Method for producing lipid bilayer membrane particle and solution composition

Abstract

A solution containing lipid bilayer membrane particles is filtered by passing it in the planar direction of a filter membrane in the presence of a nonionic surfactant at a concentration of 0.02-0.08% (w / v).

Description

Method for producing lipid bilayer particles and solution composition

[0001] This disclosure relates to a method for producing lipid bilayer particles and a solution composition.

[0002] Extracellular vesicles (EVs) are a general term for vesicles that have a lipid bilayer structure. Extracellular vesicles are broadly classified into three types based on differences in their intracellular production mechanisms: exosomes, microvesicles, and apoptotic bodies. These extracellular vesicles are expected to have applications in the diagnosis and treatment of diseases. Disease diagnosis using extracellular vesicles utilizes the miRNA contained within them as a diagnostic marker. Disease treatment using extracellular vesicles is being researched for various diseases based on the biological functions of extracellular vesicles.

[0003] Patent Document 1 discloses a method for purifying extracellular vesicles by combining cation exchange chromatography, anion exchange chromatography, and mixed-mode chromatography. Furthermore, Patent Document 1 discloses that, after purifying extracellular vesicles using the various chromatography methods described above, the solution is further concentrated into additive-free phosphate-buffered saline by tangential flow filtration and buffer exchange.

[0004] Furthermore, Patent Document 2 discloses a method for purifying a solution containing extracellular vesicles by adsorbing them onto a column using ion exchange chromatography and eluting the extracellular vesicles from the column. Methods for purifying a solution containing extracellular vesicles include size exclusion chromatography, ultrafiltration, and tangential flow filtration. In particular, Patent Document 2 discloses that in tangential flow filtration, the substitution solvent for purification may contain 0.1% by mass of a nonionic surfactant (e.g., polyoxyethylene polyoxypropylene copolymer).

[0005] Patent Document 1: International Publication No. 2020 / 191369 Patent Document 2: International Publication No. 2023 / 238837

[0006] Incidentally, as disclosed in Patent Documents 1 and 2, when the present inventors attempted to filter lipid bilayer particles, such as extracellular vesicles, by passing them through a filtration filter in the in-plane direction, a problem was found in that the number of lipid bilayer particles decreased. Therefore, the present disclosure aims to provide a method for producing lipid bilayer particles and a solution composition that prevents the decrease of lipid bilayer particles even when filtering by passing the lipid bilayer particles through a filtration filter in the in-plane direction, and that has an excellent recovery rate for lipid bilayer particles.

[0007] To achieve the above-mentioned objectives, the present inventors conducted diligent studies and found that the reduction of lipid bilayer particles can be prevented by passing the lipid bilayer particles through a filtration filter in the in-plane direction in the presence of 0.02 (w / v)% to 0.08 (w / v)% of a nonionic surfactant, thus completing this disclosure. This disclosure includes the following: <1> A method for producing lipid bilayer particles, comprising filtering a solution containing lipid bilayer particles through a filtration filter in the in-plane direction in the presence of 0.02 (w / v)% to 0.08 (w / v)% of a nonionic surfactant. <2> The method for producing lipid bilayer particles according to <1>, wherein the filtration is further carried out in the presence of a chelating agent. <3> The method for producing lipid bilayer particles according to <1> or <2>, wherein the nonionic surfactant is an ether-type nonionic surfactant or an ester-ether-type nonionic surfactant. <4> The method for producing lipid bilayer particles according to any one of <1> to <3>, wherein the nonionic surfactant is a copolymer of polyoxyethylene and polyoxypropylene. <5> The method for producing lipid bilayer particles according to <2>, wherein the chelating agent is at least one selected from the group consisting of ethylenediaminetetraacetic acid, glycol etherdiaminetetraacetic acid, and salts thereof. <6> The method for producing lipid bilayer particles according to any one of <1> to <5>, wherein the filtration is tangential flow filtration. <7> The method for producing lipid bilayer particles according to any one of <1> to <6>, wherein the lipid bilayer particles are at least one selected from the group consisting of extracellular vesicles, viruses, and artificially produced particles. <8> A solution composition used in a filtration method in which a solution containing lipid bilayer particles is passed through a filtration filter in the in-plane direction, comprising a nonionic surfactant at a concentration of 0.02 (w / v)% to 0.08 (w / v)% at the time of use. <9> The solution composition according to <8>, further comprising a chelating agent. <10> The solution composition according to <8> or <9>, wherein the nonionic surfactant is an ether-type nonionic surfactant or an ester-ether-type nonionic surfactant. <11> The solution composition according to any one of <8> to <10>, wherein the nonionic surfactant is a copolymer of polyoxyethylene and polyoxypropylene.<12> The solution composition according to <9>, wherein the chelating agent is at least one selected from the group consisting of ethylenediaminetetraacetic acid, glycol etherdiaminetetraacetic acid, and salts thereof. <13> The solution composition according to any one of <8> to <12>, wherein the filtration method is a tangential flow filtration method. <14> The solution composition according to any one of <8> to <13>, wherein the lipid bilayer particles are at least one selected from the group consisting of extracellular vesicles, viruses, and artificially produced particles.

[0008] According to the method for producing lipid bilayer particles and the solution composition of this disclosure, even when the lipid bilayer particles are filtered by passing them through the in-plane direction of a filtration filter, the reduction of lipid bilayer particles can be prevented, and the recovery rate of lipid bilayer particles can be improved.

[0009] Figure 1 is a characteristic diagram showing the recovery rates calculated for Examples 1 to 7 and Comparative Examples 1 to 4.

[0010] The embodiments of this disclosure are described below. The description is illustrative and does not limit the scope of this disclosure.

[0011] In the following embodiments, the components (including elemental steps, etc.) are not essential unless otherwise explicitly stated. The same applies to numerical values ​​and their ranges, and these do not limit the disclosure. For example, the disclosure allows for additions, omissions, substitutions, and changes to numbers, quantities, locations, ratios, materials, compositions, types, and sequences, etc., without departing from the intent of the disclosure.

[0012] In this disclosure, numerical ranges indicated using "~" include the numbers before and after "~" as the minimum and maximum values, respectively. In numerical ranges described in stages in this disclosure, the upper or lower limit of one numerical range may be replaced by the upper or lower limit of another numerical range described in stages. Also, in numerical ranges described in this disclosure, the upper or lower limit of that range may be replaced by the values ​​shown in the examples. In this disclosure, each component may contain multiple types of the corresponding substance. If multiple types of the substance corresponding to each component exist in the composition, the content or amount of each component means the total content or amount of the multiple types of substances present in the composition, unless otherwise specified. In this disclosure, "A and / or B" is synonymous with "at least one of A and B," and includes both cases where it means both A and B, or where it means either A or B.

[0013] The present disclosure's method for producing lipid bilayer particles involves filtering a solution containing lipid bilayer particles by passing it through a filter in the in-plane direction in the presence of 0.02 (w / v)% to 0.08 (w / v)% of a nonionic surfactant. The present disclosure's method for producing lipid bilayer particles can suppress the reduction of lipid bilayer particles in the solution due to filtration and improve the recovery rate of lipid bilayer particles. The reason why the presence of 0.02 (w / v)% to 0.08 (w / v)% of a nonionic surfactant in the present disclosure's method for producing lipid bilayer particles can suppress the reduction of lipid bilayer particles during the above-mentioned filtration is not clear, but it is presumed that the nonionic surfactant envelops the surface of the lipid bilayer particles and improves their hydrophilicity, thereby contributing to protection from damage caused by increased contact between the lipid bilayer particles and components such as the filter, or between lipid bilayer particles themselves, under pressurized conditions when filtering by passing the solution through the filter in the in-plane direction.

[0014] Furthermore, the solution composition of this disclosure is used in a filtration method in which a solution containing lipid bilayer particles is passed through a filtration filter in the in-plane direction, and contains a nonionic surfactant at a concentration of 0.02 (w / v)% to 0.08 (w / v)% at the time of use. That is, when the solution composition of this disclosure is mixed with a solution containing lipid bilayer particles, the concentration of the nonionic surfactant becomes 0.02 (w / v)% to 0.08 (w / v)%. For example, if the solution composition of this disclosure is mixed with a solution containing lipid bilayer particles or other solutions and diluted 10-fold, the solution composition of this disclosure is prepared to contain 0.2 (w / v)% to 0.8 (w / v)% of the nonionic surfactant before mixing.

[0015] [Lipid Bilayer Particles] In this disclosure, lipid bilayer particles are not particularly limited to particles having a lipid bilayer, and include, for example, extracellular vesicles, viruses, and artificially created particles. Extracellular vesicles refer to particles surrounded by a lipid bilayer that are released from cells and do not have a nucleus, and include exosomes, microvesicles, ectosomes, membrane particles, exosome-like vesicles, apoptotic bodies, adipose tissue, etc., but do not include viruses. Although extracellular vesicles and exosomes are sometimes defined as having the same meaning, in this disclosure, they are different from extracellular vesicles, and exosomes are included as one form of extracellular vesicles. Exosomes are approximately 30 nm to 200 nm in diameter, are formed by inward budding of the late endosomal membrane, and then fuse with the cell membrane to form a complete particle, which is released extracellularly by exocytosis. Microvesicles are approximately 100 nm to 1000 nm in diameter and are generated when the cell membrane buddings outward and separates. Apoptotic bodies are approximately 50 nm to 5000 nm in diameter and are released extracellularly when a cell undergoes apoptosis.

[0016] Examples of viruses include those with an envelope consisting of a lipid bilayer (enveloped viruses). For example, enveloped viruses include those classified into the Herpesviridae, Poxviridae, Hepadnaviridae, Flaviviridae, Togaviridae, Coronaviridae, Hepatitis D virus, Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, Bunyaviridae, Filoviridae, and Retroviridae families. Note that the envelope may also contain proteins or other materials in addition to the lipid bilayer.

[0017] Artificially produced particles are those formed by the self-assembly of lipid molecules having a hydrophilic head and a hydrophobic hydrocarbon chain in water. Artificially produced particles include liposomes, which are formed from natural lipids such as phospholipids. These artificially produced particles, such as liposomes, can encapsulate active ingredients for disease treatment within a lipid bilayer. Active ingredients include proteins, small molecule compounds, and nucleic acids such as RNA molecules. The size of artificially produced particles such as liposomes is not particularly limited and can range from approximately 100 nm to approximately 50 μm.

[0018] [Nonionic Surfactants] In this disclosure, nonionic surfactants are also referred to as nonionic surfactants. Nonionic surfactants are surfactants that have hydroxyl groups and / or ether bonds in their hydrophilic groups that do not dissociate into ions when dissolved in water. Examples of nonionic surfactants include ether type, ether ester type, ester type, and nitrogen-containing type.

[0019] Examples of ether-type nonionic surfactants include polyoxyethylene alkyl ethers, alkylphenyl ethers, alkylallylformaldehyde condensed polyoxyethylene ethers, copolymers of polyoxyethylene and polyoxypropylene, and polyoxyethylene polyoxypropylene alkyl ethers. In particular, in the method for producing lipid bilayer particles of this disclosure, it is preferable to use a copolymer of polyoxyethylene and polyoxypropylene as the nonionic surfactant. A commercially available example of a copolymer of polyoxyethylene and polyoxypropylene is Pluronic.

[0020] Examples of ester ether type nonionic surfactants include polyoxyethylene ether of glycerin ester, polyoxyethylene ether of sorbitan ester, and polyoxyethylene ether of sorbitol ester.

[0021] Examples of ester-type nonionic surfactants include polyethylene glycol fatty acid esters, glycerin esters, polyglycerin esters, sorbitan esters, propylene glycol esters, and sucrose esters.

[0022] Examples of nitrogen-containing nonionic surfactants include fatty acid alkanolamides, polyoxyethylene fatty acid amides, and polyoxyethylene alkylamides.

[0023] In addition to these, other examples of surfactants include fluorine-based surfactants.

[0024] [Filtration] In the method for producing lipid bilayer particles according to the present disclosure, a solution containing lipid bilayer particles is passed through a filtration filter in the in-plane direction to perform filtration. One embodiment of this filtration method is the tangential flow filtration method. The tangential flow filtration method is also called the cross-flow filtration method, and because the solution is passed through the filtration filter in the in-plane direction (for example, in a direction parallel to the membrane surface), filtration can be performed while suppressing the deposition of lipid bilayer particles and the like on the membrane surface of the filtration filter.

[0025] In the method for producing lipid bilayer particles according to this disclosure, the purpose of filtration is not particularly limited, and may include the removal of particles smaller than the lipid bilayer particles contained in the solution containing the lipid bilayer particles, the concentration of the lipid bilayer particles, the exchange of buffers for the solution containing the lipid bilayer particles, and the desalting of the solution containing the lipid bilayer particles. The solution containing the lipid bilayer particles contains 0.02 (w / v)% to 0.08 (w / v)% of a nonionic surfactant, and further contains a composition corresponding to the purpose of filtration described above. In particular, the solution containing the lipid bilayer particles preferably contains 0.025 (w / v)% to 0.08 (w / v)% of a nonionic surfactant, and more preferably contains 0.025 (w / v)% to 0.075 (w / v)% of a nonionic surfactant.

[0026] Furthermore, in the method for producing lipid bilayer particles according to this disclosure, filtration is preferably performed in the presence of a chelating agent. That is, the solution composition according to this disclosure preferably contains a chelating agent. By performing filtration in the presence of a chelating agent, the recovery rate of lipid bilayer particles can be further improved. It is presumed that the chelating agent acts to improve the recovery rate of lipid bilayer particles by inhibiting calcium-dependent intercellular adhesion factors, proteases, etc. The chelating agent is not particularly limited, but it is preferably one that can chelate calcium ions. In this case, the chelating agent may specifically chelate calcium ions, or it may non-selectively chelate metal ions other than calcium ions.

[0027] Specifically, examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), glycol etherdiaminetetraacetic acid (EGTA), 1,2-bis(o-aminophenoxide)ethane-N,N,N',N'-tetraacetic acid (BAPTA), N'-(2-hydroxyethyl)ethylenediamine-N,N,N'-triacetic acid (HEDTA), nitrilotriacetic acid (NTA), and sodium citrate. Among these, it is preferable that the chelating agent be at least one selected from the group consisting of EDTA, EGTA, and their salts. Furthermore, it is preferable to use EDTA in the form of salts such as EDTA-2K, EDTA-3K, EDTA-4K, EDTA-2Na, EDTA-3Na, and EDTA-4Na. One type of these EDTA salts may be used, or a mixture of multiple types may be used.

[0028] In a solution containing lipid bilayer particles used for filtration, the concentration of the chelating agent is not particularly limited, but can be 0.0006 (w / v)% to 0.05 (w / v)%, preferably 0.0007 (w / v)% to 0.05 (w / v)%, more preferably 0.0008 (w / v)% to 0.05 (w / v)%, even more preferably 0.001 (w / v)% to 0.05 (w / v)%, even more preferably 0.002 (w / v)% to 0.05 (w / v)%, and even more preferably 0.003 (w / v)% to 0.05 (w / v)%. Furthermore, in a solution containing lipid bilayer particles, the upper limit of the concentration of the chelating agent is not particularly limited, but can be, for example, 0.04 (w / v)%, and preferably 0.03 (w / v)%. By setting the concentration of the chelating agent within this range in a solution containing lipid bilayer particles used for filtration, the reduction of lipid bilayer particles during filtration can be more effectively prevented, further improving the recovery rate of lipid bilayer particles.

[0029] In the method for producing lipid bilayer particles according to the present disclosure, the filtration filter is not particularly limited, but a nominal molecular weight cutoff (NMWC) of, for example, 50 kDa to 1000 kDa can be used, preferably 50 kDa to 750 kDa, more preferably 50 kDa to 500 kDa, even more preferably 50 kDa to 300 kDa, and even more preferably 50 kDa to 100 kDa.

[0030] Furthermore, the solution containing lipid bilayer particles used for filtration preferably contains a buffering agent. The term "buffering agent" is not particularly limited and refers to any compound that exhibits pH buffering properties when in solution. Examples of buffering agents include weakly acidic compounds, combinations of weakly acidic compounds and their salts, weakly basic compounds, and combinations of weakly basic compounds and their salts. pH buffering properties refer to the ability to suppress rapid changes in pH and maintain the pH of the solution within a specific range, even when other acids or bases are added to the solution to a certain extent. Therefore, a solution containing a buffering agent can maintain its pH within a specific range even when acids or bases are added within a predetermined range.

[0031] Buffering agents include, but are not limited to, citrate, phosphate, succinate, cacodylate, 2-morpholinoethanesulfonic acid (referred to as MES), 2,2-bis(hydroxymethyl)-2,2',2''-nitrilotriethanol, N-(2-acetamide)iminodiacetic acid, N-(2-acetamide)-2-aminoethanesulfonic acid, 1,4-piperazinedietanesulfonic acid, imidazole, hydroxypropanesulfonic acid, 3-morpholinopropanesulfonic acid (referred to as MOPS), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (referred to as TES), 4-(N-morpholino)butanesulfonic acid, 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (referred to as TAPS), 2 -Hydroxy-N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (referred to as TAPSO), 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, piperazine-1,4-bis(2-hydroxypropanesulfonic acid), triethanolamine, 4-(2-hydroxyethyl)-1-piperazine-1-propanesulfonic acid, tris(hydroxymethyl)aminomethane (referred to as Tris), tricine, glycylglycine, bicine, 4-(2-hydroxy Examples include ethyl)piperazine-1-ethanesulfonic acid (referred to as HEPES), 2-amino-2-methyl-1,3-propanediol, 2-(hydroxyethyl)amino-1-propanesulfonic acid, boric acid, 2-cyclohexylaminoethanesulfonic acid (referred to as CHES), glycine, sodium 3-cyclohexylamino-2-hydroxypropanesulfonate, 3-cyclohexylaminopropanesulfonic acid, 4-(cyclohexylamino)-1-butanesulfonic acid, etc.

[0032] The method for producing lipid bilayer particles according to the present disclosure, as described above, prevents a reduction in the amount of lipid bilayer particles contained in the solution due to the filtration described above, and improves the recovery rate of lipid bilayer particles after filtration. The recovery rate is calculated as the ratio of the amount of lipid bilayer particles remaining in the filtered solution to the amount of lipid bilayer particles contained in the solution before filtration. The amount of lipid bilayer particles in the solution is not particularly limited, but can be measured using proteins specifically present on the surface of the lipid bilayer particles as an indicator. Specifically, a labeled antibody against the protein can be used to quantitatively measure the lipid bilayer particles based on the absorbance derived from the label. In particular, according to the method for producing lipid bilayer particles according to the present disclosure, the recovery rate measured as described above can be, for example, 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 93% or more, even more preferably 95% or more, even more preferably 97% or more, and most preferably 98% or more.

[0033] [Other steps] The method for producing lipid bilayer particles according to the present disclosure may include other steps in addition to the filtration described above. For example, the method for producing lipid bilayer particles according to the present disclosure may include culturing cells that produce lipid bilayer particles and recovering the lipid bilayer particles from the cultured cells.

[0034] <Cell Culture> In this disclosure, the cells are not particularly limited and primary cultured cells, cultured cell lines, recombinant cultured cell lines, etc., can be used. The origin of the cells is not particularly limited and includes mammals such as humans, chimpanzees, monkeys, cattle, horses, pigs, dogs, cats, rabbits, rats, mice, and hamsters; birds such as chickens, etc. Cells hybridized from two or more cells of different species may also be used. The organs and tissues from which the cells originate are not particularly limited and include, for example, the hematopoietic and lymphatic system, the vascular system, the brain and nervous system, bone marrow, muscle tissue, thymus, salivary glands, oral cavity, esophagus, stomach, liver, gallbladder, spleen, small intestine, large intestine, rectum, skin, cornea, lungs, thyroid gland, mammary organs, uterus, cervix, ovaries, testes, pancreas, kidneys, adrenal cortex, bladder, placenta, umbilical cord, fetus, tail, mesenchymal stem cells, cancer cells, etc.

[0035] Furthermore, examples of cells include pluripotent stem cells such as embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic tumor cells (EC cells), embryonic germ cells (EG cells), nuclear transfer ES cells, and somatic cell-derived ES cells; mesenchymal stem cells (MSCs) such as bone marrow-derived mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, and umbilical cord-derived mesenchymal stem cells; hematopoietic stem cells, other stromal-derived stem cells, Muse cells, and neural stem cells; and various stem cells such as progenitor cells and fibroblasts in various tissues such as liver, pancreas, adipose tissue, bone tissue, cartilage tissue, and nerve tissue. These cells may also be activated by stimulation with cytokines such as IFNγ, TNFα, IL1β, and IL6.

[0036] Furthermore, the cells may include cells containing viruses. There are no particular restrictions on the cells from which the virus is derived, as long as the desired virus can proliferate. Preferably, these are packaging cells into which a portion of the genes necessary for virus production have been introduced, and which alone do not produce the virus. These are eukaryotic cells, and preferably include HEK293 cells, HEK293T cells, HEK293F cells, HEK293FT cells, G3T-hi cells, Sf9 cells, and commercially available virus-producing cell lines, which have high transfection efficiency.

[0037] As for cell culture, the conditions usually used for culturing the target cells can be used as they are or modified according to the cell type. As the medium used for cell culture, either a serum medium or a serum-free medium may be used. Examples of the medium include Eagle's medium, Dulbecco's modified Eagle's medium (low glucose or high glucose), Eagle's MEM medium, αMEM medium, IMDM medium, Ham's F10 medium, Ham's F12 medium, RPMI1640 medium, etc. Additives may be added to the medium as necessary. Examples of the additives include vitamins such as vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin D; coenzymes such as folic acid; amino acids such as glycine, alanine, arginine, asparagine, glutamine, isoleucine, leucine; sugars or organic acids as carbon sources such as lactic acid; growth factors such as EGF, FGF, PGDF, TGF-β; interleukins such as IL-1, IL-6; cytokines such as TNF-α, TNF-β, leptin; metal transporters such as transferrin; metal ions such as iron ions, selenium ions, zinc ions; SH reagents such as β-mercaptoethanol, glutathione; proteins such as albumin, etc.

[0038] The cell culture conditions are not particularly limited, and conditions suitable for each cell may be used. Usually, cell culture is carried out at a temperature in the range of 30°C to 40°C, preferably 36°C to 37°C, and the pH is in the range of 6.2 to 7.7, preferably 7.4, and the CO ２ concentration can be carried out in an environment of 4% to 10% by volume, preferably 5% to 7% by volume.

[0039] <Recovery of Lipid Bilayer Particles> The method for producing lipid bilayer particles according to this disclosure may include recovering the lipid bilayer particles from cells that produce the lipid bilayer particles prior to the filtration described above. For the recovery of lipid bilayer particles, if the lipid bilayer particles are released extracellularly, such as into extracellular vesicles, the culture supernatant of cell culture is used. For the recovery of lipid bilayer particles, if the lipid bilayer particles, such as viruses, are intracellular, the cells are subjected to lysis, disruption, or other treatments, and the solution after the lysis or disruption treatment is used. The culture supernatant can be obtained, for example, by filtering or centrifuging the culture medium used for cell culture. Examples of cell disruption treatments include mechanical stirring, ultrasonic disruption, and freeze-thaw cycles. Examples of cell lysis treatments include solution extraction, chemical treatments that appropriately adjust the pH and salt concentration of the solution to be extracted, and combinations thereof. For example, if the lipid bilayer particles are viruses, the viruses can be recovered by contacting the virus-containing cells with a surfactant.

[0040] Furthermore, a process may be performed to separate components other than lipid bilayer particles contained in the culture supernatant, lysis solution, and lysation solution from the lipid bilayer particles. This process may include ultrafiltration (a different type of filtration than that described above), ultracentrifugation, polymer precipitation, affinity purification, and column chromatography. Affinity purification methods include purification using a substance that binds to molecules present on the surface of lipid bilayer particles. When the lipid bilayer particles are extracellular vesicles, for example, a method using a phosphatidylserine-binding substance (PS affinity method) can be used. The phosphatidylserine-binding substance is preferably Tim protein, more preferably Tim4 protein, Tim3 protein, or Tim1 protein, and even more preferably Tim4 protein. Examples of chromatographic methods include affinity chromatography, ion exchange chromatography, size exclusion chromatography, hydrophobic chromatography, gel filtration chromatography, reverse-phase chromatography, and immobilized metal ion affinity chromatography. Preferably, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, and combinations thereof (also referred to as multimode chromatography, mixed-mode chromatography, etc.), and size exclusion chromatography are used. Multimode chromatography (mixed-mode chromatography) utilizes multiple different separation modes (ion exchange, hydrophobic bonding, affinity bonding, size exclusion, etc.).

[0041] Specifically, when the lipid bilayer particles are extracellular vesicles, affinity chromatography using the recovered culture supernatant and a column that specifically binds to extracellular vesicles can be applied. A column that specifically binds to extracellular vesicles is, for example, a column having a phosphatidylserine-binding substance that binds to phosphatidylserine present on the surface of extracellular vesicles in a calcium-dependent manner, such as a column having the Tim4 protein (T cell immunoglobulin and mucin domain-containing molecule 4 protein). As chromatography based on such a principle, MassivEV manufactured by Fujifilm Wako Pure Chemical Corporation can be used. ＴＭ EV Purification Column PS / MassivEV ＴＭ A Purification Buffer Set can be used.

[0042] In particular, when using a column having the Tim4 protein that binds to phosphatidylserine present on the surface of extracellular vesicles in a calcium-dependent manner, the culture supernatant is passed through the column. Thereby, the lipid bilayer particles contained in the culture supernatant are affinity adsorbed to the column. Thereafter, column washing using a washing solution and elution of the lipid bilayer particles using an eluent are performed. In this case, the eluent contains a chelating agent such as EDTA in order to capture calcium ions and elute the lipid bilayer particles bound to the Tim4 protein.

[0043] Therefore, when using a column having the Tim4 protein, lipid bilayer particles can be recovered as a solution containing the above-described chelating agent. Therefore, when passing and filtering a solution containing lipid bilayer particles in the in-plane direction of a filtration filter, it is also preferable to consider the chelating agent brought in from the solution in which the lipid bilayer particles were recovered.

[0044] Hereinafter, the present disclosure will be described in more detail with reference to examples, but the technical scope of the present disclosure is not limited to the following examples.

[0045] (1) Cell culture Poietics, which are bone marrow-derived mesenchymal stem cells ＴＭHuman mesenchymal stem cells (Lonza) were cultured using MSCulture containing 15% FBS (Selborne Biological Services) ＴＭ as a growth medium. Subsequently, the cultured bone marrow-derived mesenchymal stem cells were adjusted to a cell count of 1.125 × 10 ６ and seeded into a 225 cm ２ cell culture flask (Corning International). The cells were cultured for 72 hours in a cell culture incubator set at 5% CO ２ , 37 °C until the cell count reached 6.75 × 10 ６ .

[0046] (2) Production of extracellular vesicles The bone marrow-derived mesenchymal stem cells proliferated in (1) were replaced with 90 mL of EV-Up ＴＭ MSC-exclusive exosome production medium (FUJIFILM Wako Pure Chemical Corporation), and cultured for 120 hours in a cell culture incubator set at 5% CO ２ , 37 °C. Subsequently, the obtained culture supernatant was filtered through a filter with a pore size of 0.2 μm polyethersulfone membrane to recover the culture supernatant.

[0047] (3) Obtaining extracellular vesicles by phosphatidylserine (PS) affinity method From 200 mL of the culture supernatant recovered in (2), MassivEV ＴＭ EV Purification Column PS / MassivEV ＴＭ Purification Buffer Set (FUJIFILM Wako Pure Chemical Corporation) was used to isolate extracellular vesicles according to the procedure described in the instruction manual attached to the kit. Instead of the eluent attached to the kit, 4 mL of HEPES-buffered saline (HBS) containing 0.3 (w / v)% EDTA (10 mM EDTA) (FUJIFILM Wako Pure Chemical Corporation) was used to elute the extracellular vesicles. The solution used for elution was prepared using EDTA·2Na, and 0.3 (w / v)% EDTA is the concentration as EDTA. Hereinafter, the obtained solution is referred to as the "extracellular vesicle solution".

[0048] (4) Buffer exchange of extracellular vesicle solution using TFF method 1 mL of the extracellular vesicle solution recovered in (3) was buffer-exchanged using tangential flow filtration (TFF method). The buffer exchange was performed using a KR2i TFF system (Repligen, NMCO: 100 kDa) equipped with a MICROKROS 20CM 100K MPES 0.5MM MLL X FLL 1 / PK, which is a 100 kDa cutoff hollow fiber filter. Specifically, the line inside the instrument was first filled with 3 mL of the exchange buffer described in conditions 1 to 10 of Table 1 (buffers containing HBS, Pluronic F68 (BASF), and / or EDTA (Fujifilm Wako Pure Chemical Industries, Ltd.)). Next, 1 mL of extracellular vesicle solution was added, and then, under TFF conditions of a flow rate of 18 mL / min and an intermembrane pressure difference (TMP) of 3 psi, the exchange buffer described in conditions 1 to 10 was replaced with 40 mL of buffer by diafiltration. Finally, 4 mL of extracellular vesicle solution was recovered.

[0049]

[0050] The extracellular vesicle solution recovered in (3) contained 0.3 (w / v)% EDTA. As explained in (4), the extracellular vesicle solution recovered in (3) underwent buffer exchange, so the solution after buffer exchange will contain the EDTA that was originally present in the extracellular vesicle solution recovered in (3). Specifically, the buffer exchange in (4) dilutes the extracellular vesicle solution recovered in (3) by a factor of 4096. Therefore, the 0.3 (w / v)% EDTA contained in the extracellular vesicle solution recovered in (3) will result in 0.000073 (w / v)% EDTA being carried over to the solution after buffer exchange in (4). The EDTA concentrations listed in Table 1 do not take this carried-over amount into account and represent the EDTA concentration in the buffer used in (4).

[0051] [Changes in extracellular vesicle volume before and after buffer exchange] To measure the amount of extracellular vesicles before and after buffer exchange, the exosome marker CD63 was used in PS Capture. ＴＭThe absorbance was determined using the Exosome ELISA Kit (Streptavidin HRP) (Fujifilm Wako Pure Chemical Corporation) according to the procedure described in the instructions attached to the kit. This kit captures extracellular vesicles with the PS-binding protein Tim4 protein and detects them with labeled biotinylated anti-CD63 antibody.

[0052] Then, the absorbance of a solution containing 1 mL of extracellular vesicles and 3 mL of the replacement buffer shown in Table 1 was added. The relative absorbance (%) of the extracellular vesicle solution after buffer exchange was calculated, with the absorbance before buffer exchange set to 100%. The calculated relative value was defined as the recovery rate of extracellular vesicles after TFF treatment.

[0053] The recovery rate results are shown in Figure 1 and Table 2.

[0054]

[0055] In Figure 1, the vertical axis shows the recovery rate of extracellular vesicle solutions treated with TFF under the conditions of Examples 1 to 7 and Comparative Examples 1 to 4. In Comparative Example 4, the recovery rate of extracellular vesicles was remarkably low at 36.76% when no nonionic surfactant was included. This is presumed to be because, in TFF, pressure is applied in the direction of the inner surface of the filter, causing the extracellular vesicles to be damaged by contact between the filter and other components. From the results of Comparative Example 1, Examples 1 to 3, and Comparative Example 2, it was found that when the concentration of the nonionic surfactant Pluronic F68 was in the range of 0.02 (w / v)% to 0.08 (w / v)%, the recovery rate of extracellular vesicles improved compared to Comparative Example 4 (no additive).

[0056] Furthermore, the results from Examples 4 to 7 showed that when the nonionic surfactant Pluronic F68 is included in addition to the chelating agent EDTA, the recovery rate is significantly improved. The improvement in recovery rate shown in Examples 4 to 7 could not be predicted from the results of Comparative Example 3, which showed that EDTA alone does not contribute to improving the recovery rate.

[0057] The disclosure of Japanese Patent Application No. 2024-171231, filed on 30 September 2024, is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards described herein are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard were specifically and individually noted to be incorporated by reference.

Claims

1. A method for producing lipid bilayer particles, comprising filtering a solution containing lipid bilayer particles by passing it through a filter in the in-plane direction in the presence of 0.02 (w / v)% to 0.08 (w / v)% of a nonionic surfactant.

2. The method for producing lipid bilayer particles according to claim 1, wherein the filtration is further carried out in the presence of a chelating agent.

3. The method for producing lipid bilayer particles according to claim 1, wherein the nonionic surfactant is an ether-type nonionic surfactant or an ester-ether-type nonionic surfactant.

4. The method for producing lipid bilayer particles according to claim 1, wherein the nonionic surfactant is a copolymer of polyoxyethylene and polyoxypropylene.

5. The method for producing lipid bilayer particles according to claim 2, wherein the chelating agent is at least one selected from the group consisting of ethylenediaminetetraacetic acid, glycol etherdiaminetetraacetic acid, and salts thereof.

6. The method for producing lipid bilayer particles according to claim 1, wherein the filtration is tangential flow filtration.

7. The method for producing lipid bilayer particles according to claim 1, wherein the lipid bilayer particles are at least one selected from the group consisting of extracellular vesicles, viruses, and artificially produced particles.

8. A solution composition used in a filtration method in which a solution containing lipid bilayer particles is passed through a filtration filter in the in-plane direction, comprising a nonionic surfactant at a concentration of 0.02 (w / v)% to 0.08 (w / v)% at the time of use.

9. The solution composition according to claim 8, further comprising a chelating agent.

10. The solution composition according to claim 8, wherein the nonionic surfactant is an ether-type nonionic surfactant or an ester-ether-type nonionic surfactant.

11. The solution composition according to claim 8, wherein the nonionic surfactant is a copolymer of polyoxyethylene and polyoxypropylene.

12. The solution composition according to claim 9, wherein the chelating agent is at least one selected from the group consisting of ethylenediaminetetraacetic acid, glycol etherdiaminetetraacetic acid, and salts thereof.

13. The solution composition according to claim 8, wherein the filtration method is a tangential flow filtration method.

14. The solution composition according to claim 8, wherein the lipid bilayer particles are at least one selected from the group consisting of extracellular vesicles, viruses, and artificially produced particles.

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