WO2023026315A1

WO2023026315A1 - Development of quality control technology for pharmaceutical formulation having rna encapsulated in lipid membrane

Info

Publication number: WO2023026315A1
Application number: PCT/JP2021/030716
Authority: WO
Inventors: 雄介佐藤; 精一西澤; 良一永富
Original assignee: 国立大学法人東北大学
Priority date: 2021-08-23
Filing date: 2021-08-23
Publication date: 2023-03-02
Also published as: JPWO2023026315A1

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

Development of quality control technology for formulations in which RNA is encapsulated in lipid membranes

The present invention relates to a quality inspection method for formulations in which RNA is encapsulated in a lipid membrane.

In recent years, the development of nucleic acid drugs using RNA such as siRNA (small interfering ribonucleic acid) and mRNA has attracted attention.

For example, RNAi drugs use short (approximately 20 to 25 nucleotides) double-stranded RNA (siRNA) that is complementary to target mRNA in cells to suppress the production of disease-causing proteins. There is a drug that utilizes the principle of interference, and Onpattro (trademark) by Alrylam Inc. in the United States was approved by the FDA in 2018 and by the Ministry of Health, Labor and Welfare in 2019 as a therapeutic agent for transthyretin-type familial amyloid polyneuropathy. Since siRNA is extremely unstable, it is rapidly degraded by degradative enzymes when administered into the body. In this therapeutic agent, siRNA is encapsulated in lipid nanoparticles (LNPs), a type of lipid membrane, to enhance its stability in the body. Onpattro™ should be used immediately after preparation of the diluted solution and, if stored, should be used within 16 hours, including the time of administration. Thus, RNAi medicines are prone to quality deterioration.

RNA vaccines, which are another nucleic acid medicine, are also called mRNA (messenger RNA) vaccines. They administer mRNA, which is a chemically synthesized part of the RNA sequence of a virus, to humans. protein is produced and induces both cellular and humoral immunity against this antigenic protein.

RNA vaccines make it easier for human cells to produce virus-specific antigens than conventional antigenic proteins or attenuated viruses. can respond by designing and manufacturing RNA vaccines. 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 new coronavirus Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), which has been prevalent in China since the end of 2019. ing.

The mRNA molecules of RNA vaccines are also extremely susceptible to degradation. The main structure of RNA vaccines currently being developed is the mRNA-LNP structure, which has an mRNA part corresponding to a part of the viral RNA sequence and a lipid nanoparticle (LNP) part that wraps the mRNA. Yes, LNP enhances mRNA stability. However, current RNA vaccines still require low-temperature distribution and storage. For example, BNT162b2 from Pfizer, USA 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 should be transported and stored at -20°C ± 5°C, and the storage period after thawing is 30 days in the refrigerator (4-8°C).

Long-term storage of RNA vaccines at room temperature, freeze-drying and reconstitution of RNA vaccines, etc. have been reported as factors that affect the stability and efficacy of vaccines (Non-Patent Document 1).

However, little attention has been paid to how the stability of formulations in which RNA is encapsulated in lipid membranes, such as RNAi drugs and RNA vaccines, changes during storage. almost unreported. For example, conventional methods for evaluating the physical properties of RNA vaccines include methods using photodynamic scattering, electron microscopy, and chromatograms, but these methods have major problems such as cost, speed, and throughput.

The problem to be solved by the present invention is to provide a method for rapidly and highly sensitively testing the quality of formulations in which RNA is encapsulated in lipid membranes.

As a result of intensive studies to solve the above problems, the present inventors found that a specific compound that binds to the RNA portion of a formulation in which RNA is encapsulated in a lipid membrane and a lipid membrane in a formulation in which RNA is encapsulated in a lipid membrane. Rapidly and highly sensitively inspect the quality of formulations in which RNA is encapsulated in lipid membranes by examining changes in at least one of RNA and lipid membranes in the formulation using a fluorescent dye that binds to the part of The present inventors have found that it is possible to achieve the present invention.

The present invention includes the embodiments described below.

Section 1. A method for inspecting the quality of a formulation in which RNA is encapsulated in a lipid membrane,
A step of contacting a compound that binds to the RNA of a formulation comprising RNA and a lipid membrane enveloping the RNA with the formulation;
A method comprising: contacting a fluorescent dye that binds to the lipid membrane of the formulation with the formulation; and detecting at least one of a compound bound to the RNA and the fluorescent dye bound to the lipid membrane.

Section 2. After the detecting step, the quality of the formulation is evaluated based on the change in luminescence intensity derived from the compound bound to RNA, the change in luminescence intensity derived from the fluorochrome bound to the lipid membrane, or both. Item 1. The method of Item 1, further comprising the step of:

Section 3. Item 3. The method according to

Item

1 or 2, wherein the compound that binds to RNA is a fluorescent molecule that is impermeable to lipid membranes and capable of binding to RNA.

Section 4. Item 3. The method according to

Item

1 or 2, wherein the compound that binds to RNA is a fluorescent molecule that is permeable to lipid membranes and capable of binding to RNA.

Item 5. In the step of evaluating, if the fluorescence signal derived from the compound that binds to the RNA increases, or the fluorescence signal derived from the fluorochrome decreases, or both, the quality of the preparation deteriorates. 4. The method of any one of clauses 1-3, comprising assessing that the

Item 6. Item 6. The method according to any one of Items 1 to 5, wherein the fluorescent dye that binds to lipid membranes is a peptidic fluorescent dye that binds to lipid packing defects.

Item 7. The step of contacting the compound 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 include the compound that binds to the RNA and the fluorescence that binds to the lipid membrane. Item 7. The method according to any one of Items 1 to 6, which is carried out by adding a dye and a sample containing the formulation.

Item 8. A kit for testing the quality of a formulation in which RNA is encapsulated in a lipid membrane,
a compound that binds to the RNA in a formulation comprising RNA and a lipid membrane enveloping the RNA;
a fluorescent dye that binds to the lipid membrane of the formulation.

According to the present invention, the quality of formulations in which RNA is encapsulated in lipid membranes can be tested quickly and with high sensitivity.

Schematic diagram for explaining the principle of an RNA vaccine quality inspection method using two types of probes. (A) Schematic representation of RNA vaccine, (B) Detection of lipid membranes by lipid membrane-binding probes, (C) Detection of mRNA by RNA-binding probes. Schematic diagram illustrating an example of a pattern of degraded RNA vaccines and a detection method thereof. Schematic diagram illustrating another example of the pattern of degraded RNA vaccine and its detection method. Fluorescence response to model vaccines. (A) Vaccine detection by lipid membrane-binding probe, (B) Vaccine detection by RNA-binding probe. Concentration dependence of fluorescence response to model vaccines. (A) A concentration-dependent increase in the fluorescence response of the model vaccine sample mixed with ApoC-NR, (B) a graph showing the fluorescence intensity ratio F/F ₀ of each sample in FIG. 4 (A), ( C) Increase in fluorescence response of model vaccine sample mixed with TO-PRO-1 depending on concentration of model vaccine, (D) Graph showing fluorescence intensity ratio F/ _F0 of each sample in FIG. 4(C). Fluorescence response to a model vaccine spiked with RNA.

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

The term "alkyl" refers to a branched, unbranched or cyclic saturated hydrocarbon. In some embodiments, alkyl groups have, for example, 1-20 carbon atoms, often 1-12 carbon atoms or 1-6 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 may contain one or more non-carbon and non-hydrogen atoms such as oxygen, nitrogen, sulfur, halogens and phosphorus.

The term "alkenyl" refers to a branched or unbranched unsaturated hydrocarbon having a carbon-carbon ^sp2 double bond. In some embodiments, alkenyl groups can have, for example, 2-10 carbon atoms, or 2-6 carbon atoms. In other embodiments, alkenyl groups have 2-4 carbon atoms. Examples of alkenyl include, but are not limited to, ethylene or vinyl, allyl, cyclopentenyl, 5-hexenyl, and the like. An alkenyl can be unsubstituted or substituted. Substituted alkenyl groups may contain one or more non-carbon and non-hydrogen atoms such as oxygen, nitrogen, sulfur, halogens and phosphorous.

The term "alkynyl" refers to a branched or unbranched unsaturated hydrocarbon chain having a carbon-carbon sp triple bond. In some embodiments, alkynyl groups can have, for example, 2-10 carbon atoms, or 2-6 carbon atoms. In other embodiments, alkynyl groups can have from 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 etc. An alkynyl can be unsubstituted or substituted. Substituted alkynyl groups may contain one or more non-carbon and non-hydrogen atoms such as oxygen, nitrogen, sulfur, halogens and phosphorous.

The term "alkoxy" refers to alkyl-O- (alkyl is defined herein). In some embodiments, alkoxy groups have 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- Including dimethylbutoxy and the like. An alkoxy can be unsubstituted or substituted. A substituted alkoxy group may contain an oxygen attached to the substituted alkyl group.

The term "aryl" refers to an aromatic hydrocarbon group derived by removing one hydrogen atom from one carbon atom of the parent aromatic ring. Aryl groups can have 6 to 18 carbon atoms, 6 to 14 carbon atoms, or 6 to 10 carbon atoms. Aryl groups can have a monocyclic ring (eg, phenyl) or multiple condensed rings (fused rings) in which at least one ring is aromatic (eg, 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, aryl groups can be substituted with one or more substituents (as described above) to give various substituted aryls, such as pentafluorophenyl or p-trifluoromethylphenyl.

The term "amino" refers to _-NH2 . Amino groups can be optionally substituted as defined for the term "substituted." For example, an amino group can be _-NR2 , where R is a group enumerated in the definition of "substituted". For example, the group _-NR2 can be defined as "alkylamino" (at least one R is alkyl and the other R is alkyl or hydrogen) and/or "acylamino" (-N(R)C(=O) R) (each R is independently hydrogen, alkyl, alkaryl or aryl). The amino group can be either a primary amine ( _NH2 ), a secondary amine (NHR), or a tertiary amine.

First, with reference to FIGS. 1(A) to 1(C), the principle of the method for inspecting the quality of the formulation in which the RNA of the present invention is encapsulated in a lipid membrane will be described. As shown in FIG. 1(A)(a), an RNA vaccine (1) (hereinafter simply referred to as "RNA vaccine ) comprises an mRNA (2) portion corresponding to a portion of the viral RNA sequence and a lipid membrane (3) portion encapsulating the mRNA (2). mRNA (2) may be part of any viral RNA sequence.

Viruses include coronavirus (including SARS-CoV-2), rhinovirus, norovirus, enterovirus, rotavirus, influenza virus, measles virus, rubella virus, hepatitis A virus, hepatitis C virus, HIV virus, and Japanese encephalitis. viruses, yellow fever virus, dengue virus, Zika virus, Lassa virus, Ebola virus, etc., but are not limited thereto.

When an RNA vaccine (1) is administered, mRNA is translated into a protein within the cells of a subject such as a human, and the produced protein functions as an antigen to induce an immune response or replace defective proteins. can function as proteins. RNA vaccines of the mRNA-LNP structure, which have a portion of the mRNA corresponding to a portion of the viral RNA sequence and a portion of the lipid nanoparticles (LNPs) encasing the mRNA, are well known. In some embodiments, the mRNA (2) portion of the RNA vaccine comprises, in order from the 5' end to the 3' end, the 5' cap structure, the untranslated region, the coding sequence of the mRNA, the untranslated region, and the 3' poly It consists of an mRNA construct with an A tail. In some embodiments, part of the lipid membrane (3) consists of LNP with phospholipids, cholesterol, polyethylene glycol and cationic lipids.

Figures 1 (A) and (b) are schematic diagrams of the RNA vaccine (1) in Figure 1 (A) (a). In RNA vaccine (1) immediately after production, mRNA (2) is encapsulated inside lipid membrane (3). Hereinafter, in this specification, "immediately after production" refers to within one day after production. “Preparation in which RNA is encapsulated in a lipid membrane immediately after production” refers to a preparation that has not been frozen within one day of production.

The method for inspecting the quality of the RNA vaccine of the present invention uses two types of probes. One is a lipid membrane-binding probe (4) that binds to the lipid membrane (3) (Fig. 1 (B)), and the other is an RNA-binding probe (5) that binds to mRNA (2). (Fig. 1(C)).

Regarding the quality deterioration of RNA vaccines, the inventors considered that there are aspects shown in Figure 1 (B) and Figure 1 (C) below, and investigated detection of each.

In the example of FIG. 1(B), the lipid membrane-binding probe (4) can bind to liposomes, which are freshly prepared lipid membranes (3) of small size, ie, high curvature (FIG. 1(B)). B) (a)), the longer the storage period of the RNA vaccine (1) or the repeated thawing and freezing, the larger the size of the lipid membrane (3), that is, the lower the curvature. The number of lipid membrane-binding probes (4) that can bind to (3) decreases (Fig. 1 (B) (b)). That is, by examining the signal intensity associated with the binding of the lipid membrane-binding probe (4) to the lipid membrane (3), changes in the structure of the lipid membrane (3) can be detected.

In some cases, it is conceivable that the size of the lipid membrane (3) may become smaller. Even in such a case, structural changes in 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).

In the example of FIG. 1(C), when the RNA vaccine (1) is just manufactured, the mRNA (2) is wrapped inside the lipid membrane (3), so in the solution outside the RNA vaccine (1) There is no mRNA (2) in , and the RNA-binding probe (5) cannot detect mRNA (Fig. 1 (C) (a), but the storage period of the RNA vaccine (1) is prolonged, As part of the lipid membrane (3) is damaged by repeated thawing and freezing, and mRNA (2) leaks out, it binds to the RNA-binding probe (5) outside the RNA vaccine (1). The number of mRNA (2) increases (Fig. 1 (C) (b)).In other words, by examining the signal intensity associated with the binding of RNA-binding probe (5) to mRNA (2), leaked mRNA ( 2) can be detected Thus, the quality of the RNA vaccine (1) can be tested using the lipid membrane-binding probe (4) and the RNA-binding probe (5).

Next, we will explain some examples of patterns of degraded RNA vaccines and their detection methods. As shown in FIG. 2(a), the RNA vaccine (1) immediately after production has a small lipid membrane (3) in size (large curvature), and the mRNA (2) is completely encapsulated in the lipid membrane (3). ing. 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).

As shown in Fig. 2(b), mRNA (2) has not yet leaked out of the lipid membrane (3), but when the size of the lipid membrane (3) increases due to quality deterioration, the lipid membrane (3) The curvature changes from the curvature of the lipid membrane (3) in Fig. 2(a) (pattern (A)). Therefore, the number of lipid membrane-binding probes (4) that can bind to the lipid membrane (3) is reduced, and the signal intensity associated with the binding of the lipid membrane-binding probes (4) is lower than in the case of FIG. 2(b). decrease with time. On the other hand, the signal intensity accompanying the binding of the RNA-binding probe (5) is not observed because the mRNA (2) has not yet leaked out from the lipid membrane (3).

When the size of the lipid membrane (3) is reduced, 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) increases. 2(b).

As shown in Fig. 2(c), the size of the lipid membrane (3) is maintained, but holes and breaks occur in the lipid membrane (3), and mRNA (2) leaks out of the lipid membrane (3). When there is (pattern (B)), the curvature of the lipid membrane (3) may be almost unchanged, but usually the lipid membrane-binding probe (4) can bind to the lipid membrane (3) due to the appearance of holes or breaks 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). Since the mRNA (2) leaks out of the RNA vaccine (1), signal intensity associated with the binding of the RNA-binding probe (5) is observed. A compound that is impermeable to lipid membranes and capable of binding to RNA is preferably used for detecting RNA that has leaked out of RNA vaccine (1).

As used herein, the term "permeability" refers to permeation or permeation of RNA-binding probes into lipid membranes. Permeable lipid membranes refer to both live and dead cell lipid membranes. Permeable lipid membranes include lipid nanoparticles used in RNA vaccines and the like, membranes composed of lipid bilayers of liposomes, and the like.

As shown in FIG. 2(d), when the size of lipid membrane (3) increases and mRNA (2) leaks from lipid membrane (3) (pattern (C)), lipid membrane (3) The curvature of is changed from the curvature of the lipid membrane (3) in Fig. 2(a). Therefore, the number of lipid membrane-binding probes (4) that can bind to the lipid membrane (3) is reduced, and the signal intensity associated with the binding of the lipid membrane-binding probes (4) is lower than in the case of FIG. 2(b). decrease with time. In addition, since mRNA (2) leaks out of RNA vaccine (1), signal intensity associated with binding of RNA-binding probe (5) is observed.

2(b)-(d) are summarized, detecting changes in at least one of the signal intensity associated with the binding of the lipid membrane-binding probe (4) and the signal intensity associated with the binding of the RNA-binding probe (5). This makes it possible to rapidly and highly sensitively inspect the quality of RNA vaccine (1), including what kind of structural changes have occurred.

In order to facilitate understanding of the invention, the method for inspecting the quality of RNA vaccines has been described above with reference to FIGS. can be inspected.

According to the 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, wherein a compound that binds to RNA in a formulation comprising RNA and a lipid membrane enclosing the RNA is Contacting with the formulation, contacting a fluorescent dye that binds to the lipid membrane of the formulation with the formulation, and detecting at least one of a compound bound to RNA and a fluorescent dye bound to the lipid membrane A method of comprising is provided.

RNA includes mRNA, siRNA, tRNA, rRNA, dsRNA (double-stranded RNA), miRNA (single-stranded RNA consisting of 19-23 nucleotides), and the like. dsRNAs include siRNAs (small double-stranded RNAs consisting of 21-23 base pairs).

In a preferred embodiment, the formulation 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 lipid membrane-encapsulated formulation is an RNAi drug in which siRNA is encapsulated in liposomes.

In some embodiments, it is preferable that the compound that binds to RNA is a compound that is impermeable to lipid membranes, in that RNA leaked into the solution can be detected accurately.

A specific example of a lipid membrane-impermeable, RNA-binding compound is TO-PRO-1 (trademark) (3-METHYL-2-([1-[ 3-(TRIMETHYLAMMONIO)PROPYL]-4(1H)-QUINOLINYLIDENE]METHYL)-1,3-BENZOTHIAZOL-3-IUM DIODIDE, ThermoFisher) and the like.

In some other embodiments, the compound that binds RNA is a compound that is permeable to lipid membranes in order to ensure that the quality of the formulation is preserved, i.e. that the RNA is encapsulated in the lipid membrane. be.

The detection of leaked mRNA by the lipid membrane-impermeable RNA-binding probe (5) was described with reference to the example of FIG. Fig. 3 shows the detection of the encapsulated mRNA.

According to the example in Figure 3, when the RNA vaccine (1) has just been produced, the mRNA (2) is wrapped inside the lipid membrane (3). Since a certain amount of the RNA-binding probe (5) permeates the lipid membrane (3) into the interior, the endogenous mRNA can be detected with a predetermined fluorescence intensity by the RNA-binding probe (5) (Fig. 3(a)). ). Part of the lipid membrane (3) is damaged due to deterioration in the quality of the RNA vaccine (1), and as the mRNA (2) leaks out, the mRNA that binds to the RNA-binding probe (5) existing outside the lipid membrane 2) increases in number and can be observed as a very high fluorescence intensity (Fig. 3(b)). Thus, RNA vaccines (1) can be tested for quality even with RNA-binding probes (5) that are permeable to lipid membranes.

In some other embodiments, as another method of using the lipid membrane-permeable RNA-binding probe (5), the manufactured RNA vaccine (1) transfers the mRNA (2) inside the lipid membrane (3). You can also check if it is included. When the RNA vaccine (1) is produced, if the mRNA (2) cannot be encapsulated inside the lipid membrane (3), the quality of the vaccine will naturally be questioned, but the permeable RNA-binding probe (5) can be used to judge the quality. Specifically, the fluorescence intensity is obtained in advance when the permeable RNA-binding probe (5) is brought into contact with an RNA vaccine that does not have quality problems, and the RNA vaccine (1) to be tested is subjected to RNA detection. A binding probe (5) is brought into contact. If the mRNA (2) is not encapsulated inside the lipid membrane (3) and the mRNA (2) is leaked, the number of mRNA (2) that binds to the permeable RNA-binding probe (5) is It increases and becomes a value higher than the previously acquired fluorescence intensity. In addition, it is also assumed that the mRNA (2) is not encapsulated inside the lipid membrane (3) and is not present outside the lipid membrane (3) for some reason during the manufacturing process of the vaccine. In that case, the quality cannot be determined with the above-mentioned lipid membrane-impermeable RNA-binding probe (5), but by using the lipid membrane-permeable RNA-binding probe (5), in the normal case, It becomes a value lower than the fluorescence intensity acquired in advance, and thereby it can be determined that the mRNA (2) is not included inside the lipid membrane (3).

As described above, by using the permeable RNA-binding probe (5), it is possible not only to determine the quality of the vaccine over time, but also to easily determine the quality of the vaccine at the time of vaccine production.

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

A specific example of a compound that is permeable to lipid membranes and capable of binding to RNA is the compound of formula (1). The compound of formula (1) itself shows almost no fluorescence, but upon binding to a nucleic acid, the fluorescence intensity in the vicinity of a specific wavelength increases remarkably. It is preferable in that it can be observed as a bright spot with high intensity in a limited system.

In formula (1), ring A is

and
^R1 , ^R2 , ^R3 , ^R4 , ^R5 , ^R6 , ^R7 , ^R8 , ^R9 , ^R10 and ^R11 are independently hydrogen, hydroxy, thiol, halo, alkyl, alkenyl group, alkynyl group, alkoxy group, aryl group and amino group.

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, hydroxy group, thiol group, halo, alkyl group, alkenyl group, alkynyl group, alkoxy group, aryl group and amino group.

Herein, moieties of the groups R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ and R ¹¹ of formula (1) are substituted Where possible, the term “substituted” refers to one or more (e.g., 1, 2, 3, 4, 5 or 6) of the groups indicated in the expression using “substituted”; 1, 2 or 3 in the form of 1, 2 or 3; It indicates that it can be substituted with a suitable group. Suitable substituents for substituted groups 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, Mention may be made of heteroarylsulfonyl, heterocyclesulfinyl, heterocyclesulfonyl, phosphate, sulfate, hydroxylamine, hydroxyl(alkyl)amine and cyano.

In some embodiments, ^R1 , ^R2 , ^R3 , ^R4 , ^R6 , ^R7 , ^R8 , ^R9 , ^R10 and ^R11 are independently hydrogen, hydroxy, thiol, halo, Alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, aryl groups, amino groups, and ^R5 is an alkyl group.

In some embodiments, ^R1 , ^R2 , ^R3 , ^R4 , ^R5 , ^R6 , ^R7 , ^R8 , ^R9 , ^R10 and ^R11 are independently hydrogen, hydroxy, halo, It is an alkyl group, an aryl group, or an amino group.

In some embodiments, ^R1 , ^R2 , ^R3 , ^R4 , ^R6 , ^R7 , ^R8 , ^R9 , ^R10 and ^R11 are independently hydrogen, hydroxy, thiol, halo, Alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, aryl groups, and amino groups, all of which are unsubstituted, and ^R5 is also an unsubstituted alkyl group.

In some preferred embodiments, R ¹ , R ² , R ³ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ and R ¹¹ are hydrogen and R ⁴ is C 1-6 alkyl. and R ⁵ is methyl.

In some preferred embodiments, the compound of formula (1) above is a compound of formula (1-I). Since the compound of formula (1-I) is a combination of Benzo[c,d]Indole and OxazoloPyridine, it may be referred to as BIOP hereinafter.

The compound (BIOP) of formula (1-I) is Benzo[c,d]indole-2(1H)-one(1) Taking as an example the case where it is used as a starting material, it can be produced by the following scheme.

First, using Benzo[c,d]indole-2(1H)-one (compound 1), 1-methylbenzo[c,d]indol was synthesized according to J. Med. Chem., 2016, 59, 1565-1579. -2(1H)-one (compound 2) is obtained. 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. The solution is filtered at room temperature and the solid obtained is washed with 1,4-dioxane to give 1-methylbenzo[c,d]indol-2(1H)-thione crude product 3. Crude 3 (1.71 g) is then added with iodomethane and heated to reflux overnight. After concentrating the solution under reduced pressure, diethyl ether was added and sonicated for 30 minutes. The resulting solid is collected by filtration to give 1-methyl-2(methylthio)benzo[c,d]indole-1-ium (compound 4). Next, 4-methyl-2-methyl-oxazolo[4,5]pyridinium iodide (compound 5, 193 mg, 0.70 mmol) obtained by reacting 2-methyl-oxazolo[4,5-b]pyridine with iodomethane and 0.5 mL of tetraethylamine is added to an acetonitrile solution containing compound 4 (162 mg, 0.47 mmol). The solution is stirred and heated to reflux (60° C.) for 1 hour, and then cooled to room temperature. Then diethyl ether is added and the precipitate obtained is filtered off. After that, water is added, filtered again, and dried to obtain compound 6 (BIOP).

The compounds of formula (1) above are 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. may

The compound of formula (1) above preferably emits little or no fluorescence in the absence of a nucleic acid. Fluorescence can be measured by illuminating the compound with the appropriate wavelength and monitoring the emitted fluorescence. The compound preferably fluoresces more strongly 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 the compound concentration constant and the RNA and DNA concentrations constant. Higher RNA/DNA fluorescence ratios are better for RNA detection in the presence of DNA. The RNA/DNA fluorescence ratio is preferably 1 or greater, more preferably greater than 1, 1.2 or greater, 1.5 or greater, and 2 or greater. Compounds of formula (1) above meet the criteria for being suitable for RNA detection.

The compounds of formula (1) above can also be characterized by their maximum excitation and emission wavelengths. For example, maximum excitation can be from about 450 nm to about 650 nm. Maximum excitation between these values is 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 even between any two of these values. good. For example, maximum emission may be from about 500 nm to about 675 nm. 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 of these values.

The compound of formula (1) above can be used as a fluorescent dye because it emits fluorescence upon binding to a nucleic acid. In addition, the compound of formula (1) above can detect RNA with high sensitivity. A fluorescent dye containing the compound of the formula (1) or a fluorescent dye consisting of the compound of the formula (1) has a clear response performance, so it can be suitably used for detecting the RNA portion of an RNA vaccine.

Another specific example of a compound that is permeable to lipid membranes and capable of binding to RNA is the compound of formula (2) shown below:

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, hydroxy group , thiol, halo, alkyl, alkenyl, alkynyl, alkoxy, aryl and amino groups.

As used herein, the radical moieties of 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 of formula (2) are When capable of being substituted, the term “substituted” means one or more (e.g., 1, 2, 3, 4, 5 or 6; 1, 2 or 3 in some embodiments; can be substituted with known suitable groups. Suitable substituents for substituted groups 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, Mention may be made of heteroarylsulfonyl, heterocyclesulfinyl, heterocyclesulfonyl, phosphate, sulfate, hydroxylamine, hydroxyl(alkyl)amine and cyano.

In some embodiments, ^R1 , ^R2 , ^R3 ^, ^R4 , ^R5 , R6, ^R7 , ^R8 , ^R9 , ^R10 , ^R11 , and ^R12 are independently hydrogen, hydroxy groups, thiol groups, halo, alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, aryl groups, amino groups, and ^R6 is an alkyl group.

In some embodiments, ^R1 , ^R2 , ^R3 ^, ^R4 , ^R5 , R6, ^R7 , ^R8 , ^R9 , ^R10 , ^R11 , and ^R12 are independently hydrogen, hydroxy group, halo, alkyl group, aryl group, or amino group.

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, hydroxy groups, thiol groups, halo, alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, aryl groups, amino groups, all of which are unsubstituted, and ^R6 is also unsubstituted.

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

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

The preparation of the compound of formula (1) is analogous until the crude product of compound 4 is obtained. Then, an acetonitrile solution containing 1,4-dimethylquinolin-1-ium (compound 7, 258 mg, 0.91 mmol) obtained by reaction of 4-methylquinoline with iodomethane and the crude product of compound 4 (194 mg, 0.57 mmol) Add 0.5 mL of tetraethylamine to the The solution is heated under reflux with stirring for 1 hour, and then cooled to room temperature. After that, diethyl ether can be added to the solution and a precipitate can be obtained by filtration. The obtained precipitate is washed with water and then dissolved in methanol. Diethyl ether is then added and the resulting precipitate is dried to give compound 8 (BIQ).

Compounds of formula (2) above are 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. may

The compound of formula (2) above can be used as a fluorescent dye because it emits fluorescence upon binding to a nucleic acid. In addition, the compound of formula (2) above can detect RNA with high sensitivity. Since 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, it can be suitably used for detecting the RNA portion of an RNA vaccine.

On the other hand, in some embodiments, fluorescent dyes that bind to the lipid membrane of RNA vaccines include peptide fluorescent dyes that can selectively bind to lipid bilayer structures, cell membrane staining dyes, and the like.

Peptidic fluorescent dyes that can bind highly selectively to lipid membranes include exosome-targeting binding peptides that the inventors previously developed (98th Annual Meeting of the Chemical Society of Japan (2018 (4G3-02)), The 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 curved lipid bilayer membrane with a diameter of about 100 nm, and 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 base as a binding motif for lipid packing defects, to which an environment-responsive fluorescent dye is linked. For example, since mRNA vaccines have a diameter similar to that of exosomes, it is expected that such α-helical peptide-based fluorescent dyes can selectively bind to vaccine lipid membranes. It is expected that an α-helix peptide-based fluorescent dye can selectively bind to the liposome lipid membrane of an RNAi drug encapsulated in the liposome lipid membrane.

In the design of peptidic fluorescent dyes that can selectively bind to the lipid bilayer membrane structure, the peptide portion is designed to recognize the lipid packing structure of the lipid membrane of pharmaceuticals such as vaccines, and environment-responsive fluorescence The dye is attached to a position that does not interfere with the binding of the peptide to the lipid membrane, such as the N-terminus or C-terminus of the peptide.

　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), string-like N-terminus of GMAP-210 (H. sapiens), sterol transporter Keslp (S. cerevisiae), SpoVM ( B. subtilis), Epsin (H. sapiens), Binl/amphiphysin II (H. sapiens), BRAP/Bin2-derived H0-NBAR, myristoylated Arf 1 (B. taurus), melittin (A. mellifera), etc. -Helix peptides or peptides based on these α-helix peptides (for example, part of the α-helix peptide, 1 to several, more specifically, about 1 to 5 amino acids of the α-helix peptide are mutated amino acids), but are not limited to these. Various peptide sequences can be appropriately selected by those skilled in the art as the peptide of the peptidic fluorescent dye that can selectively bind to the lipid membrane structure of the RNA vaccine.

Any dye whose fluorescence intensity is weak in a hydrophilic environment but increases in a hydrophobic environment can be used as the environment-responsive fluorescent dye, and such dyes are known in the art. Examples of environment-responsive fluorescent dyes include dansyl dyes and derivatives thereof, dapoxyl dyes and derivatives thereof, benzophenoxazine dyes and derivatives thereof, and the like. Specific examples of environmentally responsive fluorescent dyes include 1-anilinonaphthalene-8-sulfonic acid (ANS), N-methyl-2-alilinonaphthalene-6-sulfonic acid (MANS), 2-p-toluidinyl Allylnaphthalenesulfonic acids such as naphthalene-6-sulfonic acid (TNS); dimethylaminonaphthalenesulfonic acid; benzofurazan derivatives such as nitrobenzofurazan (NBD); )phenyl]-2- oxazolyl); Dapoxyl derivatives such as Dapoxyl sulfonyl chloride, Dapoxyl succinimidyl ester, Dapoxyl 3-sulfonamidopropionic acid, Dapoxyl (2-bromoacetamidoethyl) sulfonamide, Dapoxyl (2-aminoethyl) sulfonamide; dansyl chloride, dansyl sulfonamide, Dansyl pigments such as dansylaminoethyl-3-phosphate, 1-dansylsulfonamide-3-N,N-dimethylaminopropane, dansylcholine, dansylgalactoside, dansyllysine and dansylphosphatidylethanolamine; Benzophenoxazine derivatives and the like can be exemplified.

In line with this principle, it is possible to produce a peptidic fluorescent dye that binds highly selectively to the lipid membrane of RNA vaccines and exhibits fluorescence response.

Preferred embodiments of peptidic probes that can selectively bind to the lipid membrane structure of RNA vaccines include amphipathic α-helix peptide probes such as the compound of formula (9) below.

The peptidic probe of formula (9) above can be produced as follows.

First, after extending the peptide chain using an amino acid protected with an Fmoc group, it is produced by linking a fluorescent dye Nile Red derivative. Specifically, Fmoc-protected amino acids were added to Rink-Amide-ChemMatrix resin (Biotage) [1-(1-cyano-ethoxy-2-oxoethylideneamino-oxy)-dimethylamino-morpholinomethylene]methanaminiumhexafluorophosphate (COMU) and diisopropylethylamine( DIEA). Subsequently, J. Chem. Soc., Perkin Trans., 1997, 1, 1051-1058. -2-yl)oxy)hexanoic acid). Then, after cleaving from the resin and deprotection, the obtained crude product is purified by reverse-phase HPLC to obtain a peptidic fluorescent probe.

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 environment-responsive dyes are characterized by low fluorescence intensity in hydrophilic fields and high intensity in hydrophobic fields, so fluorescence can be detected without the need for washing or other operations. On the other hand, with fluorescent probes using non-environmentally responsive dyes, it is impossible to distinguish between non-binding and binding to the lipid membrane of preparations such as vaccines, so operations such as washing are required. Environmentally responsive dyes such as the compound of formula (9) are preferred, but the use of non-environmentally responsive dyes may be considered depending on the purpose and application.

As the cell membrane staining dye, a known dye capable of staining the cell membrane can be used, and a fat-soluble dye that stains the lipid bilayer membrane is particularly preferable. Such fat-soluble dyes include fat-soluble carbocyanine dyes such as PIC series from Takara Bio Inc. and SP-DiOC1 8 (3) in the Molecular Probes (registered trademark) series from Thermo Fisher Scientific. Polaric (registered trademark) manufactured by Kagaku Co., Ltd., and the like can be mentioned. Fat-soluble carbocyanine dyes are typically carbocyanine dyes with long hydrocarbon chains. In addition to these, cell membrane staining 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, may be used. Soluble dyes can be used as well.

The step of contacting the RNA vaccine with a compound that binds to RNA may be performed before, at the same time, or after the step of contacting the RNA vaccine with a fluorescent dye that binds to the lipid membrane of the RNA vaccine, but is performed at the same time. This is advantageous in that the inspection method time can be shortened.

When the step of contacting a compound that binds to RNA with an RNA vaccine and the step of contacting a fluorescent dye that binds to a lipid membrane with an RNA vaccine are performed simultaneously, a sample containing the RNA vaccine is prepared, and the compound that binds to mRNA; It is preferably carried out by adding a fluorescent dye that binds to lipid membranes to one sample containing the RNA vaccine. A sample containing an RNA vaccine may be a stock solution or a diluted solution of a commercially available or manufactured RNA vaccine, and may contain any additive, including substances that inhibit degradation of RNA.

Each of the step of contacting a compound that binds to RNA with an RNA vaccine and the step of contacting a fluorescent dye that binds to a lipid membrane with an RNA vaccine may be performed at any suitable temperature and time. Typically, the temperature is ambient or room temperature. Examples of such temperatures 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. Temperatures above about 42°C and below about 20°C are also applicable depending on the sample tested. The time is not particularly limited, and may be any time suitable for detecting changes in fluorescence. Examples of length of time are 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. minutes, about 360 minutes, about 420 minutes, about 480 minutes, about 540 minutes, about 600 minutes, and ranges between any two of these. Further extension of the time is also possible depending on the sample to be tested.

The concentrations of the compound of formula (1) and the compound of formula (2) are not particularly limited, and may be any concentration at which fluorescence excitation and emission signals can be appropriately detected in the presence of RNA. An example concentration range includes about 10 nM to 1 mM. Examples of concentrations include about 10 nM, about 100 nM, about 1 μM, about 10 μM, about 100 μM, about 1 mM and ranges between any two of these.

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. Before detection, unbound fluorescent probe may be removed by ultrafiltration or the like. Removal of unbound fluorescent probe allows detection with a higher S/N ratio.

Suitable fluorescent irradiation devices 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 micro-fluorescence spectrophotometers.

The step of detecting at least one of the compound bound to RNA and the fluorescent dye bound to the lipid membrane may be performed by visual inspection or by using various measuring instruments. Examples of instrumentation include CCD cameras, video cameras, photographic film, laser scanner devices, fluorescence intensity meters, photodiodes, quantum counters, epifluorescence microscopes, scanning microscopes, flow cytometers, fluorescence microplate readers, or photomultiplier tubes. and other amplifiers.

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

In the method of the first aspect of the present invention, after the step of detecting, the change in luminescence intensity derived from the compound bound to RNA, the change in luminescence intensity derived from the fluorescent dye bound to the lipid membrane, or both A step of evaluating the quality of the formulation in which the RNA is encapsulated in a lipid membrane may be further included.

In some embodiments, the step of assessing increases the luminescence intensity or fluorescence signal from the compound bound to the RNA, or decreases the luminescence intensity or fluorescence signal from the fluorochrome bound to the lipid membrane, Or, in the case of both, it includes evaluating that the quality of the preparation in which the RNA is encapsulated in the lipid membrane is degraded.

In some embodiments, the luminescence intensity or fluorescence signal derived from the compound bound to RNA and the luminescence intensity or fluorescence signal derived from the fluorochrome bound to the lipid membrane for a predetermined amount of a preparation with no quality problems Each is defined as a reference value, and for a formulation encapsulated in a certain lipid membrane (referred to as a test formulation), the luminescence intensity or fluorescence signal derived from the compound bound to the RNA in the formulation and the binding to the RNA in the formulation The luminescence intensity or fluorescence signal derived from the compound is measured, and the quality of the test preparation is maintained if the measured values are within the predetermined thresholds relative to the reference values, and the test preparation is rejected if the measured values are outside the thresholds. including assessing that the quality of Preparations with no quality problems include preparations immediately after manufacturing.

In some embodiments, the compound that binds RNA is impermeable to lipid membranes, and the step of assessing increases the luminescence intensity or fluorescence signal from the compound that binds RNA or binds to the lipid membrane. When the luminescence intensity or fluorescence signal derived from the fluorescent dye is decreased, or both, it is evaluated that the quality of the preparation in which the RNA is encapsulated in the lipid membrane is degraded.

In some embodiments, the compound that binds RNA is permeable to the lipid membrane, and the step of assessing increases the luminescence intensity or fluorescence signal from the compound bound to the RNA that has leaked out of the lipid membrane, A reduction in luminescence intensity and/or fluorescence signal from membrane-bound fluorochromes includes assessing a formulation in which RNA is encapsulated in a lipid membrane as degraded.

In some embodiments, the compound that binds to RNA is permeable to the lipid membrane, and the step of evaluating is not partially or entirely encapsulated within the lipid membrane, and the RNA that has leaked out of the lipid membrane. When the luminescence intensity or fluorescence signal derived from the bound compound increases and the luminescence intensity or fluorescence signal derived from the fluorescent dye bound to the lipid membrane is normal, part or all of the RNA is bound to the lipid membrane. This includes quality evaluation as a formulation that is not encapsulated and leaks out of the lipid membrane. The normal value refers to a value within the range of luminescence intensity or fluorescence signal obtained from a preparation with no quality problem, for example, derived from a fluorescent dye bound to a lipid membrane in a preparation in which RNA is encapsulated in a lipid membrane immediately after production. luminescence intensity or fluorescence signal value or lesser value.

In some embodiments, the compound that binds RNA is permeable to the lipid membrane, and the assessing is derived from the compound that binds RNA as the RNA is not completely encapsulated within the lipid membrane. When the luminescence intensity or fluorescence signal decreases and the luminescence intensity or fluorescence signal derived from the fluorescent dye bound to the lipid membrane is normal, the RNA is not inherently encapsulated in the lipid membrane (therefore, the RNA is not leaking out of the lipid membrane), including evaluating the quality as a formulation. A normal value includes, for example, a value of luminescence intensity or a fluorescence signal derived from a fluorescent dye bound to a lipid membrane in a formulation in which RNA is encapsulated in a lipid membrane immediately after production, or a value smaller than that.

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

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

In some embodiments, the compound that binds RNA is impermeable to the lipid membrane, and the step of evaluating is based on the luminescence intensity or fluorescence signal from the compound bound to the RNA that has leaked out of the lipid membrane. RNA is encapsulated in the lipid membrane if the luminescence intensity or fluorescence signal from the fluorochrome bound to the lipid membrane decreases relative to the second reference value, or both including assessing the quality of the manufactured product as degraded. The first reference value and the second reference value are as described.

In some embodiments, the compound that binds RNA is permeable to the lipid membrane, and the step of evaluating is derived from the compound bound to the RNA that has leaked out of the lipid membrane without the RNA being encapsulated in the lipid membrane. When the luminescence intensity or fluorescence signal increases relative to the first reference value, and the luminescence intensity or fluorescence signal derived from the fluorochrome bound to the lipid membrane does not change relative to the second reference value, This includes evaluating the quality of preparations in which the RNA is not encapsulated in the lipid membrane and is leaking out of the lipid membrane.

In some embodiments, the compound that binds RNA is permeable to the lipid membrane, and the step of assessing is the luminescence intensity from the compound that binds RNA, as the RNA is not encapsulated within the lipid membrane. Or the fluorescence signal decreases with respect to the first reference value, and when the emission intensity or fluorescence signal derived from the fluorescent dye bound to the lipid membrane does not change with respect to the second reference value, the RNA is This includes evaluating the quality of preparations that are not inherently encapsulated in lipid membranes (thus, RNA has not leaked out of lipid membranes).

In some embodiments, the step of evaluating is that the luminescence intensity or fluorescence signal derived from the compound bound to the RNA does not change significantly relative to the first reference value and the fluorescent dye derived from the lipid membrane bound Evaluating that the quality of the preparation in which the RNA is encapsulated in the lipid membrane is maintained when the emitted light intensity or fluorescence signal does not significantly change with respect to the second reference value. The first reference value and the second reference value are as described. For example, both the luminescence intensity or fluorescence signal (i) derived from the compound bound to the RNA and the luminescence intensity or fluorescence signal (ii) derived from the fluorochrome bound to the lipid membrane are equal to the first reference value and For each of the second reference values, (luminescence intensity or fluorescence signal (i) - first reference value) / first reference value * 100 is ± 10% or less, and (luminescence intensity or fluorescence signal (ii) - second reference value)/second reference value*100 is ±10% or less, it is evaluated that the quality of the formulation in which RNA is encapsulated in the lipid membrane is maintained. Alternatively, for example, both the luminescence intensity or fluorescence signal (i) and the luminescence intensity or fluorescence signal (ii) are compared with each of the first reference value and the second reference value, and statistically determined by statistical analysis. When there is no significant difference (p is 0.05 or more), it is evaluated that the quality of the preparation in which RNA is encapsulated in the lipid membrane is maintained.

In some embodiments, the evaluating step includes evaluating in chronological order. Specifically, the step of evaluating comprises a luminescence intensity or fluorescence signal derived from a compound bound to RNA at a first time point and a compound bound to RNA at a second time point later than the first time point. When the luminescence intensity or fluorescence signal at the second time point is increased relative to the luminescence intensity or fluorescence signal at the first time point, the RNA is present in the lipid membrane Evaluate that the quality of the formulation encapsulated in is degraded, or the luminescence intensity or fluorescence signal derived from the fluorochrome bound to the lipid membrane at the first time point and the second time point later than the first time point The luminescence intensity or fluorescence signal derived from the fluorochrome bound to the lipid membrane at the time point is compared with the luminescence intensity or fluorescence signal derived from the fluorochrome bound to the lipid membrane at the second time point. If the luminescence intensity or fluorescence signal is reduced, the quality of the formulation in which the RNA is encapsulated in the lipid membrane is reduced, or if both are the case, the RNA is encapsulated in the lipid membrane. This includes assessing the quality of the drug product as degraded.

In some embodiments, the compound that binds RNA is impermeable to the lipid membrane, and the step of assessing is the luminescence intensity or fluorescence signal from the compound that binds RNA at a first time point; The luminescence intensity or fluorescence signal from the compound bound to the RNA at a second time point later than the first time point is compared, and the luminescence intensity or fluorescence signal at the second time point is equal to the luminescence intensity at the first time point. A formulation in which RNA is encapsulated in a lipid membrane is assessed as degraded if there is an increase in intensity or fluorescence signal, or from the fluorescent dye bound to the lipid membrane at the first time point. and the emission intensity or fluorescence signal derived from the fluorescent dye bound to the lipid membrane at a second time point later than the first time point, and binding to the lipid membrane at the second time point. If the luminescence intensity or fluorescence signal derived from the fluorochrome obtained is decreased relative to the luminescence intensity or fluorescence signal at the first time point, it is evaluated that the quality of the formulation in which RNA is encapsulated in the lipid membrane is degraded. or both, the quality of preparations in which RNA is encapsulated in lipid membranes is evaluated as being degraded.

In some embodiments, the compound that binds RNA is permeable to the lipid membrane, and the step of assessing is the luminescence intensity or fluorescence signal from the compound that binds RNA at a first time point; The emission intensity or fluorescence signal from the compound bound to the RNA at a second time point later than the time point of Or assess the quality of the formulation in which the RNA is encapsulated in the lipid membrane is degraded if it is increased relative to the fluorescence signal, or is derived from the fluorescent dye bound to the lipid membrane at the first time point The luminescence intensity or fluorescence signal is compared with the luminescence intensity or fluorescence signal derived from the fluorochrome bound to the lipid membrane at a second time point later than the first time point, and the luminescence intensity or fluorescence signal derived from the fluorochrome bound to the lipid membrane at the second time point. When the luminescence intensity or fluorescence signal derived from the fluorescent dye is reduced with respect to the luminescence intensity or fluorescence signal at the first time point, it is evaluated that the quality of the formulation in which RNA is encapsulated in the lipid membrane is degraded. or both, the quality of formulations in which RNA is encapsulated in lipid membranes is evaluated as being degraded.

In some embodiments, the compound that binds RNA is permeable to lipid membranes, and the step of assessing is the luminescence intensity or fluorescence signal from the compound that binds RNA in normal formulations and the Compare the luminescence intensity or fluorescence signal derived from the compound bound to the RNA of the preparation, and compare the luminescence intensity or fluorescence signal derived from the fluorochrome bound to the lipid membrane in the normal preparation with that of the preparation to be tested. Compared to the luminescence intensity or fluorescence signal derived from the fluorochrome bound to the lipid membrane, the luminescence intensity or fluorescence signal of the preparation under test is increased relative to the luminescence intensity or fluorescence signal of the normal preparation, and the lipid If the emission intensity or fluorescence signal from the membrane-bound fluorochrome does not change with respect to the emission intensity or fluorescence signal in the normal formulation, the formulation is of failed or degraded quality. (For example, some or all of the RNA is not encapsulated in the lipid membrane and is leaking out of the lipid membrane). For example, the luminescence intensity or fluorescence signal of the preparation to be tested increases with respect to the luminescence intensity or fluorescence signal of a normal preparation, and the luminescence intensity or fluorescence signal originates from the fluorescent dye bound to the lipid membrane of the preparation to be tested. (ii) relative to the corresponding luminescence intensity or fluorescence signal in the normal preparation (luminescence intensity or fluorescence signal derived from the fluorochrome bound to the lipid membrane under test (ii) - the lipid membrane of the normal preparation luminescence intensity or fluorescence signal derived from the bound fluorescent dye)/(luminescence intensity or fluorescence signal derived from the fluorochrome bound to the lipid membrane of the normal preparation) * 100 is ±10% or less. The membrane-encapsulated formulation is evaluated as poor or degraded. Alternatively, for example, the formulation luminescence intensity or fluorescence signal under test is increased with respect to the luminescence intensity or fluorescence signal in the normal formulation, and the luminescence intensity or fluorescence signal (ii) increases relative to the corresponding luminescence in the normal formulation If there is no statistically significant difference (p is 0.05 or more) for the intensity or fluorescence signal, the quality of the formulation in which RNA is encapsulated in a lipid membrane is considered to be poor or degraded. evaluate.

In some embodiments, the compound that binds RNA is permeable to lipid membranes, and the step of assessing is the luminescence intensity or fluorescence signal from the compound that binds RNA in normal formulations and the Compare the luminescence intensity or fluorescence signal derived from the compound bound to the RNA of the preparation, and compare the luminescence intensity or fluorescence signal derived from the fluorochrome bound to the lipid membrane in the normal preparation with that of the preparation to be tested. Compared to the luminescence intensity or fluorescence signal derived from the fluorochrome bound to the lipid membrane, the luminescence intensity or fluorescence signal of the preparation under test is reduced relative to the luminescence intensity or fluorescence signal of the normal preparation, and the lipid If the emission intensity or fluorescence signal from the membrane-bound fluorochrome does not change with respect to the emission intensity or fluorescence signal in the normal formulation, the formulation is of failed or degraded quality. (For example, RNA is not naturally encapsulated in lipid membranes). For example, the luminescence intensity or fluorescence signal of the preparation to be tested is reduced with respect to the luminescence intensity or fluorescence signal of the normal preparation, and the luminescence intensity or fluorescence signal is derived from the fluorescent dye bound to the lipid membrane of the preparation to be tested. (ii) relative to the corresponding luminescence intensity or fluorescence signal in the normal preparation (luminescence intensity or fluorescence signal derived from the fluorochrome bound to the lipid membrane under test (ii) - the lipid membrane of the normal preparation luminescence intensity or fluorescence signal derived from the bound fluorescent dye)/(luminescence intensity or fluorescence signal derived from the fluorochrome bound to the lipid membrane of the normal preparation) * 100 is ±10% or less. The membrane-encapsulated formulation is evaluated as poor or degraded. Alternatively, for example, the formulation luminescence intensity or fluorescence signal under test is increased with respect to the luminescence intensity or fluorescence signal in the normal formulation, and the luminescence intensity or fluorescence signal (ii) increases relative to the corresponding luminescence in the normal formulation If there is no statistically significant difference (p is 0.05 or more) for the intensity or fluorescence signal, the quality of the formulation in which RNA is encapsulated in a lipid membrane is considered to be poor or degraded. evaluate.

In some embodiments, the step of evaluating comprises luminescence intensity or fluorescence signal from the compound bound to the RNA at a first time point and binding to the RNA at a second time point later than the first time point. If the luminescence intensity or fluorescence signal at the second time point is not significantly changed with respect to the luminescence intensity or fluorescence signal at the first time point, And the luminescence intensity or fluorescence signal derived from the fluorochrome bound to the lipid membrane at the first time point, and the luminescence intensity or fluorescence signal derived from the fluorochrome bound to the lipid membrane at a second time point later than the first time point or If the luminescence intensity or fluorescence signal derived from the fluorochrome bound to the lipid membrane at the second time point is not significantly changed relative to the luminescence intensity or fluorescence signal at the first time point 2) includes evaluating whether the quality of formulations in which RNA is encapsulated in lipid membranes is maintained.

According to the 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, wherein the kit binds to the RNA of a formulation comprising RNA and a lipid membrane enveloping the RNA. A kit is provided that includes a compound 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 described for the compound and fluorescent dye of the first embodiment, respectively.

In some embodiments, the kit may include a container containing a compound that binds to RNA. Alternatively or additionally, a container containing a fluorescent dye that binds to the lipid membrane may be further provided.

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

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

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

In some embodiments, the kit may further comprise water, buffers, buffer salts, surfactants, surfactants, salts, polysaccharides or other materials commonly used in bioassays. .
In some embodiments, the kit may further comprise solvents such as aqueous, non-aqueous, or aqueous/non-aqueous solvent systems.

In the instruction manual of the kit, the step of evaluating the quality of the RNA-encapsulated lipid membrane-encapsulated formulation described in the method for inspecting the quality of the RNA-encapsulated lipid membrane-encapsulated formulation of the first aspect of the present invention is described. may also be listed.

In addition, information on the luminescence intensity or fluorescence signal when the quality of the preparation in which the RNA is encapsulated in the lipid membrane is maintained when using a measuring instrument may be described.

Using the kit for inspecting the quality of the formulation in which the RNA is encapsulated in a lipid membrane according to the second embodiment, the mRNA in the formulation comprising mRNA and a lipid membrane enclosing the mRNA is encapsulated in the lipid membrane. Contacting the binding compound with a formulation in which RNA is encapsulated in a lipid membrane, and contacting a fluorescent dye that binds to the lipid membrane of the formulation in which RNA is encapsulated in a lipid membrane with the formulation in which RNA is encapsulated in a lipid membrane. and the step of detecting at least one of the compound bound to the RNA and the fluorescent dye bound to the lipid membrane. The quality of preparations in which RNA is encapsulated in lipid membranes can be evaluated based on changes in luminescence intensity, changes in luminescence intensity derived from fluorescent dyes bound to lipid membranes, or both.

In a preferred embodiment of the second aspect, the formulation 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 part of the sequence of the viral RNA. In another preferred embodiment, the lipid membrane-encapsulated formulation is an RNAi drug in which siRNA is encapsulated in liposomes. siRNAs are short (about 20-25 bases) double-stranded RNAs that are complementary to any target mRNA in the cell.

The method of the first aspect and the kit of the second aspect enable rapid and highly sensitive quality evaluation of preparations in which RNA is encapsulated in lipid membranes, so that RNA is encapsulated in lipid membranes at medical and research sites. can be used for quality inspection.

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

Example 1 Quality inspection of model vaccine using lipid membrane-binding probe and RNA-binding probe (method)
・Production method of model vaccine Lipocasulator (FD-UPL (lipid composition DSPC:Cholesterol = 70:30 (molar ratio)), Xygieia bioscience) with 20μM RNA (5'-CCC GUC ACA CCA UGG CCC UUC GGGG CUG GGG UGA) AGU CGGG-3′ (SEQ ID NO: 1), 41-base length: an oligonucleic acid designed with reference to a part of E. coli ribosomal RNA) was added, mixed by inversion, and then incubated at room temperature for 15 minutes. While stirring this solution, a special solution for encapsulating oligonucleotides (Xygieia Bioscience) was added. After that, a model vaccine (mean diameter 154 nm, measured by Nanosight (Malvern Panalytical NTA5330)) was obtained by purifying with VIVASPIN6 (Sartorius Japan).

- Sample preparation method with and without Triton The above model vaccine was used as a sample. A model vaccine was mixed with a highly curved membrane-binding probe ApoC-NR (excitation wavelength 552 nm) or an RNA-binding probe TO-PRO-1 (excitation wavelength 508.5 nm), and fluorescence spectra in PBS buffer were measured. In addition, in order to examine the fluorescence response to the denatured model vaccine, a sample obtained by mixing the model vaccine with a final concentration of 0.1% Triton-X 100 (Nacalai Tesque) was also used. The concentration of the model vaccine was 3.6 × 10 ¹¹ cells/mL, and the concentration of each probe ([ApoC-NR], [TO-PRO-1]) was 2.0 µM. In addition, the experiment using TO-PRO-1 was measured after incubating at room temperature for 30 minutes after mixing the sample and the probe. All measurements were performed at room temperature (25°C).

(result)
As shown in Fig. 4(A), the fluorescence intensity at 622 nm of the sample in which the model vaccine was mixed with ApoC-NR (Graph of Model vaccine) shows that the fluorescence intensity at 622 nm of the sample containing only ApoC-NR without the model vaccine (Graph of Probe ) increased 426-fold. Also, the addition of Triton-X (surfactant) significantly reduced the fluorescence response of the sample containing the model vaccine (Graph of Model vaccine (+ Triton)), 622 nm compared to the sample containing only ApoC-NR (Probe). was a 28-fold increase in This decrease in fluorescence response is due to denaturation of the liposome structure.

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

Example 2 Concentration-dependent fluorescence detection of model vaccine (Method)
The model vaccine described in Example 1 was used. A model vaccine was mixed with ApoC-NR or TO-PRO-1, and fluorescence spectra in PBS buffer were measured. The concentrations of model vaccines were 1.8×10 ¹⁰ , 3.6×10 ¹⁰ , 7.1×10 ¹⁰ , 1.4×10 ¹¹ , and 3.6×10 ¹¹ cells/mL, and the concentrations of various probes were 2.0 μM. In the experiment using TO-PRO-1, measurement was performed after mixing the sample and probe and incubating at room temperature for 30 minutes. All measurements were performed at room temperature (25°C).

(result)
As shown in FIGS. 5 (A) and (B), in the sample in which ApoC-NR was mixed with the model vaccine, the fluorescence response increased in a concentration-dependent manner (analysis wavelength 622 nm, F ₀ : model vaccine Fluorescence intensity at 622 nm of a sample containing only ApoC-NR without ApoC-NR).
The limit of detection for the model vaccine calculated using the linear range (1.8-7.1 x ¹⁰¹⁰ cells/mL) was 3.4 x ¹⁰⁷ cells/mL.
As shown in FIGS. 5(C) and 5(D), in the sample in which TO-PRO-1 was mixed with the model vaccine, the fluorescence response increased depending on the concentration of the model vaccine (analysis wavelength 538 nm, F ₀ : model Fluorescence intensity at 538 nm of samples containing only TO-PRO-1 without vaccine).
The limit of detection for the model vaccine calculated using the linear range (1.8-14 x ¹⁰¹⁰ cells/mL) was 1.6 x ¹⁰⁹ cells/mL.

Example 3 Effect of RNA Addition on Vaccine Fluorescence Ratio (Method)
The model vaccine described in Example 1 was used. A model vaccine was mixed with ApoC-NR or TO-PRO-1, and fluorescence spectra in PBS buffer were measured. In addition, a model vaccine mixed with a final concentration of 4.0 μM RNA was also used to examine changes in the fluorescence response of various probes due to the addition of RNA. The concentration of the model vaccine was 3.6×10 ¹¹ cells/mL, and the concentration of each probe was 2.0 μM. In the experiment using TO-PRO-1, measurement was performed after mixing the sample and probe and incubating at room temperature for 30 minutes. All measurements were performed at room temperature (25°C). The ratio of the fluorescence response (F) for the sample containing the model vaccine and RNA to the fluorescence response for the sample containing the model vaccine but no RNA (F _{model/vaccine} ) was calculated. F _{model/vaccine} was calculated when ApoC-NR and TO-PRO-1 were used as probes (ApoC-NR: excitation wavelength 552 nm, analysis wavelength 622 nm, TO-PRO-1: excitation wavelength 508.5 nm, analysis wavelength 538 nm).

(result)
As shown in FIG. 6, in the sample mixed with ApoC-NR, F/F _{model/vaccine,} which is the fluorescence response change associated with the addition of RNA, was as low as about 1.1 times. In the sample mixed with TO-PRO-1, F/F _{model/vaccine} , which is the change in fluorescence response, increased 7.9 times with the addition of RNA. This suggests that only TO-PRO-1 responds to RNA leakage.

Claims

A method for inspecting the quality of a formulation in which RNA is encapsulated in a lipid membrane,
A step of contacting a compound that binds to the RNA of a formulation comprising RNA and a lipid membrane enveloping the RNA with the formulation;
A method comprising: contacting a fluorescent dye that binds to the lipid membrane of the formulation with the formulation; and detecting at least one of a compound bound to the RNA and the fluorescent dye bound to the lipid membrane.
After the detecting step, the quality of the formulation is evaluated based on the change in luminescence intensity derived from the compound bound to RNA, the change in luminescence intensity derived from the fluorochrome bound to the lipid membrane, or both. 2. The method of claim 1, further comprising the step of:
The method according to claim 1 or 2, wherein the compound that binds to RNA is a fluorescent molecule that is impermeable to lipid membranes and capable of binding to RNA.
The method according to claim 1 or 2, wherein the compound that binds to RNA is a fluorescent molecule that is permeable to lipid membranes and capable of binding to RNA.
In the step of evaluating, if the fluorescence signal derived from the compound that binds to the RNA increases, or the fluorescence signal derived from the fluorochrome decreases, or both, the quality of the preparation deteriorates. A method according to any one of claims 1 to 3, comprising evaluating that the
The method according to any one of claims 1 to 5, wherein the fluorescent dye that binds to lipid membranes is a peptidic fluorescent dye that binds to lipid packing defects.
The step of contacting the compound 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 include the compound that binds to the RNA and the fluorescence that binds to the lipid membrane. A method according to any one of claims 1 to 6, carried out by adding a dye to a sample containing said formulation.
A kit for testing the quality of a formulation in which RNA is encapsulated in a lipid membrane,
a compound that binds to the RNA in a formulation comprising RNA and a lipid membrane enveloping the RNA;
a fluorescent dye that binds to the lipid membrane of the formulation.