Development of quality control technology for pharmaceuticals containing RNA encapsulated in lipid membranes

Abstract

Provided is a method for inspecting, with high sensitivity, the quality of a pharmaceutical formulation having RNA encapsulated in a lipid membrane. This method for inspecting the quality of a pharmaceutical formulation having RNA encapsulated in a lipid membrane includes: a step for bringing a compound into contact with a pharmaceutical formulation comprising RNA and a lipid membrane that encloses the RNA, the compound binding to the RNA of the pharmaceutical formulation; a step for bringing a fluorescent dye that binds to the lipid membrane of the pharmaceutical formulation into contact with the pharmaceutical formulation; and a step for detecting the compound bonded to the RNA and / or the fluorescent dye bonded to the lipid membrane.

Description

[Technical field]

[0001] The present invention relates to a method for inspecting the quality of a preparation in which RNA is encapsulated in a lipid membrane. [Background technology]

[0002] The development of nucleic acid medicines using RNA such as siRNA (small interfering ribonucleic acid) and mRNA has been attracting attention in recent years.

[0003] For example, RNAi medicines use the principle of RNA interference to suppress the production of disease-causing proteins using short (approximately 20 to 25 bases) double-stranded RNA (called siRNA) complementary to the target mRNA in cells. Onpattro (trademark) from Allyram, Inc., USA, was approved by the FDA in 2018 and by the Ministry of Health, Labor and Welfare in 2019 as a treatment for transthyretin-type familial amyloid polyneuropathy. siRNA is very unstable, so when administered into the body, it is quickly degraded by degradative enzymes. In this treatment, siRNA is encapsulated in lipid nanoparticles (LNP), a type of lipid membrane, to increase its stability in the body. Onpattro (trademark) must be used promptly after preparation of the diluted solution, and if stored, it must be used within 16 hours, including the time of administration. Thus, RNAi medicines are prone to quality deterioration.

[0004] Another type of nucleic acid medicine, RNA vaccines, also known as messenger RNA (mRNA) vaccines, involve administering mRNA, a chemically synthesized version of part of the viral RNA sequence, to humans. The mRNA is taken up into human cells and translated to produce viral proteins, which then induce both cellular and humoral immunity against the antigen proteins.

[0005] RNA vaccines are easier to produce virus-specific antigens in human cells than conventional antigen proteins or attenuated viruses, and even if a mutant virus appears, an RNA vaccine can be quickly designed and manufactured by simply changing the mRNA sequence. For this reason, development in the field of vaccine medicine, including the prevention of infectious diseases caused by viruses, is progressing rapidly. For example, Pfizer's BNT162b2 and Moderna's mRNA-1273 are known as mRNA vaccines against the novel coronavirus Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), which has been prevalent in China since the end of 2019.

[0006] In RNA vaccines, the mRNA molecules are also highly susceptible to degradation. The main structure of RNA vaccines currently being developed is an mRNA-LNP structure that has an mRNA portion that corresponds to a part of the RNA sequence of the virus and a lipid nanoparticle (LNP) portion that encapsulates the mRNA, and the stability of the mRNA is increased by the LNP. However, current RNA vaccines still require distribution and storage at low temperatures. For example, Pfizer's BNT162b2 must be transported and stored at -75°C ± 15°C, and the storage period after thawing is 5 days in a refrigerator (4-8°C). Moderna's mRNA-127 must be transported and stored at -20°C ± 5°C, and the storage period after thawing is 30 days in a refrigerator (4-8°C).

[0007] Factors that have been reported to affect the stability, efficacy, etc. of vaccines include long-term storage of RNA vaccines at room temperature and freeze-drying and reconstitution of RNA vaccines (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] L. Schoenmaker et al. International Journal of Pharmaceutics 601 (2021) 120586 Summary of the Invention [Problem to be solved by the invention]

[0009] However, little attention has been paid to how the stability of preparations in which RNA is encapsulated in a lipid membrane, such as RNAi drugs and RNA vaccines, changes during storage, and few methods have been reported for objectively evaluating the quality of such preparations. For example, methods for evaluating the physical properties of conventional RNA vaccines include dynamic light scattering, electron microscopy, and chromatograms, but these methods have significant issues in terms of cost, speed, throughput, etc.

[0010] An object of the present invention is to provide a method for quickly and sensitively testing the quality of a preparation in which RNA is encapsulated in a lipid membrane. [Means for solving the problem]

[0011] As a result of intensive research to solve the above problems, the inventors have discovered that the quality of a formulation in which RNA is encapsulated in a lipid membrane can be rapidly and sensitively examined by examining changes in at least one of the RNA and the lipid membrane in the formulation using a specific compound that binds to the RNA portion of the formulation in which RNA is encapsulated in a lipid membrane, and a fluorescent dye that binds to the lipid membrane portion of the formulation in which RNA is encapsulated in a lipid membrane, and have thus completed the present invention.

[0012] The present invention encompasses the embodiments described below.

[0013] Item 1. A method for inspecting the quality of a preparation in which RNA is encapsulated in a lipid membrane, comprising: A step of contacting a preparation comprising RNA and a lipid membrane surrounding the RNA with a fluorescent molecule that is permeable to the lipid membrane and binds to the RNA; contacting the formulation with a fluorescent dye that binds to the lipid membrane of the formulation; and The fluorescent molecule bound to the RNA and the fluorescent color bound to the lipid membrane The material Detecting step , The fluorescence intensity of the fluorescent molecule bound to the RNA and the peptide fluorescent dye bound to the lipid membrane of the preparation having no quality problem, which was previously measured, A step of evaluating the quality of the preparation based on a change in luminescence intensity resulting from the fluorescent molecule bound to the RNA and a change in luminescence intensity resulting from the fluorescent dye bound to the lipid membrane. Including fruit, The evaluating step evaluates the quality of the preparation as being deteriorated when the fluorescence intensity derived from the fluorescent molecule bound to the RNA increases or decreases relative to the pre-measured emission intensity, the fluorescence intensity derived from the fluorescent dye increases or decreases relative to the pre-measured emission intensity, or both. method.

[0018] term 2 The fluorescent dye that binds to the lipid membrane is a peptide-based fluorescent dye that binds to lipid packing defects. 1 The method according to term 3 The steps of contacting the preparation with a fluorescent molecule that binds to the RNA and contacting the preparation with a fluorescent dye that binds to the lipid membrane bind to the RNA. Fluorescent molecules Item 1, which is carried out by adding a fluorescent dye that binds to the lipid membrane to a sample containing the preparation. Mata 2 The method according to term 4 A method for testing the quality of a formulation in which RNA is encapsulated in a lipid membrane, comprising: The present invention relates to a preparation comprising an RNA and a lipid membrane enveloping the RNA, and the preparation is impermeable to the lipid membrane and binds to the RNA. Fluorescent molecules with the formulation; contacting the preparation with a peptidic fluorescent dye that binds to lipid packing defects in the lipid membrane of the preparation; and Binding to the RNA Fluorescent molecules and a peptide-based fluorescent dye bound to the lipid packing defect of the lipid membrane. The material Detecting step , The fluorescence intensity of the fluorescent molecule bound to the RNA of the preparation having no quality problem and the peptide fluorescent dye bound to the lipid packing defect of the lipid membrane, which were previously measured, evaluating the quality of the preparation based on a change in luminescence intensity resulting from the fluorescent molecule bound to the RNA and a change in luminescence intensity resulting from the peptidic fluorescent dye bound to the lipid packing defect of the lipid membrane; Including fruit, The evaluating step evaluates the quality of the preparation as being deteriorated when the fluorescence intensity derived from the fluorescent molecule bound to the RNA increases relative to the pre-measured emission intensity, or the fluorescence intensity derived from the fluorescent dye increases or decreases relative to the pre-measured emission intensity, or both. method. term5 . A method for testing the quality of a formulation in which RNA is encapsulated in a lipid membrane, comprising the steps of: A step of contacting a preparation comprising RNA and a lipid membrane surrounding the RNA with a fluorescent molecule that is permeable to the lipid membrane and binds to the RNA; contacting the preparation with a peptidic fluorescent dye that binds to lipid packing defects in the lipid membrane of the preparation; and detecting a fluorescent molecule bound to the RNA and a peptidic fluorescent dye bound to a lipid packing defect in the lipid membrane; The fluorescence intensity of the fluorescent molecule bound to the RNA of the preparation having no quality problem and the peptide fluorescent dye bound to the lipid packing defect of the lipid membrane, which were previously measured, evaluating the quality of the preparation based on a change in luminescence intensity derived from the fluorescent molecule bound to the RNA and a change in luminescence intensity derived from the peptidic fluorescent dye bound to the lipid packing defect of the lipid membrane; Including, The evaluating step evaluates the quality of the preparation as being deteriorated when the luminescence intensity derived from the fluorescent molecule bound to the RNA increases or decreases relative to the luminescence intensity measured in advance, the fluorescence intensity derived from the fluorescent dye increases or decreases relative to the luminescence intensity measured in advance, or both. method.

[0019] term 6 . Binds to the RNA Fluorescent molecules and contacting the preparation with a peptidic fluorescent dye that binds to lipid packing defects in the lipid membrane, thereby binding to the RNA. Fluorescent molecules and a peptide-based fluorescent dye that binds to lipid packing defects in the lipid membrane are added to a sample containing the preparation. 5 or 6 The method described above.

[0020] term 7 A kit for inspecting the quality of a formulation in which RNA is encapsulated in a lipid membrane, the kit comprising a fluorescent molecule that is permeable to the lipid membrane of a formulation comprising RNA and a lipid membrane encapsulating the RNA and binds to the RNA, and a fluorescent dye that binds to the lipid membrane of the formulation. term 8 A kit for inspecting the quality of a preparation in which RNA is encapsulated in a lipid membrane, the kit comprising an RNA and a lipid membrane encapsulating the RNA, and a method for detecting a binding site of the RNA in the preparation. Fluorescent molecules and a peptidic fluorescent dye that binds to a lipid packing defect in the lipid membrane of the preparation. Effect of the Invention

[0021] According to the present invention, the quality of a preparation in which RNA is encapsulated in a lipid membrane can be examined rapidly and with high sensitivity. [Brief description of the drawings]

[0022] [Figure 1] Schematic diagram explaining the principle of the method for testing the quality of RNA vaccines using two types of probes: (A) Schematic diagram of RNA vaccine, (B) Detection of lipid membranes with a lipid membrane-binding probe, and (C) Detection of mRNA with an RNA-binding probe. [Diagram 2] Schematic diagram illustrating examples of patterns of compromised RNA vaccines and how they can be detected. [Diagram 3] Schematic diagram illustrating another example of the pattern of compromised RNA vaccines and how they can be detected. [Figure 4] Fluorescence response to model vaccines. (A) Vaccine detection by lipid membrane-binding probe, (B) vaccine detection by RNA-binding probe. [Diagram 5] Concentration dependence of the fluorescence response to the model vaccine. (A) Concentration-dependent increase in the fluorescence response of the model vaccine sample mixed with ApoC-NR, (B) Graph showing the fluorescence intensity ratio F / F0 of each sample in Figure 4(A), (C) Concentration-dependent increase in the fluorescence response of the model vaccine sample mixed with TO-PRO-1, (D) Graph showing the fluorescence intensity ratio F / F0 of each sample in Figure 4(C). [Figure 6] Fluorescence response to a sample in which a model vaccine was mixed with RNA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] As used herein, the term "halo" refers to fluoro, chloro, bromo, and iodo groups.

[0024] The term "alkyl" refers to a branched, unbranched, or cyclic saturated hydrocarbon. In some embodiments, an alkyl group has, for example, 1 to 20 carbon atoms, and often has 1 to 12 carbon atoms or 1 to 6 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl (t-butyl), 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, heptyl, octyl, decyl, dodecyl, and the like. Alkyl can be unsubstituted or substituted. Substituted alkyl groups can contain one or more non-carbon and non-hydrogen atoms, such as oxygen, nitrogen, sulfur, halogens, and phosphorus.

[0025] The term "alkenyl" refers to a carbon-carbon sp 2 It refers to a branched or unbranched unsaturated hydrocarbon having a double bond. In some embodiments, the alkenyl group can have, for example, 2 to 10 carbon atoms, or 2 to 6 carbon atoms. In other embodiments, the alkenyl group has 2 to 4 carbon atoms. Examples of alkenyl include, but are not limited to, ethylene or vinyl, allyl, cyclopentenyl, 5-hexenyl, and the like. The alkenyl can be unsubstituted or substituted. Substituted alkenyl groups can contain one or more non-carbon and non-hydrogen atoms, such as oxygen, nitrogen, sulfur, halogens, and phosphorus.

[0026] The term "alkynyl" refers to a branched or unbranched unsaturated hydrocarbon chain having a carbon-carbon sp triple bond. In some embodiments, the alkynyl group can have, for example, 2 to 10 carbon atoms, or 2 to 6 carbon atoms. In other embodiments, the alkynyl group can have 2 to 4 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1-octynyl, and the like. The alkynyl can be unsubstituted or substituted. Substituted alkynyl groups can contain one or more non-carbon and non-hydrogen atoms, such as oxygen, nitrogen, sulfur, halogens, and phosphorus.

[0027] The term "alkoxy" refers to alkyl-O-, where alkyl is defined herein. In some embodiments, an alkoxy group has 1-12 carbon atoms or 1-6 carbon atoms. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexyloxy, 1,2-dimethylbutoxy, and the like. An alkoxy can be unsubstituted or substituted. A substituted alkoxy group can include an oxygen bonded to a substituted alkyl group.

[0028] The term "aryl" refers to an aromatic hydrocarbon group derived by removing a hydrogen atom from a carbon atom of a parent aromatic ring. An aryl group can have 6 to 18 carbon atoms, 6 to 14 carbon atoms, or 6 to 10 carbon atoms. An aryl group can have a single ring (e.g., phenyl) or multiple condensed rings (fused rings) in which at least one ring is aromatic (e.g., naphthyl, dihydrophenanthrenyl, fluorenyl, or anthryl). Typical aryl groups include, but are not limited to, radicals derived from benzene, naphthalene, anthracene, biphenyl, and the like. An aryl can be unsubstituted or substituted. For example, an aryl group can be substituted with one or more substituents (as described above) to give a variety of substituted aryls, such as pentafluorophenyl or p-trifluoromethylphenyl, and the like.

[0029] The term "amino" refers to -NH2. The amino group may be optionally substituted as defined for the term "substituted." For example, the amino group may be -NR2 (wherein R is a group recited in the definition of "substituted"). For example, the group -NR2 may include "alkylamino" (wherein at least one R is alkyl and the other R is alkyl or hydrogen) and / or "acylamino" (-N(R)C(=O)R) (wherein each R is independently hydrogen, alkyl, alkaryl, or aryl). The amino group may be a primary amine (NH2), a secondary amine (NHR), or a tertiary amine.

[0030] First, the principle of the method of inspecting the quality of a formulation in which RNA is encapsulated in a lipid membrane of the present invention will be described with reference to Figures 1(A)-(C). As shown in Figure 1(A)(a), an RNA vaccine (1) (hereinafter, sometimes simply referred to as "RNA vaccine") as a formulation in which RNA is encapsulated in a lipid membrane (hereinafter, sometimes simply referred to as "formulation") comprises a portion of mRNA (2) corresponding to a portion of the RNA sequence of a virus, and a portion of lipid membrane (3) encapsulating mRNA (2). mRNA (2) may be a portion of the RNA sequence of any virus.

[0031] Viruses include, but are not limited to, coronaviruses (including SARS-CoV-2), rhinoviruses, noroviruses, enteroviruses, rotaviruses, influenza viruses, measles viruses, rubella viruses, hepatitis A viruses, hepatitis C viruses, HIV viruses, Japanese encephalitis viruses, yellow fever viruses, dengue viruses, Zika viruses, Lassa viruses, and Ebola viruses.

[0032] When an RNA vaccine (1) is administered, the mRNA is translated in the cells of a subject, such as a human, to produce a protein, which can function as an antigen to induce an immune response or as a replacement protein for a defective protein. RNA vaccines having an mRNA-LNP structure, which has a portion of the mRNA corresponding to a part of the RNA sequence of a virus and a portion of a lipid nanoparticle (LNP) that encapsulates the mRNA, are well known. In some embodiments, the mRNA (2) portion of the RNA vaccine consists of an mRNA construct with, in order from the 5' end to the 3' end, a 5' cap structure, a non-translated region, a coding sequence of the mRNA, a non-translated region, and a 3' polyA tail. In some embodiments, the lipid membrane (3) portion consists of an LNP with phospholipids, cholesterol, polyethylene glycol, and cationic lipids.

[0033] FIG. 1(A)(b) is a schematic diagram of the RNA vaccine (1) in FIG. 1(A)(a). In the RNA vaccine (1) immediately after production, mRNA (2) is encapsulated inside a lipid membrane (3). Hereinafter, in this specification, "immediately after production" refers to within one day after production. "A preparation in which RNA is encapsulated in a lipid membrane immediately after production" refers to a preparation that is produced within one day and has not been subjected to a freezing process.

[0034] The method for testing the quality of an RNA vaccine of the present invention uses two types of probes: a lipid membrane-binding probe (4) that binds to a lipid membrane (3) (FIG. 1(B)), and an RNA-binding probe (5) that binds to mRNA (2) (FIG. 1(C)).

[0035] Regarding the deterioration of the quality of RNA vaccines, the inventors considered that there are the modes shown in Figure 1(B) and Figure 1(C) below, and investigated how to detect each of them.

[0036] In the example of Figure 1(B), the lipid membrane-binding probe (4) can bind to liposomes that have a small size, i.e., a lipid membrane (3) with a high curvature, shortly after production (Figure 1(B)(a)). However, when the storage period of the RNA vaccine (1) is extended or when it is repeatedly thawed and frozen, and the size of the lipid membrane (3) increases, i.e., the curvature becomes lower, the number of lipid membrane-binding probes (4) that can bind to the lipid membrane (3) decreases (Figure 1(B)(b)). In other words, by examining the signal intensity associated with the binding of the lipid membrane-binding probe (4) to the lipid membrane (3), a change in the structure of the lipid membrane (3) can be detected.

[0037] In some cases, the size of the lipid membrane (3) may become smaller. In that case, the number of lipid membrane-binding probes that can bind to the lipid membrane (3) may increase, resulting in a stronger signal intensity. Even in that case, a change in the structure of the lipid membrane (3) can be detected by examining the signal intensity of the lipid membrane-binding probe (4) that binds to the lipid membrane (3).

[0038] In the example of Figure 1(C), when the RNA vaccine (1) is newly produced, the mRNA (2) is enclosed inside the lipid membrane (3), so that there is no mRNA (2) in the solution outside the RNA vaccine (1), and the mRNA cannot be detected by the RNA-binding probe (5) (Figure 1(C)(a)). However, as the storage period of the RNA vaccine (1) is extended or the RNA vaccine (1) is repeatedly thawed and frozen, part of the lipid membrane (3) is damaged, and as the mRNA (2) leaks out, the number of mRNAs (2) that bind to the RNA-binding probe (5) outside the RNA vaccine (1) increases (Figure 1(C)(b)). In other words, by examining the signal intensity associated with the binding of the RNA-binding probe (5) to the mRNA (2), the leaked mRNA (2) can be detected. In this way, the quality of the RNA vaccine (1) can be inspected using the lipid membrane-binding probe (4) and the RNA-binding probe (5).

[0039] Next, we will explain some examples of patterns of RNA vaccines with reduced quality and how to detect them. As shown in Figure 2(a), immediately after production, the size of the lipid membrane (3) of the RNA vaccine (1) is small (its curvature is large), and the mRNA (2) is completely enclosed in the lipid membrane (3). At this time, the signal intensity associated with the binding of the lipid membrane-binding probe (4) is large, and no signal intensity associated with the binding of the RNA-binding probe (5) is observed outside the RNA vaccine (1).

[0040] As shown in Figure 2(b), when the size of the lipid membrane (3) increases due to quality deterioration, even though the mRNA (2) has not yet leaked out of the lipid membrane (3), the curvature of the lipid membrane (3) changes from that of the lipid membrane (3) in Figure 2(a) (Pattern (A)). As a result, the number of lipid membrane-binding probes (4) that can bind to the lipid membrane (3) decreases, and the signal intensity associated with the binding of the lipid membrane-binding probes (4) decreases compared to the case of Figure 2(b). On the other hand, the signal intensity associated with the binding of the RNA-binding probe (5) is not observed because the mRNA (2) has not yet leaked out of the lipid membrane (3).

[0041] In addition, when the size of the lipid membrane (3) becomes smaller, the number of lipid membrane-binding probes (4) that can bind to the lipid membrane (3) increases, and the signal intensity of the lipid membrane-binding probes (4) is thought to be increased compared to the case of Figure 2(b).

[0042] As shown in FIG. 2(c), the size of the lipid membrane (3) is maintained, but when holes or gaps are formed in the lipid membrane (3) and mRNA (2) leaks out of the lipid membrane (3) (pattern (B)), the curvature of the lipid membrane (3) may not change much. However, the number of lipid membrane-binding probes (4) that can bind to the lipid membrane (3) usually decreases due to the appearance of holes or gaps, and the signal intensity associated with the binding of the lipid membrane-binding probes (4) decreases compared to the case of FIG. 2(b). Since mRNA (2) leaks out of the RNA vaccine (1), the signal intensity associated with the binding of the RNA binding probe (5) is observed. To detect RNA that has leaked out of the RNA vaccine (1), a compound that is impermeable to lipid membranes and can bind to RNA is preferably used.

[0043] In this specification, "permeable" refers to the permeation or penetration of an RNA-binding probe into a lipid membrane. The permeable lipid membrane refers to both the lipid membrane of a living cell and the lipid membrane of a dead cell. The permeable lipid membrane also includes lipid nanoparticles used in RNA vaccines and the like, and membranes made of lipid bilayers of liposomes.

[0044] As shown in FIG. 2(d), when the size of the lipid membrane (3) increases and the mRNA (2) leaks out of the lipid membrane (3) (pattern (C)), the curvature of the lipid membrane (3) changes from that of the lipid membrane (3) in FIG. 2(a). As a result, the number of lipid membrane-binding probes (4) that can bind to the lipid membrane (3) decreases, and the signal intensity associated with the binding of the lipid membrane-binding probe (4) decreases compared to the case of FIG. 2(b). In addition, since the mRNA (2) leaks out of the RNA vaccine (1), the signal intensity associated with the binding of the RNA binding probe (5) is observed.

[0045] To summarize Figures 2(b)-(d), by detecting at least one change in the signal intensity associated with the binding of the lipid membrane-binding probe (4) or the signal intensity associated with the binding of the RNA-binding probe (5), the quality of the RNA vaccine (1), including what structural changes have occurred, can be rapidly and sensitively tested.

[0046] In order to facilitate understanding of the invention, a method for testing the quality of an RNA vaccine has been described above with reference to Figures 1 and 2, but the quality of an RNAi drug in which siRNA is encapsulated in a liposomal lipid membrane can also be tested using the same principle.

[0047] According to a first aspect of the present invention, there is provided a method for inspecting the quality of a formulation in which RNA is encapsulated in a lipid membrane, the method comprising the steps of: contacting the formulation with a compound that binds to RNA in the formulation comprising RNA and a lipid membrane encapsulating the RNA; contacting the formulation with a fluorescent dye that binds to the lipid membrane of the formulation; and detecting at least one of the compound bound to the RNA and the fluorescent dye bound to the lipid membrane.

[0048] Examples of RNA include mRNA, siRNA, tRNA, rRNA, dsRNA (double-stranded RNA), miRNA (single-stranded RNA consisting of 19-23 nucleotides), etc. dsRNA includes siRNA (low molecular weight double-stranded RNA consisting of 21 to 23 base pairs).

[0049] In a preferred embodiment, the preparation in which RNA is encapsulated in a lipid membrane is an RNA vaccine in which mRNA is encapsulated in a lipid membrane.In another preferred embodiment, the preparation in which RNA is encapsulated in a lipid membrane is an RNAi drug in which siRNA is encapsulated in a liposome.

[0050] In some embodiments, a compound that binds to RNA is preferably a compound that is impermeable to lipid membranes, from the viewpoint of enabling accurate detection of RNA that has leaked into the solution.

[0051] Specific examples of compounds that are impermeable to lipid membranes and bind to RNA include TO-PRO-1 (trademark) (3-METHYL-2-([1-[3-(TRIMETHYLAMMONIO)PROPYL]-4(1H)-QUINOLINYLIDENE]METHYL)-1,3-BENZOTHIAZOL-3-IUM DIIODIDE, ThermoFisher), which is commercially available as a membrane-impermeable fluorescent probe.

[0052] [ka]

[0053] In some alternative embodiments, the compound that binds to RNA is a compound that is permeable to lipid membranes in order to ensure that the integrity of the formulation is maintained, i.e., the RNA is encapsulated in the lipid membrane.

[0054] The detection of leaked mRNA using the lipid membrane-impermeable RNA-binding probe (5) has been described with reference to the example in Figure 1(C), and the detection of encapsulated mRNA using the lipid membrane-permeable RNA-binding probe (5) is shown in Figure 3.

[0055] In the example of Figure 3, when an RNA vaccine (1) is produced, mRNA (2) is enclosed inside a lipid membrane (3). A certain amount of the RNA-binding probe (5) penetrates the lipid membrane (3) to the inside, so that the endogenous mRNA can be detected by the RNA-binding probe (5) with a certain fluorescence intensity (Figure 3(a)). As the quality of the RNA vaccine (1) deteriorates and part of the lipid membrane (3) is damaged, and mRNA (2) leaks out, the number of mRNA (2) that binds to the RNA-binding probe (5) present outside the lipid membrane increases, and can be observed as a very high fluorescence intensity (Figure 3(b)). In this way, the quality of an RNA vaccine (1) can be inspected even by using an RNA-binding probe (5) that is permeable to lipid membranes.

[0056] In some other embodiments, as another method of using the lipid membrane-permeable RNA-binding probe (5), it is also possible to examine whether the manufactured RNA vaccine (1) contains mRNA (2) inside the lipid membrane (3). When the RNA vaccine (1) is manufactured, if the mRNA (2) cannot be contained inside the lipid membrane (3), there will naturally be problems with the quality of the vaccine, but the quality can be judged by using the permeable RNA-binding probe (5). Specifically, the fluorescence intensity when the permeable RNA-binding probe (5) is brought into contact with an RNA vaccine with no quality problems is obtained in advance, and the RNA-binding probe (5) is brought into contact with the RNA vaccine (1) to be inspected. If the mRNA (2) cannot be contained inside the lipid membrane (3) and the mRNA (2) leaks out, the number of mRNA (2) that binds to the permeable RNA-binding probe (5) increases, resulting in a higher value of fluorescence intensity than that obtained in advance. It is also possible that, for some reason during the vaccine manufacturing process, no mRNA (2) is encapsulated inside the lipid membrane (3) and is not present outside the lipid membrane (3). In such cases, the quality cannot be determined using the aforementioned lipid membrane-impermeable RNA-binding probe (5), but by using a lipid membrane-permeable RNA-binding probe (5), the normal value of the fluorescence intensity will be lower than the previously obtained value, making it possible to determine that no mRNA (2) is encapsulated inside the lipid membrane (3).

[0057] As described above, by using the permeable RNA-binding probe (5), not only can the quality of a vaccine be determined over time, but also the quality of a vaccine can be easily determined at the time of vaccine production.

[0058] The permeable RNA-binding probe (5) and the non-permeable RNA-binding probe (5) are preferably used separately, but may be used together in a form of use.

[0059] A specific example of a compound that is permeable to lipid membranes and can bind to RNA is the compound of formula (1). The compound of formula (1) is preferable in that it hardly exhibits fluorescence itself, but the fluorescence intensity at a specific wavelength increases significantly upon binding to nucleic acid, and therefore the fluorescence intensity is strong, and it is observed as a bright spot with strong intensity in a spatially limited system such as a vaccine.

[0060] [ka]

[0061] In formula (1), ring A is

[0062] [ka]

[0063] and R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 and R 11 are independently hydrogen, a hydroxyl group, a thiol group, a halo, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group.

[0064] In formula (1), R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 and R 11 are independently hydrogen, a hydroxyl group, a thiol group, a halo, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group.

[0065] In the present specification, R in formula (1) 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 and R 11 Where a portion of the group can be substituted, the term "substituted" indicates that one or more (e.g., 1, 2, 3, 4, 5 or 6; in some embodiments 1, 2 or 3; in other embodiments 1 or 2) hydrogens of the group designated with the phrase "substituted" can be replaced with a group selected from the listed designated groups or with a suitable group known to one of ordinary skill in the art, provided that such replacement results in a stable compound. Suitable substituents for substituted groups can include alkyl, alkenyl, alkynyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino, trifluoromethylthio, difluoromethyl, acetylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl, heteroarylsulfinyl, heteroarylsulfonyl, heterocyclesulfinyl, heterocyclesulfonyl, phosphate, sulfate, hydroxylamine, hydroxyl(alkyl)amine, and cyano.

[0066] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 8 , R 9 , R 10 and R 11 are independently hydrogen, a hydroxyl group, a thiol group, a halo group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group; R5 is an alkyl group.

[0067] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 and R 11 are independently hydrogen, a hydroxy group, a halo, an alkyl group, an aryl group, or an amino group.

[0068] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 8 , R 9 , R 10 and R 11 are independently hydrogen, a hydroxyl group, a thiol group, a halo group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group, all of which are unsubstituted; R 5 is also an unsubstituted alkyl group.

[0069] In some preferred embodiments, R 1 , R 2 , R 3 , R 6 , R 7 , R 8 , R 9 , R 10 and R 11 is hydrogen, and R 4 is an alkyl group having 1 to 6 carbon atoms; R 5 is methyl.

[0070] In some preferred embodiments, the compound of formula (1) is a compound of formula (1-I). The compound of formula (1-I) is a combination of Benzo[c,d]Indole and OxazoloPyridine, and is therefore hereinafter also referred to as BIOP.

[0071] [ka]

[0072] The compound (BIOP) of formula (1-I) can be produced by the following scheme, for example, using Benzo[c,d]indole-2(1H)-one (1) as a starting material.

[0073] [ka]

[0074] First, 1-methylbenzo[c,d]indol-2(1H)-one (compound 1) is obtained according to J. Med. Chem., 2016, 59, 1565-1579. A 1,4-dioxane solution containing the obtained compound 2 (0.55 g, 3.0 mmol) and Lawesson's reagent (2.43 g, 6.0 mmol) is stirred at 100 °C overnight. This solution is filtered at room temperature, and the obtained solid is washed with 1,4-dioxane to obtain 1-methylbenzo[c,d]indol-2(1H)-thione crude product 3. Next, iodomethane is added to the crude product 3 (1.71 g) and heated to reflux overnight. This solution is concentrated under reduced pressure, and then diethyl ether is added and sonicated for 30 minutes. The resulting solid is filtered to obtain 1-methyl-2(methylthio)benzo[c,d]indole-1-ium (compound 4). Next, 0.5 mL of tetraethylamine is added to an acetonitrile solution containing 4-methyl-2-methyl-oxazolo[4,5]pyridinium iodide (compound 5, 193 mg, 0.70 mmol) obtained by the reaction of 2-methyl-oxazolo[4,5-b]pyridine with iodomethane and compound 4 (162 mg, 0.47 mmol). This solution is stirred and heated to reflux (60°C) for 1 hour, then cooled to room temperature. Diethyl ether is then added and the resulting precipitate is filtered. Water is then added, filtered again, and dried to obtain compound 6 (BIOP).

[0075] The compounds of formula (1) above may be covalently attached to other molecules such as antibodies, proteins, peptides, polypeptides, amino acids, enzymes, nucleic acids, lipids, polysaccharides, drugs, beads, solid supports (e.g. glass or plastic), etc.

[0076] The compound of formula (1) preferably emits little or no fluorescence in the absence of nucleic acid. Fluorescence can be measured by irradiating the compound with an appropriate wavelength and monitoring the emitted fluorescence. The compound preferably emits stronger fluorescence in the presence of RNA than in the presence of DNA. Fluorescence in the presence of RNA relative to fluorescence in the presence of DNA is measured with constant compound concentration and constant RNA and DNA concentrations. The higher the RNA / DNA fluorescence ratio, the more suitable it is for RNA detection in the presence of DNA. The RNA / DNA fluorescence ratio is preferably 1 or more, more preferably greater than 1, 1.2 or more, 1.5 or more, and 2 or more. The compound of formula (1) meets the criteria for suitability for RNA detection.

[0077] The compound of formula (1) above may also be characterized by maximum excitation and emission wavelengths. For example, the maximum excitation may be about 450 nm to about 650 nm. The maximum excitation between these values ​​may be about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, and between any two values ​​thereof. For example, the maximum emission may be about 500 nm to about 675 nm. The maximum emission between these values ​​may be about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 675 nm, and between any two values ​​thereof.

[0078] The compound of formula (1) emits fluorescence by binding to nucleic acid, and can be used as a fluorescent dye. The compound of formula (1) can detect RNA with high sensitivity. The fluorescent dye containing the compound of formula (1) or the fluorescent dye consisting of the compound of formula (1) has a clear response performance, and can be suitably used for detecting the RNA portion of an RNA vaccine.

[0079] Another specific example of the compound that is permeable to lipid membranes and can bind to RNA is the compound of formula (2) shown below:

[0080] [ka]

[0081] In formula (2), R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are independently hydrogen, a hydroxyl group, a thiol group, a halo, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group.

[0082] In this specification, R in formula (2) 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12Where a portion of the group can be substituted, the term "substituted" indicates that one or more (e.g., 1, 2, 3, 4, 5 or 6; in some embodiments 1, 2 or 3; in other embodiments 1 or 2) hydrogens of the group designated with the phrase "substituted" can be replaced with a group selected from the listed designated groups or with a suitable group known to one of ordinary skill in the art, provided that such replacement results in a stable compound. Suitable substituents for substituted groups can include alkyl, alkenyl, alkynyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino, trifluoromethylthio, difluoromethyl, acetylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl, heteroarylsulfinyl, heteroarylsulfonyl, heterocyclesulfinyl, heterocyclesulfonyl, phosphate, sulfate, hydroxylamine, hydroxyl(alkyl)amine, and cyano.

[0083] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are independently hydrogen, a hydroxyl group, a thiol group, a halo group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group; R 6 is an alkyl group.

[0084] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are independently hydrogen, a hydroxy group, a halo, an alkyl group, an aryl group, or an amino group.

[0085] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are independently hydrogen, a hydroxyl group, a thiol group, a halo group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group, all of which are unsubstituted; R 6 is also non-substitutive.

[0086] In some preferred embodiments, the compound of formula (2) above is a compound of formula (2-I) (monomethine cyanine dye (BIQ)).

[0087] [ka]

[0088] The compound of formula (2-I) above can be produced by the method disclosed in Anal. Chem. 2019, 91, 14254-14260.

[0089] [ka]

[0090] The process is the same as that of the compound of formula (1) until the crude product of compound 4 is obtained. Then, 0.5 mL of tetraethylamine is added to an acetonitrile solution containing 1,4-dimethylquinolin-1-ium (compound 7, 258 mg, 0.91 mmol) obtained by the reaction of 4-methylquinoline with iodomethane and the crude product of compound 4 (194 mg, 0.57 mmol). This solution is reacted for 1 hour under heating and reflux while stirring, and then cooled to room temperature. Then, diethyl ether is added to the solution, and a precipitate can be obtained by filtration. The obtained precipitate is washed with water and then dissolved in methanol. Then, diethyl ether is added, and the obtained precipitate is dried to obtain compound 8 (BIQ).

[0091] The compounds of formula (2) above may be covalently attached to other molecules such as antibodies, proteins, peptides, polypeptides, amino acids, enzymes, nucleic acids, lipids, polysaccharides, drugs, beads, solid supports (e.g. glass or plastic), etc.

[0092] The compound of formula (2) emits fluorescence by binding to nucleic acid, and can be used as a fluorescent dye. The compound of formula (2) can detect RNA with high sensitivity. The fluorescent dye containing the compound of formula (2) or the fluorescent dye consisting of the compound of formula (2) has a clear response performance, and can be suitably used for detecting the RNA portion of an RNA vaccine.

[0093] On the other hand, in some embodiments, fluorescent dyes that bind to the lipid membrane of an RNA vaccine include peptide-based fluorescent dyes and cell membrane staining dyes that can selectively bind to lipid bilayer structures.

[0094] Examples of peptide-based fluorescent dyes that can bind highly selectively to lipid membranes include binding peptides that target exosomes, which the inventors previously developed (98th Annual Meeting of the Chemical Society of Japan (2018 (4G3-02)), 68th Annual Meeting of the Japan Society for Analytical Chemistry (2019 (D1101, D1102, L2006)), RSC Advances, 10, 38323-38327 (2020). [DOI:10.1039 / d0ra07763a]). This is based on the fact that exosomes have a highly curvature lipid bilayer membrane with a diameter of about 100 nm, and that hydrophobic regions called lipid packing defects appear locally on the surface of the lipid bilayer membrane. The binding peptide uses an amphipathic α-helical peptide as a binding motif for lipid packing defects, and an environmentally responsive fluorescent dye is linked to this. For example, since mRNA vaccines have a diameter similar to that of exosomes, it is expected that such fluorescent dyes based on α-helical peptides can selectively bind to the lipid membrane of the vaccine. It is also expected that fluorescent dyes based on α-helical peptides can selectively bind to the lipid membrane of the liposome for RNAi drugs encapsulated in the liposome lipid membrane.

[0095] In designing a peptide-based fluorescent dye capable of selectively binding to a lipid bilayer structure, the peptide portion is designed to recognize the lipid packing structure of the lipid membrane of a formulation such as a vaccine, and the environmentally responsive fluorescent dye is attached to a position such as the N-terminus or C-terminus of the peptide that does not interfere with the binding between the peptide and the lipid membrane.

[0096] The design of amphipathic α-helical peptides that bind to lipid packing defects is described in FEBS Lett., 2010, 584, 1840-1847. Such α-helical peptides include ArfGAPl (R. norvegicus), nucleoporin Nupl33 (H. sapiens), α-synuclein (H. sapiens), DivIVA (B. subtillis), dAmph (D. melanogaster), endophilin Al (R. norvegicus), Sarlp (S. cervisiae), antimicrobial magainin 2 (X. laevis), the string-like N-terminus of GMAP-210 (H. sapiens), the sterol transporter Keslp (S. cerevisiae), SpoVM (B. subtilis), Epsin (H. sapiens), Binl / amphiphysin II (H. sapiens), H0-NBAR from BRAP / Bin2, myristoylated Arf 1 (B. taurus), and melittin (A. mellifera). or peptides based on these α-helical peptides (for example, a part of an α-helical peptide, or an α-helical peptide in which one to several, more specifically about one to five amino acids have been mutated).Those skilled in the art can appropriately select various peptide sequences as peptides of peptidic fluorescent dyes that can selectively bind to the lipid membrane structure of an RNA vaccine.

[0097] As the environmentally responsive fluorescent dye, any dye that has a weak fluorescence intensity in a hydrophilic field but increases in intensity in a hydrophobic field can be used, and such dyes are known in the art. Examples of the environmentally responsive fluorescent dye include dansyl dye and its derivatives, Dapoxyl dye and its derivatives, and benzophenoxazine dye and its derivatives. Specific examples of environmentally responsive fluorescent dyes include allylnaphthalenesulfonic acids such as 1-anilinonaphthalene-8-sulfonic acid (ANS), N-methyl-2-anilinonaphthalene-6-sulfonic acid (MANS), and 2-p-toluidinylnaphthalene-6-sulfonic acid (TNS); dimethylaminonaphthalenesulfonic acid; benzofurazan derivatives such as nitrobenzofurazan (NBD); Dapoxyl dye (Benzenesulfonic acid, 4-[5-[4-(dimethylamino)phenyl]-2-oxazolyl); Dapoxyl sulfonyl chloride, Dapoxyl succinimidyl ester, Dapoxyl 3-sulfonamidopropionic acid, Dapoxyl (2-bromoacetamidoethyl) sulfonamide, and Dapoxyl (2-aminoethyl) Examples include dapoxyl derivatives such as sulfonamide; dansyl dyes such as dansyl chloride, dansylsulfonamide, dansylaminoethyl-3-phosphate, 1-dansylsulfonamide-3-N,N-dimethylaminopropane, dansylcholine, dansylgalactoside, dansyllysine, and dansylphosphatidylethanolamine; and benzophenoxazine derivatives such as Nile Red and Nile Blue.

[0098] Based on this principle, it is possible to prepare a peptide-based fluorescent dye that binds highly selectively to the lipid membrane of an RNA vaccine and exhibits a fluorescent response.

[0099] A preferred embodiment of a peptide probe capable of selectively binding to a lipid membrane structure of an RNA vaccine includes an amphipathic α-helical peptide probe such as a compound of formula (9) below.

[0100] [ka]

[0101] The peptide probe of the above formula (9) can be produced as follows.

[0102] First, the peptide chain is extended using amino acids protected with Fmoc groups, and then fluorescent dye Nile Red derivatives are linked to produce the probe. Specifically, Fmoc-protected amino acids are linked to Rink-Amide-ChemMatrix resin (Biotage) using [1-(1-cyano-ethoxy-2-oxoethylideneamino-oxy)-dimethylamino-morpholino methylene]methanaminium hexafluorophosphate (COMU) and diisopropylethylamine (DIEA). Next, Nile Red derivatives (6-((9-(diethylamino)-5-oxo-5H-benzo[a]phenoxazin-2-yl)oxy)hexanoic acid) synthesized according to J.Chem.Soc.,Perkin Trans., 1997, 1, 1051-1058. are linked. After that, the probe is cut out from the resin and deprotected, and the resulting crude product is purified by reversed-phase HPLC to obtain a peptide-based fluorescent probe.

[0103] The fluorescent dyes used in the peptide probes described above are not limited to environmentally responsive dyes, and non-environmentally responsive dyes such as fluorescein dyes and their derivatives, and rhodamine dyes and their derivatives can also be used. However, fluorescent probes using environmentally responsive dyes have the characteristic that the fluorescence intensity is low in hydrophilic fields and high in hydrophobic fields, so that the fluorescence can be detected without the need for operations such as washing. On the other hand, fluorescent probes using non-environmentally responsive dyes require operations such as washing because it is impossible to distinguish between when they are unbound and when they are bound to the lipid membrane of a preparation such as a vaccine. Environmentally responsive dyes such as the compound of formula (9) are preferable, but the use of non-environmentally responsive dyes may be considered depending on the purpose and use.

[0104] As the cell membrane staining dye, a known dye capable of staining the cell membrane can be used, and in particular, a fat-soluble dye that stains the lipid bilayer membrane is preferred. Such fat-soluble dyes include fat-soluble carbocyanine dyes such as SP-DiOC1 8 (3) in the PIC series of Takara Bio Inc. and the Molecular Probes (registered trademark) series of Thermo Fisher Scientific Co., Ltd., and Polaric (registered trademark) of Goryo Chemical Co., Ltd. The fat-soluble carbocyanine dye is usually a carbocyanine dye having a long hydrocarbon chain. In addition to these, fat-soluble dyes such as Sudan III, Sudan II (Oil Red O), Sudan Black B (SBB), Nile Blue, Fat Red, and Lipid Crimson, which are conventionally used for lipid staining, can also be used as the cell membrane staining dye.

[0105] The step of contacting a compound that binds to RNA with the RNA vaccine may be carried out before, simultaneously with, or after the step of contacting a fluorescent dye that binds to the lipid membrane of the RNA vaccine with the RNA vaccine, but carrying them out simultaneously is advantageous in that it shortens the time required for the testing method.

[0106] When the step of contacting the RNA vaccine with the compound that binds to RNA and the step of contacting the RNA vaccine with the fluorescent dye that binds to lipid membrane are carried out simultaneously, it is preferable to carry out the steps by preparing a sample containing the RNA vaccine, and adding the compound that binds to mRNA and the fluorescent dye that binds to lipid membrane to one sample containing the RNA vaccine. The sample containing the RNA vaccine may be a stock solution or dilution of the commercially available or manufactured RNA vaccine, and may contain any additive including a substance that inhibits the degradation of RNA.

[0107] Each of the steps of contacting the RNA vaccine with a compound that binds to RNA and contacting the RNA vaccine with a fluorescent dye that binds to a lipid membrane may be carried out at any suitable temperature and time. Typically, the temperature is room temperature or room temperature. Examples of the temperature include about 20°C, about 25°C, about 30°C, about 35°C, about 37°C, about 40°C, about 42°C, and ranges between any two of these values. Temperatures of about 42°C or higher and temperatures of about 20°C or lower are also applicable depending on the test sample. The time is not particularly limited and may be any time suitable for detecting a change in fluorescence. Examples of time lengths include about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 180 minutes, about 240 minutes, about 300 minutes, about 360 minutes, about 420 minutes, about 480 minutes, about 540 minutes, about 600 minutes, and ranges between any two of these values. Further extensions of time are possible depending on the sample being tested.

[0108] The concentration of the compound of formula (1) and the compound of formula (2) is not particularly limited, and may be any concentration that allows appropriate detection of fluorescent excitation and emission signals in the presence of RNA. An example of the concentration range is about 10 nM to 1 mM. An example of the concentration is about 10 nM, about 100 nM, about 1 μM, about 10 μM, about 100 μM, about 1 mM, and a range between any two of these values.

[0109] After the two contact steps, at least one of the compound bound to the RNA and the fluorescent dye bound to the lipid membrane is detected. Note that, before detection, unbound fluorescent probes may be removed by ultrafiltration or the like. Removing the unbound fluorescent probes enables detection with a higher S / N ratio.

[0110] Suitable illumination devices for fluorescence include portable ultraviolet lamps, mercury arc lamps, xenon lamps, lasers (such as argon and YAG lasers), and laser diodes. These illumination sources are typically optically integrated into laser scanners, fluorescence microplate readers, or standard or microfluorimeters.

[0111] The step of detecting at least one of the compound bound to the RNA and the fluorescent dye bound to the lipid membrane may be performed by visual inspection or by using various measuring instruments, such as a CCD camera, a video camera, a photographic film, a laser scanner device, a fluorescence intensity meter, a photodiode, a quantum counter, an epifluorescence microscope, a scanning microscope, a flow cytometer, a fluorescence microplate reader, or an amplification device such as a photomultiplier tube.

[0112] The detection step may be performed at a single time point, at multiple time points, or continuously.

[0113] The method of the first aspect of the present invention may further comprise, after the detection step, a step of evaluating the quality of the preparation in which the RNA is encapsulated in the lipid membrane based on a change in luminescence intensity derived from the compound bound to the RNA, a change in luminescence intensity derived from the fluorescent dye bound to the lipid membrane, or both.

[0114] In some embodiments, the evaluating step includes evaluating the quality of the formulation in which RNA is encapsulated in a lipid membrane as being degraded if there is an increase in luminescence intensity or fluorescent signal from a compound bound to the RNA, a decrease in luminescence intensity or fluorescent signal from a fluorescent dye bound to the lipid membrane, or both.

[0115] In some embodiments, the luminescence intensity or fluorescent signal derived from a compound bound to RNA and the luminescence intensity or fluorescent signal derived from a fluorescent dye bound to a lipid membrane are determined as reference values ​​for a given amount of a formulation with no quality problems, and the luminescence intensity or fluorescent signal derived from a compound bound to RNA in a formulation (called a test formulation) encapsulated in a lipid membrane and the luminescence intensity or fluorescent signal derived from a compound bound to RNA in the formulation are measured, and if the measured values ​​are within a predetermined threshold value for each of the reference values, the quality of the test formulation is maintained, and if they are outside the threshold value, the quality of the test formulation is evaluated as having deteriorated. Note that a formulation with no quality problems includes a formulation immediately after production.

[0116] In some embodiments, the compound that binds to the RNA is impermeable to the lipid membrane, and the evaluating step includes evaluating the quality of the formulation in which the RNA is encapsulated in the lipid membrane as being degraded if the luminescence intensity or fluorescent signal from the compound that binds to the RNA increases, the luminescence intensity or fluorescent signal from the fluorescent dye bound to the lipid membrane decreases, or both.

[0117] In some embodiments, the compound that binds to the RNA is permeable to the lipid membrane, and the evaluating step includes evaluating that the quality of the formulation in which the RNA is encapsulated in the lipid membrane is degraded when the luminescence intensity or fluorescent signal derived from the compound that binds to the RNA that has leaked out of the lipid membrane increases, or the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane decreases, or both.

[0118] In some embodiments, the compound that binds to the RNA is permeable to the lipid membrane, and the evaluating step includes evaluating the quality of the preparation as one in which some or all of the RNA is not encapsulated in the lipid membrane and has leaked out of the lipid membrane when some or all of the RNA is not encapsulated in the lipid membrane, the luminescence intensity or fluorescent signal derived from the compound bound to the RNA leaked from the lipid membrane increases, and the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane is a normal value. The normal value refers to a value within the range of luminescence intensity or fluorescent signal obtained from a preparation with no quality problem, and includes, for example, the value of luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane in a preparation in which RNA is encapsulated in the lipid membrane immediately after production, or a value smaller than that.

[0119] In some embodiments, the compound that binds to the RNA is permeable to the lipid membrane, and the evaluating step includes evaluating the quality of the preparation as one in which the RNA is not inherently encapsulated in the lipid membrane (and therefore the RNA has not leaked out of the lipid membrane) when the luminescence intensity or fluorescent signal derived from the compound bound to the RNA decreases due to the RNA not being completely encapsulated in the lipid membrane, and the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane is a normal value. The normal value includes, for example, the value of the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane in a preparation in which the RNA is encapsulated in the lipid membrane immediately after production, or a value smaller than that.

[0120] In some embodiments, the evaluating step includes evaluating that the quality of the formulation in which RNA is encapsulated in a lipid membrane is maintained when the luminescence intensity or fluorescent signal derived from the compound bound to the RNA does not change significantly and the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane does not change significantly. For example, when both the luminescence intensity or fluorescent signal derived from the compound bound to the RNA and the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane are (fluorescence signal after change-fluorescence signal before change) / fluorescence signal before change*100 is ±10% or less, the quality of the formulation in which RNA is encapsulated in a lipid membrane is evaluated to be maintained. Alternatively, for example, the luminescence intensity or fluorescent signal derived from the compound bound to the RNA and the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane are compared with the fluorescent signal after change and the fluorescent signal before change, and when there is no statistically significant difference by statistical analysis (p is 0.05 or more), the quality of the formulation in which RNA is encapsulated in a lipid membrane is evaluated to be maintained.

[0121] In some embodiments, the evaluating step includes evaluating that the quality of the formulation in which RNA is encapsulated in a lipid membrane is degraded when the luminescence intensity or fluorescent signal derived from the compound bound to the RNA increases relative to a first reference value, or the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane decreases relative to a second reference value, or both. The first reference value may be (i) the value of the luminescence intensity or fluorescent signal derived from the compound bound to the RNA when the method of the first aspect is performed (particularly under the same conditions as the test formulation) on a formulation in which RNA is encapsulated in a lipid membrane immediately after production, (ii) the value of the luminescence intensity or fluorescent signal derived from the compound bound to the RNA when the method of the first aspect is performed (particularly under the same conditions as the test formulation) on a formulation in which RNA is encapsulated in a lipid membrane within the appropriate refrigerated storage period indicated in the manufacturer's instructions (manual) after being frozen and stored according to the manufacturer's instructions (manual), or (iii) a predetermined value at which the quality of the formulation in which RNA is encapsulated in a lipid membrane can be considered to be maintained. The second reference value may be (i) the value of the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane when the method of the first embodiment is performed (particularly under the same conditions as the test preparation) on a preparation in which RNA is encapsulated in a lipid membrane immediately after production, (ii) the value of the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane when the method of the first embodiment is performed (particularly under the same conditions as the test preparation) on a preparation in which RNA is encapsulated in a lipid membrane that has been frozen and stored in accordance with the manufacturer's instructions (manual) and then thawed within the appropriate refrigerated storage period indicated in the manufacturer's instructions (manual), or (iii) a predetermined value at which the quality of the preparation in which RNA is encapsulated in a lipid membrane can be considered to be maintained.

[0122] In some embodiments, the compound that binds to the RNA is impermeable to the lipid membrane, and the evaluating step includes evaluating the quality of the formulation in which the RNA is encapsulated in the lipid membrane to be degraded when the luminescence intensity or fluorescent signal derived from the compound that binds to the RNA that has leaked from the lipid membrane increases relative to a first reference value, or the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane decreases relative to a second reference value, or both. The first reference value and the second reference value are as described above.

[0123] In some embodiments, the compound that binds to the RNA is permeable to the lipid membrane, and the evaluating step includes evaluating the quality of the formulation as one in which the RNA is not encapsulated in the lipid membrane and has leaked out of the lipid membrane when the RNA is not encapsulated in the lipid membrane, the luminescence intensity or fluorescent signal derived from the compound bound to the RNA that has leaked out of the lipid membrane increases relative to a first reference value, and the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane does not change relative to a second reference value.

[0124] In some embodiments, the compound that binds to the RNA is permeable to the lipid membrane, and the evaluating step includes evaluating the quality of the formulation as one in which the RNA is not inherently encapsulated in the lipid membrane (and therefore the RNA has not leaked out of the lipid membrane) when the luminescence intensity or fluorescent signal derived from the compound bound to the RNA decreases relative to a first reference value in association with the RNA not being encapsulated in the lipid membrane, and the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane does not change relative to a second reference value.

[0125] In some embodiments, the evaluating step includes evaluating that the quality of the formulation in which RNA is encapsulated in a lipid membrane is maintained when the luminescence intensity or fluorescent signal derived from the compound bound to the RNA does not change significantly with respect to the first reference value, and the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane does not change significantly with respect to the second reference value. The first reference value and the second reference value are as described above. For example, when both the luminescence intensity or fluorescent signal (i) derived from the compound bound to the RNA and the luminescence intensity or fluorescent signal (ii) derived from the fluorescent dye bound to the lipid membrane are within ±10% or less of the first reference value and the second reference value, respectively, (luminescence intensity or fluorescent signal (i) - first reference value) / first reference value*100 and (luminescence intensity or fluorescent signal (ii) - second reference value) / second reference value*100 are within ±10% or less of the first reference value and the second reference value, respectively, the quality of the formulation in which RNA is encapsulated in a lipid membrane is evaluated to be maintained. Instead, for example, both the luminescence intensity or fluorescence signal (i) and the luminescence intensity or fluorescence signal (ii) are compared with the first reference value and the second reference value, respectively, and when statistical analysis reveals no statistically significant difference (p is 0.05 or more), it is evaluated that the quality of the formulation in which RNA is encapsulated in a lipid membrane is maintained.

[0126] In some embodiments, the evaluation step includes a step of evaluating in a time series.Specifically, the evaluation step includes: comparing the luminescence intensity or fluorescent signal derived from the compound bound to RNA at a first time point with the luminescence intensity or fluorescent signal derived from the compound bound to RNA at a second time point later than the first time point, and evaluating the quality of the formulation in which RNA is encapsulated in lipid membrane to be reduced when the luminescence intensity or fluorescent signal at the second time point is increased relative to the luminescence intensity or fluorescent signal at the first time point; comparing the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to lipid membrane at a first time point with the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to lipid membrane at a second time point later than the first time point, and evaluating the quality of the formulation in which RNA is encapsulated in lipid membrane to be reduced when the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to lipid membrane at the second time point is decreased relative to the luminescence intensity or fluorescent signal at the first time point; or evaluating the quality of the formulation in which RNA is encapsulated in lipid membrane to be reduced when both of them are true.

[0127] In some embodiments, the compound that binds to the RNA is impermeable to the lipid membrane, and the evaluating step includes comparing the luminescence intensity or fluorescent signal derived from the compound bound to the RNA at a first time point with the luminescence intensity or fluorescent signal derived from the compound bound to the RNA at a second time point that is later than the first time point, and evaluating the quality of the formulation in which the RNA is encapsulated in the lipid membrane to be degraded if the luminescence intensity or fluorescent signal at the second time point is increased relative to the luminescence intensity or fluorescent signal at the first time point, or comparing the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane at the first time point with the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane at a second time point that is later than the first time point, and evaluating the quality of the formulation in which the RNA is encapsulated in the lipid membrane to be degraded if the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane at the second time point is decreased relative to the luminescence intensity or fluorescent signal at the first time point, or evaluating the quality of the formulation in which the RNA is encapsulated in the lipid membrane to be degraded if both are true.

[0128] In some embodiments, the compound that binds to the RNA is permeable to the lipid membrane, and the evaluating step includes comparing the luminescence intensity or fluorescent signal derived from the compound bound to the RNA at a first time point with the luminescence intensity or fluorescent signal derived from the compound bound to the RNA at a second time point that is later than the first time point, and evaluating the quality of the formulation in which the RNA is encapsulated in the lipid membrane to be degraded if the luminescence intensity or fluorescent signal at the second time point is increased relative to the luminescence intensity or fluorescent signal at the first time point, or comparing the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane at the first time point with the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane at a second time point that is later than the first time point, and evaluating the quality of the formulation in which the RNA is encapsulated in the lipid membrane to be degraded if the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane at the second time point is decreased relative to the luminescence intensity or fluorescent signal at the first time point, or evaluating the quality of the formulation in which the RNA is encapsulated in the lipid membrane to be degraded if both are true.

[0129] In some embodiments, the compound that binds to RNA is permeable to lipid membranes, and the evaluating step includes comparing the luminescence intensity or fluorescent signal derived from the compound bound to RNA in a normal preparation with the luminescence intensity or fluorescent signal derived from the compound bound to RNA in the preparation being tested, and comparing the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane in the normal preparation with the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane in the preparation being tested, and evaluating that the quality of the preparation has failed or deteriorated (e.g., some or all of the RNA is not encapsulated in the lipid membrane and has leaked out of the lipid membrane) if the luminescence intensity or fluorescent signal of the preparation being tested is increased relative to the luminescence intensity or fluorescent signal in the normal preparation and the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane is unchanged relative to the luminescence intensity or fluorescent signal in the normal preparation. For example, when the luminescence intensity or fluorescence signal of the preparation to be tested is increased relative to the luminescence intensity or fluorescence signal of a normal preparation, and the luminescence intensity or fluorescence signal (ii) derived from the fluorescent dye bound to the lipid membrane of the preparation to be tested is (luminescence intensity or fluorescence signal (ii) derived from the fluorescent dye bound to the lipid membrane of the preparation to be tested - luminescence intensity or fluorescence signal derived from the fluorescent dye bound to the lipid membrane of the normal preparation) / (luminescence intensity or fluorescence signal derived from the fluorescent dye bound to the lipid membrane of the normal preparation) * 100 is ± 10% or less relative to the corresponding luminescence intensity or fluorescence signal in the normal preparation, the quality of the preparation in which RNA is encapsulated in a lipid membrane is evaluated to be poor or deteriorated. Alternatively, for example, when the luminescence intensity or fluorescence signal of the preparation to be tested is increased relative to the luminescence intensity or fluorescence signal of a normal preparation, and the luminescence intensity or fluorescence signal (ii) is not statistically significantly different from the corresponding luminescence intensity or fluorescence signal in the normal preparation by statistical analysis (p is 0.05 or more), the quality of the preparation in which RNA is encapsulated in a lipid membrane is evaluated to be poor or deteriorated.

[0130] In some embodiments, the compound that binds to RNA is permeable to lipid membranes, and the evaluating step includes comparing the luminescence intensity or fluorescent signal derived from the compound bound to RNA in a normal formulation with the luminescence intensity or fluorescent signal derived from the compound bound to RNA in the formulation being tested, and comparing the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane in the normal formulation with the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane in the formulation being tested, and evaluating that the quality of the formulation has failed or been reduced (e.g., RNA is not inherently encapsulated in the lipid membrane) if the luminescence intensity or fluorescent signal of the formulation being tested is decreased relative to the luminescence intensity or fluorescent signal in the normal formulation and the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane is unchanged relative to the luminescence intensity or fluorescent signal in the normal formulation. For example, when the luminescence intensity or fluorescence signal of the preparation to be tested is decreased relative to the luminescence intensity or fluorescence signal of a normal preparation, and the luminescence intensity or fluorescence signal (ii) derived from the fluorescent dye bound to the lipid membrane of the preparation to be tested is (luminescence intensity or fluorescence signal (ii) derived from the fluorescent dye bound to the lipid membrane of the preparation to be tested - luminescence intensity or fluorescence signal derived from the fluorescent dye bound to the lipid membrane of the normal preparation) / (luminescence intensity or fluorescence signal derived from the fluorescent dye bound to the lipid membrane of the normal preparation) * 100 is ± 10% or less relative to the corresponding luminescence intensity or fluorescence signal of the normal preparation, the quality of the preparation in which RNA is encapsulated in a lipid membrane is evaluated to be poor or deteriorated. Alternatively, for example, when the luminescence intensity or fluorescence signal of the preparation to be tested is increased relative to the luminescence intensity or fluorescence signal of a normal preparation, and the luminescence intensity or fluorescence signal (ii) is not statistically significantly different from the corresponding luminescence intensity or fluorescence signal of the normal preparation by statistical analysis (p is 0.05 or more), the quality of the preparation in which RNA is encapsulated in a lipid membrane is evaluated to be poor or deteriorated.

[0131] In some embodiments, the evaluating step includes comparing the luminescence intensity or fluorescent signal derived from the compound bound to the RNA at a first time point with the luminescence intensity or fluorescent signal derived from the compound bound to the RNA at a second time point that is later than the first time point, and evaluating that the quality of the formulation in which RNA is encapsulated in a lipid membrane is maintained if the luminescence intensity or fluorescent signal at the second time point has not changed significantly compared to the luminescence intensity or fluorescent signal at the first time point, and comparing the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane at the first time point with the luminescence intensity or fluorescent signal derived from the fluorescent dye bound to the lipid membrane at a second time point that is later than the first time point, and evaluating that the quality of the formulation in which RNA is encapsulated in a lipid membrane has been maintained if the luminescence intensity or fluorescent signal at the second time point has not changed significantly compared to the luminescence intensity or fluorescent signal at the first time point.

[0132] According to a second aspect of the present invention, there is provided a kit for inspecting the quality of a formulation in which RNA is encapsulated in a lipid membrane, the kit comprising a compound that binds to the RNA in a formulation comprising RNA and a lipid membrane encapsulating the RNA, and a fluorescent dye that binds to the lipid membrane of the formulation. The compound that binds to RNA and the fluorescent dye that binds to lipid membranes are as explained for the compound and fluorescent dye of the first embodiment, respectively.

[0133] In some embodiments, the kit may include a container housing a compound that binds to RNA. Alternatively, or additionally, the kit may further include a container housing a fluorescent dye that binds to lipid membranes.

[0134] In some embodiments, the kit may further comprise a pipette, dropper, or other sample manipulation substrate.

[0135] In some embodiments, the kit may further comprise instructions for detecting a compound that binds RNA, a fluorescent dye that binds to lipid membranes, or both.

[0136] In some embodiments, the kit may further comprise a positive control and / or a negative control sample. The positive control sample may be a formulation in which RNA is encapsulated in a lipid membrane or a construct having a similar structure to the formulation in which RNA is encapsulated in a lipid membrane. The negative control sample may be a sample that does not contain the formulation in which RNA is encapsulated in a lipid membrane.

[0137] In some embodiments, the kit may further comprise water, buffers, buffer salts, detergents, surfactants, salts, polysaccharides, or other materials commonly used in bioassays. In some embodiments, the kit may further comprise a solvent, such as an aqueous, non-aqueous, or aqueous / non-aqueous solvent system.

[0138] The instructions for the kit may also include a step for evaluating the quality of a formulation in which RNA is encapsulated in a lipid membrane, as described in relation to the method for inspecting the quality of a formulation in which RNA is encapsulated in a lipid membrane of the first aspect of the present invention.

[0139] Furthermore, information on the luminescence intensity or fluorescent signal when a measuring device is used and the quality of the formulation in which RNA is encapsulated in a lipid membrane is maintained may also be provided.

[0140] Using the kit for inspecting the quality of a formulation in which RNA is encapsulated in a lipid membrane of the second embodiment, a user of the kit can perform the steps of contacting a formulation in which RNA is encapsulated in a lipid membrane with a compound that binds to the mRNA in the formulation, which comprises mRNA and a lipid membrane that encapsulates the mRNA, contacting a formulation in which RNA is encapsulated in a lipid membrane with a fluorescent dye that binds to the lipid membrane of the formulation in which RNA is encapsulated in a lipid membrane, and detecting at least one of the compound that binds to the RNA and the fluorescent dye that binds to the lipid membrane, based on the instructions, and then evaluate the quality of the formulation in which RNA is encapsulated in a lipid membrane based on a change in luminescence intensity derived from the compound that binds to the RNA, a change in luminescence intensity derived from the fluorescent dye that binds to the lipid membrane, or both.

[0141] In a preferred embodiment of the second aspect, the preparation in which RNA is encapsulated in a lipid membrane is an RNA vaccine in which mRNA is encapsulated in a lipid membrane. The mRNA may contain a part of the sequence of a viral RNA. In another preferred embodiment, the preparation in which RNA is encapsulated in a lipid membrane is an RNAi drug in which siRNA is encapsulated in a liposome. siRNA is a short (about 20 to 25 bases) double-stranded RNA complementary to any target mRNA in a cell.

[0142] The method of the first embodiment and the kit of the second embodiment enable rapid and sensitive quality evaluation of a preparation in which RNA is encapsulated in a lipid membrane, and therefore can be used for quality testing of preparations in which RNA is encapsulated in a lipid membrane in medical and research settings.

[0143] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these. EXAMPLES

[0144] Example 1 Quality inspection of a model vaccine using lipid membrane-binding probes and RNA-binding probes (method) -How to make a model vaccine 20μM RNA (5'-CCC GUC ACA CCA UGG CCC UUC GGGG CUG GGG UGA AGU CGGG-3' (SEQ ID NO: 1), 41 base length: oligonucleotide designed with reference to a part of Escherichia coli ribosomal RNA) was added to Lipocapsulator (FD-U PL (lipid composition DSPC:Cholesterol = 70:30 (molar ratio)), Xygieia bioscience), mixed by inversion, and incubated at room temperature for 15 minutes. While stirring this solution, a dedicated solution for encapsulating oligonucleotides (Xygieia bioscience) was added. Then, the model vaccine (average diameter 154 nm, measured by Nanosight (Malvern Panalytical NTA5330)) was obtained by purifying it with VIVASPIN6 (Sartorius Japan).

[0145] How to prepare samples with and without Triton The above model vaccine was used as a sample. The model vaccine was mixed with the high curvature membrane-binding probe ApoC-NR (excitation wavelength 552 nm) or the RNA-binding probe TO-PRO-1 (excitation wavelength 508.5 nm), and the fluorescence spectrum was measured in PBS buffer. In addition, to examine the fluorescence response to the denatured model vaccine, a sample was also used in which the model vaccine was mixed with a final concentration of 0.1% Triton-X 100 (Nacalai Tesque). The concentration of the model vaccine was 3.6 × 10 11 The concentration of each probe ([ApoC-NR], [TO-PRO-1]) was 2.0 μM / mL. In the experiment using TO-PRO-1, the sample and probe were mixed and incubated at room temperature for 30 minutes before measurement. All measurements were performed at room temperature (25°C).

[0146] (result) As shown in Figure 4(A), in the sample in which the model vaccine was mixed with ApoC-NR (Model vaccine graph), the fluorescence intensity at 622 nm was 426 times higher than that of the sample containing only ApoC-NR without the model vaccine (Probe graph). In addition, the fluorescence response of the sample containing the model vaccine was significantly reduced by the addition of Triton-X (surfactant) (Model vaccine (+ Triton) graph), and was 28 times higher at 622 nm than that of the sample containing only ApoC-NR (Probe). This reduction in the fluorescence response is due to the denaturation of the liposome structure.

[0147] As shown in Figure 4(B), the fluorescence intensity at 538 nm in the sample in which the model vaccine was mixed with TO-PRO-1 (Model vaccine graph) was 172 times higher than that in the sample containing only TO-PRO-1 without the model vaccine (Probe graph). In addition, the fluorescence response of the sample containing the model vaccine was significantly increased by the addition of Triton-X (surfactant) (Model vaccine (+ Triton) graph), which was 379 times higher at 538 nm than that of the sample containing only TO-PRO-1 (Probe). This increase in fluorescence response is due to the denaturation of the liposome structure and the leakage of the encapsulated RNA.

[0148] Example 2 Concentration-dependent fluorescence detection of model vaccine (method) The model vaccine described in Example 1 was used. The model vaccine was mixed with ApoC-NR or TO-PRO-1, and the fluorescence spectrum was measured in PBS buffer. The concentration of the model vaccine was 1.8 × 10 10 , 3.6 × 10 10 , 7.1 × 10 10 , 1.4 × 10 11 ,3.6 × 10 11 The concentration of each probe was 2.0 μM. In the experiment using TO-PRO-1, the sample and the probe were mixed and incubated at room temperature for 30 minutes before measurement. All measurements were performed at room temperature (25°C).

[0149] (result) As shown in Figures 5(A) and (B), in samples in which the model vaccine was mixed with ApoC-NR, the fluorescence response increased depending on the concentration of the model vaccine (analysis wavelength 622 nm, F0: fluorescence intensity at 622 nm of a sample containing only ApoC-NR without the model vaccine). Linear area (1.8-7.1 × 10 10 The detection limit of the model vaccine calculated using the 100 μg / mL antibody was 3.4 × 10 7 The number of cells was 1 / mL. As shown in Figures 5(C) and (D), in samples in which the model vaccine was mixed with TO-PRO-1, the fluorescence response increased in a concentration-dependent manner depending on the model vaccine (analysis wavelength: 538 nm, F0: fluorescence intensity at 538 nm for a sample containing only TO-PRO-1 but not the model vaccine). Linear area (1.8-14 × 10 10 The detection limit of the model vaccine calculated using the 1.6 × 10 9 The number of cells was 1 / mL.

[0150] Example 3 Effect of RNA addition on the fluorescence ratio of the vaccine (method) The model vaccine described in Example 1 was used. The model vaccine was mixed with ApoC-NR or TO-PRO-1, and the fluorescence spectrum was measured in PBS buffer. In addition, a sample in which the model vaccine was mixed with RNA at a final concentration of 4.0 μM was also used to examine the change in the fluorescence response of various probes due to the addition of RNA. The concentration of the model vaccine was 3.6 × 10 11 The concentration of each probe was 2.0 μM / mL. In the experiment using TO-PRO-1, the sample and probe were mixed and incubated at room temperature for 30 minutes, and then measurements were performed. All measurements were performed at room temperature (25°C). The fluorescence response to a sample containing the model vaccine and RNA (F) was compared with the fluorescence response to a sample containing the model vaccine but not RNA (F model / vaccine The ratio of F was calculated when the probe was ApoC-NR and when the probe was TO-PRO-1. model / vaccine was calculated (ApoC-NR: excitation wavelength 552 nm, analysis wavelength 622 nm, TO-PRO-1: excitation wavelength 508.5 nm, analysis wavelength 538 nm).

[0151] (result) As shown in Figure 6, in the sample mixed with ApoC-NR, the change in fluorescence response due to the addition of RNA, F / F model / vaccine In the sample mixed with TO-PRO-1, the change in the fluorescence response, F / F model / vaccinewas increased by 7.9-fold, suggesting that only TO-PRO-1 was responsible for RNA leakage.

Claims

1. A method for testing the quality of a formulation in which RNA is encapsulated in a lipid membrane, comprising the steps of: A step of contacting a preparation comprising RNA and a lipid membrane surrounding the RNA with a fluorescent molecule that is permeable to the lipid membrane and binds to the RNA; contacting the formulation with a fluorescent dye that binds to the lipid membrane of the formulation; and detecting the fluorescent molecule bound to the RNA and the fluorescent dye bound to the lipid membrane; The fluorescence intensity of the fluorescent molecule bound to the RNA and the peptide fluorescent dye bound to the lipid membrane of the preparation having no quality problem, which was previously measured, A step of evaluating the quality of the preparation based on a change in luminescence intensity resulting from the fluorescent molecule bound to the RNA and a change in luminescence intensity resulting from the fluorescent dye bound to the lipid membrane. Including, The evaluating step evaluates the quality of the preparation as being deteriorated when the fluorescence intensity derived from the fluorescent molecule bound to the RNA increases or decreases relative to the pre-measured emission intensity, the fluorescence intensity derived from the fluorescent dye increases or decreases relative to the pre-measured emission intensity, or both. method.

2. The method of claim 1, wherein the fluorescent dye that binds to the lipid membrane is a peptidic fluorescent dye that binds to a lipid packing defect.

3. The method according to claim 1 or 2, wherein the step of contacting the fluorescent molecule that binds to the RNA with the preparation and the step of contacting the fluorescent dye that binds to the lipid membrane with the preparation are carried out by adding the fluorescent molecule that binds to the RNA and the fluorescent dye that binds to the lipid membrane to a single sample containing the preparation.

4. A method for testing the quality of a formulation in which RNA is encapsulated in a lipid membrane, comprising the steps of: A step of contacting a preparation comprising RNA and a lipid membrane surrounding the RNA with a fluorescent molecule that is non-permeable to the lipid membrane and binds to the RNA; contacting the preparation with a peptidic fluorescent dye that binds to lipid packing defects in the lipid membrane of the preparation; and detecting a fluorescent molecule bound to the RNA and a peptidic fluorescent dye bound to a lipid packing defect in the lipid membrane; The fluorescence intensity of the fluorescent molecule bound to the RNA of the preparation having no quality problem and the peptide fluorescent dye bound to the lipid packing defect of the lipid membrane, which were previously measured, evaluating the quality of the preparation based on a change in luminescence intensity resulting from the fluorescent molecule bound to the RNA and a change in luminescence intensity resulting from the peptidic fluorescent dye bound to the lipid packing defect of the lipid membrane; Including, The evaluating step evaluates the quality of the preparation as being deteriorated when the fluorescence intensity derived from the fluorescent molecule bound to the RNA increases relative to the pre-measured emission intensity, or the fluorescence intensity derived from the fluorescent dye increases or decreases relative to the pre-measured emission intensity, or both. method.

5. A method for testing the quality of a formulation in which RNA is encapsulated in a lipid membrane, comprising the steps of: A step of contacting a preparation comprising RNA and a lipid membrane surrounding the RNA with a fluorescent molecule that is permeable to the lipid membrane and binds to the RNA; contacting the preparation with a peptidic fluorescent dye that binds to lipid packing defects in the lipid membrane of the preparation; and detecting a fluorescent molecule bound to the RNA and a peptidic fluorescent dye bound to a lipid packing defect in the lipid membrane; The fluorescence intensity of the fluorescent molecule bound to the RNA of the preparation having no quality problem and the peptide fluorescent dye bound to the lipid packing defect of the lipid membrane, which were previously measured, evaluating the quality of the preparation based on a change in luminescence intensity derived from the fluorescent molecule bound to the RNA and a change in luminescence intensity derived from the peptidic fluorescent dye bound to the lipid packing defect of the lipid membrane; Including, The evaluating step evaluates the quality of the preparation as being deteriorated when the luminescence intensity derived from the fluorescent molecule bound to the RNA increases or decreases relative to the luminescence intensity measured in advance, the fluorescence intensity derived from the fluorescent dye increases or decreases relative to the luminescence intensity measured in advance, or both. method.

6. The method according to claim 4 or 5, wherein the steps of contacting a fluorescent molecule that binds to the RNA with the preparation and contacting a peptidic fluorescent dye that binds to lipid packing defects in the lipid membrane with the preparation are carried out by adding a fluorescent molecule that binds to the RNA and a peptidic fluorescent dye that binds to lipid packing defects in the lipid membrane to a single sample containing the preparation.

7. A kit for testing the quality of a formulation in which RNA is encapsulated in a lipid membrane, comprising: A preparation comprising RNA and a lipid membrane surrounding the RNA, and a fluorescent molecule that is permeable to the lipid membrane and binds to the RNA; A fluorescent dye that binds to the lipid membrane of the formulation.

8. A kit for testing the quality of a formulation in which RNA is encapsulated in a lipid membrane, comprising: A fluorescent molecule that binds to the RNA in a preparation comprising RNA and a lipid membrane that encases the RNA; a peptidic fluorescent dye that binds to a lipid packing defect in the lipid membrane of the formulation; A kit equipped with

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