Method for concentrating lipid nanoparticle-containing liquid, solvent exchange method for lipid nanoparticle-containing liquid, and method for producing lipid nanoparticle concentrated liquid

Abstract

Provided is a method for concentrating a lipid nanoparticle-containing liquid, the method comprising a step for filtering a lipid nanoparticle-containing liquid with a polyacrylonitrile-based ultrafiltration membrane.

Description

Method for concentrating lipid nanoparticle-containing liquid, method for solvent exchange of lipid nanoparticle-containing liquid, and method for producing concentrated lipid nanoparticle liquid

[0001] The present invention relates to a method for concentrating a lipid nanoparticle-containing liquid, a method for solvent exchange of a lipid nanoparticle-containing liquid, and a method for producing a lipid nanoparticle-concentrated liquid.

[0002] Lipid nanoparticles, such as liposomes and lipid nanoparticles (LNPs), are structures formed from lipid molecules and can be spherical or other shapes. The term "lipid" refers to a hydrocarbon-based hydrophobic molecule with a molecular weight of several hundred, which has a hydrophilic moiety (such as an amino group) at one end. In aqueous solutions, the hydrophobic moiety is stabilized by the hydrophilic moiety to form "oil droplet-like structures (such as lipid nanoparticles (LNPs))" or "vesicles (such as liposomes) made of lipid bilayer membranes" (see Figure 1). A typical example of an "oil droplet-like structure" is an "emulsion." A typical example of a "vesicle made of lipid bilayer membranes" is a "cell membrane."

[0003] The lipid nanoparticles referred to in this embodiment refer to structures (in one embodiment, spherical structures) with particle sizes of about 30 to 200 nm (in one embodiment, particle diameters of about 30 to 200 nm). Lipid nanoparticles can be used to incorporate nucleic acid drugs or the like into the body and inject drugs into the body by subcutaneous / muscular injection, intravenous injection, or the like. A representative example is ONPATTRO (registered trademark), a therapeutic agent for familial transthyretin amyloidosis, which encapsulates small interfering RNA (siRNA) that targets transthyretin as an active ingredient (lipid nanoparticle (LNP) type; see Non-Patent Documents 1, 2, and 4). COVID-19 vaccines also use similar lipid nanoparticle (LNP) technology, encapsulating messenger RNA encoding SARS-CoV-2 antigens as the active ingredient. In addition to COVID-19 vaccines, much research and development is underway on formulations that encapsulate nucleic acid-based drugs inside lipid nanoparticles (LNP) (see Non-Patent Documents 1 and 2).

[0004] Furthermore, vesicles (liposomes) formed by lipid bilayer membranes are usually filled with an aqueous liquid (aqueous phase), which can be used to incorporate pharmaceuticals into the vesicles. These vesicles containing pharmaceuticals can be injected into the body via subcutaneous / muscular or intravenous injection. Much research and development is underway into pharmaceuticals that incorporate pharmaceuticals with various medicinal properties, such as cancer prevention and treatment, into the aqueous phase of liposomes for infusion into the body. Additionally, lipid nanoparticles also include a group of structures formed by lipid molecules known in the biological context as "exosomes."

[0005] In general, lipid nanoparticles (LNPs) are composed of an ionized lipid (a pH-sensitive lipid (a lipid having a tertiary amino group or the like in the hydrophilic portion)) as one of the main components, a phospholipid (a lipid having a neutrally charged zwitterion containing a cationic group (a quaternary amino group or the like) and an anionic group (a phosphate group or the like) in the hydrophilic portion is often used), cholesterol, and a polyethylene glycol lipid (see Non-Patent Documents 1 and 5). For example, when an ionized lipid (pH-sensitive lipid) has a tertiary amino group, it is neutral when the surrounding pH environment is neutral, but becomes positively charged when the surrounding pH environment becomes acidic. In other words, the surface charge of a lipid nanoparticle (LNP) is neutral when the surrounding pH environment is neutral, but often becomes positively charged when the surrounding pH environment becomes acidic. Liposomes are often composed primarily of neutral phospholipids (lipids having electrically neutral zwitterions containing a cationic group (such as a quaternary amino group) and an anionic group (such as a phosphate group) in the hydrophilic portion) (see Non-Patent Documents 6 and 7). As a result, the surface charge of liposomes is often neutral. However, for the same reasons as those mentioned above, there are also liposomes with negatively charged surfaces and liposomes with positively charged surfaces.

[0006] Lipid nanoparticle (LNP) type lipid nanoparticle medicines are a relatively new modality (medical procedure), and their practical application began with the aforementioned ONPATTRO (registered trademark), which was approved by the U.S. Food and Drug Administration (FDA) in 2018. It has since been used worldwide as a COVID-19 vaccine since 2020. The active ingredients of nucleic acid medicines (siRNA and mRNA) encapsulated in the lipid nanoparticles (LNP) of ONPATTRO (registered trademark) and COVID-19 vaccines are susceptible to degradation in the body, so encapsulating them in lipid nanoparticles (LNP) allows for highly reliable delivery to the body. Ionizable lipids (pH-sensitive lipids) are said to hold the key to reliably encapsulating nucleic acid drugs in lipid nanoparticles (LNPs) and controlling their release in vivo.

[0007] When lipid nanoparticles are used as pharmaceuticals, separation, purification, and concentration of lipid nanoparticles are important from the viewpoint of ensuring safety and efficacy. Currently, research and development of membrane separation (e.g., membrane filtration) is being conducted as one of the methods for separating, purifying, and concentrating lipid nanoparticles. Membrane separation is a simple method for achieving separation, purification, and concentration.

[0008] Polyacrylonitrile-based materials are used as membrane materials for membrane filtration. Polyacrylonitrile-based materials are excellent in mechanical strength (physical strength and durability) and chemical strength (chemical resistance, etc.), and are therefore used as membrane materials for industrial water treatment and the like (see Non-Patent Document 3 and Patent Document 1). Polyacrylonitrile-based materials are also being investigated as membrane materials when mixed with various copolymers (see Patent Document 2).

[0009] International Publication No. 98 / 58728 JP 11-033375

[0010] Hiroki Tanaka, Hidetoshi Akita, PHARM TECH JAPAN, 39 (2023) 368-373 Hidetoshi Akita, New DDS Technology and Formulation for New Modality Drugs, Technical Information Association, pp. 265-276, 2023 Noboru Kubota, Membrane, Vol. 33, pp. 239-246, 2008 Yuka Yasuoka, PharmaStyle, No. 3, December 2020, 18-23 Junichi Mineno, Shigetomo Tsujihata, Pharmaceutical and Medical Device Regulatory Science, Vol. 54, No. 4, pp. 295-299, 2023; Naoko Suzuki and Masahiro Otaki, Journal of Human Environmental Engineering, Vol. 6, No. 2, pp. 220-221, 2004; Yusuke Oda, Ryo Suzuki, Kazuo Maruyama, Drug Delivery System, 31-4, pp. 370-371, 2016; Michio Mineshima, Journal of the Japanese Society of Nephrology, Vol. 55 (No. 4), pp. 515-522, 2013

[0011] Membrane filtration of lipid nanoparticle-containing liquids has been difficult due to frequent membrane clogging. Furthermore, even if lipid nanoparticle-containing liquids can be filtered through membranes, the lipid nanoparticles subjected to membrane filtration are prone to denaturation, deterioration, and damage. Therefore, there is a need for a membrane filtration technology that can filter lipid nanoparticles stably with minimal membrane clogging, has a high lipid nanoparticle recovery rate, and allows for easy concentration.

[0012] The present invention aims to provide a method for concentrating a lipid nanoparticle-containing liquid, which allows stable filtration of the lipid nanoparticle-containing liquid with little clogging of the membrane, has a high recovery rate of lipid nanoparticles, and allows for easy concentration.

[0013] The present invention is as follows. <<Aspect 1>> A method for concentrating a lipid nanoparticle-containing liquid, comprising a step of filtering the lipid nanoparticle-containing liquid through a polyacrylonitrile-based ultrafiltration membrane. <<Aspect 2>> A method for concentrating a lipid nanoparticle-containing liquid according to Aspect 1, wherein the polyacrylonitrile-based ultrafiltration membrane contains a basic copolymer component. <<Aspect 3>> A method for concentrating a lipid nanoparticle-containing liquid according to Aspect 1 or 2, wherein the polyacrylonitrile-based ultrafiltration membrane has a small pore layer on the membrane surface on the side that comes into contact with the lipid nanoparticle-containing liquid. <<Aspect 4>> A method for concentrating a lipid nanoparticle-containing liquid according to Aspect 1, wherein the polyacrylonitrile-based ultrafiltration membrane is a hollow fiber membrane. <<Aspect 5>> A method for concentrating a lipid nanoparticle-containing liquid according to Aspect 2, wherein the polyacrylonitrile-based ultrafiltration membrane is a hollow fiber membrane. <<Aspect 6>> A method for concentrating a lipid nanoparticle-containing liquid according to Aspect 3, wherein the polyacrylonitrile-based ultrafiltration membrane is a hollow fiber membrane. <<Aspect 7>> A method for concentrating a lipid nanoparticle-containing liquid according to Aspect 2 or 5, wherein the lipid nanoparticles comprise an ionized lipid having a tertiary amino group as the lipid. <<Aspect 8>> A method for concentrating a lipid nanoparticle-containing liquid according to Aspect 3 or 6, wherein the lipid nanoparticles comprise an ionized lipid having a tertiary amino group as the lipid. <<Aspect 9>> A method for solvent exchange of a lipid nanoparticle-containing liquid, comprising a step of adding to the lipid nanoparticle-containing liquid an amount of a solvent different from the solvent components of the lipid nanoparticle-containing liquid removed by filtration through a polyacrylonitrile-based ultrafiltration membrane, in an amount equal to all or part of the amount lost by the filtration. <<Aspect 10>> A method for solvent exchange of a lipid nanoparticle-containing liquid according to Aspect 9, wherein the polyacrylonitrile-based ultrafiltration membrane comprises a basic copolymer component. <<Aspect 11>> A method for solvent exchange of a lipid nanoparticle-containing liquid according to Aspect 9 or 10, wherein the polyacrylonitrile-based ultrafiltration membrane has a small pore layer on the membrane surface on the side that comes into contact with the lipid nanoparticle-containing liquid. <<Aspect 12>> The method for solvent exchange of a lipid nanoparticle-containing liquid according to Aspect 9, wherein the polyacrylonitrile-based ultrafiltration membrane is a hollow fiber membrane. <<Aspect 13>> The method for solvent exchange of a lipid nanoparticle-containing liquid according to Aspect 10, wherein the polyacrylonitrile-based ultrafiltration membrane is a hollow fiber membrane.<<Aspect 14>> A method for solvent exchange of a lipid nanoparticle-containing liquid according to Aspect 11, wherein the polyacrylonitrile-based ultrafiltration membrane is a hollow fiber membrane. <<Aspect 15>> A method for solvent exchange of a lipid nanoparticle-containing liquid according to Aspect 10 or 13, wherein the lipid nanoparticles contain an ionized lipid having a tertiary amino group as the lipid. <<Aspect 16>> A method for solvent exchange of a lipid nanoparticle-containing liquid according to Aspect 11 or 14, wherein the lipid nanoparticles contain an ionized lipid having a tertiary amino group as the lipid. <<Aspect 17>> A method for producing a concentrated lipid nanoparticle liquid, comprising a step of filtering a lipid nanoparticle-containing liquid with a polyacrylonitrile-based ultrafiltration membrane.

[0014] According to the present invention, lipid nanoparticle-containing liquid can be filtrated stably with little clogging of the membrane, and the lipid nanoparticles can be easily concentrated with a high recovery rate.

[0015] FIG. 1 is an example of a simple schematic diagram of lipid nanoparticles. FIG. 1A is a schematic diagram of lipid nanoparticles forming oil droplet-like structures. FIG. 1 is an example of a simple schematic diagram of lipid nanoparticles. FIG. 1B is a schematic diagram of lipid nanoparticles forming vesicles consisting of two lipid molecules. FIG. 2 is an electron microscope photograph of a polyacrylonitrile-based ultrafiltration membrane prepared in Production Example 1 of a polyacrylonitrile-based ultrafiltration membrane. FIG. 2A is an electron microscope photograph of a circular cross section of a polyacrylonitrile-based ultrafiltration membrane cut perpendicular to the longitudinal direction of the hollow fiber membrane. FIG. 2 is an electron microscope photograph of a polyacrylonitrile-based ultrafiltration membrane prepared in Production Example 1 of a polyacrylonitrile-based ultrafiltration membrane. FIG. 2B is an electron microscope photograph of the inner surface of a polyacrylonitrile-based ultrafiltration membrane. FIG. 2 is an electron microscope photograph of a polyacrylonitrile-based ultrafiltration membrane prepared in Production Example 1 of a polyacrylonitrile-based ultrafiltration membrane. 2C is an electron microscope photograph of the membrane cross section on the inner surface side of a polyacrylonitrile-based ultrafiltration membrane. FIG. 2 is an electron microscope photograph of a polyacrylonitrile-based ultrafiltration membrane produced in Production Example 1 of a polyacrylonitrile-based ultrafiltration membrane. FIG. 2D is an electron microscope photograph of the central part (central part of the thick part) of the membrane cross section of a polyacrylonitrile-based ultrafiltration membrane. FIG. 3 is an electron microscope photograph of a polyacrylonitrile-based ultrafiltration membrane produced in Production Example 2 of a polyacrylonitrile-based ultrafiltration membrane. FIG. 3A is an electron microscope photograph of a circular cross section of a polyacrylonitrile-based ultrafiltration membrane cut perpendicular to the longitudinal direction of the hollow fiber membrane. FIG. 3 is an electron microscope photograph of a polyacrylonitrile-based ultrafiltration membrane produced in Production Example 2 of a polyacrylonitrile-based ultrafiltration membrane. FIG. 3B is an electron microscope photograph of the inner surface of a polyacrylonitrile-based ultrafiltration membrane. 3 is an electron microscope photograph of a polyacrylonitrile-based ultrafiltration membrane produced in Production Example 2 of a polyacrylonitrile-based ultrafiltration membrane. FIG. 3C is an electron microscope photograph of a membrane cross section on the inner surface side of a polyacrylonitrile-based ultrafiltration membrane. FIG. 3 is an electron microscope photograph of a polyacrylonitrile-based ultrafiltration membrane produced in Production Example 2 of a polyacrylonitrile-based ultrafiltration membrane. FIG. 3D is an electron microscope photograph of the central part (central part of the thick part) of the membrane cross section of a polyacrylonitrile-based ultrafiltration membrane. FIG. 4 is an external view photograph of a mini-module produced in a production example of a mini-module.FIG. 5 is a photograph showing the appearance of a mini-module prepared in a mini-module manufacturing example with a pressure sensor connected thereto. FIG. 6 is a photograph showing a filtration experiment in which a "pressure sensor-connected mini-module" is connected to a syringe containing a liquid to be filtered and set in a syringe pump, and filtration (total filtration) is performed using the syringe pump. FIG. 7 shows the measurement results of the filtration pressure per elapsed time measured in Reference Examples 1 and 2. FIG. 7A shows the measurement results of the filtration pressure per elapsed time of filtration operation C in Reference Example 1. FIG. 7B shows the measurement results of the filtration pressure per elapsed time of the first filtration operation D in Reference Example 1. FIG. 7 shows the measurement results of the filtration pressure per elapsed time measured in Reference Examples 1 and 2. FIG. 7C shows the measurement results of the filtration pressure per elapsed time of filtration operation C in Reference Example 2. FIG. 7 shows the measurement results of the filtration pressure per elapsed time measured in Reference Examples 1 and 2. 7D shows the measurement results of the filtration pressure per elapsed time of the first filtration operation D in Reference Example 2. FIG. 8 is an electron microscope photograph of a polysulfone-based ultrafiltration membrane produced in a Production Example. FIG. 8A is an electron microscope photograph of a circular cross section of a polysulfone-based ultrafiltration membrane cut perpendicular to the longitudinal direction of the hollow fiber membrane. FIG. 8 is an electron microscope photograph of a polysulfone-based ultrafiltration membrane produced in a Production Example. FIG. 8B is an electron microscope photograph of the inner surface of a polysulfone-based ultrafiltration membrane. FIG. 8 is an electron microscope photograph of a polysulfone-based ultrafiltration membrane produced in a Production Example. FIG. 8C is an electron microscope photograph of a membrane cross section on the inner surface side of a polysulfone-based ultrafiltration membrane. FIG. 8 is an electron microscope photograph of a polysulfone-based ultrafiltration membrane produced in a Production Example. FIG. 8D is an electron microscope photograph of the central portion (central portion of the thick portion) of the membrane cross section of a polysulfone-based ultrafiltration membrane.

[0016] Hereinafter, a detailed description will be given of an embodiment of the present invention (hereinafter also referred to as the present embodiment). Note that the present invention is not limited to the embodiment described below, and various modifications can be made within the scope of the embodiment.

[0017] <Polyacrylonitrile-based ultrafiltration membrane> The polyacrylonitrile-based ultrafiltration membrane of this embodiment is preferably an ultrafiltration membrane containing a polyacrylonitrile-based polymer in which 70% by weight or more of acrylonitrile and a copolymerization component for acrylonitrile are copolymerized.In one aspect, it is more preferable that the ultrafiltration membrane contains a polyacrylonitrile-based polymer in which 70% by weight or more of acrylonitrile and one or more vinyl compounds are copolymerized as a copolymerization component.In addition, the polyacrylonitrile-based polymer may not contain a copolymerization component for acrylonitrile, and may be a homopolymer of acrylonitrile.

[0018] Acrylonitrile provides the resulting polymer with mechanical and chemical strength and some hydrophilic properties. Furthermore, copolymerization components can be used with acrylonitrile to add or strengthen various properties and functions.

[0019] As the copolymerization component, vinyl compounds are preferred. Examples of vinyl compounds that can be copolymerized with acrylonitrile include acrylic acid, methyl acrylate, ethyl acrylate, itaconic acid, vinyl acetate, sodium acrylic sulfonate, sodium methallyl sulfonate, sodium parastyrene sulfonate, hydroxyethyl methacrylate, ethyl methacrylate trimethylammonium chloride, ethyl methacrylate triethylammonium chloride and vinylpyrrolidone. Among these, polyacrylonitrile-based polymers containing basic copolymerization components (e.g., ethyl methacrylate trimethylammonium chloride and ethyl methacrylate triethylammonium chloride, etc.) can be particularly preferably used as a material for polyacrylonitrile-based ultrafiltration membranes for filtration and concentration of lipid nanoparticle-containing liquids.

[0020] The lipid nanoparticles in the lipid nanoparticle-containing solution may be positively charged depending on the surrounding environment, such as pH (particularly when the lipid nanoparticles are of the lipid nanoparticle (LNP) type (particularly when the lipid nanoparticles have an ionized lipid (pH-sensitive lipid) having a tertiary amino group)). In such cases, when the polyacrylonitrile-based ultrafiltration membrane contains a basic copolymer component, the membrane (for example, the membrane surface, particularly in the case of a hollow fiber membrane, the inner surface of the membrane) is positively charged (or weakly negatively charged, or neutral in charge), the attraction between the positively charged lipid nanoparticles and the membrane is reduced (or repulsion occurs). As a result, adhesion of lipid nanoparticles to the membrane surface is suppressed, which is thought to increase filtration stability and lipid nanoparticle recovery rate. The charged state of solid surfaces such as lipid nanoparticles and membranes is sometimes expressed (measured) as "zeta potential". Lipid nanoparticles (LNPs), a new modality (medical procedure), are composed of one of their main components, an ionized lipid (a pH-sensitive lipid (e.g., a lipid having a tertiary amino group in the hydrophilic portion)), which becomes positively charged in the surrounding pH environment (e.g., an acidic environment). As a result, the LNP itself often becomes positively charged. Therefore, an ultrafiltration membrane containing a polyacrylonitrile-based polymer containing a basic copolymer component is particularly suitable for membrane filtration of lipid nanoparticle-containing liquids.

[0021] The zeta potential of a membrane made of polyacrylonitrile is usually negative ("Correct Procedures for Filtration Scale-Up and Collection of Successful Cases," published by Technical Information Association, August 2014, p. 266; Artificial Organs, Vol. 22, No. 1 (1993), pp. 74-78). In order to shift this negative charge as much as possible to the positive side (or to make it positively charged), it is effective to increase the content of the basic copolymer component in the polyacrylonitrile polymer, while it is preferable not to include an excessive amount of the basic copolymer component in order to ensure the water resistance of the polyacrylonitrile polymer material.

[0022] The polyacrylonitrile polymer may not contain a copolymerization component with acrylonitrile, and may be a homopolymer of acrylonitrile. From the viewpoint of positively charging the membrane, the polyacrylonitrile ultrafiltration membrane of this embodiment preferably contains a basic copolymerization component. The ratio of the basic copolymerization component in the polyacrylonitrile polymer material is preferably 15% by weight or less, more preferably 1% by weight or more and 10% by weight or less. In addition, when the ratio of the basic copolymerization component in the polyacrylonitrile polymer is known, the ratio of the basic copolymerization component in the polyacrylonitrile polymer may be the ratio of the basic copolymerization component in the polyacrylonitrile polymer. However, when the ratio of the basic copolymerization component is unknown, it can be measured by NMR analysis (nuclear magnetic resonance analysis) or IR analysis (infrared spectroscopy analysis).

[0023] Lipid nanoparticle-containing liquids are often prepared by mixing a solution in which multiple lipids, including pH-responsive lipids, are dissolved in an organic solvent such as alcohol with water (buffer solution) in which a medicinal component such as a nucleic acid drug is dissolved (see Non-Patent Document 5). The polyacrylonitrile polymer material has a certain degree of resistance to organic solvents such as alcohol, and therefore is suitable for the filtration and concentration of lipid nanoparticle-containing liquids, which often contain organic solvents.

[0024] The ultrafiltration membrane has a molecular weight cutoff of 1,000 or more but less than 500,000, preferably 3,000 or more but less than 100,000. Membranes with too small a molecular weight cutoff tend to have low permeability. Furthermore, membranes with too large a molecular weight cutoff tend to have a risk of reduced filtration stability and reduced lipid nanoparticle recovery due to all or part of the lipid nanoparticles penetrating the membrane. The membrane fractionation (rejection) performance can be evaluated by the rejection rate of solutes such as dextran.

[0025] Specifically, the ultrafiltration membrane of this embodiment can be a membrane that has a rejection rate of 90% or less for dextrans with an average molecular weight of 10,000 and a rejection rate of 50% or more for dextrans with an average molecular weight of 2,000,000, preferably a rejection rate of 50% or less for dextrans with an average molecular weight of 10,000 and a rejection rate of 70% or more for dextrans with an average molecular weight of 2,000,000, more preferably a rejection rate of 20% or less for dextrans with an average molecular weight of 10,000 and a rejection rate of 70% or more for dextrans with an average molecular weight of 2,000,000. In one aspect, the rejection rate of dextrans with an average molecular weight of 10,000 by the ultrafiltration membrane of this embodiment is 90% or less, or 50% or less, or 20% or less, or 15% or less, or 10% or less, or 5% or less, or less than 5%.

[0026] As dextran having an average molecular weight of 10,000 and dextran having an average molecular weight of 2,000,000, Dextran T-10 and Dextran T-2000, both manufactured by Pharmacosmos, can be used, respectively.

[0027] The solute rejection rate of a membrane is measured as follows: A 0.1 wt % aqueous solution of dextran is filtered through the membrane at a filtration pressure of 0.5 bar, a linear velocity of 0.1 m / sec, and room temperature (25°C). The solute rejection rate (%) can be calculated by 100 x {1 - (dextran concentration in the permeated liquid) / (dextran concentration in the liquid supplied to the membrane)}. The dextran concentration in the liquid that has permeated the membrane (permeated liquid) can be calculated, for example, from the refractive index of the permeated liquid measured with a refractometer and a calibration curve of dextran concentration.

[0028] Typically, the cross-sectional structure of an ultrafiltration membrane often includes a dense layer with relatively small pores (small pore layer) that contributes to the membrane's fractionation performance (solute blocking performance), and a porous layer with relatively large pores (large pore layer) that contributes little to the membrane's fractionation performance but contributes to ensuring the membrane's mechanical strength (pressure resistance, etc.). The "small pore layer" is necessary to ensure the membrane's fractionation performance, but reduces the permeability of the filtrate (performance in terms of filtration speed). On the other hand, the "large pore layer" contributes little to the fractionation performance, but does not significantly reduce the permeability. Therefore, from the perspective of balancing "ensuring fractionation performance," "ensuring permeability," and "ensuring mechanical strength," ultrafiltration membranes often include both a "small pore layer" and a "large pore layer." Although the pore sizes of the "small pore layer" and the "large pore layer" are relative, in the present disclosure, the "small pore layer" of an ultrafiltration membrane is typically a layer having a pore size of about 30 nm to 1 nm, and the "large pore layer" of an ultrafiltration membrane is a layer having a pore size of about 0.1 μm or more. The pore sizes of the "large pore layer" of an ultrafiltration membrane and the "small pore layer" of an ultrafiltration membrane are measured by observing the cross section and / or surface of the ultrafiltration membrane with an electron microscope. Note that the pore sizes of the "small pore layer" and the "large pore layer" do not necessarily change discontinuously, but may change continuously. Generally, in many ultrafiltration membranes, a "small pore layer" is present on the membrane surface, and a "large pore layer" is often present within the thick portion of the membrane cross section (see, for example, Patent Document 1).

[0029] In this embodiment, the lipid nanoparticles are concentrated by an ultrafiltration membrane and then recovered as a concentrate. Therefore, it is preferable that the lipid nanoparticles are blocked at the surface of the membrane and do not penetrate into the thick part of the membrane. Therefore, it is preferable that the "small pore layer" is present at least on the membrane surface on the side that comes into contact with the lipid nanoparticle-containing liquid to be filtered. Note that in this embodiment, it is acceptable for the "small pore layer" to be present on the membrane surface on the side that does not come into contact with the lipid nanoparticle-containing liquid to be filtered, or within the thick part of the membrane cross section. The location of the "small pore layer" on the membrane cross section can be confirmed by electron microscopic observation of the membrane cross section. In one aspect, the polyacrylonitrile-based ultrafiltration membrane has a small pore layer on the membrane surface on the side that comes into contact with the lipid nanoparticle-containing liquid.

[0030] The polyacrylonitrile-based ultrafiltration membrane of this embodiment can be obtained by forming the polyacrylonitrile-based polymer material. The polyacrylonitrile-based ultrafiltration membrane of this embodiment can be produced, for example, in the same manner as in Example 1 described in WO 98 / 58728.

[0031] Lipid nanoparticle-containing liquid Lipid nanoparticles, such as liposomes and lipid nanoparticles (LNPs), are structures formed from lipid molecules, and their shapes include spherical shapes. The term "lipid" as used herein refers to a molecule with a molecular weight of several hundred, which has a hydrophilic portion 10 (such as an amino group) of a lipid molecule at one end of a hydrocarbon-based hydrophobic molecule. In aqueous solutions, the hydrophobic portion 11 of the lipid molecule is stabilized by the hydrophilic portion to form "oil droplet-like structures" (such as lipid nanoparticles (LNPs)) or "vesicles consisting of lipid bilayer membranes" (such as liposomes) (see Figures 1A and 1B). Figure 1A is a schematic diagram of lipid nanoparticles forming an oil droplet-like structure. Since the external environment of the lipid nanoparticle 110 is an aqueous liquid (e.g., aqueous phase 120), the lipid molecules constituting the lipid nanoparticle 110 form an "oil droplet-like structure" by arranging the hydrophobic portion 11 of the lipid molecule inward and the hydrophilic portion 10 of the lipid molecule outward (see FIG. 1A). The exterior of the lipid nanoparticle 110 that has formed an "oil droplet-like structure" is an aqueous phase 120, and the interior of the lipid nanoparticle 110 is an oil phase 13 (note that fine aqueous phases may be partially encapsulated within the oil phase 13 (see, for example, FIG. 3 of Non-Patent Document 4 and FIG. 3 of Non-Patent Document 5)). By encapsulating a drug 12 (e.g., messenger RNA, etc.) inside the lipid nanoparticle 110 that has formed an "oil droplet-like structure," the drug 12 can be stabilized even in an aqueous liquid (e.g., aqueous phase 120). FIG. 1B is a schematic diagram of a lipid nanoparticle forming a vesicle consisting of a lipid bilayer membrane. Lipid nanoparticles 110 form a bilayer (e.g., a lipid bilayer) in which the hydrophilic portions 10 of the lipid molecules face each other and the hydrophobic portions 11 of the lipid molecules face each other, forming a vesicle made of a lipid bilayer (see FIG. 1B). Inside the lipid nanoparticle 110 that has formed a "lipid bilayer," there is an aqueous phase 14 formed by the hydrophilic portions 10 of the lipid molecules and in which a pharmaceutical agent 12 is encapsulated, and an oil phase 13 formed by the hydrophobic portions 11 of the lipid molecules (see FIG. 1B). A typical example of an "oil droplet-like structure" is an "emulsion." A typical example of a "vesicle made of a lipid bilayer" is a "cell membrane."

[0032] Here, the lipid nanoparticles referred to in this embodiment refer to structures (in one embodiment, spherical structures) formed from lipid molecules with particle sizes of about 30 to 200 nm (in one embodiment, particle diameters of about 30 to 200 nm), and include lipid nanoparticles (LNPs), liposomes, and exosomes. Lipid nanoparticles contain nucleic acid drugs or the like inside, and the drugs can be injected into the body by subcutaneous / muscular injection, intravenous injection, or the like.

[0033] As the medicinal active ingredient and pharmaceutical agent to be contained in the lipid nanoparticles, nucleic acid pharmaceuticals, anticancer drugs such as doxorubicin (Doxil), antibiotics such as AmBisome, etc. are preferred.

[0034] A lipid nanoparticle-containing liquid is prepared, for example, by the following steps 1) to 3). 1) A medicinal active ingredient is dissolved in pH-buffered water (e.g., acetate buffer). The pH-buffered water (buffer) can be selected appropriately depending on the medicinal active ingredient. 2) A lipid nanoparticle component such as lipid is dissolved in an organic solvent. The organic solvent is preferably a water-miscible organic solvent, such as alcohol (e.g., ethanol, isopropyl alcohol, and tertiary butyl alcohol). 3) The solution prepared in 1) is mixed with the solution prepared in 2).

[0035] Examples of lipids (particularly ionizable lipids (pH-sensitive lipids) used in lipid nanoparticle (LNP) types, etc.) include hydrocarbon compounds having a hydrophilic moiety (a cationic group such as a tertiary amino group) at one end, as described in, for example, "Pharmaceutical Research (2023) 40:27-46" and "Acc. Chem. Res., 2022, 55, 1, 2-12." Representative examples include SM-102, DLin-MC3-DMA, and DODAP.

[0036] In addition, components other than lipids can be dissolved in a water-miscible organic solvent such as alcohol together with the lipids in the above procedure 2) to prepare lipid nanoparticles containing components other than lipids. These components other than lipids contribute to the stability of the lipid nanoparticles and can be added depending on the purpose. Examples of lipid nanoparticle components other than lipids include the following. For example, lipid nanoparticle components other than lipids include phospholipids, sterols, and polyethylene glycol-bound lipids, and these components may be used alone or in combination.

[0037] When components other than lipids are added, the composition ratios (in one embodiment, molar ratios) are 30% to 70% lipid, 0% to 20% phospholipid, 30% to 50% sterol, and approximately 0.5% to 3.0% polyethylene glycol-conjugated lipid. The composition ratios of the lipid nanoparticles are preferably 40 to 60%, more preferably 45 to 55%, 5 to 15%, more preferably 7.5 to 12.5%, 35 to 45% sterol, and 1.0 to 2.5% polyethylene glycol-conjugated lipid.

[0038] As the phospholipid, neutral phospholipids and the like are preferred. Examples of neutral phospholipids include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), DMPC (dimyristoylphosphatidylcholine), DPPC (dipalmitoylphosphatidylcholine), DSPC (distearoylphosphatidylcholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine), SOPC (1-stearoyl-2-oleoylphosphatidylcholine), POPC (1-palmitoyl-2-oleoylphosphatidylcholine), DMPE (dimyristylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), POPE (1-palmitoyl-2-oleoylphosphatidylethanolamine), and SOPE (1-stearoyl-2-oleoylphosphatidylethanolamine). Examples of sterols include cholesterol, stigmasterol, and β-sitosterol. Examples of polyethylene glycol-conjugated lipids include 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (PEG number average molecular weight (Mn) is 2000) (DMG-PEG2000), DMG-PEG5000 (same as above, PEG number average molecular weight (Mn) is 5000), DSG-PEG2000 (distearoyl-rac-glycerol polyethylene glycol (PEG number average molecular weight (Mn) is 2000), and DSG-PEG5000 (same as above, PEG number average molecular weight (Mn) is 5000).

[0039] For example, when a lipid nanoparticle-containing liquid in which a medicinal active ingredient is incorporated into lipid nanoparticles, prepared by the procedures 1) to 3) above, is to be injected into a living body, it is usually preferable to perform the following procedures: (1) "Solvent exchange" procedure: This is a procedure in which the solvent components and their concentrations (composition) of the lipid nanoparticle-containing liquid are adjusted to the desired final composition (in one embodiment, the ratio of phosphate buffer to the solvent of the lipid nanoparticle-containing liquid is 95% by volume or more and the ratio of organic solvent to 1% by volume or less), taking into consideration the safety of the lipid nanoparticle-containing liquid when injected into a living body and the stability of the lipid nanoparticles in the lipid nanoparticle-containing liquid.

[0040] (2) "Concentration" treatment: This is a treatment to increase the lipid nanoparticle concentration in the lipid nanoparticle-containing liquid to a concentration at which the medicinal efficacy of the pharmaceutical active ingredients contained in the lipid nanoparticles can be expected. After the "concentration" treatment, the lipid nanoparticle-containing liquid may be diluted as appropriate before use. Here, in the present disclosure, the "lipid nanoparticle concentration" in the lipid nanoparticle-containing liquid is expressed as the "total molar concentration of lipid nanoparticle components (lipids, phospholipids, sterols, polyethylene glycol-bound lipids, etc.)" in the lipid nanoparticle-containing liquid. The "lipid nanoparticle concentration" in the lipid nanoparticle-containing liquid of this embodiment is approximately 0.1 mM to 40 mM.

[0041] (1) In the "solvent exchange" treatment, for example, a solvent (in one embodiment, PBS solution (phosphate buffered saline; pH 7.4) and Tris buffered saline (TBS), etc.) different from the solvent components of the lipid nanoparticle-containing liquid removed by filtration or the like (in one embodiment, the organic solvent and the pH buffered water used in 1) above) is added to the lipid nanoparticle-containing liquid in an amount equal to all or part of the amount lost by filtration. By performing the "solvent exchange" treatment, the solvent composition at the time of preparation of the lipid nanoparticles, which contains a large amount of organic solvents such as alcohol and the pH buffered water used in 1) above, i.e., at the time of 3) above, is changed to a solvent composition with a large proportion of different solvents other than the organic solvent and the pH buffered water used in 1) above. In one aspect, the final concentrations of the different solvents and organic solvents other than the pH-buffered water (such as acetate buffer) used in 1) above are, respectively, 95% by volume or more, 97% by volume or more, or 99% by volume or more for phosphate buffer and TBS, etc., in the solvent of the lipid nanoparticle-containing liquid, and 1% by volume or less for the organic solvent.

[0042] Each of the above-mentioned "solvent exchange" and "concentration" treatments can be carried out using "membrane filtration", for example, as in the following (A) to (C) treatments. (A) For example, the liquid obtained in 3) above is filtered using a polyacrylonitrile-based ultrafiltration membrane. The lipid nanoparticles are blocked by the ultrafiltration membrane and concentrated without permeating the membrane. The solvent components of the lipid nanoparticle-containing liquid, including organic solvents, are permeated through the membrane and removed. In one embodiment, the (A) treatment corresponds to a volume reduction treatment of the solvent components of the lipid nanoparticle-containing liquid carried out before the (B) treatment. (B) Next, for example, a solvent different from the solvent components of the lipid nanoparticle-containing liquid removed by filtration (in one embodiment, phosphate buffered saline (PBS), Tris buffered saline (TBS), etc. at pH 7.4) is added to the lipid nanoparticle-containing liquid in an amount equal to all or part of the volume lost by filtration. That is, a solvent different from the components of the lipid nanoparticle-containing liquid removed by filtration in (A) may be added to the lipid nanoparticle-containing liquid in an amount equivalent to all or part of the volume lost by filtration. By the treatment (B), the concentration of the pH buffered water and organic solvent used in the above 1) in the solvent of the lipid nanoparticle-containing liquid is reduced. The treatments (A) and / or (B) may be performed once or multiple times until the concentration of the solvent components of the lipid nanoparticle-containing liquid (in one embodiment, the organic solvent and phosphate buffer) reaches the desired final concentration. (C) Once the solvent components of the lipid nanoparticle-containing liquid reach the desired final concentration, filtration is performed using a polyacrylonitrile-based ultrafiltration membrane to concentrate the lipid nanoparticles to the desired concentration.

[0043] In the above case, the processes (A) and (B) correspond to "solvent exchange" processes, and the process (C) corresponds to "concentration" processes. By the above processes, it is possible to obtain a lipid nanoparticle-containing liquid in which the solvent components of the lipid nanoparticle-containing liquid are concentrated to a desired final concentration and the lipid nanoparticles are concentrated to a desired concentration.

[0044] Common "filtration" operations include "pressure filtration" and "dialysis." "Pressure filtration" is an operation in which a liquid to be filtered (e.g., a liquid to be filtered) is pressurized or negatively pressurized (suctioned) against a membrane. Solutes and suspended matter larger than the membrane's "pore size" are blocked at the membrane surface, while solutes and solvents smaller than the membrane's "pore size" are permeated and discharged. Therefore, in "pressure filtration," solutes and suspended matter larger than the membrane's "pore size" are concentrated, and the volume of the "liquid to be filtered" is reduced. On the other hand, "dialysis" is an operation in which a solute diffuses from a liquid with a higher concentration to a liquid with a lower concentration according to the difference in solute concentration between two liquids in contact with the membrane surface (in the case of a hollow fiber membrane, the liquid in contact with the inner surface of the membrane and the liquid in contact with the outer surface of the membrane). In this case, the volume of the "liquid to be filtered" is not actively reduced. In this embodiment, particularly in "solvent exchange," the above-mentioned "pressure filtration" is usually carried out in order to "reduce the volume of the liquid by a filtration operation and add new solvent in an amount that corresponds to all or part of the volume that was lost," as described above. The "filtration" in this embodiment usually refers to "pressure filtration," but depending on the embodiment, "filtration" by "dialysis" may also be carried out as appropriate.

[0045] The above processes (A) to (C) may be carried out by combining the individual "batch operations" of (A) to (C), or may be carried out continuously using a "diafiltration" method. In the "diafiltration" method, for example, the solution to be filtered is pumped from a "container containing the solution to be filtered" to a membrane and filtered through the membrane, while the solution to be filtered permeates through the membrane. A "solvent exchange" treatment is carried out continuously by continuously adding a "solvent different from the solvent contained in the lipid nanoparticle-containing solution" in an amount equivalent to the amount of liquid removed by permeation to the "container containing the solution to be filtered." Once the "solvent exchange" treatment is completed, the "solvent introduction" is stopped, and the method continues up to the "concentration" treatment.

[0046] In addition, when performing the above (A), before performing the above (A) treatment, the lipid nanoparticle-containing liquid obtained in 3) above can be pre-diluted with the "desired solvent" (e.g., PBS, etc.) that you want to include in the solvent of the lipid nanoparticle-containing liquid. By pre-diluting, the liquid volume of the lipid nanoparticle-containing liquid (the liquid volume treated in (A) above) increases, but the solvent components contained in the lipid nanoparticle-containing liquid obtained in 3) above have the advantage of being able to approach the desired final concentration at an early stage. When pre-dilution is performed with the "desired solvent", the stability of the lipid nanoparticles in the liquid (shape stability, etc.) is often increased in the solvent contained in the lipid nanoparticle-containing liquid, and the filtration stability through the membrane is also increased. Therefore, although the amount of liquid treated in (A) above increases, it may be preferable overall in performing (A).

[0047] The above membrane filtration operation can be carried out by either "total filtration" or "cross-flow filtration." "Total filtration" is a filtration method also known as "dead-end filtration," in which the solution to be filtered is filtered without circulating. "Cross-flow filtration" is a method in which the solution to be filtered is filtered while circulating it. Since the solution to be filtered flows parallel to the membrane surface, it has the advantage that dirt is less likely to adhere to the membrane surface. However, "cross-flow filtration" requires equipment such as a pump or other liquid delivery equipment, and tends to generate a large amount of liquid that cannot be filtered (dead volume). From the viewpoint of ease of membrane filtration operation and minimizing "dead volume" as much as possible, "total filtration" is preferred.

[0048] <Hollow fiber membrane> The polyacrylonitrile-based ultrafiltration membrane of this embodiment can be in the form of a flat membrane, a tubular membrane, or a hollow fiber membrane. The hollow fiber membrane is preferred because it is easy to perform quantitative concentration operations, easy to perform filtration operations (e.g., easy to handle) regardless of whether it is small-scale or large-scale, and easy to make compact equipment (large membrane area per unit volume).

[0049] A "hollow fiber membrane" is a "thin tubular membrane" having an outer diameter of about 5 mm to 0.2 mm and an outer-to-inner diameter ratio (outer diameter ÷ inner diameter) of about 2.5 to 1.2. The "membrane surface" includes an "outer surface" and an "inner surface." The smaller the outer diameter, the easier it is to increase the membrane area per unit volume, and the greater the ratio of the outer diameter to the inner diameter, the easier it is to increase the membrane's mechanical strength (pressure resistance, etc.). A small inner diameter increases the resistance to liquid flow through the hollow portion of the hollow fiber membrane (the tubular portion of the lumen surrounded by the inner surface). From the viewpoint of the balance between the membrane area per unit volume, mechanical strength, and resistance to liquid flow, the outer diameter of the hollow fiber membrane is preferably about 2.0 mm to 0.5 mm, and the ratio of the outer diameter to the inner diameter is preferably about 2.0 to 1.3. From the viewpoint of mechanical strength (pressure resistance), the ratio of the outer diameter to the inner diameter is preferably greater than 1.5, and more preferably greater than 1.5 but not greater than 2.0.

[0050] As mentioned above, the "filtration" in this embodiment is assumed to be "pressure filtration," so the mechanical strength (pressure resistance) of the membrane is important. It is preferable that the "filtration pressure" be at least 0.2 MPa. One way to ensure pressure resistance is to adjust the ratio of the outer diameter to the inner diameter of the hollow fiber membrane. Note that, if the ratio of the outer diameter to the inner diameter of the hollow fiber membrane is large, the membrane thickness of the hollow fiber membrane becomes relatively thick, increasing the filtration resistance of the membrane and potentially detrimental to the membrane's permeability. However, ensuring pressure resistance is also important. Therefore, it is important to set the ratio of the outer diameter to the inner diameter of the hollow fiber membrane from the perspective of balancing these two factors (membrane permeability and pressure resistance). On the other hand, since dialysis membranes used in artificial kidneys and the like are not intended for (and therefore do not need to be emphasized as) pressure filtration, it is generally not necessary to set a value for the ratio of the outer diameter to the inner diameter of the dialysis membrane, and the value of the ratio of the outer diameter to the inner diameter of the dialysis membrane is small (see, for example, Non-Patent Document 8).

[0051] When "concentrating valuables" using a hollow fiber membrane (when the purpose of filtration is to obtain the liquid that did not permeate the membrane, rather than the liquid that did permeate the membrane), it is preferable to perform filtration by applying pressure from the inner surface side to the outer surface side, so that the "concentrated liquid" is present on the inner surface side of the hollow fiber membrane (internal pressure filtration), from the viewpoint of adjusting and increasing the concentration level. Furthermore, when the lipid nanoparticles are valuables (e.g., pharmaceuticals) that are to be concentrated and recovered by filtration, "internal pressure filtration" is preferred.

[0052] <Method for concentrating lipid nanoparticle-containing liquid> The method for concentrating lipid nanoparticle-containing liquid of this embodiment includes a step (filtration step) of filtering the lipid nanoparticle-containing liquid using the polyacrylonitrile-based ultrafiltration membrane of this embodiment. In one aspect, the method for concentrating lipid nanoparticle-containing liquid of this embodiment also includes a step (solvent exchange step) of adding a solvent different from the solvent component of the lipid nanoparticle-containing liquid removed by filtration to the lipid nanoparticle-containing liquid in an amount equal to all or part of the amount lost by filtration. The solvent exchange step is a step that is optionally performed depending on the need for solvent exchange.

[0053] In the method for concentrating a lipid nanoparticle-containing liquid, the lipid nanoparticles of this embodiment, the lipid nanoparticle-containing liquid of this embodiment, and the polyacrylonitrile-based ultrafiltration membrane of this embodiment are the same as described above. Also, the step of filtering the lipid nanoparticle-containing liquid with the polyacrylonitrile-based ultrafiltration membrane of this embodiment in the concentration method of this embodiment is the same as the "concentration" treatment. Furthermore, the step of adding a solvent different from the solvent component of the lipid nanoparticle-containing liquid removed by filtration in an amount equal to all or part of the amount lost by filtration to the lipid nanoparticle-containing liquid is the same as the "solvent exchange" treatment.

[0054] According to the concentration method of this embodiment, the lipid nanoparticle-containing liquid can be filtrated stably with little clogging of the membrane, the lipid nanoparticle recovery rate is high, and the concentration can be easily performed. Furthermore, according to the concentration method of this embodiment, the lipid nanoparticle concentration can be increased to a concentration at which the medicinal efficacy of the medicinal active ingredients contained in the lipid nanoparticles can be expected.

[0055] <Method for Solvent Exchange of Lipid Nanoparticle-Containing Liquid> The method for solvent exchange of lipid nanoparticle-containing liquid of this embodiment is a method comprising a step of adding a solvent different from the solvent component of the lipid nanoparticle-containing liquid removed by filtration using a polyacrylonitrile-based ultrafiltration membrane to the lipid nanoparticle-containing liquid in an amount equal to all or part of the amount lost by filtration (solvent exchange step). In one aspect, the method for solvent exchange of lipid nanoparticle-containing liquid of this embodiment comprises a step of filtering the lipid nanoparticle-containing liquid using the polyacrylonitrile-based ultrafiltration membrane of this embodiment (filtration step).

[0056] In the solvent exchange method for lipid nanoparticle-containing liquid, the polyacrylonitrile-based ultrafiltration membrane of this embodiment, the lipid nanoparticles of this embodiment, the lipid nanoparticle-containing liquid of this embodiment, and the solvent of the lipid nanoparticle-containing liquid are the same as described above.In addition, the solvent exchange method of this embodiment involves adding a solvent different from the solvent component of the lipid nanoparticle-containing liquid removed by filtration to the lipid nanoparticle-containing liquid in an amount equal to all or part of the amount lost by filtration, which is the same as the "solvent exchange" process.Furthermore, the process of filtering the lipid nanoparticle-containing liquid with the polyacrylonitrile-based ultrafiltration membrane of this embodiment is the same as the "concentration" process.

[0057] According to the solvent exchange method of this embodiment, the concentration of organic solvents such as alcohol mixed in the lipid nanoparticle-containing liquid can be reduced to a concentration that is safe for injection into a living body, and the solvent components of the lipid nanoparticle-containing liquid can be adjusted to the desired final concentration so that the lipid nanoparticles can be stably present in the lipid nanoparticle-containing liquid. Furthermore, according to the concentration method and solvent exchange method of this embodiment, the denaturation and deterioration / breakage of the lipid nanoparticles can be suppressed, and the lipid nanoparticles can be easily concentrated with a high recovery rate. In particular, when lipid nanoparticles are used as pharmaceuticals, it is important to have a low risk of denaturation, deterioration, and breakage in order to ensure their safety. Furthermore, because pharmaceuticals are expensive and valuable, a high recovery rate of lipid nanoparticles is particularly important.

[0058] <Method for producing lipid nanoparticle concentrate> The method for producing a lipid nanoparticle concentrate of this embodiment is a method comprising a step of filtering a lipid nanoparticle-containing liquid with the polyacrylonitrile-based ultrafiltration membrane of this embodiment (filtration step). In one aspect, the method for producing a lipid nanoparticle concentrate of this embodiment comprises a step of adding a solvent different from the solvent components of the lipid nanoparticle-containing liquid removed by filtration to the lipid nanoparticle-containing liquid in an amount equal to all or part of the amount lost by filtration (solvent exchange step).

[0059] In the method for producing a lipid nanoparticle concentrated solution, the lipid nanoparticles of this embodiment, the lipid nanoparticle-containing solution of this embodiment, and the polyacrylonitrile-based ultrafiltration membrane of this embodiment are the same as those described above. Also, the process of filtering the lipid nanoparticle-containing solution using the polyacrylonitrile-based ultrafiltration membrane of this embodiment in the production method of this embodiment is the same as the "concentration" process. Furthermore, the process of adding a solvent different from the solvent component of the lipid nanoparticle-containing solution removed by filtration to the lipid nanoparticle-containing solution in an amount equal to all or part of the amount lost by filtration is the same as the "solvent exchange" process. The solvent exchange process is an optional process.

[0060] According to the production method of this embodiment, a lipid nanoparticle concentrate can be produced easily while suppressing denaturation, deterioration, and damage of the lipid nanoparticles, and achieving a high recovery rate of the lipid nanoparticles.

[0061] In the present disclosure, a high recovery rate of lipid nanoparticles means that the recovery rate of lipid nanoparticles concentrated through a method for concentrating a lipid nanoparticle-containing liquid is 70% or higher.

[0062] Hereinafter, the embodiments of the present invention will be described in more detail with reference to examples, but the embodiments of the present invention are not limited to these examples.

[0063] (Production Example 1 of Polyacrylonitrile-Based Ultrafiltration Membrane) According to Example 1 described in WO 98 / 58728, a polyacrylonitrile-based ultrafiltration membrane was prepared, and a polyacrylonitrile-based ultrafiltration hollow fiber membrane was obtained, consisting of a polyacrylonitrile-based polymer material containing 91.5% by weight of acrylonitrile, 8.0% by weight of methyl acrylate, and 0.5% by weight of sodium methallylsulfonate. The obtained membrane had an outer diameter of 1.35 mm, an inner diameter of 0.75 mm, a rejection rate of dextran T-10 of less than 5%, and a rejection rate of dextran T-2000 of 95%. The pure water permeability of the obtained membrane was 400 L / m at 25 ° C. ２ (internal surface area) / h / 0.1 MPa.

[0064] (Production Example 2 of Polyacrylonitrile-Based Ultrafiltration Membrane) As the polyacrylonitrile-based polymer material, a polymer material consisting of 89.0 wt% acrylonitrile, 5.0 wt% methyl acrylate, and 6.0 wt% ethyl methacrylate trimethylammonium chloride was used, except that DMSO was used as the solvent for the polymer material and a 50 wt% DMSO aqueous solution was used as the internal solution. A polyacrylonitrile-based ultrafiltration hollow fiber membrane was produced in the same manner as in Example 1 described in WO 98 / 58728. The produced membrane had an outer diameter of 1.35 mm, an inner diameter of 0.75 mm, and a rejection rate of dextran T-10 of 10% or less and a rejection rate of dextran T-2000 of 98% or more. The pure water permeability of the obtained membrane was 300 L / m at 25 ° C. ２ (internal surface area) / h / 0.1 MPa.

[0065] The outer diameter and inner diameter of the polyacrylonitrile-based ultrafiltration membranes prepared in Production Examples 1 and 2 were determined by the method described in WO 98 / 58728. The rejection rates of dextran T-10 and dextran T-2000 of the polyacrylonitrile-based ultrafiltration membranes prepared in Production Examples 1 and 2 were determined by the following method. 0.1 wt % aqueous solutions of dextran T-10 and dextran T-2000 were filtered through the polyacrylonitrile-based ultrafiltration membranes prepared in Production Examples 1 and 2 at a filtration pressure of 0.5 bar, a linear velocity of 0.1 m / sec, and room temperature (25°C). The dextran concentration in the liquid that permeated the membrane (permeated liquid) was determined from the refractive index of the permeated liquid measured with a refractometer and a calibration curve of dextran concentration. The dextran concentrations in the permeate and the liquid supplied to the membrane were applied to the formula: Solute rejection (%) = 100 × {1 - (dextran concentration in permeate) / (dextran concentration in liquid supplied to the membrane)} to determine the rejection rates of dextran T-10 and dextran T-2000 by the membrane. The pure water permeability at 25°C of the polyacrylonitrile-based ultrafiltration membranes prepared in Production Examples 1 and 2 was determined by the method described in WO 98 / 58728.

[0066] (Production Example of Polysulfone-Based Ultrafiltration Membrane) According to Example 1 of JP-A-62-201602, a polysulfone-based ultrafiltration hollow fiber membrane made of polysulfone resin was obtained, having an outer diameter of 1.10 mm, an inner diameter of 0.60 mm, and a rejection rate of dextran T-10 of 5% or less and a rejection rate of dextran T-2000 of 98% or more. The pure water permeability of the obtained membrane was 500 L / m at 25°C. ２ (internal surface area) / h / 0.1 MPa.

[0067] (Mini-Module Manufacturing Example) Ten polyacrylonitrile-based ultrafiltration hollow fiber membranes (e.g., hollow fiber membranes 25) obtained in Manufacturing Example 2 were inserted into a cylindrical transparent plastic container 26 made of polycarbonate resin, and both ends of the hollow fiber membranes 25 were sealed with seals 24 (e.g., epoxy resin) to produce a polyacrylonitrile-based ultrafiltration hollow fiber membrane mini-module 27 having a hollow fiber membrane length of approximately 11 cm (effective length contributing to filtration: approximately 9 cm) (see FIG. 4). In the mini-module 27 of FIG. 4, multiple hollow fiber membranes 25 are housed in the plastic container 26. Both ends of the hollow fiber membranes 25 are adhesively fixed to the plastic container 26 by the seals 24. Two end nozzles (e.g., end nozzle 1: 20 and end nozzle 2: 21) are provided at both ends of the plastic container 26. There are two side nozzles (e.g., side nozzle 1: 22 and side nozzle 2: 23) on the side of the plastic container 26. These side nozzles serve as outlets for the liquid that has permeated through the hollow fiber membrane 25. These end nozzles serve as inlets for the liquid to be filtered (e.g., a liquid containing lipid nanoparticles) when the liquid is filtered through the hollow fiber membrane 25. The fabricated mini-module 27 was stored with pure water sealed in the hollow portion of the hollow fiber membrane 25 and in the plastic container 26.

[0068] (Components of lipid nanoparticle-containing liquid) (Ionized lipids (pH-sensitive lipids): all of the following examples have a tertiary amino group and are positively charged at acidic pH.) SM-102 (manufactured by Cayman Chemical) DODAP (manufactured by Polysciences) DLin-MC3-DMA (manufactured by Cayman Chemical) (Phospholipid) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (product name: COATSOME MC-8080, manufactured by NOF CORPORATION) (Sterol) Cholesterol (manufactured by Sigma-Aldrich) (Polyethylene glycol-conjugated lipid) 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (PEG number average molecular weight (Mn) is 2000) (DMG-PEG2000) (product name: SUNBRIGHT GM-020, manufactured by NOF CORPORATION) (Fluorescent dye) DiI (manufactured by Thermo Fisher Scientific) (Buffer) Acetic acid buffer (pH 5.0, 6.25 mM) was prepared as follows: the required amount of sodium acetate was measured and dissolved in pure water, and a 5N aqueous solution of NaOH was added dropwise with stirring to adjust the pH to 5.0. PBS (phosphate buffered saline, pH 7.4) (product name: Dulbecco's Phosphate Buffered Saline D-PBS(-) (1x), manufactured by Nacalai Tesque) All of the lipid nanoparticles produced in this example were lipid nanoparticles (LNP) type, and all of them used lipids having a tertiary amino group as ionizable lipids, so that they are lipid nanoparticles that become positively charged when the pH is acidic.

[0069] (Lipid Nanoparticle-Containing Liquid Production Example 1) A lipid nanoparticle-containing ethanol solution was prepared in which the molar ratio of SM-102 / 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) / cholesterol / 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (PEG number average molecular weight (Mn) 2000) (DMG-PEG2000) was 50 / 10 / 38.5 / 1.5 and the total concentration was 4.0 mM. In addition, to label the lipid nanoparticles, the fluorescent dye DiI was added in an amount of 0.5 mol% of the total solute. 750 μL of this lipid nanoparticle-containing ethanol solution and 2250 μL of acetate buffer (6.25 mM, pH 5.0) were mixed using a NanoAssemblr® ultrafast nanomedicine production device (manufactured by Precision NanoSystems). At this time, the flow rates of the ethanol solution containing lipid nanoparticles and the acetate buffer solution were 1.0 mL / min and 3.0 mL / min, respectively (i.e., the ethanol solution containing lipid nanoparticles and the acetate buffer solution were mixed in a volume ratio of 1:3), yielding 3.0 mL of lipid nanoparticle-containing liquid (hereinafter referred to as lipid nanoparticle-containing liquid 1).

[0070] Here, in the present disclosure, the "lipid nanoparticle concentration" in the lipid nanoparticle-containing liquid is expressed as the "total molar concentration of the lipid nanoparticle components (lipids, phospholipids, sterols, polyethylene glycol-bound lipids, etc.)," ​​and the lipid nanoparticle concentration in lipid nanoparticle-containing liquid 1 was 1 mM.

[0071] (Lipid nanoparticle-containing liquid production example 2) An ethanol solution containing lipid nanoparticles was prepared, with a molar ratio of SM-102 / DSPC / cholesterol / DMG-PEG2000 of 50 / 10 / 38.5 / 1.5 and a total concentration of 4.0 mM. In addition, to label the lipid nanoparticles, the fluorescent dye DiI was added in an amount of 0.5 mol % of the total solute. 750 μL of the ethanol solution containing the lipid nanoparticles and 2250 μL of acetate buffer (6.25 mM, pH 5.0) containing messenger RNA (TriLink, CleanCap (registered trademark) FLuc mRNA (5-methoxy Uridine: 5 moU) − (L-7202)) at a concentration of 0.04 μg / μL were mixed using a NanoAssemblr (registered trademark) ultrafast nanomedicine production device (Precision NanoSystems). At this time, the flow rates of the ethanol solution containing lipid nanoparticles and the acetate buffer solution containing messenger RNA were 1.0 mL / min and 3.0 mL / min, respectively (i.e., the ethanol solution containing lipid nanoparticles and the acetate buffer solution containing messenger RNA were mixed in a volume ratio of 1:3), yielding 3.0 mL of lipid nanoparticle-containing liquid (hereinafter referred to as lipid nanoparticle-containing liquid 2). The lipid nanoparticle concentration in lipid nanoparticle-containing liquid 2 was 1 mM.

[0072] (Lipid Nanoparticle-Containing Liquid Production Example 3) An ethanol solution containing lipid nanoparticles was prepared, in which the molar ratio of SM-102 / DSPC / cholesterol / DMG-PEG2000 was 50 / 10 / 38.5 / 1.5 and the total concentration was 4.0 mM. In addition, to label the lipid nanoparticles, the fluorescent dye DiI was added in an amount of 0.5 mol% of the total solutes. 750 μL of this ethanol solution containing lipid nanoparticles and 2250 μL of acetate buffer (6.25 mM, pH 5.0) were mixed using a NanoAssemblr (registered trademark) ultrafast nanomedicine production device (manufactured by Precision NanoSystems). At this time, the flow rates of the ethanol solution containing lipid nanoparticles and the acetate buffer solution were 1.0 mL / min and 3.0 mL / min, respectively (i.e., the ethanol solution containing lipid nanoparticles and the acetate buffer solution were mixed in a volume ratio of 1:3), and 3.0 mL of lipid nanoparticle-containing solution was obtained. The obtained lipid nanoparticle-containing solution was diluted with 12 mL of PBS and transferred to an Amicon Ultra 4 (manufactured by Millipore). The transferred lipid nanoparticle-containing solution was subjected to ultrafiltration under centrifugation conditions (25 ° C, 1000 G, approximately 15 minutes). The lipid nanoparticle-containing solution was concentrated to approximately 1500 μL, and then the obtained concentrate was diluted to 15 mL using PBS and again concentrated to approximately 1000 μL by ultrafiltration under the same centrifugation conditions (25 ° C, 1000 G, approximately 15 minutes). Finally, the mixture was diluted with PBS so that the total concentration of SM-102, DSPC, cholesterol, and DMG-PEG2000 was 1 mM, yielding the desired lipid nanoparticle-containing liquid (hereinafter, lipid nanoparticle-containing liquid 3). The lipid nanoparticle concentration in lipid nanoparticle-containing liquid 3 was 1 mM.

[0073] (Lipid Nanoparticle-Containing Liquid Production Example 4) An ethanol solution containing lipid nanoparticles was prepared, with a molar ratio of DODAP / DSPC / cholesterol / DMG-PEG2000 of 50 / 10 / 38.5 / 1.5 and a total concentration of 4.0 mM. Furthermore, to label the lipid nanoparticles, the fluorescent dye DiI was added in an amount of 0.5 mol% of the total solutes. 750 μL of this ethanol solution containing lipid nanoparticles and 2250 μL of acetate buffer (6.25 mM, pH 5.0) were mixed using a NanoAssemblr® ultrafast nanomedicine production device (manufactured by Precision NanoSystems). At this time, the flow rates of the ethanol solution containing lipid nanoparticles and the acetate buffer solution were 1.0 mL / min and 3.0 mL / min, respectively (i.e., the ethanol solution containing lipid nanoparticles and the acetate buffer solution were mixed in a volume ratio of 1:3), and 3.0 mL of lipid nanoparticle-containing solution was obtained. The obtained lipid nanoparticle-containing solution was diluted with 12 mL of PBS and transferred to an Amicon Ultra 4 (manufactured by Millipore). The transferred lipid nanoparticle-containing solution was subjected to ultrafiltration under centrifugation conditions (25 ° C, 1000 G, approximately 15 minutes). The lipid nanoparticle-containing solution was concentrated to approximately 1500 μL, and then the obtained concentrate was diluted to 15 mL using PBS and again concentrated to approximately 1000 μL by ultrafiltration under the same centrifugation conditions (25 ° C, 1000 G, approximately 15 minutes). Finally, the mixture was diluted with PBS so that the total concentration of DODAP, DSPC, cholesterol, and DMG-PEG2000 was 1 mM, yielding the desired lipid nanoparticle-containing liquid (hereinafter, lipid nanoparticle-containing liquid 4). The lipid nanoparticle concentration in lipid nanoparticle-containing liquid 4 was 1 mM.

[0074] (Lipid Nanoparticle-Containing Liquid Production Example 5) An ethanol solution containing lipid nanoparticles was prepared, with a molar ratio of DLin-MC3-DMA / DSPC / cholesterol / DMG-PEG2000 of 50 / 10 / 38.5 / 1.5, for a total concentration of 4.0 mM. Furthermore, to label the lipid nanoparticles, the fluorescent dye DiI was added in an amount of 0.5 mol% of the total solutes. 750 μL of this ethanol solution containing lipid nanoparticles and 2250 μL of acetate buffer (6.25 mM, pH 5.0) were mixed using a NanoAssemblr® ultrafast nanomedicine production device (manufactured by Precision NanoSystems). At this time, the flow rates of the ethanol solution containing lipid nanoparticles and the acetate buffer solution were 1.0 mL / min and 3.0 mL / min, respectively (i.e., the lipid-containing ethanol solution and the acetate buffer solution were mixed in a volume ratio of 1:3), and 3.0 mL of lipid nanoparticle-containing solution was obtained. The obtained lipid nanoparticle-containing solution was diluted with 12 mL of PBS and transferred to an Amicon Ultra 4 (manufactured by Millipore). The transferred lipid nanoparticle-containing solution was subjected to ultrafiltration under centrifugation conditions (25 ° C, 1000 G, approximately 15 minutes). The lipid nanoparticle-containing solution was concentrated to approximately 1500 μL, and then the obtained concentrate was diluted to 15 mL using PBS and again concentrated to approximately 1000 μL by ultrafiltration under the same centrifugation conditions (25 ° C, 1000 G, approximately 15 minutes). Finally, the mixture was diluted with PBS so that the total concentration of DLin-MC3-DMA, DSPC, cholesterol, and DMG-PEG2000 was 1 mM, yielding the desired lipid nanoparticle-containing solution (hereinafter, lipid nanoparticle-containing solution 5). The lipid nanoparticle concentration in lipid nanoparticle-containing solution 5 was 1 mM.

[0075] (Lipid nanoparticle-containing liquid production example 6) An ethanol solution containing lipid nanoparticles was prepared, with a molar mixture ratio of SM-102 / DSPC / cholesterol / DMG-PEG2000 of 50 / 10 / 38.5 / 1.5 and a total concentration of 20 mM. In addition, to label the lipid nanoparticles, the fluorescent dye DiI was added in an amount of 0.5 mol % of the total solute. 1000 μL of this ethanol solution containing lipid nanoparticles and 3000 μL of acetate buffer (6.25 mM, pH 5.0) containing messenger RNA (TriLink, CleanCap (registered trademark) Fluc mRNA (5-methoxy Uridine: 5 moU) - (L7202)) at a concentration of 0.04 μg / μL, NanoAssemblr (registered trademark) ultrafast nanomedicine production device (Precision NanoSystems) was mixed using. At this time, the flow rates of the ethanol solution containing lipid nanoparticles and the acetate buffer solution were 1.0 mL / min and 3.0 mL / min, respectively (i.e., the ethanol solution containing lipid nanoparticles and the acetate buffer solution were mixed in a volume ratio of 1:3), and 4.0 mL of lipid nanoparticle-containing liquid (hereinafter, lipid nanoparticle-containing liquid 6) was obtained. The lipid nanoparticle concentration in the lipid nanoparticle-containing solution 6 was 5 mM.

[0076] (Lipid Nanoparticle-Containing Liquid Production Example 7) An ethanol solution containing lipid nanoparticles was prepared, in which the molar ratio of SM-102 / DSPC / cholesterol / DMG-PEG2000 was 50 / 10 / 38.5 / 1.5 and the total concentration was 20 mM. Furthermore, to label the lipid nanoparticles, the fluorescent dye DiI was added in an amount of 0.5 mol % of the total solutes. 1000 μL of this ethanol solution containing lipid nanoparticles and 3000 μL of acetate buffer (6.25 mM, pH 5.0) were mixed using a NanoAssemblr (registered trademark) ultrafast nanomedicine production device (manufactured by Precision NanoSystems). At this time, the flow rates of the ethanol solution containing lipid nanoparticles and the acetate buffer solution were 1.0 mL / min and 3.0 mL / min, respectively (i.e., the ethanol solution containing lipid nanoparticles and the acetate buffer solution were mixed at a volume ratio of 1:3), yielding 4.0 mL of lipid nanoparticle-containing liquid (hereinafter referred to as lipid nanoparticle-containing liquid 7). Lipid nanoparticle-containing liquid 7 had the same composition as lipid nanoparticle-containing liquid 6 except that it did not contain messenger RNA, and the lipid nanoparticle concentration was 5 mM.

[0077] Example 1: An injection needle (gauge: 21G) was inserted into both ends of an approximately 20 cm long polyacrylonitrile-based ultrafiltration hollow fiber membrane obtained in Production Example 1 of a polyacrylonitrile-based ultrafiltration membrane. 3.0 mL of each of lipid nanoparticle-containing solutions 1 to 3 was placed in a plastic syringe, and the syringe was connected to the injection needle at one end of the hollow fiber membrane. The lipid nanoparticle-containing solution was injected from the syringe into the hollow portion of the hollow fiber membrane by manually pushing the syringe. When the lipid nanoparticle-containing solution reached the injection needle at the other end, the injection of the lipid nanoparticle-containing solution from the syringe was stopped, and the end of the injection needle at the other end was plugged. Next, by manually pushing the syringe containing the lipid nanoparticle-containing solution, the lipid nanoparticle-containing solution was filtered from the inner surface side of the polyacrylonitrile-based ultrafiltration hollow fiber membrane using a total filtration method. Filtration was performed until the amount of lipid nanoparticle-containing solution remaining in the syringe was 1.5 mL. The entire amount of permeate that permeated through the hollow fiber membrane was sampled. After filtration was completed, the stopper of the injection needle at the other end was removed, and the syringe containing the lipid nanoparticle-containing solution was pulled, and the lipid nanoparticle-containing solution concentrated in the hollow portion of the hollow fiber membrane was collected in the syringe containing the lipid nanoparticle-containing solution. In addition, a syringe containing 1.0 mL of a solvent having the same composition as the solvent contained in the lipid nanoparticle-containing solution was connected to the injection needle at the other end from which the stopper had been removed, and the remaining liquid of the concentrated lipid nanoparticle-containing solution that remained attached to the inner surface of the hollow portion of the hollow fiber membrane was also collected by injecting it from the syringe into the hollow portion of the hollow fiber membrane.

[0078] The lipid nanoparticle concentration in the resulting recovered solution and permeate was measured by quantifying the fluorescent dye (DiI) used to label the lipid nanoparticles. The volume of the recovered solution was adjusted to 3.0 mL so that it was the same volume as the volume at the start of filtration. A 0.2% (w / v) aqueous solution of Triton X-100 (MP biomedicals) was added to the sample after adjustment, and diluted to 0.5 mM, which is the theoretical value when all lipids are recovered. In addition, a lipid nanoparticle solution that had not been subjected to membrane filtration (no loss in principle) was diluted to a final concentration of 0.5 mM, and this solution was diluted with a 0.2% (w / v) Triton X-100 solution to 0.250 mM, 0.125 mM, 0.063 mM, and 0.031 mM, and a calibration curve was created. The fluorescence intensity of DiI present in these solutions was measured using Infinite 200 PRO (Ex 550 nm, Em 595 nm) manufactured by TECAN, and the amount of fluorescent dye present in each solution was calculated from the calibration curve, which was used as the recovery rate of lipid nanoparticles by the concentration method using a polyacrylonitrile-based ultrafiltration hollow fiber membrane and the amount of lipid nanoparticles that leaked from the hollow fiber membrane into the permeate.

[0079] The results of performing the above operation on lipid nanoparticle-containing solutions 1 to 3 are shown below. Lipid nanoparticle-containing solution 1: lipid nanoparticle recovery rate 70%, and the amount of lipid nanoparticles leaked into the permeate: zero. Lipid nanoparticle-containing solution 2: lipid nanoparticle recovery rate 80%, and the amount of lipid nanoparticles leaked during permeation: zero. Lipid nanoparticle-containing solution 3: lipid nanoparticle recovery rate 90%, and the amount of lipid nanoparticles leaked into the permeate: zero.

[0080] For all lipid nanoparticle-containing liquids, the filtration operation itself by manually pushing the syringe could be performed without any particular problems. On the other hand, the ease of filtration (minimum force required for manual pushing) was in the order of lipid nanoparticle-containing liquid 3, lipid nanoparticle-containing liquid 2, and lipid nanoparticle-containing liquid 1.

[0081] In Example 1, the recovery rate of lipid nanoparticles was high (no leakage) for any lipid nanoparticle-containing liquid, and furthermore, the filtration operation was easy, allowing the lipid nanoparticles to be easily concentrated.

[0082] The results of Example 1 demonstrate that the method for concentrating a lipid nanoparticle-containing liquid of this embodiment, which includes a step of filtering the lipid nanoparticle-containing liquid through a polyacrylonitrile-based ultrafiltration membrane, is advantageous.

[0083] (Example 2) Using the polyacrylonitrile-based ultrafiltration hollow fiber membrane having a length of approximately 20 cm obtained in Polyacrylonitrile-based Ultrafiltration Membrane Production Example 2, filtration operations were carried out on the lipid nanoparticle-containing liquids prepared in Lipid Nanoparticle-containing Liquids 1 to 5 in the same manner as in Example 1. The results are shown below.

[0084] Lipid nanoparticle-containing liquid 1: Lipid nanoparticle recovery rate 80%, and the amount of lipid nanoparticles leaked into the permeate: zero. Lipid nanoparticle-containing liquid 2: Lipid nanoparticle recovery rate 80%, and the amount of lipid nanoparticles leaked into the permeate: zero. Lipid nanoparticle-containing liquid 3: Lipid nanoparticle recovery rate 92%, and the amount of lipid nanoparticles leaked into the permeate: zero. Lipid nanoparticle-containing liquid 4: Lipid nanoparticle recovery rate 92%, and the amount of lipid nanoparticles leaked into the permeate: zero. Lipid nanoparticle-containing liquid 5: Lipid nanoparticle recovery rate 92%, and the amount of lipid nanoparticles leaked into the permeate: zero.

[0085] For any lipid nanoparticle-containing liquid, the filtration operation itself by manually pushing the syringe could be performed without any particular problems, and for lipid nanoparticle-containing liquids 1 to 3, filtration was slightly easier than with the membrane obtained in Production Example 1 of the polyacrylonitrile-based ultrafiltration membrane (the force required for manual pushing was slightly smaller). Furthermore, the degree of ease of filtration (smaller force required for manual pushing) was, as in Example 1, in the order of lipid nanoparticle-containing liquid 3, lipid nanoparticle-containing liquid 2, and lipid nanoparticle-containing liquid 1. The degree of ease of filtration (smaller force required for manual pushing) for lipid nanoparticle-containing liquids 3 to 5 was approximately the same.

[0086] Furthermore, when the membrane was cut after the filtration operation of the lipid nanoparticle-containing liquid 1 was completed and the inner surface was visually observed, it was found to be colored red due to the adhesion of lipid nanoparticles labeled with a red fluorescent dye. It was confirmed that the coloration of the inner surface of the membrane after the filtration operation could be removed by injecting ethyl alcohol into the hollow part of the membrane from a syringe needle at another end of the membrane. This indicates that the polyacrylonitrile-based ultrafiltration membrane used in this embodiment can be washed with ethyl alcohol even if lipid nanoparticles adhere to the membrane surface.

[0087] In Example 2, regardless of the lipid nanoparticle-containing liquid, the recovery rate of lipid nanoparticles was high (there was no leakage), and furthermore, the filtration operation was easy, allowing the lipid nanoparticles to be easily concentrated.

[0088] The results of Example 2 demonstrate that the method for concentrating a lipid nanoparticle-containing liquid of this embodiment, which includes a step of filtering the lipid nanoparticle-containing liquid through a polyacrylonitrile-based ultrafiltration membrane, is advantageous.

[0089] (Example 3) The lipid nanoparticle-containing liquid was subjected to a filtration operation, a PBS solution addition operation, and a recovery operation as follows.

[0090] Filtration Operation A: A syringe needle (gauge: 21G) was inserted into both ends of the approximately 20 cm long polyacrylonitrile-based ultrafiltration hollow fiber membrane obtained in Production Example 2 of the polyacrylonitrile-based ultrafiltration membrane and attached. 3.0 mL of lipid nanoparticle-containing liquid 2 was placed in a plastic syringe, and the syringe was connected to the syringe needle at one end of the hollow fiber membrane. The lipid nanoparticle-containing liquid 2 was injected from the syringe into the hollow portion of the hollow fiber membrane by manually pushing the syringe. When the lipid nanoparticle liquid reached the syringe at the other end, the injection of the lipid nanoparticle-containing liquid from the syringe was stopped, and the end of the syringe needle at the other end was plugged. Next, by manually pushing the syringe containing the lipid nanoparticle-containing liquid, the lipid nanoparticle-containing liquid was filtered from the inner surface side of the polyacrylonitrile-based ultrafiltration hollow fiber membrane using a total filtration method. Filtration was performed until the amount of lipid nanoparticle-containing liquid remaining in the syringe was 1.5 mL. The entire amount of the permeate that had permeated through the hollow fiber membrane was sampled.

[0091] - PBS solution addition operation A After completion of filtration operation A, the plug of the injection needle at the other end was removed, and 1.5 mL of PBS solution (phosphate buffered saline, pH 7.4, Dulbecco's phosphate buffered saline D-PBS(-)(1x) manufactured by Nacalai Tesque) was injected as a solvent different from the solvent of lipid nanoparticle-containing liquid 2. At this point, a total of 3 mL of lipid nanoparticle-containing liquid, in which the ratio of PBS to the solvent contained in the lipid nanoparticle-containing liquid is 50% by volume, is present in the syringe containing the lipid nanoparticle-containing liquid and in the hollow part of the hollow fiber membrane.

[0092] Filtration Operation B: The empty syringe used to inject the PBS solution was removed from the injection needle, and the needle was capped. Next, the syringe containing the lipid nanoparticle-containing solution was manually pressed, and the lipid nanoparticle-containing solution was filtered from the inner surface side of the polyacrylonitrile-based ultrafiltration hollow fiber membrane using a total filtration method. Filtration was continued until the volume of the lipid nanoparticle-containing solution remaining in the syringe was 1.5 mL. The entire amount of the permeated liquid that had permeated through the hollow fiber membrane was sampled.

[0093] Following this filtration operation B, the PBS solution addition operation A, the filtration operation B, the PBS solution addition operation A, the filtration operation B, the PBS solution addition operation A, and the filtration operation B were carried out in this order.

[0094] - Recovery operation A The stopper of the injection needle was removed, and the syringe containing the lipid nanoparticle-containing solution 2 was pulled, and the lipid nanoparticle-containing solution concentrated in the hollow portion of the hollow fiber membrane was collected into the syringe containing the lipid nanoparticle-containing solution. In addition, a syringe containing 1.5 mL of the PBS solution was connected to the other end of the injection needle from which the stopper was removed, and the remaining liquid of the concentrated lipid nanoparticle-containing solution adhering to the inner surface of the hollow portion of the hollow fiber membrane was also collected. Through the above operations, 3 mL of lipid nanoparticle-containing solution (hereinafter referred to as the sample of Example 3) having a PBS ratio of 97% by volume and an ethanol ratio of 0.8% by volume as the final composition of the solvent was obtained.

[0095] Analysis of the obtained sample of Example 3 revealed that the lipid nanoparticle recovery rate was 86%, the amount of lipid nanoparticles leaked into the permeate was zero, the nucleic acid encapsulation rate in the lipid nanoparticles was 90%, the average particle size of the lipid nanoparticles was 58 nm, and the polydispersity index of the lipid nanoparticle particle size was 0.11. The measurement of the lipid nanoparticle recovery rate in the recovery solution and the amount of lipid nanoparticles leaked into the permeate was performed in the same manner as in Example 1.

[0096] The nucleic acid encapsulation rate serves as an indicator of whether degradation or damage has occurred in the lipid nanoparticles. If the nucleic acid encapsulation rate is 90% or higher, it can be considered that degradation or damage to the lipid nanoparticles has not occurred during the membrane filtration operation. Furthermore, the polydispersity index of the particle size of the lipid nanoparticles serves as an indicator of whether denaturation of the lipid nanoparticles, such as aggregation or destruction, has occurred in the lipid nanoparticles that have undergone operations such as concentration, and takes a value between 0 and 1.0. A polydispersity index of the particle size of the lipid nanoparticles of 0.20 or less is an indication that denaturation such as aggregation or destruction has not occurred. In this disclosure, the average particle size of the lipid nanoparticles and the polydispersity index of the particle size refer to "the absolute values ​​of the average particle size and the polydispersity index of the particle size of the lipid nanoparticles contained in the lipid nanoparticle-containing liquid that has undergone operations such as concentration."

[0097] The nucleic acid encapsulation rate within lipid nanoparticles was measured using the following method. The prepared samples were diluted to a theoretical value of 1 μg / mL of nucleic acid. Triton X-100 was added to the diluted samples to a concentration of 0.2% (w / v) to destroy the lipid nanoparticles. A sample with destroyed lipid nanoparticles (a sample in which the lipid nanoparticle structure was destroyed and the nucleic acid within the lipid nanoparticles was released into the solution; hereinafter, "destroyed sample") and a sample in which the lipid nanoparticles were not destroyed (hereinafter, "non-destroyed sample") were prepared. A fluorescent dye (RiboGreen) was diluted 200-fold in the final solution of each of these samples. 50 μL of these samples were transferred to a black 96-well plate, and the fluorescence of the RiboGreen contained in the nucleic acid was measured using a TECAN Infinite 200 PRO at an excitation wavelength of 484 nm and an emission wavelength of 535 nm. A nucleic acid calibration curve was created in the range of 0 μg / mL to 2 μg / mL, and the nucleic acid concentration in these samples was determined. The proportion of nucleic acid encapsulated in lipid nanoparticles (nucleic acid encapsulation rate) was determined by comparing the quantitative values ​​of the disrupted sample and the non-disrupted sample.

[0098] The mean particle size and polydispersity index of the lipid nanoparticles were measured by the following method. The mean particle size and polydispersity index of the lipid nanoparticles of the sample were measured by dynamic scattering (software: Zetasizer Nano; Malvern). When measuring the mean particle size of the sample, the sample was diluted with PBS to a sample concentration of 0.2 mM, and measured using the "size(quartz) SOP" (measurement program) in the software.

[0099] It can be seen that the recovery rate of lipid nanoparticles in Example 3 was high, and there was no leakage of lipid nanoparticles into the permeate, and lipid nanoparticles were recovered. Furthermore, since the nucleic acid encapsulation rate in Example 3 was high, it can be seen that there was almost no deterioration or damage of the lipid nanoparticles during the membrane filtration operation. Furthermore, since the polydispersity index of the particle size of the lipid nanoparticles was 0.20 or less, it can be seen that there was no aggregation or destruction of the lipid nanoparticles during the membrane filtration operation, and there was no denaturation of the lipid nanoparticles.

[0100] The results of Example 3 show that the solvent exchange method for lipid nanoparticle-containing liquid of this embodiment, which includes a step of adding a solvent different from the solvent component of the lipid nanoparticle-containing liquid removed by filtration to the lipid nanoparticle-containing liquid in an amount equal to all or part of the weight loss due to filtration, and the concentration method for lipid nanoparticle-containing liquid of this embodiment, which includes a step of filtering the lipid nanoparticle-containing liquid with a polyacrylonitrile-based ultrafiltration membrane, are advantageous.

[0101] (Example 4) The lipid nanoparticle-containing liquid was subjected to a filtration operation, a PBS solution addition operation, and a recovery operation as follows.

[0102] - Filtration operation C After draining (discharging) the pure water sealed in the hollow portion of the hollow fiber membrane of the mini-module produced in the mini-module production example and in the plastic container, a plastic syringe (male luer lock) containing 4.0 mL of lipid nanoparticle-containing liquid 2 was connected to the end nozzle 1 (male luer lock) via a female luer lock joint. The lipid nanoparticle-containing liquid 2 was injected from the syringe into the hollow portion of the hollow fiber membrane in the mini-module by pushing the syringe by hand. When the lipid nanoparticle-containing liquid reached the end nozzle 2, the injection of the lipid nanoparticle-containing liquid from the syringe was stopped, and the end nozzle 2 (male luer lock) was plugged. Since the empty volume of the hollow portion of the hollow fiber membrane etc. was about 0.8 mL, the amount of lipid nanoparticle-containing liquid in the syringe became about 3.2 mL when the plug was inserted. Next, by manually pushing the syringe containing the lipid nanoparticle-containing liquid, the lipid nanoparticle-containing liquid was filtered through the polyacrylonitrile-based ultrafiltration hollow fiber membrane in the mini-module from the inner surface side of the hollow fiber membrane using a total filtration method. Filtration was continued until the amount of lipid nanoparticle-containing liquid remaining in the syringe became zero. The entire amount of permeated liquid that permeated through the hollow fiber membrane was sampled.

[0103] - PBS solution addition operation B After completion of the filtration operation C, the plug of the end nozzle 2 was removed, and the end nozzle 2 was connected to a plastic syringe (male luer lock) containing 3.2 mL of PBS solution (phosphate buffered saline, pH 7.4, Dulbecco's phosphate buffered saline D-PBS (-) (1x) manufactured by Nacalai Tesque) via a female luer lock joint, and the total amount of 3.2 mL of PBS solution was injected toward the hollow portion of the hollow fiber membrane in the mini module. At this point, a total of 4.0 mL of lipid nanoparticle-containing liquid, which is a solvent composition with a PBS ratio of 80% by volume, is present in the syringe containing the lipid nanoparticle-containing liquid 2 and in the hollow portion of the hollow fiber membrane.

[0104] Filtration Operation D The empty syringe and female Luer lock joint used to inject the PBS solution were removed from the end nozzle 2, and the end nozzle 2 was plugged. Then, by manually pushing the syringe containing the lipid nanoparticle-containing liquid, the lipid nanoparticle-containing liquid was filtered through the polyacrylonitrile-based ultrafiltration hollow fiber membrane in the mini module using a total filtration method from the inner surface side of the hollow fiber membrane. Filtration was continued until the amount of lipid nanoparticle-containing liquid remaining in the syringe reached zero. The entire amount of permeated liquid that had permeated through the hollow fiber membrane was sampled.

[0105] Following this filtration operation D, the following operations were performed in this order: PBS solution addition operation B, filtration operation D, PBS solution addition operation B, filtration operation D, PBS solution addition operation B, and filtration operation D. At this point, 0.8 mL of lipid nanoparticle-containing liquid, in which the PBS ratio in the solvent was 99.8% by volume and the ethanol ratio was 0.04% by volume, was present in the mini-module.

[0106] Recovery Operation B: The lipid nanoparticle-containing liquid 2 present in the hollow portion of the hollow fiber membrane in the mini-module was recovered. First, the stopper of the end nozzle 2 was removed, and the empty syringe containing the lipid nanoparticle-containing liquid was pulled to recover the lipid nanoparticle-containing liquid present in the hollow portion of the hollow fiber membrane in the mini-module into the syringe containing the lipid nanoparticle-containing liquid. Next, a syringe (male luer lock) containing 1.0 mL of the PBS solution was connected to the end nozzle 2 via a female luer lock joint, and the PBS solution was injected from the syringe into the hollow portion of the hollow fiber membrane of the mini-module, thereby recovering the remaining lipid nanoparticle-containing liquid adhering to the inner surface of the hollow fiber membrane. A syringe (male luer lock) containing several mL of air was connected to the end nozzle 2 via a female luer lock joint, and air was injected from the syringe into the hollow portion of the hollow fiber membrane of the mini-module, thereby recovering the remaining concentrated lipid nanoparticle-containing liquid adhering to the inner surface of the hollow fiber membrane. The amount of the lipid nanoparticle-containing liquid collected was approximately 2 mL.

[0107] Analysis of the lipid nanoparticle-containing liquid recovered by the lipid nanoparticle-containing liquid concentration method and solvent exchange method of Example 4 was performed in the same manner as in Example 3. The lipid nanoparticle recovery rate was 87%, the nucleic acid encapsulation rate in the lipid nanoparticles was 94%, the average particle diameter of the lipid nanoparticles was 52 nm, and the polydispersity index of the lipid nanoparticle particle diameter was 0.14. The amount of lipid nanoparticle leakage in the permeate was zero. Furthermore, according to the method for concentrating the lipid nanoparticle-containing liquid of Example 4, the volume of the lipid nanoparticle-containing liquid was 4 mL before filtration, and the volume of the lipid nanoparticle-containing liquid finally recovered after the filtration operation was 2 mL. In addition, the lipid nanoparticle concentration in the lipid nanoparticle-containing liquid 2 before concentration in Example 4 was 1 mM, but the lipid nanoparticle concentration recovered after concentration in Example 4 was approximately 2 mM. Therefore, according to the method for concentrating the lipid nanoparticle-containing liquid of Example 4, it was possible to easily perform a 2-fold concentration of the lipid nanoparticle-containing liquid.

[0108] It can be seen that the recovery rate of lipid nanoparticles in Example 4 was high, and there was no leakage of lipid nanoparticles into the permeate, and lipid nanoparticles were recovered. Furthermore, since the nucleic acid encapsulation rate in Example 4 was high, it can be seen that there was almost no deterioration or damage of the lipid nanoparticles during the membrane filtration operation. Furthermore, since the polydispersity index of the particle size of the lipid nanoparticles was 0.20 or less, it can be seen that there was no aggregation or destruction of the lipid nanoparticles during the membrane filtration operation, and there was no denaturation of the lipid nanoparticles.

[0109] The results of Example 4 show that the solvent exchange method for lipid nanoparticle-containing liquid of this embodiment, which includes a step of adding a solvent different from the solvent component of the lipid nanoparticle-containing liquid removed by filtration to the lipid nanoparticle-containing liquid in an amount equal to all or part of the volume lost by filtration, and the method for concentrating lipid nanoparticle-containing liquid of this embodiment using a module including a polyacrylonitrile-based ultrafiltration membrane, are advantageous.

[0110] (Example 5) The method for concentrating the lipid nanoparticle-containing liquid and the method for solvent exchange were carried out in the same manner as in Example 4, except that the filtration operation, the PBS solution addition operation, and the recovery operation were carried out on the lipid nanoparticle-containing liquid 6. As a result, approximately 2 mL of the lipid nanoparticle-containing liquid was finally recovered, similar to Example 4.

[0111] Analysis of the recovered lipid nanoparticle-containing liquid in the same manner as in Example 4 revealed that the lipid nanoparticle recovery rate was 87%, the nucleic acid encapsulation rate in the lipid nanoparticles was 96%, the average particle size of the lipid nanoparticles was 56 nm, and the polydispersity index of the lipid nanoparticle particle size was 0.14. The amount of lipid nanoparticles leaked into the permeate was zero. Furthermore, according to the method for concentrating the lipid nanoparticle-containing liquid of Example 5, the volume of the lipid nanoparticle-containing liquid was 4 mL before filtration, and the volume of the lipid nanoparticle-containing liquid finally recovered after the filtration operation was 2 mL. In addition, the lipid nanoparticle concentration in the lipid nanoparticle-containing liquid 6 before concentration in Example 5 was 5 mM, but the lipid nanoparticle concentration recovered after concentration in Example 5 was approximately 10 mM. Therefore, according to the method for concentrating the lipid nanoparticle-containing liquid of Example 5, the lipid nanoparticle-containing liquid could be easily concentrated twice.

[0112] It can be seen that the recovery rate of lipid nanoparticles in Example 5 was high, and that lipid nanoparticles were recovered without leakage into the permeate. Furthermore, since the nucleic acid encapsulation rate in Example 5 was high, it can be seen that there was almost no deterioration or damage to the lipid nanoparticles during the membrane filtration operation. Furthermore, since the polydispersity index of the particle size of the lipid nanoparticles was 0.20 or less, it can be seen that there was no aggregation or destruction of the lipid nanoparticles during the membrane filtration operation, and that there was no denaturation of the lipid nanoparticles.

[0113] The results of Example 5 show that the solvent exchange method for lipid nanoparticle-containing liquid of this embodiment, which includes a step of adding a solvent different from the solvent component of the lipid nanoparticle-containing liquid removed by filtration to the lipid nanoparticle-containing liquid in an amount equal to all or part of the volume lost by filtration, and the method for concentrating lipid nanoparticle-containing liquid of this embodiment using a module including a polyacrylonitrile-based ultrafiltration membrane, are advantageous.

[0114] The results of Examples 1 to 5 above demonstrate that the method for concentrating a lipid nanoparticle-containing liquid and the method for solvent exchange of a lipid nanoparticle-containing liquid according to the present embodiment are effective regardless of the concentration of lipid nanoparticles in the lipid nanoparticle-containing liquid. In the present disclosure, a "low lipid nanoparticle concentration" in a lipid nanoparticle-containing liquid is approximately 0.1 mM or more and 4 mM or less, and a "high lipid nanoparticle concentration" in a lipid nanoparticle-containing liquid is approximately more than 4 mM and 40 mM or less.

[0115] (Comparative Example 1) For the approximately 20 cm long polysulfone-based ultrafiltration hollow fiber membrane produced in the production example, except that a syringe needle (gauge: 23 G) was used, filtration operations were performed on lipid nanoparticle-containing solutions 1 and 3 in the same manner as in Example 1. However, for all lipid nanoparticle-containing solutions, the filtration resistance of the hollow fiber membrane increased significantly after filtration began, and filtration was attempted using a force equal to or greater than that in Examples 1 and 2, but filtration could not be performed until the amount of lipid nanoparticle-containing solution remaining in the syringe reached 1.5 mL.

[0116] The present inventors speculate that the reason for the increased filtration resistance in polysulfone-based ultrafiltration hollow fiber membrane is as follows.When polysulfone is used as the hollow fiber membrane material, it is said to be more hydrophobic (lipophilic) than when polyacrylonitrile is used as the hollow fiber membrane material, so the present inventors speculate that the oily lipid nanoparticles adhere firmly to the surface of the hollow fiber membrane, blocking the pores on the membrane surface, which increases the filtration resistance.On the other hand, when polyacrylonitrile is used as the hollow fiber membrane material, it is a material with a certain degree of hydrophilicity, so the oily lipid nanoparticles are less likely to adhere to the surface of the hollow fiber membrane, so it is speculated that the pores on the membrane surface are not blocked, and filtration can be carried out quickly.

[0117] Reference Example: Measurement of filtration pressure during filtration operation using the mini-module produced in the mini-module manufacturing example was performed. The measurement of filtration pressure during filtration operation using the mini-module will be described with reference to FIGS. 5 and 6 . To measure the filtration pressure, a diaphragm (DPS-P, Surpass Industrial Co., Ltd.), which is the mounting portion of the pressure sensor 31, was connected to one end of the mini-module 30 using a luer lock system (see FIG. 5 ; the mini-module with the diaphragm of the pressure sensor connected will be referred to as the "mini-module with pressure sensor" hereinafter). The "mini-module with pressure sensor 42" was connected to a syringe 40 containing a liquid to be filtered (liquid containing lipid nanoparticles) set in a syringe pump 41 (Legato 110, KD Scientific), and the diaphragm of the pressure sensor 43 was connected to an amplifier (DPS-APS-P10, Surpass Industrial Co., Ltd.) of the pressure sensor 43. The filtration pressure during filtration was measured while total filtration was performed by quantitative filtration using the syringe pump 41 (see FIG. 6 ). The pressure sensor 43 was connected to a PC using a dedicated adapter (V-KIT, Surpass Industrial Co., Ltd.), and the pressure was recorded using software (Sensor Viewer, Surpass Industrial Co., Ltd.) All operations were carried out at room temperature (about 25°C), as in the other examples.

[0118] (Reference Example 1) A filtration operation of the lipid nanoparticle-containing liquid was performed in the same manner as in Example 4, except that a "mini-module with pressure sensor" was used as the mini-module, and lipid nanoparticle-containing liquid 1 (4.0 mL) was used as the lipid nanoparticle-containing liquid, and filtration was performed using a syringe pump instead of manually pushing the syringe. The filtration pressure during the filtration operation was measured. The quantitative filtration rate using the syringe pump was 1.0 mL / min. The measurement results of the filtration pressure in "filtration operation C" of Reference Example 1 (the amount of liquid in the syringe at the start of the filtration operation was 4.0 mL) are shown in Figure 7A. Filtration was completed in approximately 180 seconds, and the filtration pressure was within 90 kPa. The time required for the filtration operation when "filtration operation C" of lipid nanoparticle-containing liquid 1 (the amount of liquid in the syringe at the start of the filtration operation was 4.0 mL) was performed by "manually pushing the syringe" as performed in Examples 1 to 5 was 2 to 3 minutes. Fig. 7B shows the measurement results of the filtration pressure in the "first filtration operation D" (the amount of liquid in the syringe at the start of the filtration operation was 4.0 mL) in Reference Example 1. The filtration was completed in about 180 seconds, and the filtration pressure was within 20 kPa.

[0119] (Reference Example 2) A "mini-module with pressure sensor" was used as the mini-module, and lipid nanoparticle-containing liquid 7 (4.0 mL) was used as the lipid nanoparticle-containing liquid. The filtration operation of the lipid nanoparticle-containing liquid was performed in the same manner as in Example 4, except that filtration was performed using a syringe pump instead of manually pushing the syringe. The filtration pressure during the filtration operation was measured. The quantitative filtration rate using the syringe pump was 1.0 mL / min. The measurement results of the filtration pressure in "filtration operation C" of Reference Example 2 (the amount of liquid in the syringe at the start of the filtration operation was 4.0 mL) are shown in Figure 7C. Filtration was completed in approximately 180 seconds, and the filtration pressure was within 350 kPa. The time required for the filtration operation when "filtration operation C" of lipid nanoparticle-containing liquid 7 (the amount of liquid in the syringe at the start of the filtration operation was 4.0 mL) was performed by "manually pushing the syringe" as performed in Examples 1 to 5 was approximately 15 minutes. FIG. 7D shows the measurement results of the filtration pressure in the "first filtration operation D" (the volume of liquid in the syringe at the start of the filtration operation was 4.0 mL) in Reference Example 2. Filtration was completed in approximately 180 seconds, and the filtration pressure was within 180 kPa. When the "first filtration operation D" of lipid nanoparticle-containing liquid 7 (the volume of liquid in the syringe at the start of filtration was 4.0 mL) was performed by "pushing the syringe by hand," as was done in Examples 1 to 5, the time required for the filtration operation was approximately 4 to 5 minutes.

[0120] As an addendum, from the results of Reference Examples 1 and 2, it is estimated that the filtration pressure when "pushing the syringe by hand" performed in Examples 1 to 5 and Comparative Example 1 was about 100 to 150 kPa, or a pressure lower than that. Also, as an addendum, it was easier to filter by "pushing the syringe by hand" a lipid nanoparticle-containing liquid to which nucleic acid such as messenger RNA was added (e.g., lipid nanoparticle-containing liquid 6, etc.) than a lipid nanoparticle-containing liquid to which nucleic acid such as messenger RNA was not added (e.g., lipid nanoparticle-containing liquid 7). For example, when "filtration operation C" of lipid nanoparticle-containing liquid 7 (4.0 mL) was performed by "pushing the syringe by hand" as in Examples 1 to 5, the time required for the filtration operation was approximately 15 minutes (see Reference Example 2), but when "filtration operation C" of lipid nanoparticle-containing liquid 6 (4.0 mL) was performed by "pushing the syringe by hand" as in Examples 1 to 5 (i.e., "filtration operation C" in Example 5), the time required for the filtration operation was approximately 5 minutes.

[0121] (Reference Example 3) Filtration pressure measurement was carried out in the same manner as in Reference Example 1, except that pure water was placed in the syringe instead of the lipid nanoparticle-containing liquid. The filtration pressure was 10 kPa or less even 180 seconds after the start of filtration.

[0122] By using the method of the present invention, when lipid nanoparticle-containing liquids used in pharmaceuticals, foods, etc. are subjected to membrane filtration, the lipid nanoparticle-containing liquid can be filtrated stably with little clogging of the membrane. Furthermore, by using the method of the present invention, denaturation and deterioration / breakage of lipid nanoparticles can be suppressed, the recovery rate of lipid nanoparticles is high, and concentration (and solvent exchange) can be easily performed, making it possible to use the method in industrial fields such as the pharmaceutical industry, food industry, and chemical industry.

[0123] 10 Hydrophilic part of lipid molecule 11 Hydrophobic part of lipid molecule 12 Pharmaceutical (messenger RNA, etc.) 13 Oil phase 14 Aqueous phase in lipid bilayer membrane 110 Lipid nanoparticle 120 Aqueous phase 20 End nozzle 1 21 End nozzle 2 22 Side nozzle 1 23 Side nozzle 2 24 Seal part (epoxy resin) 25 Hollow fiber membrane 26 Plastic container (polycarbonate resin) 27, 30 Mini module 31 Pressure sensor (mounting part) 40 Syringe 41 Syringe pump 42 Mini module with pressure sensor 43 Pressure sensor (diaphragm part and amplifier part)

Claims

1. A method for concentrating a lipid nanoparticle-containing liquid, comprising a step of filtering the lipid nanoparticle-containing liquid through a polyacrylonitrile-based ultrafiltration membrane.

2. A method for concentrating a lipid nanoparticle-containing liquid as described in claim 1, wherein the polyacrylonitrile-based ultrafiltration membrane contains a basic copolymer component.

3. A method for concentrating a lipid nanoparticle-containing liquid as described in claim 2, wherein the polyacrylonitrile-based ultrafiltration membrane has a small pore layer on the membrane surface on the side that comes into contact with the lipid nanoparticle-containing liquid.

4. A method for concentrating a lipid nanoparticle-containing liquid as described in claim 1, wherein the polyacrylonitrile-based ultrafiltration membrane is a hollow fiber membrane.

5. A method for concentrating a lipid nanoparticle-containing liquid as described in claim 2, wherein the polyacrylonitrile-based ultrafiltration membrane is a hollow fiber membrane.

6. A method for concentrating a lipid nanoparticle-containing liquid as described in claim 3, wherein the polyacrylonitrile-based ultrafiltration membrane is a hollow fiber membrane.

7. A method for concentrating a lipid nanoparticle-containing liquid according to claim 2 or 5, wherein the lipid nanoparticles contain an ionized lipid having a tertiary amino group as the lipid.

8. A method for concentrating a lipid nanoparticle-containing liquid according to claim 3 or 6, wherein the lipid nanoparticles contain an ionized lipid having a tertiary amino group as the lipid.

9. A method for solvent exchange of a lipid nanoparticle-containing liquid, comprising the step of adding a solvent different from the solvent component of the lipid nanoparticle-containing liquid removed by filtration through a polyacrylonitrile-based ultrafiltration membrane to the lipid nanoparticle-containing liquid in an amount equal to all or part of the weight loss due to the filtration.

10. A method for solvent exchange of a lipid nanoparticle-containing liquid as described in claim 9, wherein the polyacrylonitrile-based ultrafiltration membrane contains a basic copolymer component.

11. A method for solvent exchange of lipid nanoparticle-containing liquid described in claim 10, wherein the polyacrylonitrile-based ultrafiltration membrane has a small pore layer on the membrane surface on the side that comes into contact with the lipid nanoparticle-containing liquid.

12. A method for solvent exchange of a lipid nanoparticle-containing liquid according to claim 9, wherein the polyacrylonitrile-based ultrafiltration membrane is a hollow fiber membrane.

13. A method for solvent exchange of a lipid nanoparticle-containing liquid described in claim 10, wherein the polyacrylonitrile-based ultrafiltration membrane is a hollow fiber membrane.

14. A method for solvent exchange of a lipid nanoparticle-containing liquid described in claim 11, wherein the polyacrylonitrile-based ultrafiltration membrane is a hollow fiber membrane.

15. A method for solvent exchange of a lipid nanoparticle-containing liquid described in claim 10 or 13, wherein the lipid nanoparticles contain an ionized lipid having a tertiary amino group as the lipid.

16. A method for solvent exchange of a lipid nanoparticle-containing liquid according to claim 11 or 14, wherein the lipid nanoparticles contain an ionized lipid having a tertiary amino group as the lipid.

17. A method for producing a concentrated lipid nanoparticle solution, comprising a step of filtering a lipid nanoparticle-containing solution through a polyacrylonitrile-based ultrafiltration membrane.

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