Method for determining at least one parameter of a sample composition comprising nucleic acids such as RNA and optionally particles

Abstract

The present disclosure relates generally to the field of analyzing nucleic acids, such as RNA, and in particular to determining at least one parameter of a sample composition comprising nucleic acids, especially RNA, and optionally particles.

Description

Technical Field

[0001] The present disclosure relates generally to the field of analyzing nucleic acids, such as RNA, and in particular to determining at least one parameter of a sample composition comprising nucleic acids, especially RNA, and optionally particles. Background Art

[0002] The use of recombinant nucleic acids (such as DNA or RNA) to deliver exogenous genetic information to target cells is well known. Advantages of using RNA include transient expression and non-transformation properties. RNA does not need to enter the cell nucleus for expression and cannot integrate into the host genome, thereby eliminating various risks such as tumorigenesis.

[0003] Recombinant nucleic acids can be administered naked to a subject in need thereof; however, recombinant nucleic acids are typically administered using pharmaceutical compositions. For example, RNA can be delivered via so-called nanoparticle formulations, which contain RNA and a nanoparticle-forming vehicle, such as a cationic lipid, a mixture of a cationic lipid and a helper lipid, or a cationic polymer.

[0004] The fate of such nanoparticle formulations is controlled by various key factors (e.g., the integrity and concentration of nucleic acids in the nanoparticles; the amount of free nucleic acids; the size, size distribution, quantitative size distribution, and morphology of the nanoparticles; etc.). For example, these factors are referred to as specific properties that should be analyzed and specified in the 2018 FDA "Liposome Drug Products Guidance." The current limitation of the clinical application of nanoparticle formulations may be the lack of homogeneous, pure, and well-characterized nanoparticle formulations. This is also because all existing technologies for determining these factors have some shortcomings.

[0005] For example, the technology (such as based on dye, gel electrophoresis, microchannel electrophoresis or capillary electrophoresis (CE)) that is currently used to determine the integrity and / or concentration of nucleic acids in nanoparticles is labor-intensive, expensive, uses sample preparation steps to cause illusions, can not provide enough information and / or can not analyze a large number of samples. In a kind of technology that currently uses dye (such as fluorescent dye), dye itself can cause difference, and this can affect the reliability of measurement result. In addition, most of the technology based on gel electrophoresis requires multiple washing steps, uses special running buffer that increases program length, and owing to the special precautions taken using toxic reagents. For example, agarose gel technology is subject to the influence of multiple parameters (such as, the quality of agarose, the casting of gel, dye / sensitivity (needing a larger amount of sample), exposure time, processing raw data and optical density determination software standardization evaluation (28S / 18S method)), making this technology unreliable. Compared with agarose gel electrophoresis, the technology based on microchannel, chip-based electrophoresis or capillary electrophoresis provides faster running time and improved data quality, but needs manual processing to start and load gel, label and sample to system. CE instruments lack the sensitivity, dynamic range, and separation quality required for adequate RNA quality / quantity analysis.

[0006] In addition, a key challenge in characterizing nanoparticle preparations is the quantitative determination of the size distribution of the particles contained in the preparation. This is especially true for particles with a diameter less than about 500nm (i.e., the range relevant to most pharmaceutical products). Another unmet need is to determine the size distribution of nanoparticle preparations, where the size distribution is wide or complex (particularly asymmetric). All prior arts that can be used to characterize nanoparticle preparations have certain shortcomings: for example, they do not provide direct quantitative information about size, or they only measure a small, optionally unrepresentative sample subset, or are very different in sensitivity (or other parameters, for example, relative to the refractive index gradient of the bulk phase) to particles of different sizes, which strongly affects the size distribution obtained.

[0007] For size measurement of particles in the lower submicron range (about 100 nm), several techniques exist, such as dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), electron microscopy (EM) and size exclusion chromatography (SEC)-UV.

[0008] DLS provides information on the diffusion constant of the nanoparticles, from which the hydrodynamic radius R is calculated using the Stokes-Einstein equation. h. However, DLS only provides average data, from which the particle size is numerically calculated using certain algorithms. In order to obtain quantitatively reliable numbers, the nanoparticle preparation should be unimodal and monodisperse, which is not the case for many products, including nanoparticle pharmaceutical preparations. The most widely used algorithm in DLS is the so-called cumulative analysis (DE Koppel, J. Chem. Phys. 57 (1972) 4814-4820), which, as a premise, only assumes a unimodal size distribution and only provides physically meaningful numbers when the polydispersity is below a certain threshold. Other algorithms for DLS (see, for example, Provencher, SW, Comput. Phys. Commun. 1982, 27, 229–242) provide size curves, which mainly depend on the fitting parameters, and several very different spectra can correspond to the same data set. This analysis is hindered by the fact that the light scattering intensity of large particles is much higher than that of smaller particles, which makes it difficult to determine the fraction of smaller particles in the presence of much larger particles.

[0009] NTA is a method for determining particle size from their diffusion constants by observing scattered light from a very small (diluted) subset of a sample with a microscope over time. In principle, NTA can provide a quantitative size distribution spectrum; however, only very diluted samples can be measured, and the particles must be present in a relatively small size range, that is, because the scattering intensity is much higher, very small particles cannot be determined against a much larger background. Therefore, NTA is not suitable as a conventional method for determining the quantitative size distribution of pharmaceutical preparations. In addition, compared with other techniques (e.g., DLS), the statistical standard deviation of NTA is higher. This is a direct result of the low particle amount analyzed by NTA by one to three orders of magnitude. In particular, trace particles (e.g., aggregates) with biological effects may be underestimated or even undetectable by NTA. NTA requires several time-consuming optimization steps (e.g., video capture settings, different sample dilutions, etc.) to determine the settings suitable for accurate measurement. Typically, samples for NTA measurements must be diluted 10-1000 times, which may cause problems, especially aggregation or decomposition of particles depending on concentration. Due to all these shortcomings, it is difficult to establish NTA as a quality control method.

[0010] EM provides quantitative information about the size, shape and morphology of individual particles, but the number of particles that can be analyzed is even lower than NTA. Therefore, EM has similar or the same disadvantages as NTA in that the measured particles may not be representative of the entire sample. Other major disadvantages of this technique are high cost, complex sample preparation and long turnaround time for analyzing samples. This is why EM is not commonly used as a GMP method. Another problem with EM is that the fixation of the sample can lead to artifacts (e.g., shrinkage, aggregation, etc.). If the sample is not fixed (e.g., in Cryo-EM), the sample may have low contrast and cannot be analyzed.

[0011] Other separation techniques such as SEC-UV are not suitable because the interaction with the column matrix can cause problems (for example, adsorption or elution delay). Due to the limited size range of SEC columns, nanoparticles can not fully disperse, or nanoparticles can not be separated from aggregates. In addition, in the sense that quality or particle number are directly related to particle size, SEC-UV does not provide quantitative size distribution. Other dispersion methods such as analytical ultracentrifugation (AUC) only allow indirect size measurement, for example, based on sedimentation coefficient, wherein several assumptions must be made to calculate size distribution. In addition, AUC is both expensive and time-consuming, and it is not a common method in conventional quality control. Therefore, AUC is also not suitable for determining quantitative size spectrum as conventional quality control method.

[0012] In view of the above, in order to ensure the reproducible quality of nanoparticle formulations, advanced analytical methods for in-depth particle characterization are needed. In particular, there is a need for an improved method for analyzing nanoparticle formulations containing nucleic acids, especially RNA, wherein the method preferably (i) provides information on the characteristics of the formulation (such as the quantitative size distribution of the particles contained in the formulation (especially for particles with a diameter of less than 500 nm); (ii) provides information on the composition characteristics of the particles (for example, the amount of nucleic acid, especially RNA, contained in the particles, especially as a function of the particle size, such as the ratio of the amount of nucleic acid, especially RNA, contained in the particles to the amount of particle-forming compound, especially lipids and / or polymers, such as cationic lipids to cationic polymers, especially as a function of the particle size); (iii) is compatible with GMP; (iv) does not rely on the use of dyes; (v) is semi-automatic; and / or (vi) can be used to analyze changes in one or more reactions when the composition is prepared and / or stored. The composition comprises nucleic acid (such as RNA) and optionally particles, and is preferably characterized by the following parameters: nucleic acid (such as RNA) integrity; total amount of nucleic acid (such as RNA); amount of free nucleic acid (such as RNA); amount of nucleic acid (such as RNA) associated with the particles; size of the particles containing nucleic acid (such as RNA) (e.g., based on the radius of gyration (R) of the particles containing nucleic acid (such as RNA); and the effect of stress on the composition (e.g., salt concentration; temperature; pH or buffer concentration; light / radiation; oxygen; shear force; pressure; freeze / thaw cycles; drying / reconstitution cycles; addition of excipients (e.g., stabilizers and / or chelators); type and / or source of particle-forming compounds (especially lipids and / or polymers); charge ratio; and / or ratio of nucleic acid (such as RNA) to particle-forming compounds (especially lipids and / or polymers). Preferably, the method provides data on one or more of the following parameters: nucleic acid (such as RNA) integrity; total amount of nucleic acid (such as RNA); amount of free nucleic acid (such as RNA); amount of nucleic acid (such as RNA) associated with the particles; size of the particles containing nucleic acid (such as RNA) (e.g., based on the radius of gyration (R) of the particles containing nucleic acid (such as RNA)). g ) and / or the hydrodynamic radius (R) of particles containing nucleic acids (such as RNA) h )); size distribution of particles containing nucleic acids (such as RNA) (e.g., based on R g or R h values); quantitative size distribution of particles containing nucleic acids (such as RNA) (e.g., based on R g or R h value); molecular weight of nucleic acid (such as RNA); and / or shape of the particle containing nucleic acid (such as RNA) (e.g., shape and / or form factor). Optionally, other parameters may include one or more of the following: amount of surface nucleic acid (such as amount of surface RNA), amount of encapsulated nucleic acid (such as amount of encapsulated RNA), amount of accessible nucleic acid (such as amount of accessible RNA), size of nucleic acid (especially RNA) (e.g., based on R g or R h values), size distribution of nucleic acids (especially RNA) (e.g., based on Rg or R h values), quantitative size distribution of nucleic acids (especially RNA) (e.g., based on R g or R h value), nucleic acid (especially RNA) encapsulation efficiency, the ratio of the amount of nucleic acid (such as RNA) bound to the particles to the total amount of particle-forming compounds (especially lipids and / or polymers) in the particles, the ratio of the amount of positively charged parts of the particle-forming compounds (especially lipids and / or polymers) in the particles to the amount of nucleic acid (such as RNA) bound to the particles, and the charge ratio (N / P ratio) of the amount of positively charged parts of the particle-forming compounds (especially lipids and / or polymers) in the particles to the amount of negatively charged parts of nucleic acid (such as RNA) bound to the particles.

[0013] The inventors have surprisingly found that the methods and uses described herein meet the above requirements. Summary of the Invention

[0014] In a first aspect, the present disclosure provides a method for determining one or more parameters of a sample composition, wherein the sample composition comprises nucleic acid (such as RNA) and optionally particles, the method comprising:

[0015] (a) subjecting at least a portion of the sample composition to field-flow fractionation, thereby fractionating components contained in the sample composition according to their size to produce one or more sample fractions;

[0016] (b) measuring at least one signal selected from a UV signal, a fluorescence signal, and a refractive index (RI) signal, and optionally measuring a light scattering (LS) signal, of at least one of the one or more sample fractions obtained from step (a); and

[0017] (c) calculating the one or more parameters from the at least one signal selected from the UV signal, the fluorescence signal and the RI signal, and optionally from the LS signal, wherein the one or more parameters include the integrity of the nucleic acid (such as RNA), the total amount of nucleic acid (such as RNA), the amount of free nucleic acid (such as RNA), the amount of nucleic acid (such as RNA) bound to the particle, the size of the particle containing the nucleic acid (such as RNA) (particularly, based on the radius of gyration (R) of the particle containing the nucleic acid (such as RNA) g ) and / or the hydrodynamic radius (R h )), size distribution of particles containing nucleic acids (such as RNA) (e.g., based on the R g or R h values) and quantitative size distribution of particles containing nucleic acids (such as RNA) (e.g., based on the R values ​​of particles containing nucleic acids (especially RNA) g or Rh In general, the size distribution and / or quantitative size distribution of particles containing nucleic acids (such as RNA) can be given as the number of particles containing nucleic acids (such as RNA), the molar amount of particles containing nucleic acids (such as RNA) or the mass of particles containing nucleic acids (such as RNA), each as a function of their size. Additional optional parameters include the molecular weight of the nucleic acids (especially RNA), the amount of surface nucleic acids (such as the amount of surface RNA), the amount of encapsulated nucleic acids (such as the amount of encapsulated RNA), the amount of accessible nucleic acids (such as the amount of accessible RNA), the size of the nucleic acids (especially RNA) (in particular, based on the R g and / or R h values), size distribution of nucleic acids (especially RNA) (e.g., based on the R g or R h values), quantitative size distribution of nucleic acids (especially RNA) (e.g., based on the R g or R h value), shape factor, form factor and nucleic acid (especially RNA) encapsulation efficiency. In general, the size distribution and / or quantitative size distribution of nucleic acids (especially RNA) can be given as the number of nucleic acid (especially RNA) molecules, the molar amount of nucleic acids (especially RNA) or the mass of nucleic acids (especially RNA), each as a function of their size. Other additional optional parameters include the ratio of the amount of nucleic acid (such as RNA) associated with the particle to the total amount of particle-forming compounds (especially lipids and / or polymers) in the particle, wherein the ratio can be given as a function of the particle size; the ratio of the amount of positively charged parts of the particle-forming compounds (especially lipids and / or polymers) in the particle to the amount of nucleic acid (such as RNA) associated with the particle, wherein the ratio can be given as a function of the particle size; and the charge ratio of the amount of positively charged parts of the particle-forming compounds (especially lipids and / or polymers) in the particle to the amount of negatively charged parts of nucleic acids (such as RNA) associated with the particle, wherein the charge ratio is usually expressed as N / P ratio and can be given as a function of the particle size.

[0018] In a first subgroup of the first aspect, the method comprises:

[0019] (a) subjecting at least a portion of the sample composition to field-flow fractionation, thereby fractionating components contained in the sample composition according to their size to produce one or more sample fractions;

[0020] (b) measuring at least a UV signal and optionally a light scattering (LS) signal of at least one of the one or more sample fractions obtained from step (a); and

[0021] (c) calculating the one or more parameters from the UV signal and optionally from the LS signal.

[0022] In a second and preferred subgroup of the first aspect, the method is for determining one or more parameters of a sample composition, wherein the sample composition comprises RNA and optionally particles, the method comprising:

[0023] (a) subjecting at least a portion of the sample composition to field-flow fractionation, thereby fractionating components contained in the sample composition according to their size to produce one or more sample fractions;

[0024] (b) measuring at least one signal selected from a UV signal, a fluorescence signal, and a refractive index (RI) signal, and optionally measuring a light scattering (LS) signal, of at least one of the one or more sample fractions obtained from step (a); and

[0025] (c) calculating the one or more parameters from the at least one signal selected from the group consisting of the UV signal, the fluorescence signal and the RI signal, and optionally from the LS signal.

[0026] In a third and more preferred subgroup of the first aspect, the method is for determining one or more parameters of a sample composition, wherein the sample composition comprises RNA and optionally particles, the method comprising:

[0027] (a) subjecting at least a portion of the sample composition to field-flow fractionation, thereby fractionating components contained in the sample composition according to their size to produce one or more sample fractions;

[0028] (b) measuring at least a UV signal and optionally a light scattering (LS) signal of at least one of the one or more sample fractions obtained from step (a); and

[0029] (c) calculating the one or more parameters from the UV signal and optionally from the LS signal.

[0030] In one embodiment of the first aspect (particularly, in one embodiment of the first, second or third subgroup of the first aspect), the R based on the particle containing nucleic acid (such as RNA) g In another embodiment of the first aspect (particularly, in another embodiment of the first, second or third subgroup of the first aspect), the size, size distribution and / or quantitative size distribution of the nucleic acid (such as RNA) containing particle is calculated based on the R value of the nucleic acid (such as RNA) containing particle. hIn another embodiment of the first aspect (particularly, in another embodiment of the first, second or third subgroup of the first aspect), the size, size distribution and / or quantitative size distribution of the nucleic acid (such as RNA) containing particle is calculated based on the R value of the nucleic acid (such as RNA) containing particle. g values ​​and respectively based on the R of particles containing nucleic acids (such as RNA) h The size, size distribution and / or quantitative size distribution of the nucleic acid (e.g., RNA) containing particles is calculated using the R value (i.e., this embodiment generates two data sets of size, size distribution and / or quantitative size distribution of the nucleic acid (e.g., RNA) containing particles, one based on R g value, a value based on R h value).

[0031] In an embodiment of the first aspect (particularly, in an embodiment of the first, second or third subgroup of the first aspect), wherein the one or more parameters include the size, size distribution and / or quantitative size distribution of the nucleic acid (such as RNA), the R g In another embodiment of the first aspect (particularly, in another embodiment of the first, second or third subgroup of the first aspect), wherein the one or more parameters include the size, size distribution and / or quantitative size distribution of the nucleic acid (such as RNA), the R value of the nucleic acid (such as RNA) is calculated. h In another embodiment of the first aspect (particularly, in another embodiment of the first, second or third subgroup of the first aspect), wherein the one or more parameters include the size, size distribution and / or quantitative size distribution of the nucleic acid (such as RNA), the R value of the nucleic acid (such as RNA) is calculated. g values ​​and respectively based on the R h The size, size distribution and / or quantitative size distribution of the nucleic acid (e.g., RNA) is calculated using the R value (i.e., this embodiment generates two data sets of size, size distribution and / or quantitative size distribution of nucleic acids (e.g., RNA), one based on R g value, a value based on R h value).

[0032] In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), the field-flow fractionation is preferably a flow field-flow fractionation, such as an asymmetric flow field-flow fractionation (AF4) or a hollow fiber flow field-flow fractionation (HF5).

[0033] In one embodiment of the first aspect (particularly, in one embodiment of the first, second or third subgroup of the first aspect), step (a) is performed using a membrane having a molecular weight cut-off (MW) suitable for preventing nucleic acids (especially RNA) from penetrating the membrane, preferably a membrane with a MW cut-off in the range of 2kDa-30kDa, such as a membrane with a MW cut-off of 10kDa.

[0034] In one embodiment of the first aspect (particularly, in one embodiment of the first, second or third subgroup of the first aspect), step (a) is performed using a polyethersulfone (PES) or regenerated cellulose membrane.

[0035] In one embodiment of the first aspect (particularly, in one embodiment of the first, second or third subgroup of the first aspect), step (a) is performed using: (I) a cross flow rate of up to 8 mL / min, preferably up to 4 mL / min, more preferably up to 2 mL / min, e.g., a cross flow profile; and / or (II) an injection flow rate in the range of 0.05-0.35 mL / min, preferably in the range of 0.10-0.30 mL / min, more preferably in the range of 0.15-0.25 mL / min; and / or (III) a detector flow rate in the range of 0.30-0.70 mL / min, preferably in the range of 0.40-0.60 mL / min, more preferably in the range of 0.45-0.55 mL / min.

[0036] In one embodiment of the first aspect (particularly, in one embodiment of the first, second or third subgroup of the first aspect), the crossflow velocity profile preferably comprises a fractionation stage that allows the components contained in the control or sample composition to be fractionated / separated by size to produce one or more sample fractions. Preferably, the crossflow velocity varies during this fractionation stage (e.g., starting from a value (e.g., about 1 to about 4 mL / min) and then decreasing to a lower value (e.g., about 0 to about 0.1 mL / min), or starting from a value (e.g., about 0 to about 0.1 mL / min) and then increasing to a higher value (e.g., about 1 to about 4 mL / min)), which can be varied in any manner, for example, continuously (e.g., linearly or exponentially) or stepwise. Preferably, the crossflow velocity profile comprises a fractionation stage in which the crossflow velocity varies continuously (preferably exponentially), starting from a value (e.g., about 1 to about 4 mL / min) and then decreasing to a lower value (e.g., about 0 to about 0.1 mL / min). The fractionation stage can have any length suitable for fractionating / separating the components contained in the sample composition by their size, for example, from about 5 min to about 60 min, such as from about 10 min to about 50 min, from about 15 min to about 45 min, from about 20 min to about 40 min, or from about 25 min to about 35 min, or about 30 min. The cross-flow profile can contain additional stages (e.g., 1, 2, 3 or 4 stages) that can be before and / or after the fractionation stage (e.g., 1 before the fractionation stage and 1, 2 or 3 after the fractionation stage), and can be used to separate the non-nucleic acid (especially non-RNA) components (e.g., proteins, polypeptides, mononucleotides, etc.) contained in the sample composition from the nucleic acid (especially RNA) contained in the sample composition, to concentrate the nucleic acid (especially RNA) contained in the sample composition and / or to regenerate the field-flow fractionation device (e.g., to remove all components bound to the membrane of the device). Preferably, the cross-flow rate of the additional stages is constant for each additional stage, and the length of each of the additional stages is independently in the range of about 5 min to about 60 min (such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min or about 25 min to about 35 min, or about 30 min) for each of the additional stages.For example, the cross flow rate profile can contain (i) a first additional stage before the fractionation stage, wherein the cross flow rate of the first additional stage is constant and is the same as the cross low rate at the beginning of the fractionation stage (the length of the first additional stage can be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 10 min or about 20 min or about 30 min); (ii) a second additional stage after the fractionation stage, wherein the cross flow rate of the second additional stage is constant and is the same as the cross low rate at the end of the fractionation stage (the length of the second additional stage can be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 10 min or about 20 min or about 30 min); min, about 20 min to about 40 min or about 25 min to about 35 min, or about 10 min or about 20 min or about 30 min); and optionally (iii) a third additional stage after the second additional stage, wherein the cross-flow rate of the third additional stage is constant and different from that of the second additional stage (the length of the third additional stage may be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min or about 25 min to about 35 min, or about 10 min or about 20 min or about 30 min).In embodiments wherein the cross-flow velocity profile comprises a graded stage, wherein the cross-flow velocity varies continuously (preferably exponentially) starting at one value (e.g., from about 1 to about 4 mL / min) and then decreasing to a lower value (e.g., from about 0 to about 0.1 mL / min), preferably the cross-flow velocity profile further comprises (i) a first additional stage before the graded stage, wherein the cross-flow velocity of the first additional stage is constant and is the same as the cross-flow velocity at the beginning of the graded stage (e.g., from about 1 to about 4 mL / min) (the length of the first additional stage may be in the range of from about 5 min to about 30 min, such as from about 6 min to about 25 min, from about 7 min to about 20 min, or from about 8 min to about 15 min, or from about 10 min to about 12 min, or about 5 min, or about 10 min, or about 12 min); (ii) a second additional stage after the graded stage, wherein the cross-flow velocity of the second additional stage is constant and is the same as the cross-flow velocity at the beginning of the graded stage (e.g., from about 1 to about 4 mL / min); and (iii) a third additional stage after the second additional stage, wherein the crossover flow rate of the third additional stage is constant and lower than that of the second additional stage (for example, the crossover flow rate of the third additional stage is 0) (the length of the third additional stage may be in the range of about 5 min to about 30 min, such as about 6 min to about 25 min, about 7 min to about 20 min, or about 8 min to about 15 min, or about 10 min to about 12 min, or about 5 min, about 10 min, or about 12 min). Preferred examples of such cross-flow profiles are as follows: 1.0-2.0 mL / min for 10 min, exponential gradient from 1.0-2.0 mL / min to 0.01-0.07 mL / min in 30 min; 0.01-0.07 mL / min for 30 min; and 0 mL / min for 10 min.

[0037] In one embodiment of the first aspect (particularly in one embodiment of the first, second or third subgroup of the first aspect), the integrity of a control nucleic acid (particularly RNA) is used to calculate the integrity of the nucleic acid (particularly RNA) contained in the sample composition.

[0038] In a first specific example of this embodiment of the first aspect, the integrity of the control nucleic acid (especially RNA) is determined by the following steps:

[0039] (a') subjecting at least part of a control composition containing a control nucleic acid, in particular RNA, to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0040] (b') measuring at least one signal selected from a UV signal, a fluorescence signal, and a refractive index (RI) signal of at least one of the one or more control fractions obtained from step (a');

[0041] (c'1) calculating the area from the maximum height of a UV, fluorescence or RI peak to the end of the UV, fluorescence or RI peak from at least one signal selected from the UV signal, fluorescence signal and RI signal obtained in step (b'), thereby obtaining A 50％ (control);

[0042] (c'2) calculating the total area of ​​one peak used in step (c'1) from at least one signal selected from the UV signal, the fluorescence signal, and the RI signal obtained in step (b'), thereby obtaining A 100％ (control); and

[0043] (c'3) Determine A 50％ (control) and A 100％ (control), thereby obtaining the integrity of the control nucleic acid (especially RNA) (I(control)).

[0044] In this first example, the integrity of the nucleic acids (especially RNA) contained in the sample composition can be calculated by the following steps:

[0045] (c1) calculating the area from the maximum height of the sample UV, fluorescence or RI peak corresponding to the control UV, fluorescence or RI peak used in step (c'1) to the end of the sample UV, fluorescence or RI peak from at least one signal selected from the UV signal, fluorescence signal and RI signal obtained in step (b), thereby obtaining A 50％ (sample);

[0046] (c2) calculating the total area of ​​the sample UV, fluorescence or RI peak used in step (c1) from the sample UV, fluorescence or RI signal obtained in step (b), thereby obtaining A 100％ (sample);

[0047] (c3) Determine A 50％ (sample) and A 100％ (samples), thereby obtaining I(sample); and

[0048] (c4) determining the ratio between I(sample) and I(control) to obtain the integrity of the nucleic acids (especially RNA) contained in the sample composition.

[0049] In a second specific example of this embodiment of the first aspect, the integrity of the control nucleic acid (especially RNA) is determined by the following steps:

[0050] (a") subjecting at least part of a control composition containing a control nucleic acid, in particular RNA, to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0051] (b") measuring at least one signal selected from a UV signal, a fluorescence signal, and an RI signal of at least one of the one or more control fractions obtained from step (a"); and

[0052] (c") determining the height of a UV, fluorescence or RI peak (H(control)) from at least one signal selected from the UV signal, fluorescence signal and RI signal obtained in step (b"), thereby obtaining the integrity of the control nucleic acid (especially RNA).

[0053] In this second example, the integrity of the nucleic acids (especially RNA) contained in the sample composition can be calculated by the following steps:

[0054] (c1′) determining the height (H(sample)) of the sample UV, fluorescence or RI peak corresponding to the control UV, fluorescence or RI peak used in step (c″) from at least one signal selected from the UV signal, fluorescence signal and RI signal obtained in step (b); and

[0055] (c2') determining the ratio between H(sample) and H(control) to obtain the integrity of the nucleic acids (especially RNA) contained in the sample composition.

[0056] In a third specific example of this embodiment of the first aspect (relating to the first subgroup of the first aspect), the integrity of the control nucleic acid (especially RNA) is determined by the following steps:

[0057] (a') subjecting at least part of a control composition containing a control nucleic acid, in particular RNA, to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0058] (b') measuring the UV signal of at least one of the one or more control fractions obtained from step (a');

[0059] (c'1) Calculating the area from the maximum height of a UV peak to the end of the UV peak from the UV signal obtained in step (b') to obtain A 50％ (control);

[0060] (c'2) Calculating the total area of ​​one peak used in step (c'1) from the UV signal obtained in step (b') to obtain A 100％ (control); and

[0061] (c'3) Determine A 50％ (control) and A 100％ (control), thereby obtaining the integrity of the control nucleic acid (especially RNA) (I(control)).

[0062] In this third example, the integrity of the nucleic acid (especially RNA) contained in the sample composition can be calculated by the following steps:

[0063] (c1) calculating the area from the maximum height of the sample UV peak corresponding to the control UV peak used in step (c'1) to the end of the sample UV peak from the UV signal obtained in step (b), thereby obtaining A 50％ (sample);

[0064] (c2) calculating the total area of ​​the sample UV peak used in step (c1) from the sample UV signal obtained in step (b) to obtain A 100％ (sample);

[0065] (c3) Determine A 50％ (sample) and A 100％ (samples), thereby obtaining I(sample); and

[0066] (c4) determining the ratio between I(sample) and I(control) to obtain the integrity of the nucleic acids (especially RNA) contained in the sample composition.

[0067] In a fourth specific example of this embodiment of the first aspect (relating to the first subgroup of the first aspect), the integrity of the control nucleic acid (especially RNA) is determined by the following steps:

[0068] (a") subjecting at least part of a control composition containing a control nucleic acid, in particular RNA, to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0069] (b") measuring the UV signal of at least one of the one or more control fractions obtained from step (a"); and

[0070] (c") determining the height of a UV peak (H(control)) from the UV signal obtained in step (b"), thereby obtaining the integrity of the control nucleic acid (especially RNA).

[0071] In this fourth example, the integrity of the nucleic acid (especially RNA) contained in the sample composition can be calculated by the following steps:

[0072] (c1′) determining the height of the sample UV peak (H(sample)) corresponding to the control UV peak used in step (c”) from the UV signal obtained in step (b); and

[0073] (c2') determining the ratio between H(sample) and H(control) to obtain the integrity of the nucleic acids (especially RNA) contained in the sample composition.

[0074] In a fifth specific example of this embodiment of the first aspect (relating to the second subgroup of the first aspect), the integrity of the control RNA is determined by the following steps:

[0075] (a') subjecting at least part of a control composition comprising a control RNA to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0076] (b') measuring at least one signal selected from a UV signal, a fluorescence signal, and an RI signal of at least one of the one or more control fractions obtained in step (a');

[0077] (c'1) calculating the area from the maximum height of a UV, fluorescence or RI peak to the end of the UV, fluorescence or RI peak from at least one signal selected from the UV signal, fluorescence signal and RI signal obtained in step (b'), thereby obtaining A 50％ (control);

[0078] (c'2) calculating the total area of ​​one peak used in step (c'1) from at least one signal selected from the UV signal, the fluorescence signal, and the RI signal obtained in step (b'), thereby obtaining A 100％ (control); and

[0079] (c'3) Determine A 50％ (control) and A 100％ (control) to obtain the integrity of the control RNA (I(control)).

[0080] In this fifth example, the integrity of the RNA contained in the sample composition can be calculated by the following steps:

[0081] (c1) calculating the area from the maximum height of the sample UV, fluorescence or RI peak corresponding to the control UV, fluorescence or RI peak used in step (c'1) to the end of the sample UV, fluorescence or RI peak from at least one signal selected from the UV signal, fluorescence signal and RI signal obtained in step (b), thereby obtaining A 50％ (sample);

[0082] (c2) calculating the total area of ​​the sample UV, fluorescence or RI peak used in step (c1) from the sample UV, fluorescence or RI signal obtained in step (b), thereby obtaining A 100％ (sample);

[0083] (c3) Determine A 50％ (sample) and A 100％ (samples), thereby obtaining I(sample); and

[0084] (c4) determining the ratio between I(sample) and I(control) to obtain the integrity of the RNA contained in the sample composition.

[0085] In a sixth specific example of this embodiment of the first aspect (relating to the second subgroup of the first aspect), the integrity of the control RNA is determined by the following steps:

[0086] (a") subjecting at least part of a control composition comprising a control RNA to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0087] (b") measuring at least one signal selected from a UV signal, a fluorescence signal, and an RI signal of at least one of the one or more control fractions obtained from step (a"); and

[0088] (c") determining the height of a UV, fluorescence or RI peak (H(control)) from at least one signal selected from the UV signal, fluorescence signal and RI signal obtained in step (b"), thereby obtaining the integrity of the control RNA.

[0089] In this sixth example, the integrity of the RNA contained in the sample composition can be calculated by the following steps:

[0090] (c1′) determining the height (H(sample)) of the sample UV, fluorescence or RI peak corresponding to the control UV, fluorescence or RI peak used in step (c″) from at least one signal selected from the UV signal, fluorescence signal and RI signal obtained in step (b); and

[0091] (c2') Determine the ratio between H(sample) and H(control) to obtain the integrity of the RNA contained in the sample composition.

[0092] In a seventh specific example of this embodiment of the first aspect (relating to the third subgroup of the first aspect), the integrity of the control RNA is determined by the following steps:

[0093] (a') subjecting at least part of a control composition comprising a control RNA to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0094] (b') measuring the UV signal of at least one of the one or more control fractions obtained from step (a');

[0095] (c'1) Calculating the area from the maximum height of a UV peak to the end of the UV peak from the UV signal obtained in step (b') to obtain A 50％ (control);

[0096] (c'2) Calculating the total area of ​​one peak used in step (c'1) from the UV signal obtained in step (b') to obtain A 100％ (control); and

[0097] (c'3) Determine A 50％ (control) and A 100％ (control) to obtain the integrity of the control RNA (I(control)).

[0098] In this seventh example, the integrity of the RNA contained in the sample composition can be calculated by the following steps:

[0099] (c1) calculating the area from the maximum height of the sample UV peak corresponding to the control UV peak used in step (c'1) to the end of the sample UV peak from the UV signal obtained in step (b), thereby obtaining A 50％ (sample);

[0100] (c2) calculating the total area of ​​the sample UV peak used in step (c1) from the sample UV signal obtained in step (b) to obtain A 100％ (sample);

[0101] (c3) Determine A 50％ (sample) and A 100％ (samples), thereby obtaining I(sample); and

[0102] (c4) determining the ratio between I(sample) and I(control) to obtain the integrity of the RNA contained in the sample composition.

[0103] In an eighth specific example of this embodiment of the first aspect (relating to the third subgroup of the first aspect), the integrity of the control RNA is determined by the following steps:

[0104] (a") subjecting at least part of a control composition comprising a control RNA to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0105] (b") measuring the UV signal of at least one of the one or more control fractions obtained from step (a"); and

[0106] (c") Determine the height of a UV peak (H(control)) from the UV signal obtained in step (b") to obtain the integrity of the control RNA.

[0107] In this eighth example, the integrity of the RNA contained in the sample composition can be calculated by the following steps:

[0108] (c1′) determining the height of the sample UV peak (H(sample)) corresponding to the control UV peak used in step (c”) from the UV signal obtained in step (b); and

[0109] (c2') Determine the ratio between H(sample) and H(control) to obtain the integrity of the RNA contained in the sample composition.

[0110] In one embodiment of the first aspect (particularly in one embodiment of the second or third subgroup of the first aspect), the amount of nucleic acid (especially RNA) is determined by using (i) a nucleic acid extinction coefficient (especially an RNA extinction coefficient) or (ii) a nucleic acid calibration curve (especially an RNA calibration curve).

[0111] In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), the sample composition comprises nucleic acids (especially RNA) and particles, such as lipid complex particles and / or lipid nanoparticles and / or polyplex particles and / or lipid multiplex particles and / or virus-like particles, and the nucleic acids (especially RNA) are associated with the particles.

[0112] In one embodiment of the first aspect (particularly in one embodiment of the first, second or third subgroup of the first aspect), the amount of total nucleic acid (particularly the amount of total RNA) is determined by: (i) treating at least a portion of the sample composition with a releasing agent; (ii) performing steps (a) to (c) with at least the portion obtained from step (i); and (iii) determining the amount of nucleic acid (particularly RNA) as described herein (e.g., by using (i) a nucleic acid extinction coefficient (particularly an RNA extinction coefficient) or (ii) a nucleic acid calibration curve (particularly an RNA calibration curve). In this embodiment, in step (a) of the method of the first aspect, field-flow fractionation is preferably performed using a liquid phase containing a releasing agent.

[0113] In one embodiment of the first aspect (particularly, in one embodiment of the first, second or third subgroup of the first aspect), the release agent is (i) a surfactant, such as an anionic surfactant (e.g., sodium lauryl sulfate), a zwitterionic surfactant (e.g., n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), or (ii) a surfactant. 3-14)), a cationic surfactant, a nonionic surfactant or a mixture thereof; (ii) an alcohol, such as a fatty alcohol (e.g., ethanol), or a mixture of alcohols; or (iii) a combination of (i) and (ii).

[0114] In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), the amount of free nucleic acid, in particular RNA, is determined by: performing steps (a) to (c) without adding a releasing agent, in particular in the absence of any releasing agent; and determining the amount of nucleic acid, in particular RNA, as described herein (e.g., using (i) a nucleic acid extinction coefficient, in particular an RNA extinction coefficient, or (ii) a nucleic acid calibration curve, in particular an RNA calibration curve).

[0115] In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), the amount of nucleic acids (in particular RNA) associated with the particles is determined by subtracting the amount of free nucleic acids (in particular RNA) as determined herein (e.g., by performing steps (a) to (c) without adding a releasing agent, in particular in the absence of any releasing agent; and determining the amount of nucleic acids (in particular RNA) as described herein (e.g., using (i) a nucleic acid extinction coefficient (in particular an RNA extinction coefficient) or (ii) a nucleic acid calibration curve (in particular an RNA calibration curve))) from the amount of total nucleic acids (in particular RNA) as determined herein (e.g., by (i) treating at least a portion of the sample composition with a releasing agent; (ii) performing steps (a) to (c) with at least the portion obtained from step (i); and (iii) determining the amount of nucleic acids (in particular RNA) as described herein (e.g., using (i) a nucleic acid extinction coefficient (in particular an RNA extinction coefficient) or (ii) a nucleic acid calibration curve (in particular an RNA calibration curve))).

[0116] In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), step (b) further comprises measuring an LS signal of at least one of the one or more sample fractions obtained from step (a), such as a dynamic light scattering (DLS) signal and / or a static light scattering (SLS), for example, a multi-angle light scattering (MALS) signal.

[0117] In one embodiment of the first aspect (particularly, in one embodiment of the first, second or third subgroup of the first aspect), the radius of gyration (R g ) value and / or hydrodynamic radius (R h ) value to determine the size of particles containing nucleic acids (especially RNA). In one embodiment of the first aspect (particularly in one embodiment of the first, second or third subgroup of the first aspect), step (b) comprises measuring a dynamic light scattering (DLS) signal of at least one of the one or more sample fractions obtained from step (a), and step (c) comprises calculating R from the DLS signal. h In one embodiment of the first aspect (particularly, in one embodiment of the first, second or third subgroup of the first aspect), step (b) comprises measuring a static light scattering (SLS), such as a MALS signal, of at least one of the one or more sample fractions obtained in step (a), and step (c) comprises calculating R from said SLS signal. gIn one embodiment of the first aspect (particularly, in one embodiment of the first, second or third subgroup of the first aspect), step (b) comprises measuring a dynamic light scattering (DLS) signal and a static light scattering (SLS), such as a MALS signal, of at least one of the one or more sample fractions obtained in step (a), and step (c) comprises calculating R g and R h This latter embodiment generates two data sets of particle sizes containing nucleic acids (such as RNA), namely, one based on R g value, a value based on R h value.

[0118] In one embodiment of the first aspect (particularly, in one embodiment of the first, second or third subgroup of the first aspect), the R g or R h value (e.g., by calculating R from the SLS signal obtained in step (b) g value or calculate R by the DLS signal obtained from step (b) h In a first example of this embodiment (a first subgroup of the first aspect), the size distribution of particles containing nucleic acids (particularly RNA) is determined by plotting the UV signal obtained from step (b) against the R value determined as described herein. g or R h value (e.g., by calculating R from the SLS signal obtained in step (b) g value or calculate R by the DLS signal obtained from step (b) h In a second example of this embodiment (relating to the second subgroup of the first aspect), the size distribution of particles containing nucleic acids (especially RNA) is determined by plotting the at least one signal selected from the UV signal, the fluorescence signal and the RI signal obtained in step (b) against the R value determined as described herein. g or R h value (e.g., by calculating R from the SLS signal obtained in step (b) g value or calculate R by the DLS signal obtained from step (b) h In a third example of this embodiment (a third subgroup of the first aspect), the size distribution of the particles containing RNA is determined by plotting the UV signal obtained from step (b) against the R value determined as described herein. g or R h value (e.g., by calculating R from the SLS signal obtained in step (b) g value or calculate R by the DLS signal obtained from step (b) hIn each of the first, second and third examples above, the size distribution of the particles containing RNA can be determined by plotting the values ​​of R g Value, R h The size distribution of particles containing nucleic acids (especially RNA) is determined based on the values ​​of R and R, or both. g Value and R h The size distribution of particles containing nucleic acids (especially RNA) is determined based on the values ​​of R. g The size distribution of the values ​​and an R-based h The size distribution of values.

[0119] In one embodiment of the first aspect (particularly, in one embodiment of the first, second or third subgroup of the first aspect), the UV, fluorescence or RI signal is converted into a cumulative weight fraction and the cumulative weight fraction is converted to R g or R h The values ​​are plotted and displayed as R g or R h The quantitative size distribution of particles containing nucleic acids, particularly RNA, is calculated from a plot of the UV, fluorescence or RI signal as a function of the value of the UV, fluorescence or RI signal. In a first example of this embodiment (the first subgroup of the first aspect), the UV signal is converted into a cumulative weight fraction and the cumulative weight fraction is plotted against the R g or R h The values ​​are plotted and displayed as R g or R h The quantitative size distribution of particles containing nucleic acids, particularly RNA, is calculated from a plot of the UV signal as a function of the UV value of the UV signal. In a second example of this embodiment (relating to the second subgroup of the first aspect), the UV, fluorescence or RI signal is converted into a cumulative weight fraction and the cumulative weight fraction is plotted against the R value. g or R h The values ​​are plotted and displayed as R g or R h The quantitative size distribution of the RNA-containing particles is calculated from a plot of the UV, fluorescence or RI signal as a function of the value of the UV, fluorescence or RI signal. In a third example of this embodiment (the third subgroup with respect to the first aspect), the size distribution of the RNA-containing particles is calculated by converting the UV signal to a cumulative weight fraction and plotting the cumulative weight fraction against the R value. g or R h The values ​​are plotted and displayed as R g or R h The quantitative size distribution of the RNA-containing particles can be calculated from a plot of the UV signal as a function of the value of R. g Value, R h The quantitative size distribution of particles containing nucleic acids (especially RNA) is determined based on the values ​​of R and R, or both. gValue and R h The quantitative size distribution of particles containing nucleic acids (especially RNA) is determined based on the values ​​of R. g The quantitative size distribution of the values ​​and an R h Quantitative size distribution of values.

[0120] In one embodiment of the first aspect (particularly in one embodiment of the first, second or third subgroup of the first aspect), the quantitative size distribution comprises D10, D50 and / or D90 values ​​(e.g., based on R g or R h value). If in R g Value and R h The quantitative size distribution of particles containing nucleic acids (especially RNA) is determined based on the values ​​of R. g The D10, D50 and / or D90 values ​​and a set of R-based h The D10, D50 and / or D90 values ​​of the

[0121] In an embodiment of the first aspect (in particular, in an embodiment of the first, second or third subgroup of the first aspect), the one or more parameters comprise (or are) at least two, preferably at least three, parameters as specified herein (including additional optional parameters), in particular at least two, preferably at least three, parameters selected from the group consisting of: the amount of free nucleic acid (in particular RNA), the amount of nucleic acid (in particular RNA) bound to the particles, the size distribution of the particles containing nucleic acid (in particular RNA) (in particular, based on the radius of gyration (R g ) and / or the hydrodynamic radius (R h )) and quantitative size distribution of particles containing nucleic acids (especially RNA) (e.g., based on R g or R h value). If in R g Value and R h The size distribution of particles containing nucleic acids (especially RNA) is determined based on the values ​​of R. g value, a value based on R h However, according to the present invention, these two data sets of the size distribution of particles containing nucleic acids, in particular RNA, are considered as only one parameter (instead of two parameters). Furthermore, if the fractogram obtained by field-flow fractionation shows more than one particle peak, the determination of the size distribution of each particle peak is considered as only one parameter (instead of one parameter for each particle peak). The same applies to the determination of the size distribution of particles containing nucleic acids, in particular RNA, in the Rg Value and R h The quantitative size distribution of particles containing nucleic acids, in particular RNA, is determined based on the values.

[0122] In an embodiment of the first aspect (particularly in an embodiment of the first, second or third subgroup of the first aspect, particularly in a preferred embodiment of the third subgroup of the first aspect), the one or more parameters comprise a quantitative size distribution of the particles containing nucleic acids, especially RNA (e.g. based on the radius of gyration (R g ) and / or the hydrodynamic radius (R h )) and optionally at least one parameter, such as at least two parameters, of the remaining parameters specified herein (including additional optional parameters); preferably these remaining parameters are selected from the group consisting of: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to the particles, and the size distribution of the particles containing nucleic acid (especially RNA) (e.g. based on R g or R h In one embodiment of the first aspect (particularly in one embodiment of the first, second or third subgroup of the first aspect, and particularly in a preferred embodiment of the third subgroup of the first aspect), the one or more parameters comprise a quantitative size distribution of particles containing nucleic acids, especially RNA (e.g. based on R g or R h value) and at least one parameter, such as at least two parameters, selected from the group consisting of the amount of free nucleic acid, in particular RNA, the amount of nucleic acid, in particular RNA, bound to the particles, and the size distribution of the particles containing nucleic acid, in particular RNA (e.g. based on R g or R h In one embodiment of the first aspect (particularly in one embodiment of the first, second or third subgroup of the first aspect, and particularly in a preferred embodiment of the third subgroup of the first aspect), the one or more parameters comprise a quantitative size distribution of particles containing nucleic acids, especially RNA (e.g. based on R g or R h value), the amount of free nucleic acid (especially RNA) and the amount of nucleic acid (especially RNA) bound to particles. g Value and R h The quantitative size distribution of particles containing nucleic acids (especially RNA) is determined based on the values ​​of R. g value, a value based on R hHowever, according to the present invention, these two data sets for the quantitative size distribution of particles containing nucleic acids, in particular RNA, are considered to be only one parameter (instead of two parameters). Furthermore, if the fractionation profile obtained by field-flow fractionation shows more than one particle peak, the determination of the quantitative size distribution for each particle peak is considered to be only one parameter (instead of one parameter for each particle peak). The same applies to the determination of the quantitative size distribution for particles containing nucleic acids, in particular RNA, in the R g Value and R h The size distribution of particles containing nucleic acids, in particular RNA, is determined based on the values.

[0123] In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), the amount of nucleic acid (in particular RNA), in particular free nucleic acid (in particular RNA), is determined by measuring the UV signal, for example, at a wavelength in the range of 260 nm-280 nm, such as at a wavelength of 260 nm or 280 nm, and using the extinction coefficient of nucleic acid (in particular RNA) at the corresponding wavelength (e.g., 260 nm or 280 nm).

[0124] In one embodiment of the first aspect (particularly in one embodiment of the first, second or third subgroup of the first aspect, and particularly in a preferred embodiment of the third subgroup of the first aspect), the size distribution of the particles containing nucleic acid (particularly RNA) (e.g., based on R g or R h values) and / or quantitative size distribution of particles containing nucleic acids, in particular RNA (e.g., based on R g or R h In a preferred embodiment of the third subgroup of the first aspect, the (quantitative) size distribution of the RNA-containing particles (e.g., based on R g or R h value) is in the range of 10-1000 nm, such as in the range of 15-500 nm, 20-450 nm, 25-400 nm, 30-350 nm, 40-300 nm or 50-250 nm.

[0125] In one embodiment of the first aspect (particularly in one embodiment of the first, second or third subgroup of the first aspect), the nucleic acid (especially RNA) has a length of 10-15,000 nucleotides, such as 40-15,000 nucleotides, 100-12,000 nucleotides or 200-10,000 nucleotides.

[0126] In one embodiment of the first aspect (particularly in one embodiment of the first subgroup of the first aspect), the nucleic acid is RNA. In this embodiment and in the embodiments of the second or third subgroup of the first aspect, the RNA is preferably mRNA or in vitro transcribed RNA, particularly in vitro transcribed mRNA.

[0127] In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), measuring at least one signal selected from UV signals, fluorescence signals and RI signals, optionally LS signals, such as SLS, for example, MALS signals and / or DLS signals, is performed online, and / or step (c) is performed online.

[0128] In an embodiment of the first aspect (particularly in an embodiment of the first, second or third subgroup of the first aspect), the one or more parameters are determined in one cycle of steps (a) to (c).

[0129] In one embodiment of the first aspect (particularly, in one embodiment of the first, second or third subgroup of the first aspect), prior to subjecting at least a portion of the sample composition to field-flow fractionation, the at least a portion of the sample composition is diluted with a solvent or solvent mixture that prevents the formation of particle aggregates. In one embodiment, the solvent mixture is a mixture of water and an organic solvent such as formamide.

[0130] In an embodiment of the first aspect (particularly in an embodiment of the first, second or third subgroup of the first aspect), measuring the UV signal is performed by using circular dichroism (CD) spectroscopy.

[0131] In a second aspect, the present disclosure provides a method of analyzing the effect of varying one or more reaction conditions in providing a composition comprising a nucleic acid (such as RNA) and optionally a particle, the method comprising:

[0132] (A) providing a first composition comprising a nucleic acid (such as RNA) and optionally a particle;

[0133] (B) providing a second composition comprising nucleic acid (such as RNA) and optionally particles, wherein providing the second composition differs from providing the first composition only in one or more reaction conditions;

[0134] (C) subjecting a portion of the first composition to the method of the first aspect, thereby determining one or more parameters of the first composition;

[0135] (D) subjecting a corresponding portion of the second composition to the method used in step (C), thereby determining one or more parameters of the second composition; and

[0136] (E) comparing one or more parameters of the first composition obtained in step (C) with the corresponding one or more parameters of the second composition obtained in step (D).

[0137] In one embodiment of the second aspect, the one or more parameters include nucleic acid (e.g., RNA) integrity, the total amount of nucleic acid (e.g., RNA), the amount of free nucleic acid (e.g., RNA), the amount of nucleic acid (e.g., RNA) associated with particles, the size of the particles containing nucleic acid (e.g., RNA) (particularly, based on the radius of gyration (R) of the particles containing nucleic acid (e.g., RNA)), the amount of free nucleic acid (e.g., RNA ... g ) and / or the hydrodynamic radius (R) of particles containing nucleic acids (such as RNA) h )), size distribution of particles containing nucleic acids (such as RNA) (e.g., based on the R g or R h values) and quantitative size distribution of particles containing nucleic acids (e.g., RNA) (e.g., based on the R values ​​of particles containing nucleic acids (e.g., RNA)). g or R h In general, the size distribution and / or quantitative size distribution of particles containing nucleic acids (such as RNA) can be given as the number of particles containing nucleic acids (such as RNA), the molar amount of particles containing nucleic acids (such as RNA) or the mass of particles containing nucleic acids (such as RNA), each as a function of their size. Additional optional parameters include the molecular weight of the nucleic acids (especially RNA), the amount of surface nucleic acids (such as the amount of surface RNA), the amount of encapsulated nucleic acids (such as the amount of encapsulated RNA), the amount of accessible nucleic acids (such as the amount of accessible RNA), the size of the nucleic acids (especially RNA) (in particular, based on the R g and / or R h values), size distribution of nucleic acids (especially RNA) (e.g., based on the R g or R h values), quantitative size distribution of nucleic acids (especially RNA) (e.g., based on the R gor R h value), shape factor, form factor and nucleic acid (especially RNA) encapsulation efficiency. In general, the size distribution and / or quantitative size distribution of nucleic acids (especially RNA) can be given as the number of nucleic acid (especially RNA) molecules, the molar amount of nucleic acids (especially RNA) or the mass of nucleic acids (especially RNA), each as a function of their size. Other additional optional parameters include the ratio of the amount of nucleic acid (such as RNA) associated with the particle to the total amount of particle-forming compounds (especially lipids and / or polymers) in the particle, wherein the ratio can be given as a function of the particle size; the ratio of the amount of positively charged parts of the particle-forming compounds (especially lipids and / or polymers) in the particle to the amount of nucleic acid (such as RNA) associated with the particle, wherein the ratio can be given as a function of the particle size; and the charge ratio of the amount of positively charged parts of the particle-forming compounds (especially lipids and / or polymers) in the particle to the amount of negatively charged parts of nucleic acids (such as RNA) associated with the particle, wherein the charge ratio is usually expressed as N / P ratio and can be given as a function of the particle size.

[0138] In one embodiment of the second aspect, the one or more parameters comprise (or are) at least two, preferably at least three, parameters as specified herein (including additional optional parameters), in particular at least two, preferably at least three, parameters selected from the group consisting of: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to the particles, the size distribution of the particles containing nucleic acid (especially RNA) (e.g., based on the R g or R h values), quantitative size distribution of particles containing nucleic acids, especially RNA (e.g., based on the R values ​​of particles containing nucleic acids, such as RNA) g or R h In one embodiment of the second aspect, the one or more parameters include (or are) at least two, preferably at least three, parameters selected from the group consisting of: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to particles, the size distribution of particles containing nucleic acid (especially RNA) (e.g., based on the R g or R h values) and quantitative size distribution of particles containing nucleic acids, especially RNA (e.g., based on the R g or R h value).

[0139] In one embodiment of the second aspect, the method of the first aspect used in steps (C) and (D) is a method comprising:

[0140] (a) subjecting at least a portion of a composition (e.g., the first composition for step (C) or the second composition for step (D)) to field-flow fractionation, thereby fractionating components contained in the composition according to their size to produce one or more composition fractions;

[0141] (b) measuring at least one signal selected from a UV signal, a fluorescence signal and a refractive index (RI) signal, and optionally measuring a light scattering (LS) signal of at least one of the one or more composition fractions obtained from step (a); and

[0142] (c) calculating the one or more parameters from the at least one signal selected from the group consisting of the UV signal, the fluorescence signal and the RI signal, and optionally from the LS signal.

[0143] In a first subgroup of the second aspect, the method of the first aspect used in steps (C) and (D) is a method comprising:

[0144] (a) subjecting at least a portion of a composition (e.g., the first composition for step (C) or the second composition for step (D)) to field-flow fractionation, thereby fractionating components contained in the composition according to their size to produce one or more fractions;

[0145] (b) measuring at least a UV signal and optionally a light scattering (LS) signal of at least one of the one or more fractions obtained from step (a); and

[0146] (c) calculating the one or more parameters from the UV signal and optionally from the LS signal.

[0147] In a second and preferred subgroup of the second aspect, the method of the first aspect used in steps (C) and (D) is a method for determining one or more parameters of a sample composition (e.g., the first composition used in step (C) or the second composition used in step (D)), wherein the sample composition comprises RNA and optionally particles, the method comprising:

[0148] (a) subjecting at least a portion of the sample composition to field-flow fractionation, thereby fractionating components contained in the sample composition according to their size to produce one or more sample fractions;

[0149] (b) measuring at least one signal selected from a UV signal, a fluorescence signal, and a refractive index (RI) signal, and optionally measuring a light scattering (LS) signal, of at least one of the one or more sample fractions obtained from step (a); and

[0150] (c) calculating the one or more parameters from the at least one signal selected from the group consisting of the UV signal, the fluorescence signal and the RI signal, and optionally from the LS signal.

[0151] In a third and more preferred subgroup of the second aspect, the method of the first aspect used in steps (C) and (D) is a method for determining one or more parameters of a sample composition (e.g., the first composition used in step (C) or the second composition used in step (D)), wherein the sample composition comprises RNA and optionally particles, the method comprising:

[0152] (a) subjecting at least a portion of the sample composition to field-flow fractionation, thereby fractionating components contained in the sample composition according to their size to produce one or more sample fractions;

[0153] (b) measuring at least a UV signal and optionally a light scattering (LS) signal of at least one of the one or more sample fractions obtained from step (a); and

[0154] (c) calculating the one or more parameters from the UV signal and optionally from the LS signal.

[0155] In one embodiment of the second aspect (particularly in one embodiment of the first, second or third subgroup of the second aspect), the R based on the particle containing nucleic acid (such as RNA) g In another embodiment of the second aspect (particularly, in another embodiment of the first, second or third subgroup of the second aspect), the size, size distribution and / or quantitative size distribution of the particles containing nucleic acids (such as RNA) are calculated based on the R value of the particles containing nucleic acids (such as RNA). h In another embodiment of the second aspect (particularly, in another embodiment of the first, second or third subgroup of the second aspect), the size, size distribution and / or quantitative size distribution of the particles containing nucleic acids (such as RNA) are calculated based on the R value of the particles containing nucleic acids (such as RNA). g values ​​and respectively based on the R of particles containing nucleic acids (such as RNA) h The size, size distribution and / or quantitative size distribution of particles containing nucleic acids (such as RNA) are calculated using the R-values ​​(i.e., this embodiment generates two data sets of sizes, size distributions and / or quantitative size distributions of particles containing nucleic acids (such as RNA), one based on R g value, a value based on R h value).

[0156] In an embodiment of the second aspect (particularly, in an embodiment of the first, second or third subgroup of the second aspect), wherein the one or more parameters include the size, size distribution and / or quantitative size distribution of the nucleic acid (such as RNA), the R g In another embodiment of the second aspect (particularly, in another embodiment of the first, second or third subgroup of the second aspect), wherein the one or more parameters include the size, size distribution and / or quantitative size distribution of the nucleic acid (such as RNA), the R value of the nucleic acid (such as RNA) is calculated. h In another embodiment of the second aspect (particularly, in another embodiment of the first, second or third subgroup of the second aspect), wherein the one or more parameters include the size, size distribution and / or quantitative size distribution of the nucleic acid (such as RNA), the R value of the nucleic acid (such as RNA) is calculated. g values ​​and respectively based on the R h The size, size distribution and / or quantitative size distribution of nucleic acids (such as RNA) are calculated using the R-values ​​(i.e., this embodiment generates two data sets of sizes, size distributions and / or quantitative size distributions of nucleic acids (such as RNA), one based on R g value, a value based on R h value).

[0157] In one embodiment of the second aspect (particularly in one embodiment of the first, second or third subgroup of the second aspect), the one or more parameters include nucleic acid (particularly RNA) integrity, the total amount of nucleic acid (particularly RNA), the amount of free nucleic acid (particularly RNA), the amount of nucleic acid associated with particles (particularly RNA), the size of the particles containing nucleic acid (particularly RNA) (particularly based on the radius of gyration (R g ) and / or the hydrodynamic radius (R h )), size distribution of particles containing nucleic acids (especially RNA) (e.g., based on R g or R h values) and quantitative size distribution of particles containing nucleic acids (especially RNA) (e.g., based on R g or R h value), and optionally the molecular weight of a nucleic acid (especially RNA).

[0158] In an embodiment of the second aspect (particularly in an embodiment of the first, second or third subgroup of the second aspect), the one or more parameters include (or are) at least two, preferably at least three, parameters selected from the group consisting of: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to particles, the size distribution of particles containing nucleic acid (especially RNA) (e.g., based on the R g or R h values), quantitative size distribution of particles containing nucleic acids, especially RNA (e.g., based on R values ​​of particles containing nucleic acids, especially RNA) g or R h In one embodiment of the first, second or third subgroup of the second aspect, the one or more parameters include (or are) at least two, preferably at least three, parameters selected from the group consisting of: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to particles, the size distribution of particles containing nucleic acid (especially RNA) (e.g., based on the R g or R h values) and the quantitative size distribution of particles containing nucleic acids, in particular RNA (e.g., based on the R values ​​of particles containing nucleic acids, in particular RNA). g or R h value). If in R g Value and R h The size distribution of particles containing nucleic acids (especially RNA) is determined based on the values ​​of R. g value, a value based on R h However, according to the present invention, these two data sets of the size distribution of particles containing nucleic acids, in particular RNA, are considered as only one parameter (instead of two parameters). Furthermore, if the fractionation pattern obtained by field-flow fractionation shows more than one particle peak, the determination of the size distribution of each particle peak is considered as only one parameter (instead of one parameter for each particle peak). The same applies to the determination of the size distribution of particles containing nucleic acids, in particular RNA, in the R g Value and R h The quantitative size distribution of particles containing nucleic acids, in particular RNA, is determined based on the values.

[0159] In an embodiment of the second aspect (particularly in an embodiment of the first, second or third subgroup of the second aspect, particularly in a preferred embodiment of the third subgroup of the second aspect), the one or more parameters comprise a quantitative size distribution of the particles containing nucleic acids, especially RNA (e.g. based on the radius of gyration (R g) and / or the hydrodynamic radius (R h )) and optionally at least one parameter, such as at least two parameters, of the remaining parameters specified herein (including additional optional parameters); preferably these remaining parameters are selected from the group consisting of: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to the particles, and the size distribution of the particles containing nucleic acid (especially RNA) (e.g. based on R g or R h In one embodiment of the second aspect (particularly in one embodiment of the first, second or third subgroup of the second aspect, and particularly in preferred embodiments of the third subgroup of the second aspect), the one or more parameters comprise a quantitative size distribution of particles containing nucleic acid (particularly RNA) (e.g. based on R g or R h value) and at least one parameter, such as at least two parameters, selected from the group consisting of the amount of free nucleic acid, in particular RNA, the amount of nucleic acid, in particular RNA, bound to the particles, and the size distribution of the particles containing nucleic acid, in particular RNA (e.g. based on R g or R h In one embodiment of the second aspect (particularly in one embodiment of the first, second or third subgroup of the second aspect, and particularly in preferred embodiments of the third subgroup of the second aspect), the one or more parameters comprise a quantitative size distribution of particles containing nucleic acid (particularly RNA) (e.g. based on R g or R h value), the amount of free nucleic acid (especially RNA) and the amount of nucleic acid (especially RNA) bound to particles. g Value and R h The quantitative size distribution of particles containing nucleic acids (especially RNA) is determined based on the values ​​of R. g value, a value based on R h However, according to the present invention, these two data sets for the quantitative size distribution of particles containing nucleic acids, in particular RNA, are considered to be only one parameter (instead of two parameters). Furthermore, if the fractionation profile obtained by field-flow fractionation shows more than one particle peak, the determination of the quantitative size distribution for each particle peak is considered to be only one parameter (instead of one parameter for each particle peak). The same applies to the determination of the quantitative size distribution for particles containing nucleic acids, in particular RNA, in the R g Value and R h The size distribution of particles containing nucleic acids, in particular RNA, is determined based on the values.

[0160] In an embodiment of the second aspect (particularly in an embodiment of the first, second or third subgroup of the second aspect), the one or more parameters are determined in one cycle of steps (a) to (c).

[0161] In one embodiment of the second aspect (particularly, in one embodiment of the first, second or third subgroup of the second aspect), the one or more reaction conditions include any one of: salt concentration / ionic strength; temperature; pH or buffer concentration; light / radiation; oxygen; shear force; pressure; freeze / thaw cycles; drying / reconstitution cycles; addition of excipients (e.g., stabilizers and / or chelating agents); type and / or source of particle-forming compounds (particularly lipids and / or polymers); charge ratio; physical state; and the ratio of nucleic acid (particularly RNA) to particle-forming compounds (particularly lipids and / or polymers constituting the particles). Exemplary salt concentrations include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 mM salt, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 mM NaCl. Exemplary temperature conditions include low temperature (e.g., -20°C), ambient temperature or room temperature, moderate temperature (e.g., 30°C) or high temperature (e.g., 50°C). Exemplary conditions regarding the type and / or source of the particle-forming compound are cationic lipids versus cationic polymers, cationic lipids versus zwitterionic lipids, or PEGylated lipids versus non-PEGylated lipids. Exemplary charge ratios of positive to negative charges in the nucleic acid (especially RNA) particles are from about 6: 1 to about 1: 2, such as from about 5: 1 to about 1.2: 2, from about 4: 1 to about 1.4: 2, from about 3: 1 to about 1.6: 2, from about 2: 1 to about 1.8: 2, or from about 1.6: 1 to about 1: 1. Exemplary ratios of nucleic acid (especially RNA) to particle-forming compounds (especially lipids and / or polymers comprising the particle) include ratios of nucleic acid (especially RNA) to total lipids in the range of about 1: 100 to about 10: 1 (w / w).

[0162] In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect), the field-flow fractionation is preferably a flow field-flow fractionation, such as an asymmetric flow field-flow fractionation (AF4) or a hollow fiber flow field-flow fractionation (HF5).

[0163] In one embodiment of the second aspect (particularly, in one embodiment of the first, second or third subgroup of the second aspect), step (a) is performed using a membrane having a molecular weight cut-off (MW) suitable for preventing nucleic acids (especially RNA) from penetrating the membrane, preferably a membrane with a MW cut-off in the range of 2kDa-30kDa, such as a membrane with a MW cut-off of 10kDa.

[0164] In one embodiment of the second aspect (particularly, in one embodiment of the first, second or third subgroup of the second aspect), step (a) is performed using a polyethersulfone (PES) or regenerated cellulose membrane.

[0165] In one embodiment of the second aspect (particularly, in one embodiment of the first, second or third subgroup of the second aspect), step (a) is performed using: (I) a cross flow rate of up to 8 mL / min, preferably up to 4 mL / min, more preferably up to 2 mL / min, for example, a cross flow profile; and / or (II) an injection flow rate in the range of 0.05-0.35 mL / min, preferably in the range of 0.10-0.30 mL / min, more preferably in the range of 0.15-0.25 mL / min; and / or (III) a detector flow rate in the range of 0.30-0.70 mL / min, preferably in the range of 0.40-0.60 mL / min, more preferably in the range of 0.45-0.55 mL / min.

[0166] In one embodiment of the second aspect (particularly, in one embodiment of the first, second, or third subgroup of the second aspect), the crossflow profile preferably comprises a fractionation stage that allows the components contained in a composition (e.g., a control or sample composition, particularly the first composition used in step (C) or the second composition used in step (D)) to be fractionated / separated by size to produce one or more composition fractions. Preferably, the crossflow rate varies during this fractionation stage (e.g., starting from a value (e.g., about 1 to about 4 mL / min) and then decreasing to a lower value (e.g., about 0 to about 0.1 mL / min), or starting from a value (e.g., about 0 to about 0.1 mL / min) and then increasing to a higher value (e.g., about 1 to about 4 mL / min)), which can be varied in any manner, for example, continuously (e.g., linearly or exponentially) or stepwise. Preferably, the crossflow profile comprises a fractionation stage in which the crossflow rate varies continuously (preferably exponentially), starting from a value (e.g., about 1 to about 4 mL / min) and then decreasing to a lower value (e.g., about 0 to about 0.1 mL / min). The fractionation stage can have any length that is suitable for the component contained in the composition to be graded / separated by its size, for example, from about 5 min to about 60 min, such as from about 10 min to about 50 min, from about 15 min to about 45 min, from about 20 min to about 40 min, or from about 25 min to about 35 min, or about 30 min. The cross-flow profile can contain additional stages (for example, 1, 2, 3 or 4 stages), which can be before and / or after the fractionation stage (for example, 1 before the fractionation stage and 1, 2 or 3 after the fractionation stage), and can be used for separating the non-nucleic acid (especially non-RNA) components (for example, proteins, polypeptides, mononucleotides etc.) contained in the composition from the nucleic acid (especially RNA) contained in the composition, to concentrate the nucleic acid (especially RNA) contained in the composition and / or regenerate the field-flow fractionation device (for example, to remove all components bound to the membrane of the device). Preferably, the cross-flow rate of the additional stages is constant for each additional stage, and the length of each of the additional stages is independently in the range of about 5 min to about 60 min (such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min or about 25 min to about 35 min, or about 30 min) for each of the additional stages.For example, the cross flow rate profile can contain (i) a first additional stage before the fractionation stage, wherein the cross flow rate of the first additional stage is constant and is the same as the cross low rate at the beginning of the fractionation stage (the length of the first additional stage can be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 10 min or about 20 min or about 30 min); (ii) a second additional stage after the fractionation stage, wherein the cross flow rate of the second additional stage is constant and is the same as the cross low rate at the end of the fractionation stage (the length of the second additional stage can be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 10 min or about 20 min or about 30 min); and optionally (iii) a third additional stage following the second additional stage, wherein the cross-flow rate of the third additional stage is constant and different from that of the second additional stage (the length of the third additional stage may be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min or about 25 min to about 35 min, or about 10 min or about 20 min or about 30 min).In embodiments wherein the cross-flow velocity profile comprises a graded stage, wherein the cross-flow velocity varies continuously (preferably exponentially) starting at one value (e.g., from about 1 to about 4 mL / min) and then decreasing to a lower value (e.g., from about 0 to about 0.1 mL / min), preferably the cross-flow velocity profile further comprises (i) a first additional stage before the graded stage, wherein the cross-flow velocity of the first additional stage is constant and is the same as the cross-flow velocity at the beginning of the graded stage (e.g., from about 1 to about 4 mL / min) (the length of the first additional stage may be in the range of from about 5 min to about 30 min, such as from about 6 min to about 25 min, from about 7 min to about 20 min, or from about 8 min to about 15 min, or from about 10 min to about 12 min, or about 5 min, or about 10 min, or about 12 min); (ii) a second additional stage after the graded stage, wherein the cross-flow velocity of the second additional stage is constant and is the same as the cross-flow velocity at the beginning of the graded stage (e.g., from about 1 to about 4 mL / min); and (iii) a third additional stage after the second additional stage, wherein the crossover flow rate of the third additional stage is constant and lower than that of the second additional stage (for example, the crossover flow rate of the third additional stage is 0) (the length of the third additional stage may be in the range of about 5 min to about 30 min, such as about 6 min to about 25 min, about 7 min to about 20 min, or about 8 min to about 15 min, or about 10 min to about 12 min, or about 5 min, about 10 min, or about 12 min). Preferred examples of such cross-flow profiles are as follows: 1.0-2.0 mL / min for 10 min, exponential gradient from 1.0-2.0 mL / min to 0.01-0.07 mL / min in 30 min; 0.01-0.07 mL / min for 30 min; and 0 mL / min for 10 min.

[0167] In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect), the integrity of the control nucleic acid (in particular RNA) is used to calculate the integrity of the nucleic acid (in particular RNA) contained in the sample composition (e.g., the first composition used in step (C) or the second composition used in step (D)).

[0168] In a first specific example of this embodiment of the second aspect, the integrity of the control nucleic acid (especially RNA) is determined by the following steps:

[0169] (a') subjecting at least part of a control composition containing a control nucleic acid, in particular RNA, to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0170] (b') measuring at least one signal selected from a UV signal, a fluorescence signal, and a refractive index (RI) signal of at least one of the one or more control fractions obtained from step (a');

[0171] (c'1) calculating the area from the maximum height of a UV, fluorescence or RI peak to the end of the UV, fluorescence or RI peak from at least one signal selected from the UV signal, fluorescence signal and RI signal obtained in step (b'), thereby obtaining A 50％ (control);

[0172] (c'2) calculating the total area of ​​one peak used in step (c'1) from at least one signal selected from the UV signal, the fluorescence signal, and the RI signal obtained in step (b'), thereby obtaining A 100％ (control); and

[0173] (c'3) Determine A 50％ (control) and A 100％ (control), thereby obtaining the integrity of the control nucleic acid (especially RNA) (I(control)).

[0174] In this first example, the integrity of the nucleic acid (especially RNA) contained in the sample composition (e.g., the first composition used in step (C) or the second composition used in step (D)) can be calculated by the following steps:

[0175] (c1) calculating the area from the maximum height of the sample UV, fluorescence or RI peak corresponding to the control UV, fluorescence or RI peak used in step (c'1) to the end of the sample UV, fluorescence or RI peak from at least one signal selected from the UV signal, fluorescence signal and RI signal obtained in step (b), thereby obtaining A 50％ (sample);

[0176] (c2) calculating the total area of ​​the sample UV, fluorescence or RI peak used in step (c1) from the sample UV, fluorescence or RI signal obtained in step (b), thereby obtaining A 100％ (sample);

[0177] (c3) Determine A 50％ (sample) and A 100％ (samples), thereby obtaining I(sample); and

[0178] (c4) determining the ratio between I(sample) and I(control) to obtain the integrity of the nucleic acids (especially RNA) contained in the sample composition.

[0179] In a second specific example of this embodiment of the second aspect, the integrity of the control nucleic acid (especially RNA) is determined by the following steps:

[0180] (a") subjecting at least part of a control composition containing a control nucleic acid, in particular RNA, to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0181] (b") measuring at least one signal selected from a UV signal, a fluorescence signal, and an RI signal of at least one of the one or more control fractions obtained from step (a"); and

[0182] (c") determining the height of a UV, fluorescence or RI peak (H(control)) from at least one signal selected from the UV signal, fluorescence signal and RI signal obtained in step (b"), thereby obtaining the integrity of the control nucleic acid (especially RNA).

[0183] In this second example, the integrity of the nucleic acid (especially RNA) contained in the sample composition (e.g., the first composition used in step (C) or the second composition used in step (D)) can be calculated by the following steps:

[0184] (c1′) determining the height (H(sample)) of the sample UV, fluorescence or RI peak corresponding to the control UV, fluorescence or RI peak used in step (c″) from at least one signal selected from the UV signal, fluorescence signal and RI signal obtained in step (b); and

[0185] (c2') determining the ratio between H(sample) and H(control) to obtain the integrity of the nucleic acids (especially RNA) contained in the sample composition.

[0186] In a third specific example of this embodiment of the second aspect (relating to the first subgroup of the second aspect), the integrity of the control nucleic acid (especially RNA) is determined by the following steps:

[0187] (a') subjecting at least part of a control composition containing a control nucleic acid, in particular RNA, to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0188] (b') measuring the UV signal of at least one of the one or more control fractions obtained from step (a');

[0189] (c'1) Calculating the area from the maximum height of a UV peak to the end of the UV peak from the UV signal obtained in step (b') to obtain A 50％ (control);

[0190] (c'2) Calculating the total area of ​​one peak used in step (c'1) from the UV signal obtained in step (b') to obtain A 100％ (control); and

[0191] (c'3) Determine A 50％ (control) and A 100％ (control), thereby obtaining the integrity of the control nucleic acid (especially RNA) (I(control)).

[0192] In this third example, the integrity of the nucleic acid (especially RNA) contained in the sample composition (e.g., the first composition used in step (C) or the second composition used in step (D)) can be calculated by the following steps:

[0193] (c1) calculating the area from the maximum height of the sample UV peak corresponding to the control UV peak used in step (c'1) to the end of the sample UV peak from the UV signal obtained in step (b), thereby obtaining A 50％ (sample);

[0194] (c2) calculating the total area of ​​the sample UV peak used in step (c1) from the sample UV signal obtained in step (b) to obtain A 100％ (sample);

[0195] (c3) Determine A 50％ (sample) and A 100％ (samples), thereby obtaining I(sample); and

[0196] (c4) determining the ratio between I(sample) and I(control) to obtain the integrity of the nucleic acids (especially RNA) contained in the sample composition.

[0197] In a fourth specific example of this embodiment of the second aspect (relating to the first subgroup of the second aspect), the integrity of the control nucleic acid (especially RNA) is determined by the following steps:

[0198] (a") subjecting at least part of a control composition containing a control nucleic acid, in particular RNA, to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0199] (b") measuring the UV signal of at least one of the one or more control fractions obtained from step (a"); and

[0200] (c") determining the height of a UV peak (H(control)) from the UV signal obtained in step (b"), thereby obtaining the integrity of the control nucleic acid (especially RNA).

[0201] In this fourth example, the integrity of the nucleic acid (especially RNA) contained in the sample composition (e.g., the first composition used in step (C) or the second composition used in step (D)) can be calculated by the following steps:

[0202] (c1′) determining the height of the sample UV peak (H(sample)) corresponding to the control UV peak used in step (c”) from the UV signal obtained in step (b); and

[0203] (c2') determining the ratio between H(sample) and H(control) to obtain the integrity of the nucleic acids (especially RNA) contained in the sample composition.

[0204] In a fifth specific example of this embodiment of the second aspect (relating to the second subgroup of the second aspect), the integrity of the control RNA is determined by the following steps:

[0205] (a') subjecting at least part of a control composition comprising a control RNA to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0206] (b') measuring at least one signal selected from a UV signal, a fluorescence signal, and an RI signal of at least one of the one or more control fractions obtained in step (a');

[0207] (c'1) calculating the area from the maximum height of a UV, fluorescence or RI peak to the end of the UV, fluorescence or RI peak from at least one signal selected from the UV signal, fluorescence signal and RI signal obtained in step (b'), thereby obtaining A 50％ (control);

[0208] (c'2) calculating the total area of ​​one peak used in step (c'1) from at least one signal selected from the UV signal, the fluorescence signal, and the RI signal obtained in step (b'), thereby obtaining A 100％ (control); and

[0209] (c'3) Determine A 50％ (control) and A 100％ (control) to obtain the integrity of the control RNA (I(control)).

[0210] In this fifth example, the integrity of the RNA contained in the sample composition (e.g., the first composition used in step (C) or the second composition used in step (D)) can be calculated by the following steps:

[0211] (c1) calculating the area from the maximum height of the sample UV, fluorescence or RI peak corresponding to the control UV, fluorescence or RI peak used in step (c'1) to the end of the sample UV, fluorescence or RI peak from at least one signal selected from the UV signal, fluorescence signal and RI signal obtained in step (b), thereby obtaining A 50％ (sample);

[0212] (c2) calculating the total area of ​​the sample UV, fluorescence or RI peak used in step (c1) from the sample UV, fluorescence or RI signal obtained in step (b), thereby obtaining A 100％ (sample);

[0213] (c3) Determine A 50％ (sample) and A 100％ (samples), thereby obtaining I(sample); and

[0214] (c4) determining the ratio between I(sample) and I(control) to obtain the integrity of the RNA contained in the sample composition.

[0215] In a sixth specific example of this embodiment of the second aspect (relating to the second subgroup of the second aspect), the integrity of the control RNA is determined by the following steps:

[0216] (a") subjecting at least part of a control composition comprising a control RNA to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0217] (b") measuring at least one signal selected from a UV signal, a fluorescence signal, and an RI signal of at least one of the one or more control fractions obtained from step (a"); and

[0218] (c") determining the height of a UV, fluorescence or RI peak (H(control)) from at least one signal selected from the UV signal, fluorescence signal and RI signal obtained in step (b"), thereby obtaining the integrity of the control RNA.

[0219] In this sixth example, the integrity of the RNA contained in the sample composition (e.g., the first composition used in step (C) or the second composition used in step (D)) can be calculated by the following steps:

[0220] (c1′) determining the height (H(sample)) of the sample UV, fluorescence or RI peak corresponding to the control UV, fluorescence or RI peak used in step (c″) from at least one signal selected from the UV signal, fluorescence signal and RI signal obtained in step (b); and

[0221] (c2') Determine the ratio between H(sample) and H(control) to obtain the integrity of the RNA contained in the sample composition.

[0222] In a seventh specific example of this embodiment of the second aspect (relating to the third subgroup of the second aspect), the integrity of the control RNA is determined by the following steps:

[0223] (a') subjecting at least part of a control composition comprising a control RNA to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0224] (b') measuring the UV signal of at least one of the one or more control fractions obtained from step (a');

[0225] (c'1) Calculating the area from the maximum height of a UV peak to the end of the UV peak from the UV signal obtained in step (b') to obtain A 50％ (control);

[0226] (c'2) Calculating the total area of ​​one peak used in step (c'1) from the UV signal obtained in step (b') to obtain A 100％ (control); and

[0227] (c'3) Determine A 50％ (control) and A 100％ (control) to obtain the integrity of the control RNA (I(control)).

[0228] In this seventh example, the integrity of the RNA contained in the sample composition (e.g., the first composition used in step (C) or the second composition used in step (D)) can be calculated by the following steps:

[0229] (c1) calculating the area from the maximum height of the sample UV peak corresponding to the control UV peak used in step (c'1) to the end of the sample UV peak from the UV signal obtained in step (b), thereby obtaining A 50％ (sample);

[0230] (c2) calculating the total area of ​​the sample UV peak used in step (c1) from the sample UV signal obtained in step (b) to obtain A 100％ (sample);

[0231] (c3) Determine A 50％ (sample) and A 100％ (samples), thereby obtaining I(sample); and

[0232] (c4) determining the ratio between I(sample) and I(control) to obtain the integrity of the RNA contained in the sample composition.

[0233] In an eighth specific example of this embodiment of the second aspect (relating to the third subgroup of the second aspect), the integrity of the control RNA is determined by the following steps:

[0234] (a") subjecting at least part of a control composition comprising a control RNA to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0235] (b") measuring the UV signal of at least one of the one or more control fractions obtained from step (a"); and

[0236] (c") Determine the height of a UV peak (H(control)) from the UV signal obtained in step (b") to obtain the integrity of the control RNA.

[0237] In this eighth example, the integrity of the RNA contained in the sample composition (e.g., the first composition used in step (C) or the second composition used in step (D)) can be calculated by the following steps:

[0238] (c1′) determining the height of the sample UV peak (H(sample)) corresponding to the control UV peak used in step (c”) from the UV signal obtained in step (b); and

[0239] (c2') Determine the ratio between H(sample) and H(control) to obtain the integrity of the RNA contained in the sample composition.

[0240] In one embodiment of the second aspect (particularly in one embodiment of the first, second or third subgroup of the second aspect), the amount of nucleic acid (especially RNA) is determined by using (i) a nucleic acid extinction coefficient (especially an RNA extinction coefficient) or (ii) a nucleic acid calibration curve (especially an RNA calibration curve).

[0241] In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect), the sample composition (e.g., the first composition used in step (C) or the second composition used in step (D)) comprises nucleic acids (especially RNA) and particles, such as lipid complex particles and / or lipid nanoparticles and / or polyplex particles and / or lipid multiplex particles and / or virus-like particles, to which the nucleic acids (especially RNA) are associated.

[0242] In one embodiment of the second aspect (particularly in one embodiment of the first, second or third subgroup of the second aspect), the amount of total nucleic acids (particularly the amount of total RNA) is determined by: (i) treating at least a portion of the sample composition (e.g., the first composition used in step (C) or the second composition used in step (D)) with a releasing agent; (ii) performing steps (a) to (c) with at least the portion obtained from step (i); and (iii) determining the amount of nucleic acids (particularly RNA) as described herein (e.g., using (i) a nucleic acid extinction coefficient (particularly an RNA extinction coefficient) or (ii) a nucleic acid calibration curve (particularly an RNA calibration curve)). In this embodiment, in step (a) of the method of the second aspect, field-flow fractionation is preferably performed using a liquid phase containing a releasing agent.

[0243] In one embodiment of the second aspect (particularly, in one embodiment of the first, second or third subgroup of the second aspect), the release agent is (i) a surfactant, such as an anionic surfactant (e.g., sodium lauryl sulfate), a zwitterionic surfactant (e.g., n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), or a surfactant such as (ii) a surfactant. 3-14)), a cationic surfactant, a nonionic surfactant or a mixture thereof; (ii) an alcohol, such as a fatty alcohol (e.g., ethanol), or a mixture of alcohols; or (iii) a combination of (i) and (ii).

[0244] In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect), the amount of free nucleic acid, in particular RNA, is determined by: performing steps (a) to (c) without adding a releasing agent, in particular in the absence of any releasing agent; and determining the amount of nucleic acid, in particular RNA, as described herein (e.g., using (i) a nucleic acid extinction coefficient, in particular an RNA extinction coefficient, or (ii) a nucleic acid calibration curve, in particular an RNA calibration curve).

[0245] In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect), the amount of nucleic acids (in particular RNA) associated with the particles is determined by subtracting the amount of free nucleic acids (in particular RNA) as determined herein (e.g., by performing steps (a) to (c) without adding a releasing agent, in particular in the absence of any releasing agent; and determining the amount of nucleic acids (in particular RNA) as described herein (e.g., using (i) a nucleic acid extinction coefficient (in particular an RNA extinction coefficient) or (ii) a nucleic acid calibration curve (in particular an RNA calibration curve))) from the amount of total nucleic acids (in particular RNA) as determined herein (e.g., by (i) treating at least a portion of the sample composition (e.g., the first composition for step (C) or the second composition for step (D)) with a releasing agent; (ii) performing steps (a) to (c) with at least the portion obtained from step (i); and (iii) determining the amount of nucleic acids (in particular RNA) as described herein (e.g., using (i) a nucleic acid extinction coefficient (in particular an RNA extinction coefficient) or (ii) a nucleic acid calibration curve (in particular an RNA calibration curve))).

[0246] In one embodiment of the second aspect (in particular, in one embodiment of the second or third subgroup of the second aspect), step (b) further comprises measuring an LS signal, such as dynamic light scattering (DLS) and / or static light scattering (SLS), e.g., a multi-angle light scattering (MALS) signal, of at least one of the one or more sample fractions obtained from step (a).

[0247] In one embodiment of the second aspect (particularly, in one embodiment of the second or third subgroup of the second aspect), the radius of gyration (R g ) value and / or hydrodynamic radius (R h ) value to determine the size of particles containing nucleic acids (especially RNA). In one embodiment of the second aspect (particularly in one embodiment of the first, second or third subgroup of the second aspect), step (b) comprises measuring a dynamic light scattering (DLS) signal of at least one of the one or more sample fractions obtained from step (a), and step (c) comprises calculating R from the DLS signal. h In one embodiment of the second aspect (particularly in one embodiment of the first, second or third subgroup of the second aspect), step (b) comprises measuring a static light scattering (SLS) such as a MALS signal of at least one of the one or more sample fractions obtained in step (a), and step (c) comprises calculating R from said SLS signal. gIn one embodiment of the second aspect (particularly, in one embodiment of the first, second or third subgroup of the second aspect), step (b) comprises measuring a dynamic light scattering (DLS) signal and a static light scattering (SLS), such as a MALS signal, of at least one of the one or more sample fractions obtained in step (a), and step (c) comprises calculating R g and R h This latter embodiment generates two data sets of particle sizes containing nucleic acids (such as RNA), namely, one based on R g value, a value based on R h value.

[0248] In one embodiment of the second aspect (particularly, in one embodiment of the first, second or third subgroup of the second aspect), the R g or R h value (e.g., by calculating R from the SLS signal obtained in step (b) g value or calculate R by the DLS signal obtained from step (b) h In a first example of this embodiment (relating to the first subgroup of the second aspect), the size distribution of particles containing nucleic acids, particularly RNA, is determined by plotting the UV signal obtained from step (b) against the R value determined as described herein. g or R h value (e.g., by calculating R from the SLS signal obtained in step (b) g value or calculate R by the DLS signal obtained from step (b) h In a second example of this embodiment (relating to the second subgroup of the second aspect), the size distribution of particles containing nucleic acids (particularly RNA) is determined by plotting the at least one signal selected from the UV signal, the fluorescence signal and the RI signal obtained in step (b) against the R value determined as described herein. g or R h value (e.g., by calculating R from the SLS signal obtained in step (b) g value or calculate R by the DLS signal obtained from step (b) h In a third example of this embodiment (relating to the third subgroup of the second aspect), the size distribution of the particles containing RNA is determined by plotting the UV signal obtained from step (b) against the R value determined as described herein. g or R h value (e.g., by calculating R from the SLS signal obtained in step (b) g value or calculate R by the DLS signal obtained from step (b) hIn each of the first, second and third examples above, the size distribution of the particles containing RNA can be determined by plotting the values ​​of R g Value, R h The size distribution of particles containing nucleic acids (especially RNA) is determined based on the values ​​of R and R, or both. g Value and R h The size distribution of particles containing nucleic acids (especially RNA) is determined based on the values ​​of R. g The size distribution of the values ​​and an R-based h The size distribution of values.

[0249] In one embodiment of the second aspect (particularly, in one embodiment of the first, second or third subgroup of the second aspect), the UV, fluorescence or RI signal is converted into a cumulative weight fraction and the cumulative weight fraction is converted to R g or R h The values ​​are plotted and displayed as R g or R h The quantitative size distribution of particles containing nucleic acids, particularly RNA, is calculated from a plot of the UV, fluorescence or RI signal as a function of the value of the UV, fluorescence or RI signal. In a first example of this embodiment (the first subgroup relating to the second aspect), the UV signal is converted into a cumulative weight fraction and the cumulative weight fraction is plotted against the R g or R h The values ​​are plotted and displayed as R g or R h The quantitative size distribution of particles containing nucleic acids, particularly RNA, is calculated from a plot of the UV signal as a function of the UV value of the UV signal. In a second example of this embodiment (relating to the second subgroup of the second aspect), the UV, fluorescence or RI signal is converted into a cumulative weight fraction and the cumulative weight fraction is plotted against the R value. g or R h The values ​​are plotted and displayed as R g or R h The quantitative size distribution of the RNA-containing particles is calculated from a plot of the UV, fluorescence or RI signal as a function of the value of the UV, fluorescence or RI signal. In a third example of this embodiment (the third subgroup of the second aspect), the size distribution of the RNA-containing particles is calculated by converting the UV signal to a cumulative weight fraction and plotting the cumulative weight fraction against the R value. g or R h The values ​​are plotted and displayed as R g or R h The quantitative size distribution of the RNA-containing particles can be calculated from a plot of the UV signal as a function of the value of R. g Value, R h The quantitative size distribution of particles containing nucleic acids (especially RNA) is determined based on the values ​​of R and R, or both. gValue and R h The quantitative size distribution of particles containing nucleic acids (especially RNA) is determined based on the values ​​of R. g The quantitative size distribution of the values ​​and an R h Quantitative size distribution of values.

[0250] In one embodiment of the second aspect (particularly in one embodiment of the first, second or third subgroup of the second aspect), the quantitative size distribution comprises D10, D50 and / or D90 values. g Value and R h The quantitative size distribution of particles containing nucleic acids (especially RNA) is determined based on the values ​​of R. g The D10, D50 and / or D90 values ​​and a set of R-based h The D10, D50 and / or D90 values ​​of the

[0251] In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect), the amount of nucleic acid (in particular RNA), in particular free nucleic acid (in particular RNA), is determined by measuring the UV signal, for example, at a wavelength in the range of 260 nm-280 nm, such as at a wavelength of 260 nm or 280 nm, and using the extinction coefficient of nucleic acid (in particular RNA) at the corresponding wavelength (e.g., 260 nm or 280 nm).

[0252] In one embodiment of the second aspect (particularly in one embodiment of the first, second or third subgroup of the second aspect, and particularly in a preferred embodiment of the third subgroup of the second aspect), the size distribution of the particles containing nucleic acid (particularly RNA) (e.g., based on R g or R h values) and / or quantitative size distribution of particles containing nucleic acids, in particular RNA (e.g., based on R g or R h In a preferred embodiment of the third subgroup of the second aspect, the (quantitative) size distribution of the RNA-containing particles (e.g., based on R g or R hvalue) is in the range of 10-1000 nm, for example, in the range of 15-500 nm, 20-450 nm, 25-400 nm, 30-350 nm, 40-300 nm or 50-250 nm.

[0253] In one embodiment of the second aspect (particularly in one embodiment of the first, second or third subgroup of the second aspect), the nucleic acid (especially RNA) has a length of 10-15,000 nucleotides, such as 40-15,000 nucleotides, 100-12,000 nucleotides or 200-10,000 nucleotides.

[0254] In one embodiment of the second aspect (particularly in one embodiment of the first subgroup of the second aspect), the nucleic acid is RNA. In this embodiment and in the second or third subgroup of embodiments of the second aspect, the RNA is preferably mRNA or in vitro transcribed RNA, particularly in vitro transcribed mRNA.

[0255] In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect), measuring at least one signal selected from UV signals, fluorescence signals and RI signals, optionally LS signals, such as SLS, for example, MALS signals and / or DLS signals, is performed online, and / or step (c) is performed online.

[0256] In one embodiment of the second aspect (particularly, in one embodiment of the first, second, or third subgroup of the second aspect), prior to subjecting at least a portion of the sample composition (e.g., the first composition used in step (C) or the second composition used in step (D)) to field-flow fractionation, at least a portion of the sample composition is diluted with a solvent or solvent mixture that prevents the formation of particle aggregates. In one embodiment, the solvent mixture is a mixture of water and an organic solvent such as formamide.

[0257] In an embodiment of the second aspect (particularly in an embodiment of the first, second or third subgroup of the second aspect), measuring the UV signal is performed by using circular dichroism (CD) spectroscopy.

[0258] It will be appreciated that any embodiment described herein in the context of the first aspect may also apply to any embodiment of the second aspect.

[0259] In a third aspect, the present disclosure provides for use of field-flow fractionation in determining one or more parameters of a sample composition comprising nucleic acids (such as RNA) and optionally particles, wherein the one or more parameters include nucleic acid (such as RNA) integrity, the total amount of nucleic acids (such as RNA), the amount of free nucleic acids (such as RNA), the amount of nucleic acids (such as RNA) associated with particles, the size of the particles containing nucleic acids (such as RNA) (particularly, based on the radius of gyration (R) of the particles containing nucleic acids (such as RNA)), the size of the particles containing nucleic acids (such as RNA ... g ) and / or the hydrodynamic radius (R) of particles containing nucleic acids (such as RNA) h )), size distribution of particles containing nucleic acids (such as RNA) (e.g., based on the R g or R h values) and quantitative size distribution of particles containing nucleic acids (e.g., RNA) (e.g., based on the R values ​​of particles containing nucleic acids (e.g., RNA)). g or R h In general, the size distribution and / or quantitative size distribution of particles containing nucleic acids (such as RNA) can be given as the number of particles containing nucleic acids (such as RNA), the molar amount of particles containing nucleic acids (such as RNA) or the mass of particles containing nucleic acids (such as RNA), each as a function of their size. Additional optional parameters include the molecular weight of the nucleic acids (such as RNA), the amount of surface nucleic acids (such as the amount of surface RNA), the amount of encapsulated nucleic acids (such as the amount of encapsulated RNA), the amount of accessible nucleic acids (such as the amount of accessible RNA), the size of the nucleic acids (especially RNA) (in particular, based on the R of the particles containing nucleic acids (such as RNA)). g and / or R h values), size distribution of nucleic acids (especially RNA) (e.g., based on the R g or R h values), quantitative size distribution of nucleic acids (especially RNA) (e.g., based on the R g or R hvalue), shape factor, form factor and nucleic acid (especially RNA) encapsulation efficiency. In general, the size distribution and / or quantitative size distribution of nucleic acids (especially RNA) can be given as the number of nucleic acid (especially RNA) molecules, the molar amount of nucleic acids (especially RNA) or the mass of nucleic acids (especially RNA), each as a function of their size. Other additional optional parameters include the ratio of the amount of nucleic acid (such as RNA) associated with the particle to the total amount of particle-forming compounds (especially lipids and / or polymers) in the particle, wherein the ratio can be given as a function of the particle size; the ratio of the amount of positively charged parts of the particle-forming compounds (especially lipids and / or polymers) in the particle to the amount of nucleic acid (such as RNA) associated with the particle, wherein the ratio can be given as a function of the particle size; and the charge ratio of the amount of positively charged parts of the particle-forming compounds (especially lipids and / or polymers) in the particle to the amount of negatively charged parts of nucleic acids (such as RNA) associated with the particle, wherein the charge ratio is usually expressed as N / P ratio and can be given as a function of the particle size.

[0260] In an embodiment of the third aspect, the field-flow fractionation comprises:

[0261] (a) subjecting at least a portion of the sample composition to field-flow fractionation, thereby fractionating components contained in the sample composition according to their size to produce one or more sample fractions;

[0262] (b) measuring at least one signal selected from a UV signal, a fluorescence signal, and a refractive index (RI) signal, and optionally measuring a light scattering (LS) signal, of at least one of the one or more sample fractions obtained from step (a); and

[0263] (c) calculating the one or more parameters from the at least one signal selected from the group consisting of the UV signal, the fluorescence signal and the RI signal, and optionally from the LS signal.

[0264] In the first subgroup of the third aspect, the field-flow grading comprises:

[0265] (a) subjecting at least a portion of the sample composition to field-flow fractionation, thereby fractionating components contained in the sample composition according to their size to produce one or more sample fractions;

[0266] (b) measuring at least a UV signal and optionally a light scattering (LS) signal of at least one of the one or more sample fractions obtained from step (a); and

[0267] (c) calculating the one or more parameters from the UV signal and optionally from the LS signal.

[0268] In a second and preferred subgroup of the third aspect, the use is for determining one or more parameters of a sample composition, wherein the sample composition comprises RNA and optionally particles, and the field-flow fractionation comprises:

[0269] (a) subjecting at least a portion of the sample composition to field-flow fractionation, thereby fractionating components contained in the sample composition according to their size to produce one or more sample fractions;

[0270] (b) measuring at least one signal selected from a UV signal, a fluorescence signal, and a refractive index (RI) signal, and optionally measuring a light scattering (LS) signal, of at least one of the one or more sample fractions obtained from step (a); and

[0271] (c) calculating the one or more parameters from the at least one signal selected from the group consisting of the UV signal, the fluorescence signal and the RI signal, and optionally from the LS signal.

[0272] In a third and more preferred subgroup of the third aspect, the use is for determining one or more parameters of a sample composition, wherein the sample composition comprises RNA and optionally particles, and the field-flow fractionation comprises:

[0273] (a) subjecting at least a portion of the sample composition to field-flow fractionation, thereby fractionating components contained in the sample composition according to their size to produce one or more sample fractions;

[0274] (b) measuring at least a UV signal and optionally a light scattering (LS) signal of at least one of the one or more sample fractions obtained from step (a); and

[0275] (c) calculating the one or more parameters from the UV signal and optionally from the LS signal.

[0276] In one embodiment of the third aspect (particularly, in one embodiment of the first, second or third subgroup of the third aspect), the R based on the particle containing nucleic acid (such as RNA) g In another embodiment of the third aspect (particularly, in another embodiment of the first, second or third subgroup of the third aspect), the size, size distribution and / or quantitative size distribution of the particles containing nucleic acids (such as RNA) are calculated based on the R value of the particles containing nucleic acids (such as RNA). h In another embodiment of the third aspect (particularly, in another embodiment of the first, second or third subgroup of the third aspect), the size, size distribution and / or quantitative size distribution of the particles containing nucleic acids (such as RNA) are calculated based on the R value of the particles containing nucleic acids (such as RNA). g values ​​and respectively based on the R of particles containing nucleic acids (such as RNA)h The size, size distribution and / or quantitative size distribution of particles containing nucleic acids (such as RNA) are calculated using the R-values ​​(i.e., this embodiment generates two data sets of sizes, size distributions and / or quantitative size distributions of particles containing nucleic acids (such as RNA), one based on R g value, a value based on R h value).

[0277] In one embodiment of the third aspect (particularly, in one embodiment of the first, second or third subgroup of the third aspect), wherein the one or more parameters include the size, size distribution and / or quantitative size distribution of the nucleic acid (such as RNA), the R g In another embodiment of the third aspect (particularly, in another embodiment of the first, second or third subgroup of the third aspect), wherein the one or more parameters include the size, size distribution and / or quantitative size distribution of the nucleic acid (such as RNA), the R value of the nucleic acid (such as RNA) is calculated. h In another embodiment of the third aspect (particularly, in another embodiment of the first, second or third subgroup of the third aspect), wherein the one or more parameters include the size, size distribution and / or quantitative size distribution of the nucleic acid (such as RNA), the R value of the nucleic acid (such as RNA) is calculated. g values ​​and respectively based on the R h The size, size distribution and / or quantitative size distribution of nucleic acids (such as RNA) are calculated using the R-values ​​(i.e., this embodiment generates two data sets of sizes, size distributions and / or quantitative size distributions of nucleic acids (such as RNA), one based on R g value, a value based on R h value).

[0278] In one embodiment of the third aspect (particularly, in one embodiment of the first, second or third subgroup of the third aspect), the field-flow fractionation is a flow field-flow fractionation, such as an asymmetric flow field-flow fractionation (AF4) or a hollow fiber flow field-flow fractionation (HF5).

[0279] In one embodiment of the third aspect (in particular, in one embodiment of the first, second or third subgroup of the third aspect), the field-flow fractionation uses a membrane having a molecular weight cutoff (MW) suitable for preventing nucleic acids (especially RNA) from penetrating the membrane, preferably a membrane with a MW cutoff in the range of 2kDa-30kDa, such as a membrane with a MW cutoff of 10kDa.

[0280] In one embodiment of the third aspect (particularly, in one embodiment of the first, second or third subgroup of the third aspect), the field-flow fractionation uses a polyethersulfone (PES) or regenerated cellulose membrane.

[0281] In one embodiment of the third aspect (particularly, in one embodiment of the first, second or third subgroup of the third aspect), step (a) is performed using: (I) a cross flow rate of up to 8 mL / min, preferably up to 4 mL / min, more preferably up to 2 mL / min, for example, a cross flow profile; and / or (II) an injection flow rate in the range of 0.05-0.35 mL / min, preferably in the range of 0.10-0.30 mL / min, more preferably in the range of 0.15-0.25 mL / min; and / or (III) a detector flow rate in the range of 0.30-0.70 mL / min, preferably in the range of 0.40-0.60 mL / min, more preferably in the range of 0.45-0.55 mL / min.

[0282] In one embodiment of the third aspect (particularly, in one embodiment of the first, second or third subgroup of the third aspect), the crossflow velocity profile preferably comprises a fractionation stage that allows the components contained in the control or sample composition to be fractionated / separated by size to produce one or more sample fractions. Preferably, the crossflow velocity changes during this fractionation stage (e.g., starting from a value (e.g., about 1 to about 4 mL / min) and then decreasing to a lower value (e.g., about 0 to about 0.1 mL / min), or starting from a value (e.g., about 0 to about 0.1 mL / min) and then increasing to a higher value (e.g., about 1 to about 4 mL / min)), which can be varied in any manner, for example, continuously (e.g., linearly or exponentially) or stepwise. Preferably, the crossflow velocity profile comprises a fractionation stage in which the crossflow velocity changes continuously (preferably exponentially), starting from a value (e.g., about 1 to about 4 mL / min) and then decreasing to a lower value (e.g., about 0 to about 0.1 mL / min). The fractionation stage can have any length suitable for fractionating / separating the components contained in the sample composition by their size, for example, from about 5 min to about 60 min, such as from about 10 min to about 50 min, from about 15 min to about 45 min, from about 20 min to about 40 min, or from about 25 min to about 35 min, or about 30 min. The cross-flow profile can contain additional stages (e.g., 1, 2, 3 or 4 stages) that can be before and / or after the fractionation stage (e.g., 1 before fractionation and 1, 2 or 3 after fractionation), and can be used to separate the non-nucleic acid (especially non-RNA) components (e.g., proteins, polypeptides, mononucleotides, etc.) contained in the sample composition from the nucleic acid (especially RNA) contained in the sample composition, to concentrate the nucleic acid (especially RNA) contained in the sample composition and / or to regenerate the field-flow fractionation device (e.g., to remove all components bound to the membrane of the device). Preferably, the cross-flow rate of the additional stages is constant for each additional stage, and the length of each of the additional stages is independently in the range of about 5 min to about 60 min (such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min or about 25 min to about 35 min, or about 30 min) for each of the additional stages.For example, the cross flow rate profile can contain (i) a first additional stage before the fractionation stage, wherein the cross flow rate of the first additional stage is constant and is the same as the cross low rate at the beginning of the fractionation stage (the length of the first additional stage can be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 10 min or about 20 min or about 30 min); (ii) a second additional stage after the fractionation stage, wherein the cross flow rate of the second additional stage is constant and is the same as the cross low rate at the end of the fractionation stage (the length of the second additional stage can be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 10 min or about 20 min or about 30 min); and optionally (iii) a third additional stage following the second additional stage, wherein the cross-flow rate of the third additional stage is constant and different from that of the second additional stage (the length of the third additional stage may be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min or about 25 min to about 35 min, or about 10 min or about 20 min or about 30 min).In embodiments wherein the cross-flow velocity profile comprises a graded stage, wherein the cross-flow velocity varies continuously (preferably exponentially) starting at one value (e.g., from about 1 to about 4 mL / min) and then decreasing to a lower value (e.g., from about 0 to about 0.1 mL / min), preferably the cross-flow velocity profile further comprises (i) a first additional stage before the graded stage, wherein the cross-flow velocity of the first additional stage is constant and is the same as the cross-flow velocity at the beginning of the graded stage (e.g., from about 1 to about 4 mL / min) (the length of the first additional stage may be in the range of from about 5 min to about 30 min, such as from about 6 min to about 25 min, from about 7 min to about 20 min, or from about 8 min to about 15 min, or from about 10 min to about 12 min, or about 5 min, or about 10 min, or about 12 min); (ii) a second additional stage after the graded stage, wherein the cross-flow velocity of the second additional stage is constant and is the same as the cross-flow velocity at the beginning of the graded stage (e.g., from about 1 to about 4 mL / min); and (iii) a third additional stage after the second additional stage, wherein the crossover flow rate of the third additional stage is constant and lower than that of the second additional stage (for example, the crossover flow rate of the third additional stage is 0) (the length of the third additional stage may be in the range of about 5 min to about 30 min, such as about 6 min to about 25 min, about 7 min to about 20 min, or about 8 min to about 15 min, or about 10 min to about 12 min, or about 5 min, about 10 min, or about 12 min). Preferred examples of such cross-flow profiles are as follows: 1.0-2.0 mL / min for 10 min, exponential gradient from 1.0-2.0 mL / min to 0.01-0.07 mL / min in 30 min; 0.01-0.07 mL / min for 30 min; and 0 mL / min for 10 min.

[0283] In one embodiment of the third aspect (particularly in one embodiment of the first, second or third subgroup of the third aspect), the integrity of a control nucleic acid (particularly RNA) is used to determine the integrity of the nucleic acid (particularly RNA) contained in the sample composition.

[0284] In a first specific example of this embodiment of the third aspect, the integrity of the control nucleic acid (especially RNA) is determined by the following steps:

[0285] (a') subjecting at least part of a control composition containing a control nucleic acid, in particular RNA, to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0286] (b') measuring at least one signal selected from a UV signal, a fluorescence signal, and a refractive index (RI) signal of at least one of the one or more control fractions obtained from step (a');

[0287] (c'1) calculating the area from the maximum height of a UV, fluorescence or RI peak to the end of the UV, fluorescence or RI peak from at least one signal selected from the UV signal, fluorescence signal and RI signal obtained in step (b'), thereby obtaining A 50％ (control);

[0288] (c'2) calculating the total area of ​​one peak used in step (c'1) from at least one signal selected from the UV signal, the fluorescence signal, and the RI signal obtained in step (b'), thereby obtaining A 100％ (control); and

[0289] (c'3) Determine A 50％ (control) and A 100％ (control), thereby obtaining the integrity of the control nucleic acid (especially RNA) (I(control)).

[0290] In this first example, the integrity of the nucleic acids (especially RNA) contained in the sample composition can be calculated by the following steps:

[0291] (c1) calculating the area from the maximum height of the sample UV, fluorescence or RI peak corresponding to the control UV, fluorescence or RI peak used in step (c'1) to the end of the sample UV, fluorescence or RI peak from at least one signal selected from the UV signal, fluorescence signal and RI signal obtained in step (b), thereby obtaining A 50％ (sample);

[0292] (c2) calculating the total area of ​​the sample UV, fluorescence or RI peak used in step (c1) from the sample UV, fluorescence or RI signal obtained in step (b), thereby obtaining A 100％ (sample);

[0293] (c3) Determine A 50％ (sample) and A 100％ (samples), thereby obtaining I(sample); and

[0294] (c4) determining the ratio between I(sample) and I(control) to obtain the integrity of the nucleic acids (especially RNA) contained in the sample composition.

[0295] In a second specific example of this embodiment of the third aspect, the integrity of the control nucleic acid (especially RNA) is determined by the following steps:

[0296] (a") subjecting at least part of a control composition containing a control nucleic acid, in particular RNA, to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0297] (b") measuring at least one signal selected from a UV signal, a fluorescence signal, and an RI signal of at least one of the one or more control fractions obtained from step (a"); and

[0298] (c") determining the height of a UV, fluorescence or RI peak (H(control)) from at least one signal selected from the UV signal, fluorescence signal and RI signal obtained in step (b"), thereby obtaining the integrity of the control nucleic acid (especially RNA).

[0299] In this second example, the integrity of the nucleic acids (especially RNA) contained in the sample composition can be calculated by the following steps:

[0300] (c1′) determining the height (H(sample)) of the sample UV, fluorescence or RI peak corresponding to the control UV, fluorescence or RI peak used in step (c″) from at least one signal selected from the UV signal, fluorescence signal and RI signal obtained in step (b); and

[0301] (c2') determining the ratio between H(sample) and H(control) to obtain the integrity of the nucleic acids (especially RNA) contained in the sample composition.

[0302] In a third specific example of this embodiment of the third aspect (relating to the first subgroup of the third aspect), the integrity of the control nucleic acid (especially RNA) is determined by the following steps:

[0303] (a') subjecting at least part of a control composition containing a control nucleic acid, in particular RNA, to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0304] (b') measuring the UV signal of at least one of the one or more control fractions obtained from step (a');

[0305] (c'1) Calculating the area from the maximum height of a UV peak to the end of the UV peak from the UV signal obtained in step (b') to obtain A 50％ (control);

[0306] (c'2) Calculating the total area of ​​one peak used in step (c'1) from the UV signal obtained in step (b') to obtain A 100％ (control); and

[0307] (c'3) Determine A 50％ (control) and A 100％ (control), thereby obtaining the integrity of the control nucleic acid (especially RNA) (I(control)).

[0308] In this third example, the integrity of the nucleic acid (especially RNA) contained in the sample composition can be calculated by the following steps:

[0309] (c1) calculating the area from the maximum height of the sample UV peak corresponding to the control UV peak used in step (c'1) to the end of the sample UV peak from the UV signal obtained in step (b), thereby obtaining A 50％ (sample);

[0310] (c2) calculating the total area of ​​the sample UV peak used in step (c1) from the sample UV signal obtained in step (b) to obtain A 100％ (sample);

[0311] (c3) Determine A 50％ (sample) and A 100％ (samples), thereby obtaining I(sample); and

[0312] (c4) determining the ratio between I(sample) and I(control) to obtain the integrity of the nucleic acids (especially RNA) contained in the sample composition.

[0313] In a fourth specific example of this embodiment of the third aspect (relating to the first subgroup of the third aspect), the integrity of the control nucleic acid (especially RNA) is determined by:

[0314] (a") subjecting at least part of a control composition containing a control nucleic acid, in particular RNA, to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0315] (b") measuring the UV signal of at least one of the one or more control fractions obtained from step (a"); and

[0316] (c") determining the height of a UV peak (H(control)) from the UV signal obtained in step (b"), thereby obtaining the integrity of the control nucleic acid (especially RNA).

[0317] In this fourth example, the integrity of the nucleic acid (especially RNA) contained in the sample composition can be calculated by the following steps:

[0318] (c1′) determining the height of the sample UV peak (H(sample)) corresponding to the control UV peak used in step (c”) from the UV signal obtained in step (b); and

[0319] (c2') determining the ratio between H(sample) and H(control) to obtain the integrity of the nucleic acids (especially RNA) contained in the sample composition.

[0320] In a fifth specific example of this embodiment of the third aspect (relating to the second subgroup of the third aspect), the integrity of the control RNA is determined by the following steps:

[0321] (a') subjecting at least part of a control composition comprising a control RNA to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0322] (b') measuring at least one signal selected from a UV signal, a fluorescence signal, and an RI signal of at least one of the one or more control fractions obtained in step (a');

[0323] (c'1) calculating the area from the maximum height of a UV, fluorescence or RI peak to the end of the UV, fluorescence or RI peak from at least one signal selected from the UV signal, fluorescence signal and RI signal obtained in step (b'), thereby obtaining A 50％ (control);

[0324] (c'2) calculating the total area of ​​one peak used in step (c'1) from at least one signal selected from the UV signal, the fluorescence signal, and the RI signal obtained in step (b'), thereby obtaining A 100％ (control); and

[0325] (c'3) Determine A 50％ (control) and A 100％ (control) to obtain the integrity of the control RNA (I(control)).

[0326] In this fifth example, the integrity of the RNA contained in the sample composition can be calculated by the following steps:

[0327] (c1) calculating the area from the maximum height of the sample UV, fluorescence or RI peak corresponding to the control UV, fluorescence or RI peak used in step (c'1) to the end of the sample UV, fluorescence or RI peak from at least one signal selected from the UV signal, fluorescence signal and RI signal obtained in step (b), thereby obtaining A 50％ (sample);

[0328] (c2) calculating the total area of ​​the sample UV, fluorescence or RI peak used in step (c1) from the sample UV, fluorescence or RI signal obtained in step (b), thereby obtaining A 100％ (sample);

[0329] (c3) Determine A 50％ (sample) and A 100％ (samples), thereby obtaining I(sample); and

[0330] (c4) determining the ratio between I(sample) and I(control) to obtain the integrity of the RNA contained in the sample composition.

[0331] In a sixth specific example of this embodiment of the third aspect (relating to the second subgroup of the third aspect), the integrity of the control RNA is determined by the following steps:

[0332] (a") subjecting at least part of a control composition comprising a control RNA to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0333] (b") measuring at least one signal selected from a UV signal, a fluorescence signal, and an RI signal of at least one of the one or more control fractions obtained from step (a"); and

[0334] (c") determining the height of a UV, fluorescence or RI peak (H(control)) from at least one signal selected from the UV signal, fluorescence signal and RI signal obtained in step (b"), thereby obtaining the integrity of the control RNA.

[0335] In this sixth example, the integrity of the RNA contained in the sample composition can be calculated by the following steps:

[0336] (c1′) determining the height (H(sample)) of the sample UV, fluorescence or RI peak corresponding to the control UV, fluorescence or RI peak used in step (c″) from at least one signal selected from the UV signal, fluorescence signal and RI signal obtained in step (b); and

[0337] (c2') Determine the ratio between H(sample) and H(control) to obtain the integrity of the RNA contained in the sample composition.

[0338] In a seventh specific example of this embodiment of the third aspect (relating to the third subgroup of the third aspect), the integrity of the control RNA is determined by the following steps:

[0339] (a') subjecting at least part of a control composition comprising a control RNA to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0340] (b') measuring the UV signal of at least one of the one or more control fractions obtained from step (a');

[0341] (c'1) Calculating the area from the maximum height of a UV peak to the end of the UV peak from the UV signal obtained in step (b') to obtain A 50％ (control);

[0342] (c'2) Calculating the total area of ​​one peak used in step (c'1) from the UV signal obtained in step (b') to obtain A 100％ (control); and

[0343] (c'3) Determine A 50％ (control) and A 100％ (control) to obtain the integrity of the control RNA (I(control)).

[0344] In this seventh example, the integrity of the RNA contained in the sample composition can be calculated by the following steps:

[0345] (c1) calculating the area from the maximum height of the sample UV peak corresponding to the control UV peak used in step (c'1) to the end of the sample UV peak from the UV signal obtained in step (b), thereby obtaining A 50％ (sample);

[0346] (c2) calculating the total area of ​​the sample UV peak used in step (c1) from the sample UV signal obtained in step (b) to obtain A 100％ (sample);

[0347] (c3) Determine A 50％ (sample) and A 100％ (samples), thereby obtaining I(sample); and

[0348] (c4) determining the ratio between I(sample) and I(control) to obtain the integrity of the RNA contained in the sample composition.

[0349] In an eighth specific example of this embodiment of the third aspect (relating to the third subgroup of the third aspect), the integrity of the control RNA is determined by the following steps:

[0350] (a") subjecting at least part of a control composition comprising a control RNA to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0351] (b") measuring the UV signal of at least one of the one or more control fractions obtained from step (a"); and

[0352] (c") Determine the height of a UV peak (H(control)) from the UV signal obtained in step (b") to obtain the integrity of the control RNA.

[0353] In this eighth example, the integrity of the RNA contained in the sample composition can be calculated by the following steps:

[0354] (c1′) determining the height of the sample UV peak (H(sample)) corresponding to the control UV peak used in step (c”) from the UV signal obtained in step (b); and

[0355] (c2') Determine the ratio between H(sample) and H(control) to obtain the integrity of the RNA contained in the sample composition.

[0356] In one embodiment of the third aspect (in particular, in one embodiment of the first, second or third subgroup of the third aspect), the amount of nucleic acid (especially RNA) is determined by using (i) a nucleic acid extinction coefficient (especially an RNA extinction coefficient) or (ii) a nucleic acid calibration curve (especially an RNA calibration curve).

[0357] In one embodiment of the third aspect (in particular, in one embodiment of the first, second or third subgroup of the third aspect), the sample composition comprises nucleic acids (especially RNA) and particles, such as lipid complex particles and / or lipid nanoparticles and / or polyplex particles and / or lipid multiplex particles and / or virus-like particles, and the nucleic acids (especially RNA) are associated with the particles.

[0358] In one embodiment of the third aspect (in particular, in one embodiment of the first, second or third subgroup of the third aspect), the amount of total nucleic acid (in particular RNA) is determined by: (i) treating at least a portion of the sample composition with a releasing agent; (ii) performing steps (a) to (c) with at least the portion obtained from step (i); and (iii) determining the amount of nucleic acid (in particular RNA) as described herein (e.g., by using (i) a nucleic acid extinction coefficient (in particular an RNA extinction coefficient) or (ii) a nucleic acid calibration curve (in particular an RNA calibration curve). In this embodiment, in step (a) of the method of the first aspect, field-flow fractionation is preferably performed using a liquid phase containing a releasing agent.

[0359] In one embodiment of the third aspect (particularly, in one embodiment of the first, second or third subgroup of the third aspect), the release agent is (i) a surfactant, such as an anionic surfactant (e.g., sodium lauryl sulfate), a zwitterionic surfactant (e.g., n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), 3-14)), a cationic surfactant, a nonionic surfactant or a mixture thereof; (ii) an alcohol, such as a fatty alcohol (e.g., ethanol), or a mixture of alcohols; or (iii) a combination of (i) and (ii).

[0360] In one embodiment of the third aspect (in particular, in one embodiment of the first, second or third subgroup of the third aspect), the amount of free nucleic acid, in particular RNA, is determined by: performing steps (a) to (c) without adding a releasing agent, in particular in the absence of any releasing agent; and determining the amount of nucleic acid, in particular RNA, as described herein (e.g., using (i) a nucleic acid extinction coefficient, in particular an RNA extinction coefficient, or (ii) a nucleic acid calibration curve, in particular an RNA calibration curve).

[0361] In one embodiment of the third aspect (in particular, in one embodiment of the first, second or third subgroup of the third aspect), the amount of nucleic acids (in particular RNA) bound to the particles is determined by subtracting the amount of free nucleic acids (in particular RNA) as determined herein (e.g. by performing steps (a) to (c) without adding a releasing agent, in particular in the absence of any releasing agent; and determining the amount of nucleic acids (in particular RNA) as described herein (e.g. using (i) a nucleic acid extinction coefficient (in particular an RNA extinction coefficient) or (ii) a nucleic acid calibration curve (in particular an RNA calibration curve))) from the amount of total nucleic acids (in particular RNA) as determined herein (e.g. by (i) treating at least a portion of the sample composition with a releasing agent; (ii) performing steps (a) to (c) with at least the portion obtained from step (i); and (iii) determining the amount of nucleic acids (in particular RNA) as described herein (e.g. using (i) a nucleic acid extinction coefficient (in particular an RNA extinction coefficient) or (ii) a nucleic acid calibration curve (in particular an RNA calibration curve))).

[0362] In one embodiment of the third aspect (in particular, in one embodiment of the first, second or third subgroup of the third aspect), step (b) further comprises measuring an LS signal of at least one of the one or more sample fractions obtained from step (a), such as a dynamic light scattering (DLS) signal and / or a static light scattering (SLS), for example, a multi-angle light scattering (MALS) signal.

[0363] In one embodiment of the third aspect (particularly, in one embodiment of the first, second or third subgroup of the third aspect), the radius of gyration (R g ) value and / or hydrodynamic radius (R h ) value to determine the size of particles containing nucleic acids (especially RNA). In one embodiment of the third aspect (particularly, in one embodiment of the first, second or third subgroup of the third aspect), step (b) comprises measuring a dynamic light scattering (DLS) signal of at least one of the one or more sample fractions obtained in step (a), and step (c) comprises calculating R from the DLS signal. h In one embodiment of the third aspect (particularly, in one embodiment of the first, second or third subgroup of the third aspect), step (b) comprises measuring a static light scattering (SLS), such as a MALS signal, of at least one of the one or more sample fractions obtained in step (a), and step (c) comprises calculating R from said SLS signal. gIn one embodiment of the third aspect (particularly, in one embodiment of the first, second or third subgroup of the third aspect), step (b) comprises measuring a dynamic light scattering (DLS) signal and a static light scattering (SLS), such as a MALS signal, of at least one of the one or more sample fractions obtained in step (a), and step (c) comprises calculating R g and R h This latter embodiment generates two data sets of particle sizes containing nucleic acids (such as RNA), namely, one based on R g value, a value based on R h value.

[0364] In one embodiment of the third aspect (particularly, in one embodiment of the first, second or third subgroup of the third aspect), the R g or R h value (e.g., by calculating R from the SLS signal obtained in step (b) g value or calculate R by the DLS signal obtained from step (b) h In a first example of this embodiment (relating to the first subgroup of the third aspect), the size distribution of particles containing nucleic acids (particularly RNA) is determined by plotting the UV signal obtained from step (b) against the R value determined as described herein. g or R h value (e.g., by calculating R from the SLS signal obtained in step (b) g value or calculate R by the DLS signal obtained from step (b) h In a second example of this embodiment (relating to the second subgroup of the third aspect), the size distribution of particles containing nucleic acids (especially RNA) is determined by plotting the at least one signal selected from the UV signal, the fluorescence signal and the RI signal obtained in step (b) against the R value determined as described herein. g or R h value (e.g., by calculating R from the SLS signal obtained in step (b) g value or calculate R by the DLS signal obtained from step (b) h In a third example of this embodiment (a third subgroup of the third aspect), the size distribution of the particles containing RNA is determined by plotting the UV signal obtained from step (b) against the R value determined as described herein. g or R h value (e.g., by calculating R from the SLS signal obtained in step (b) g value or calculate R by the DLS signal obtained from step (b) hIn each of the first, second and third examples above, the size distribution of the particles containing RNA can be determined by plotting the values ​​of R g Value, R h The size distribution of particles containing nucleic acids (especially RNA) is determined based on the values ​​of R and R, or both. g Value and R h The size distribution of particles containing nucleic acids (especially RNA) is determined based on the values ​​of R. g The size distribution of the values ​​and an R-based h The size distribution of values.

[0365] In one embodiment of the third aspect (particularly, in one embodiment of the first, second or third subgroup of the third aspect), the UV, fluorescence or RI signal is converted into a cumulative weight fraction and the cumulative weight fraction is converted to R g or R h The values ​​are plotted and displayed as R g or R h The quantitative size distribution of particles containing nucleic acids, particularly RNA, is calculated from a plot of the UV, fluorescence or RI signal as a function of the value of the UV, fluorescence or RI signal. In a first example of this embodiment (relating to the first subgroup of the third aspect), the UV signal is converted into a cumulative weight fraction and the cumulative weight fraction is plotted against the R g or R h The values ​​are plotted and displayed as R g or R h In a second example of this embodiment (relating to the second subgroup of the third aspect), the quantitative size distribution of particles containing nucleic acids (especially RNA) is calculated by converting the UV, fluorescence or RI signal into cumulative weight fractions and plotting the cumulative weight fractions against R g or R h The values ​​are plotted and displayed as R g or R h The quantitative size distribution of the RNA-containing particles is calculated from a plot of the UV, fluorescence or RI signal as a function of the value of the UV, fluorescence or RI signal. In a third example of this embodiment (a third subgroup relating to the third aspect), the size distribution of the RNA-containing particles is calculated by converting the UV signal to a cumulative weight fraction and plotting the cumulative weight fraction against the R value. g or R h The values ​​are plotted and displayed as R g or R h The quantitative size distribution of the RNA-containing particles can be calculated from a plot of the UV signal as a function of the value of R. g Value, R h The quantitative size distribution of particles containing nucleic acids (especially RNA) is determined based on the values ​​of R and R, or both. gValue and R h The quantitative size distribution of particles containing nucleic acids (especially RNA) is determined based on the values ​​of R. g The quantitative size distribution of the values ​​and an R h Quantitative size distribution of values.

[0366] In one embodiment of the third aspect (particularly, in one embodiment of the first, second or third subgroup of the third aspect), the quantitative size distribution comprises D10, D50 and / or D90 values. g Value and R h The quantitative size distribution of particles containing nucleic acids (especially RNA) is determined based on the values ​​of R. g The D10, D50 and / or D90 values ​​and a set of R-based h The D10, D50 and / or D90 values ​​of the

[0367] In an embodiment of the third aspect (in particular, in an embodiment of the first, second or third subgroup of the third aspect), the one or more parameters comprise (or are) at least two, preferably at least three, parameters as specified herein (including additional optional parameters), in particular at least two, preferably at least three, parameters selected from the group consisting of: the amount of free nucleic acid (in particular RNA), the amount of nucleic acid (in particular RNA) bound to the particles, the size distribution of the particles containing nucleic acid (in particular RNA) (in particular, based on the radius of gyration (R g ) and / or the hydrodynamic radius (R h )) and quantitative size distribution of particles containing nucleic acids (especially RNA) (e.g., based on R g or R h value). If in R g Value and R h The size distribution of particles containing nucleic acids (especially RNA) is determined based on the values ​​of R. g value, a value based on R h However, according to the present invention, these two data sets of the size distribution of particles containing nucleic acids, in particular RNA, are considered as only one parameter (instead of two parameters). Furthermore, if the fractionation pattern obtained by field-flow fractionation shows more than one particle peak, the determination of the size distribution of each particle peak is considered as only one parameter (instead of one parameter for each particle peak). The same applies to the determination of the size distribution of particles containing nucleic acids, in particular RNA, in the R g Value and R h The quantitative size distribution of particles containing nucleic acids, in particular RNA, is determined based on the values.

[0368] In one embodiment of the third aspect (particularly in one embodiment of the first, second or third subgroup of the third aspect, especially in a preferred embodiment of the third subgroup of the third aspect), the one or more parameters comprise a quantitative size distribution of the particles containing nucleic acids, especially RNA (e.g. based on the radius of gyration (R g ) and / or the hydrodynamic radius (R h )) and optionally at least one parameter, such as at least two parameters, of the remaining parameters specified herein (including additional optional parameters); preferably these remaining parameters are selected from the group consisting of: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to the particles, and the size distribution of the particles containing nucleic acid (especially RNA) (e.g. based on R g or R h In one embodiment of the third aspect (particularly in one embodiment of the first, second or third subgroup of the third aspect, and particularly in a preferred embodiment of the third subgroup of the third aspect), the one or more parameters comprise a quantitative size distribution of particles containing nucleic acid (particularly RNA) (e.g. based on R g or R h value) and at least one parameter, such as at least two parameters, selected from the group consisting of the amount of free nucleic acid, in particular RNA, the amount of nucleic acid, in particular RNA, bound to the particles, and the size distribution of the particles containing nucleic acid, in particular RNA (e.g. based on R g or R h In one embodiment of the third aspect (particularly in one embodiment of the first, second or third subgroup of the third aspect, and particularly in a preferred embodiment of the third subgroup of the third aspect), the one or more parameters comprise a quantitative size distribution of particles containing nucleic acid (particularly RNA) (e.g. based on R g or R h value), the amount of free nucleic acid (especially RNA) and the amount of nucleic acid (especially RNA) bound to particles. g Value and R h The quantitative size distribution of particles containing nucleic acids (especially RNA) is determined based on the values ​​of R. g value, a value based on R hHowever, according to the present invention, these two data sets for the quantitative size distribution of particles containing nucleic acids, in particular RNA, are considered to be only one parameter (instead of two parameters). Furthermore, if the fractionation profile obtained by field-flow fractionation shows more than one particle peak, the determination of the quantitative size distribution for each particle peak is considered to be only one parameter (instead of one parameter for each particle peak). The same applies to the determination of the quantitative size distribution for particles containing nucleic acids, in particular RNA, in the R g Value and R h The size distribution of particles containing nucleic acids, in particular RNA, is determined based on the values.

[0369] In an embodiment of the third aspect (particularly in an embodiment of the first, second or third subgroup of the third aspect), the one or more parameters are determined in one cycle of steps (a) to (c).

[0370] In one embodiment of the third aspect (in particular, in one embodiment of the first, second or third subgroup of the third aspect), the amount of nucleic acid (in particular RNA), in particular free nucleic acid (in particular RNA), is determined by measuring the UV signal, for example, at a wavelength in the range of 260 nm-280 nm, such as at a wavelength of 260 nm or 280 nm, and using the extinction coefficient of nucleic acid (in particular RNA) at the corresponding wavelength (e.g., 260 nm or 280 nm).

[0371] In one embodiment of the third aspect (particularly, in one embodiment of the first, second or third subgroup of the third aspect, and particularly in a preferred embodiment of the third subgroup of the third aspect), the size distribution of the particles containing nucleic acid (particularly RNA) (e.g., based on R g or R h values) and / or quantitative size distribution of particles containing nucleic acids, in particular RNA (e.g., based on R g or R h In a preferred embodiment of the third subgroup of the third aspect, the (quantitative) size distribution of the RNA-containing particles (e.g., based on R g or R hvalue) is in the range of 10-1000 nm, for example, in the range of 15-500 nm, 20-450 nm, 25-400 nm, 30-350 nm, 40-300 nm or 50-250 nm.

[0372] In one embodiment of the third aspect (particularly in one embodiment of the first, second or third subgroup of the third aspect), the nucleic acid (especially RNA) has a length of 10-15,000 nucleotides, such as 40-15,000 nucleotides, 100-12,000 nucleotides or 200-10,000 nucleotides.

[0373] In one embodiment of the third aspect (particularly, in one embodiment of the first subgroup of the third aspect), the nucleic acid is RNA. In this embodiment and in the second or third subgroup of the third aspect, the RNA is preferably mRNA or in vitro transcribed RNA, particularly in vitro transcribed mRNA.

[0374] In one embodiment of the third aspect (in particular, in one embodiment of the first, second or third subgroup of the third aspect), measuring at least one signal selected from UV signals, fluorescence signals and RI signals, optionally LS signals, such as SLS, for example, MALS signals and / or DLS signals, is performed online, and / or step (c) is performed online.

[0375] In one embodiment of the third aspect (particularly, in one embodiment of the first, second or third subgroup of the third aspect), prior to subjecting at least a portion of the sample composition to field-flow fractionation, the at least a portion of the sample composition is diluted with a solvent or solvent mixture that prevents the formation of particle aggregates. In one embodiment, the solvent mixture is a mixture of water and an organic solvent such as formamide.

[0376] In an embodiment of the third aspect (particularly, in an embodiment of the first, second or third subgroup of the third aspect), measuring the UV signal is performed by using circular dichroism (CD) spectroscopy.

[0377] It will be appreciated that any embodiment described herein in the context of the first or second aspect may also apply to any embodiment of the third aspect.

[0378] Further implementation plans are as follows:

[0379] 1. A method for determining one or more parameters of a sample composition, wherein the sample composition comprises RNA and optionally particles, the method comprising:

[0380] (a) subjecting at least a portion of the sample composition to field-flow fractionation, thereby fractionating components contained in the sample composition according to their size to produce one or more sample fractions;

[0381] (b) measuring at least a UV signal and optionally a light scattering (LS) signal of at least one of the one or more sample fractions obtained from step (a); and

[0382] (c) calculating said one or more parameters from said UV signal and optionally from said LS signal,

[0383] The one or more parameters include RNA integrity, the total amount of RNA, the amount of free RNA, the amount of RNA associated with particles, the size of particles containing RNA, the size distribution of particles containing RNA, and the quantitative size distribution of particles containing RNA.

[0384] 2. The method of item 1, wherein the field-flow fractionation is flow field-flow fractionation, such as asymmetric flow field-flow fractionation (AF4) or hollow fiber flow field-flow fractionation (HF5).

[0385] 3. The method of claim 1 or 2, wherein step (a) is performed using a membrane having a molecular weight cutoff (MW) suitable for preventing RNA from penetrating the membrane, preferably a membrane with a MW cutoff in the range of 2 kDa to 30 kDa, such as a membrane with a MW cutoff of 10 kDa.

[0386] 4. The method of any one of items 1 to 3, wherein step (a) is performed using a polyethersulfone (PES) or regenerated cellulose membrane.

[0387] 5. The method of any one of items 1 to 4, wherein step (a) is performed using a cross-flow rate of up to 8 mL / min, preferably up to 4 mL / min, more preferably up to 2 mL / min.

[0388] 6. The method of any one of items 1-5, wherein step (a) is performed using the following cross-flow profile: 1.0-2.0 mL / min for 10 min, an exponential gradient from 1.0-2.0 mL / min to 0.01-0.07 mL / min over 30 min; 0.01-0.07 mL / min for 30 min; and 0 mL / min for 10 min.

[0389] 7. The method of any one of items 1 to 6, wherein step (a) is performed using an injection flow rate in the range of 0.05-0.35 mL / min, preferably in the range of 0.10-0.30 mL / min, more preferably in the range of 0.15-0.25 mL / min.

[0390] 8. The method of any one of items 1 to 7, wherein step (a) is performed using a detector flow rate in the range of 0.30-0.70 mL / min, preferably in the range of 0.40-0.60 mL / min, more preferably in the range of 0.45-0.55 mL / min.

[0391] 9. The method of any one of items 1 to 8, wherein the integrity of the control RNA is used to calculate the integrity of the RNA contained in the sample composition.

[0392] 10. The method of claim 9, wherein the integrity of the control RNA is determined by:

[0393] (a') subjecting at least part of a control composition comprising a control RNA to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0394] (b') measuring the UV signal of at least one of the one or more control fractions obtained from step (a');

[0395] (c'1) Calculating the area from the maximum height of a UV peak to the end of the UV peak from the UV signal obtained in step (b') to obtain A 50％ (control);

[0396] (c'2) Calculating the total area of ​​one peak used in step (c'1) from the UV signal obtained in step (b') to obtain A 100％ (control); and

[0397] (c'3) Determine A 50％ (control) and A 100％ (control) to obtain the integrity of the control RNA (I(control)).

[0398] 11. The method of claim 10, wherein the integrity of the RNA contained in the sample composition is calculated by the following steps:

[0399] (c1) calculating the area from the maximum height of the sample UV peak corresponding to the control UV peak used in step (c'1) to the end of the sample UV peak from the sample UV signal obtained in step (b), thereby obtaining A 50％ (sample);

[0400] (c2) calculating the total area of ​​the sample UV peak used in step (c1) from the sample UV signal obtained in step (b) to obtain A 100％ (sample);

[0401] (c3) Determine A 50％(sample) and A 100％ (samples), thereby obtaining I(sample); and

[0402] (c4) determining the ratio between I(sample) and I(control) to obtain the integrity of the RNA contained in the sample composition.

[0403] 12. The method of claim 9, wherein the integrity of the computational control RNA is determined by:

[0404] (a") subjecting at least part of a control composition comprising a control RNA to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0405] (b") measuring the UV signal of at least one of the one or more control fractions obtained from step (a"); and

[0406] (c") Determine the height of a UV peak (H(control)) from the UV signal obtained in step (b") to obtain the integrity of the control RNA.

[0407] 13. The method of claim 12, wherein the integrity of the RNA contained in the sample composition is calculated by the following steps:

[0408] (c1′) determining the height of the sample UV peak (H(sample)) corresponding to the control UV peak used in step (c”) from the UV signal obtained in step (b); and

[0409] (c2') Determine the ratio between H(sample) and H(control) to obtain the integrity of the RNA contained in the sample composition.

[0410] 14. The method of any one of items 1 to 13, wherein the amount of RNA is determined by using (i) the RNA extinction coefficient or (ii) an RNA calibration curve.

[0411] 15. The method of any one of items 1 to 14, wherein the sample composition comprises RNA and particles, such as lipid complex particles and / or lipid nanoparticles and / or polyplex particles and / or lipid multiplex particles and / or virus-like particles, to which the RNA is bound.

[0412] 16. The method of claim 15 , wherein the amount of total RNA is determined by: (i) treating at least a portion of the sample composition with a releasing agent; (ii) performing steps (a)-(c) using at least the portion obtained from step (i); and (iii) determining the amount of RNA as described in claim 14 .

[0413] 17. The method of claim 16, wherein in step (a), field-flow fractionation is performed using a liquid phase containing a releasing agent.

[0414] 18. The method of item 16 or 17, wherein the release agent is (i) a surfactant, such as an anionic surfactant (e.g., sodium lauryl sulfate), a zwitterionic surfactant (e.g., n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), 3-14)), a cationic surfactant, a nonionic surfactant or a mixture thereof; (ii) an alcohol, such as a fatty alcohol (e.g., ethanol), or a mixture of alcohols; or (iii) a combination of (i) and (ii).

[0415] 19. The method of any one of items 15-18, wherein the amount of free RNA is determined by: performing steps (a)-(c) without adding a releasing agent, in particular in the absence of any releasing agent; and determining the amount of RNA as described in item 14.

[0416] 20. The method of any one of items 15-19, wherein the amount of RNA bound to the particles is determined by subtracting the amount of free RNA determined as in item 19 from the amount of total RNA determined as in any one of items 16-18.

[0417] 21. The method of any one of items 15-20, wherein step (b) further comprises measuring an LS signal of at least one of the one or more sample fractions obtained from step (a), such as a dynamic light scattering (DLS) signal and / or a static light scattering (SLS), for example, a multi-angle light scattering (MALS) signal.

[0418] 22. The method of claim 21, wherein the radius of gyration (R g ) value and / or hydrodynamic radius (R h ) values ​​to determine the size of the RNA-containing particles.

[0419] 23. The method of item 21, wherein the experimentally determined R g and / or R h The values ​​are smoothed, preferably by dividing the experimentally determined or calculated R g or R h Fit the values ​​to a polynomial or linear function and recalculate R based on the polynomial or linear fit g or R h value.

[0420] 24. The method of any one of items 21 to 23, wherein the UV signal obtained from step (b) is compared to the R g or R hThe values ​​were plotted to determine the size distribution of the RNA-containing particles.

[0421] 25. The method of any one of items 21 to 24, wherein the UV signal is converted into a cumulative weight fraction and the cumulative weight fraction is plotted against R g or R h The values ​​are plotted and displayed as R g The quantitative size distribution of RNA-containing particles was calculated from a plot of the UV signal as a function of the values.

[0422] 26. The method of claim 25, wherein the quantitative size distribution comprises D10, D50 and / or D90 values.

[0423] 27. The method of any one of items 22 to 26, wherein step (b) comprises measuring a dynamic light scattering (DLS) signal of at least one of the one or more sample fractions obtained from step (a), and step (c) comprises calculating R from the DLS signal. h value.

[0424] 28. The method of any one of items 15-27, wherein the one or more parameters include (or are) at least two, preferably at least three, parameters selected from the following: the amount of free RNA, the amount of RNA bound to particles, the size distribution of particles containing RNA, and the quantitative size distribution of particles containing RNA.

[0425] 29. The method of any one of items 15 to 28, wherein the amount of RNA, in particular free RNA, is determined by measuring the UV signal at 260 nm and using the RNA extinction coefficient at 260 nm or by measuring the UV signal at 280 nm and using the RNA extinction coefficient at 280 nm.

[0426] 30. The method of any one of items 1 to 29, wherein the size distribution of the RNA-containing particles and / or the quantitative size distribution of the RNA-containing particles is in the range of 20-1500 nm, such as 30-1200 nm, 40-1100 nm, 50-1000, 60-900 nm, 70-800 nm, 80-700 nm, 90-600 nm or 100-500 nm, for example in the range of 10-1000 nm, 15-500 nm, 20-450 nm, 25-400 nm, 30-350 nm, 40-300 nm or 50-250 nm.

[0427] 31. The method of any one of items 1 to 30, wherein the RNA is 10-15,000 nucleotides in length, such as 40-15,000 nucleotides, 100-12,000 nucleotides, or 200-10,000 nucleotides in length.

[0428] 32. The method according to any one of items 1 to 31, wherein the RNA is in vitro transcribed RNA, in particular in vitro transcribed mRNA.

[0429] 33. The method of any one of items 1 to 32, wherein measuring the UV signal, optionally the LS signal, such as SLS, e.g., MALS signal and / or DLS signal, is performed online, and / or step (c) is performed online.

[0430] 34. The method of any one of items 15-33, wherein before subjecting at least a portion of the sample composition to field-flow fractionation, at least a portion of the sample composition is diluted with a solvent or solvent mixture that is capable of preventing the formation of particle aggregates.

[0431] 35. The process of claim 34, wherein the solvent mixture is a mixture of water and an organic solvent such as formamide.

[0432] 35a. The method of any one of items 1 to 35, wherein measuring the UV signal is performed by using circular dichroism (CD) spectroscopy.

[0433] 36. A method of analyzing the effect of varying one or more reaction conditions in providing a composition comprising RNA and optionally particles, the method comprising:

[0434] (A) providing a first composition comprising RNA and optionally particles;

[0435] (B) providing a second composition comprising RNA and optionally particles, wherein providing the second composition differs from providing the first composition only in one or more reaction conditions;

[0436] (C) subjecting a portion of the first composition to the method of any one of items 1-35 and 35a, thereby determining one or more parameters of the first composition;

[0437] (D) subjecting a corresponding portion of the second composition to the method used in step (C), thereby determining one or more parameters of the second composition; and

[0438] (E) comparing one or more parameters of the first composition obtained in step (C) with the corresponding one or more parameters of the second composition obtained in step (D).

[0439] 37. The method of claim 36, wherein the one or more reaction conditions include any one of: salt concentration / ionic strength (e.g., 2 mM NaCl or 100 mM NaCl); temperature (e.g., low temperature (e.g., -20°C) or high temperature (e.g., 50°C)); pH or buffer concentration; light / radiation; oxygen; shear force; pressure; freeze / thaw cycles; drying / recovery cycles; addition of excipients (e.g., stabilizers and / or chelating agents); type and / or source of particle-forming compounds (particularly lipids and / or polymers, e.g., cationic lipids versus cationic polymers, cationic lipids versus zwitterionic lipids, or PEGylated lipids versus non-PEGylated lipids); charge ratio; physical state; and the ratio of RNA to particle-forming compounds (particularly lipids and / or polymers).

[0440] 38. Use of field-flow fractionation in determining one or more parameters of a sample composition comprising RNA and optionally particles, wherein the one or more parameters include RNA integrity, the total amount of RNA, the amount of free RNA, the amount of RNA associated with particles, the size of the RNA-containing particles (such as the hydrodynamic radius of the RNA-containing particles), the size distribution of the RNA-containing particles, and the quantitative size distribution of the RNA-containing particles.

[0441] 39. The use according to item 38, wherein the field-flow classification comprises:

[0442] (a) subjecting at least a portion of the sample composition to field-flow fractionation, thereby fractionating components contained in the sample composition according to their size to produce one or more sample fractions;

[0443] (b) measuring at least a UV signal and optionally a light scattering (LS) signal of at least one of the one or more sample fractions obtained from step (a); and

[0444] (c) calculating the one or more parameters from the UV signal and optionally from the LS signal.

[0445] 40. The use of item 38 or 39, wherein the field-flow fractionation is flow field-flow fractionation, such as asymmetric flow field-flow fractionation (AF4) or hollow fiber flow field-flow fractionation (HF5).

[0446] 41. The use of any one of items 38-40, wherein the field-flow fractionation uses a membrane with a molecular weight cut-off (MW) suitable for preventing RNA from penetrating the membrane, preferably a membrane with a MW cut-off in the range of 2kDa-30kDa, such as a membrane with a MW cut-off of 10kDa.

[0447] 42. The use according to any one of items 38 to 41, wherein the field-flow fractionation uses a polyethersulfone (PES) or regenerated cellulose membrane.

[0448] 43. The use according to any one of items 39 to 42, wherein step (a) is carried out using:

[0449] (I) a crossflow rate of up to 8 mL / min, preferably up to 4 mL / min, more preferably up to 2 mL / min, such as the following crossflow profile: 1.0-2.0 mL / min for 10 min, an exponential gradient from 1.0-2.0 mL / min to 0.01-0.07 mL / min over 30 min; 0.01-0.07 mL / min for 30 min; and 0 mL / min for 10 min; and / or

[0450] (II) an injection rate in the range of 0.05-0.35 mL / min, preferably in the range of 0.10-0.30 mL / min, more preferably in the range of 0.15-0.25 mL / min; and / or

[0451] (III) A detector flow rate in the range of 0.30-0.70 mL / min, preferably in the range of 0.40-0.60 mL / min, more preferably in the range of 0.45-0.55 mL / min.

[0452] 44. The use according to any one of items 38 to 43, wherein the integrity of the RNA contained in the sample composition is determined using the integrity of a control RNA.

[0453] 45. The method of claim 44, wherein the integrity of the control RNA is determined by:

[0454] (a') subjecting at least part of a control composition comprising a control RNA to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0455] (b') measuring the UV signal of at least one of the one or more control fractions obtained from step (a');

[0456] (c'1) Calculating the area from the maximum height of a UV peak to the end of the UV peak from the UV signal obtained in step (b') to obtain A 50％ (control);

[0457] (c'2) Calculating the total area of ​​one peak used in step (c'1) from the UV signal obtained in step (b') to obtain A 100％ (control); and

[0458] (c'3) Determine A 50％ (control) and A100％ (control) to obtain the integrity of the control RNA (I(control)).

[0459] 46. ​​The method of claim 45, wherein the integrity of the RNA contained in the sample composition is calculated by the following steps:

[0460] (c1) calculating the area from the maximum height of the sample UV peak corresponding to the control UV peak used in step (c'1) to the end of the sample UV peak from the sample UV signal obtained in step (b), thereby obtaining A 50％ (sample);

[0461] (c2) calculating the total area of ​​the sample UV peak used in step (c1) from the sample UV signal obtained in step (b) to obtain A 100％ (sample);

[0462] (c3) Determine A 50％ (sample) and A 100％ (samples), thereby obtaining I(sample); and

[0463] (c4) determining the ratio between I(sample) and I(control) to obtain the integrity of the RNA contained in the sample composition.

[0464] 47. The method of claim 44, wherein the integrity of the control RNA is determined by the following steps:

[0465] (a") subjecting at least part of a control composition comprising a control RNA to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions;

[0466] (b") measuring the UV signal of at least one of the one or more control fractions obtained from step (a"); and

[0467] (c") Determine the height of a UV peak (H(control)) from the UV signal obtained in step (b") to obtain the integrity of the control RNA.

[0468] 48. The method according to claim 47, wherein the integrity of the RNA contained in the sample composition is calculated by the following steps:

[0469] (c1′) determining the height of the sample UV peak (H(sample)) corresponding to the control UV peak used in step (c”) from the UV signal obtained in step (b); and

[0470] (c2') Determine the ratio between H(sample) and H(control) to obtain the integrity of the RNA contained in the sample composition.

[0471] 49. The use according to any one of items 38 to 48, wherein the amount of RNA is determined by using (i) the RNA extinction coefficient or (ii) an RNA calibration curve.

[0472] 50. The use of any one of items 39-49, wherein the sample composition comprises RNA and particles, such as lipid complex particles and / or lipid nanoparticles and / or polyplex particles and / or lipid multiplex particles and / or virus-like particles, the RNA being associated with the particles and / or the particles containing RNA.

[0473] 51. The use of item 50, wherein the amount of total RNA is determined by: (i) treating at least a portion of the sample composition with a releasing agent; (ii) performing steps (a)-(c) using at least the portion obtained from step (i); and (iii) determining the amount of RNA as described in item 49.

[0474] 52. The use according to claim 51, wherein in step (a), field-flow fractionation is performed using a liquid phase containing a releasing agent.

[0475] 53. The use of item 51 or 52, wherein the release agent is (i) a surfactant, such as an anionic surfactant (e.g., sodium lauryl sulfate), a zwitterionic surfactant (e.g., n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), 3-14)), a cationic surfactant, a nonionic surfactant or a mixture thereof; (ii) an alcohol, such as a fatty alcohol (e.g., ethanol), or a mixture of alcohols; or (iii) a combination of (i) and (ii).

[0476] 54. The use of any one of items 50-53, wherein the amount of free RNA is determined by: performing steps (a)-(c) without adding a releasing agent, in particular in the absence of any releasing agent; and determining the amount of RNA as described in item 49.

[0477] 55. The use of any one of items 50-54, wherein the amount of RNA bound to the particles is determined by subtracting the amount of free RNA determined as in item 53 from the amount of total RNA determined as in item 51-53.

[0478] 56. The use of any one of items 50-55, wherein step (b) further comprises measuring an LS signal of at least one of the one or more sample fractions obtained from step (a), such as a dynamic light scattering (DLS) signal and / or a static light scattering (SLS), for example, a multi-angle light scattering (MALS) signal.

[0479] 57. The use of item 56, wherein the radius of gyration (R g ) value and / or hydrodynamic radius (R h ) values ​​to determine the size of the RNA-containing particles.

[0480] 58. The use of item 57, wherein the experimentally determined R g and / or R h The values ​​are smoothed, preferably by dividing the experimentally determined or calculated R g or R h Fit the values ​​to a polynomial or linear function and recalculate R based on the polynomial or linear fit g or R h value.

[0481] 59. The use of any one of items 56 to 58, wherein the UV signal obtained from step (b) is compared with the R g or R h The values ​​were plotted to determine the size distribution of the RNA-containing particles.

[0482] 60. The use of any one of items 56 to 59, wherein the UV signal is converted into a cumulative weight fraction and the cumulative weight fraction is converted to R g or R h The values ​​are plotted and displayed as R g or R h The quantitative size distribution of RNA-containing particles was calculated from a plot of the UV signal as a function of the values.

[0483] 61. The use of item 60, wherein the quantitative size distribution comprises D10, D50 and / or D90 values.

[0484] 62. The use of any one of items 57-61, wherein step (b) further comprises measuring a dynamic light scattering (DLS) signal of at least one of the one or more sample fractions obtained from step (a), and step (c) comprises calculating R from the DLS signal. h value.

[0485] 63. The use of any one of items 50-62, wherein the one or more parameters include (or are) at least two, preferably at least three, parameters selected from the following: the amount of free RNA, the amount of RNA associated with particles, the size distribution of particles containing RNA, and the quantitative size distribution of particles containing RNA.

[0486] 64. The use of any one of items 50 to 63, wherein the amount of RNA, in particular free RNA, is determined by measuring the UV signal at 260 nm and using the RNA extinction coefficient at 260 nm or by measuring the UV signal at 280 nm and using the RNA extinction coefficient at 280 nm.

[0487] 65. The use of any of items 38 to 64, wherein the size distribution of the RNA-containing particles and / or the quantitative size distribution of the RNA-containing particles is in the range of 20-1500 nm, such as 30-1200 nm, 40-1100 nm, 50-1000, 60-900 nm, 70-800 nm, 80-700 nm, 90-600 nm or 100-500 nm, for example in the range of 10-1000 nm, 15-500 nm, 20-450 nm, 25-400 nm, 30-350 nm, 40-300 nm or 50-250 nm.

[0488] 66. The use of any one of items 38 to 64, wherein the RNA has a length of 10-15,000 nucleotides, such as 40-15,000 nucleotides, 100-12,000 nucleotides, or 200-10,000 nucleotides.

[0489] 67. The use according to any one of items 38 to 65, wherein the RNA is in vitro transcribed RNA, in particular in vitro transcribed mRNA.

[0490] 68. The use of any one of items 39-67, wherein measuring the UV signal, optionally the LS signal, such as SLS, for example, MALS signal and / or DLS signal, is performed online, and / or step (c) is performed online.

[0491] 69. The use of any one of items 39-68, wherein before subjecting at least part of the sample composition to field-flow fractionation, at least part of the sample composition is diluted with a solvent or solvent mixture that is capable of preventing the formation of particle aggregates.

[0492] 70. The use according to item 69, wherein the solvent mixture is a mixture of water and an organic solvent such as formamide.

[0493] 70a. The use of any one of items 39-70, wherein measuring the UV signal is performed by using circular dichroism (CD) spectroscopy.

[0494] In a fourth aspect, the present disclosure provides a data processing device / system comprising means for performing any method of the present disclosure, in particular the method of the first aspect (e.g., the method defined in any of items 1-35 and 35a) and / or the method of the second aspect (e.g., the method defined in item 36 or 37).

[0495] In a fifth aspect, the present disclosure provides a computer program suitable for performing any method of the present disclosure, in particular the method of the first aspect (for example, the method defined in any of items 1-35 and 35a) and / or the method of the second aspect (for example, the method defined in item 36 or 37).

[0496] In a sixth aspect, the present disclosure provides a computer-readable storage medium or data carrier comprising the program of the fifth aspect of the present disclosure.

[0497] Other aspects of the disclosure are disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0498] Figure 1 Shown is the time-flow spectrum of an asymmetric flow field-flow fractionation (AF4) separation with a detector flow (Vd) of 0.5 mL / min and a crossflow (Vx) starting at 1.5 mL / min and decreasing exponentially to 0.04 mL / min.

[0499] Figure 2 An overview of the preferred calculation procedure for estimating relative RNA integrity is shown. Example of an AF4 fractionation profile after RNA isolation using UV signal at 260 nm. A) Limits for calculation of control RNA. B) Limits for calculation of total RNA peak area. C) Limits for calculation of slightly degraded RNA. D) Limits for calculation of total slightly degraded RNA peak area.

[0500] Figure 3 RNA quantification without standards is shown: A) RNA stock solution at different injection volumes was analyzed by AF4-UV-RI. The peak area under the curve (UV solid line; RI dashed line) was plotted against injection volume and fitted with a linear regression. B) Serial dilutions of RNA were measured by AF4-UV-RI using the same injection volume and analyzed as in A).

[0501] Figure 4 shows the quantification of degraded RNA using AF4-UV-RI without standards: A) Representative AF4 fractionation profiles showing different thermally degraded RNAs and untreated RNA (curves represent UV signals at 260 nm). B) Applying the Lambert-Beer law, the UV signals of different degraded RNAs are directly related to their concentration.

[0502] Figure 5 Shown is a representative fractionation profile obtained from a sample particle composition (containing lipid and RNA at a molar ratio of 1.3 / 2) separated by the AF4 method disclosed herein. The solid line represents the light scattering (LS) signal at a 90° angle and indicates the particle peak (t = ~35 min), while the dashed line represents the UV signal (recorded at 260 nm) and reflects bound (t = ~38 min) and unbound RNA (t = ~20 min).

[0503] Figure 6 shows quantitative RNA integrity measurements of non-formulated RNA: A) Representative AF4 fractionation profiles of different thermally degraded RNAs (n=3; curves represent UV signals at 260 nm). B) Summary of the average calculated relative integrity of four thermally degraded RNAs of different lengths (RNA#1-4; size: 986-1688 nt). Error bars represent standard deviations (n=3). C) Representative AF4 fractionation profiles obtained from RNA#2 using different ratios (untreated, fully thermally degraded, and a 50:50 mixture of untreated and fully thermally degraded). D) Validation experiment: Different RNAs (RNA#1-3, 986-1688 nt) were thermally degraded and mixed in a specified manner (for AF4 fractionation profile of mixture 4, see Figure 6C ) and analyzed by AF4-UV measurement. The bar graph represents the relative RNA integrity determined using the AF4 method disclosed herein (dark, medium and light grey bars) compared to the theoretically calculated value (black bars).

[0504] Figure 7 demonstrates the suitability of UV signals for RNA quantification compared to using fluorescent dyes for RNA quantification (proof of concept). A) The UV peak and fluorescence (FS) integral of a sample composition (comprising RNA and fluorescently labeled particles) correlate with the corresponding amount of total RNA in the sample composition. B) The calculated ratio of UV to FS peak areas for the sample composition was found to be constant over a wide mass range (1-15 μg total RNA in the sample composition).

[0505] FIG8 shows UV ratio as a parameter for RNA sample composition. A) UV peak integrals for free RNA and particle-bound RNA relative to the corresponding nominal total RNA amount contained in the sample composition. B) Calculation of UV ratio for free RNA (peak) and bound RNA (peak).

[0506] Figure 9 shows proof of concept for quantifying particle size distribution by AF4-UV-MALS. A) Representative AF4 fractionation profile of a sample composition (RNA and Atto594-labeled particles): the dashed line represents the UV trace recorded at 260 nm, and the solid line represents the fluorescence signal (FS) emitted at 624 nm. B) The UV / FS ratio (dashed line) was calculated and plotted against the radius of gyration (R g ) and the recorded UV signal (highlighted grey peak) from the particle peak fraction (elution time: 22-60 min). R changes below 50% of the UV / FS ratio were plotted. g The region is highlighted (box). R between 50 and 300 nm g The UV / FS ratio varies little over the range and gives reliable magnitude values. g The value is affected by the RNA signal. A larger R g The values ​​are affected by scattering. In general, these affected R g Values ​​were less than 10% of the total signal. C) Quantitative quality parameters (D10, D50, D90) were calculated based on the cumulative weight fraction analysis using the fluorescence emission at 624 nm and the UV signal at 260 nm.

[0507] Figure 10 A representative AF4 fractionation profile of the sample composition (RNA and particles) is shown, using the LS signal at 90° and UV detection at 260 nm. Calculated radius of gyration (R g ) values ​​(grey squares) are derived from multi-angle light scattering (MALS) using Berry plots, while the hydrodynamic radius (R h ) values ​​were derived from online dynamic light scattering (DLS; grey circles).

[0508] Figure 11 Figure 4 shows the quantification of particle size distribution in a complex sample composition using AF4-UV-MALS. A) shows the AF4-UV-MALS elution profile of the sample composition (RNA and particles), the UV signal at 260 nm for RNA detection (dashed line), and the light scattering signal at 90° (solid line). The corresponding radius of gyration (R) from the MALS signal is shown. g ) values ​​are shown as black dots. B) Experimentally determined RMS values ​​of the particle peak (elution time: 26-55 min) were fitted to a polynomial equation (light grey line). C) UV signal (solid line) was plotted as the R of the polynomial fit. g Function of value (see Figure 11 B), and the corresponding cumulative weight fraction is plotted as a function of the UV signal (dashed line).

[0509] FIG12 shows the separation and qualitative analysis of different sample compositions (prepared by mixing lipids and RNA at different lipid / RNA ratios (0.1-0.9) with 100 mM NaCl) using the AF4 method disclosed herein. A) For each different sample composition, an overlay shows the UV signal (at 260 nm), the light scattering signal (at 90°), and the corresponding radius of gyration (R) calculated using a Berry plot. g ) value. B) R calculated from the MALS signal g The values ​​are plotted against the appropriate cumulative weight fraction analysis and the corresponding D90 values ​​are then calculated. g (D90) values ​​are plotted as a function of lipid / RNA ratio with 100 mM NaCl (black dots) or without NaCl (open dots).

[0510] Figure 13 It is shown that by dividing the hydrodynamic radius (R h ) value and R g The values ​​are correlated to estimate the “shape factor.” These values ​​are fit to a linear regression and the resulting slope provides information about the particle shape.

[0511] Figure 14 Figure 2 shows the separation and characterization of different particle compositions (LPX, LNP, polyplex particles (PLX), liposomes, VLPs + LPX) by the AF4 method disclosed herein. Shown is the AF4-UV-MALS-DLS separation / detection. The LS at a 90° angle is shown as a solid line and represents the particle peak. The dashed line represents the UV signal recorded at 260 nm (for RNA detection). The radius of gyration (R g ) values ​​(black dots) are derived from multi-angle light scattering (MALS) signals using Zimm plots. Dynamic light scattering (DLS; gray dots) provides the hydrodynamic radius (R h). Individual particle peak fractions are highlighted by grey bars. A) Representative fractionation profile of an LPX sample containing lipids and RNA in a molar ratio of 1.3 / 2 after AF4-UV-MALS-DLS separation / detection. B) Representative fractionation profile of a composition comprising two types of particles (short RNA-LPX:VLP, 1:1 mixture). C) Representative fractionation profile of a liposome sample (positively charged liposomes composed of DOTMA and DOPE in a molar ratio of 2 / 1). D) Representative fractionation profile of an LPX sample (positively charged LPX containing DOTMA and cholesterol, and RNA in a molar ratio of 4 / 1). E) Representative fractionation profile of a lipid nanoparticle (LNP) sample composed of DODMA, cholesterol, DOPE, PEG (molar ratios of 1.2 / 1.44 / 0.3 / 0.06), and RNA in a molar ratio of 3 / 1. F) Representative fractionation profiles of particles containing JetPEI polymer and IVT-RNA or saRNA at a particle to RNA ratio of 12 / 1.

[0512] Figure 15 Shown is an analysis of RNA behavior in the presence of ions (sodium chloride). Shown is an exemplary AF4 fractionation pattern (light scattering signal at 90°) from non-formulated RNA at different sodium chloride concentrations (0-50 mM). Radius of gyration (R g ) values ​​were derived from multi-angle light scattering (MALS) using Zimm plots.

[0513] Figure 16 shows the characterization of RNA after treatment with sodium chloride. A) shows R derived from cumulative weight fraction analysis at different sodium chloride concentrations (0-50 mM). g (D50) value. B) RNA R g (D50) value (from Figure 16A ) versus sodium chloride concentration and calculate the ratio (mM sodium chloride to nm R g The linear fit of the ratio from 0 to 10 mM NaCl values ​​is represented by the bold line, while the dotted line represents the fit from 10 to 50 mM NaCl. The grey and black lines represent examples measured with two different RNA concentrations.

[0514] Figure 17Quantification of free / unbound RNA in a complex sample composition is shown. A) Using the AF4 method disclosed herein, different amounts of free RNA (1-15 μg) were detected in a composition without particles by UV absorption at 260 nm. The RNA amount was plotted against the area under the curve of the respective UV peak (AUC*min) to generate a linear calibration curve. B) Different amounts of particle compositions (containing 1-15 μg total RNA) were analyzed by the AF4 method. The superimposed AF4 fractionation profile shows the UV signal at 260 nm. The first peak (elution time: ~20 min) corresponds to free RNA, while the second peak (elution time: ~38 min) corresponds to particles (bound RNA). The amount of free, unbound RNA in the particle composition can be calculated relative to a reference RNA (=100%) (see Figure 17 A). C) To demonstrate the linearity of the method, free RNA (see Figure 17 B) and reference, naked RNA (see Figure 17 A) UV peak integral plotted as a function of different RNA amounts (1-15 μg). D) As a second, preferred procedure for quantifying free RNA (direct method), an unbound RNA peak is defined and the RNA amount can be calculated directly using the specific extinction coefficient of RNA.

[0515] Figure 18 Analysis of the amount of free RNA in sample compositions with different physicochemical behaviors is shown. AF4-UV fractionation profiles are shown for A) particle compositions without NaCl ((DOTMA / DOPE 2 / 1) / RNA complexes mixed at variable charge ratios (0.1-0.9)) or B) particle compositions with 100 mM NaCl. C) Graph of the calculated percentage of unbound RNA (mol / mol) with 100 mM NaCl (black circles) and without NaCl (open circles) using AF4-UV detection at 260 nm. All mixtures were prepared in duplicate and measured at least in duplicate. Error bars represent standard deviations. D) Graph of the unbound RNA concentration (μg / mL) with 100 mM NaCl (black circles) and without NaCl (open circles) calculated using the extinction coefficient of RNA at 260 nm.

[0516] Figure 19Quantification of total RNA in a particle composition is shown. A) AF4 fractionation profile of Zwittergent-treated, naked RNA isolated by the AF4 method disclosed herein. The UV signal at 260 nm is represented by the black line, and the LS signal at 90° is represented by the dashed line. B) Representative fractionation profile of a particle composition with UV detection (solid line), free RNA (highlighted in gray), and bound RNA (second peak), with the LS signal at a 90° angle (dashed line). C) Corresponding AF4 fractionation profile of an RNA composition in which the particles have been solubilized using a releasing agent (a liquid phase containing 0.1% Zwittergent), with UV detection (solid line) and light scattering at 90° (dashed line). D) Direct quantification of naked and total RNA after treatment with a releasing agent (Zwittergent).

[0517] Figure 20 shows the integrity of free and total RNA in a sample composition containing RNA and particles. A) UV traces of particles separated using the AF4 method disclosed herein, wherein the particles have different RNA integrity (untreated RNA: solid black line; partially heat-degraded RNA: dotted line; mixture (50% untreated and 50% fully degraded mixed in a defined manner): dashed line; fully degraded RNA in the particles: solid gray line). B) Quantification of intact free RNA (dark gray) as well as total free RNA (black) and fully degraded (light gray) free RNA in the particles. C) UV trace of dissolved particles after AF4 separation (using a releasing agent in the liquid phase). D) Determined integrity of free and total RNA analysis in the particles measured by AF4-UV. The bar graph represents the relative RNA integrity of the free RNA (gray bars) compared to the determined integrity of the total RNA value in the particles (black bars).

[0518] Figure 21 A scheme showing how to determine different fractions of RNA (total, bound, encapsulated, accessible, surface, and unbound RNA) by the AF4 method disclosed herein. For example, the AF4 method can be used to quantify accessible RNA and / or surface RNA using the fluorescence emission signal of an intercalating dye (e.g., GelRED). Quantitative combinations of free (unbound), total, and accessible RNA can be used to calculate encapsulated, bound, and surface RNA. The fluorescence emission of GelRED at 600 nm is enhanced by intercalating RNA.

[0519] Figure 22 shows (A) the linearity of fluorescence detection using the AF4 method disclosed herein; (B) a bar graph showing the relative amounts of accessible (black bars) and encapsulated (grey bars) RNA; and (C) a comparison of the relative amounts of free RNA in particle compositions, where the amounts were determined using different RNA detection methods: UV absorbance at 260 nm (black bars) and fluorescence emission signal (FS) at 600 nm (grey bars).

[0520] Figure 23 Figure 2 shows the analysis of RNA integrity using the AF4 method disclosed herein without the use of reference RNA. A) Shown is an exemplary AF4 fractionation profile of a long saRNA with LS signal at 90° (dotted line) and UV signal at 260 nm (solid line). The thick black line represents the molecular weight curve derived from the MALS signal. B) For better overview, Figure 23 In the upper panel of B, only the molecular weight curve from (A) is shown as a solid line. The limits for the total RNA peak (Peak 1) were set based on the total UV peak signal (i.e., from t = 10 min to t = 40 min). Here, the limits for the "intact" RNA peak (Peak 2) were set by the first derivative of the molecular weight curve (derived from MALS) as follows. The first derivative of the molecular weight curve was calculated ( Figure 23 B (dotted line in the figure below). The more horizontal part of the molecular weight curve reflects the retention time, where the fraction of undegraded RNA is present. On this basis, the integration limit can be selected and the amount of undegraded RNA in the sample can be calculated.

[0521] FIG24 shows quantitative analysis of free and bound RNA using UV to determine particle size distribution, specifically the cumulative RNA weight fraction, the RNA mass in the RNA lipid complex (LPX) fractions, and the RNA copy number per LPX fraction. A) Representative AF4 fractionation profile of an RNA LPX sample composition is shown with the LS signal at 90° (solid line) and the UV signal at 260 nm (dashed line). The UV signal shows two peaks, the first representing the amount of free, unbound RNA and the second from the RNA-containing LPX nanoparticles. The UV signal directly represents the amount of RNA in the different fractions as a function of elution time. The radius of gyration (R) is the radius of gyration of the sample. g , thick line) is derived from the MALS signal. B) shows the Figure 24A The UV signal at 260 nm (dashed line) is shown, and the solid line shows the cumulative weight fraction based on the area under the UV signal. C) shows the weight fraction of the particles of different R g The amount of RNA bound in the RNALPX sample composition was calculated using the absorbance at 260 nm in the fraction (Δt = 1 min). g The RNA amount of the fraction was calculated using only the LPX peak (i.e., Figure 24A and 24B The second peak starts at t = 24 min and ends at t = 60 min. D) shows the peak from Figure 24C The results shown are calculated for each R g Calculated RNA copy number of fractions (bars, left y-axis). g The calculated particle numbers of the fractions are represented by the corresponding dotted curves (second right y-axis).

[0522] Figure 25 illustrates the feasibility of using circular dichroism (CD) spectroscopy in the AF4 method disclosed herein. A) Shown is a representative AF4 fractionation profile of an RNA lipid complex (LPX) preparation with an LS signal at a 90° angle (solid line) and a CD signal recorded at 260 nm (dotted line), where the latter represents unbound RNA (first peak; t = 18 min) and bound RNA (second peak; t = 35 min). B) Calibration curves for naked RNA generated using UV detection at 260 nm and CD detection at 260 nm in parallel. The peak area under the curve (CD: solid squares and solid line; UV: solid triangles and dotted line) is plotted against the amount of injected RNA. The ratio of the peak area of ​​the CD and UV signals is shown as points (second right y-axis). C) Analysis of different amounts of RNA LPX samples (2-15 μg) using the AF4 method. The area under the curve (AUC) of the CD signal from the appropriate naked RNA was correlated with the appropriate total AUC CD signal, where the respective CD peak AUC values ​​were plotted against the amount of RNA, resulting in a linear fit (R 2 = 0.998). The relative amounts (%) of unbound RNA (open squares) and bound RNA (open circles) in the RNA LPX sample compositions were determined by relating the amounts of unbound RNA and bound RNA to the amount of total RNA. Detailed Description of the Invention

[0524] Although the present disclosure is further described in more detail below, it should be understood that the present disclosure is not limited to the specific methods, protocols and reagents described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the present disclosure, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0525] The elements of the present disclosure are described in more detail below. These elements are listed with specific embodiments, however, it should be understood that they can be combined in any manner and in any number to produce additional embodiments. The various described examples and preferred embodiments should not be understood to limit the present disclosure to only the explicitly described embodiments. Such descriptions should be understood to support and cover embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. In addition, unless the context indicates otherwise, any permutation and combination of all described elements in this application should be considered to be disclosed by the description of this application. For example, if in a preferred embodiment of the method of the present disclosure AF4 is used as field-flow fractionation, and in another preferred embodiment of the method of the present disclosure the nucleic acid (such as RNA) is in vitro transcribed RNA, then in another preferred embodiment of the method of the present disclosure AF4 is used as field-flow fractionation and the nucleic acid (such as RNA) is in vitro transcribed RNA.

[0526] Preferably, the terms used herein are as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", HGW Leuenberger, B. Nagel, and H. Definitions as described in Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0527] The practice of the present disclosure will employ, unless otherwise indicated, conventional chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques explained in the literature in the art (see, e.g., Organikum, Deutscher Verlag der Wissenschaften, Berlin 1990; Streitwieser / Heathcook, "Organische Chemie", VCH, 1990; Beyer / Walter, "Lehrbuch der Organischen Chemie", S. Hirzel Verlag Stuttgart, 1988; Carey / Sundberg, "Organische Chemie", VCH, 1995; March, "Advanced Organic Chemistry", John Wiley & Sons, 1985; Chemie Lexikon, Falbe / Regitz (Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0528] Throughout the specification and appended claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" should be understood to mean the inclusion of the stated members, integers or steps or groups of members, integers or steps but not the exclusion of any other members, integers or steps or groups of members, integers or steps. The term "consisting essentially of means the exclusion of other members, integers or steps with any substantive meaning. The term "comprise" encompasses the term "consisting essentially of", which in turn encompasses the term "consisting of". Thus, the term "comprises" may be replaced with the term "consisting essentially of" or "consisting of" at each occurrence in this application. Likewise, the term "consisting essentially of" may be replaced with the term "consisting of" at each occurrence in this application.

[0529] The terms "a," "an," and "the," and similar designations used in the context of describing the present disclosure (particularly in the context of the claims) should be understood to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. The enumeration of ranges of values ​​herein is merely a shorthand method of referring individually to each different value that falls within the range. Unless otherwise indicated herein, each individual value is incorporated into this specification as if it were individually stated herein. Unless otherwise indicated herein or clearly contradicted by the context, all methods described herein can be performed in any suitable order. The use of any and all examples or exemplary language (e.g., "such as") provided herein is solely for the purpose of better illustrating the present disclosure and does not limit the scope of the disclosure otherwise claimed. No language in this specification should be interpreted as implying any unclaimed element that is essential to the practice of the present disclosure.

[0530] As used herein, "and / or" is taken as specific disclosure of each of the two specified features or components, with or without the other. For example, "X and / or Y" is taken as specific disclosure of each of (i) X, (ii) Y, and (iii) X and Y, as if each were individually listed herein.

[0531] In the context of this disclosure, the term "about" refers to an interval of accuracy that one of ordinary skill in the art will understand still ensures the technical effect of the feature under consideration. The term generally refers to a deviation from the numerical value shown of ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, and, for example, ±0.01%. As one of ordinary skill will understand, the specific deviation from the numerical value of a given technical effect will depend on the nature of the technical effect. For example, natural or biotechnological effects can generally have greater such deviations than artificial or engineered effects.

[0532] Recitation of ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein.

[0533] The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended solely to better illustrate the invention and does not limit the scope of the invention otherwise claimed. No language in this specification should be construed as implying any non-claimed element as essential to the practice of the invention.

[0534] Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein shall be construed as an admission that the present invention is not entitled to priority from any prior application.

[0535] definition

[0536] In the following, definitions applicable to all aspects of this disclosure are provided. Unless otherwise indicated, the following terms have the following meanings. Any undefined terms have their generally accepted meanings.

[0537] As used herein, terms such as "reduce" or "inhibit" refer to the ability to cause an overall decrease, for example, a decrease of about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, or about 75% or more in levels. The term "inhibit" or similar phrases include complete or substantially complete inhibition, i.e., a decrease to 0 or substantially to 0.

[0538] In one embodiment, terms such as "increase" or "enhance" relate to an increase or enhancement of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 80% or at least about 100%.

[0539] As used herein, "physiological pH" refers to a pH of about 7.5.

[0540] As used in this disclosure, "% w / v" refers to weight percent by volume, which is a unit of concentration that measures the amount of solute in grams (g) expressed as a percentage of the total volume of a solution in milliliters (mL).

[0541] The term "ionic strength" refers to the mathematical relationship between the number of different ionic species in a particular solution and their respective charges. S Mathematically, it is expressed as follows

[0542]

[0543] where c is the molar concentration of a particular ion species and z is the absolute value of its charge. The sum Σ is taken over all the different ions (i) in the solution.

[0544] According to the present disclosure, in one embodiment, the term "ionic strength" refers to the presence of monovalent ions. With respect to the presence of divalent ions, particularly divalent cations, their concentration or effective concentration (presence of free ions) is, in one embodiment, sufficiently low to prevent RNA degradation due to the presence of chelating agents. In one embodiment, the concentration or effective concentration of divalent ions is below the catalytic level of hydrolysis of phosphodiester bonds between RNA nucleotides. In one embodiment, the concentration of free divalent ions is 20 μM or less. In one embodiment, there are no or substantially no free divalent ions.

[0545] "Osmolality" refers to the concentration of a specific solute expressed as osmoles of solute per kilogram of solvent.

[0546] The term "freezing" involves the solidification of a liquid, usually with the removal of heat.

[0547] The term "lyophilizing" or "lyophilization" refers to the freeze-drying of a substance by freezing it and then reducing the surrounding pressure to allow the freezing medium in the substance to sublime directly from the solid phase to the liquid phase.

[0548] The term "spray drying" refers to spray drying of a substance by mixing a (heated) gas with a fluid that is atomized (sprayed) within a vessel (spray dryer), where the solvent from the formed droplets evaporates, resulting in a dry powder.

[0549] The term "reconstitution" relates to the addition of a solvent, such as water, to the dried product to return it to a liquid state, such as its original liquid state.

[0550] The term "recombinant" in the context of the present disclosure means "produced by genetic engineering." In one embodiment, a "recombinant object" in the context of the present disclosure is not naturally occurring.

[0551] As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that exists in an organism (including a virus) and can be isolated from a natural source and has not been intentionally modified by humans in a laboratory is naturally occurring. The term "found in nature" means "existing in nature" and includes known objects as well as objects that have not yet been discovered and / or isolated from nature but may be discovered and / or isolated from natural sources in the future.

[0552] As used herein, the terms "room temperature" and "ambient temperature" are used interchangeably herein and refer to a temperature of at least about 15° C., preferably about 15° C. to about 35° C., about 15° C. to about 30° C., about 15° C. to about 25° C., or about 17° C. to about 22° C. Such temperatures include 15° C., 16° C., 17° C., 18° C., 19° C., 20° C., 21° C., and 22° C.

[0553] The term "ethanol injection technique" refers to a process in which an ethanol solution containing lipids is rapidly injected through a needle into an aqueous solution. This action disperses the lipids throughout the solution and promotes lipid structure formation, such as lipid vesicle formation such as liposome formation. In general, the nucleic acid (especially RNA) lipid complex particles described herein can be obtained by adding nucleic acid (especially RNA) to a colloidal liposomal dispersion. In one embodiment, using the ethanol injection technique, such a colloidal liposomal dispersion is formed as follows: an ethanol solution containing lipids, such as cationic lipids (such as DOTMA) and additional lipids is injected into an aqueous solution under stirring. In one embodiment, the nucleic acid (especially RNA) lipid complex particles described herein can be obtained without an extrusion step.

[0554] The term EDTA refers to ethylenediaminetetraacetic acid disodium salt. All concentrations are given in relation to EDTA disodium salt.

[0555] The term "alkyl" refers to a monovalent group of a saturated straight or branched hydrocarbon. Preferably, the alkyl group comprises 1-12 (e.g., 1-10) carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), more preferably 1-8 carbon atoms, such as 1-6 or 1-4 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, isopropyl (also referred to as 2-propyl or 1-methylethyl), butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, 1,2-dimethyl-propyl, isopentyl (iso-amyl), n-hexyl, isohexyl, sec-hexyl, n-heptyl, isoheptyl, n-octyl, 2-ethyl-hexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, etc.

[0556] According to the present disclosure, the term "peptide" includes oligopeptides and polypeptides and refers to a substance comprising about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids interconnected by peptide bonds. The term "protein" refers to large peptides, particularly peptides having at least about 151 amino acids, but the terms "peptide" and "protein" are generally used as synonyms herein.

[0557] According to the present disclosure, preferably once taken up or introduced, i.e., transfected or transduced into a cell, a nucleic acid such as RNA (preferably mRNA) encoding a peptide or protein results in expression of the peptide or protein, which cell may be present in vitro or in a subject. The cell may express the encoded peptide or protein intracellularly (e.g., in the cytoplasm and / or in the nucleus), may secrete the encoded peptide or protein, or may express it on the surface.

[0558] According to the present disclosure, terms such as "nucleic acid expression" and "nucleic acid encoding" or similar terms are used interchangeably herein and, with respect to a particular peptide or polypeptide, mean that the nucleic acid can be expressed to produce the peptide or polypeptide if present in an appropriate environment, preferably within a cell.

[0559] According to the present disclosure, the part or fragment of a peptide or protein preferably has at least one functional property of the peptide or protein from which it is derived. This functional property includes pharmacological activity, interaction with other peptides or proteins, enzymatic activity, interaction with antibodies, and selective binding of nucleic acids. For example, a pharmacologically active fragment of a peptide or protein has at least one pharmacological activity of the peptide or protein from which the fragment is derived. The part or fragment of a peptide or protein preferably comprises a sequence of at least 6, particularly at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, or at least 50 continuous amino acids of the peptide or protein. The part or fragment of a peptide or protein preferably comprises a sequence of up to 8, particularly up to 10, up to 12, up to 15, up to 20, up to 30, or up to 55 continuous amino acids of the peptide or protein.

[0560] According to the disclosure, the analog of a peptide or protein is a modified form of the described peptide or protein derived therefrom, and has at least one functional property of the described peptide or protein. For example, the pharmacologically active analog of a peptide or protein has at least one pharmacological activity of the peptide or protein derived therefrom. This type of modification includes any chemical modification, and includes single or multiple replacements, deletions and / or additions of any molecule (such as carbohydrate, lipid and / or protein or peptide) relevant to the protein or peptide. In one embodiment, " analog " of a protein or peptide includes those modified forms caused by glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introducing protection / blocking groups, protease cleavage or being attached to an antibody or another cellular ligand. The term " analog " also extends to all functional chemical equivalents of the described protein and peptide.

[0561] According to the present disclosure, "antigen" encompasses any substance that can trigger an immune response and / or any substance to which an immune response or immune mechanism, such as a cellular response, is directed. This also includes situations in which the antigen is processed into antigenic peptides and the immune response or immune mechanism is directed against one or more antigenic peptides, particularly if presented in the context of an MHC molecule. In particular, "antigen" relates to any substance, preferably a peptide or protein, that specifically reacts with an antibody or T lymphocyte (T cell). According to the present invention, the term "antigen" includes any molecule comprising at least one epitope, such as a T cell epitope. Preferably, an antigen in the context of the present disclosure is a molecule that, optionally after processing, induces an immune response, preferably specific to an antigen (including cells expressing the antigen). In one embodiment, the antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such an antigen.

[0562] According to the present disclosure, any suitable antigen can be used, which is a candidate for an immune response, wherein the immune response can be a humoral as well as a cellular immune response. In the context of some embodiments of the present disclosure, the antigen is preferably presented by a cell, preferably by an antigen presenting cell, in the context of an MHC molecule, which results in an immune response against the antigen. The antigen is preferably a product corresponding to or derived from a naturally occurring antigen. Such naturally occurring antigens may include or may be derived from allergens, viruses, bacteria, fungi, parasites and other infectious agents and pathogens, or the antigen may also be a tumor antigen. According to the present invention, the antigen may correspond to a naturally occurring product, for example, a viral protein or a portion thereof.

[0563] In a preferred embodiment, the antigen is a tumor antigen, i.e., a part of a tumor cell, in particular, one that occurs primarily intracellularly or as a tumor cell surface antigen. In another embodiment, the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, for example, from a virus, bacteria, unicellular organism, or parasite, such as a viral antigen such as a viral ribonucleoprotein or coat protein. In particular, the antigen should be presented by an MHC molecule, which leads to modulation, in particular, activation of immune system cells (preferably CD4+ and CD8+ lymphocytes), in particular by modulation of the activity of the T cell receptor.

[0564] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule containing an epitope that stimulates the host's immune system to produce a cellular antigen-specific immune response and / or a humoral antibody response against the disease. Disease-associated antigens include pathogen-associated antigens, i.e., antigens associated with microbial infection, typically microbial antigens (such as bacterial or viral antigens), or antigens associated with cancer, typically tumors, such as tumor antigens.

[0565] The term "tumor antigen" refers to a component of a cancer cell, which may originate from the cytoplasm, cell surface, or cell nucleus. In particular, it refers to antigens that are produced intracellularly or present as surface antigens on tumor cells. For example, tumor antigens include carcinoembryonic antigen, α1-fetoprotein, isoferritin and fetosulfuron, α2-H-ferritin and γ-fetoprotein, as well as various viral tumor antigens. According to the present disclosure, tumor antigens preferably include any antigen that is characteristic of a tumor or cancer, as well as tumor cells or cancer cells, in terms of type and / or expression level.

[0566] The term "viral antigen" refers to any viral component that has antigenic properties, ie, is capable of eliciting an immune response in an individual. A viral antigen may be a viral ribonucleoprotein or an envelope protein.

[0567] The term "bacterial antigen" refers to any bacterial component that has antigenic properties, ie, is capable of eliciting an immune response in an individual. Bacterial antigens may be derived from the cell wall or cytoplasmic membrane of the bacteria.

[0568] The term "epitope" refers to an antigenic determinant in a molecule such as an antigen, that is, a part or fragment of a molecule recognized by the immune system, for example, that of a molecule recognized by an antibody T cell or B cell, particularly when presented in the context of an MHC molecule. The epitope of an albumen preferably comprises a continuous or discontinuous portion of the albumen, and preferably has a length of about 5 to about 100, preferably about 5 to about 50, more preferably about 8 to about 0, most preferably about 10 to about 25 amino acids, for example, the epitope can preferably have a length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids. Particularly preferred epitopes in the context of the present invention are T cell epitopes.

[0569] Terms such as "epitope", "antigenic fragment", "immunogenic peptide" and "antigenic peptide" are used interchangeably herein and preferably relate to an incomplete representation of an antigen that is preferably capable of eliciting an immune response against the antigen or a cell expressing or containing or preferably presenting the antigen. Preferably, the term relates to an immunogenic portion of an antigen. Preferably, it is a portion of an antigen that is recognized (i.e., specifically bound) by a T cell receptor, particularly if presented in the context of an MHC molecule. Certain preferred immunogenic portions bind to MHC class I or class II molecules.

[0570] The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by a T cell when presented in the context of an MHC molecule. The term "major histocompatibility complex" and the abbreviation "MHC" include MHC class I and MHC class II molecules and relate to a gene complex present in all vertebrates. MHC proteins or molecules are important for signal transduction between lymphocytes and antigen presenting cells or diseased cells in an immune response, wherein MHC proteins or molecules bind to peptide epitopes and present them for recognition by T cell receptors on T cells. MHC-encoded proteins are expressed on the cell surface and display autoantigens (peptide fragments from the cell itself) and non-self antigens (e.g., fragments of invading microorganisms) to T cells. In the case of class I MHC / peptide complexes, binding peptides are typically about 8 to about 10 amino acids long, although longer or shorter peptides may be effective. In the case of class II MHC / peptide complexes, binding peptides are typically about 10 to about 25 amino acids long, particularly about 13 to about 18 amino acids long, while longer or shorter peptides may be effective.

[0571] The term "target" shall mean a substance, such as a cell or tissue, that is the target of an immune response, such as a cellular immune response. Targets include cells that present an antigen or an antigenic epitope (i.e., a peptide fragment derived from an antigen). In one embodiment, a target cell is a cell that expresses an antigen and preferably presents the antigen using class I MHC.

[0572] The term "portion" refers to a fraction. With respect to a specific structure such as an amino acid sequence or a protein, the term "portion" thereof may refer to a continuous or discontinuous portion of said structure.

[0573] The terms "part" and "fragment" are used interchangeably herein to refer to a continuous element. For example, a portion of a structure, such as an amino acid sequence or a protein, refers to a continuous element of the structure. When used in the context of a composition, the term "part" refers to a portion of the composition. For example, a portion of a composition can be any portion from 0.1% to 99.9% (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%) of the composition.

[0574] "Antigen processing" refers to the degradation of an antigen into processing products that are fragments of the antigen (e.g., degradation of a protein into peptides), and the association (e.g., by binding) of one or more of these fragments with an MHC molecule for presentation by a cell (preferably a specific T-cell antigen-presenting cell).

[0575] "Antigen-reactive CTL" refers to a CD8 + T-cells, which present antigens together with class I MHC on the surface of antigen-presenting cells.

[0576] According to the present invention, CTL reactivity can include sustained calcium flux, cell division, production of cytokines such as IFN-γ and TNF-α, upregulation of activation markers such as CD44 and CD69, and specific cytolytic killing of target cells expressing tumor antigens. CTL reactivity can also be determined using artificial reporters that accurately indicate CTL reactivity.

[0577] The terms "immune response" and "immune reaction" are used interchangeably herein in their conventional sense to refer to the overall body response to an antigen, and preferably to a cellular immune response, a humoral immune response, or both. According to the present invention, the terms "immune response to" or "immune response against" with respect to a substance (such as an antigen, cell or tissue) relate to an immune response (such as a cellular response) against said substance. The immune response may comprise one or more responses selected from the group consisting of: the production of antibodies against one or more antigens, and the production of antigen-specific T lymphocytes (preferably CD4 T lymphocytes). + and CD8 +T lymphocytes, more preferably CD8 + T lymphocytes), which can be detected in various proliferation or cytokine production assays in vitro.

[0578] In the context of the present invention, the terms "induce an immune response" and "trigger an immune response" and similar terms refer to the induction of an immune response, preferably a cellular immune response, a humoral immune response or the induction of both. The immune response can be protective / preventative / prophylactic and / or therapeutic. The immune response can be directed against any immunogen or antigen or antigenic peptide, preferably against a tumor-associated antigen or a pathogen-associated antigen (e.g., an antigen of a virus (e.g., influenza virus (A, B or C), CMV or RSV)). In this context, "induce" can mean that there is no immune response to a specific antigen or pathogen before induction, but it can also mean that there is a certain level of immune response to a specific antigen or pathogen before induction, and that the immune response is enhanced after induction. Therefore, in this context, "induce an immune response" also includes "enhance an immune response". Preferably, after inducing an immune response in an individual, the individual is protected from developing a disease such as an infectious disease or a cancerous disease, or the disease condition is improved by inducing an immune response.

[0579] The terms "cellular immune response," "cellular response," "cell-mediated immunity," or similar terms are meant to include cellular responses directed against cells characterized by expression of antigen and / or presentation of antigen using class I or class II MHC. The cellular response involves cells called T cells or T lymphocytes, which act as "helpers" or "killers." Helper T cells (also known as CD4 + T cells) play a central role in regulating immune responses, while killer cells (also known as cytotoxic T cells, cytolytic T cells, CD8 + T cells or CTLs) kill cells such as diseased cells.

[0580] The term "humoral immune response" refers to the process by which living organisms produce antibodies in response to substances and organisms, which ultimately neutralize and / or eliminate the substances and organisms. The specificity of the antibody response is mediated by T and / or B cells through membrane-associated receptors that bind to a single specific antigen. After binding to the appropriate antigen and receiving various other activation signals, B lymphocytes divide to produce memory B cells and antibody-secreting plasma cell clones, each clone producing antibodies that recognize the same antigenic epitope as its antigen receptor. Memory B lymphocytes remain dormant until they are subsequently activated by their specific antigen. These lymphocytes provide the cellular basis of memory and lead to an escalation of the antibody response when exposed to the specific antigen again.

[0581] As used herein, the term "antibody" refers to an immunoglobulin molecule that is capable of specifically binding to an epitope on an antigen. In particular, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, and any combination of the foregoing. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain comprises a light chain variable region (VL) and a light chain constant region (CL). The variable and constant regions are also referred to herein as variable domains and constant domains, respectively. The VH and VL regions can be further subdivided into highly variable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of VH are called HCDR1, HCDR2 and HCDR3, and the CDRs of VL are called LCDR1, LCDR2 and LCDR3. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL), wherein CH can be further subdivided into a constant domain CHI, a hinge region and constant domains CH2 and CH3 (arranged in the following order from amino terminus to carboxyl terminus: CH1, CH2, CH3). The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. An antibody can be a complete immunoglobulin derived from a natural or recombinant source, and can be an immunologically active portion of a complete immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single-chain antibodies and humanized antibodies.

[0582] The term "immunoglobulin" refers to a protein of the immunoglobulin superfamily, preferably to an antigen receptor such as an antibody or a B cell receptor (BCR). Immunoglobulins are characterized by domains, i.e., immunoglobulin domains having a characteristic immunoglobulin (Ig) fold. The term encompasses membrane-bound immunoglobulins as well as soluble immunoglobulins. Membrane-bound immunoglobulins are also called surface immunoglobulins or membrane immunoglobulins, which are generally part of the BCR. Soluble immunoglobulins are generally referred to as antibodies. Immunoglobulins generally comprise several chains, usually two identical heavy chains and two identical light chains linked by disulfide bonds. These chains are primarily composed of immunoglobulin domains, such as V L (Variable light chain) domain, C L (Constant light chain) domain, V H(variable heavy chain) domain and C H (Constant heavy chain) domain C H 1. C H 2. C H 3 and C H 4. There are five types of mammalian immunoglobulin heavy chains, namely, α, δ, ε, γ, and μ, which give rise to different classes of antibodies, namely, IgA, IgD, IgE, IgG, and IgM. In contrast to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins contain a transmembrane domain and a short cytoplasmic domain at their carboxyl termini. In mammals, there are two types of light chains, namely, λ and κ. Immunoglobulin chains contain variable and constant regions. The constant region is largely conserved among different immunoglobulin isotypes, while the variable portion is highly diverse and is responsible for antigen recognition.

[0583] The terms "vaccination" and "immunization" describe the process of treating an individual for therapeutic or prophylactic reasons and involve the procedure of administering to an individual one or more immunogens or antigens or derivatives thereof, particularly in the form of RNA encoding the same, as described herein, and stimulating an immune response against the one or more immunogens or antigens or cells characterized by presenting the one or more immunogens or antigens.

[0584] "Cells characterized by presenting antigens" or "cells presenting antigens" or "MHC molecules presenting antigens on the surface of antigen-presenting cells" or similar expressions refer to cells, such as diseased cells, in particular tumor cells, or antigen-presenting cells, which present antigens or antigenic peptides directly or after processing in the context of MHC molecules (preferably MHC class I and / or MHC class II molecules, most preferably MHC class I molecules).

[0585] In the context of this disclosure, the term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA can be translated into a peptide or protein.

[0586] With respect to RNA, the terms "expression" or "translation" refer to the process in the cell's ribosomes by which an mRNA chain directs the assembly of an amino acid sequence to produce a peptide or protein.

[0587] As used herein, the terms "optional" or "optionally" mean that the subsequently described event, circumstance or condition may or may not occur, and that the description includes instances where said event, circumstance or condition occurs and instances where it does not.

[0588] The radius of gyration of the particle about its axis of rotation (abbreviated herein as R g) is the radial distance from the axis of rotation of a point such that if the entire mass of the particle were assumed to be concentrated at that point, its moment of inertia about a given axis would be the same as its actual mass distribution. Mathematically, R g is the root mean square distance of the particle's components from its center of mass or a given axis. For example, for a macromolecule composed of n mass elements, the mass is m i (i=1,2,3,…,n), located at a fixed distance s from the center of mass i Department, R g is the s of all mass elements i 2 The square root of the mass mean and can be calculated as follows:

[0589]

[0590] The radius of gyration can be determined experimentally or calculated, for example, by using light scattering. In particular, for small scattering vectors The structure function S is defined as follows:

[0591]

[0592] where N is the number of components (Guinier's law).

[0593] The "D10 value," particularly with respect to the quantitative size distribution of particles, is the diameter below which 10% of the particles have a diameter smaller than this value. The D10 value is a method of describing the proportion of the smallest particles within a population of particles (e.g., within a particle peak obtained from field-flow fractionation).

[0594] The "D50 value," particularly with respect to a quantitative size distribution of particles, is the diameter at which 50% of the particles have a diameter smaller than this value. The D50 value is a method of describing the average particle size of a population of particles (e.g., within a particle peak obtained from field-flow fractionation).

[0595] The "D90 value," particularly with respect to a quantitative size distribution of particles, is the diameter at which 90% of the particles have a diameter smaller than this value. The "D95," "D99," and "D100" values ​​have corresponding meanings. The D90, D95, D99, and D100 values ​​are a way of describing the proportion of the largest particles within a particle population (e.g., within a particle peak obtained from field-flow fractionation).

[0596] The "hydrodynamic radius" (sometimes called the "Stokes radius" or "Stokes-Einstein" radius) of a particle is the radius of a hypothetical hard sphere that diffuses at the same speed as the particle. The hydrodynamic radius is related to the mobility of the particle, taking not only size into account but also solvent effects. For example, a smaller charged particle with greater hydration may have a larger hydrodynamic radius than a larger charged particle with lesser hydration. This is because the smaller particle drags more water molecules as it moves through the solution. Because the actual size of the particle in the solvent cannot be directly measured, the hydrodynamic radius can be defined by the Stokes-Einstein equation:

[0597]

[0598] where k B is the Boltzmann constant; T is the temperature; η is the viscosity of the solvent; and D is the diffusion coefficient. The diffusion coefficient can be determined experimentally, for example, by using dynamic light scattering (DLS). Therefore, one method for determining the hydrodynamic radius of a particle or a population of particles (such as the hydrodynamic radius of a particle contained in a sample or control composition disclosed herein or the hydrodynamic radius of a particle peak obtained by field-flow fractionation of such a sample or control composition) is to measure the DLS signal of the particle or the population of particles (such as the DLS signal of a particle contained in a sample or control composition disclosed herein or the DLS signal of a particle peak obtained by field-flow fractionation of such a sample or control composition).

[0599] As used herein, the term "shape factor" refers to the g Values ​​(such as recalculated R g value) and hydrodynamic radius (R h ) value. It can be calculated by g Values ​​(such as recalculated R g value) to the hydrodynamic radius (R h ) values ​​and fitting the data points to a function (e.g., a linear function) to determine or calculate.

[0600] As used herein, the term "form factor" refers to the hydrodynamic radius (R h ) value and R g Values ​​(such as recalculated R g value). It can be calculated by h ) value pair R g Values ​​(such as recalculated R g values) and fitting the data points to a function (e.g., a linear function) to determine or calculate.

[0601] As used herein, the expression "nucleic acid encapsulation efficiency" refers to the ratio of the amount of encapsulated nucleic acid contained in a sample or control composition comprising nucleic acid and particles to the total amount of nucleic acid contained in the sample or control composition. For example, where the nucleic acid is RNA, the expression "RNA encapsulation efficiency" as used herein refers to the ratio of the amount of encapsulated RNA contained in a sample or control composition comprising RNA and particles to the total amount of RNA contained in the sample or control composition.

[0602] As used herein, the term "membrane" refers to a size selective barrier that allows molecules below a certain size (referred to as "cut-off" (e.g., molecular weight cut-off (MW))) to pass through but prevents molecules above the certain size (i.e., cut-off, such as MW cut-off). Preferably, the membrane is synthetic. Examples of membranes suitable for the methods and / or uses of the present disclosure include ultrafiltration membranes, polyethersulfone (PES) membranes, regenerated cellulose membranes, polyvinylidene fluoride (PVDF) membranes, and other ultrafiltration membranes.

[0603] The term "aggregate" as used herein relates to a cluster of particles in which the particles are identical or very similar and adhere to each other non-covalently (eg, through ionic interactions, H-bridge interactions, dipole interactions, and / or van der Waals interactions).

[0604] As used herein, the expression "light scattering" refers to the physical process whereby light is forced to deviate from a straight-line trajectory into one or more paths due to local inhomogeneities in a medium through which the light passes.

[0605] The term "UV" stands for ultraviolet light, and designates a band of the electromagnetic spectrum with wavelengths from 10 nm to 400 nm, ie, shorter than visible light but longer than X-rays.

[0606] As used herein, the term "circular dichroism spectroscopy" or "CD spectroscopy" refers to spectroscopy using circularly polarized light. Preferably, CD spectroscopy involves the differential absorption of left-handed and right-handed light.

[0607] The term "UV CD light" or "UV CD signal" refers to circularly polarized light having a wavelength from 10 nm to 400 nm, ie shorter than visible light but longer than X-rays.

[0608] The expression "multi-angle light scattering" or "MALS" as used herein relates to a technique for measuring light scattered by a sample into multiple angles. "Multi-angle" in this context means that the scattered light can be detected at different discrete angles during the measurement, for example by a single detector that is movable over a range that includes a selected specific angle or by an array of detectors that are fixed at specific angular positions. In a preferred embodiment, the light source used in MALS is a laser source (MALLS: Multi-angle Laser Light Scattering). Based on the MALS signal of a composition comprising particles and by using an appropriate form (e.g. a Zimm plot, a Berry plot or a Debye plot), the radius of gyration (R g ), and thus determine the size of the particles. Preferably, the Zimm diagram is a graphical representation using the following equation:

[0609]

[0610] Where c is the mass concentration of particles in the solvent (g / mL); A2 is the second virial coefficient (mol·mL / g 2 ); P(θ) is a form factor related to the angular dependence of the scattered light intensity; R θ is the excess Rayleigh ratio (cm -1 ); K* is an optical constant equal to 4π 2 η o (dn / dc) 2 λ0 -4 N A -1 , where η o is the refractive index of the solvent at the wavelength of the incident radiation (vacuum), λ0 is the wavelength of the incident radiation (vacuum) (nm), N A is Avogadro's number (mol -1 ), dn / dc is the differential refractive index increment (mL / g) (see, for example, Buchholz et al. (Electrophoresis 22 (2001), 4118-4128); BH Zimm (J. Chem. Phys. 13 (1945), 141; P. Debye (J. Appl. Phys. 15 (1944): 338; and W. Burchard (Anal. Chem. 75 (2003), 4279-4291). Preferably, the Berry diagram is calculated as follows:

[0611]

[0612] Where c, R θ and K* are as defined above. Preferably, the Debye graph is calculated as follows:

[0613]

[0614] Where c, R θ and K* are as defined above. Although nucleic acids (especially RNA) themselves are not particles in the sense of the definition provided above, the size of nucleic acids (especially RNA) can also be determined using any of the above-mentioned formats (e.g., Zimm plots, Berry plots, or Debye plots), assuming that the nucleic acid (especially RNA) is in the form of a random coil. Therefore, in one embodiment of the methods and / or uses of the present disclosure, the size of nucleic acids (such as RNA) based on R g In another embodiment of the methods and / or uses of the present disclosure, the size, size distribution and / or quantitative size distribution of nucleic acids (such as RNA) are calculated based on the R h In another embodiment of the methods and / or uses of the present disclosure, the size, size distribution and / or quantitative size distribution of nucleic acids (such as RNA) are calculated based on the R g values ​​and respectively based on the R h The size, size distribution and / or quantitative size distribution of nucleic acids (such as RNA) are calculated using the R-values ​​(i.e., this embodiment generates two data sets of sizes, size distributions and / or quantitative size distributions of nucleic acids (such as RNA), one based on R g value, a value based on R h value).

[0615] As used herein, the expression "dynamic light scattering" or "DLS" refers to a technique for determining the size and size distribution spectrum of particles (particularly with respect to the hydrodynamic radius of the particles). A monochromatic light source, typically a laser, is directed into a sample through a polarizer. The scattered light then passes through a second polarizer, where it is detected, and the resulting image is projected onto a screen. Particles in the solution are struck by the light and diffract it in all directions. The diffracted light from the particles can interfere constructively (bright areas) or destructively (dark areas). This process is repeated at short time intervals, and the resulting speckle pattern set is analyzed by an autocorrelator that compares the light intensity at each point over time.

[0616] As used herein, the expression "static light scattering" or "SLS" refers to a technique for determining the size and size distribution spectrum of particles (particularly with respect to the radius of gyration of the particles and / or the molar mass of the particles). High-intensity monochromatic light, typically a laser, is emitted into a solution containing the particles. One or more detectors are used to measure the scattered intensity at one or more angles. Angular dependence is required to obtain an accurate measurement of the molar mass and radius size of all macromolecules. Therefore, simultaneous measurements at several angles relative to the direction of the incident light, known as multi-angle light scattering (MALS) or multi-angle laser light scattering (MALLS), are generally considered to be the standard practice of static light scattering.

[0617] The expressions "elution time" and "retention time" are used interchangeably herein and relate to the time it takes for a particular analyte to pass through a system (eg, from the injection point of a field-flow fractionation device to a detector) under set conditions.

[0618] The expression "continuously changes" means that the change from one value to a different value is performed steadily, ie without any jumps. Examples of continuous changes are linear changes or exponential changes (such as linear gradients or exponential gradients).

[0619] The expression "stepwise change" means that the change from one value to a different value is not continuous, but rather jumps from a first specific value to a second specific value, thereby omitting at least one of the values ​​between the first and second values. An example of a stepwise change is a flow rate profile starting from a first value (e.g., 10 mL / min) and ending at a second value (e.g., 0 mL / min), wherein during this profile, the flow rate may be only integers (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 mL / min), thereby omitting values ​​between these integers.

[0620] As used herein, the expression "providing a composition comprising nucleic acid (such as RNA) and optionally particles" means providing such a composition by any means, for example, it may be prepared, processed (such as purified and / or dried) and / or stored.

[0621] Nucleic Acids

[0622] According to the present disclosure, the term "nucleic acid" includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof and modified forms thereof. The term includes genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. According to the present disclosure, nucleic acids can exist as single-stranded or double-stranded and linear or covalently circular closed molecules. According to the present disclosure, nucleic acids can be isolated. According to the present disclosure, the term "isolated nucleic acid" means that the nucleic acid: (i) is amplified in vitro, for example by polymerase chain reaction (PCR) for DNA or in vitro transcription for RNA (using, for example, RNA polymerase), (ii) is produced by cloning recombination, (iii) is purified, for example by cutting and separating by gel electrophoresis, or (iv) is synthesized, for example, by chemical synthesis.

[0623] The term "nucleoside" (abbreviated herein as "N") refers to compounds that can be considered nucleotides without a phosphate group. A nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose), while a nucleotide consists of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.

[0624] The five standard nucleosides that typically make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine, and guanosine. The five nucleosides are often abbreviated by their single-letter codes, U, A, T, C, and G, respectively. However, thymidine is more commonly written as "dT" (the "d" stands for "deoxy") because it contains a 2'-deoxyribofuranose moiety instead of the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) but not ribonucleic acid (RNA). Conversely, uridine is found in RNA but not DNA. The remaining three nucleosides can be found in both RNA and DNA. In RNA, they would be represented as A, C, and G, while in DNA, they would be represented as dA, dC, and dG.

[0625] The modified purine (A or G) or pyrimidine (C, T or U) base moiety is preferably modified with one or more alkyl groups, more preferably one or more C 1-4 alkyl, even more preferably one or more methyl groups. Specific examples of modified purine or pyrimidine base moieties include N 7 -alkyl-guanine, N 6 -alkyl-adenine, 5-alkyl-cytosine, 5-alkyl-uracil and N(1)-alkyl-uracil, such as N 7 -C 1-4 Alkyl-guanine, N 6 -C 1-4 Alkyl-adenine, 5-C 1-4 Alkyl-cytosine, 5-C 1-4 Alkyl-uracil and N(1)-C 1-4Alkyl-uracil, preferably N 7 -methyl-guanine, N 6 -methyl-adenine, 5-methyl-cytosine, 5-methyl-uracil and N(1)-methyl-uracil.

[0626] In the present disclosure, the term "DNA" refers to a nucleic acid molecule comprising deoxyribonucleotide residues. In preferred embodiments, DNA contains all or most of the deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide lacking a hydroxyl group at the 2'-position of a β-D-ribofuranosyl group. DNA encompasses, but is not limited to, double-stranded DNA, single-stranded DNA, isolated DNA such as partially purified DNA, substantially pure DNA, synthetic DNA, recombinantly produced DNA, and modified DNA that is different from naturally occurring DNA by adding, deleting, replacing, and / or changing one or more nucleotides. Such changes can refer to the addition of non-nucleotide substances to internal DNA nucleotides or the ends of DNA. It is also contemplated herein that the nucleotides in the DNA can be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the present disclosure, it is believed that these altered DNAs are analogs of naturally occurring DNA. A molecule contains "mostly deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule is greater than 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in the molecule is the sum of all nucleotide residues (regardless of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0627] In one embodiment, the DNA is recombinant DNA and can be obtained by cloning nucleic acids, particularly cDNA. cDNA can be obtained by reverse transcription of RNA.

[0628] In the present disclosure, the term "RNA" refers to a nucleic acid molecule comprising ribonucleotide residues. In a preferred embodiment, RNA contains all or most of the ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2'-position of a β-D-ribofuranosyl group. RNA encompasses, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that is different from naturally occurring RNA by adding, deleting, replacing and / or changing one or more nucleotides. Such changes can refer to the addition of non-nucleotide substances to internal RNA nucleotides or the ends of RNA. It is also contemplated herein that the nucleotides in RNA can be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered / modified nucleotides can be referred to as analogs of naturally occurring nucleotides, and the corresponding RNA containing such altered / modified nucleotides (i.e., altered / modified RNA) can be referred to as analogs of naturally occurring RNA. A molecule contains "mostly ribonucleotide residues" if the content of ribonucleotide residues in the molecule is greater than 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (regardless of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0629] According to the present disclosure, "RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA) or microRNA (miRNA)), activating RNA (such as small activating RNA) and immunostimulatory RNA (isRNA).

[0630] As used herein, the term "in vitro transcription" or "IVT" refers to transcription (i.e., production of RNA) performed in a cell-free manner. That is, IVT does not utilize living / cultured cells, but rather utilizes transcription machinery extracted from cells (e.g., cell lysate or isolated components thereof, including RNA polymerase (preferably T7, T3, or SP6 polymerase)).

[0631] According to the present disclosure, the term "mRNA" means "messenger RNA" and refers to a "transcript" that can be produced using a DNA template and can encode a peptide or protein. Typically, an mRNA comprises a 5'-UTR, a peptide / protein coding region, and a 3'-UTR. In the context of the present disclosure, mRNA is preferably produced from a DNA template by in vitro transcription (IVT). As indicated above, in vitro transcription methods are known to those skilled in the art, and various in vitro transcription kits are commercially available.

[0632] mRNA is single-stranded but may contain self-complementary sequences that allow part of the mRNA to fold and pair with itself to form a double helix.

[0633] According to the present disclosure, "dsRNA" means double-stranded RNA, and is RNA having two partially or fully complementary strands.

[0634] The length of the RNA can vary from 10 nucleotides to 15,000, such as 40-15,000, 100-12,000 or 200-10,000 nucleotides. In one embodiment, the RNA is an inhibitory RNA and has a length of 10-100 nucleotides (such as at most 90 nucleotides, at most 80 nucleotides, at most 70 nucleotides, at most 60 nucleotides, at most 50 nucleotides, at most 45 nucleotides, at most 40 nucleotides, at most 35 nucleotides, at most 30 nucleotides, at most 25 nucleotides or at most 20 nucleotides). In one embodiment, the RNA encodes a peptide or protein and has a length of at least 45 nucleotides (e.g., at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides), preferably up to 15,000, such as up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides or up to 10,000 nucleotides.

[0635] In certain embodiments of the present disclosure, the RNA is an mRNA associated with an RNA transcript encoding a peptide or protein. As established in the art, mRNA generally contains a 5' untranslated region (5'-UTR), a peptide coding region, and a 3' untranslated region (3'-UTR). In some embodiments, the RNA is produced by in vitro transcription or chemical synthesis. In one embodiment, mRNA is produced by in vitro transcription using a DNA template. In vitro transcription methods are known to those skilled in the art; see, for example, Molecular Cloning: A Laboratory Manual, 2 nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989. In addition, various in vitro transcription kits are commercially available, for example, from Thermo Fisher Scientific (such as TranscriptAid TM T7 test kit, T7 test kit, New England BioLabs Inc. (eg HiScribe TM T7 kit, HiScribe TM T7 ARCA mRNA kit), Promega (such as RiboMAX TM 、 systems), Jena Bioscience (such as SP6 or T7 transcription kits), and Epicentre (such as AmpliScribe TM In order to provide a modified RNA, corresponding modified nucleotides, such as modified naturally occurring nucleotides, non-naturally occurring nucleotides and / or modified non-naturally occurring nucleotides, can be incorporated during synthesis (preferably in vitro transcription), or the modification is achieved after transcription and / or added to the RNA.

[0636] In one embodiment, the RNA is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of an appropriate DNA template. The promoter used to control transcription can be any promoter for any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly a cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA can be obtained by reverse transcription of RNA.

[0637] In the context of the present disclosure, the RNA, preferably mRNA, contains one or more modifications, for example, to increase its stability and / or increase translation efficiency and / or reduce immunogenicity and / or reduce cytotoxicity.For example, in order to increase the expression of RNA (especially mRNA), it can be modified in the coding region, i.e., the sequence of the peptide or protein encoded for expression, preferably without changing the sequence of the expressed peptide or protein. Such modifications are described, for example, in WO 2007 / 036366 and PCT / EP2019 / 056502, and include the following: 5'-cap structure; naturally occurring poly (A) tail extension or truncation; 5'- and / or 3'- untranslated region (UTR) changes, such as introducing UTRs unrelated to the coding region of the RNA; replacing one or more naturally occurring nucleotides with synthetic nucleotides; and codon optimization (for example, changing, preferably increasing the GC content of RNA). According to the present disclosure, the term "modification" in the context of modified RNA (preferably mRNA) preferably relates to any modification of the RNA (preferably mRNA) that is not naturally occurring in the RNA.

[0638] In some embodiments, the RNA (preferably mRNA) according to the present disclosure comprises a 5'-cap structure. In one embodiment, the RNA (preferably mRNA) does not have an uncapped 5'-triphosphate. In one embodiment, the RNA (preferably mRNA) may comprise a conventional 5'-cap and / or a 5'-cap analog. The term "conventional 5'-cap" refers to a cap structure found at the 5'-end of an mRNA molecule, generally consisting of guanosine 5'-triphosphate (Gppp), which is linked to the 5'-end of the next nucleotide of the mRNA via its triphosphate portion (i.e., guanosine is linked to the rest of the mRNA via a 5' to 5' triphosphate bond). Guanosine may be present at position N 7 Methylation (resulting in cap structure m 7 Gppp). The term "5'-cap analogue" refers to a 5'-cap that is based on a conventional 5'-cap but has been modified in the m 7 The 2'- or 3'-position of the guanosine structure is modified to prevent the 5'-cap analog from incorporating in the reverse direction (such 5'-cap analogs are also called anti-reverse cap analogs (ARCA)). Particularly preferred 5'-cap analogs are those with one or more substitutions at the bridging and non-bridging oxygens of the phosphate bridge, such as phosphorothioate-modified 5'-cap analogs at the β-phosphate (e.g., m2 7,2'OG(5')ppSp(5')G (referred to as beta-S-ARCA or β-S-ARCA)), as described in PCT / EP2019 / 056502, the entire disclosure of which is incorporated herein by reference. Providing an RNA (preferably an mRNA) having a 5'-cap structure as described herein can be achieved by in vitro transcription of a DNA template in the presence of a corresponding 5'-cap compound, wherein the 5'-cap structure is co-transcriptionally incorporated into the resulting RNA chain, or the RNA (preferably mRNA) can be produced, for example, by in vitro transcription, and the 5'-cap structure can be post-transcriptionally attached to the RNA using a capping enzyme, such as a capping enzyme of vaccinia virus.

[0639] In some embodiments, the RNA (preferably mRNA) according to the present disclosure comprises a member selected from m2 7,2'O G(5')ppSp(5')G (especially its D1 diastereomer), m2 7,3'O G(5')ppp(5')G and m2 7,3'-O Gppp(m1 2'-O )ApG 5'-cap structure.

[0640] In some embodiments, the RNA (preferably mRNA) comprises cap0, cap1 or cap2, preferably cap1 or cap2. According to the present disclosure, the term "cap0" means the structure "m 7 GpppN", wherein N is any nucleoside with an OH moiety at position 2'. According to the present disclosure, the term "cap1" means a structure "m 7 GpppNm", wherein Nm is any nucleoside with an OCH3 moiety at position 2'. According to the present disclosure, the term "cap2" means a structure "m 7 GpppNmNm", wherein each Nm is independently any nucleoside with an OCH3 moiety at the 2' position.

[0641] The D1 diastereomer of beta-S-ARCA (β-S-ARCA) has the following structure:

[0642]

[0643] "D1 diastereomer of β-S-ARCA" or "β-S-ARCA (D1)" is the diastereomer of β-S-ARCA that elutes first on the HPLC column compared to the D2 diastereomer of β-S-ARCA (β-S-ARCA (D2)), thus exhibiting a shorter retention time. The HPLC is preferably analytical HPLC. In one embodiment, a Supelcosil LC-18-T RP column, preferably with dimensions of 5 μm and 4.6 x 250 mm, is used for the separation, allowing for a flow rate of 1.3 ml / min. In one embodiment, a methanol gradient in ammonium acetate is used, for example, a linear gradient of 0-25% methanol in 0.05 M ammonium acetate, pH 5.9, over 15 minutes. UV detection (VWD) can be performed at 260 nm, and fluorescence detection (FLD) can be performed with excitation at 280 nm and detection at 337 nm.

[0644] Component of Cap1, 5'-cap analog M2 7,3'-O Gppp(m1 2'-O )ApG (also known as m2 7,3'O G(5')ppp(5')m 2'-O ApG) has the following structure:

[0645]

[0646] An exemplary capO RNA comprising β-S-ARCA and RNA has the following structure:

[0647]

[0648] Including m2 7,3'O An exemplary cap0 RNA of G(5')ppp(5')G and RNA has the following structure:

[0649]

[0650] Including m2 7,3'-O Gppp(m1 2'-O )ApG and an exemplary cap1 RNA has the following structure:

[0651]

[0652] As used herein, the term "poly (A) tail" or "poly-A sequence" refers to an uninterrupted or interrupted sequence of adenylate residues, which is typically located at the 3'-end of an RNA molecule. Poly-A tails or poly-A sequences are known to those skilled in the art and may follow the 3'-UTR in the RNA described herein. An uninterrupted poly-A tail is characterized by continuous adenylate residues. In nature, an uninterrupted poly-A tail is typical. The RNA disclosed herein may have a poly-A tail that is attached to the free 3'-end of the RNA by a template-independent RNA polymerase after transcription, or a poly-A tail that is encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0653] A poly-A tail of approximately 120 A nucleotides has been shown to have beneficial effects on RNA levels in transfected eukaryotic cells and on the levels of proteins translated from open reading frames present upstream (5') of the poly-A tail (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0654] The poly-A tail can be of any length. In some embodiments, the poly-A tail comprises, consists essentially of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, particularly about 120 A nucleotides. In this context, "consisting essentially of" means that the majority of the nucleotides in the poly-A tail are A nucleotides, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the nucleotides in the poly-A tail are A nucleotides, but it is permitted that the remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylic acid), G nucleotides (guanylic acid), or C nucleotides (cytidylic acid). In this context, "consisting of" means that all nucleotides in the poly-A tail, i.e., 100% of the nucleotides in the poly-A tail are A nucleotides. The term "A nucleotide" or "A" refers to adenosine.

[0655] In some embodiments, a poly-A tail is attached during RNA transcription, e.g., during the preparation of in vitro transcribed RNA, based on a DNA template containing repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence encoding the poly-A tail (coding strand) is referred to as a poly(A) box.

[0656] In some embodiments, the poly (A) box present in the DNA coding chain is essentially composed of dA nucleotides, but is interrupted by a random sequence of 4 nucleotides (dA, dC, dG and dT). The length of such a random sequence can be 5-50, 10-30 or 10-20 nucleotides. Such a box is disclosed in WO 2016 / 005324 A1, which is incorporated herein by reference. Any poly (A) box disclosed in WO 2016 / 005324 A1 can be used in the present invention. Such a poly (A) box is encompassed, which is essentially composed of dA nucleotides, but is interrupted by a random sequence with an equal distribution of 4 nucleotides (dA, dC, dG, dT) and having, for example, a length of 5-50 nucleotides, exhibits constant proliferation of plasmid DNA in Escherichia coli (E. coli) at the DNA level, and is still associated with the beneficial properties of supporting RNA stability and translation efficiency at the RNA level. Thus, in some embodiments, the poly-A tail contained in the RNA molecules described herein consists essentially of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such a random sequence can be 5-50, 10-30, or 10-20 nucleotides in length.

[0657] In some embodiments, the poly-A tail is not flanked at its 3'-end by nucleotides other than A nucleotides, ie, the poly-A tail is not masked or followed by nucleotides other than A at its 3'-end.

[0658] In some embodiments, RNA according to the present disclosure comprises 5'-UTR and / or 3'-UTR. The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or to a corresponding region in an RNA molecule such as an mRNA molecule. An untranslated region (UTR) can be present at the 5' (upstream) (5'-UTR) and / or the 3' (downstream) (3'-UTR) of an open reading frame. If present, the 5'-UTR is located at the 5'-end, upstream of the start codon of the protein coding region. The 5'-UTR is located downstream of the 5'-cap (if present), for example, directly adjacent to the 5'-cap. If present, the 3'-UTR is located at the 3'-end, downstream of the stop codon of the protein coding region, but the term "3'-UTR" preferably does not include a poly-A sequence. Therefore, the 3'-UTR is located upstream of the poly-A sequence (if present), for example, directly adjacent to the poly (A) sequence. Incorporating a 3'-UTR into the 3'-untranslated region of an RNA (preferably mRNA) molecule can result in an increase in translation efficiency. A synergistic effect can be achieved by incorporating two or more such 3'-UTRs (which are preferably arranged in a head-to-tail orientation; see, for example, Holtkamp et al., Blood 108, 4009-4017 (2006)). The 3'-UTRs can be autologous or heterologous to the RNA (preferably mRNA) into which they are introduced. In a specific embodiment, the 3'-UTR is derived from a globin gene or mRNA, such as an α2-globin, α1-globin or β-globin, preferably a β-globin, more preferably a gene or mRNA for human β-globin. For example, RNA (preferably mRNA) can be modified by replacing an existing 3'-UTR with one or more, preferably two copies of a 3'-UTR or inserting one or more, preferably two copies of a 3'-UTR derived from a globin gene, such as α2-globin, α1-globin or β-globin, preferably β-globin, more preferably human β-globin.

[0659] The RNA (preferably mRNA) may have modified ribonucleotides to increase its stability and / or reduce immunogenicity and / or reduce cytotoxicity. For example, in one embodiment, the uridine in the RNA described herein is replaced (partially or completely, preferably completely) by a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.

[0660] In some embodiments, the modified uridine replacing uridine is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), 5-methyl-uridine (m5U), and combinations thereof.

[0661] In some embodiments, the modified nucleoside replacing (partially or completely, preferably completely) the uridine in the RNA may be any one or more of the following: 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxoacetic acid (cmo5U), uridine 5-oxoacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5- Propynyl-uridine, 1-propynyl-pseudouridine, 5-taurine methyl-uridine (τm5U), 1-taurine methyl-pseudouridine, 5-taurine methyl-2-thio-uridine (τm5s2U), 1-taurine methyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m5Um), 2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2′-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm5Um), 3 , 2′-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2′-F-arabino-uridine, 2′-F-uridine, 2′-OH-arabino-uridine, 5-(2-methoxycarbonylvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

[0662] RNA (preferably mRNA) modified with pseudouridine (partially or completely, preferably completely replacing uridine) is referred to herein as "Ψ-modified", while the term "m1Ψ-modified" means that the RNA (preferably mRNA) contains N(1)-methylpseudouridine (partially or completely, preferably completely replacing uridine). In addition, the term "m5U-modified" means that the RNA (preferably mRNA) contains 5-methyluridine (partially or completely, preferably completely replacing uridine). Such Ψ- or m1Ψ- or m5U-modified RNAs exhibit reduced immunogenicity compared to their unmodified forms and are therefore preferred in applications where the induction of an immune response is avoided or minimized.

[0663] The codons of the RNA (preferably mRNA) of the present disclosure can be further optimized, for example, to increase the GC content of the RNA and / or replace rare codons in the cell (or subject), wherein the peptide or protein of interest is expressed by codons that are synonymous with frequent codons in the cell (or subject).

[0664] The combination of the above modifications, i.e., incorporation of a 5'-cap structure, incorporation of a poly-A sequence, exposure of a poly-A sequence, alteration of the 5'- and / or 3'-UTR (e.g., incorporation of one or more 3'-UTRs), replacement of one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine and / or pseudouridine (Ψ) or N(1)-methylpseudouridine (m1Ψ) or 5-methyluridine (m5U) for uridine) and codon optimization has a synergistic effect on improving the stability of RNA (preferably mRNA) and the efficiency of translation. Therefore, in a preferred embodiment, the RNA (preferably mRNA) according to the present disclosure contains a combination of at least two, at least three, at least four or all five of the above modifications, i.e., (i) incorporation of a 5'-cap structure, (ii) incorporation of a poly-A sequence, exposure of a poly-A sequence; (iii) alteration of the 5'- and / or 3'-UTR (e.g., incorporation of one or more 3'-UTRs); (iv) replacement of one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine and / or pseudouridine (Ψ) or N(1)-methylpseudouridine (m1Ψ) or 5-methyluridine (m5U) for uridine), and (v) codon optimization.

[0665] In one embodiment, the RNA according to the present disclosure comprises a nucleic acid sequence encoding a peptide or protein, preferably a pharmaceutically active peptide or protein.

[0666] In a preferred embodiment, the RNA according to the present disclosure comprises a nucleic acid sequence encoding a peptide or protein, preferably a pharmaceutically active peptide or protein, and is capable of expressing said peptide or protein, particularly if transferred into a cell or a subject. Thus, the RNA according to the present invention preferably contains a coding region (open reading frame (ORF)) encoding a peptide or protein, preferably a pharmaceutically active peptide or protein. In this regard, an "open reading frame" or "ORF" is a continuous stretch of codons that begins with a start codon and ends with a stop codon.

[0667] According to the present disclosure, the term "pharmaceutically active peptide or protein" refers to a peptide or protein that can be used to treat an individual, wherein the expression of the peptide or protein would be beneficial, for example, to improve the symptoms of a disease or condition. Preferably, the pharmaceutically active peptide or protein has therapeutic or palliative properties and can be administered to improve, alleviate, relieve, reverse, delay the onset of, or reduce the severity of one or more symptoms of a disease or condition. Preferably, when administered to an individual in a therapeutically effective amount, the pharmaceutically active peptide or protein has a positive or beneficial effect on the individual's condition or disease state. Pharmaceutically active peptides or proteins can have preventive properties and can be used to delay the onset of a disease or condition or to reduce the severity of such a disease or condition. The term "pharmaceutically active peptide or protein" includes complete proteins or polypeptides and can also refer to pharmaceutically active fragments thereof. It can also include pharmaceutically active analogs of the peptide or protein.

[0668] Specific examples of pharmaceutically active peptides and proteins include, but are not limited to, cytokines, hormones, adhesion molecules, immunoglobulins, immunoactive compounds, growth factors, protease inhibitors, enzymes, receptors, apoptosis regulators, transcription factors, tumor suppressor proteins, structural proteins, reprogramming factors, genome engineering proteins, and blood proteins.

[0669] The term "cytokine" refers to a protein with a molecular weight of about 5-20 kDa and involved in cell signaling (e.g., paracrine, endocrine and / or autocrine signaling). In particular, when released, cytokines affect the behavior of cells around their release site. Examples of cytokines include lymphokines, interleukins, chemokines, interferons and tumor necrosis factor (TNF). According to the present disclosure, cytokines do not include hormones or growth factors. The difference between cytokines and hormones is that (i) they usually work at more variable concentrations than hormones, and (ii) are generally produced by a wide range of cells (almost all nucleated cells can produce cytokines). Interferons are generally characterized by antiviral, antiproliferative and immunomodulatory activities. Interferons are proteins that change and regulate gene transcription in cells by binding to interferon receptors on the surface of regulated cells, thereby preventing viral replication in cells. Interferons can be divided into two types. IFN-γ is the only type II interferon; the others are all type I interferons. Specific examples of cytokines include erythropoietin (EPO), colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF), bone morphogenetic protein (BMP), interferon α (IFNα), interferon β (IFNβ), interferon γ (INFγ), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 10 (IL-10) and interleukin 11 (IL-11).

[0670] The term "hormone" refers to a class of signaling molecules produced by glands, wherein signaling generally includes the following steps: (i) synthesis of the hormone in a specific tissue; (ii) storage and secretion of the hormone; (iii) transport to its target; (iv) binding of the hormone via receptors; (v) relaying and amplification of the signal; and (vi) breakdown of the hormone. Hormones differ from cytokines in that (1) hormones generally act at less variable concentrations and (2) are generally produced by specific types of cells. In one embodiment, a "hormone" is a peptide or protein hormone, such as insulin, vasopressin, prolactin, adrenocorticotropic hormone (ACTH), thyroid hormone, growth hormone (such as human growth hormone or bovine somatotropin), oxytocin, atrial natriuretic peptide (ANP), glucagon, somatostatin, cholecystokinin, gastrin, and leptin.

[0671] The term "adhesion molecule" refers to a protein that is located on the cell surface and is involved in the binding of cells to other cells or to the extracellular matrix (ECM). Adhesion molecules are typically transmembrane receptors and can be divided into calcium-independent (e.g., integrins, immunoglobulin superfamily, lymphocyte homing receptors) and calcium-dependent (cadherins and selectins). Specific examples of adhesion molecules are integrins, lymphocyte homing receptors, selectins (e.g., P-selectin), and addressins.

[0672] Integrins are also involved in signal transduction. In particular, when bound to a ligand, integrins regulate cell signaling pathways, for example, pathways of transmembrane protein kinases such as receptor tyrosine kinases (RTKs). This regulation can lead to cell growth, division, survival or differentiation, or apoptosis. Specific examples of integrins include: α1β1, α2β1, α3β1, α4β1, α5β1, α6β1, α7β1, α L β2, α M β2, α IIb β3, α V β1, α V β3, α V β5, α V β6, α V β8 and α6β4.

[0673] The term "immunoglobulin" or "immunoglobulin superfamily" refers to molecules involved in the recognition,...

Claims

1. A method for determining the RNA integrity of a sample composition, wherein the sample composition comprises RNA and particles containing RNA, the method comprising: (a) subjecting at least a portion of the sample composition to field-flow fractionation, thereby fractionating components contained in the sample composition according to their size to produce one or more sample fractions; (b) measuring a UV signal of at least one of the one or more sample fractions obtained from step (a); and (c) calculating the RNA integrity from the UV signal, wherein the integrity of the control RNA is used to calculate the integrity of the RNA contained in the sample composition, in (A) Determine the integrity of the control RNA by following these steps: (a') subjecting at least a portion of a control composition containing a control RNA to field-flow fractionation, thereby fractionating components contained in the control composition according to their size to produce one or more control fractions; (b') measuring the UV signal of at least one of the one or more control fractions obtained from step (a'); (c'1) calculating the area from the maximum height of a UV peak to the end of the UV peak from the UV signal obtained in step (b') to obtain A 50％，对照 ; (c'2) Calculating the total area of ​​one peak used in step (c'1) from the UV signal obtained in step (b') to obtain A 100％，对照 ;as well as (c'3) Determine A 50％，对照 and A 100％，对照 The ratio between , thereby obtaining the integrity of the control RNA, I 对照 ,and The integrity of the RNA contained in the sample composition was calculated by the following steps: (c1) calculating the area from the maximum height of the sample UV peak corresponding to the control UV peak used in step (c'1) to the end of the sample UV peak from the sample UV signal obtained in step (b), thereby obtaining A 50％，样品 ; (c2) calculating the total area of ​​the sample UV peak used in step (c1) from the sample UV signal obtained in step (b) to obtain A 100％，样品 ; (c3) Determine A 50％，样品 and A 100％，样品 The ratio between them is obtained 样品 ; as well as (c4) Determine I 样品 and I 对照 , thereby obtaining the integrity of the RNA contained in the sample composition; or (B) Determine the integrity of the calculated control RNA by the following steps: (a") subjecting at least a portion of a control composition comprising a control RNA to field-flow fractionation, thereby fractionating components contained in the control composition according to their size to produce one or more control fractions; (b") measuring the UV signal of at least one of the one or more control fractions obtained from step (a"); as well as (c") Determine the height of a UV peak, H, from the UV signal obtained in step (b"). 对照 , thereby obtaining the integrity of the control RNA, and The integrity of the RNA contained in the sample composition was calculated by the following steps: (c1′) determining the height of the sample UV peak corresponding to the reference UV peak used in step (c″) from the UV signal obtained in step (b), H 样品 ;as well as (c2') Determine H 样品 and H 对照 The ratio between them is thus determined, thereby obtaining the integrity of the RNA contained in the sample composition.

2. The method of claim 1, wherein the field-flow fractionation is flow field-flow fractionation.

3. The method of claim 2, wherein the field-flow fractionation is an asymmetric flow field-flow fractionation or a hollow fiber flow field-flow fractionation.

4. The method of claim 1, wherein step (a) is performed using a membrane having a molecular weight cutoff suitable for preventing RNA from penetrating the membrane, or step (a) is performed using a membrane having a molecular weight cutoff in the range of 2 kDa-30 kDa.

5. The method of claim 4, wherein step (a) is performed using a polyethersulfone or regenerated cellulose membrane.

6. The method of claim 1, wherein step (a) is performed using a cross flow rate of up to 8 mL / min.

7. The method of claim 6, wherein step (a) is performed using a cross flow rate of up to 4 mL / min or up to 2 mL / min.

8. The method of claim 1, wherein step (a) is performed using the following cross-flow profile: 1.0-2.0 mL / min for 10 min, an exponential gradient from 1.0-2.0 mL / min to 0.01-0.07 mL / min in 30 min; 0.01-0.07 mL / min for 30 min; and 0 mL / min for 10 min.

9. The method of claim 1, wherein step (a) is performed using an injection flow rate in the range of 0.05-0.35 mL / min.

10. The method of claim 9, wherein step (a) is performed using an injection flow rate in the range of 0.10-0.30 mL / min or in the range of 0.15-0.25 mL / min.

11. The method of claim 1, wherein step (a) is performed using a detector flow rate in the range of 0.30-0.70 mL / min.

12. The method of claim 11, wherein step (a) is performed using a detector flow rate in the range of 0.40-0.60 mL / min or in the range of 0.45-0.55 mL / min.

13. The method of claim 1, wherein the sample composition comprises RNA and lipid complex particles and / or lipid nanoparticles and / or polyplex particles and / or lipid multiplex particles and / or virus-like particles, and the RNA is bound to the particles.

14. The method of claim 1, wherein the RNA is 10-15,000 nucleotides in length.

15. The method of claim 1, wherein the RNA is in vitro transcribed RNA or in vitro transcribed mRNA.

16. The method of claim 1, wherein measuring the UV signal is performed online, and / or step (c) is performed online.

17. The method of claim 13, wherein prior to subjecting at least a portion of the sample composition to field-flow fractionation, at least a portion of the sample composition is diluted with a solvent or solvent mixture that is capable of preventing the formation of particle aggregates.

18. The process of claim 17, wherein the solvent mixture is a mixture of water and an organic solvent or a mixture of water and formamide.

19. The method of claim 1, wherein measuring the UV signal is performed by using circular dichroism spectroscopy.

20. A method of analyzing the effect of changing one or more reaction conditions in providing a composition comprising RNA and particles comprising RNA, the method comprising: (A) providing a first composition comprising RNA and particles comprising the RNA; (B) providing a second composition comprising RNA and particles comprising RNA, wherein providing the second composition differs from providing the first composition only in one or more reaction conditions; (C) subjecting a portion of the first composition to the method of any one of claims 1 to 19, thereby determining the RNA integrity of the first composition; (D) subjecting a corresponding portion of the second composition to the method used in step (C), thereby determining the RNA integrity of the second composition; and (E) The RNA integrity of the first composition obtained in step (C) is compared with the RNA integrity of the second composition obtained in step (D).

21. The method of claim 20, wherein the one or more reaction conditions include any of: salt concentration / ionic strength; temperature; pH or buffer concentration; light / radiation; oxygen; shear force; pressure; freeze / thaw cycles; dry / reconstitute cycles; addition of excipients; type and / or source of particle-forming compound; charge ratio; physical state; and the ratio of RNA to particle-forming compound.

22. Use of field-flow fractionation for determining RNA integrity of a sample composition comprising RNA and particles containing RNA, wherein the field-flow fractionation comprises: (a) subjecting at least a portion of the sample composition to field-flow fractionation, thereby fractionating components contained in the sample composition according to their size to produce one or more sample fractions; (b) measuring a UV signal of at least one of the one or more sample fractions obtained from step (a); and (c) calculating the RNA integrity from the UV signal, wherein the integrity of the control RNA is used to determine the integrity of the RNA contained in the sample composition, in (A) Determine the integrity of the control RNA by following these steps: (a') subjecting at least a portion of a control composition containing a control RNA to field-flow fractionation, thereby fractionating components contained in the control composition according to their size to produce one or more control fractions; (b') measuring the UV signal of at least one of the one or more control fractions obtained from step (a'); (c'1) calculating the area from the maximum height of a UV peak to the end of the UV peak from the UV signal obtained in step (b') to obtain A 50％，对照 ; (c'2) Calculating the total area of ​​one peak used in step (c'1) from the UV signal obtained in step (b') to obtain A 100％，对照 ;as well as (c'3) Determine A 50％，对照 and A 100％，对照 The ratio between , thereby obtaining the integrity of the control RNA, I 对照 ,and The integrity of the RNA contained in the sample composition was calculated by the following steps: (c1) calculating the area from the maximum height of the sample UV peak corresponding to the control UV peak used in step (c'1) to the end of the sample UV peak from the sample UV signal obtained in step (b), thereby obtaining A 50％，样品 ; (c2) calculating the total area of ​​the sample UV peak used in step (c1) from the sample UV signal obtained in step (b) to obtain A 100％，样品 ; (c3) Determine A 50％，样品 and A 100％，样品 The ratio between them is obtained 样品 ; as well as (c4) Determine I 样品 and I 对照 , thereby obtaining the integrity of the RNA contained in the sample composition; or (B) Calculate the integrity of the control RNA by the following steps: (a") subjecting at least a portion of a control composition comprising a control RNA to field-flow fractionation, thereby fractionating components contained in the control composition according to their size to produce one or more control fractions; (b") measuring the UV signal of at least one of the one or more control fractions obtained from step (a"); as well as (c") Determine the height of a UV peak, H, from the UV signal obtained in step (b"). 对照 , thereby obtaining the integrity of the control RNA, and The integrity of the RNA contained in the sample composition was calculated by the following steps: (c1′) determining the height of the sample UV peak corresponding to the reference UV peak used in step (c″) from the UV signal obtained in step (b), H 样品 ;as well as (c2') Determine H 样品 and H 对照 The ratio between them is thus determined, thereby obtaining the integrity of the RNA contained in the sample composition.

23. The use according to claim 22, wherein the field-flow fractionation is flow field-flow fractionation.

24. The use according to claim 23, wherein the field-flow fractionation is an asymmetric flow field-flow fractionation or a hollow fiber flow field-flow fractionation.

25. The use of claim 22, wherein the field-flow fractionation uses a membrane having a molecular weight cutoff suitable for preventing RNA from penetrating the membrane, or the field-flow fractionation uses a membrane having a molecular weight cutoff in the range of 2 kDa-30 kDa.

26. The use according to claim 25, wherein the field-flow fractionation uses a polyethersulfone or regenerated cellulose membrane.

27. The method of claim 22, wherein step (a) is performed using: (I) a cross flow rate of up to 8 mL / min, or the following cross flow rate profile: 1.0-2.0 mL / min for 10 min, exponential gradient from 1.0-2.0 mL / min to 0.01-0.07 mL / min over 30 min; 0.01-0.07 mL / min for 30 min; and 0 mL / min for 10 min; and / or (II) an injection rate in the range of 0.05-0.35 mL / min; and / or (III) Detector flow rate in the range of 0.30-0.70 mL / min.

28. The use according to claim 22, wherein the sample composition comprises RNA and lipid complex particles and / or lipid nanoparticles and / or polyplex particles and / or lipid polyplex particles and / or virus-like particles, and the RNA is bound to the particles.

29. The use of claim 22, wherein the RNA is 10-15,000 nucleotides in length.

30. The use of claim 22, wherein the RNA is in vitro transcribed RNA or in vitro transcribed mRNA.

31. The use of claim 22, wherein measuring the UV signal is performed online, and / or step (c) is performed online.

32. The use of claim 22, wherein prior to subjecting at least a portion of the sample composition to field-flow fractionation, said at least a portion of the sample composition is diluted with a solvent or solvent mixture that is capable of preventing the formation of particle aggregates.

33. The use according to claim 32, wherein the solvent mixture is a mixture of water and an organic solvent or a mixture of water and formamide.

34. The use of claim 22, wherein measuring the UV signal is performed by using circular dichroism spectroscopy.

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