a combination thereof, in an amount of about 5 % (w/v) to about 15% (w/v), based on the total volume of the aqueous formulation, a surfactant, a salt, and a buffering agent having a buffering capacity at a pH equal to or less than about 7.0; and optionally adjusting the pH to be equal to or less than about 7.0; thereby providing the aqueous formulation.

[0042] In the context of this method, the step of adjusting the pH is optional in that this step does not need to be conducted if the pH is already equal to or less than about 7.0 after the dissolving step, that is, the pH does not need to be adjusted after the dissolving step. The pH may be adjusted by methods known in the art, for example using hydrochloric, sulphuric, nitric, phosphoric, citric acid or a combination thereof.

[0043] The buffering agent used to prepare the aqueous formulation may be provided in any suitable form, including as a salt or a hydrate. In some embodiments, the buffering agent is histidine. In these embodiments, the histidine may be provided as a salt such as histidine hydrochloride.

[0044] The present invention also provides a method of preparing an EV formulation comprising: providing the aqueous formulation described herein or the aqueous formulation prepared by the method described herein; and combining the aqueous formulation and an EV; thereby providing the EV formulation. [0045] The step of combining the aqueous formulation and an EV to provide the EV formulation may be prepared by methods known in the art, including the methods described herein. The EV formulation may be prepared by directly combining the EV and the aqueous formulation, for example by dispersing the EV in the aqueous formulation. The EV may be provided in a concentrated saline solution to which the other components of the aqueous formulation may be added to provide the EV formulation. Alternatively, the EV may be provided in another formulation and buffer exchanged into the EV formulation, for example by size exclusion chromatography or dialysis.

Applications

[0046] The aqueous formulation of the invention may be suitable for preserving biological material such as an EV. As shown in the Examples, aqueous formulations in accordance with the disclosure herein containing EVs were capable of preserving EVs over an extended period of time, where the EVs were shown to be effective after freezing and thawing in the aqueous formulation.

[0047] Accordingly, in one aspect there is provided a method of preserving an EV comprising: combining an EV and the aqueous formulation described herein to provide an EV formulation; and freezing the EV formulation to provide a preserved EV formulation.

[0048] The step of freezing the EV formulation may comprise storing the EV formulation at a temperature sufficient to freeze the formulation. In some embodiments, the freezing temperature is equal to or less than about 0 °C, equal to or less than about -5 °C, equal to or less than about -20 °C, equal to or less than about -80 °C, or equal to or less than about -150 °C. In some embodiments, the freezing temperature is from about 0 °C to about -180 °C, from about -5 °C to about -180 °C, from about -20 °C to about -180 °C, from about -80 °C to about -180 °C, or from about -150 °C to about -180 °C. Any minimum and maximum amount can be combined to form a range provided that the range is within about 0 °C to about -180 °C, such as a range of from about -20 °C to about -150 °C. The rate of freezing may be uncontrolled, for example by passively storing the EV formulation at a freezing temperature, or may be controlled, for example by actively cooling (eg snap freezing) the EV formulation to the freezing temperature.

[0049] The EV formulation may be stored at the freezing temperature for a duration of time such that the structural integrity of the EV is preserved. In some embodiments, the EV formulation is stored at the freezing temperature for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, or at least about 14 days. In some embodiments, the EV formulation is stored at the freezing temperature for up to about 1 week, up to about 2 weeks, up to about 3 weeks, up to about 4 weeks, up to about 5 weeks, up to about 6 weeks, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months, up to about 12 months, up to about 15 months, up to about 18 months, up to about 21 months, or up to about 24 months. Any minimum and maximum amount can be combined to form a range provided that the range is within about 1 day to about 24 months, such as a range of from about 7 days to about 12 months.

[0050] The method may further comprise thawing the preserved EV formulation. It will be understood that the thawing step relates to thawing a frozen EV formulation. Accordingly, the step of thawing may comprise storing the (frozen) preserved EV formulation at a temperature sufficient to thaw the formulation. In some embodiments, the thawing temperature is equal to or above about -5 °C, equal to or above about 0 °C, equal to or above about 4 °C, equal to or above about 20 °C, or equal to or above about 25 °C. In some embodiments, the thawing temperature is from about -5 °C to about 37 °C, from about 0 °C to about 37 °C, from about 4 °C to about 37 °C, from about 20 °C to about 37 °C, or from about 25 °C to about 37 °C. Any minimum and maximum amount can be combined to form a range provided that the range is within about -4 °C to about 37 °C, such as a range of from about 0 °C to about 25 °C. The rate of thawing may be uncontrolled, for example by passively storing the (frozen) EV formulation at the thawing temperature, or may be controlled, for example by actively warming (eg by heating in a water bath) the EV formulation to the thawing temperature. [0051] The thawed EV formulation may be stored at a temperature and/or for a duration of time such that the structural integrity of the EV is preserved, prior to, for example, transferring the preserved EV to a new formulation (which may be the aqueous formulation described herein or another suitable formulation) or administering the preserved EV formulation to a subject. The method of transferring the preserved EV formulation to a new formulation include methods known in the art, including the methods described herein. The preserved EV formulation may be precipitated or centrifuged to provide an EV pellet, wherein the EV pellet may be dispersed in a new formulation. The preserved EV formulation may be buffer exchanged into a new formulation, for example by size exclusion chromatography or dialysis. Advantageously, the thawed EV formulation may be directly administered to a subject, without the need to transfer the preserved EV to a new formulation. Further, the thawed EV formulation may advantageously allow the EV to be maintained at a temperature and for a duration that is effective and practical for timely administration of the EV formulation to a subject.

[0052] In some embodiments, the thawed EV formulation is stored at a temperature of from about -5 °C to about 10 °C, from about 0 °C to about 10 °C, from about 0 °C to about 5 °C, or from about 0 °C to about 4 °C. Any minimum and maximum amount can be combined to form a range provided that the range is within about -5 °C to about 10 °C, such as a range of from about 0 °C to about 10 °C. In some embodiments, the thawed EV formulation is maintained at a temperature of about 4 °C. In some embodiments, the thawed EV formulation is stored at the temperature for up to about 1 hour, up to about 2 hours, up to about 3 hours, up to about 4 hours, up to about 5 hours, up to about 6 hours, up to about 7 hours, up to about 8 hours, up to about 9 hours, up to about 10 hours, up to about 11 hours, up to about 12 hours, up to about 14 hours, up to about 18 hours, up to about 20 hours, up to about 22 hours, up to about 24 hours, up to about 28 hours, up to about 32 hours, up to about 36 hours, up to about 40 hours, up to about 44 hours, up to about 48 hours, up to about 54 hours, up to about 60 hours, up to about 66 hours, or up to about 72 hours.

[0053] The stability of an EV formulation may be assessed by methods known in the art, including the methods described herein.

[0054] The aqueous formulation of the invention may be suitable for delivering an EV to a subject in need thereof. [0055] Accordingly, in one aspect there is provided a method of administering an EV to a subject comprising: providing an EV formulation comprising an EV and the aqueous formulation described herein; and administering the EV formulation to the subject.

[0056] Advantageously, the EV formulation may be suitable for use as an injectable formulation. Accordingly, in some embodiments, the EV formulation is an injectable formulation. In some embodiments, the EV formulation is for use as an injectable formulation.

[0057] The EV formulation may also be suitable for administration by injection. Examples of suitable routes of administration include parenteral administration, such as subcutaneous, intradermal, intramuscular and intravenous administration. Accordingly, in some embodiments, the EV formulation is administered by injection. In some embodiments, the EV formulation is administered parenterally, including subcutaneously, intramuscularly, or intravenously.

[0058] The EV formulation may be the preserved EV formulation described herein. Accordingly, in some embodiments, the EV formulation is the preserved EV formulation described herein, which has been frozen and thawed. As discussed above, advantageously, the thawed EV formulation may be directly administered to a subject, without the need to transfer the preserved EV to a new formulation.

Examples

[0059] The invention will be further described by way of non-limiting example(s). It will be understood to persons skilled in the art of the invention that many modifications may be made without departing from the spirit and scope of the invention.

Example 1. Aqueous formulations

[0060] To identify aqueous formulations which may be suitable for preserving EVs, the aqueous formulations in Table 1 were prepared using standard techniques. In brief, the aqueous formulations were prepared by dissolving histidine (as histidine hydrochloride salt), NaCI, polysorbate 20 (PS20) and sucrose/trehalose in the appropriate volume of MilliQ (pure) water, and adjusting the pH of the formulation.

Table 1. Aqueous formulations

a Calculated based on the equation Osm = 2[His] + 2[NaCI] + [PS20] + [Sugar], based on the addition of histidine as the hydrochloride salt.

[0061] The aqueous formulation in Table 2 was prepared as a comparative formulation.

Table 2. Comparative aqueous formulation

[0062] Formulations containing purified EVs were prepared by mixing purified EVs of interest with an aqueous formulation, both in amounts suitable to provide a desired concentration of purified EVs in the formulation. Example 2. Methods for stability assessments

[0063] The suitability of the formulations of Table 1 for storing and preserving EVs may be assessed by various techniques, including size exclusion chromatography (SEC), bicinchoninic acid (BCA) assay, and microfluidic resistive pulse sensing analysis.

[0064] For the SEC experiments, 15 mI_ of each sample was injected onto a Superose 6 Increase column (3.2/300) from Cytiva. The flow rate was set at 0.15 ml_ min^-1. The Shimadzu BioHPLC was run in isocratic mode with Buffer A: 1xPBS (phosphate- buffered saline), pH 7.4, 100 nm filtered. The column oven temperature was set at 30 °C. Total run duration was 30 min. Data was collected using a PDA. The PDA (UV detector) was set to scan from 190 - 800 nm. The SEC trace was segmented into 5 areas, based on retention time 6 mins-Peak A-7.2 mins-Peak B-8.4 mins-Peak C-9.8 mins-Peak D-11 mins-Tail-12.5 mins. EVs are known to elute in the void peak (Peak A), whilst smaller particles and free protein would be retained on the column and elute later. The areas for each peak were calculated by the LabSolutions software using the manual integration function. The average of three injections were used to determine % area of each peak. Blank injections were used between samples to reduce carry over. SEC may be used to identify changes in the distribution of composition of an EV sample. By way of example, particle rupture may be identified by a drop in Peak A, and a resultant increase in the later peaks. Protein aggregation may result in a shift in the opposite direction.

[0065] BCA assays were conducted using the Pierce™ BCA Protein Assay Kit (Thermo Fisher), following the manufacturer’s instructions. Briefly, bovine serum albumin (BSA) standards were prepared by diluting a stock solution of 2 mg/ml_ BSA with MilliQ water. Concentrations were made to 15.625, 31.25, 62.5, 125, 250, 500 and 1000 pg/mL by serial dilution. A ratio of 4:1 of RIPA buffer (Thermo Fisher) to samples was prepared and incubated for 5 minutes. Formulation controls were also treated equally. Samples were spun down at 13,000 rpm, 7 minutes at 4°C. Working reagent was prepared by mixing 50 parts Solution A and 1 part Solution B. 25 mI_ of each standard (triplicate) and samples (triplicate) were added into a flat bottom 96-well plate. 200 mI_ of working reagent was added to each well. The plate was incubated at 37 °C for 30 minutes in BMG Labtech CLARIOstar® Plus reader prewarmed to 37 °C.

Reading was taken at the wavelength of 562 nm. The results were analysed using the MARS software associated with the CLARIOstar plate reader and results are presented as pg/mL. The BCA assay may be used to identify changes in total protein concentration. By way of example, if protein aggregation and/or precipitation occurs during EV storage, a decrease in the total protein concentration may be observed.

[0066] The microfluidic resistive pulse sensing analyses were performed using a Spectradyne particle size analyser. The Spectradyne particle size analyser combines microfluidic technology and nanotechnology to detect and measure particles (eg EVs) in a formulation. Spectradyne implements microfluidic resistive pulse sensing (MRPS), which uses electrical sensing to measure the diameter of each particle as it passes through a nanoconstriction, providing real-time sizing and concentration information. For the microfluidic resistive pulse sensing analyses, 10 pL of each sample was routinely diluted in PBST, 0.02 urn filtered. 5 pL was aliquoted into a CS-400 cartridge and loaded onto the Spectradyne particle size analyser. The acquisition time was set at 10 s. The first acquisition of each sample was excluded from processing. Where possible, datasets for each sample are collected until the apparent error of the particle analysis is less than 1 % based on the default processing conditions. The total concentration of particles was calculated by deriving the area under the curve from 65 nm - 400 nm using the analysis software, Viewer. Three sizing and concertation standards are analysed on the same day with the same chipset. These standards have the following parameters: 93.5 nm 2.2 x 10¹³ particle rmL¹, 150.4 nm 5.35 x10¹² particle mL¹, 197.6 nm 2.36x10¹² particle mL¹. These standards were analysed at a 1 :5,000 dilution on a CS-400 cartridge. Post-processing of the data was conducted which included the subtraction of background particles, correcting for the diameter factor (based on the standards), correcting for the concentration factor (based on the standards) and finally multiplying by the dilution factor.

Example 3. Stability assays using Formulations 2 and 3 containing PLX0

[0067] The stability of Plexaris (purified EVs derived from platelets, PLX0) in Formulations 2 and 3 of Table 1 was investigated by SEC, BCA assay, and microfluidic resistive pulse sensing analysis in line with the methods described in Example 2.

[0068] PLX0 was formulated in Formulations 2 and 3 of Table 1 to give a final PLX0 total protein concentration of 0.2 mg/mL. The PLX0 formulations were stored at -80°C and assessed at various time points (0 weeks, 1 week, 2 weeks), the frozen formulations being thawed at the time points for assessment. A PLXO formulation that was not frozen was also assessed.

[0069] The SEC results are provided in Table 3. Figure 1 shows an illustrative SEC spectrum (214 nm) of the Formulation 3, 0 weeks, no freeze/thaw sample, denoting Peaks A-D and Tail areas.

Table 3. Size exclusion chromatography (SEC) results for Formulations 2 and 3

[0070] As shown in Table 3, the % of Peaks A-D and Tail areas did not substantially vary under the different storage conditions. These results indicate that the sample was stable for at least 1 week in Formulation 2, and at least 2 weeks in Formulation 3, based on SEC analysis. In comparison, samples based on Formulation C1 were found to fail the SEC analysis after 1 week. In particular, a change in Peak C was observed during freezing (0 weeks) from 48.8 ± 21.34 to 54.3 ± 5.26, and then after 1 week to 44.9 ± 8.98. These area differences primarily came from changes in Peak D. The sudden changes in peak areas indicated that Formulation C1 was not stable up to 1 week. [0071] The results of the BCA assay are provided in Table 4. As shown in Table 4, the total protein concentration did not substantially vary under the different storage conditions. Therefore, the results show that the storage of platelet EVs at -80°C for up to 2 weeks did not adversely affect the protein concentration, indicating that minimal to no protein aggregation and/or precipitation occurred during EV storage. These results indicate that the sample was stable for at least 2 weeks, based on BCA analysis.

Table 4. Bicinchoninic acid (BCA) assay results for Formulation 3

[0072] The microfluidic resistive pulse sensing analyses results are provided in Table 5. As shown in Table 5, the particle size and concentration did not substantially vary under the different storage conditions. Therefore, the results show that the storage of platelet EVs at -80°C for up to 2 weeks in Formulation 2 or 3 did not adversely affect the particle concentration or distribution. These results indicate that the samples were stable for at least 2 weeks, based on microfluidic resistive pulse sensing analysis.

Table 5. Microfluidic resistive pulse sensing analysis results for Formulations 2 and 3

[0073] The above results provide an indication that aqueous formulations according to the present invention may be useful for preserving EVs over an extended period of time, such that the integrity of the EVs is maintained.

Example 4. Proliferation assay using Formulations 2 and 3 containing PLXO

[0074] The activity of Plexaris (purified EVs derived from platelets, PLXO) in Formulation 3 of Table 1 was investigated in a normal human dermal fibroblast (NHDF) cell proliferation assay.

[0075] The NHDF cell proliferation assay was performed according to the following procedure. Normal Human Dermal Fibroblast (NHDF) cells from Lonza were cultured in T25 flask in Fibroblast Growth Medium-2 (FGM™-2) with 2 % FBS (Growth Media) added the week before commencing the proliferation assay. The cells were detached with TrypLe (2 mL/T25 flask) for 5 min at 37 °C. The cells were centrifuged at 230-250 xg for 5 min, and then resuspended in 1 mL of Full Media. 5000 cells/well were added into the xCELLigence E-plate. Cells were incubated overnight to allow attachment. Each sample to be analysed was diluted with Fibroblast Growth Medium-2 (FGM™-2) with 0.2 % FBS (Basal Media, n=3). Growth Media was discarded, and the freshly prepared media were added into the wells accordingly. The plate was placed back into the incubator and the experiment was resumed. The cell index was recorded with the RTCA software, once every hour for 100 hours subsequently. The cell index data was normalized to the timepoint two points before the removal of the plate for treatment. The cell indices after 72 hr of incubation with each sample were plotted with Graph Pad Prism. [0076] PLXO was formulated in Formulation 3 of Table 1 to give a final PLXO total protein concentration of 0.2 mg/mL The PLXO formulations were stored at -80°C and assessed at various time points (0 weeks, 1 week), the frozen formulations being thawed at the time points for assessment. The thawed formulations were diluted 1 :10 in Fibroblast Growth Media with 0.2% FCS. Formulations containing 1 :10 Formulation 3 alone in Fibroblast Growth Media with 0.2% FCS and control samples containing either 2% FCS or 0.2% FCS alone were also assessed.

[0077] Figure 2 illustrates proliferation results of the PLXO samples after 1 week storage, Formulation 3 alone in Fibroblast Growth Media with 0.2% FCS and control 2% FCS and 0.2% FCS samples. The data in Figure 2 is normalised to a maximum attachment of 0.2% FCS. The results show that 1 :10 PLX in Formulation 3 in Fibroblast Growth Media with 2% FCS (empty triangles) displayed growth in proliferation over time and a similar increase in cell index over time to positive control 2% FCS alone (empty circles). The 1 :10 Formulation 3 alone in Fibroblast Growth Media with 0.2% FCS (filled triangles) displayed a comparable cell index to negative control 0.2% FCS alone (filled circles). These results indicate that storage of platelet EVs at -80°C for 1 week did not adversely affect the efficacy of the EVs on cells at 1 :10 dilution.

[0078] The activity of PLXO in Formulation 2 of Table 1 was also assessed following the procedure described above, except that Formulation 2 was used instead of Formulation 3. The PLXO in Formulation 2 samples also displayed growth in the proliferation assay.

[0079] The above results provide an indication that aqueous formulations according to the present invention may be useful for preserving EVs over an extended period of time, such that the integrity and efficacy of the EVs is maintained.

Example 5. Stability assay using Formulation 5

[0080] The stability of Plexaris (purified EVs derived from platelets, PLXO) in Formulation 5 of Table 1 was investigated by microfluidic resistive pulse sensing analysis in line with the methods described in Example 2.

[0081] PLXO was formulated in Formulation 5 of Table 1 to give a final PLXO total protein concentration of 0.3 mg/mL. The PLXO formulations were stored at -80°C and assessed at various time points (2 days, 27 days, and 7 months and 3 weeks), the frozen formulations being thawed at the time points for assessment.

[0082] The microfluidic resistive pulse sensing analysis results are provided in Table 6. As shown in Table 6, the particle size and concentration did not substantially vary under the different storage conditions. Therefore, the results show that the storage of platelet EVs at -80°C for up to 7 months and 3 weeks in Formulation 5 did not adversely affect the particle concentration or distribution. These results indicate that the samples were stable for at least 7 months and 3 weeks, based on microfluidic resistive pulse sensing analysis.

Table 6. Microfluidic resistive pulse sensing analysis results for Formulation FI

Example 6. Stability assays using Formulation 5 containing PLX1

[0083] The stability of Plexaris (purified EVs derived from platelets, autologous, PLX1) in Formulation 5 of Table 1 was investigated by BCA assay and microfluidic resistive pulse sensing analysis in line with the methods described in Example 2.

[0084] PLX1 was formulated in Formulation 5 of Table 1 to give a final PLX0 total protein concentration of 0.3 mg/mL. The PLX1 formulations were stored at -80°C and assessed at various time points (0 months, 3 months, 6 months, 12 months), the frozen formulations being thawed at the time points for assessment.

[0085] The results of the BCA assay are provided in Table 7. As shown in Table 7, the total protein concentration did not substantially vary under the different storage conditions. Therefore, the results show that the storage of platelet EVs at -80°C for up to 12 months did not adversely affect the protein concentration, indicating that minimal to no protein aggregation and/or precipitation occurred during EV storage. These results indicate that the sample was stable for at least 12 months, based on BCA analysis.

Table 7. BCA assay results for Formulation 5 containing PLX1

[0086] The microfluidic resistive pulse sensing analysis results are provided in Table 8. As shown in Table 8, the particle size and concentration did not substantially vary under the different storage conditions. Therefore, the results show that the storage of platelet EVs at -80°C for up to 7 months and 3 weeks in Formulation 5 did not adversely affect the particle concentration or distribution. These results indicate that the samples were stable for at least 12 months, based on microfluidic resistive pulse sensing analysis.

Table 8. Microfluidic resistive pulse sensing analysis results for Formulation 5 containing PLX1

Example 7. Proliferation assays using Formulation 5 containing PLX1

[0087] The activity of Plexaris (purified EVs derived from platelets, autologous, PLX1 ) in Formulation 5 of Table 1 was investigated in a NHDF cell proliferation assay.

[0088] The NHDF proliferation assay was performed as follows. Normal Human Dermal Fibroblast (NHDF) cells from Lonza were cultured in T75 flask in Fibroblast Growth Medium-2 (FGM™-2) with 2 % FBS (Growth Media) added the week before commencing the proliferation assay. The cells were detached with TrypLe (2 ml_/T75 flask) for 5 min at 37 °C. The cells were centrifuged at 230-250 xg for 5 min, and then resuspended in 1 ml_ of Growth Media. 5000 cells/well were added into the xCELLigence E-plate. Cells were incubated overnight to allow attachment. After 24 hr, Growth Media was removed and wells were washed with Fibroblast Growth Medium-2 (FGM™-2) with 0.1 % FBS (Basal Media). Cells were then incubated for a further 14 - 18 hr in Basal Media (starvation period). Each sample to be analysed was diluted with Fibroblast Growth Medium-2 (FGM™-2) with 0.1 % FBS (Basal Media, n=5). Placebo samples constituted formulation alone (without API). Basal media was discarded, and the freshly prepared media were added into the wells accordingly. The plate was placed back into the incubator and the experiment was resumed. The cell index was recorded with the RTCA software, once every hour for 100 hours subsequently. The cell index data was normalized to the timepoint prior to the addition of the treatment. The cell indices for each sample were plotted with GraphPad Prism.

[0089] PLX1 was formulated in Formulation 5 of Table 1 to give a final PLX0 concentration of 15 pg rmL¹ or 7.5 pg rmL¹. The PLX0 formulations were stored at -80°C and assessed at various time points (0 months, 6 months, 12 months), the frozen formulations being thawed at the time points for assessment.

[0090] Figure 3 illustrates proliferation results of the PLX1 samples after storing for 0 months (Figure 3A), 6 months (Figure 3B) and 12 months (Figure 3C). The data in Figure 3 is normalised to the time point prior to the addition of the treatment, as described in the procedure above. The results show that PLX1 in each sample demonstrated cell proliferation activity. These results indicate that PLX1 retained bioactivity after 12 months storage in Formulation 5 at -80°C.

Example 8. Proliferation assays using Formulation 5 containing PLX2 [0091] The activity of Plexaris (purified EVs derived from platelets, allogeneic, PLX2) in Formulation 5 of Table 1 was investigated in a NHDF cell proliferation assay.

[0092] The NFIDF cell proliferation assay was performed as follows. Normal Fluman Dermal Fibroblast cells from Lonza were cultured in T75 flask in Fibroblast Growth Medium-2 (FGM™-2) with 2 % FBS (Growth Media) added the week before commencing the proliferation assay. The cells were detached with TrypLe (2 ml_/T75 flask) for 5 min at 37 °C. The cells were centrifuged at 230-250 xg for 5 min, and then resuspended in 1 ml_ of Growth Media. 5000 cells/well were added into the xCELLigence E-plate. Cells were incubated overnight to allow attachment. After 24 hr, Growth Media was removed and wells were washed with Fibroblast Growth Medium-2 (FGM™-2) with 0.1 % FBS (Basal Media). Cells were then incubated for a further 14 - 18 hr in Basal Media (starvation period). Each sample to be analysed was diluted with Fibroblast Growth Medium-2 (FGM™-2) with 0.1 % FBS (Basal Media, n=5). Placebo samples constituted formulation alone (without API). Basal media was discarded, and the freshly prepared media were added into the wells accordingly. The plate was placed back into the incubator and the experiment was resumed. The cell index was recorded with the RTCA software, once every hour for 100 hours subsequently. The cell index data was normalized to the timepoint prior to the addition of the treatment. The cell indices for each sample were plotted with Graph Pad Prism.

[0093] PLX2 was formulated in Formulation 5 of Table 1 to give a final PLX2 concentration of 15 pg rmL¹ or 7.5 pg rmL¹. The PLX0 formulations were stored at -80°C and assessed at various time points (0 months, 6 months, 12 months), the frozen formulations being thawed at the time points for assessment.

[0094] Figure 4 illustrates proliferation results of samples after storing for 0 months (Figure 4A) and 6 months (Figure 4B) at -80°C, freeze/thaw. The data in Figure 4 is normalised to the time point prior to the addition of the treatment, as described in the procedure above. As shown in the Figures, PLX2 in both 0 month and 6 month samples produced significantly higher cell indexes compared to both 0.1% FBS basal media and placebo controls at 36 hours post-treatment (p < 0.05). The results show that PLX2 at 15 pg/rmL promotes higher NHDF cell proliferation compared to negative controls at 36 hours post-treatment. These results provide an indication PLX2 retained bioactivity after 6 months storage in Formulation 5 at -80°C. Example 9. Stability assays using Formulations 6 and 7 containing PLX

[0095] The stability of Plexaris (purified EVs derived from platelets, PLX) in Formulations 6 and 7 of Table 1 was investigated by BCA assay and microfluidic resistive pulse sensing analysis in line with the methods described in Example 2.

[0096] PLX was formulated in Formulations 6 and 7 of Table 1 to give a final PLX total protein concentration of 0.3 mg/mL. The PLX0 formulations were stored at -80°C and assessed at various time points (0 weeks, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months, 9 months), the frozen formulations being thawed at the time points for assessment.

[0097] The results of the BCA assay are provided in Table 9. As shown in Table 9, the total protein concentration did not substantially vary under the different storage conditions. Therefore, the results show that the storage of platelet EVs at -80°C for up to 9 months in Formulation 6 or 7 did not adversely affect the protein concentration, indicating that minimal to no protein aggregation and/or precipitation occurred during EV storage. These results indicate that the samples were stable for at least 9 months, based on BCA analysis.

Table 9. BCA assay results for Formulations 6 and 7 containing PLX

[0098] The microfluidic resistive pulse sensing analysis results up to 6 months are provided in Table 10. As shown in Table 10, the particle size and concentration did not substantially vary under the different storage conditions. Therefore, the results show that the storage of platelet EVs at -80°C for up 6 months in Formulation 6 or 7 did not adversely affect the particle concentration or distribution. These results indicate that the samples were stable for at least 6 months, based on microfluidic resistive pulse sensing analysis.

Table 10. Microfluidic resistive pulse sensing analysis results for Formulations 6 and 7 containing PLX

Example 10. Stability assays using Formulations 5 and 6 containing CEV

[0099] The stability of Cevaris (purified exosomes derived from bone marrow, CEV) in Formulations 5 and 6 of Table 1 was investigated by BCA assay and microfluidic resistive pulse sensing analysis in line with the methods described in Example 2.

[0100] CEV was formulated in Formulations 5 and 6 of Table 1 to give a final CEV total protein concentration of 1 mg/mL The CEV formulations were stored at -80°C and assessed at various time points (0 weeks, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months), the frozen formulations being thawed at the time points for assessment.

[0101] The results of the BCA assay for samples stored between 0 weeks to 3 months are provided in Table 11. As shown in Table 11 , the total protein concentration did not substantially vary under the different storage conditions. Therefore, the results show that the storage of bone marrow exosomes at -80°C for up to 3 months in Formulation 5 or 6 did not adversely affect the protein concentration, indicating that minimal to no protein aggregation and/or precipitation occurred during EV storage. These results indicate that the samples were stable for at least 3 months, based on BCA analysis.

Table 11. BCA assay results for Formulations 5 and 6 containing CEV

[0102] Table 12 provides the microfluidic resistive pulse sensing analysis results for CEV in Formulation 5 stored up to 3 months and CEV in Formulation 6 for up to 6 months. As shown in Table 12, the particle size and concentration did not substantially vary under the different storage conditions. Therefore, the results show that the storage of bone marrow exosomes at -80°C for up to 3 months in Formulation 5 or up to 6 months in Formulation 6 did not adversely affect the particle concentration or distribution. These results indicate that the samples were stable for at least 3 months in Formulation 5 and at least to 6 months in Formulation 6, based on microfluidic resistive pulse sensing analysis. Table 12. Microfluidic resistive pulse sensing analysis results for Formulations 5 and 6 containing CEV

[0103] The above results provide an indication that aqueous formulations according to the present invention may be useful for preserving EVs over an extended period of time, such that the integrity and efficacy of the EVs is maintained, even at high EV total protein concentrations of 1 mg/mL

Claims

1. An aqueous formulation comprising: a polyol, a sugar, or a combination thereof, in an amount of about 5 % (w/v) to about 15% (w/v), based on the total volume of the aqueous formulation; a surfactant; a salt; and a buffering agent having a buffering capacity at a pH equal to or less than about 7.0; wherein the aqueous formulation has a pH of equal to or less than about 7.0.

2. The aqueous formulation according to claim 1 when used as a buffer solution for an extracellular vesicle (EV).

3. The aqueous formulation according to claim 1 or claim 2, wherein the polyol, sugar or combination thereof is selected from mannitol, sorbitol, sucrose, trehalose, mannose, dextran, and combinations thereof.

4. The aqueous formulation according to any one of claims 1 to 3, wherein the buffering agent is selected from one or more of histidine, citric acid, acetic acid, succinic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 2-(N- morpholino)ethanesulfonic acid (MES).

5. The aqueous formulation according to any one of claims 1 to 4 wherein the buffering agent is histidine.

6. The aqueous formulation according to any one of claims 1 to 5, wherein the buffering agent is present in a concentration of from about 10 mM to about 30 mM.

7. The aqueous formulation according to any one of claims 1 to 6, wherein the surfactant is a non-ionic surfactant.

8. The aqueous formulation according to any one of claims 1 to 7, wherein the surfactant is selected from a polysorbate, a poloxamer, and combinations thereof.

9. The aqueous formulation according to claim 8, wherein one or both of the following apply: the polysorbate is selected from polysorbate 20 and polysorbate 80; the poloxamer is poloxamer 188.

10. The aqueous formulation according to any one of claims 1 to 8, wherein the surfactant is present in an amount of from about 0.001 % (w/v) to about 0.02 % (w/v), based on the total volume of the aqueous formulation.

11. The aqueous formulation according to any one of claims 1 to 10, wherein the salt is selected from sodium chloride, potassium chloride, and combinations thereof.

12. The aqueous formulation according to any one of claims 1 to 11 , wherein the salt is present in a concentration of from about 120 mM to about 180 mM.

13. The aqueous formulation according to any one of claims 1 to 12, wherein the aqueous formulation comprises: a sugar selected from sucrose, trehalose, or a combination thereof; polysorbate 20; sodium chloride; and histidine.

14. The aqueous formulation according to any one of claims 1 to 13, wherein the aqueous formulation comprises: a sugar selected from sucrose, trehalose, or a combination thereof, in an amount of about 5 % (w/v) to about 15% (w/v), based on the total volume of the aqueous formulation; polysorbate 20 in an amount of from about 0.001 % (w/v) to about 0.02 % (w/v), based on the total volume of the aqueous formulation; sodium chloride in a concentration of from about 120 mM to about 180 mM; and histidine in a concentration of from about 10 mM to about 30 mM.

15. The aqueous formulation according to any one of claims 1 to 14, wherein the aqueous formulation has a pH of from about 5.0 to about 7.0.

16. The aqueous formulation according to any one of claims 1 to 15, wherein the aqueous formulation further comprises an EV.

17. A method of preserving an EV comprising: combining an EV and the aqueous formulation according to any one of claims 1 to 16 to provide an EV formulation; and freezing the EV formulation to provide a preserved EV formulation.

18. The method according to claim 17, further comprising thawing the preserved EV formulation.

19. A preserved EV formulation prepared by the method according to claim 17 or claim 18.

20 A method of administering an EV to a subject comprising: providing an EV formulation comprising an EV and the aqueous formulation according to any one of claims 1 to 16; and administering the EV formulation to the subject.

21. The method according to claim 20, wherein the EV formulation is administered by injection.

22. The method according to claim 20 or claim 21 , wherein the EV formulation is the preserved EV formulation according to claim 19.