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

Abstract

To provide a method for determining at least one parameter of a sample composition comprising a nucleic acid, such as RNA, and optionally particles.SOLUTION: There is provided a method for determining integrity of RNA contained in a sample composition. Therein the sample composition comprises RNA and RNA-containing particles. The method comprises the steps of: (a) subjecting at least a part of the sample composition to field-flow fractionation, thereby fractionating components contained in the sample composition by their size so as to produce one or more sample fractions; (b) measuring at least one UV signal of at least one of the one or more sample fractions obtained from the step (a); and (c) calculating RNA integrity from the UV signal. Therein the integrity of the RNA contained in the sample composition is calculated using integrity of a control RNA.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] The use of recombinant nucleic acid (such as DNA or RNA) for delivering exogenous genetic information to target cells is well known.The advantages of using RNA include transient expression and non-transforming properties.RNA does not need to enter the nucleus for expression, and furthermore, it cannot be integrated into the host genome, thereby eliminating various risks such as carcinogenesis.

[0003] Recombinant nucleic acid can be administered to the subject who needs it in naked form, but usually, recombinant nucleic acid is administered using pharmaceutical composition.For example, RNA can be delivered by so-called nanoparticle formulation, which comprises RNA and nanoparticle-forming vehicle, for example, cationic lipid, mixture of cationic lipid and helper lipid, or cationic polymer.

[0004] The fate of such nanoparticle formulations is controlled by a variety of important 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 nanoparticles, etc.). These factors have been cited in the FDA "Liposome Drug Products Guidance" since 2018 as specific attributes that should be analyzed and identified. A limitation to the clinical application of current nanoparticle formulations may lie in the lack of uniform, pure, and well-characterized nanoparticle formulations. This is also due to the fact that all current techniques for determining these factors have some drawbacks.

[0005] For example, current techniques for determining the integrity and / or concentration of nucleic acids in nanoparticles (e.g., methods based on dyes, gel electrophoresis, microchannel electrophoresis, or capillary electrophoresis (CE)) are labor-intensive, costly, utilize artifact-inducing sample preparation steps, fail to provide sufficient information, and / or are unable to analyze large numbers of samples. One current technique that uses dyes (e.g., fluorescent dyes) is that the dye itself can introduce variances that can affect the reliability of the measurement results. In addition, many gel electrophoresis-based techniques require special care due to multiple washing steps, the use of special running buffers that increase the length of the procedure, and the use of toxic reagents. For example, agarose gel techniques are affected by multiple parameters [e.g., agarose quality, gel casting, dye / sensitivity (larger sample amounts required), exposure time, raw data processing, and standardized evaluation by densitometry software (28S / 18S method)], which make this technique unreliable. Techniques based on microchannel, chip-based, or capillary electrophoresis offer faster run times and improved data quality compared to agarose gel electrophoresis, but require hands-on processing for priming and loading gels, markers, and samples into the system. CE instruments lack the sensitivity, dynamic range, and separation quality required for adequate RNA quality / quantitative analysis.

[0006] Furthermore, one of the important issues for characterizing nanoparticle formulations is the quantitative determination of the size distribution of particles contained in the formulation.This is particularly true for particles with diameters less than about 500 nm, i.e., in the range suitable for most pharmaceutical products.Another unmet need is the determination of the size distribution of nanoparticle formulations whose size distribution is broad or complex (especially asymmetric).All existing techniques available for characterizing nanoparticle formulations have certain drawbacks, for example, they do not provide direct quantitative information on size, or they measure only a small, not adequately representative, subset of samples, or have very different sensitivities to particles with different sizes (or other parameters, such as the refractive index gradient relative to the bulk phase), which strongly affects the size distribution obtained.

[0007] For sizing particles in the lower submicron range (approximately 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 is calculated using the Stokes-Einstein equation to determine the hydrodynamic radius, R hDLS provides information about the diffusion constant of nanoparticles, from which particle size is calculated using a specific algorithm. However, DLS only provides average data, from which particle size is numerically calculated using a specific algorithm. To obtain quantitatively reliable numbers, nanoparticle preparations must be monomodal 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 assumes only a monomodal size distribution and provides physically meaningful numbers only 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 are highly dependent on the fitting parameters, and several very different profiles may correspond to the same data set. Such analysis is hindered by the fact that the light scattering intensity for large particles is much higher than for smaller particles, making 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 the diffusion constant of a very small (diluted) subset of a sample by microscopically observing the scattered light from that subset over time. While NTA can, in principle, provide a quantitative size distribution profile, it can only measure very diluted samples, and particles must exist in a relatively small size range. This means that very small particles cannot be determined in the context of much larger particles due to their much higher scattering intensity. Therefore, NTA is not a formally applicable method for determining quantitative size distributions for pharmaceutical formulations. Furthermore, the statistical standard deviation of NTA is high compared to other techniques (e.g., DLS). This is a direct result of the 1-3 orders of magnitude lower amount of particles analyzed by NTA. In particular, particles with marginal biological impact (e.g., aggregates) 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 identify the appropriate settings for accurate measurements. Typically, samples for NTA measurements must be diluted 10-1000 times, which can cause problems, especially with regard to concentration-dependent particle aggregation or disassembly. All these disadvantages make NTA difficult to establish 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 smaller than NTA. Therefore, EM has similar or identical disadvantages to NTA in that the particles measured may not be representative of the entire sample. Further major drawbacks of this technique are its high cost, complex sample preparation, and the long turnaround time required to analyze the sample. This explains why EM is not commonly used as a GMP method. Another problem with EM is that sample fixation can cause artifacts (e.g., shrinkage, clumping, etc.). If the sample is not fixed (e.g., Cryo-EM), the sample may have low contrast and may not be analyzed.

[0011] Other separation techniques, such as SEC-UV, are not applicable because interactions with the column matrix can cause problems (e.g., absorption or delay in elution). Nanoparticles may not be sufficiently dispersed due to the limited size range of the SEC column, or nanoparticles may not be separated from aggregates. Furthermore, SEC-UV does not provide a quantitative size distribution in the sense that mass or particle number is directly correlated with particle size. Other dispersive methods, such as analytical ultracentrifugation (AUC), only allow indirect size measurements, for example, based on the sedimentation coefficient, in which case several assumptions must be made to calculate the size profile. In addition, AUC is expensive and time-consuming, and it is not a common method in formal quality control. Therefore, AUC is also not suitable for determining quantitative size profiles as a formal quality control method. Summary of the Invention [Problem to be solved by the invention]

[0012] In view of the above, there is a need for advanced analytical methods for detailed particle characterization to ensure the reproducible quality of nanoparticle formulations. In particular, there is a need for improved methods for analyzing nanoparticle formulations containing nucleic acids (especially RNA), which preferably (i) provide information about the characteristics of the formulation (e.g., the quantitative size distribution of the particles contained in the formulation (especially for particles with a diameter of less than 500 nm)); (ii) provide information about the characteristics of the particle composition (e.g., the amount of nucleic acid (especially RNA) contained in the particles, particularly as a function of particle size, e.g., the ratio of the amount of nucleic acid (especially RNA) contained in the particles to the amount of particle-forming compounds (especially lipids and / or polymers, e.g., cationic lipids to cationic polymers), particularly as a function of particle size); (iii) are GMP-compatible; (iv) do not rely on the use of dyes; (v) are semi-automated; and / or (vi) are useful when preparing and / or storing compositions containing nucleic acids (e.g., RNA) and, where appropriate, particles. The method can be used to analyze the effect of altering one or more reaction conditions (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 (e.g., lipids and / or polymers); charge ratio; and / or ratio of nucleic acid (e.g., RNA) to particle-forming compounds (e.g., lipids and / or polymers)). Preferably, the method provides data regarding one or more of the following parameters: nucleic acid (e.g., RNA) integrity; total amount of nucleic acid (e.g., RNA); amount of free nucleic acid (e.g., RNA); amount of nucleic acid (e.g., RNA) bound to particles; size of nucleic acid (e.g., RNA)-containing particles (e.g., radius of gyration (R) of nucleic acid (e.g., RNA)-containing particles). g ) and / or the hydrodynamic radius (R h based on the size distribution of nucleic acid (e.g., RNA)-containing particles (e.g., R g Or R h quantitative size distribution of nucleic acid (e.g., RNA)-containing particles (e.g., based on R gOr R h value); molecular weight of the nucleic acid (e.g., RNA); and / or shape (e.g., shape and / or form factor) of the nucleic acid (e.g., RNA)-containing particle. Optionally, the additional parameters can include one or more of the following: amount of surface nucleic acid (e.g., amount of surface RNA), amount of encapsulated nucleic acid (e.g., amount of encapsulated RNA), amount of accessible nucleic acid (e.g., amount of accessible RNA), size of the nucleic acid (particularly RNA) (e.g., R g or R h value), size distribution of nucleic acids (e.g., R g or R h values), quantitative size distribution of nucleic acids (e.g., R g or R h values), nucleic acid (particularly RNA) encapsulation efficiency, the ratio of the amount of nucleic acid (e.g., RNA) bound to the particle to the total amount of particle-forming compounds (particularly lipids and / or polymers) in the particle, the ratio of the amount of positively charged moieties of particle-forming compounds (particularly lipids and / or polymers) in the particle to the amount of nucleic acid (e.g., RNA) bound to the particle, and the charge ratio (N / P ratio) of the amount of positively charged moieties of particle-forming compounds (particularly lipids and / or polymers) in the particle to the amount of negatively charged moieties of nucleic acid (e.g., RNA) bound to the particle.

[0013] The inventors have surprisingly found that the methods and uses described herein meet the above requirements. [Means for solving the problem]

[0014] In a first aspect, the present disclosure provides a method for determining one or more parameters of a sample composition, the sample composition comprising nucleic acid (e.g., RNA) and optionally particles, the method comprising: (a) subjecting at least a portion of the sample composition to field-flow fractionation to size-fractionate components contained in the sample composition to produce one or more sample fractions; (b) measuring at least one signal selected from the group consisting of a UV signal, a fluorescence signal, and a refractory index (RI) signal, and optionally a light scattering (LS) signal, of at least one of the one or more sample fractions obtained from step (a); (c) calculating one or more parameters from at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal, and optionally from an LS signal; Including, The one or more parameters may include nucleic acid (e.g., RNA) integrity, total amount of nucleic acid (e.g., RNA), amount of free nucleic acid (e.g., RNA), amount of nucleic acid (e.g., RNA) bound to particles, size of nucleic acid (e.g., RNA)-containing particles [particularly, radius of gyration (R) of nucleic acid (e.g., RNA)-containing particles], and the like. g ) and / or the hydrodynamic radius (R h ) based on the size distribution of nucleic acid (e.g., RNA)-containing particles [e.g., the R g Value or R h values], and quantitative size distribution of nucleic acid (e.g., RNA)-containing particles [e.g., R g Value or R h The present invention provides a method for determining the size distribution and / or quantitative size distribution of nucleic acid (e.g., RNA)-containing particles, the number of nucleic acid (e.g., RNA)-containing particles, the molar amount of nucleic acid (e.g., RNA)-containing particles, or the mass of nucleic acid (e.g., RNA)-containing particles (each as a function of particle size). Optional additional parameters include the molecular weight of the nucleic acid (e.g., RNA), the amount of surface nucleic acid (e.g., the amount of surface RNA), the amount of encapsulated nucleic acid (e.g., the amount of encapsulated RNA), the amount of accessible nucleic acid (e.g., the amount of accessible RNA), the size of the nucleic acid (e.g., RNA) [e.g., the R of the nucleic acid (e.g., RNA) g Value and / or R h value], size distribution of nucleic acids (especially RNA) [e.g., R g Value or R hvalues], quantitative size distribution of nucleic acids (especially RNA) [e.g., R g Value or R h Based on the value], shape factor, form factor, and nucleic acid (especially RNA) encapsulation efficiency. Usually, the size distribution and / or quantitative size distribution of nucleic acid (especially RNA) can be given as the number of nucleic acid (especially RNA) molecules, the molar amount of nucleic acid (especially RNA), or the mass of nucleic acid (especially RNA) (each as a function of particle size). In addition, optional additional parameters include the ratio of the amount of nucleic acid (e.g., RNA) bound to particles to the total amount of particle-forming compounds (especially lipids and / or polymers) in particles, which can be given as a function of particle size; the ratio of the amount of positively charged moieties of particle-forming compounds (especially lipids and / or polymers) in particles to the amount of nucleic acid (e.g., RNA) bound to particles, which can be given as a function of particle size; and the charge ratio of the amount of positively charged moieties of particle-forming compounds (especially lipids and / or polymers) in particles to the amount of negatively charged moieties of nucleic acid (e.g., RNA) bound to particles, which is usually expressed as N / P ratio and can be given as a function of particle size.

[0015] In a first subgroup of the first embodiment, the method comprises: (a) subjecting at least a portion of the sample composition to field-flow fractionation to size-fractionate components contained in the sample composition to produce one or more sample fractions; (b) measuring at least the UV signal, and optionally the light scattering (LS) signal, of at least one of the one or more sample fractions obtained from step (a); (c) calculating one or more parameters from the UV signal and, optionally, from the LS signal; Includes.

[0016] In a second and preferred subgroup of the first aspect, the method is for determining one or more parameters of a sample composition, the sample composition comprising RNA, and optionally particles, and the method comprises: (a) subjecting at least a portion of the sample composition to field-flow fractionation to size-fractionate components contained in the sample composition to produce one or more sample fractions; (b) measuring at least one signal selected from the group consisting of a UV signal, a fluorescence signal, and a refractive index (RI) signal, and optionally a light scattering (LS) signal, of at least one of the one or more sample fractions obtained from step (a); (c) calculating one or more parameters from at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal, and optionally from an LS signal; Includes.

[0017] In a third and more preferred subgroup of the first aspect, the method is for determining one or more parameters of a sample composition, the sample composition comprising RNA, and optionally particles, and the method comprises: (a) subjecting at least a portion of the sample composition to field-flow fractionation to size-fractionate components contained in the sample composition to produce one or more sample fractions; (b) measuring at least the UV signal, and optionally the light scattering (LS) signal, of at least one of the one or more sample fractions obtained from step (a); (c) calculating one or more parameters from the UV signal and, optionally, from the LS signal; Includes.

[0018] In one embodiment of the first aspect (particularly in one 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 (e.g., RNA)-containing particles is determined by the Rg In another embodiment of the first aspect (particularly 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 (e.g., RNA)-containing particles is calculated based on the R value of the nucleic acid (e.g., RNA)-containing particles. h In another embodiment of the first aspect (particularly 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 (e.g., RNA)-containing particles is calculated based on the R value of the nucleic acid (e.g., RNA)-containing particles. g Based on the R value, and separately, the R of the nucleic acid (e.g., RNA)-containing particles h values [i.e., this embodiment results in two data sets for the size, size distribution, and / or quantitative size distribution of nucleic acid (e.g., RNA)-containing particles, one of which is R g Based on the value, and one is R h value-based].

[0019] In one embodiment of the first aspect (particularly in one embodiment of the first, second or third subgroup of the first aspect) where the one or more parameters include size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA), the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA) may be determined by the R g In other embodiments of the first aspect (particularly other embodiments of the first, second, or third subgroups of the first aspect) where the one or more parameters include the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA), the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA) is calculated based on the R hIn other embodiments of the first aspect (particularly other embodiments of the first, second, or third subgroups of the first aspect) where the one or more parameters include the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA), the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA) is calculated based on the R g Based on the value, and separately, the R h [i.e., this embodiment provides two data sets for nucleic acid (e.g., RNA) size, size distribution, and / or quantitative size distribution, one of which is R g Based on the value, and one is R h value-based].

[0020] In one embodiment of the first aspect (particularly in one embodiment of the first, second or third subgroup of the first aspect), the field-flow fractionation is preferably flow field-flow fractionation, such as asymmetric flow field-flow fractionation (AF4) or hollow fiber flow field-flow fractionation (HF5).

[0021] 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 carried out using a membrane with a molecular weight (MW) cut-off suitable to prevent nucleic acids (especially RNA) from passing through, preferably a membrane with a MW cut-off in the range of 2 kDa to 30 kDa, for example a MW cut-off of 10 kDa.

[0022] 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 carried out using a polyethersulfon (PES) or regenerated cellulose membrane.

[0023] 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 carried out using: (I) a cross-flow rate, e.g., a cross-flow rate profile, of up to 8 mL / min, preferably up to 4 mL / min, more preferably up to 2 mL / min; and / or (II) an inject flow 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 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.

[0024] In one embodiment of the first aspect (particularly in one embodiment of the first, second, or third subgroup of the first aspect), the cross-flow rate profile preferably includes a fractionation phase in which components contained in the control or sample composition are fractionated / separated by size to generate one or more sample fractions. The cross-flow rate preferably varies during this fractionation phase (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)). The variation may be continuous (e.g., linear or exponential) or stepwise. Preferably, the cross-flow rate profile includes a fractionation phase in which the cross-flow rate varies continuously (preferably exponentially) starting from a certain 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 phase may have any length suitable for fractionating / separating components contained in the sample composition by size, such as about 5 to about 60 minutes, e.g., about 10 to about 50 minutes, about 15 to about 45 minutes, about 20 to about 40 minutes, or about 25 to about 35 minutes, or about 30 minutes. The cross-flow rate profile may contain additional phases (e.g., 1, 2, 3, or 4 phases), which may precede and / or follow the fractionation phase (e.g., 1 phase before and 1, 2, or 3 phases after), and which may function to separate non-nucleic acid (especially non-RNA) components contained in the sample composition (e.g., proteins, polypeptides, mononucleotides, etc.) from nucleic acids (especially RNA) contained in the sample composition, to focus the nucleic acids (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 these additional phases is constant for each additional phase, and the length of each additional phase is, independently, within the range of about 5 minutes to about 60 minutes (about 10 minutes to about 50 minutes, about 15 minutes to about 45 minutes, about 20 minutes to about 40 minutes, or about 25 minutes to about 35 minutes, or about 30 minutes). For example, the cross flow rate profile may include: (i) a first additional phase preceding a fractionation phase, in which the cross flow rate of the first additional phase is constant and begins at the same cross flow rate at which the fractionation phase begins; (ii) a second additional phase following the fractionation phase, wherein the cross-flow rate of the second additional phase is constant and is the same cross-flow rate at which the fractionation phase ends (the length of the second additional phase may be in the range of about 5 minutes to about 60 minutes, e.g., about 10 minutes to about 50 minutes, about 15 minutes to about 45 minutes, about 20 minutes to about 40 minutes, or about 25 minutes to about 35 minutes, or about 10 minutes, or about 20 minutes, or about 30 minutes); (iii) a second additional phase following the fractionation phase, wherein the cross-flow rate of the second additional phase is constant and is the same cross-flow rate at which the fractionation phase ends (the length of the second additional phase may be in the range of about 5 minutes to about 60 minutes, e.g., about 10 minutes to about 50 minutes, about 15 minutes to about 45 minutes, or about 20 minutes to about 40 minutes, or about 25 minutes to about 35 minutes, or about 10 minutes, or about 20 minutes, or about 30 minutes); and optionally (iii) a third additional phase following the second additional phase, wherein the cross-flow rate of said third additional phase is constant and different from the cross-flow rate of the second additional phase (the length of the third additional phase may be in the range of about 5 minutes to about 60 minutes, e.g., about 10 minutes to about 50 minutes, about 15 minutes to about 45 minutes, about 20 minutes to about 40 minutes, or about 25 minutes to about 35 minutes, or about 10 minutes, or about 20 minutes, or about 30 minutes).In embodiments where the cross-flow rate profile contains a fractionation phase and the cross-flow rate varies continuously (preferably exponentially) starting from a certain 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 cross-flow rate profile may further include: (i) a first additional phase preceding the fractionation phase, wherein the cross-flow rate of said first additional phase is constant and at the same cross-flow rate (e.g., about 1 to about 4 mL / min) at which the fractionation phase begins (the length of the first additional phase may be in the range of about 5 minutes to about 30 minutes, e.g., about 6 minutes to about 25 minutes, about 7 minutes to about 20 minutes, or about 8 minutes to about 15 minutes, or about 10 minutes to about 12 minutes, or about 5 minutes, or about 10 minutes, or about 12 minutes); (ii) a second additional phase following the fractionation phase, wherein the cross-flow rate of said second additional phase is constant and at the same cross-flow rate (e.g., about 1 to about 4 mL / min) at which the fractionation phase begins (the length of the first additional phase may be in the range of about 5 minutes to about 30 minutes, e.g., about 6 minutes to about 25 minutes, about 7 minutes to about 20 minutes, or about 8 minutes to about 15 minutes, or about 10 minutes to about 12 minutes); a second additional phase having the same cross-flow rate (e.g., about 0.01 to about 0.1 mL / min) at which the fractionation phase ends (the length of the second additional phase may be in the range of about 5 minutes to about 60 minutes, e.g., about 10 minutes to about 50 minutes, about 15 minutes to about 45 minutes, about 20 minutes to about 40 minutes, or about 25 minutes to about 35 minutes, or about 30 minutes); and optionally (iii) a third additional phase following the second additional phase, wherein the cross-flow rate of the third additional phase is Preferably, the cross-flow rate profile includes a third additional phase whose cross-flow rate is constant and lower than that of the second additional phase (e.g., the cross-flow rate of the third additional phase is 0). (The length of the third additional phase may be within the range of about 5 minutes to about 30 minutes, e.g., about 6 minutes to about 25 minutes, about 7 minutes to about 20 minutes, about 8 minutes to about 15 minutes, about 10 minutes to about 12 minutes, or about 5 minutes, about 10 minutes, or about 12 minutes.) Preferred examples of such cross-flow rate profiles are as follows: 1.0-2.0 mL / min for 10 minutes, an exponential ramp from 1.0-2.0 mL / min to 0.01-0.07 mL / min within 30 minutes; 0.01-0.07 mL / min for 30 minutes; and 0 mL / min for 10 minutes.

[0025] 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 the nucleic acid (especially RNA) contained in the sample composition is calculated using the integrity of a control nucleic acid (especially RNA).

[0026] In a first particular example of this embodiment of the first aspect, the integrity of a control nucleic acid (in particular RNA) is determined by the following steps: (a') subjecting at least a portion of a control composition containing a control nucleic acid (especially RNA) to field-flow fractionation, particularly AF4 or HF5, to size fractionation of components contained in the control composition to produce one or more control fractions; (b') measuring at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and a refractive index (RI) signal of at least one of the one or more control fractions obtained from step (a'); (c'1) From at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained in step (b'), calculate the area from the maximum height of one UV, fluorescent, or RI peak to the end of the UV, fluorescent, or RI peak, thereby obtaining A 50% obtaining a control; (c'2) A is obtained by calculating the total area of one peak used in step (c'1) from at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained in step (b'). 100% obtaining a control; (c'3)A 50% (control) and A 100% (control) to obtain the integrity of the control nucleic acid (especially RNA) [I(control)]; is calculated by

[0027] In this first example, the integrity of the nucleic acids (especially RNA) contained in the sample composition is determined by the following steps: (c1) From at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained in step (b), calculate the area from the maximum height of the sample UV, fluorescent, or RI peak corresponding to the control UV, fluorescent, or RI peak used in step (c'1) to the end of the sample UV, fluorescent, or RI peak, thereby obtaining A 50% Obtaining a sample; (c2) A is obtained by 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 from step (b). 100% Obtaining a sample; (c3)A 50% (Sample) and A 100% obtaining I(sample) by calculating the ratio of I(sample); (c4) determining the ratio of I (sample) to I (control) to determine the integrity of the nucleic acids (especially RNA) contained in the sample composition; It can be calculated by:

[0028] In a second particular example of this embodiment of the first aspect, the integrity of a control nucleic acid (especially RNA) is determined by the following steps: (a") subjecting at least a portion of a control composition containing a control nucleic acid (especially RNA) to field-flow fractionation, particularly AF4 or HF5, to size fractionation of components contained in the control composition to produce one or more control fractions; (b") measuring at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal of at least one of the one or more control fractions obtained from step (a"); (c") determining the integrity of a control nucleic acid (especially RNA) by determining the height [H(control)] of one UV, fluorescent, or RI peak from at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained in step (b"); is calculated by

[0029] In this second example, the integrity of the nucleic acids (especially RNA) contained in the sample composition is determined by the following steps: (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 group consisting of a UV signal, a fluorescence signal, and an RI signal obtained in step (b); (c2') determining the ratio of H(sample) to H(control) to determine the integrity of the nucleic acids (especially RNA) contained in the sample composition; It can be calculated by:

[0030] In a third particular example of this embodiment of the first aspect (relating to the first subgroup of the first aspect), the integrity of the control nucleic acid (in particular RNA) is determined by the following steps: (a') subjecting at least a portion of a control composition containing a control nucleic acid (especially RNA) to field-flow fractionation, particularly AF4 or HF5, to size fractionation of components contained in the control composition to produce one or more control fractions; (b') measuring at least the UV signal of at least one of the one or more control fractions obtained from step (a'); (c'1) From the UV signal obtained in step (b'), calculate the area from the maximum height of one UV peak to the end of the UV peak to obtain A 50% obtaining a control; (c'2) A is obtained by calculating the total area of the single peak used in step (c'1) from the UV signal obtained in step (b'). 100% obtaining a control; (c'3)A 50% (control) and A 100% (control) to obtain the integrity of the control nucleic acid (especially RNA) [I(control)]; is calculated by

[0031] In this third example, the integrity of the nucleic acids (especially RNA) contained in the sample composition is determined by the following steps: (c1) From the UV signal obtained from step (b), calculate 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, thereby obtaining A 50% Obtaining a sample; (c2) A is calculated by calculating the total area of the sample UV peaks used in step (c1) from the sample UV signal obtained from step (b). 100% Obtaining a sample; (c3)A 50% (Sample) and A 100% obtaining I(sample) by calculating the ratio of I(sample); (c4) determining the ratio of I (sample) to I (control) to determine the integrity of the nucleic acids (especially RNA) contained in the sample composition; It can be calculated by:

[0032] In a fourth particular example of this embodiment of the first aspect (relating to the first subgroup of the first aspect), the integrity of the control nucleic acid (in particular RNA) is determined by the following steps: (a") subjecting at least a portion of a control composition containing a control nucleic acid (especially RNA) to field-flow fractionation, particularly AF4 or HF5, to size fractionation of components contained in the control composition to produce one or more control fractions; (b") measuring at least the UV signal of at least one of the one or more control fractions obtained from step (a"); (c") determining the integrity of a control nucleic acid (especially RNA) by determining the height of one UV peak [H(control)] from the UV signal obtained in step (b"); is calculated by

[0033] In this fourth example, the integrity of nucleic acids (especially RNA) contained in a sample composition is determined by the following steps: (c1') determining the height [H(sample)] of the sample UV peak corresponding to the control UV peak used in step (c") from the UV signal obtained in step (b); (c2') determining the ratio of H(sample) to H(control) to determine the integrity of the nucleic acids (especially RNA) contained in the sample composition; It can be calculated by:

[0034] In a fifth particular example of this embodiment of the first aspect (related to the second subgroup of the first aspect), the integrity of the control RNA is determined by the following steps: (a') subjecting at least a portion of the control composition containing the control RNA to field-flow fractionation, particularly AF4 or HF5, to size fractionate the components contained in the control composition to produce one or more control fractions; (b') measuring at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal of at least one of the one or more control fractions obtained from step (a'); (c'1) From at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained in step (b'), calculate the area from the maximum height of one UV, fluorescent, or RI peak to the end of the UV, fluorescent, or RI peak, thereby obtaining A 50% obtaining a control; (c'2) A total area of one peak used in step (c'1) is calculated from at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained in step (b'). 100% obtaining a control; (c'3)A 50% (control) and A 100% obtaining the control RNA integrity [I(control)] by calculating the ratio of I(control) to I(control); is calculated by

[0035] In this fifth example, the integrity of the RNA contained in the sample composition is determined by the following steps: (c1) From at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained in step (b), calculate the area from the maximum height of the sample UV, fluorescent, or RI peak corresponding to the control UV, fluorescent, or RI peak used in step (c'1) to the end of the sample UV, fluorescent, or RI peak, thereby obtaining A 50% Obtaining a sample; (c2) A is obtained by 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 from step (b). 100% Obtaining a sample; (c3)A 50% (Sample) and A 100% obtaining I(sample) by calculating the ratio of I(sample); (c4) determining the ratio of I (sample) to I (control) to determine the integrity of the RNA contained in the sample composition; It can be calculated by:

[0036] In a sixth particular example of this embodiment of the first aspect (related to the second subgroup of the first aspect), the integrity of the control RNA is determined by the following steps: (a") subjecting at least a portion of a control composition containing a control RNA to field-flow fractionation, particularly AF4 or HF5, to size fractionate components contained in the control composition to produce one or more control fractions; (b") measuring at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal of at least one of the one or more control fractions obtained from step (a"); (c") determining the integrity of the control RNA by determining the height [H(control)] of one UV, fluorescent, or RI peak from at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained in step (b"); is calculated by

[0037] In this sixth example, the integrity of the RNA contained in the sample composition is determined by the following steps: (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 group consisting of a UV signal, a fluorescence signal, and an RI signal obtained in step (b); (c2') determining the ratio of H(sample) to H(control) to determine the integrity of the RNA contained in the sample composition; It can be calculated by:

[0038] In a seventh particular example of this embodiment of the first aspect (related to the third subgroup of the first aspect), the integrity of the control RNA is determined by the following steps: (a') subjecting at least a portion of the control composition containing the control RNA to field-flow fractionation, particularly AF4 or HF5, to size fractionate the components contained in the control composition to produce one or more control fractions; (b') measuring at least the UV signal of at least one of the one or more control fractions obtained from step (a'); (c'1) From the UV signal obtained in step (b'), calculate the area from the maximum height of one UV peak to the end of the UV peak to obtain A 50% obtaining a control; (c'2) A is obtained by calculating the total area of the single peak used in step (c'1) from the UV signal obtained in step (b'). 100% obtaining a control; (c'3)A 50% (control) and A 100% obtaining the control RNA integrity [I(control)] by calculating the ratio of I(control) to I(control); is calculated by

[0039] In this seventh example, the integrity of the RNA contained in the sample composition is determined by the following steps: (c1) From the UV signal obtained from step (b), calculate 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, thereby obtaining A 50% Obtaining a sample; (c2) A is calculated by calculating the total area of the sample UV peaks used in step (c1) from the sample UV signal obtained from step (b). 100% Obtaining a sample; (c3)A 50% (Sample) and A 100% obtaining I(sample) by calculating the ratio of I(sample); (c4) determining the ratio of I (sample) to I (control) to determine the integrity of the RNA contained in the sample composition; It can be calculated by:

[0040] In an eighth particular example of this embodiment of the first aspect (related to the third subgroup of the first aspect), the integrity of the control RNA is determined by the following steps: (a") subjecting at least a portion of a control composition containing a control RNA to field-flow fractionation, particularly AF4 or HF5, to size fractionate components contained in the control composition to produce one or more control fractions; (b") measuring at least the UV signal of at least one of the one or more control fractions obtained from step (a"); (c") determining the integrity of the control RNA by determining the height of one UV peak [H(control)] from the UV signal obtained in step (b"); is calculated by

[0041] In this eighth example, the integrity of the RNA contained in the sample composition is determined by the following steps: (c1') determining the height [H(sample)] of the sample UV peak corresponding to the control UV peak used in step (c") from the UV signal obtained in step (b); (c2') determining the ratio of H(sample) to H(control) to determine the integrity of the RNA contained in the sample composition; It can be calculated by:

[0042] 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 decay coefficient (especially an RNA decay coefficient) or (ii) a nucleic acid calibration curve (especially an RNA calibration curve).

[0043] In one embodiment of the first aspect (particularly in one embodiment of the first, second or third subgroup of the first aspect), the sample composition comprises nucleic acid (especially RNA) and particles to which the nucleic acid (especially RNA) is associated, such as lipoplex particles and / or lipid nanoparticles and / or polyplex particles and / or lipopolyplex particles and / or virus-like particles.

[0044] 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) through (c) on at least the portion obtained from step (i); and (iii) determining the amount of nucleic acid (particularly RNA) as defined herein (e.g., by using (i) a nucleic acid decay coefficient (particularly an RNA decay 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.

[0045] 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 dodecyl sulfate), a zwitterionic surfactant [e.g., n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent® 3-14)], a cationic surfactant, a nonionic surfactant, or a mixture thereof; (ii) an alcohol, such as an aliphatic alcohol (e.g., ethanol), or a mixture of alcohols; or (iii) a combination of (i) and (ii).

[0046] 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 free nucleic acid (especially RNA) is determined by carrying out steps (a) to (c) without the addition of a releasing agent, particularly without any releasing agent; and determining the amount of nucleic acid (especially RNA) as defined herein (for example by using (i) a nucleic acid decay factor (especially an RNA decay factor) or (ii) a nucleic acid calibration curve (especially an RNA calibration curve)).

[0047] 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 nucleic acid (especially RNA) bound to the particles is determined herein [e.g., by carrying out steps (a) to (c) without the addition of a releasing agent, particularly without any releasing agent; and determining the amount of nucleic acid (especially RNA) as defined herein [e.g., by using (i) a nucleic acid decay factor (especially an RNA decay factor) or (ii) a nucleic acid calibration curve (especially an RNA calibration curve)]] by subtracting the amount of free nucleic acid (especially RNA) from the amount of total nucleic acid (especially RNA) as determined herein [e.g., (i) treating at least a portion of the sample composition with a releasing agent; (ii) carrying out steps (a) to (c) on at least the portion obtained from step (i); and (iii) determining the amount of nucleic acid (especially RNA) as defined herein [e.g., by using (i) a nucleic acid decay factor (especially an RNA decay factor) or (ii) a nucleic acid calibration curve (especially an RNA calibration curve)]].

[0048] In one embodiment of the first aspect (particularly in one embodiment of the first, second or third subgroup of the first aspect), step (b) further comprises measuring at least one LS signal, such as a dynamic light scattering (DLS) signal and / or a static light scattering (SLS), such as a multi-angle light scattering (MALS) signal, of one or more sample fractions obtained from step (a).

[0049] In one embodiment of the first aspect (particularly in one embodiment of the first, second or third subgroup of the first aspect), the size of the nucleic acid (particularly RNA) containing particles can be determined from the LS signal obtained from step (b) by measuring the radius of gyration (R g ) value and / or hydrodynamic radius (R hIn 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 sample fractions obtained from step (a), and step (c) comprises calculating an R 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 at least one static light scattering (SLS), e.g., MALS, signal of one or more sample fractions obtained from step (a), and step (c) comprises calculating an R value from the SLS signal. g 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 at least one dynamic light scattering (DLS) signal and a static light scattering (SLS), e.g., MALS, signal of one or more sample fractions obtained from step (a), and step (c) comprises calculating an R g value and R h This latter embodiment involves calculating two data sets of sizes of nucleic acid (e.g., RNA)-containing particles: R g Value-based and R h Bringing value-based.

[0050] In one embodiment of the first aspect (particularly in one embodiment of the first, second or third subgroup of the first aspect), the size distribution of the nucleic acid (particularly RNA) containing particles is determined by comparing at least one signal selected from the group consisting of a UV signal, a fluorescent signal and an RI signal obtained from step (b) with an R determined as defined herein. g Value or R h by plotting the R g or by calculating the R value from the DLS signal obtained from step (b). hIn a first example of this embodiment (pertaining to the first subgroup of the first aspect), the size distribution of nucleic acid (especially RNA) containing particles is determined by multiplying the UV signal from step (b) by the R value determined as defined herein. g Value or R h by plotting the R g or by calculating the R value from the DLS signal obtained from step (b). h In a second example of this embodiment (pertaining to the second subgroup of the first aspect), the size distribution of the RNA-containing particles is determined by multiplying at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained from step (b) by an R value determined as defined herein. g Value or R h by plotting the R g or by calculating the R value from the DLS signal obtained from step (b). h In a third example of this embodiment (pertaining to the third subgroup of the first aspect), the size distribution of the RNA-containing particles is determined by multiplying the UV signal from step (b) by the R value, determined as defined herein. g Value or R h by plotting the R g or by calculating the R value from the DLS signal obtained from step (b). h In each of the first, second, and third examples above, the size distribution of nucleic acid (especially RNA)-containing particles is determined by the R g Value, R h The size distribution of nucleic acid (especially RNA)-containing particles can be determined based on R g value and R h If it is based on the values of two data sets, namely, R g Size distribution based on values and Rh This results in a size distribution based on the value.

[0051] 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 of the nucleic acid (particularly RNA) containing particles is R g Value or R h From a plot showing the UV, fluorescence, or RI signal as a function of the value, convert the UV, fluorescence, or RI signal to a cumulative weight fraction and convert the cumulative weight fraction to R g Value or R h In a first example of this embodiment (pertaining to the first subgroup of the first aspect), the quantitative size distribution of nucleic acid (especially RNA) containing particles is calculated by plotting the R g Value or R h From the plot showing the UV signal as a function of the value, convert the UV signal to cumulative weight fraction and calculate the cumulative weight fraction as R g Value or R h In a second example of this embodiment (pertaining to the second subgroup of the first aspect), the quantitative size distribution of the RNA-containing particles is calculated by plotting the R g Value or R h From a plot showing the UV, fluorescence, or RI signal as a function of the value, convert the UV, fluorescence, or RI signal to a cumulative weight fraction and convert the cumulative weight fraction to R g Value or R h In a third example of this embodiment (pertaining to the third subgroup of the first aspect), the quantitative size distribution of the RNA-containing particles is calculated by plotting the R g Value or R h From the plot showing the UV signal as a function of the value, convert the UV signal to cumulative weight fraction and calculate the cumulative weight fraction as R g Value or R h In each of the first, second, and third examples above, the quantitative size distribution of nucleic acid (especially RNA)-containing particles is calculated by plotting the R g Value, R hThe quantitative size distribution of nucleic acid (especially RNA)-containing particles can be determined based on R g value and R h If it is based on the values of two data sets, namely, R g Quantitative size distribution and R based on values h This results in a quantitative size distribution based on the values.

[0052] 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 is measured using a D10 value, a D50 value, and / or a D90 value (e.g., R g Value or R h The quantitative size distribution of nucleic acid (especially RNA)-containing particles is based on the R g value and R h If it is based on the values of two data sets, namely, R g a set of D10, D50, and / or D90 values based on the R h The resulting set of D10, D50, and / or D90 values is based on the value.

[0053] In one embodiment of the first aspect (particularly in one embodiment of the first, second or third subgroup of the first aspect), the one or more parameters are at least two, preferably at least three parameters (including additional parameters as needed) as defined herein, in particular the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to the particles, the size distribution of the nucleic acid (especially RNA)-containing particles [in particular the radius of gyration (R) of the nucleic acid (especially RNA)-containing particles], g ) and / or the hydrodynamic radius (R h ) ], and quantitative size distribution of nucleic acid (especially RNA)-containing particles (e.g., R g Value or R h The size distribution of nucleic acid (especially RNA)-containing particles comprises (or is) at least two, preferably at least three parameters selected from the group consisting of: Rg value and R h If it is based on the values of two data sets, namely, R g Value-based and R h However, according to the present invention, these two data sets for the size distribution of nucleic acid (especially RNA)-containing particles are considered as only one parameter (not two parameters). Also, when a fractogram obtained by field-flow fractionation shows multiple particle peaks, the size distribution obtained for each particle peak is considered as only one parameter (not one parameter for each particle peak). The quantitative size distribution of nucleic acid (especially RNA)-containing particles is considered as R g value and R h The same is true for value-based situations.

[0054] 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 include a quantitative size distribution of the nucleic acid (especially RNA)-containing particles [e.g., a radius of gyration (R g ) and / or the hydrodynamic radius (R h ) )], and optionally at least one parameter, e.g., at least two parameters, of the remaining parameters (including additional parameters as needed) defined herein; preferably, the remaining parameters include 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 nucleic acid (especially RNA)-containing particles (e.g., R g Value or R hIn one embodiment of the first aspect (particularly in an 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 are selected from the group consisting of a quantitative size distribution (e.g., R g Value or R h values), as well as the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to the particles, and the size distribution of nucleic acid (especially RNA)-containing particles (e.g., R g Value or R h In one embodiment of the first aspect (particularly in an 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 are based on a quantitative size distribution (e.g., R g Value or R h The quantitative size distribution of nucleic acid (especially RNA)-containing particles includes the amount of nucleic acid (especially RNA) bound to the particle, based on the R g value and R h If it is based on the values of two data sets, namely, R g Value-based and R h However, according to the present invention, these two data sets for the quantitative size distribution of nucleic acid (especially RNA)-containing particles are considered as only one parameter (not two parameters). Also, in the case where a fractogram obtained by field-flow fractionation shows multiple particle peaks, the quantitative size distribution obtained for each particle peak is considered as only one parameter (not one parameter for each particle peak). When the size distribution of nucleic acid (especially RNA)-containing particles is R g value and R h The same is true for value-based situations.

[0055] 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 nucleic acid (especially RNA), in particular free nucleic acid (especially RNA), is determined by measuring the UV signal, for example at a wavelength in the range of 260 nm to 280 nm, for example at a wavelength of 260 nm or 280 nm, and using the nucleic acid (especially RNA) extinction coefficient at the corresponding wavelength (e.g. 260 nm or 280 nm).

[0056] 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 nucleic acid (especially RNA) containing particles (e.g., R g Value or R h values) and / or quantitative size distribution of nucleic acid (especially RNA)-containing particles (e.g., R g Value or R h value) is in the range of 10 to 2000 nm, preferably 20 to 1500 nm, for example, 30 to 1200 nm, 40 to 1100 nm, 50 to 1000, 60 to 900 nm, 70 to 800 nm, 80 to 700 nm, 90 to 600 nm, or 100 to 500 nm, or for example, 10 to 1000 nm, 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm, or 50 to 250 nm. In a preferred embodiment of the third subgroup of the first aspect, the (quantitative) size distribution (e.g., R g Value or R h value) is in the range of 10 to 1000 nm, for example, in the range of 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm, or 50 to 250 nm.

[0057] 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 (particularly RNA) has a length of 10 to 15,000 nucleotides, for example 40 to 15,000 nucleotides, 100 to 12,000 nucleotides, or 200 to 10,000 nucleotides.

[0058] 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 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.

[0059] In one embodiment of the first aspect (particularly in one embodiment of the first, second or third subgroup of the first aspect), measuring at least one signal selected from the group consisting of a UV signal, a fluorescence signal and an RI signal, optionally an LS signal, such as an SLS, such as an MALS signal, and / or a DLS signal, is performed online and / or step (c) is performed online.

[0060] In one embodiment of the first aspect (particularly in one embodiment of the first, second or third subgroup of the first aspect), the one or more parameters are determined by one cycle of steps (a) to (c).

[0061] 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, at least a portion of the sample composition is diluted with a solvent or solvent mixture capable of preventing the formation of particle aggregates. In one embodiment, the solvent mixture is a mixture of water and an organic solvent, such as formamide.

[0062] In one embodiment of the first aspect (particularly in one embodiment of the first, second or third subgroup of the first aspect), measuring the UV signal is carried out by using circular dichroism (CD) spectroscopy.

[0063] In a second aspect, the present disclosure provides a method for analyzing the effect of altering one or more reaction conditions when providing a composition comprising nucleic acid (e.g., RNA) and optionally particles, the method comprising: (A) providing a first composition comprising nucleic acid (e.g., RNA) and, optionally, particles; (B) providing a second composition comprising nucleic acid (e.g., RNA) and optionally particles, which differs from the first composition only in one or more reaction conditions; (C) determining one or more parameters of the first composition by subjecting a portion of the first composition to the method of the first aspect; (D) determining one or more parameters of the second composition by subjecting a corresponding portion of the second composition to the method used in step (C); (E) comparing one or more parameters of the first composition obtained in step (C) with corresponding one or more parameters of the second composition obtained in step (D); The present invention provides a method comprising:

[0064] In one embodiment of the second aspect, the one or more parameters include nucleic acid (e.g., RNA) integrity, total amount of nucleic acid (e.g., RNA), amount of free nucleic acid (e.g., RNA), amount of nucleic acid (e.g., RNA) bound to particles, size of nucleic acid (e.g., RNA)-containing particles [particularly, radius of gyration (R) of nucleic acid (e.g., RNA)-containing particles], g ) and / or the hydrodynamic radius (R h ) )], size distribution of nucleic acid (e.g., RNA)-containing particles [e.g., R g Value or R hvalues], and quantitative size distribution of nucleic acid (e.g., RNA)-containing particles [e.g., R g Value or R h Typically, the size distribution and / or quantitative size distribution of nucleic acid (e.g., RNA)-containing particles can be given as the number of nucleic acid (e.g., RNA)-containing particles, the molar amount of nucleic acid (e.g., RNA)-containing particles, or the mass of nucleic acid (e.g., RNA)-containing particles (each as a function of particle size). Additional parameters as needed include the molecular weight of the nucleic acid (e.g., RNA), the amount of surface nucleic acid (e.g., the amount of surface RNA), the amount of encapsulated nucleic acid (e.g., the amount of encapsulated RNA), the amount of accessible nucleic acid (e.g., the amount of accessible RNA), the size of the nucleic acid (e.g., RNA) [e.g., the R of the nucleic acid (e.g., RNA)]. g Value and / or R h value], size distribution of nucleic acids (e.g., RNA) [e.g., R g Value or R h values], quantitative size distribution of nucleic acids (e.g., RNA) [e.g., R g Value or R hBased on the value], shape factor, form factor, and nucleic acid (especially RNA) encapsulation efficiency. Usually, the size distribution and / or quantitative size distribution of nucleic acid (especially RNA) can be given as the number of nucleic acid (especially RNA) molecules, the molar amount of nucleic acid (especially RNA), or the mass of nucleic acid (especially RNA) (each as a function of particle size). In addition, optional additional parameters include the ratio of the amount of nucleic acid (e.g., RNA) bound to particles to the total amount of particle-forming compounds (especially lipids and / or polymers) in particles, which can be given as a function of particle size; the ratio of the amount of positively charged moieties of particle-forming compounds (especially lipids and / or polymers) in particles to the amount of nucleic acid (e.g., RNA) bound to particles, which can be given as a function of particle size; and the charge ratio of the amount of positively charged moieties of particle-forming compounds (especially lipids and / or polymers) in particles to the amount of negatively charged moieties of nucleic acid (e.g., RNA) bound to particles, which is usually expressed as N / P ratio and can be given as a function of particle size.

[0065] In one embodiment of the second aspect, the one or more parameters are at least two, preferably at least three parameters (including additional parameters as needed) as defined herein, in particular the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to the particles, the size distribution of the nucleic acid (especially RNA)-containing particles [e.g., the R of the nucleic acid (e.g. RNA)-containing particles], and the like. g Value or R h values], quantitative size distribution of nucleic acid (e.g., RNA)-containing particles [e.g., R g Value or R hIn 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 the particles, the size distribution of the nucleic acid (especially RNA)-containing particles [e.g., the R g Value or R h values], and quantitative size distribution of nucleic acid (especially RNA)-containing particles [e.g., R g Value or R h The method comprises (or is) at least two, preferably at least three parameters selected from the group consisting of: [based on the value of

[0066] In one embodiment of the second aspect, the method of the first aspect used in steps (C) and (D) comprises: (a) subjecting at least a portion of the composition (e.g., the first composition for step (C) or the second composition for step (D)) to field-flow fractionation to size-fractionate components contained in the composition to produce one or more composition fractions; (b) measuring at least one signal selected from the group consisting of a UV signal, a fluorescence signal, and a refractive index (RI) signal, and optionally a light scattering (LS) signal, of at least one of the one or more composition fractions obtained from step (a); (c) calculating one or more parameters from at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal, and optionally from an LS signal; The method includes:

[0067] In a first subgroup of the second embodiment, the method of the first embodiment used in steps (C) and (D) comprises: (a) subjecting at least a portion of the composition (e.g., the first composition for step (C) or the second composition for step (D)) to field-flow fractionation to size-fractionate components contained in the composition to produce one or more fractions; (b) measuring at least the UV signal, and optionally the light scattering (LS) signal, of at least one of the fractions obtained from step (a); (c) calculating one or more parameters from the UV signal and, optionally, from the LS signal; The method includes:

[0068] 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., a first composition for step (C) or a second composition for step (D)), wherein the sample composition comprises RNA, and optionally particles, and the method comprises: (a) subjecting at least a portion of the sample composition to field-flow fractionation to size-fractionate components contained in the sample composition to produce one or more sample fractions; (b) measuring at least one signal selected from the group consisting of a UV signal, a fluorescence signal, and a refractive index (RI) signal, and optionally a light scattering (LS) signal, of at least one of the one or more sample fractions obtained from step (a); (c) calculating one or more parameters from at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal, and optionally from an LS signal; Includes.

[0069] 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., a first composition for step (C) or a second composition for step (D)), wherein the sample composition comprises RNA, and optionally particles, and the method comprises: (a) subjecting at least a portion of the sample composition to field-flow fractionation to size-fractionate components contained in the sample composition to produce one or more sample fractions; (b) measuring at least the UV signal, and optionally the light scattering (LS) signal, of at least one of the one or more sample fractions obtained from step (a); (c) calculating one or more parameters from the UV signal and, optionally, from the LS signal; Includes.

[0070] In one embodiment of the second aspect (particularly in one embodiment of the first, second, or third subgroup of the second aspect), the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA)-containing particles is determined by the R g In another embodiment of the second aspect (particularly another embodiment of the first, second, or third subgroup of the second aspect), the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA)-containing particles is calculated based on the R value of the nucleic acid (e.g., RNA)-containing particles. h In another embodiment of the second aspect (particularly another embodiment of the first, second, or third subgroup of the second aspect), the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA)-containing particles is calculated based on the R value of the nucleic acid (e.g., RNA)-containing particles. g Based on the R value, and separately, the R of the nucleic acid (e.g., RNA)-containing particles hvalues [i.e., this embodiment results in two data sets for the size, size distribution, and / or quantitative size distribution of nucleic acid (e.g., RNA)-containing particles, one of which is R g Based on the value, and one is R h value-based].

[0071] In one embodiment of the second aspect (particularly in one embodiment of the first, second, or third subgroup of the second aspect) where the one or more parameters include the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA), the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA) may be determined by the R g In other embodiments of the second aspect (particularly other embodiments of the first, second, or third subgroups of the second aspect) in which the one or more parameters include the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA), the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA) is calculated based on the R h In other embodiments of the second aspect (particularly other embodiments of the first, second, or third subgroups of the second aspect) in which the one or more parameters include the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA), the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA) is calculated based on the R g Based on the value, and separately, the R h [i.e., this embodiment provides two data sets for nucleic acid (e.g., RNA) size, size distribution, and / or quantitative size distribution, one of which is R g Based on the value, and one is R h value-based].

[0072] 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 (especially RNA) integrity, total amount of nucleic acid (especially RNA), amount of free nucleic acid (especially RNA), amount of nucleic acid (especially RNA) bound to the particle, size of the nucleic acid (especially RNA) containing particle [particularly radius of gyration (R) of the nucleic acid (especially RNA) containing particle]. g ) and / or the hydrodynamic radius (R h ) based on the size distribution of nucleic acid (especially RNA)-containing particles (e.g., R g Value or R h values), and quantitative size distributions of nucleic acid (especially RNA)-containing particles (e.g., R g Value or R h This includes molecular weights of nucleic acids (especially RNA), as well as molecular weights of nucleic acids (especially RNA), where appropriate.

[0073] 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 the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to the particles, the size distribution of the nucleic acid (especially RNA)-containing particles [e.g., the R g Value or R h values], quantitative size distribution of nucleic acid (especially RNA)-containing particles [e.g., R g Value 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 the particles, the size distribution of the nucleic acid (especially RNA)-containing particles [e.g., the R g Value or R hvalues], and quantitative size distribution of nucleic acid (especially RNA)-containing particles [e.g., R g Value or R h The size distribution of nucleic acid (especially RNA)-containing particles comprises (or is) at least two, preferably at least three parameters selected from the group consisting of: R g value and R h If it is based on the values of two data sets, namely, R g Value-based and R h However, according to the present invention, these two data sets for the size distribution of nucleic acid (especially RNA)-containing particles are considered as only one parameter (not two parameters). Also, when a fractogram obtained by field-flow fractionation shows multiple particle peaks, the size distribution obtained for each particle peak is considered as only one parameter (not one parameter for each particle peak). The quantitative size distribution of nucleic acid (especially RNA)-containing particles is considered as R g value and R h The same is true for value-based situations.

[0074] 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 one or more parameters include a quantitative size distribution of the nucleic acid (especially RNA)-containing particles [e.g., a radius of gyration (R g ) and / or the hydrodynamic radius (R h ) )], and optionally at least one parameter, e.g., at least two parameters, of the remaining parameters (including additional parameters as needed) defined herein; preferably, the remaining parameters include 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 nucleic acid (especially RNA)-containing particles (e.g., Rg Value or R h In one embodiment of the second aspect (particularly in an 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 one or more parameters are selected from the group consisting of a quantitative size distribution (e.g., R g Value or R h values), as well as the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to the particles, and the size distribution of nucleic acid (especially RNA)-containing particles (e.g., R g Value or R h In one embodiment of the second aspect (particularly in an 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 one or more parameters are based on a quantitative size distribution (e.g., R g Value or R h The quantitative size distribution of nucleic acid (especially RNA)-containing particles includes the amount of nucleic acid (especially RNA) bound to the particle, based on the R g value and R h If it is based on the values of two data sets, namely, R g Value-based and R h However, according to the present invention, these two data sets for the quantitative size distribution of nucleic acid (especially RNA)-containing particles are considered as only one parameter (not two parameters). Also, in the case where a fractogram obtained by field-flow fractionation shows multiple particle peaks, the quantitative size distribution obtained for each particle peak is considered as only one parameter (not one parameter for each particle peak). When the size distribution of nucleic acid (especially RNA)-containing particles is R g value and R hThe same is true for value-based situations.

[0075] 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 are determined by one cycle of steps (a) to (c).

[0076] 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 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 chelators); type and / or source of particle-forming compounds (particularly lipids and / or polymers); charge ratio; physical state; and ratio of nucleic acid (especially RNA) to particle-forming compounds (particularly lipids and / or polymers that make up 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, e.g., 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 (e.g., −20° C.), ambient or room temperature, medium (e.g., 30° C.), or high (e.g., 50° C.) temperature. Exemplary conditions regarding the type and / or source of particle-forming compounds include 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 nucleic acid (especially RNA) particles are about 6:1 to about 1:2, e.g., about 5:1 to about 1.2:2, about 4:1 to about 1.4:2, about 3:1 to about 1.6:2, about 2:1 to about 1.8:2, or about 1.6:1 to about 1:1. Exemplary ratios of nucleic acid (especially RNA) to particle-forming compounds (especially lipids and / or polymers constituting the particles) include ratios of nucleic acid (especially RNA) to total lipids in the range of about 1:100 to about 10:1 (w / w).

[0077] In one embodiment of the second aspect (particularly in one embodiment of the first, second or third subgroup of the second aspect), the field-flow fractionation is preferably flow field-flow fractionation, such as asymmetric flow field-flow fractionation (AF4) or hollow fiber flow field-flow fractionation (HF5).

[0078] 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 carried out using a membrane with a molecular weight (MW) cut-off suitable to prevent nucleic acids (especially RNA) from passing through, preferably a membrane with a MW cut-off in the range of 2 kDa to 30 kDa, for example a MW cut-off of 10 kDa.

[0079] 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 carried out using a polyethersulfone (PES) or regenerated cellulose membrane.

[0080] 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 carried out using: (I) a cross-flow rate, e.g., a cross-flow rate profile, of up to 8 mL / min, preferably up to 4 mL / min, more preferably up to 2 mL / min; and / or (II) an inject flow 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 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.

[0081] In one embodiment of the second aspect (particularly in one embodiment of the first, second, or third subgroup of the second aspect), the cross-flow rate profile preferably includes a fractionation phase in which components contained in a composition (e.g., a control composition or a sample composition, particularly the first composition for step (C) or the second composition for step (D)) can be fractionated / separated by size to generate one or more composition fractions. The cross-flow rate preferably changes during this fractionation phase (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)). The change may be continuous (e.g., linear or exponential) or stepwise. Preferably, the cross-flow rate profile includes a fractionation phase in which the cross-flow rate varies continuously (preferably exponentially) starting from a certain 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 phase may have any length suitable for fractionating / separating components contained in the composition by size, such as about 5 to about 60 minutes, e.g., about 10 to about 50 minutes, about 15 to about 45 minutes, about 20 to about 40 minutes, or about 25 to about 35 minutes, or about 30 minutes. The cross-flow rate profile may contain additional phases (e.g., 1, 2, 3, or 4 phases), which may precede and / or follow the fractionation phase (e.g., 1 phase before and 1, 2, or 3 phases after), and which may function to separate non-nucleic acid (especially non-RNA) components contained in the composition (e.g., proteins, polypeptides, mononucleotides, etc.) from nucleic acids (especially RNA) contained in the composition, to focus the nucleic acids (especially RNA) contained in the 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 these additional phases is constant for each additional phase, and the length of each additional phase is, independently, within the range of about 5 minutes to about 60 minutes (about 10 minutes to about 50 minutes, about 15 minutes to about 45 minutes, about 20 minutes to about 40 minutes, or about 25 minutes to about 35 minutes, or about 30 minutes). For example, the cross-flow rate profile may include: (i) a first additional phase preceding a fractionation phase, wherein the cross-flow rate of the first additional phase is constant and is the cross-flow rate at which the fractionation phase begins (the length of the first additional phase may be in the range of about 5 minutes to about 60 minutes, e.g., about 10 minutes to about 50 minutes, about 15 minutes to about 45 minutes, about 20 minutes to about 40 minutes, or about 25 minutes to about 35 minutes, or about 10 minutes, or about 20 minutes, or about 30 minutes); (ii) a second additional phase following a fractionation phase, wherein the cross-flow rate of the second additional phase is constant and is the cross-flow rate at which the fractionation phase ends (the length of the second additional phase may be in the range of about 5 minutes to about 60 minutes, e.g., about 10 minutes to about 50 minutes, about 15 minutes to about 45 minutes, about 20 minutes to about 40 minutes, or about 25 minutes to about 35 minutes, or about 10 minutes, or about 20 minutes, or about 30 minutes); and optionally (iii) a third additional phase following the second additional phase, wherein the cross-flow rate of said third additional phase is constant and different from the cross-flow rate of the second additional phase (the length of the third additional phase may be in the range of about 5 minutes to about 60 minutes, e.g., about 10 minutes to about 50 minutes, about 15 minutes to about 45 minutes, about 20 minutes to about 40 minutes, or about 25 minutes to about 35 minutes, or about 10 minutes, or about 20 minutes, or about 30 minutes).In embodiments where the cross-flow rate profile includes a fractionation phase and the cross-flow rate varies continuously (preferably exponentially) starting from a certain 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 cross-flow rate profile can further include: (i) a first additional phase preceding the fractionation phase, wherein the cross-flow rate of said first additional phase is constant and is the cross-flow rate at which the fractionation phase begins (e.g., about 1 to about 4 mL / min) (the length of the first additional phase can be in the range of about 5 minutes to about 30 minutes, e.g., about 6 minutes to about 25 minutes, about 7 minutes to about 20 minutes, or about 8 minutes to about 15 minutes, or about 10 minutes to about 12 minutes, or about 5 minutes, or about 10 minutes, or about 12 minutes); (ii) a second additional phase following the fractionation phase, wherein the cross-flow rate of said second additional phase is constant and a cross-flow rate (e.g., about 0.01 to about 0.1 mL / min) at which the fractionation phase ends (the length of the second additional phase may be in the range of about 5 minutes to about 60 minutes, e.g., about 10 minutes to about 50 minutes, about 15 minutes to about 45 minutes, about 20 minutes to about 40 minutes, or about 25 minutes to about 35 minutes, or about 30 minutes); and optionally (iii) a third additional phase following the second additional phase, wherein the cross-flow rate of the third additional phase is Preferably, the cross-flow rate profile includes a third additional phase whose cross-flow rate is constant and lower than that of the second additional phase (e.g., the cross-flow rate of the third additional phase is 0). (The length of the third additional phase may be within the range of about 5 minutes to about 30 minutes, e.g., about 6 minutes to about 25 minutes, about 7 minutes to about 20 minutes, about 8 minutes to about 15 minutes, about 10 minutes to about 12 minutes, or about 5 minutes, about 10 minutes, or about 12 minutes.) Preferred examples of such cross-flow rate profiles are as follows: 1.0-2.0 mL / min for 10 minutes, an exponential ramp from 1.0-2.0 mL / min to 0.01-0.07 mL / min within 30 minutes; 0.01-0.07 mL / min for 30 minutes; and 0 mL / min for 10 minutes.

[0082] In one embodiment of the second aspect (particularly in one embodiment of the first, second or third subgroup of the second aspect), the integrity of the nucleic acid (especially RNA) contained in the sample composition (e.g., the first composition for step (C) or the second composition for step (D)) is calculated using the integrity of a control nucleic acid (especially RNA).

[0083] In a first particular example of this embodiment of the second aspect, the integrity of a control nucleic acid (in particular RNA) is determined by the following steps: (a') subjecting at least a portion of a control composition containing a control nucleic acid (especially RNA) to field-flow fractionation, particularly AF4 or HF5, to size fractionation of components contained in the control composition to produce one or more control fractions; (b') measuring at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and a refractive index (RI) signal of at least one of the one or more control fractions obtained from step (a'); (c'1) From at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained in step (b'), calculate the area from the maximum height of one UV, fluorescent, or RI peak to the end of the UV, fluorescent, or RI peak, thereby obtaining A 50% obtaining a control; (c'2) A is obtained by calculating the total area of one peak used in step (c'1) from at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained in step (b'). 100% obtaining a control; (c'3)A 50% (control) and A 100% (control) to obtain the integrity of the control nucleic acid (especially RNA) [I(control)]; is calculated by

[0084] In this first example, the integrity of the nucleic acids (especially RNA) contained in the sample composition (e.g., the first composition for step (C) or the second composition for step (D)) is determined by the following steps: (c1) From at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained in step (b), calculate the area from the maximum height of the sample UV, fluorescent, or RI peak corresponding to the control UV, fluorescent, or RI peak used in step (c'1) to the end of the sample UV, fluorescent, or RI peak, thereby obtaining A 50% Obtaining a sample; (c2) A is obtained by 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 from step (b). 100% Obtaining a sample; (c3)A 50% (Sample) and A 100% obtaining I(sample) by calculating the ratio of I(sample); (c4) determining the ratio of I (sample) to I (control) to determine the integrity of the nucleic acids (especially RNA) contained in the sample composition; It can be calculated by:

[0085] In a second particular example of this embodiment of the second aspect, the integrity of a control nucleic acid (especially RNA) is determined by the following steps: (a") subjecting at least a portion of a control composition containing a control nucleic acid (especially RNA) to field-flow fractionation, particularly AF4 or HF5, to size fractionation of components contained in the control composition to produce one or more control fractions; (b") measuring at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal of at least one of the one or more control fractions obtained from step (a"); (c") determining the integrity of a control nucleic acid (especially RNA) by determining the height [H(control)] of one UV, fluorescent, or RI peak from at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained in step (b"); is calculated by

[0086] In this second example, the integrity of the nucleic acids (especially RNA) contained in the sample composition (e.g., the first composition for step (C) or the second composition for step (D)) is determined by the following steps: (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 group consisting of a UV signal, a fluorescence signal, and an RI signal obtained in step (b); (c2') determining the ratio of H(sample) to H(control) to determine the integrity of the nucleic acids (especially RNA) contained in the sample composition; It can be calculated by:

[0087] In a third particular example of this embodiment of the second aspect (relating to the first subgroup of the second aspect), the integrity of the control nucleic acid (in particular RNA) is determined by the following steps: (a') subjecting at least a portion of a control composition containing a control nucleic acid (especially RNA) to field-flow fractionation, particularly AF4 or HF5, to size fractionation of components contained in the control composition to produce one or more control fractions; (b') measuring at least the UV signal of at least one of the one or more control fractions obtained from step (a'); (c'1) From the UV signal obtained in step (b'), calculate the area from the maximum height of one UV peak to the end of the UV peak to obtain A 50% obtaining a control; (c'2) A is obtained by calculating the total area of the single peak used in step (c'1) from the UV signal obtained in step (b'). 100% obtaining a control; (c'3)A 50% (control) and A 100% (control) to obtain the integrity of the control nucleic acid (especially RNA) [I(control)]; is calculated by

[0088] In this third example, the integrity of the nucleic acids (especially RNA) contained in the sample composition (e.g., the first composition for step (C) or the second composition for step (D)) is determined by the following steps: (c1) From the UV signal obtained from step (b), calculate 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, thereby obtaining A 50% Obtaining a sample; (c2) A is calculated by calculating the total area of the sample UV peaks used in step (c1) from the sample UV signal obtained from step (b). 100% Obtaining a sample; (c3)A 50% (Sample) and A 100% obtaining I(sample) by calculating the ratio of I(sample); (c4) determining the ratio of I (sample) to I (control) to determine the integrity of the nucleic acids (especially RNA) contained in the sample composition; It can be calculated by:

[0089] In a fourth particular example of this embodiment of the second aspect (relating to the first subgroup of the second aspect), the integrity of the control nucleic acid (in particular RNA) is determined by the following steps: (a") subjecting at least a portion of a control composition containing a control nucleic acid (especially RNA) to field-flow fractionation, particularly AF4 or HF5, to size fractionation of components contained in the control composition to produce one or more control fractions; (b") measuring at least the UV signal of at least one of the one or more control fractions obtained from step (a"); (c") determining the integrity of a control nucleic acid (especially RNA) by determining the height of one UV peak [H(control)] from the UV signal obtained in step (b"); is calculated by

[0090] In this fourth example, the integrity of the nucleic acids (especially RNA) contained in the sample composition (e.g., the first composition for step (C) or the second composition for step (D)) is determined by the following steps: (c1') determining the height [H(sample)] of the sample UV peak corresponding to the control UV peak used in step (c") from the UV signal obtained in step (b); (c2') determining the ratio of H(sample) to H(control) to determine the integrity of the nucleic acids (especially RNA) contained in the sample composition; It can be calculated by:

[0091] In a fifth particular 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: (a') subjecting at least a portion of the control composition containing the control RNA to field-flow fractionation, particularly AF4 or HF5, to size fractionate the components contained in the control composition to produce one or more control fractions; (b') measuring at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal of at least one of the one or more control fractions obtained from step (a'); (c'1) From at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained in step (b'), calculate the area from the maximum height of one UV, fluorescent, or RI peak to the end of the UV, fluorescent, or RI peak, thereby obtaining A 50% obtaining a control; (c'2) A total area of one peak used in step (c'1) is calculated from at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained in step (b'). 100% obtaining a control; (c'3)A 50% (control) and A 100% obtaining the control RNA integrity [I(control)] by calculating the ratio of I(control) to I(control); is calculated by

[0092] In this fifth example, the integrity of the RNA contained in the sample composition (e.g., the first composition for step (C) or the second composition for step (D)) is determined by the following steps: (c1) From at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained in step (b), calculate the area from the maximum height of the sample UV, fluorescent, or RI peak corresponding to the control UV, fluorescent, or RI peak used in step (c'1) to the end of the sample UV, fluorescent, or RI peak, thereby obtaining A 50% Obtaining a sample; (c2) A is obtained by 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 from step (b). 100% Obtaining a sample; (c3)A 50% (Sample) and A 100% obtaining I(sample) by calculating the ratio of I(sample); (c4) determining the ratio of I (sample) to I (control) to determine the integrity of the RNA contained in the sample composition; It can be calculated by:

[0093] In a sixth particular 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: (a") subjecting at least a portion of a control composition containing a control RNA to field-flow fractionation, particularly AF4 or HF5, to size fractionate components contained in the control composition to produce one or more control fractions; (b") measuring at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal of at least one of the one or more control fractions obtained from step (a"); (c") determining the integrity of the control RNA by determining the height [H(control)] of one UV, fluorescent, or RI peak from at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained in step (b"); is calculated by

[0094] In this sixth example, the integrity of the RNA contained in the sample composition (e.g., the first composition for step (C) or the second composition for step (D)) is determined by the following steps: (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 group consisting of a UV signal, a fluorescence signal, and an RI signal obtained in step (b); (c2') determining the ratio of H(sample) to H(control) to determine the integrity of the RNA contained in the sample composition; It can be calculated by:

[0095] In a seventh particular example of this embodiment of the second aspect (related to the third subgroup of the second aspect), the integrity of the control RNA is determined by the following steps: (a') subjecting at least a portion of the control composition containing the control RNA to field-flow fractionation, particularly AF4 or HF5, to size fractionate the components contained in the control composition to produce one or more control fractions; (b') measuring at least the UV signal of at least one of the one or more control fractions obtained from step (a'); (c'1) From the UV signal obtained in step (b'), calculate the area from the maximum height of one UV peak to the end of the UV peak to obtain A 50% obtaining a control; (c'2) A is obtained by calculating the total area of the single peak used in step (c'1) from the UV signal obtained in step (b'). 100% obtaining a control; (c'3)A 50% (control) and A 100% obtaining the control RNA integrity [I(control)] by calculating the ratio of I(control) to I(control); is calculated by

[0096] In this seventh example, the integrity of the RNA contained in the sample composition (e.g., the first composition for step (C) or the second composition for step (D)) is determined by the following steps: (c1) From the UV signal obtained from step (b), calculate 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, thereby obtaining A 50% Obtaining a sample; (c2) A is calculated by calculating the total area of the sample UV peaks used in step (c1) from the sample UV signal obtained from step (b). 100% Obtaining a sample; (c3)A 50% (Sample) and A 100% obtaining I(sample) by calculating the ratio of I(sample); (c4) determining the ratio of I (sample) to I (control) to determine the integrity of the RNA contained in the sample composition; It can be calculated by:

[0097] In an eighth particular example of this embodiment of the second aspect (related to the third subgroup of the second aspect), the integrity of the control RNA is determined by the following steps: (a") subjecting at least a portion of a control composition containing a control RNA to field-flow fractionation, particularly AF4 or HF5, to size fractionate components contained in the control composition to produce one or more control fractions; (b") measuring at least the UV signal of at least one of the one or more control fractions obtained from step (a"); (c") determining the integrity of the control RNA by determining the height of one UV peak [H(control)] from the UV signal obtained in step (b"); is calculated by

[0098] In this eighth example, the integrity of the RNA contained in the sample composition (e.g., the first composition for step (C) or the second composition for step (D)) is determined by the following steps: (c1') determining the height [H(sample)] of the sample UV peak corresponding to the control UV peak used in step (c") from the UV signal obtained in step (b); (c2') determining the ratio of H(sample) to H(control) to determine the integrity of the RNA contained in the sample composition; It can be calculated by:

[0099] 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 (particularly RNA) is determined by using (i) a nucleic acid decay coefficient (particularly an RNA decay coefficient) or (ii) a nucleic acid calibration curve (particularly an RNA calibration curve).

[0100] In one embodiment of the second aspect (particularly in one embodiment of the first, second or third subgroup of the second aspect), the sample composition (e.g. the first composition for step (C) or the second composition for step (D)) comprises nucleic acid (especially RNA) and particles to which the nucleic acid (especially RNA) is bound, such as lipoplex particles and / or lipid nanoparticles and / or polyplex particles and / or lipopolyplex particles and / or virus-like particles.

[0101] 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 acid (particularly the amount of total RNA) is determined 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) on at least the portion obtained from step (i); and (iii) determining the amount of nucleic acid (particularly RNA) as defined herein (e.g., using (i) a nucleic acid decay coefficient (particularly an RNA decay 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.

[0102] 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 dodecyl sulfate), a zwitterionic surfactant [e.g., n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent® 3-14)], a cationic surfactant, a nonionic surfactant, or a mixture thereof; (ii) an alcohol, such as an aliphatic alcohol (e.g., ethanol), or a mixture of alcohols; or (iii) a combination of (i) and (ii).

[0103] 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 free nucleic acid (especially RNA) is determined by carrying out steps (a) to (c) without the addition of a releasing agent, particularly without any releasing agent; and determining the amount of nucleic acid (especially RNA) as defined herein (for example using (i) a nucleic acid decay factor (especially an RNA decay factor) or (ii) a nucleic acid calibration curve (especially an RNA calibration curve)).

[0104] 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) bound to the particles is determined herein (e.g., by carrying out steps (a)-(c) without the addition of a releasing agent, particularly without any releasing agent; and determining the amount of nucleic acid (especially RNA) as defined herein (e.g., using (i) a nucleic acid decay coefficient (especially an RNA decay coefficient) or (ii) a nucleic acid calibration curve (especially an RNA calibration curve))]). The amount of nucleic acid (especially RNA) is determined by subtracting it from the total amount of nucleic acid (especially RNA) by determining (e.g., (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 release agent; (ii) performing steps (a) to (c) on at least a portion obtained from step (i); and (iii) determining the amount of nucleic acid (especially RNA) as defined herein (e.g., using (i) a nucleic acid decay coefficient (especially an RNA decay coefficient) or (ii) a nucleic acid calibration curve (especially an RNA calibration curve))].

[0105] In one embodiment of the second aspect (particularly in one embodiment of the second or third subgroup of the second aspect), step (b) further comprises measuring at least one LS signal, such as a dynamic light scattering (DLS) and / or static light scattering (SLS), such as a multi-angle light scattering (MALS) signal, of one or more sample fractions obtained from step (a).

[0106] In one embodiment of the second aspect (particularly in one embodiment of the second or third subgroup of the second aspect), the size of the nucleic acid (particularly RNA) containing particles is determined from the LS signal obtained from step (b) by calculating the radius of gyration (R g ) value and / or hydrodynamic radius (R 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 dynamic light scattering (DLS) signal of at least one of the sample fractions obtained from step (a), and step (c) comprises calculating an R 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 at least one static light scattering (SLS), e.g., MALS, signal of one or more sample fractions obtained from step (a), and step (c) comprises calculating an R value from the SLS signal. g 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 at least one dynamic light scattering (DLS) signal and a static light scattering (SLS), e.g., MALS, signal of one or more sample fractions obtained from step (a), and step (c) comprises calculating an R g value and R h This latter embodiment involves calculating two data sets of sizes of nucleic acid (e.g., RNA)-containing particles: R g Value-based and R h Bringing value-based.

[0107] In one embodiment of the second aspect (particularly in one embodiment of the first, second or third subgroup of the second aspect), the size distribution of the nucleic acid (particularly RNA) containing particles is determined by comparing at least one signal selected from the group consisting of a UV signal, a fluorescent signal and an RI signal obtained from step (b) with an R determined as defined herein. g Value or R hby plotting the R g or by calculating the R value from the DLS signal obtained from step (b). h In a first example of this embodiment (which relates to the first subgroup of the second aspect), the size distribution of nucleic acid (especially RNA) containing particles is determined by multiplying the UV signal from step (b) by the R value, determined as defined herein. g Value or R h by plotting the R g or by calculating the R value from the DLS signal obtained from step (b). h In a second example of this embodiment (pertaining to the second subgroup of the second aspect), the size distribution of the RNA-containing particles is determined by multiplying at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained from step (b) by an R value determined as defined herein. g Value or R h by plotting the R g or by calculating the R value from the DLS signal obtained from step (b). h In a third example of this embodiment (pertaining to the third subgroup of the second aspect), the size distribution of the RNA-containing particles is determined by multiplying the UV signal from step (b) by the R value, determined as defined herein. g Value or R h by plotting the R g or by calculating the R value from the DLS signal obtained from step (b). h In each of the first, second, and third examples above, the size distribution of nucleic acid (especially RNA)-containing particles is determined by the R g Value, R hThe size distribution of nucleic acid (especially RNA)-containing particles can be determined based on R g value and R h If it is based on the values of two data sets, namely, R g Size distribution based on values and R h This results in a size distribution based on the value.

[0108] 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 of the nucleic acid (especially RNA) containing particles is R g Value or R h From a plot showing the UV, fluorescence, or RI signal as a function of the value, convert the UV, fluorescence, or RI signal to a cumulative weight fraction and convert the cumulative weight fraction to R g Value or R h In a first example of this embodiment (pertaining to the first subgroup of the second aspect), the quantitative size distribution of the nucleic acid (especially RNA) containing particles is calculated by plotting the R g Value or R h From the plot showing the UV signal as a function of the value, convert the UV signal to cumulative weight fraction and calculate the cumulative weight fraction as R g Value or R h In a second example of this embodiment (pertaining to the second subgroup of the second aspect), the quantitative size distribution of the RNA-containing particles is calculated by plotting the R g Value or R h From a plot showing the UV, fluorescence, or RI signal as a function of the value, convert the UV, fluorescence, or RI signal to a cumulative weight fraction and convert the cumulative weight fraction to R g Value or R h In a third example of this embodiment (pertaining to the third subgroup of the second aspect), the quantitative size distribution of the RNA-containing particles is calculated by plotting the R g Value or R h From the plot showing the UV signal as a function of the value, convert the UV signal to cumulative weight fraction and calculate the cumulative weight fraction as R g Value or Rh In each of the first, second, and third examples above, the quantitative size distribution of nucleic acid (especially RNA)-containing particles is calculated by plotting the R g Value, R h The quantitative size distribution of nucleic acid (especially RNA)-containing particles can be determined based on R g value and R h If it is based on the values of two data sets, namely, R g Quantitative size distribution and R based on values h This results in a quantitative size distribution based on the values.

[0109] 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 a D10 value, a D50 value, and / or a D90 value. The quantitative size distribution of the nucleic acid (especially RNA)-containing particles is g value and R h If it is based on the values of two data sets, namely, R g a set of D10, D50, and / or D90 values based on the R h The resulting set of D10, D50, and / or D90 values is based on the value.

[0110] 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), in particular free nucleic acid (especially RNA), is determined by measuring the UV signal, for example at a wavelength in the range of 260 nm to 280 nm, for example at a wavelength of 260 nm or 280 nm, and using the nucleic acid (especially RNA) extinction coefficient at the corresponding wavelength (e.g. 260 nm or 280 nm).

[0111] 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 nucleic acid (especially RNA)-containing particles (e.g., Rg Value or R h values) and / or quantitative size distribution of nucleic acid (especially RNA)-containing particles (e.g., R g Value or R h value) is in the range of 10 to 2000 nm, preferably 20 to 1500 nm, for example, 30 to 1200 nm, 40 to 1100 nm, 50 to 1000, 60 to 900 nm, 70 to 800 nm, 80 to 700 nm, 90 to 600 nm, or 100 to 500 nm, or for example, 10 to 1000 nm, 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm, or 50 to 250 nm. In a preferred embodiment of a third subgroup of the second aspect, the (quantitative) size distribution (e.g., R g Value or R h value) is in the range of 10 to 1000 nm, for example, in the range of 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm, or 50 to 250 nm.

[0112] 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 (particularly RNA) has a length of 10 to 15,000 nucleotides, for example 40 to 15,000 nucleotides, 100 to 12,000 nucleotides, or 200 to 10,000 nucleotides.

[0113] 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 embodiments of the second or third subgroup of the second aspect, the RNA is preferably mRNA or in vitro transcribed RNA, particularly in vitro transcribed mRNA.

[0114] In one embodiment of the second aspect (particularly in one embodiment of the first, second or third subgroup of the second aspect), measuring at least one signal selected from the group consisting of a UV signal, a fluorescence signal and an RI signal, optionally an LS signal, such as an SLS, such as an MALS signal, and / or a DLS signal, is performed online and / or step (c) is performed online.

[0115] 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 for step (C) or the second composition for step (D)) to field-flow fractionation, at least a portion of the sample composition is diluted with a solvent or solvent mixture capable of preventing the formation of particle aggregates. In one embodiment, the solvent mixture is a mixture of water and an organic solvent, such as formamide.

[0116] In one embodiment of the second aspect (particularly in one embodiment of the first, second or third subgroup of the second aspect), measuring the UV signal is carried out by using circular dichroism (CD) spectroscopy.

[0117] It will be understood that any embodiment described herein in the context of the first aspect may be used in any embodiment of the second aspect.

[0118] In a third aspect, the present disclosure provides the use of field-flow fractionation to determine one or more parameters of a sample composition comprising nucleic acids (e.g., RNA) and optionally particles, wherein the one or more parameters include nucleic acid (e.g., RNA) integrity, total amount of nucleic acid (e.g., RNA), amount of free nucleic acid (e.g., RNA), amount of nucleic acid (e.g., RNA) bound to particles, size of nucleic acid (e.g., RNA)-containing particles [particularly, radius of gyration (R) of nucleic acid (e.g., RNA)-containing particles]. g) and / or the hydrodynamic radius (R h ) )], size distribution of nucleic acid (e.g., RNA)-containing particles [e.g., R g Value or R h values], and quantitative size distribution of nucleic acid (e.g., RNA)-containing particles [e.g., R g Value or R h Typically, the size distribution and / or quantitative size distribution of nucleic acid (e.g., RNA)-containing particles can be given as the number of nucleic acid (e.g., RNA)-containing particles, the molar amount of nucleic acid (e.g., RNA)-containing particles, or the mass of nucleic acid (e.g., RNA)-containing particles (each as a function of particle size). Additional parameters as needed include the molecular weight of the nucleic acid (e.g., RNA), the amount of surface nucleic acid (e.g., the amount of surface RNA), the amount of encapsulated nucleic acid (e.g., the amount of encapsulated RNA), the amount of accessible nucleic acid (e.g., the amount of accessible RNA), the size of the nucleic acid (e.g., RNA) [particularly the R of the nucleic acid (e.g., RNA)-containing particles]. g Value and / or R h value], size distribution of nucleic acids (e.g., RNA) [e.g., R g Value or R h values], quantitative size distribution of nucleic acids (e.g., RNA) [e.g., R g Value or R hBased on the value], shape factor, form factor, and nucleic acid (especially RNA) encapsulation efficiency. Usually, the size distribution and / or quantitative size distribution of nucleic acid (especially RNA) can be given as the number of nucleic acid (especially RNA) molecules, the molar amount of nucleic acid (especially RNA), or the mass of nucleic acid (especially RNA) (each as a function of particle size). In addition, optional additional parameters include the ratio of the amount of nucleic acid (e.g., RNA) bound to particles to the total amount of particle-forming compounds (especially lipids and / or polymers) in particles, which can be given as a function of particle size; the ratio of the amount of positively charged moieties of particle-forming compounds (especially lipids and / or polymers) in particles to the amount of nucleic acid (e.g., RNA) bound to particles, which can be given as a function of particle size; and the charge ratio of the amount of positively charged moieties of particle-forming compounds (especially lipids and / or polymers) in particles to the amount of negatively charged moieties of nucleic acid (e.g., RNA) bound to particles, which is usually expressed as N / P ratio and can be given as a function of particle size.

[0119] In one embodiment of the third aspect, the field-flow fractionation comprises: (a) subjecting at least a portion of the sample composition to field-flow fractionation to size-fractionate components contained in the sample composition to produce one or more sample fractions; (b) measuring at least one signal selected from the group consisting of a UV signal, a fluorescence signal, and a refractive index (RI) signal, and optionally a light scattering (LS) signal, of at least one of the one or more sample fractions obtained from step (a); (c) calculating one or more parameters from at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal, and optionally from an LS signal; Includes.

[0120] In a first subgroup of the third embodiment, the field-flow fractionation comprises: (a) subjecting at least a portion of the sample composition to field-flow fractionation to size-fractionate components contained in the sample composition to produce one or more sample fractions; (b) measuring at least the UV signal, and optionally the light scattering (LS) signal, of at least one of the one or more sample fractions obtained from step (a); (c) calculating one or more parameters from the UV signal and, optionally, from the LS signal; Includes.

[0121] In a second and preferred subgroup of the third aspect, the use is for determining one or more parameters of a sample composition, the sample composition comprising RNA, and optionally particles, and the field-flow fractionation comprises: (a) subjecting at least a portion of the sample composition to field-flow fractionation to size-fractionate components contained in the sample composition to produce one or more sample fractions; (b) measuring at least one signal selected from the group consisting of a UV signal, a fluorescence signal, and a refractive index (RI) signal, and optionally a light scattering (LS) signal, of at least one of the one or more sample fractions obtained from step (a); (c) calculating one or more parameters from at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal, and optionally from an LS signal; Includes.

[0122] In a third and more preferred subgroup of the third aspect, the use is for determining one or more parameters of a sample composition, the sample composition comprising RNA, and optionally particles, and the field-flow fractionation comprises: (a) subjecting at least a portion of the sample composition to field-flow fractionation to size-fractionate components contained in the sample composition to produce one or more sample fractions; (b) measuring at least the UV signal, and optionally the light scattering (LS) signal, of at least one of the one or more sample fractions obtained from step (a); (c) calculating one or more parameters from the UV signal and, optionally, from the LS signal; Includes.

[0123] In one embodiment of the third aspect (particularly in one embodiment of the first, second, or third subgroup of the third aspect), the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA)-containing particles is determined by the R g In another embodiment of the third aspect (particularly another embodiment of the first, second, or third subgroup of the third aspect), the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA)-containing particles is calculated based on the R value of the nucleic acid (e.g., RNA)-containing particles. h In another embodiment of the third aspect (particularly another embodiment of the first, second, or third subgroup of the third aspect), the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA)-containing particles is calculated based on the R value of the nucleic acid (e.g., RNA)-containing particles. g Based on the R value, and separately, the R of the nucleic acid (e.g., RNA)-containing particles h values [i.e., this embodiment results in two data sets for the size, size distribution, and / or quantitative size distribution of nucleic acid (e.g., RNA)-containing particles, one of which is R g Based on the value, and one is R h value-based].

[0124] In one embodiment of the third aspect where the one or more parameters include the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA) (particularly in one embodiment of the first, second, or third subgroup of the third aspect), the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA) may be determined by the R g In other embodiments of the third aspect (particularly other embodiments of the first, second, or third subgroups of the third aspect) in which the one or more parameters include the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA), the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA) is calculated based on the R h In other embodiments of the third aspect (particularly other embodiments of the first, second, or third subgroups of the third aspect) in which the one or more parameters include the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA), the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA) is calculated based on the R g Based on the value, and separately, the R h [i.e., this embodiment provides two data sets for nucleic acid (e.g., RNA) size, size distribution, and / or quantitative size distribution, one of which is R g Based on the value, and one is R h value-based].

[0125] 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 flow field-flow fractionation, such as asymmetric flow field-flow fractionation (AF4) or hollow fiber flow field-flow fractionation (HF5).

[0126] 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 membrane with a molecular weight (MW) cut-off suitable to prevent nucleic acids (especially RNA) from passing through, preferably a membrane with a MW cut-off in the range of 2 kDa to 30 kDa, for example a MW cut-off of 10 kDa.

[0127] 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.

[0128] 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 carried out using: (I) a cross-flow rate, e.g., a cross-flow rate profile, of up to 8 mL / min, preferably up to 4 mL / min, more preferably up to 2 mL / min; and / or (II) an inject flow 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 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.

[0129] In one embodiment of the third aspect (particularly in one embodiment of the first, second, or third subgroup of the third aspect), the cross-flow rate profile preferably includes a fractionation phase in which components contained in the control or sample composition are fractionated / separated by size to generate one or more sample fractions. The cross-flow rate preferably varies during this fractionation phase (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)). The variation may be continuous (e.g., linear or exponential) or stepwise. Preferably, the cross-flow rate profile includes a fractionation phase in which the cross-flow rate varies continuously (preferably exponentially) starting from a certain 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 phase may have any length suitable for fractionating / separating components contained in the sample composition by size, such as about 5 to about 60 minutes, e.g., about 10 to about 50 minutes, about 15 to about 45 minutes, about 20 to about 40 minutes, or about 25 to about 35 minutes, or about 30 minutes. The cross-flow rate profile may contain additional phases (e.g., 1, 2, 3, or 4 phases), which may precede and / or follow the fractionation phase (e.g., 1 phase before and 1, 2, or 3 phases after), and which may function to separate non-nucleic acid (especially non-RNA) components contained in the sample composition (e.g., proteins, polypeptides, mononucleotides, etc.) from nucleic acids (especially RNA) contained in the sample composition, to focus the nucleic acids (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 these additional phases is constant for each additional phase, and the length of each additional phase is, independently, within the range of about 5 minutes to about 60 minutes (about 10 minutes to about 50 minutes, about 15 minutes to about 45 minutes, about 20 minutes to about 40 minutes, or about 25 minutes to about 35 minutes, or about 30 minutes). For example, the cross-flow rate profile may include: (i) a first additional phase preceding a fractionation phase, wherein the cross-flow rate of the first additional phase is constant and is the cross-flow rate at which the fractionation phase begins (the length of the first additional phase may be in the range of about 5 minutes to about 60 minutes, e.g., about 10 minutes to about 50 minutes, about 15 minutes to about 45 minutes, about 20 minutes to about 40 minutes, or about 25 minutes to about 35 minutes, or about 10 minutes, or about 20 minutes, or about 30 minutes); (ii) a second additional phase following a fractionation phase, wherein the cross-flow rate of the second additional phase is constant and is the cross-flow rate at which the fractionation phase ends (the length of the second additional phase may be in the range of about 5 minutes to about 60 minutes, e.g., about 10 minutes to about 50 minutes, about 15 minutes to about 45 minutes, about 20 minutes to about 40 minutes, or about 25 minutes to about 35 minutes, or about 10 minutes, or about 20 minutes, or about 30 minutes); and optionally (iii) a third additional phase following the second additional phase, wherein the cross-flow rate of said third additional phase is constant and different from the cross-flow rate of the second additional phase (the length of the third additional phase may be in the range of about 5 minutes to about 60 minutes, e.g., about 10 minutes to about 50 minutes, about 15 minutes to about 45 minutes, about 20 minutes to about 40 minutes, or about 25 minutes to about 35 minutes, or about 10 minutes, or about 20 minutes, or about 30 minutes).In embodiments where the cross-flow rate profile includes a fractionation phase and the cross-flow rate varies continuously (preferably exponentially) starting from a certain 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 cross-flow rate profile can further include: (i) a first additional phase preceding the fractionation phase, wherein the cross-flow rate of said first additional phase is constant and is the cross-flow rate at which the fractionation phase begins (e.g., about 1 to about 4 mL / min) (the length of the first additional phase can be in the range of about 5 minutes to about 30 minutes, e.g., about 6 minutes to about 25 minutes, about 7 minutes to about 20 minutes, or about 8 minutes to about 15 minutes, or about 10 minutes to about 12 minutes, or about 5 minutes, or about 10 minutes, or about 12 minutes); (ii) a second additional phase following the fractionation phase, wherein the cross-flow rate of said second additional phase is constant and a cross-flow rate (e.g., about 0.01 to about 0.1 mL / min) at which the fractionation phase ends (the length of the second additional phase may be in the range of about 5 minutes to about 60 minutes, e.g., about 10 minutes to about 50 minutes, about 15 minutes to about 45 minutes, about 20 minutes to about 40 minutes, or about 25 minutes to about 35 minutes, or about 30 minutes); and optionally (iii) a third additional phase following the second additional phase, wherein the cross-flow rate of the third additional phase is Preferably, the cross-flow rate profile includes a third additional phase whose cross-flow rate is constant and lower than that of the second additional phase (e.g., the cross-flow rate of the third additional phase is 0). (The length of the third additional phase may be within the range of about 5 minutes to about 30 minutes, e.g., about 6 minutes to about 25 minutes, about 7 minutes to about 20 minutes, about 8 minutes to about 15 minutes, about 10 minutes to about 12 minutes, or about 5 minutes, about 10 minutes, or about 12 minutes.) Preferred examples of such cross-flow rate profiles are as follows: 1.0-2.0 mL / min for 10 minutes, an exponential ramp from 1.0-2.0 mL / min to 0.01-0.07 mL / min within 30 minutes; 0.01-0.07 mL / min for 30 minutes; and 0 mL / min for 10 minutes.

[0130] 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 the nucleic acid (especially RNA) contained in the sample composition is determined using the integrity of a control nucleic acid (especially RNA).

[0131] In a first particular example of this embodiment of the third aspect, the integrity of a control nucleic acid (especially RNA) is determined by the following steps: (a') subjecting at least a portion of a control composition containing a control nucleic acid (especially RNA) to field-flow fractionation, particularly AF4 or HF5, to size fractionation of components contained in the control composition to produce one or more control fractions; (b') measuring at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and a refractive index (RI) signal of at least one of the one or more control fractions obtained from step (a'); (c'1) From at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained in step (b'), calculate the area from the maximum height of one UV, fluorescent, or RI peak to the end of the UV, fluorescent, or RI peak, thereby obtaining A 50% obtaining a control; (c'2) A is obtained by calculating the total area of one peak used in step (c'1) from at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained in step (b'). 100% obtaining a control; (c'3)A 50% (control) and A 100% (control) to obtain the integrity of the control nucleic acid (especially RNA) [I(control)]; is calculated by

[0132] In this first example, the integrity of the nucleic acids (especially RNA) contained in the sample composition is determined by the following steps: (c1) From at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained in step (b), calculate the area from the maximum height of the sample UV, fluorescent, or RI peak corresponding to the control UV, fluorescent, or RI peak used in step (c'1) to the end of the sample UV, fluorescent, or RI peak, thereby obtaining A 50% Obtaining a sample; (c2) A is obtained by 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 from step (b). 100% Obtaining a sample; (c3)A 50% (Sample) and A 100% obtaining I(sample) by calculating the ratio of I(sample); (c4) determining the ratio of I (sample) to I (control) to determine the integrity of the nucleic acids (especially RNA) contained in the sample composition; It can be calculated by:

[0133] In a second particular example of this embodiment of the third aspect, the integrity of a control nucleic acid (especially RNA) is determined by the following steps: (a") subjecting at least a portion of a control composition containing a control nucleic acid (especially RNA) to field-flow fractionation, particularly AF4 or HF5, to size fractionation of components contained in the control composition to produce one or more control fractions; (b") measuring at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal of at least one of the one or more control fractions obtained from step (a"); (c") determining the integrity of a control nucleic acid (especially RNA) by determining the height [H(control)] of one UV, fluorescent, or RI peak from at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained in step (b"); is calculated by

[0134] In this second example, the integrity of the nucleic acids (especially RNA) contained in the sample composition is determined by the following steps: (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 group consisting of a UV signal, a fluorescence signal, and an RI signal obtained in step (b); (c2') determining the ratio of H(sample) to H(control) to determine the integrity of the nucleic acids (especially RNA) contained in the sample composition; It can be calculated by:

[0135] In a third particular example of this embodiment of the third aspect (relating to the first subgroup of the third aspect), the integrity of the control nucleic acid (in particular RNA) is determined by the following steps: (a') subjecting at least a portion of a control composition containing a control nucleic acid (especially RNA) to field-flow fractionation, particularly AF4 or HF5, to size fractionation of components contained in the control composition to produce one or more control fractions; (b') measuring at least the UV signal of at least one of the one or more control fractions obtained from step (a'); (c'1) From the UV signal obtained in step (b'), calculate the area from the maximum height of one UV peak to the end of the UV peak to obtain A 50% obtaining a control; (c'2) A is obtained by calculating the total area of the single peak used in step (c'1) from the UV signal obtained in step (b'). 100% obtaining a control; (c'3)A 50% (control) and A 100% (control) to obtain the integrity of the control nucleic acid (especially RNA) [I(control)]; is calculated by

[0136] In this third example, the integrity of the nucleic acids (especially RNA) contained in the sample composition is determined by the following steps: (c1) From the UV signal obtained from step (b), calculate 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, thereby obtaining A 50% Obtaining a sample; (c2) A is calculated by calculating the total area of the sample UV peaks used in step (c1) from the sample UV signal obtained from step (b). 100% Obtaining a sample; (c3)A 50% (Sample) and A 100% obtaining I(sample) by calculating the ratio of I(sample); (c4) determining the ratio of I (sample) to I (control) to determine the integrity of the nucleic acids (especially RNA) contained in the sample composition; It can be calculated by:

[0137] In a fourth particular example of this embodiment of the third aspect (relating to the first subgroup of the third aspect), the integrity of the control nucleic acid (in particular RNA) is determined by the following steps: (a") subjecting at least a portion of a control composition containing a control nucleic acid (especially RNA) to field-flow fractionation, particularly AF4 or HF5, to size fractionation of components contained in the control composition to produce one or more control fractions; (b") measuring at least the UV signal of at least one of the one or more control fractions obtained from step (a"); (c") determining the integrity of a control nucleic acid (especially RNA) by determining the height of one UV peak [H(control)] from the UV signal obtained in step (b"); is calculated by

[0138] In this fourth example, the integrity of the nucleic acids (especially RNA) contained in the sample composition is determined by the following steps: (c1') determining the height [H(sample)] of the sample UV peak corresponding to the control UV peak used in step (c") from the UV signal obtained in step (b); (c2') determining the ratio of H(sample) to H(control) to determine the integrity of the nucleic acids (especially RNA) contained in the sample composition; It can be calculated by:

[0139] In a fifth particular example of this embodiment of the third aspect (related to the second subgroup of the third aspect), the integrity of the control RNA is determined by the following steps: (a') subjecting at least a portion of the control composition containing the control RNA to field-flow fractionation, particularly AF4 or HF5, to size fractionate the components contained in the control composition to produce one or more control fractions; (b') measuring at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal of at least one of the one or more control fractions obtained from step (a'); (c'1) From at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained in step (b'), calculate the area from the maximum height of one UV, fluorescent, or RI peak to the end of the UV, fluorescent, or RI peak, thereby obtaining A 50% obtaining a control; (c'2) A total area of one peak used in step (c'1) is calculated from at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained in step (b'). 100% obtaining a control; (c'3)A 50% (control) and A 100% obtaining the control RNA integrity [I(control)] by calculating the ratio of I(control) to I(control); is calculated by

[0140] In this fifth example, the integrity of the RNA contained in the sample composition is determined by the following steps: (c1) From at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained in step (b), calculate the area from the maximum height of the sample UV, fluorescent, or RI peak corresponding to the control UV, fluorescent, or RI peak used in step (c'1) to the end of the sample UV, fluorescent, or RI peak, thereby obtaining A 50% Obtaining a sample; (c2) A is obtained by 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 from step (b). 100% Obtaining a sample; (c3)A 50% (Sample) and A 100% obtaining I(sample) by calculating the ratio of I(sample); (c4) determining the ratio of I (sample) to I (control) to determine the integrity of the RNA contained in the sample composition; It can be calculated by:

[0141] In a sixth particular example of this embodiment of the third aspect (related to the second subgroup of the third aspect), the integrity of the control RNA is determined by the following steps: (a") subjecting at least a portion of a control composition containing a control RNA to field-flow fractionation, particularly AF4 or HF5, to size fractionate components contained in the control composition to produce one or more control fractions; (b") measuring at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal of at least one of the one or more control fractions obtained from step (a"); (c") determining the integrity of the control RNA by determining the height [H(control)] of one UV, fluorescent, or RI peak from at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained in step (b"); is calculated by

[0142] In this sixth example, the integrity of the RNA contained in the sample composition is determined by the following steps: (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 group consisting of a UV signal, a fluorescence signal, and an RI signal obtained in step (b); (c2') determining the ratio of H(sample) to H(control) to determine the integrity of the RNA contained in the sample composition; It can be calculated by:

[0143] In a seventh particular example of this embodiment of the third aspect (related to the third subgroup of the third aspect), the integrity of the control RNA is determined by the following steps: (a') subjecting at least a portion of the control composition containing the control RNA to field-flow fractionation, particularly AF4 or HF5, to size fractionate the components contained in the control composition to produce one or more control fractions; (b') measuring at least the UV signal of at least one of the one or more control fractions obtained from step (a'); (c'1) From the UV signal obtained in step (b'), calculate the area from the maximum height of one UV peak to the end of the UV peak to obtain A 50% obtaining a control; (c'2) A is obtained by calculating the total area of the single peak used in step (c'1) from the UV signal obtained in step (b'). 100% obtaining a control; (c'3)A 50% (control) and A 100% obtaining the control RNA integrity [I(control)] by calculating the ratio of I(control) to I(control); is calculated by

[0144] In this seventh example, the integrity of the RNA contained in the sample composition is determined by the following steps: (c1) From the UV signal obtained from step (b), calculate 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, thereby obtaining A 50% Obtaining a sample; (c2) A is calculated by calculating the total area of the sample UV peaks used in step (c1) from the sample UV signal obtained from step (b). 100% Obtaining a sample; (c3)A 50% (Sample) and A 100% obtaining I(sample) by calculating the ratio of I(sample); (c4) determining the ratio of I (sample) to I (control) to determine the integrity of the RNA contained in the sample composition; It can be calculated by:

[0145] In an eighth particular example of this embodiment of the third aspect (related to the third subgroup of the third aspect), the integrity of the control RNA is determined by the following steps: (a") subjecting at least a portion of a control composition containing a control RNA to field-flow fractionation, particularly AF4 or HF5, to size fractionate components contained in the control composition to produce one or more control fractions; (b") measuring at least the UV signal of at least one of the one or more control fractions obtained from step (a"); (c") determining the integrity of the control RNA by determining the height of one UV peak [H(control)] from the UV signal obtained in step (b"); is calculated by

[0146] In this eighth example, the integrity of the RNA contained in the sample composition is determined by the following steps: (c1') determining the height [H(sample)] of the sample UV peak corresponding to the control UV peak used in step (c") from the UV signal obtained in step (b); (c2') determining the ratio of H(sample) to H(control) to determine the integrity of the RNA contained in the sample composition; It can be calculated by:

[0147] In one embodiment of the third aspect (particularly 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 decay coefficient (especially an RNA decay coefficient) or (ii) a nucleic acid calibration curve (especially an RNA calibration curve).

[0148] In one embodiment of the third aspect (particularly in one embodiment of the first, second or third subgroup of the third aspect), the sample composition comprises nucleic acid (especially RNA) and particles to which the nucleic acid (especially RNA) is associated, such as lipoplex particles and / or lipid nanoparticles and / or polyplex particles and / or lipopolyplex particles and / or virus-like particles.

[0149] In one embodiment of the third aspect (particularly in one embodiment of the first, second, or third subgroup of the third aspect), the amount of total nucleic acid (especially RNA) is determined by: (i) treating at least a portion of the sample composition with a releasing agent; (ii) performing steps (a) through (c) on at least the portion obtained from step (i); and (iii) determining the amount of nucleic acid (especially RNA) as defined herein (e.g., by using (i) a nucleic acid decay coefficient (especially an RNA decay coefficient) or (ii) a nucleic acid calibration curve (especially 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.

[0150] 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 dodecyl sulfate), a zwitterionic surfactant [e.g., n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent® 3-14)], a cationic surfactant, a nonionic surfactant, or a mixture thereof; (ii) an alcohol, such as an aliphatic alcohol (e.g., ethanol), or a mixture of alcohols; or (iii) a combination of (i) and (ii).

[0151] In one embodiment of the third aspect (particularly in one embodiment of the first, second or third subgroup of the third aspect), the amount of free nucleic acid (especially RNA) is determined by carrying out steps (a) to (c) without the addition of a releasing agent, particularly without any releasing agent; and determining the amount of nucleic acid (especially RNA) as defined herein (for example by using (i) a nucleic acid decay factor (especially an RNA decay factor) or (ii) a nucleic acid calibration curve (especially an RNA calibration curve)).

[0152] In one embodiment of the third aspect (particularly in one embodiment of the first, second or third subgroup of the third aspect), the amount of nucleic acid (especially RNA) bound to the particles is determined herein [e.g., by carrying out steps (a) to (c) without the addition of a releasing agent, particularly without any releasing agent; and determining the amount of nucleic acid (especially RNA) as defined herein [e.g., by using (i) a nucleic acid decay factor (especially an RNA decay factor) or (ii) a nucleic acid calibration curve (especially an RNA calibration curve)]] by subtracting the amount of free nucleic acid (especially RNA) from the amount of total nucleic acid (especially RNA) as determined herein [e.g., by (i) treating at least a portion of the sample composition with a releasing agent; (ii) carrying out steps (a) to (c) on at least the portion obtained from step (i); and (iii) determining the amount of nucleic acid (especially RNA) as defined herein [e.g., by using (i) a nucleic acid decay factor (especially an RNA decay factor) or (ii) a nucleic acid calibration curve (especially an RNA calibration curve)]].

[0153] In one embodiment of the third aspect (particularly in one embodiment of the first, second or third subgroup of the third aspect), step (b) further comprises measuring at least one LS signal, such as a dynamic light scattering (DLS) signal and / or a static light scattering (SLS), such as a multi-angle light scattering (MALS) signal, of one or more sample fractions obtained from step (a).

[0154] In one embodiment of the third aspect (particularly in one embodiment of the first, second or third subgroup of the third aspect), the size of the nucleic acid (particularly RNA) containing particles can be determined from the LS signal obtained from step (b) by calculating the radius of gyration (R g ) value and / or hydrodynamic radius (R hIn 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 sample fractions obtained from step (a), and step (c) comprises calculating an R 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 at least one static light scattering (SLS), e.g., MALS, signal of one or more sample fractions obtained from step (a), and step (c) comprises calculating an R value from the SLS signal. g 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 at least one dynamic light scattering (DLS) signal and a static light scattering (SLS), e.g., MALS, signal of one or more sample fractions obtained from step (a), and step (c) comprises calculating an R g value and R h This latter embodiment involves calculating two data sets of sizes of nucleic acid (e.g., RNA)-containing particles: R g Value-based and R h Bringing value-based.

[0155] In one embodiment of the third aspect (particularly in one embodiment of the first, second or third subgroup of the third aspect), the size distribution of the nucleic acid (particularly RNA) containing particles is determined by measuring at least one signal selected from the group consisting of a UV signal, a fluorescent signal and an RI signal obtained from step (b) using an R determined as defined herein. g Value or R h by plotting the R g or by calculating the R value from the DLS signal obtained from step (b). hIn a first example of this embodiment (pertaining to the first subgroup of the third aspect), the size distribution of nucleic acid (especially RNA) containing particles is determined by multiplying the UV signal from step (b) by the R value determined as defined herein. g Value or R h by plotting the R g or by calculating the R value from the DLS signal obtained from step (b). h In a second example of this embodiment (pertaining to the second subgroup of the third aspect), the size distribution of the RNA-containing particles is determined by multiplying at least one signal selected from the group consisting of a UV signal, a fluorescent signal, and an RI signal obtained from step (b) by an R value determined as defined herein. g Value or R h by plotting the R g or by calculating the R value from the DLS signal obtained from step (b). h In a third example of this embodiment (pertaining to the third subgroup of the third aspect), the size distribution of the RNA-containing particles is determined by multiplying the UV signal from step (b) by the R value, determined as defined herein. g Value or R h by plotting the R g or by calculating the R value from the DLS signal obtained from step (b). h In each of the first, second, and third examples above, the size distribution of nucleic acid (especially RNA)-containing particles is determined by the R g Value, R h The size distribution of nucleic acid (especially RNA)-containing particles can be determined based on R g value and R h If it is based on the values of two data sets, namely, R g Size distribution based on values and Rh This results in a size distribution based on the value.

[0156] 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 of the nucleic acid (especially RNA) containing particles is R g Value or R h From a plot showing the UV, fluorescence, or RI signal as a function of the value, convert the UV, fluorescence, or RI signal to a cumulative weight fraction and convert the cumulative weight fraction to R g Value or R h In a first example of this embodiment (pertaining to the first subgroup of the third aspect), the quantitative size distribution of the nucleic acid (especially RNA) containing particles is calculated by plotting the R g Value or R h From the plot showing the UV signal as a function of the value, convert the UV signal to cumulative weight fraction and calculate the cumulative weight fraction as R g Value or R h In a second example of this embodiment (pertaining to the second subgroup of the third aspect), the quantitative size distribution of the RNA-containing particles is calculated by plotting the R g Value or R h From a plot showing the UV, fluorescence, or RI signal as a function of the value, convert the UV, fluorescence, or RI signal to a cumulative weight fraction and convert the cumulative weight fraction to R g Value or R h In a third example of this embodiment (pertaining to the third subgroup of the third aspect), the quantitative size distribution of the RNA-containing particles is calculated by plotting the R g Value or R h From the plot showing the UV signal as a function of the value, convert the UV signal to cumulative weight fraction and calculate the cumulative weight fraction as R g Value or R h In each of the first, second, and third examples above, the quantitative size distribution of nucleic acid (especially RNA)-containing particles is calculated by plotting the R g Value, R hThe quantitative size distribution of nucleic acid (especially RNA)-containing particles can be determined based on R g value and R h If it is based on the values of two data sets, namely, R g Quantitative size distribution and R based on values h This results in a quantitative size distribution based on the values.

[0157] 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 a D10 value, a D50 value, and / or a D90 value. The quantitative size distribution of the nucleic acid (especially RNA)-containing particles is g value and R h If it is based on the values of two data sets, namely, R g a set of D10, D50, and / or D90 values based on the R h The resulting set of D10, D50, and / or D90 values is based on the value.

[0158] In one embodiment of the third aspect (particularly in one embodiment of the first, second or third subgroup of the third aspect), the one or more parameters are at least two, preferably at least three parameters (including additional parameters as needed) as defined herein, in particular: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to the particles, the size distribution of the nucleic acid (especially RNA)-containing particles [in particular the radius of gyration (R) of the nucleic acid (especially RNA)-containing particles]. g ) and / or the hydrodynamic radius (R h ) ], and quantitative size distribution of nucleic acid (especially RNA)-containing particles (e.g., R g Value or R h The size distribution of nucleic acid (especially RNA)-containing particles comprises (or is) at least two, preferably at least three parameters selected from the group consisting of: R g value and R hIf it is based on the values of two data sets, namely, R g Value-based and R h However, according to the present invention, these two data sets for the size distribution of nucleic acid (especially RNA)-containing particles are considered as only one parameter (not two parameters). Also, when a fractogram obtained by field-flow fractionation shows multiple particle peaks, the size distribution obtained for each particle peak is considered as only one parameter (not one parameter for each particle peak). The quantitative size distribution of nucleic acid (especially RNA)-containing particles is considered as R g value and R h The same is true for value-based situations.

[0159] 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 include a quantitative size distribution of the nucleic acid (especially RNA)-containing particles [e.g., a radius of gyration (R g ) and / or the hydrodynamic radius (R h ) )], and optionally at least one parameter, e.g., at least two parameters, of the remaining parameters (including additional parameters as needed) defined herein; preferably, the remaining parameters are: 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 nucleic acid (especially RNA)-containing particles (e.g., R g Value or R h In one embodiment of the third aspect (particularly in an 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 are selected from the group consisting of a quantitative size distribution (e.g., R g Value or Rh values), as well as: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to the particles, and the size distribution of nucleic acid (especially RNA)-containing particles (e.g., R g Value or R h In one embodiment of the third aspect (particularly in an 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 are based on a quantitative size distribution (e.g., R g Value or R h The quantitative size distribution of nucleic acid (especially RNA)-containing particles includes the amount of nucleic acid (especially RNA) bound to the particle, based on the R g value and R h If it is based on the values of two data sets, namely, R g Value-based and R h However, according to the present invention, these two data sets for the quantitative size distribution of nucleic acid (especially RNA)-containing particles are considered as only one parameter (not two parameters). Also, in the case where a fractogram obtained by field-flow fractionation shows multiple particle peaks, the quantitative size distribution obtained for each particle peak is considered as only one parameter (not one parameter for each particle peak). When the size distribution of nucleic acid (especially RNA)-containing particles is R g value and R h The same is true for value-based situations.

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

[0161] In one embodiment of the third aspect (particularly in one embodiment of the first, second or third subgroup of the third aspect), the amount of nucleic acid (especially RNA), in particular free nucleic acid (especially RNA), is determined by measuring the UV signal, for example at a wavelength in the range of 260 nm to 280 nm, for example at a wavelength of 260 nm or 280 nm, and using the nucleic acid (especially RNA) extinction coefficient at the corresponding wavelength (e.g. 260 nm or 280 nm).

[0162] 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 nucleic acid (especially RNA)-containing particles (e.g., R g Value or R h values) and / or quantitative size distribution of nucleic acid (especially RNA)-containing particles (e.g., R g Value or R h value) is in the range of 10 to 2000 nm, preferably 20 to 1500 nm, for example, 30 to 1200 nm, 40 to 1100 nm, 50 to 1000, 60 to 900 nm, 70 to 800 nm, 80 to 700 nm, 90 to 600 nm, or 100 to 500 nm, or for example, 10 to 1000 nm, 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm, or 50 to 250 nm. In a preferred embodiment of a third subgroup of the third aspect, the (quantitative) size distribution (e.g., R g Value or R h value) is in the range of 10 to 1000 nm, for example, in the range of 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm, or 50 to 250 nm.

[0163] 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 (particularly RNA) has a length of 10 to 15,000 nucleotides, for example 40 to 15,000 nucleotides, 100 to 12,000 nucleotides, or 200 to 10,000 nucleotides.

[0164] 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 embodiments of the second or third subgroup of the third aspect, the RNA is preferably mRNA or in vitro transcribed RNA, particularly in vitro transcribed mRNA.

[0165] In one embodiment of the third aspect (particularly in one embodiment of the first, second or third subgroup of the third aspect), measuring at least one signal selected from the group consisting of a UV signal, a fluorescence signal and an RI signal, optionally an LS signal, such as an SLS, such as an MALS signal, and / or a DLS signal, is performed online and / or step (c) is performed online.

[0166] 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, at least a portion of the sample composition is diluted with a solvent or solvent mixture capable of preventing the formation of particle aggregates. In one embodiment, the solvent mixture is a mixture of water and an organic solvent, such as formamide.

[0167] In one embodiment of the third aspect (particularly in one embodiment of the first, second or third subgroup of the third aspect), measuring the UV signal is carried out by using circular dichroism (CD) spectroscopy.

[0168] It will be understood that any embodiment described herein in the context of the first or second aspect may be used in any embodiment of the third aspect.

[0169] Further embodiments are as follows: 1. A method for determining one or more parameters of a sample composition, the sample composition comprising RNA, and optionally particles, the method comprising: (a) subjecting at least a portion of the sample composition to field-flow fractionation to size-fractionate components contained in the sample composition to produce one or more sample fractions; (b) measuring at least the UV signal, and optionally the light scattering (LS) signal, of at least one of the one or more sample fractions obtained from step (a); (c) calculating one or more parameters from the UV signal and, optionally, from the LS signal; Including, The method, wherein the one or more parameters include RNA integrity, total amount of RNA, amount of free RNA, amount of RNA bound to particles, size of RNA-containing particles, size distribution of RNA-containing particles, and quantitative size distribution of RNA-containing particles.

[0170] 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).

[0171] 3. The method of item 1 or 2, wherein step (a) is carried out using a membrane with a molecular weight (MW) cutoff suitable to prevent RNA from passing through, preferably a membrane with a MW cutoff in the range of 2 kDa to 30 kDa, for example a MW cutoff of 10 kDa.

[0172] 4. The method of any one of items 1 to 3, wherein step (a) is carried out using a polyethersulfone (PES) or regenerated cellulose membrane.

[0173] 5. The method of any one of items 1 to 4, wherein step (a) is carried out using a cross-flow rate of at most 8 mL / min, preferably at most 4 mL / min, more preferably at most 2 mL / min.

[0174] 6. The method of any one of items 1 to 5, wherein step (a) is carried out using the following cross-flow rate profile: 1.0-2.0 mL / min for 10 minutes, exponential gradient from 1.0-2.0 mL / min to 0.01-0.07 mL / min within 30 minutes; 0.01-0.07 mL / min for 30 minutes; and 0 mL / min for 10 minutes.

[0175] 7. The method of any one of items 1 to 6, wherein step (a) is carried out using an inject flow in the range of 0.05 to 0.35 mL / min, preferably in the range of 0.10 to 0.30 mL / min, more preferably in the range of 0.15 to 0.25 mL / min.

[0176] 8. The method of any one of items 1 to 7, wherein step (a) is carried out using a detector flow in the range of 0.30 to 0.70 mL / min, preferably in the range of 0.40 to 0.60 mL / min, more preferably in the range of 0.45 to 0.55 mL / min.

[0177] 9. The method of any one of items 1 to 8, wherein the integrity of the RNA contained in the sample composition is calculated using the integrity of a control RNA.

[0178] 10. The integrity of the control RNA is verified by the following steps: (a') subjecting at least a portion of the control composition containing the control RNA to field-flow fractionation, particularly AF4 or HF5, to size fractionate the components contained in the control composition to produce one or more control fractions; (b') measuring at least the UV signal of at least one of the one or more control fractions obtained from step (a'); (c'1) From the UV signal obtained in step (b'), calculate the area from the maximum height of one UV peak to the end of the UV peak to obtain A 50% obtaining a control; (c'2) A is obtained by calculating the total area of the single peak used in step (c'1) from the UV signal obtained in step (b'). 100% obtaining a control; (c'3)A 50% (control) and A 100% obtaining the control RNA integrity [I(control)] by calculating the ratio of I(control) to I(control); The method of item 9 is as follows:

[0179] 11. The integrity of the RNA contained in the sample composition is determined by the following steps: (c1) From the sample UV signal obtained from step (b), calculate 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, thereby obtaining A 50% Obtaining a sample; (c2) A is calculated by calculating the total area of the sample UV peaks used in step (c1) from the sample UV signal obtained from step (b). 100% Obtaining a sample; (c3)A 50% (Sample) and A 100% obtaining I(sample) by calculating the ratio of I(sample); (c4) determining the ratio of I (sample) to I (control) to determine the integrity of the RNA contained in the sample composition; Calculated by the method in item 10.

[0180] 12. The step of calculating the integrity of the control RNA comprises the steps of: (a") subjecting at least a portion of a control composition containing a control RNA to field-flow fractionation, particularly AF4 or HF5, to size fractionate components contained in the control composition to produce one or more control fractions; (b") measuring at least the UV signal of at least one of the one or more control fractions obtained from step (a"); (c") determining the integrity of the control RNA by determining the height of one UV peak [H(control)] from the UV signal obtained in step (b"); The method of item 9 is determined by the

[0181] 13. The integrity of the RNA contained in the sample composition is determined by the following steps: (c1') determining the height [H(sample)] of the sample UV peak corresponding to the control UV peak used in step (c") from the UV signal obtained in step (b); (c2') determining the ratio of H(sample) to H(control) to determine the integrity of the RNA contained in the sample composition; Calculated by the method in item 12.

[0182] 14. The method of any one of items 1 to 13, wherein the amount of RNA is determined by using (i) an RNA decay coefficient or (ii) an RNA calibration curve.

[0183] 15. The method of any one of items 1 to 14, wherein the sample composition comprises RNA and particles to which the RNA is bound, such as lipoplex particles and / or lipid nanoparticles and / or polyplex particles and / or lipopolyplex particles and / or virus-like particles.

[0184] 16. The method of item 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) to (c) on at least the portion obtained from step (i); and (iii) determining the amount of RNA as specified in item 14.

[0185] 17. The method of item 16, wherein in step (a), field-flow fractionation is carried out using a liquid phase containing a releasing agent.

[0186] 18. The method of item 16 or 17, wherein the release agent is (i) a surfactant, such as an anionic surfactant (e.g., sodium dodecyl sulfate), a zwitterionic surfactant [e.g., n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent® 3-14)], a cationic surfactant, a nonionic surfactant, or a mixture thereof; (ii) an alcohol, such as an aliphatic alcohol (e.g., ethanol), or a mixture of alcohols; or (iii) a combination of (i) and (ii).

[0187] 19. The method of any one of items 15 to 18, wherein the amount of free RNA is determined by carrying out steps (a) to (c) without the addition of a releasing agent, in particular without any releasing agent; and determining the amount of RNA as defined in item 14.

[0188] 20. The method of any one of items 15 to 19, wherein the amount of particle-bound RNA is determined by subtracting the amount of free RNA determined by item 19 from the amount of total RNA determined by any one of items 16 to 18.

[0189] 21. The method of any one of items 15 to 20, wherein step (b) further comprises measuring at least one LS signal, such as a dynamic light scattering (DLS) signal and / or a static light scattering (SLS), such as a multi-angle light scattering (MALS) signal, of one or more sample fractions obtained from step (a).

[0190] 22. The size of the RNA-containing particles can be determined from the LS signal obtained from step (b) by calculating the radius of gyration (R g ) value and / or hydrodynamic radius (R h ) value, as determined by the method of item 21.

[0191] 23. Experimentally determined R g Value and / or R h The value is preferably experimentally determined or calculated R g Value or R h The values are fitted to a polynomial or linear function to generate an R based on the polynomial or linear fit. g Value or R h The method of item 21, where the values are smoothed by recalculating them.

[0192] 24. The size distribution of the RNA-containing particles is determined by dividing the UV signal from step (b) by the R g Value or R h 24. The method of any one of items 21 to 23, wherein the value is determined by plotting the

[0193] 25. The quantitative size distribution of RNA-containing particles is g Value or R h From the plot showing the UV signal as a function of the value, convert the UV signal to cumulative weight fraction and calculate the cumulative weight fraction as R g Value or R h 25. The method of any one of items 21 to 24, wherein the value is calculated by plotting the

[0194] 26. The method of item 25, wherein the quantitative size distribution comprises a D10 value, a D50 value, and / or a D90 value.

[0195] 27. Step (b) comprises measuring at least one dynamic light scattering (DLS) signal of one or more sample fractions obtained from step (a), and step (c) comprises determining R from the DLS signal. h 27. The method of any one of items 22 to 26, comprising calculating a value.

[0196] 28. The method of any one of items 15 to 27, wherein the one or more parameters comprise (or are) at least two, preferably at least three, parameters selected from the group consisting of the amount of free RNA, the amount of RNA bound to particles, the size distribution of RNA-containing particles, and the quantitative size distribution of RNA-containing particles.

[0197] 29. The method of any one of items 15 to 28, wherein the amount of RNA, in particular the amount of free RNA, is determined by measuring the UV signal at 260 nm and using the RNA attenuation coefficient at 260 nm, or by measuring the UV signal at 280 nm and using the RNA attenuation coefficient at 280 nm.

[0198] 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 within the range of 20 to 1500 nm, e.g., 30 to 1200 nm, 40 to 1100 nm, 50 to 1000, 60 to 900 nm, 70 to 800 nm, 80 to 700 nm, 90 to 600 nm, or 100 to 500 nm, e.g., 10 to 1000 nm, 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm, or 50 to 250 nm.

[0199] 31. The method of any one of items 1 to 30, wherein the RNA has a length of 10 to 15,000 nucleotides, e.g., 40 to 15,000 nucleotides, 100 to 12,000 nucleotides, or 200 to 10,000 nucleotides.

[0200] 32. The method of any one of items 1 to 31, wherein the RNA is in vitro transcribed RNA, in particular in vitro transcribed mRNA.

[0201] 33. The method according to any one of items 1 to 32, wherein the step of measuring the UV signal, optionally the LS signal, such as the SLS, such as the MALS signal and / or the DLS signal, is carried out online and / or step (c) is carried out online.

[0202] 34. The method of any one of items 15 to 33, 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, wherein the solvent or solvent mixture is capable of preventing the formation of particle aggregates.

[0203] 35. The process of item 34, wherein the solvent mixture is a mixture of water and an organic solvent, such as formamide.

[0204] 35a. The method of any one of items 1 to 35, wherein the step of measuring the UV signal is carried out by using circular dichroism (CD) spectroscopy.

[0205] 36. A method for analyzing the effect of altering one or more reaction conditions when a composition comprising RNA and optionally particles is provided, comprising: (A) providing a first composition comprising RNA and optionally particles; (B) providing a second composition comprising RNA and optionally particles, which differs from the first composition only in one or more reaction conditions; (C) determining one or more parameters of the first composition by subjecting a portion of the first composition to the method of any one of items 1 to 35 and 35a; (D) determining one or more parameters of the second composition by subjecting a corresponding portion of the second composition to the method used in step (C); (E) comparing one or more parameters of the first composition obtained in step (C) with corresponding one or more parameters of the second composition obtained in step (D); A method comprising:

[0206] 37. The method of item 36, wherein the one or more reaction conditions include any of the following: 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 / reconstitution cycles; addition of excipients (e.g., stabilizers and / or chelators); type and / or source of particle-forming compounds (e.g., 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 ratio of RNA to particle-forming compounds (e.g., lipids and / or polymers).

[0207] 38. Use of field-flow fractionation to determine one or more parameters of a sample composition comprising RNA and optionally particles, wherein the one or more parameters include RNA integrity, total amount of RNA, amount of free RNA, amount of RNA bound to particles, size of RNA-containing particles (e.g., hydrodynamic radius of RNA-containing particles), size distribution of RNA-containing particles, and quantitative size distribution of RNA-containing particles.

[0208] 39. Field-flow fractionation: (a) subjecting at least a portion of the sample composition to field-flow fractionation to size-fractionate components contained in the sample composition to produce one or more sample fractions; (b) measuring at least the UV signal, and optionally the light scattering (LS) signal, of at least one of the one or more sample fractions obtained from step (a); (c) calculating one or more parameters from the UV signal and, optionally, from the LS signal; Use of item 38, including:

[0209] 40. Use of item 38 or 39, wherein the field-flow fractionation is flow field-flow fractionation, for example asymmetric flow field-flow fractionation (AF4) or hollow fiber flow field-flow fractionation (HF5).

[0210] 41. Use according to any one of items 38 to 40, wherein the field-flow fractionation uses a membrane with a molecular weight (MW) cut-off suitable to prevent RNA from passing through, preferably a membrane with a MW cut-off in the range of 2 kDa to 30 kDa, for example a MW cut-off of 10 kDa.

[0211] 42. Use according to any one of items 38 to 41, wherein the field-flow fractionation uses a polyethersulfone (PES) or regenerated cellulose membrane.

[0212] 43. Step (a) is (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, with the following cross-flow rate profile: 1.0-2.0 mL / min for 10 minutes, exponentially ramping from 1.0-2.0 mL / min to 0.01-0.07 mL / min within 30 minutes; 0.01-0.07 mL / min for 30 minutes; and 0 mL / min cross-flow rate for 10 minutes; and / or (II) an inject flow rate in the range of 0.05 to 0.35 mL / min, preferably in the range of 0.10 to 0.30 mL / min, more preferably in the range of 0.15 to 0.25 mL / min; and / or (III) Detector flow rate in the range of 0.30 to 0.70 mL / min, preferably in the range of 0.40 to 0.60 mL / min, and more preferably in the range of 0.45 to 0.55 mL / min 43. Use according to any one of items 39 to 42, carried out using

[0213] 44. The use of 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.

[0214] 45. The integrity of the control RNA is verified by the following steps: (a') subjecting at least a portion of the control composition containing the control RNA to field-flow fractionation, particularly AF4 or HF5, to size fractionate the components contained in the control composition to produce one or more control fractions; (b') measuring at least the UV signal of at least one of the one or more control fractions obtained from step (a'); (c'1) From the UV signal obtained in step (b'), calculate the area from the maximum height of one UV peak to the end of the UV peak to obtain A 50% obtaining a control; (c'2) A is obtained by calculating the total area of the single peak used in step (c'1) from the UV signal obtained in step (b'). 100% obtaining a control; (c'3)A 50% (control) and A 100% obtaining the control RNA integrity [I(control)] by calculating the ratio of I(control) to I(control); Use of item 44 as required by.

[0215] 46. The integrity of the RNA contained in a sample composition is determined by the following steps: (c1) From the sample UV signal obtained from step (b), calculate 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, thereby obtaining A 50% Obtaining a sample; (c2) A is calculated by calculating the total area of the sample UV peaks used in step (c1) from the sample UV signal obtained from step (b). 100% Obtaining a sample; (c3)A 50% (Sample) and A 100% obtaining I(sample) by calculating the ratio of I(sample); (c4) determining the ratio of I (sample) to I (control) to determine the integrity of the RNA contained in the sample composition; The use of item 45, calculated by

[0216] 47. The step of calculating the integrity of the control RNA comprises the steps of: (a") subjecting at least a portion of a control composition containing a control RNA to field-flow fractionation, particularly AF4 or HF5, to size fractionate components contained in the control composition to produce one or more control fractions; (b") measuring at least the UV signal of at least one of the one or more control fractions obtained from step (a"); (c") determining the integrity of the control RNA by determining the height of one UV peak [H(control)] from the UV signal obtained in step (b"); Use of item 44, as determined by.

[0217] 48. The integrity of the RNA contained in a sample composition is determined by the following steps: (c1') determining the height [H(sample)] of the sample UV peak corresponding to the control UV peak used in step (c") from the UV signal obtained in step (b); (c2') determining the ratio of H(sample) to H(control) to determine the integrity of the RNA contained in the sample composition; The use of item 47, calculated by

[0218] 49. Use according to any one of items 38 to 48, wherein the amount of RNA is determined by using (i) an RNA decay coefficient or (ii) an RNA calibration curve.

[0219] 50. Use of any one of items 39 to 49, wherein the sample composition comprises RNA and particles to which the RNA is bound and / or in which the RNA is contained, such as lipoplex particles and / or lipid nanoparticles and / or polyplex particles and / or lipopolyplex particles and / or virus-like particles.

[0220] 51. 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) to (c) on at least the portion obtained from step (i); and (iii) determining the amount of RNA as specified in item 49.

[0221] 52. Use according to item 51, in which in step (a) the field-flow fractionation is carried out using a liquid phase containing a releasing agent.

[0222] 53. Use of item 51 or 52, wherein the release agent is (i) a surfactant, such as an anionic surfactant (e.g., sodium dodecyl sulfate), a zwitterionic surfactant [e.g., n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent® 3-14)], a cationic surfactant, a nonionic surfactant, or a mixture thereof; (ii) an alcohol, such as an aliphatic alcohol (e.g., ethanol), or a mixture of alcohols; or (iii) a combination of (i) and (ii).

[0223] 54. Use according to any one of items 50 to 53, wherein the amount of free RNA is determined by carrying out steps (a) to (c) without the addition of a releasing agent, in particular without any releasing agent; and determining the amount of RNA as defined in item 49.

[0224] 55. The use of any one of items 50 to 54, wherein the amount of particle-bound RNA is determined by subtracting the amount of free RNA determined according to item 53 from the amount of total RNA determined according to any one of items 51 to 53.

[0225] 56. Use according to any one of items 50 to 55, wherein step (b) further comprises measuring at least one LS signal, such as a dynamic light scattering (DLS) signal and / or a static light scattering (SLS), such as a multi-angle light scattering (MALS) signal, of one or more sample fractions obtained from step (a).

[0226] 57. The size of the RNA-containing particles can be determined from the LS signal obtained from step (b) by calculating the radius of gyration (R g ) value and / or hydrodynamic radius (R h ) value, using item 56.

[0227] 58. Experimentally determined R g Value and / or R h The value is preferably experimentally determined or calculated R g Value or R h The values are fitted to a polynomial or linear function to generate an R based on the polynomial or linear fit. g Value or R h The use of item 57, which is smoothed by recalculating the values.

[0228] 59. The size distribution of the RNA-containing particles is determined by dividing the UV signal obtained from step (b) by the R determined as defined in item 57. g Value or R h Use of any one of items 56 to 58, determined by plotting the value of

[0229] 60. The quantitative size distribution of RNA-containing particles is R g Value or R h From the plot showing the UV signal as a function of the value, convert the UV signal to cumulative weight fraction and calculate the cumulative weight fraction as R g Value or R h Use of any one of items 56 to 59, calculated by plotting the value against

[0230] 61. Use of item 60, wherein the quantitative size distribution includes a D10 value, a D50 value, and / or a D90 value.

[0231] 62. Step (b) further comprises measuring at least one dynamic light scattering (DLS) signal of one or more sample fractions obtained from step (a), and step (c) determining R h 62. Use of any one of items 57 to 61, including calculating a value.

[0232] 63. Use according to any one of items 50 to 62, wherein the one or more parameters comprise (or are) at least two, preferably at least three parameters selected from the group consisting of the amount of free RNA, the amount of RNA bound to particles, the size distribution of RNA-containing particles, and the quantitative size distribution of RNA-containing particles.

[0233] 64. Use according to any one of items 50 to 63, wherein the amount of RNA, in particular the amount of free RNA, is determined by measuring the UV signal at 260 nm and using the RNA attenuation coefficient at 260 nm, or by measuring the UV signal at 280 nm and using the RNA attenuation coefficient at 280 nm.

[0234] 65. Use according to any one 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 to 1500 nm, e.g., 30 to 1200 nm, 40 to 1100 nm, 50 to 1000, 60 to 900 nm, 70 to 800 nm, 80 to 700 nm, 90 to 600 nm, or 100 to 500 nm, e.g., 10 to 1000 nm, 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm, or 50 to 250 nm.

[0235] 66. Use according to any one of items 38 to 64, wherein the RNA has a length of 10 to 15,000 nucleotides, for example 40 to 15,000 nucleotides, 100 to 12,000 nucleotides, or 200 to 10,000 nucleotides.

[0236] 67. Use according to any one of items 38 to 65, wherein the RNA is in vitro transcribed RNA, in particular in vitro transcribed mRNA.

[0237] 68. Use according to any one of items 39 to 67, wherein the step of measuring the UV signal, optionally the LS signal, such as the SLS, such as the MALS signal and / or the DLS signal, is carried out online and / or step (c) is carried out online.

[0238] 69. Use according to any one of items 39 to 68, 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, said solvent or solvent mixture being capable of preventing the formation of particle aggregates.

[0239] 70. Use of item 69, wherein the solvent mixture is a mixture of water and an organic solvent, such as formamide.

[0240] 70a. Use according to any one of items 39 to 70, wherein the step of measuring the UV signal is carried out by using circular dichroism (CD) spectroscopy.

[0241] In a fourth aspect, the present disclosure provides a data processing apparatus / system comprising means for carrying out any of the methods of the present disclosure, in particular the method of the first aspect (e.g. the method defined in any one of items 1 to 35 and 35a) and / or the method of the second aspect (e.g. the method defined in item 36 or 37).

[0242] In a fifth aspect, the present disclosure provides a computer program adapted to perform any of the methods of the disclosure, in particular the method of the first aspect (e.g., the method defined in any one of items 1 to 35 and 35a) and / or the method of the second aspect (e.g., the method defined in item 36 or 37).

[0243] 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.

[0244] Further aspects of the present disclosure are disclosed herein. [Brief explanation of the drawings]

[0245] [Figure 1] Figure 1 shows the time-flow profile of an asymmetric flow field-flow fractionation (AF4) separation, where the detector flow (Vd) was 0.5 mL / min and the cross-flow (Vx) started at 1.5 mL / min and decreased exponentially to 0.04 mL / min. [Figure 2] Figure 2 shows an overview of the preferred calculation procedure for the assessment of relative RNA integrity. An example of an AF4 fractogram is shown by the UV signal at 260 nm after RNA separation. A) Limitations in the calculation of control RNA. B) Limitations in the calculation of total RNA peak area. C) Limitations in the calculation of slightly degraded RNA. D) Limitations in the calculation of slightly degraded total RNA peak area. [Figure 3] Figure 3 shows the quantification of RNA without the use of standards: A) Different injection volumes of RNA stock solution were analyzed by AF4-UV-RI. The peak area under the curve (UV, solid line; RI, dashed line) was plotted against the injection volume and a linear regression was fitted. B) Serial dilutions of RNA were measured by AF4-UV-RI at the same injection volume and analyzed as in A). [Figure 4] Figure 4 shows the quantification of degraded RNA using AF4-UV-RI and without standards: A) Representative AF4 fractograms of different heat-degraded and untreated RNAs are shown (curves represent UV signals at 260 nm). B) The UV signals of different degraded RNAs are directly correlated to their concentrations, applying the Beer-Lambert law. [Figure 5]Figure 5 shows a representative fractogram obtained from a sample particle composition (containing lipids and RNA in a 1.3 / 2 molar ratio) separated by the AF4 method disclosed herein. The solid line represents the light scattering (LS) signal at a 90° angle, which indicates the particle peak (t = approx. 35 min), while the dashed line represents the UV signal (recorded at 260 nm), which reflects bound RNA (t = approx. 38 min) and unbound RNA (t = approx. 20 min). [Figure 6-1] Figure 6 shows quantitative RNA integrity measurements of unformulated RNA: A) Representative AF4 fractograms of different pyrolyzed RNAs (n = 3; curves represent UV signals at 260 nm). B) Summary of the calculated average relative integrity of four pyrolyzed RNAs (RNAs #1-4; sizes: 986-1688 nt) varying in length. Error bars represent standard deviation (n = 3). C) Representative AF4 fractograms obtained from RNA #2 using different ratios (untreated, fully pyrolyzed, and a 50:50 mixture of untreated and fully pyrolyzed). D) Validation experiment: Different RNAs (RNAs #1-3, 986-1688 nt) were pyrolyzed and mixed in a defined manner (see Figure 6C for the AF4 fractogram of Mixture 4) and analyzed by AF4-UV measurements. The bar graphs represent the relative RNA integrity (dark grey bars, medium grey bars, and light grey bars) determined by the AF4 method disclosed herein compared to the theoretically calculated values (black bars). [Figure 6-2] Same as above. [Figure 7] Figure 7 demonstrates the suitability of UV signal for quantifying RNA in comparison with RNA quantification using fluorescent dyes (proof of concept). A) The UV peak and fluorescence (FS) integrals of the sample composition (containing RNA and fluorescently labeled particles) were correlated to the corresponding total RNA amount in the sample composition. B) The calculated ratio of the UV peak area to the FS peak area of the sample composition was found to be constant over a wide mass range (1-15 μg total RNA in the sample composition). [Figure 8]Figure 8 shows the UV ratio as a parameter for RNA sample composition. A) The UV peak integrals of free and particle-bound RNA correlate with the corresponding nominal total RNA amount contained in the sample composition. B) Calculation of the UV ratio of free RNA (peak) to bound RNA (peak). [Figure 9-1] Figure 9 shows a proof-of-concept for quantifying particle size distribution by AF4-UV-MALS. A) Representative AF4 fractogram 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 (Rg), as well as the recorded UV signal (highlighted gray peak) from the particle peak fraction (elution time: 22–60 min). Rg areas with less than 50% variation in the UV / FS ratio are highlighted (squares). In the Rg range between 50 and 300 nm, the variation in the UV / FS ratio is small and provides reliable size values. Smaller Rg values are affected by the RNA signal. Larger Rg values are affected by scattering. In total, these affected Rg values account for less than 10% of the total signal. C) Calculation of quantitative quality parameters (D10, D50, D90) based on cumulative weight fraction analysis using fluorescence emission at 624 nm and UV signal at 260 nm. [Figure 9-2] Same as above. [Figure 10] Figure 10 shows a representative AF4 fractogram of a sample composition (RNA and particles) with LS signal at 90° and UV detection at 260 nm. Calculated radius of gyration (Rg) values (gray squares) are obtained from multi-angle light scattering (MALS) using a Berry plot, and hydrodynamic radius (Rh) values are obtained from online dynamic light scattering (DLS: gray circles). [Figure 11-1]Figure 11 shows the quantification of particle size distribution in a complex sample composition using AF4-UV-MALS. A) The AF4-UV-MALS elution profile of the sample composition (RNA and particles) is shown. The UV signal at 260 nm for RNA detection (dashed line) and the light scattering signal at 90° (solid line) are shown. The corresponding radius of gyration (Rg) values from the MALS signal are shown as black dots. B) The experimentally determined RMS values of the particle peak (elution time: 26-55 min) are fitted to a polynomial equation (light gray line). C) The UV signal (solid line) is plotted as a function of the polynomial-fitted Rg values (see Figure 11B), and the corresponding cumulative weight fraction is plotted as a function of the UV signal (dashed line). [Figure 11-2] Same as above. [Figure 12-1] Figure 12 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 of the different sample compositions, the UV signal (at 260 nm), light scattering signal (at 90°), and corresponding radius of gyration (Rg) values calculated using a Berry plot are overlaid. B) The Rg values calculated from the MALS signal are plotted against the appropriate cumulative weight fraction analysis, followed by calculation of the corresponding D90 value. C) The Rg (D90) values obtained from the cumulative weight fraction analysis are plotted as a function of lipid / RNA ratio with (black dots) or without (white dots) 100 mM NaCl. [Figure 12-2] Same as above. [Figure 13] Figure 13 shows the evaluation of "shape factor" by correlating hydrodynamic radius (Rh) values to Rg values. The values are fit to a linear regression, and the resulting slope provides information about particle shape. [Figure 14-1]Figure 14 shows the separation and characterization of various particle compositions (LPX, LNP, polyplex particles (PLX), liposomes, VLP+LPX) by the AF4 method disclosed herein. AF4-UV-MALS-DLS separation / detection is shown. LS at a 90° angle is represented as a solid line, indicating the particle peak. The dashed line represents the UV signal recorded at 260 nm (for RNA detection). Radius of gyration (Rg) values (dark dots) are obtained by multi-angle light scattering (MALS) using a Zimm plot. Dynamic light scattering (DLS; gray dots) provides the hydrodynamic radius (Rh). Individual particle peak fractions are highlighted by gray bars. A) Representative fractogram of an LPX sample containing lipids and RNA at a molar ratio of 1.3 / 2 after AF4-UV-MALS-DLS separation / detection. B) Representative fractogram of a composition containing two types of particles (small RNA-LPX:VLP, 1:1 mixture). C) Representative fractogram of a liposome sample (positively charged liposomes composed of DOTMA and DOPE in a 2 / 1 molar ratio). D) Representative fractogram of an LPX sample (positively charged LPX containing DOTMA and cholesterol and RNA in a 4 / 1 molar ratio). E) Representative fractogram of a lipid nanoparticle (LNP) sample composed of DODMA, cholesterol, DOPE, PEG (1.2 / 1.44 / 0.3 / 0.06 molar ratio) and RNA in a 3 / 1 molar ratio. F) Representative fractogram of particles containing JetPEI polymer and IVT-RNA or saRNA in a 12 / 1 particle to RNA ratio. [Figure 14-2] Same as above. [Figure 14-3] Same as above. [Figure 15] Figure 15 shows the analysis of RNA behavior in the presence of ions (sodium chloride). Exemplary AF4 fractograms (light scattering signal at 90° is shown) from unformulated RNA at different sodium chloride concentrations (0-50 mM) are shown. Radius of gyration (Rg) values are obtained from multi-angle light scattering (MALS) using Zimm plots. [Figure 16]Figure 16 shows the characterization of RNA after treatment with sodium chloride. A) Rg(D50) values obtained from cumulative weight fraction analysis are shown for different sodium chloride concentrations (0-50 mM). B) RNA Rg(D50) values (from Figure 16A) were plotted against sodium chloride concentration, and the ratio (sodium chloride (mM) vs. Rg (nm)) was calculated. The linear fit of the ratio of 0-10 mM NaCl values is represented by the thick line, while the dotted line represents the fit from 10-50 mM NaCl. The gray and black lines represent examples of measurements with two different RNA concentrations. [Figure 17] Figure 17 shows the quantification of free / unbound RNA in complex sample compositions. A) Using the AF4 method disclosed herein, different amounts of free RNA (1-15 μg) were detected by UV absorbance at 260 nm in particle-free compositions. To generate a linear calibration curve, the amount of RNA was plotted against the UV peak area under the curve (AUC*min). B) Various amounts of particle compositions (containing 1-15 μg of total RNA) were analyzed by the AF4 method. The overlaid AF4 fractograms show the UV signal at 260 nm. The first peak (elution time: approximately 20 min) corresponds to free RNA, while the second peak (elution time: approximately 38 min) corresponds to particles (bound RNA). The amount of unbound free RNA in a particle composition can be calculated relative to a reference RNA (=100%) (see Figure 17A). C) To demonstrate the linearity of the method, the UV peak integrals of free RNA (see Figure 17B) as well as naked reference RNA (see Figure 17A) are plotted as a function of different RNA amounts (1-15 µg). D) As a second preferred procedure (direct method) for quantification of free RNA, the peak of unbound RNA is defined, and the RNA amount can be directly calculated using the specific extinction coefficient of the RNA. [Figure 18]Figure 18 shows the analysis of free RNA amounts in sample compositions with different physicochemical behavior. A) AF4-UV fractograms of particle compositions without NaCl ((DOTMA / DOPE2 / 1) / RNA complexes mixed at various charge ratios (0.1-0.9)) or B) particle compositions with 100 mM NaCl are shown. C) Plot of the calculated percentage of unbound RNA (mol / mol) with 100 mM NaCl (filled 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 deviation. D) Plot of the concentration of unbound RNA (μg / mL) with 100 mM NaCl (filled circles) and without NaCl (open circles), calculated using the extinction coefficient of RNA at 260 nm. [Figure 19] Figure 19 shows the quantification of total RNA in particle compositions. A) AF4 fractogram of Zwittergent-treated naked RNA separated by the AF4 method disclosed herein. The UV signal at 260 nm is represented by a black line, and the LS signal at 90° is represented by a dashed line. B) Representative fractogram of a particle composition with UV detection (solid line) of free RNA (highlighted in gray) and bound RNA (second peak), and the LS signal at a 90° angle (dashed line). C) AF4 fractogram of an RNA composition in which particles have been dissolved using a releasing agent (the liquid phase contained 0.1% Zwittergent) with UV detection (solid line) and light scattering at 90° (dashed line). D) Direct quantification of naked RNA and total RNA after treatment with a releasing agent (Zwittergent). [Figure 20-1]Figure 20 shows the integrity of free and total RNA in a sample composition containing RNA and particles. A) UV traces of particles separated with RNA that differ in RNA integrity using the AF4 method disclosed herein (untreated RNA: solid black line; partially pyrolyzed RNA: dotted line; mixture (50% untreated and 50% fully degraded mixed in a defined manner): dashed line; fully degraded RNA in particles: solid gray line). B) Quantification of intact free RNA (dark gray) and total (black) and fully degraded (light gray) free RNA in particles. C) UV traces of dissolved particles after AF4 separation (using a release agent in the liquid phase). D) The determined integrity of free and total RNA in particles analyzed by AF4-UV measurement. The bar graph represents the relative RNA integrity of free RNA (gray bars) compared to the determined integrity of total RNA values in particles (black bars). [Figure 20-2] Same as above. [Figure 21] Figure 21 shows a scheme of how different fractions of RNA (total RNA, bound RNA, encapsulated RNA, accessible RNA, surface RNA, and unbound RNA) can be determined by the AF4 method disclosed herein. For example, the AF4 method can be used to quantify accessible RNA and / or surface RNA using the fluorescent emission signal of an intercalating dye (e.g., GelRED). By using a combination of quantification of free (unbound) RNA, total RNA, and accessible RNA, encapsulated RNA, bound RNA, and surface RNA can be calculated. The fluorescent emission of GelRED at 600 nm is enhanced by its intercalation into RNA. [Figure 22-1]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 RNA (black bars) and encapsulated RNA (gray bars); 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 absorption at 260 nm (black bars) and fluorescence emission signal (FS) at 600 nm (gray bars). [Figure 22-2] Same as above. [Figure 23] Figure 23 shows the analysis of RNA integrity using the AF4 method disclosed herein without the use of a reference RNA. A) An exemplary AF4 fractogram of long saRNA is shown, with the MALS signal at 90° (dotted line) and the UV signal at 260 nm (solid line). The thick, dark line represents the molecular weight curve obtained from the MALS signal. B) For a better overview, only the molecular weight curve from (A) is shown as a solid line in the upper panel of Figure 23B. The limit for the total RNA peak (Peak 1) is set based on the total UV peak signal (i.e., t = 10 min to t = 40 min). Here, the limit for the "intact" RNA peak (Peak 2) is set by the first derivative from the molecular weight curve (obtained from MALS) as follows: The first derivative from the molecular weight curve is calculated (dotted line in the lower panel of Figure 23B). The more horizontal portion of the molecular weight curve reflects the retention time, where a fraction of undegraded RNA is present. Based on this, the integration limits can be selected, and the amount of undegraded RNA in the sample can be calculated. [Figure 24-1]Figure 24 shows quantitative analysis of free and bound RNA using UV for the determination of particle size distribution, specifically cumulative RNA weight fraction, RNA mass in RNA lipoplex (LPX) fractions, and RNA copies per LPX fraction. A) A representative AF4 fractogram for an RNA LPX sample composition is shown, with the MALS signal at 90° (solid line) and the UV signal at 260 nm (dashed line). The UV signal shows two peaks, where the first peak represents the amount of unbound free RNA and the second peak arises from 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 (Rg; thick line) is obtained from the MALS signal. B) The UV signal at 260 nm (dashed line) from Figure 24A is shown, along with the cumulative weight fraction based on the area under the UV signal as a solid line. C) The amount of bound RNA in an RNA LPX sample composition is shown by using the absorbance at 260 nm in different Rg fractions (Δt = 1 min) containing particles of a certain size. For calculation of the RNA amount in the different Rg fractions, only the LPX peak (i.e., the second peak in Figures 24A and 24B, beginning at t = ≈24 min and ending at t = ≈60 min) was used. D) The calculated number of RNA copies per Rg fraction (bar, left y-axis) calculated from the results presented in Figure 24C is shown. The calculated particle number per Rg fraction is represented by the corresponding dotted curve (second right y-axis). [Figure 24-2] Same as above. [Figure 25-1]Figure 25 demonstrates the feasibility of using circular dichroism (CD) spectroscopy in the AF4 method disclosed herein. A) A representative AF4 fractogram of an RNA lipoplex (LPX) formulation is shown, with the LS signal at a 90° angle (solid line) and the 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) A calibration curve of naked RNA was generated using simultaneous UV detection at 260 nm and CD detection at 260 nm. The peak area under the curve (CD: filled squares and solid line; UV: filled triangles and dotted line) is plotted against the amount of RNA injected. The ratio of the peak areas of the CD and UV signals is shown as points (second right y-axis). C) Different amounts of RNA LPX samples (2-15 μg) were analyzed using the AF4 method. The area under the curve (AUC) of the CD signal from the appropriate naked RNA was correlated to the appropriate total AUC CD signal, where each CD peak AUC value was plotted against the amount of RNA, resulting in a linear fit (R2 = 0.998). The relative amounts (%) of unbound RNA (open squares) and bound RNA (open circles) in the RNA LPX sample composition were determined by correlating the amounts of unbound and bound RNA to the total RNA amount. [Figure 25-2] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0246] Although the present disclosure will be further described in more detail below, it should be understood that the present disclosure is not limited to the specific methodology, 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 particular embodiments only, and is not intended to limit the scope of the present disclosure, which is limited only by the appended claims. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0247] The elements of the present disclosure are described in more detail below. The elements are described with specific embodiments. However, it should be understood that the elements can be combined in any manner and in any number to produce additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the present disclosure to only the explicitly described embodiments. The specification should be understood to supplement and encompass embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, unless the context indicates otherwise, all permutations and combinations of all elements described 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 for field-flow fractionation, and in another preferred embodiment of the method of the present disclosure, the nucleic acid (e.g., RNA) is in vitro transcribed RNA, then in a further preferred embodiment of the method of the present disclosure, AF4 is used for field-flow fractionation, and the nucleic acid (e.g., RNA) is in vitro transcribed RNA.

[0248] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)," H.G. W. Leuenberger, B. Nagel, and H. Koelbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland (1995).

[0249] The practice of the present disclosure will employ, unless otherwise indicated, conventional chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques as described in the art [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; Römpp Chemie Lexikon, Falbe / Regitz (Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular Cloning: A Laboratory Manual, 2nd Edition, J. See Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989].

[0250] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise," and variations such as "comprises" and "comprising," will be understood to mean the inclusion of the specified element, integer, or step, or group of elements, integers, or steps, and not the exclusion of any other element, integer, or step, or group of elements, integers, or steps. The term "consisting essentially of" means excluding any other element, integer, or step of any essential significance. The term "comprising" encompasses the term "consisting essentially of," which in turn encompasses the term "consisting of." Thus, in each occurrence in this application, the term "comprising" may be replaced with the term "consisting essentially of" or "consisting of." Similarly, in each occurrence in this application, the term "consisting essentially of" may be replaced with the term "consisting of."

[0251] The terms "a," "an," and "the," and similar references used in the context of describing this disclosure (particularly in the context of the claims), should be construed to encompass both the singular and the plural, unless otherwise stated herein or unless the context clearly contradicts. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise stated herein, each value is incorporated herein as if individually listed herein. All methods described herein can be performed in any suitable order unless otherwise stated herein or unless the context clearly contradicts. The use of any and all examples, or exemplary language (e.g., "for example") provided herein is intended merely to fully illustrate the disclosure and does not pose a limitation on the scope of the disclosure as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0252] As used herein, "and / or" should be understood as a specific disclosure of each of the two specified components or elements with or without the other. For example, "X and / or Y" should be understood as a specific disclosure of (i) X, (ii) Y, and (iii) each of X and Y, as if each were individually set forth herein.

[0253] In the context of the present disclosure, the term "about" refers to an interval of accuracy that a person skilled in the art would understand to still ensure the technical effect of the composition in question. The term typically indicates a deviation from the indicated numerical value 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 will be recognized by a person skilled in the art, such a specific deviation of the numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may typically have a larger deviation than an artificial or engineered technical effect.

[0254] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise stated herein, each individual value is incorporated herein as if it were individually recited herein.

[0255] The use of any and all examples or exemplary language (e.g., "for example") provided herein is intended merely to fully describe the invention and does not pose a limitation on the scope of the otherwise claimed disclosure. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0256] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, manuals, etc.), whether supra or infra, is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate its disclosure by virtue of prior invention.

[0257] definition The following provides definitions that apply to all aspects of this disclosure. The following terms have the following meanings unless otherwise specified: Any term not given a definition has its art-recognized meaning.

[0258] As used herein, the terms "reduce" or "inhibit" and the like refer to the ability to cause an overall decrease in levels, e.g., by 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. The term "inhibit" or similar phrases includes complete or essentially complete inhibition, i.e., a reduction to or essentially to zero.

[0259] The terms "increase" or "enhance" or the like in one embodiment 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%.

[0260] As used herein, "physiological pH" refers to a pH of about 7.5.

[0261] As used in this disclosure, "% w / v" refers to weight / volume percent, which is a unit of concentration that measures the amount of solute in grams (g) expressed as a percentage of the total volume of the solution in milliliters (mL).

[0262] The term "ionic strength" refers to the mathematical relationship between the number of different ionic species in a particular solution and their respective charges. Thus, ionic strength I S is the expression

[0263]

number

[0264] According to the present disclosure, the term "ionic strength" in one embodiment 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 low enough to prevent RNA degradation in one embodiment due to the presence of a chelating agent. In one embodiment, the concentration or effective concentration of divalent ions is below the catalytic level for the hydrolysis of phosphodiester bonds between RNA nucleotides. In one embodiment, the concentration of free divalent ions is 20 μM or less. In one embodiment, free divalent ions are absent or essentially absent.

[0265] "Osmolality" refers to the concentration of a particular solute expressed as the number of osmoles of solute per kilogram of solvent.

[0266] The term "freezing" refers to the solidification of a liquid, usually by removal of heat.

[0267] The terms "lyophilizing" or "lyophilization" refer to the freeze-drying of a substance by freezing the substance and then reducing the surrounding pressure to cause the freezing medium in the substance to sublimate directly from the solid phase to the gas phase.

[0268] The term "spray drying" refers to spray drying a substance by mixing a (heated) gas with an atomized (sprayed) fluid in a vessel (spray dryer), and the solvent from the droplets formed evaporates, resulting in a dry powder.

[0269] The term "reconstituting" relates to the addition of a solvent, such as water, to a dried product to return it to a liquid state, such as its original liquid state.

[0270] The term "recombinant" in the context of the present disclosure means "made by genetic engineering." In one embodiment, a "recombinant subject" in the context of the present disclosure is not naturally occurring.

[0271] The term "naturally occurring" used herein refers to the fact that an object can be found in nature.For example, naturally occurring is a peptide or nucleic acid that exists in living organisms (including viruses) and can be isolated from sources in nature and has not been intentionally modified by humans in laboratories.The term "found in nature" means "existing in nature", and includes known objects and 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.

[0272] 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 from about 15° C. to about 35° C., from about 15° C. to about 30° C., from about 15° C. to about 25° C., or from 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.

[0273] 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, promoting lipid structure formation, such as lipid vesicle formation, e.g., liposome formation. Typically, the nucleic acid (especially RNA) lipoplex particles described herein can be obtained by adding nucleic acid (especially RNA) to a colloidal liposome dispersion. Using the ethanol injection technique, such a colloidal liposome dispersion is formed, in one embodiment, as follows: an ethanol solution containing lipids, e.g., a cationic lipid such as DOTMA, and additional lipids is injected into a stirred aqueous solution. In one embodiment, the nucleic acid (especially RNA) lipoplex particles described herein can be obtained without an extrusion step.

[0274] The term EDTA refers to ethylenediaminetetraacetic acid disodium salt. All concentrations are given in terms of EDTA disodium salt.

[0275] The term "alkyl" refers to a saturated straight-chain or branched hydrocarbon monoradical. Preferably, the alkyl group contains 1 to 12 (e.g., 1 to 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 to 8 carbon atoms, e.g., 1 to 6 or 1 to 4 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, isopropyl (also called 2-propyl or 1-methylethyl), butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, 1,2-dimethylpropyl, isoamyl, n-hexyl, isohexyl, sec-hexyl, n-heptyl, isoheptyl, n-octyl, 2-ethylhexyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl, and the like.

[0276] In accordance with this 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 joined together via peptide bonds. The term "protein" refers to large peptides, particularly peptides having at least about 151 amino acids, although the terms "peptide" and "protein" are generally used interchangeably herein.

[0277] In accordance with the present disclosure, a nucleic acid, e.g., RNA (preferably mRNA), encoding a peptide or protein introduced by or into a cell, i.e., transfected or transduced, (the cell may be in vitro or in a subject) preferably results in expression of said peptide or protein. 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 its surface.

[0278] In accordance with the present disclosure, terms such as "nucleic acid expression" and "nucleic acid coding" or similar terms are used interchangeably herein and mean that, with respect to a particular peptide or polypeptide, a nucleic acid can be expressed to produce said peptide or said polypeptide if it is present in an appropriate environment, preferably intracellularly.

[0279] According to the present disclosure, a portion or fragment of a peptide or protein preferably possesses at least one functional property of the peptide or protein from which it is derived. Such functional properties include 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 possesses at least one pharmacological activity of the peptide or protein from which it is derived. A portion 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 consecutive amino acids of the peptide or protein. A portion or fragment of a peptide or protein preferably comprises a sequence of up to 8, particularly up to 10, at most 12, at most 15, at most 20, at most 30, or at most 55 consecutive amino acids of the peptide or protein.

[0280] According to the present disclosure, a peptide or protein analog is a modified form of the peptide or protein from which it is derived, retaining at least one functional property of the peptide or protein. For example, a pharmacologically active analog of a peptide or protein retains at least one pharmacological activity of the peptide or protein from which it is derived. Such modifications include any chemical modification, including single or multiple substitutions, deletions, and / or additions of any molecules associated with the protein or peptide, such as carbohydrates, lipids, and / or proteins or peptides. In one embodiment, a protein or peptide "analog" includes modifications resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protecting / blocking groups, proteolytic cleavage, or conjugation to an antibody or another cellular ligand. The term "analog" also covers all functional chemical equivalents of the proteins and peptides.

[0281] According to the present disclosure, an "antigen" encompasses any substance that will elicit an immune response and / or any substance against which an immune response or immune mechanism, e.g., a cellular response, is directed. This also includes situations in which an antigen is processed into antigenic peptides and an 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 antibodies or T lymphocytes (T cells). According to the present invention, the term "antigen" includes any molecule that contains at least one epitope, e.g., a T cell epitope. Preferably, an antigen in the context of the present disclosure is a molecule that, after appropriate processing, induces an immune response that is preferably specific to the antigen (including cells expressing the antigen). In one embodiment, the antigen is a disease-associated antigen, e.g., a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such an antigen.

[0282] In accordance with the present disclosure, any suitable antigen that is a candidate for an immune response may be used, and the immune response may be both a humoral and a cellular immune response. In the context of some embodiments of the present disclosure, the antigen is preferably presented by a cell, preferably an antigen-presenting cell, in the context of an MHC molecule to elicit 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 be derived from allergens, viruses, bacteria, fungi, parasites, and other infectious agents and pathogens, or the antigen may be a tumor antigen. In accordance with the present invention, the antigen may correspond to a naturally occurring product, for example, a viral protein or a portion thereof.

[0283] In a preferred embodiment, the antigen is a tumor antigen, i.e., a part of tumor cells, particularly, mainly present intracellularly or as a surface antigen of tumor cells.In another embodiment, the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, for example, a virus, a bacterium, a single-cell organism, or a parasite, such as a viral antigen, for example, a viral ribonucleoprotein or coat protein.In particular, the antigen should be presented by MHC molecules, which leads to the modulation, particularly activation, of cells of the immune system, preferably CD4+ and CD8+ lymphocytes, particularly through the modulation of the activity of T cell receptors.

[0284] The term "disease-associated antigen" is used in the 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 mount 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 infection by microorganisms, typically microbial antigens (e.g., bacterial antigens or viral antigens), or antigens associated with cancer, typically tumors, such as tumor antigens.

[0285] The term "tumor antigen" refers to a component of a cancer cell that may originate from the cytoplasm, cell surface, or cell nucleus. It particularly refers to an antigen produced intracellularly or as a surface antigen on a tumor cell. For example, tumor antigens include carcinoembryonal antigen, α1-fetoprotein, isoferritin and fetal sulfoglycoprotein, α2-H-iron protein and γ-fetoprotein, as well as various viral tumor antigens. According to the present disclosure, tumor antigens preferably include any antigen that is specific to a tumor or cancer and to tumor or cancer cells in terms of type and / or expression level.

[0286] The term "viral antigen" refers to any viral component that has antigenic properties, i.e., is capable of eliciting an immune response in an individual. A viral antigen may be a viral ribonucleoprotein or envelope protein.

[0287] The term "bacterial antigen" refers to any bacterial component that has antigenic properties, i.e., is capable of eliciting an immune response in an individual. Bacterial antigens can be derived from the bacterial cell wall or cytoplasmic membrane.

[0288] The term "epitope" refers to an antigenic determinant in a molecule, e.g., an antigen, i.e., a portion or fragment of a molecule that is recognized by the immune system, e.g., by antibodies, T cells, or B cells, particularly when presented in the context of an MHC molecule. Protein epitopes preferably comprise a continuous or discontinuous portion of the protein and are preferably about 5 to about 100, preferably about 5 to about 50, more preferably about 8 to about 0, and most preferably about 10 to about 25 amino acids in length; e.g., epitopes may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. It is particularly preferred that epitopes in the context of the present invention are T cell epitopes.

[0289] The terms "epitope," "fragment of an antigen," "immunogenic peptide," and "antigenic peptide," etc., are used interchangeably herein and preferably relate to an antigen, or an incomplete representation of an antigen that is preferably capable of eliciting an immune response against a cell that expresses or contains, and preferably presents, the antigen. Preferably, the term relates to an immunogenic portion of an antigen, preferably 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.

[0290] The term "T cell epitope," when presented in the context of an MHC molecule, refers to a portion or fragment of a protein that is recognized by T cells. The terms "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes present in all vertebrates, including MHC class I and MHC class II molecules. MHC proteins or molecules are important for signaling between lymphocytes and antigen-presenting or diseased cells in the immune response; they bind peptide epitopes and present them for recognition by T cell receptors on T cells. Proteins encoded by MHC are expressed on the surface of cells and present both self-antigens (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, the bound peptide is typically about 8 to about 10 amino acids in length, although longer or shorter peptides may be effective. For class II MHC / peptide complexes, the binding peptides are typically about 10 to about 25 amino acids in length, particularly about 13 to about 18 amino acids in length, although longer and shorter peptides may be effective.

[0291] The term "target" is intended to mean an agent, e.g., a cell or tissue, that is the target of an immune response, e.g., a cellular immune response. Targets include cells that present an antigen or an antigen 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 said antigen on a class I MHC.

[0292] The term "portion" refers to a fraction. With respect to a particular structure, e.g., an amino acid sequence or a protein, the term "portion" may refer to a contiguous or non-contiguous fraction of said structure.

[0293] The terms "portion" and "fragment" are used interchangeably herein and refer to a continuous element. For example, a portion of a structure, such as an amino acid sequence or protein, refers to a continuous element of said structure. When used in the context of a composition, the term "portion" refers to a portion of the composition. For example, a portion of a composition can be any portion between 0.1% and 99.9% of said composition (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%).

[0294] "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., via binding) of the fragments with one or more MHC molecules for presentation to specific T cells by a cell, preferably an antigen-presenting cell.

[0295] "Antigen-responsive CTL" refers to a CD8 CTL that is responsive to an antigen presented by class I MHC on the surface of an antigen-presenting cell, or to a peptide derived from said antigen. + It means T cells.

[0296] According to the present invention, CTL responsiveness 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 tumor antigen-expressing target cells. CTL responsiveness can also be determined using artificial reporters that accurately represent CTL responsiveness.

[0297] The terms "immune response" and "immune reaction" are used interchangeably herein in their conventional sense and refer to the integrated body's response to an antigen, preferably a cellular immune response, a humoral immune response, or both. According to the present invention, the term "immune response to" or "immune response against," in reference to an agent, e.g., an antigen, cell, or tissue, refers to an immune response, e.g., a cellular response, directed against the agent. An immune response includes the development of antibodies against one or more antigens and the production of antigen-specific T lymphocytes, preferably CD4 + and CD8 + T lymphocytes, more preferably CD8 + The antibody may comprise one or more responses selected from the group consisting of proliferation of T lymphocytes, which may be detected in vitro by various proliferation or cytokine production tests.

[0298] In the context of the present invention, the terms "inducing an immune response" and "eliciting an immune response," as well as similar terms, refer to the induction of an immune response, preferably a cellular immune response, a humoral immune response, or both. The immune response may be protective / preventive / prophylactic and / or therapeutic. The immune response may 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)). "Inducing" in this context can mean that there was no immune response against a particular antigen or pathogen before induction, but it can also mean that there was a particular level of immune response against a particular antigen or pathogen before and after induction, in which the immune response is enhanced. Thus, in this context, "inducing an immune response" also includes "enhancing 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 symptoms of the disease are ameliorated by inducing an immune response.

[0299] 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 antigens and / or presentation of antigens by class I or class II MHC. Cellular responses involve cells called T cells or T lymphocytes, which perform "helper" or "killer" functions. Helper T cells (CD4 + T cells (also called T cells) play a central role by regulating the immune response and are known as killer cells (cytotoxic T cells, cytolytic T cells, CD8 + T cells, or CTLs, kill cells, such as diseased cells.

[0300] The term "humoral immune response" refers to the process in living organisms in which antibodies are produced in response to and ultimately neutralize and / or eliminate agents 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 the appropriate antigen and receiving various other activation signals, B lymphocytes divide, giving rise to memory B cells and antibody-secreting plasma cell clones, each of which produces antibodies that recognize the same antigenic epitope recognized by its antigen receptor. Memory B lymphocytes remain quiescent until later activated by their specific antigen. These lymphocytes achieve cell-based memory and consequent recruitment in the antibody response upon re-exposure to the specific antigen.

[0301] The term "antibody" as used herein refers to an immunoglobulin molecule, which 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 is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain is composed of 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 and constant domains, respectively. The VH and VL regions can be further subdivided into regions of hypervariability (referred to as complementarity-determining regions (CDRs)), interspersed with more conserved regions (referred to as framework regions (FRs)). Each VH and each VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of the VH are referred to as HCDR1, HCDR2, and HCDR3, and the CDRs of the VL are referred to as 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), and the CH can be further subdivided into a constant domain CH1, a hinge region, and constant domains CH2 and CH3 (arranged from the amino terminus to the carboxy terminus in the following order: CH1, CH2, CH3). The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or host factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). Antibodies can be intact immunoglobulins derived from natural or recombinant sources, and can be immunologically active portions of intact immunoglobulins. Antibodies are typically tetrameric immunoglobulin molecules. Antibodies can exist in a variety of forms, including polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies and humanized antibodies.

[0302] The term "immunoglobulin" relates to proteins of the immunoglobulin superfamily, preferably antigen receptors, such as antibodies or B-cell receptors (BCRs). Immunoglobulins are characterized by structural domains, i.e., immunoglobulin domains, with a distinctive immunoglobulin (Ig) fold. The term encompasses membrane-bound and soluble immunoglobulins. Membrane-bound immunoglobulins are also called surface or membrane immunoglobulins, which are usually part of the BCR. Soluble immunoglobulins are usually called antibodies. Immunoglobulins usually contain several chains, typically two identical heavy chains and two identical light chains, which are linked via disulfide bonds. These chains are mainly composed of immunoglobulin domains, e.g., 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 immunoglobulin heavy chains in mammals: α, δ, ε, γ, and μ, which constitute the various classes of antibodies: 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 carboxy termini. In mammals, there are two types of light chains: lambda and kappa. Immunoglobulin chains contain a variable region and a constant region. The constant region is essentially conserved within the various immunoglobulin isotypes, while the variable region is highly variable and is responsible for antigen recognition.

[0303] The terms "vaccination" and "immunization" describe the process of treating an individual for therapeutic or prophylactic reasons and relate to the procedure of administering to an individual one or more immunogens or antigens or derivatives thereof, particularly in the form of RNA encoding same as described herein, to elicit an immune response against said one or more immunogens or antigens or cells characterized by the presentation of said one or more immunogens or antigens.

[0304] "Cells characterized by antigen presentation" or "cells presenting antigen" or "MHC molecules presenting antigens on the surface of antigen-presenting cells" or similar expressions refer to cells such as diseased cells, particularly tumor cells, or antigen-presenting cells that present antigens or antigenic peptides directly or following processing, in the context of MHC molecules, preferably MHC class I and / or MHC class II molecules, most preferably MHC class I molecules.

[0305] In the context of the present disclosure, the term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA, which may then be translated into peptides or proteins.

[0306] With respect to RNA, the terms "expression" or "translation" refer to the process within a cell's ribosomes by which a chain of mRNA directs the assembly of a sequence of amino acids to form a peptide or protein.

[0307] As used herein, the term "optionally" or "appropriately" means that the subsequently described event, circumstance, or condition may or may not occur, and that the description includes instances in which said event, circumstance, or condition occurs and instances in which said event, circumstance, or condition does not occur.

[0308] The "radius of gyration" (referred to herein as R) of a particle around its axis of rotation g) is the radial distance of a point from the axis of rotation at which, if the entire mass of a particle were concentrated at one point, the moment of inertia about a given axis would be the same as the actual distribution of mass. Mathematically, R g is the root mean square distance of the particle's components from the center of mass or a given axis. For example, if the particle is at a fixed distance s from the center of mass, i Mass m positioned at i For a macromolecule consisting of n mass elements (i=1, 2, 3, …, n), R g is the si for all mass elements 2 is the mass average square root of and can be calculated as follows:

[0309]

number

[0310] The radius of gyration can be determined experimentally, for example, by using light scattering, or can be calculated.

[0311]

number

[0312]

number

[0313] The "D10 value" refers specifically to the quantitative size distribution of particles, and is the diameter of 10% of the particles less than a certain value. The D10 value is a way of describing the proportion of the smallest particles within a population of particles (e.g., within a particle peak obtained from field-flow fractionation).

[0314] The "D50 value" refers, particularly to a quantitative particle size distribution, to the diameter below which 50% of the particles have a diameter. The D50 value is a way of describing the average particle size of a population of particles (e.g., within a particle peak obtained from field-flow fractionation).

[0315] A "D90 value," particularly in reference to the quantitative size distribution of particles, is the diameter of 90% of the particles less than a certain value. The "D95," "D99," and "D100" values have corresponding meanings. The D90, D95, D99, and D100 values are a means of describing the proportion of larger particles within a population of particles (e.g., within a particle peak obtained from field-flow fractionation).

[0316] The "hydrodynamic radius" of a particle (sometimes called the "Stokes radius" or "Stokes-Einstein radius") is the radius of a hypothetical hard sphere diffusing at the same rate as the particle. The hydrodynamic radius relates to the particle's ability to move, taking into account not only size but also solvent effects. For example, a smaller, more hydrated, charged particle may have a larger hydrodynamic radius than a larger, less hydrated, charged particle. This is because smaller particles drag a greater number of water molecules with them as they move through a solution. Because the actual dimensions of a particle in a solvent are not directly measurable, the hydrodynamic radius may be defined by the Stokes-Einstein equation:

[0317]

number

[0318] As used herein, the term "shape factor" refers to the R g values (e.g., recalculated R g value), the hydrodynamic radius (R h ) value. This means the ratio of g values (e.g., recalculated R g value) to the hydrodynamic radius (R h ) values and fitting the data points to a function (e.g., a linear function) or can be calculated.

[0319] As used herein, the term "form factor" refers to the hydrodynamic radius (R h ) value of R g values (e.g., recalculated R g This means the ratio of the hydrodynamic radius (R h ) value to R g values (e.g., recalculated R g The value can be plotted against the σ value and fit the data points to a function (e.g., a linear function) or can be calculated.

[0320] As used herein, expression " nucleic acid encapsulation efficiency " refers to the ratio of the amount of encapsulated nucleic acid contained in the sample or control composition that comprises nucleic acid and particles to the total amount of nucleic acid contained in the sample or control composition.For example, when nucleic acid is RNA, as used herein, expression " RNA encapsulation efficiency " refers to the ratio of the amount of encapsulated RNA contained in the sample or control composition that comprises RNA and particles to the total amount of RNA contained in the sample or control composition.

[0321] As used herein, the term "membrane" refers to a size-selective barrier that allows molecules to pass below a certain size, referred to as a "cutoff" (e.g., molecular weight (MW) cutoff), but stops molecules above that size (i.e., a cutoff, e.g., a MW cutoff). 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.

[0322] The term "aggregate" as used herein refers to a cluster of particles where the particles are identical or very similar to one another and are non-covalently attached (e.g., via ionic, H-bridge, dipole, and / or van der Waals interactions).

[0323] As used herein, the expression "light scattering" refers to the physical process by which light is forced to deviate from a straight-line trajectory by one or more paths due to localized inhomogeneities in the medium through which the light passes.

[0324] The term "UV" means ultraviolet light and refers to the band of the electromagnetic spectrum with wavelengths between 10 nm and 400 nm, i.e., shorter than visible light but longer than X-rays.

[0325] 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.

[0326] The term "UV CD light" or "UV CD signal" refers to circularly polarized light with wavelengths between 10 nm and 400 nm, i.e., shorter than visible light but longer than X-rays.

[0327] The expression "multi-angle light scattering" or MALS, as used herein, refers to a technique for measuring light scattered at multiple angles by a sample. "Multi-angle" in this context means that the scattered light can be detected at a variety of discrete angles, measured, for example, by a single detector moving over a range that includes a selected specific angle, or by an array of detectors 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 containing particles, and by using an appropriate formalism (e.g., Zimm plot, Berry plot, or Debye plot), the radius of gyration (R g ) and hence the size of the particles can be determined. Preferably, the Zimm plot is a graphical representation using the following formula:

[0328]

number

[0329]

number

[0330]

number

[0331] As used herein, the phrase "dynamic light scattering" or "DLS" refers to a technique for determining particle size and size distribution profiles, particularly with respect to the hydrodynamic radius of particles. A monochromatic light source, usually a laser, is emitted into a sample through a polarizer. The scattered light then enters through a second polarizer and is detected, and the resulting image is projected onto a screen. Particles in solution impinge on 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 set of speckle patterns is analyzed by an autocorrelator, which compares the light intensity at each spot over time.

[0332] As used herein, the phrase "static light scattering" or "SLS" refers to a technique for determining particle size and size distribution profiles, particularly with respect to the particle's radius of gyration and / or molar mass. A high-intensity monochromatic light, usually a laser, is projected into a solution containing the particles. One or many detectors are used to measure the scattered intensity at one or many angles. Angular dependence is required to obtain accurate measurements of both the molar mass and size of all macromolecular radii. Therefore, simultaneous measurements at several angles relative to the direction of incident light (known as multi-angle light scattering (MALS) or multi-angle laser light scattering (MALLS)) are usually considered the standard practice of static light scattering.

[0333] The expressions "elution time" and "retention time" are used interchangeably herein and relate to the period of time it takes for a particular analyte to pass through a system (e.g., from the injection point of a field-flow fractionation device to the detector) under set conditions.

[0334] The expression "continuous change" means that the change from one value to another is carried out continuously, i.e. without any jumps. Examples of continuous change are linear or exponential changes (e.g., linear or exponential gradients).

[0335] The expression "step change" means that the change from one value to a different value is not continuous, but skips at least one value between the first and second values by jumping from a first specific value to a second specific value. An example of a step change is a flow rate profile that starts at a first value (e.g., 10 mL / min) and ends at a second value (e.g., 0 mL / min), during which the flow rates can only be integers (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 mL / min), thereby skipping values between these integers.

[0336] As used herein, the phrase "providing a composition comprising nucleic acid (e.g., RNA) and optionally particles" means that such a composition may be provided by any means, e.g., may be prepared, processed (e.g., purified and / or dried), and / or stored.

[0337] nucleic acid According to the present disclosure, the term "nucleic acid" includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modifications thereof. The term includes genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. According to the present disclosure, a nucleic acid may exist as a single-stranded or double-stranded molecule, and may be a linear molecule or a covalently closed circular molecule. A nucleic acid may be isolated according to the present disclosure. The term "isolated nucleic acid" according to the present disclosure means that the nucleic acid has been (i) amplified in vitro, e.g., via polymerase chain reaction (PCR) for DNA or in vitro transcription (e.g., using RNA polymerase) for RNA; (ii) recombinantly produced by cloning; (iii) purified, e.g., by cleavage and separation by gel electrophoresis; or (iv) synthesized, e.g., by chemical synthesis.

[0338] The term "nucleoside" (abbreviated herein as "N") refers to a compound that can be considered a nucleotide without a phosphate group. A nucleoside is a nucleic acid base linked to a sugar (e.g., ribose or deoxyribose), while a nucleotide is composed of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.

[0339] The five standard nucleosides that typically make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine, and guanosine. The five nucleosides are commonly 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 rather than 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, these are represented as A, C, and G, but in DNA, they are represented as dA, dC, and dG.

[0340] The modified purine (A or G) or pyrimidine (C, T, or U) base moiety preferably comprises one or more alkyl groups, more preferably one or more C 1~4 and more preferably modified by one or more alkyl groups, and even more preferably by 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, e.g., 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-uracils and N(1)-C 1~4 Alkyl-uracil, preferably N 7 -methyl-guanine, N 6 -methyl-adenine, 5-methyl-cytosine, 5-methyl-uracil, and N(1)-methyl-uracil.

[0341] In this disclosure, the term "DNA" refers to a nucleic acid molecule containing deoxyribonucleotide residues. In a preferred embodiment, DNA contains all or a majority of deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide lacking a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. DNA includes, but is not limited to, double-stranded DNA, single-stranded DNA, isolated DNA, e.g., partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, and modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may refer to the addition of non-nucleotide material to internal DNA nucleotides or to the ends of the DNA. It is also contemplated herein that the nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the purposes of this disclosure, modified DNA is considered an analog of naturally occurring DNA. A molecule contains a "majority of 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 a molecule is the sum of all nucleotide residues (whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0342] In one embodiment, the DNA is recombinant DNA and may be obtained by cloning a nucleic acid in a particular cDNA, which may be obtained by reverse transcription of RNA.

[0343] In this disclosure, the term "RNA" refers to a nucleic acid molecule containing ribonucleotide residues. In a preferred embodiment, the RNA contains all or a majority of 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 includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA, e.g., partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may refer to the addition of non-nucleotide material to internal RNA nucleotides or to the ends of the RNA. It is also contemplated herein that the nucleotides in the RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For purposes of this disclosure, altered / modified nucleotides may be referred to as analogs of naturally occurring nucleotides, and the corresponding RNA containing such altered / modified nucleotides (i.e., altered / modified RNA) may be referred to as analogs of naturally occurring RNA. A molecule contains a "majority of 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, whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof.

[0344] 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 (e.g., antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)], activating RNA (e.g., small activating RNA), and immunostimulatory RNA (isRNA).

[0345] As used herein, the term "in vitro transcription" or "IVT" means that transcription (i.e., production of RNA) is performed in a cell-free manner. That is, IVT uses transcription machinery extracted from cells (e.g., cell lysates or isolated components thereof, including RNA polymerase, preferably T7, T3, or SP6 polymerase)) rather than live / cultured cells.

[0346] According to the present disclosure, the term "mRNA" refers to "messenger RNA" and relates to a "transcript" that can be produced by using a DNA template and can encode a peptide or protein. Typically, 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 by in vitro transcription (IVT) from a DNA template. As indicated above, in vitro transcription methodologies are known to those skilled in the art, and various in vitro transcription kits are commercially available.

[0347] Although mRNA is single-stranded, it may contain self-complementary sequences that allow part of the mRNA to fold back and pair with itself to form a double helix.

[0348] According to the present disclosure, "dsRNA" means double-stranded RNA, which is RNA having two partially or completely complementary strands.

[0349] The length of the RNA can vary from 10 nucleotides to 15,000, e.g., 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 (e.g., 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, e.g., up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides, or up to 10,000 nucleotides.

[0350] In certain embodiments of the present disclosure, the RNA is mRNA, which refers to RNA transcription products that code for peptides or proteins.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, 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 methodology is known to those skilled in the art; for example, see Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989. Additionally, various in vitro transcription kits are commercially available, for example, from Thermo Fisher Scientific (e.g., TranscriptAid™ T7 Kit, MEGAscript™ T7 Kit, MAXIscript™), New England BioLabs Inc. (e.g., HiScribe™ T7 Kit, HiScribe™ T7 ARCA mRNA Kit), Promega (e.g., iboMAX™, HeLaScribe™, Riboprobe™ System), Jena Bioscience (e.g., SP6 or T7 Transcription Kit), and Epicentre (e.g., AmpliScribe™). To prepare modified RNA, accordingly, modified nucleotides, such as naturally occurring modified nucleotides, non-naturally occurring nucleotides, and / or non-naturally occurring modified nucleotides, can be incorporated during synthesis (preferably in vitro transcription), or modifications can be introduced into and / or added to the transcribed RNA.

[0351] In one embodiment, the RNA is in vitro transcribed RNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling 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 a vector suitable for in vitro transcription. The cDNA can be obtained by reverse transcription of RNA.

[0352] In the context of the present disclosure, RNA, preferably mRNA, contains one or more modifications, such as 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 coding region, i.e., in the sequence that codes for expressed peptide or protein, preferably without changing the sequence of expressed peptide or protein.Such modifications are described, for example, in International Publication No. 2007 / 036366 and International Application No. PCT / EP2019 / 056502, and include: 5'-cap structure; extension or truncation of naturally occurring poly(A) tail; modification of 5'- and / or 3'-untranslated region (UTR), for example, introduction of UTR that is not related to the coding region of the RNA; substitution of one or more naturally occurring nucleotides with synthetic nucleotides; and codon optimization (for example, modifying, preferably increasing, the GC content of RNA). The term "modification" in the context of modified RNA (preferably mRNA) according to the present disclosure preferably relates to any modification of RNA (preferably mRNA) that does not naturally occur in said RNA.

[0353] In some embodiments, 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 the cap structure found on the 5'-end of an mRNA molecule, generally consisting of guanosine 5'-triphosphate (Gppp), which is linked via its triphosphate moiety to the 5'-end of the adjacent nucleotide in the mRNA (i.e., the guanosine is linked to the remainder of the mRNA via a 5'-5' triphosphate bond). The guanosine is N 7 may be methylated at position m 7 The term "5'-cap analog" is based on the traditional 5'-cap but is 7 This refers to a 5'-cap that is modified at the 2' or 3' position of the guanosine structure to prevent reverse integration of the 5'-cap analog (such 5'-cap analogs are also called anti-reverse cap analogs (ARCAs)). Particularly preferred 5'-cap analogs are those with one or more substitutions at the bridging position and a non-bridging oxygen in the phosphate bridge, such as phosphorothioate-modified 5'-cap analogs at the β-phosphate (e.g., m2 7,2’OG(5')ppSp(5')G (also referred to as beta-S-ARCA or β-S-ARCA), which is described in International Application PCT / EP2019 / 056502 (the entire disclosure of which is incorporated herein by reference). Providing RNA (preferably 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, with the 5'-cap structure being co-transcriptionally incorporated into the resulting RNA strand, or 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, for example, vaccinia virus capping enzyme.

[0354] In some embodiments, the RNA (preferably mRNA) according to the present disclosure is 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.

[0355] In some embodiments, the RNA (preferably mRNA) comprises cap0, cap1, or cap2 (preferably cap1 or cap2). In accordance with the present disclosure, the term "cap0" refers to the structure "m 7 GpppN, where N is any nucleoside having an OH moiety at the 2' position. In accordance with the present disclosure, the term "cap1" refers to the structure "m 7 GpppNm, where Nm is any nucleoside having an OCH3 moiety at the 2' position. In accordance with the present disclosure, the term "cap2" refers to the structure "m 7 GpppNmNm" where each Nm is independently any nucleoside having an OCH3 moiety at the 2' position.

[0356] The D1 diastereomer of beta-S-ARCA (β-S-ARCA) has the following structure:

[0357] [ka]

[0358] The "D1 diastereomer of beta-S-ARCA" or "beta-S-ARCA(D1)" is a diastereomer of beta-S-ARCA that elutes first from an HPLC column and therefore exhibits a shorter retention time than the D2 diastereomer of beta-S-ARCA (beta-S-ARCA(D2)). The HPLC is preferably analytical HPLC. In one embodiment, a Supelcosil LC-18-T RP column, preferably 5 μm, 4.6 × 250 mm format, is used for separation, allowing for a flow rate of 1.3 ml / min. In one embodiment, a gradient of methanol in ammonium acetate is used, e.g., a 0-25% linear gradient of methanol in 0.05 M ammonium acetate, pH 5.9, within 15 minutes. UV detection (VWD) may be performed at 260 nm, and fluorescence detection (FLD) may be performed with excitation at 280 nm and detection at 337 nm.

[0359] 5'-cap analog m2, a building block of cap1 7,3’-O Gppp(m1 2’-O )ApG(m2 7,3’O G(5')ppp(5')m 2’-O ApG (also called ApG) has the following structure:

[0360] [ka]

[0361] An exemplary cap0 RNA comprising β-S-ARCA and RNA has the following structure:

[0362] [ka]

[0363] m2 7,3’O An exemplary cap0 RNA comprising G(5')ppp(5')G and RNA has the following structure:

[0364] [ka]

[0365] m2 7,3-O Gppp(m1 2’-O ) An exemplary cap1 RNA comprising ApG and RNA has the following structure:

[0366] [ka]

[0367] As used herein, the term "poly A tail" or "poly A sequence" refers to an uninterrupted or interrupted sequence of adenylate residues 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 RNAs described herein. Uninterrupted poly A tails are characterized by consecutive adenylate residues. Uninterrupted poly A tails are typical in nature. The RNAs disclosed herein may have a poly A tail attached to the free 3'-end of the RNA by a template-independent RNA polymerase after transcription, or a poly A tail encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0368] Poly(A) tails of approximately 120 A nucleotides have been demonstrated to have a beneficial effect on the levels of RNA in transfected eukaryotic cells and on the levels of proteins translated from open reading frames located upstream (5') of the poly(A) tail (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0369] 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 most nucleotides in the poly-A tail, 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 number of nucleotides in the poly-A tail, are A nucleotides, while allowing for the remaining nucleotides to be nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, "consisting of" means that all nucleotides in the poly A tail, i.e., 100% of the number of nucleotides in the poly A tail, are A nucleotides. The term "A nucleotide" or "A" refers to adenylate.

[0370] In some embodiments, the poly(A) tail is attached during RNA transcription, e.g., during 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) cassette.

[0371] In some embodiments, a poly(A) cassette present in the coding strand of DNA consists essentially of dA nucleotides but is interrupted by random sequences of four nucleotides (dA, dC, dG, and dT). Such random sequences may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such cassettes are disclosed in International Publication No. 2016 / 005324 A1, which is incorporated herein by reference. Any poly(A) cassette disclosed in International Publication No. 2016 / 005324 A1 may be used in the present invention. A poly(A) cassette consisting essentially of dA nucleotides but interrupted by random sequences with an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of, for example, 5 to 50 nucleotides, exhibits consistent growth of plasmid DNA in E. coli at the DNA level, yet is accompanied by beneficial properties at the RNA level, including support for RNA stability and translation efficiency. Consequently, in some embodiments, the poly-A tails contained in the RNA molecules described herein consist essentially of A nucleotides, but are interrupted by random sequences of four nucleotides (A, C, G, U). Such random sequences can be 5-50, 10-30, or 10-20 nucleotides in length.

[0372] In some embodiments, no nucleotides other than A nucleotides flank the poly A tail at its 3'-end, i.e., the poly A tail is not masked or followed by nucleotides other than A at its 3'-end.

[0373] In some embodiments, the RNA according to the present disclosure comprises a 5'-UTR and / or a 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 a corresponding region in an RNA molecule, such as an mRNA molecule. The untranslated region (UTR) can be located 5' (upstream) of the open reading frame (5'-UTR) and / or 3' (downstream) of the open reading frame (3'-UTR). 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 downstream of the 5'-cap (if present), e.g., 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, although the term "3'-UTR" preferably does not include a polyA sequence. Thus, the 3'-UTR is located upstream of the polyA sequence (if present), for example, directly adjacent to the polyA sequence. The incorporation of a 3'-UTR into the 3'-untranslated region of an RNA (preferably mRNA) molecule can result in enhanced translation efficiency. The incorporation of two or more such 3'-UTRs (preferably arranged in a head-to-tail orientation; see, for example, Holtkamp et al., Blood 108, 4009-4017 (2006)) can achieve a synergistic effect. The 3'-UTR can be autologous or heterologous to the introduced RNA (preferably mRNA). In a specific embodiment, the 3'-UTR is derived from a globin gene or mRNA, such as alpha2-globin, alpha1-globin, or beta-globin, preferably beta-globin, more preferably human beta-globin. For example, the RNA (preferably mRNA) may be modified by replacing or inserting one or more, preferably two, copies of the existing 3'-UTR with a 3'-UTR derived from a globin gene, e.g., alpha2-globin, alpha1-globin, beta-globin, preferably beta-globin, more preferably human beta-globin.

[0374] 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 modified nucleoside.In some embodiments, the modified nucleoside is modified uridine.

[0375] In some embodiments, the modified uridine replacing the uridine is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), 5-methyl-uridine (m5U), and combinations thereof.

[0376] In some embodiments, the modified nucleoside that replaces (partially or completely, preferably completely) uridine in the RNA is 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), 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), bromo-uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-aminomethyl-2-thiouridine (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-taurinomethyl-uridine (τm5U), 1-taurinomethyl-uridine Methyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (m5s2U), 1-taurinomethyl-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-dihydro-pseudouridine, 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 The modified uridine may be any one or more of uridine (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-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine], or any other modified uridine known in the art.

[0377] RNA (preferably mRNA) modified with pseudouridine (partially or completely, preferably completely replacing uridine) is referred to herein as "Ψ-modified," although the term "mΨ-modified" means that the RNA (preferably mRNA) contains N(1)-methylpseudouridine (partially or completely, preferably completely replacing uridine). Furthermore, the term "m5U-modified" means that the RNA (preferably mRNA) contains 5-methyluridine (partially or completely, preferably completely replacing uridine). Such Ψ- or mΨ- or m5U-modified RNAs typically exhibit reduced immunogenicity compared to their unmodified forms and are therefore preferred in applications where induction of an immune response is to be avoided or minimized.

[0378] The codons of the RNA (preferably mRNA) of the present disclosure may be further optimized, for example, by increasing the GC content of the RNA and / or replacing codons that are rare in a cell (or subject) so that a peptide or protein of interest is expressed by codons that are frequent synonymous codons in said cell (or subject).

[0379] Combinations of the modifications described above, i.e., incorporation of a 5'-cap structure, incorporation of a polyA sequence, unmasking of a polyA sequence, alteration of the 5'- and / or 3'-UTR (e.g., incorporation of one or more 3'-UTRs), substitution of one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine, and / or pseudouridine (Ψ) or N(1)-methylpseudouridine (mΨ) or 5-methyluridine (m5U) for uridine), and codon optimization, have a synergistic effect on increasing RNA (preferably mRNA) stability and translation efficiency. Thus, in a preferred embodiment, an RNA (preferably an 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, namely: (i) incorporation of a 5'-cap structure, (ii) incorporation of a polyA sequence, unmasking of a polyA sequence; (iii) alteration of the 5'- and / or 3'-UTR (e.g., incorporation of one or more 3'-UTRs); (iv) substitution of one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine, and / or pseudouridine (Ψ) or N(1)-methylpseudouridine (mΨ) or 5-methyluridine (m5U) for uridine), and (v) codon optimization.

[0380] In one embodiment, the RNA according to the present disclosure comprises a nucleic acid sequence that encodes a peptide or protein, preferably a pharmaceutically active peptide or protein.

[0381] 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 when transferred into a cell or a subject. Thus, preferably, the RNA according to the present invention contains a coding region (open reading frame (ORF)) encoding a peptide or protein, preferably encoding a pharmaceutically active peptide or protein. In this regard, an "open reading frame" or "ORF" is a contiguous stretch of codons beginning with a start codon and ending with a stop codon.

[0382] 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, where expression of the peptide or protein is beneficial, for example, to ameliorate symptoms of a disease or disorder. Preferably, a pharmaceutically active peptide or protein has therapeutic or palliative properties and may be administered to ameliorate, alleviate, mitigate, arrest, or delay the onset of a disease or disorder, or to reduce the severity of one or more symptoms of a disease or disorder. Preferably, a pharmaceutically active peptide or protein, when administered to an individual in a therapeutically effective amount, has a positive or beneficial effect on the individual's symptoms or disease state. A pharmaceutically active peptide or protein may have prophylactic properties and may be used to delay the onset of a disease or disorder or reduce the severity of such a disease or disorder. The term "pharmaceutically active peptide or protein" includes whole proteins or polypeptides and may refer to pharmaceutically active fragments thereof. It may also include pharmaceutically active analogs of peptides or proteins.

[0383] Specific examples of pharmaceutically active peptides and proteins include, but are not limited to, cytokines, hormones, adhesion molecules, immunoglobulins, immunologically active compounds, growth factors, protease inhibitors, enzymes, receptors, apoptosis regulators, transcription factors, tumor suppressor proteins, structural proteins, reprogramming factors, genome engineering proteins, and blood proteins.

[0384] The term "cytokine" refers to proteins having a molecular weight of approximately 5 to 20 kDa and involved in cell signaling (e.g., paracrine signaling, endocrine signaling, and / or autocrine signaling). Specifically, upon release, cytokines exert effects on cellular behavior around the site of release. Examples of cytokines include lymphokines, interleukins, chemokines, interferons, and tumor necrosis factors (TNFs). According to the present disclosure, cytokines do not include hormones or growth factors. Cytokines differ from hormones in that (i) they typically operate at much more variable concentrations than hormones and (ii) they are generally produced by a wide range of cells (almost all nucleated cells can produce cytokines). Interferons are typically characterized by antiviral, antiproliferative, and immunomodulatory activities. Interferons are proteins that interfere with viral replication in cells by altering and regulating gene transcription within the cells through binding to interferon receptors on the surface of regulated cells. Interferons can be grouped into two types: IFN-gamma is the only type II interferon; all others are type I interferons. Specific examples of cytokines include erythropoietin (EPO), colony-stimulating factors (CSFs), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF), bone morphogenetic proteins (BMPs), interferon alpha (IFNα), interferon beta (IFNβ), interferon gamma (INFγ), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 10 (IL-10), and interleukin 11 (IL-11).

[0385] The term "hormone" refers to a class of signaling molecules produced by glands, and signal transduction typically involves the following steps: (i) synthesis of the hormone in specific tissues; (ii) storage and secretion; (iii) transport of the hormone to its target; (iv) binding of the hormone by a receptor; (v) signal relay and amplification; and (vi) degradation of the hormone. Hormones differ from cytokines in that (1) hormones typically operate 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 (e.g., human growth hormone or bovine growth hormone), oxytocin, atrial natriuretic peptide (ANP), glucagon, somatostatin, cholecystokinin, gastrin, and leptin.

[0386] The term "adhesion molecule" refers to a protein located on the surface of a cell and involved in binding of the cell to other cells or to the extracellular matrix (ECM). Adhesion molecules are typically transmembrane receptors and can be classified as calcium-independent (e.g., integrins, immunoglobulin superfamily, lymphocyte homing receptors) and calcium-dependent (cadherins and selectins). Specific examples of adhesion molecules include integrins, lymphocyte homing receptors, selectins (e.g., P-selectin), and addressins.

[0387] Integrins are also involved in signal transduction. In particular, immediately after ligand binding, integ...

Claims

1. 1. A method for determining one or more parameters of a sample composition, said sample composition comprising RNA and optionally particles, said method comprising: (a) subjecting at least a portion of the sample composition to field-flow fractionation to fractionate components contained in the sample composition by size to produce one or more sample fractions; (b) measuring at least the UV signal, and optionally the light scattering (LS) signal, of at least one of said one or more sample fractions obtained from step (a); (c) calculating one or more parameters from said UV signal and optionally from said LS signal; Including, The method, wherein the one or more parameters include RNA integrity, total amount of RNA, amount of free RNA, amount of RNA bound to particles, size of RNA-containing particles, size distribution of RNA-containing particles, and quantitative size distribution of RNA-containing particles.

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

3. 3. The method of claim 1 or 2, wherein step (a) is carried out using a membrane with a molecular weight (MW) cut-off suitable to prevent RNA from passing through, preferably a membrane with a MW cut-off in the range of 2 kDa to 30 kDa, for example a MW cut-off of 10 kDa.

4. 4. The method according to any one of claims 1 to 3, wherein step (a) is carried out using a polyethersulfone (PES) or regenerated cellulose membrane.

5. 5. The method of any one of claims 1 to 4, wherein step (a) is carried out using a cross-flow rate of at most 8 mL / min, preferably at most 4 mL / min, more preferably at most 2 mL / min.

6. 6. The method of any one of claims 1 to 5, wherein step (a) is carried out using the following cross-flow rate profile: 1.0-2.0 mL / min for 10 minutes, exponentially ramping from 1.0-2.0 mL / min to 0.01-0.07 mL / min within 30 minutes; 0.01-0.07 mL / min for 30 minutes; and 0 mL / min for 10 minutes.

7. 7. The method of any one of claims 1 to 6, wherein step (a) is carried out using an inject flow in the range of 0.05 to 0.35 mL / min, preferably in the range of 0.10 to 0.30 mL / min, more preferably in the range of 0.15 to 0.25 mL / min.

8. 8. The method of any one of claims 1 to 7, wherein step (a) is carried out using a detector flow in the range of 0.30 to 0.70 mL / min, preferably in the range of 0.40 to 0.60 mL / min, more preferably in the range of 0.45 to 0.55 mL / min.

9. 9. The method of claim 1, wherein the integrity of the RNA contained in the sample composition is calculated using the integrity of a control RNA.

10. The integrity of the control RNA is determined by the following steps: (a') subjecting at least a portion of a control composition containing a control RNA to field-flow fractionation, particularly AF4 or HF5, to size fractionate components contained in said control composition to produce one or more control fractions; (b') measuring at least the UV signal of at least one of the one or more control fractions obtained from step (a'); (c'1) From the UV signal obtained in step (b'), calculate the area from the maximum height of one UV peak to the end of the UV peak to obtain A ５０％ obtaining a (control); (c'2) A is obtained by calculating the total area of the one peak used in step (c'1) from the UV signal obtained in step (b'). １００％ obtaining a (control); (c'3) A ５０％ (Control) and A １００％ (control) to obtain the integrity of the control RNA [I(control)]; The method of claim 9, wherein the formula is determined by:

11. The integrity of the RNA contained in the sample composition can be determined by the following steps: (c1) From the sample UV signal obtained from step (b), calculate 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, thereby obtaining A ５０％ Obtaining a sample; (c2) calculating the total area of the sample UV peaks used in step (c1) from the sample UV signal obtained from step (b), thereby obtaining A １００％ Obtaining a sample; (c3) A ５０％ (Sample) and A １００％ obtaining I(sample) by calculating the ratio of I(sample) to I(sample); (c4) determining the ratio of I(sample) to I(control) to determine the integrity of the RNA contained in the sample composition; The method of claim 10, wherein the calculated value is:

12. The step of calculating the integrity of the control RNA comprises the steps of: (a") subjecting at least a portion of a control composition containing a control RNA to field-flow fractionation, particularly AF4 or HF5, to size fractionation of components contained in said control composition to produce one or more control fractions; (b") measuring at least the UV signal of at least one of said one or more control fractions obtained from step (a"); (c") determining the integrity of the control RNA by determining the height of one UV peak [H(control)] from the UV signal obtained in step (b"); The method of claim 9 , wherein the value is determined by

13. The integrity of the RNA contained in the sample composition can be determined by the following steps: (c1') determining the height [H(sample)] of the sample UV peak corresponding to the reference UV peak used in step (c") from the UV signal obtained in step (b); (c2') determining the ratio of H(sample) to H(control) to determine the integrity of the RNA contained in the sample composition; The method of claim 12, wherein the calculated value is:

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

15. 15. The method of any one of claims 1 to 14, wherein the sample composition comprises RNA and particles to which RNA is bound, such as lipoplex particles and / or lipid nanoparticles and / or polyplex particles and / or lipopolyplex particles and / or virus-like particles.

16. 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) to (c) on at least the portion obtained from step (i); and (iii) determining the amount of RNA as defined in claim 14.

17. 17. The method of claim 16, wherein in step (a), the field-flow fractionation is carried out using a liquid phase containing the releasing agent.

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

19. 19. The method of any one of claims 15 to 18, wherein the amount of free RNA is determined by carrying out steps (a) to (c) without the addition of a releasing agent, in particular without any releasing agent; and determining the amount of RNA as defined in claim 14.

20. 20. The method of any one of claims 15 to 19, wherein the amount of RNA bound to the particles is determined by subtracting the amount of free RNA determined by claim 19 from the amount of total RNA determined by any one of claims 16 to 18.

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

22. The size of the RNA-containing particles can be determined from the LS signal obtained from step (b) by calculating the radius of gyration (R ｇ ) value and / or hydrodynamic radius (R ｈ 22. The method of claim 21, wherein the value is determined by calculating the value of

23. The experimentally determined R ｇ value and / or said R ｈ The value is preferably experimentally determined or calculated. ｇ value or the R ｈ The values are fitted to a polynomial or linear function to obtain an R based on the polynomial or linear fit. ｇ Value or R ｈ 22. The method of claim 21, wherein the values are smoothed by recalculating them.

24. The size distribution of the RNA-containing particles is determined by dividing the UV signal from step (b) by the R determined as defined in claim 22. ｇ value or the R ｈ 24. The method of any one of claims 21 to 23, wherein the value is determined by plotting the value of

25. The quantitative size distribution of RNA-containing particles is ｇ value or the R ｈ From a plot showing the UV signal as a function of the value, the UV signal is converted to a cumulative weight fraction, and the cumulative weight fraction is converted to the R ｇ value or the R ｈ 25. The method of any one of claims 21 to 24, wherein the value is calculated by plotting the value of

26. 26. The method of claim 25, wherein the quantitative size distribution comprises a D10 value, a D50 value, and / or a D90 value.

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

28. 28. The method of any one of claims 15 to 27, wherein 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 RNA, the amount of RNA bound to particles, the size distribution of RNA-containing particles, and the quantitative size distribution of RNA-containing particles.

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

30. 30. The method of any one of claims 1 to 29, wherein the size distribution of RNA-containing particles and / or the quantitative size distribution of RNA-containing particles is in the range of 10-2000 nm, preferably 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, such as 10-1000 nm, 15-500 nm, 20-450 nm, 25-400 nm, 30-350 nm, 40-300 nm, or 50-250 nm.

31. 31. The method of any one of claims 1 to 30, wherein the RNA has a length of 10 to 15,000 nucleotides, such as 40 to 15,000 nucleotides, 100 to 12,000 nucleotides, or 200 to 10,000 nucleotides.

32. 32. The method of any one of claims 1 to 31, wherein the RNA is in vitro transcribed RNA, in particular in vitro transcribed mRNA.

33. 33. A method according to any one of claims 1 to 32, wherein the step of measuring the UV signal, optionally the LS signal, such as the SLS, such as the MALS signal and / or the DLS signal, is carried out online and / or step (c) is carried out online.

34. 34. The method of any one of claims 15 to 33, wherein 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, the solvent or solvent mixture being capable of preventing the formation of particle aggregates.

35. 37. The method of claim 36, wherein the solvent mixture is a mixture of water and an organic solvent, such as formamide.

36. 36. The method of any one of claims 1 to 35, wherein the step of measuring the UV signal is carried out by using circular dichroism (CD) spectroscopy.

37. 1. A method for analyzing the effect of altering one or more reaction conditions when a composition comprising RNA and optionally particles is provided, comprising: (A) providing a first composition comprising RNA and optionally particles; (B) providing a second composition comprising RNA and optionally particles, which differs from the first composition only in the one or more reaction conditions; (C) determining one or more parameters of the first composition by subjecting a portion of the first composition to the method of any one of claims 1 to 36; (D) determining one or more parameters of the second composition by subjecting a corresponding portion of the second composition to the method used in step (C); and (E) comparing the 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); A method comprising:

38. 38. The method of claim 37, wherein the one or more reaction conditions include any of: salt concentration / ionic strength (e.g., 2 mM NaCl or 100 mM NaCl); temperature [e.g., low (e.g., −20° C.) or high (e.g., 50° C.)]; 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 (e.g., lipids and / or polymers, e.g., cationic lipids vs. zwitterionic lipids, or pegylated lipids vs. non-pegylated lipids); charge ratio; physical state; and ratio of RNA to particle-forming compounds (e.g., lipids and / or polymers).

39. Use of field-flow fractionation to determine one or more parameters of a sample composition comprising RNA and optionally particles, wherein the one or more parameters include RNA integrity, total amount of RNA, amount of free RNA, amount of RNA bound to particles, size of RNA-containing particles (e.g., hydrodynamic radius of RNA-containing particles), size distribution of RNA-containing particles, and quantitative size distribution of RNA-containing particles.

40. The field-flow fractionation (a) subjecting at least a portion of the sample composition to field-flow fractionation to fractionate components contained in the sample composition by size to produce one or more sample fractions; (b) measuring at least the UV signal, and optionally the light scattering (LS) signal, of at least one of said one or more sample fractions obtained from step (a); (c) calculating one or more parameters from said UV signal and optionally from said LS signal; 40. The use of claim 39, comprising:

41. 41. The use according to claim 39 or 40, wherein the field-flow fractionation is a flow field-flow fractionation, such as asymmetric flow field-flow fractionation (AF4) or hollow fiber flow field-flow fractionation (HF5).

42. 42. The use according to any one of claims 39 to 41, wherein the field-flow fractionation uses a membrane with a molecular weight (MW) cut-off suitable to prevent RNA from passing through, preferably a membrane with a MW cut-off in the range of 2 kDa to 30 kDa, for example a MW cut-off of 10 kDa.

43. 43. The use according to any one of claims 39 to 42, wherein the field-flow fractionation uses a polyethersulfone (PES) or regenerated cellulose membrane.

44. Step (a) (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, with the following cross-flow rate profile: 1.0-2.0 mL / min for 10 minutes, exponentially ramping from 1.0-2.0 mL / min to 0.01-0.07 mL / min within 30 minutes; 0.01-0.07 mL / min for 30 minutes; and 0 mL / min for 10 minutes; and / or (II) an inject flow in the range of 0.05 to 0.35 mL / min, preferably in the range of 0.10 to 0.30 mL / min, more preferably in the range of 0.15 to 0.25 mL / min; and / or (III) a detector flow in the range of 0.30 to 0.70 mL / min, preferably in the range of 0.40 to 0.60 mL / min, and more preferably in the range of 0.45 to 0.55 mL / min; 44. The use according to any one of claims 40 to 43, wherein the use is carried out using

45. 45. The use according to any one of claims 39 to 44, wherein the integrity of the RNA contained in the sample composition is determined using the integrity of a control RNA.

46. The integrity of the control RNA is determined by the following steps: (a') subjecting at least a portion of a control composition containing a control RNA to field-flow fractionation, particularly AF4 or HF5, to size fractionate components contained in said control composition to produce one or more control fractions; (b') measuring at least the UV signal of at least one of the one or more control fractions obtained from step (a'); (c'1) From the UV signal obtained in step (b'), calculate the area from the maximum height of one UV peak to the end of the UV peak to obtain A ５０％ obtaining a (control); (c'2) A is obtained by calculating the total area of the one peak used in step (c'1) from the UV signal obtained in step (b'). １００％ obtaining a (control); (c'3) A ５０％ (Control) and A １００％ (control) to obtain the integrity of the control RNA [I(control)]; 46. The use according to claim 45, wherein the use is determined by

47. The integrity of the RNA contained in the sample composition can be determined by the following steps: (c1) From the sample UV signal obtained from step (b), calculate 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, thereby obtaining A ５０％ Obtaining a sample; (c2) calculating the total area of the sample UV peaks used in step (c1) from the sample UV signal obtained from step (b), thereby obtaining A １００％ Obtaining a sample; (c3) A ５０％ (Sample) and A １００％ obtaining I(sample) by calculating the ratio of I(sample) to I(sample); (c4) determining the ratio of I(sample) to I(control) to determine the integrity of the RNA contained in the sample composition; 47. The use according to claim 46, wherein the calculated value is:

48. The step of calculating the integrity of the control RNA comprises the steps of: (a") subjecting at least a portion of a control composition containing a control RNA to field-flow fractionation, particularly AF4 or HF5, to size fractionation of components contained in said control composition to produce one or more control fractions; (b") measuring at least the UV signal of at least one of said one or more control fractions obtained from step (a"); (c") determining the integrity of the control RNA by determining the height of one UV peak [H(control)] from the UV signal obtained in step (b"); 46. The use according to claim 45, wherein the

49. The integrity of the RNA contained in the sample composition can be determined by the following steps: (c1') determining the height [H(sample)] of the sample UV peak corresponding to the reference UV peak used in step (c") from the UV signal obtained in step (b); (c2') determining the ratio of H(sample) to H(control) to determine the integrity of the RNA contained in the sample composition; 49. The use according to claim 48, wherein the calculated value is:

50. 50. The use according to any one of claims 39 to 49, wherein the amount of RNA is determined by using (i) an RNA decay coefficient or (ii) an RNA calibration curve.

51. 51. The use according to any one of claims 40 to 50, wherein the sample composition comprises RNA and particles to which RNA is bound, such as lipoplex particles and / or lipid nanoparticles and / or polyplex particles and / or lipopolyplex particles and / or virus-like particles.

52. 52. The use of claim 51, 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) to (c) on at least the portion obtained from step (i); and (iii) determining the amount of RNA as defined in claim 50.

53. 53. The use according to claim 52, wherein in step (a), the field-flow fractionation is carried out using a liquid phase containing the releasing agent.

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

55. 55. Use according to any one of claims 51 to 54, wherein the amount of free RNA is determined by carrying out steps (a) to (c) without the addition of a releasing agent, in particular without any releasing agent; and determining the amount of RNA as defined in claim 50.

56. 56. The use of any one of claims 51 to 55, wherein the amount of RNA bound to the particles is determined by subtracting the amount of free RNA determined by claim 55 from the amount of total RNA determined by any one of claims 52 to 54.

57. 57. The use of any one of claims 51 to 56, wherein step (b) further comprises measuring at least one LS signal, such as a dynamic light scattering (DLS) signal and / or a static light scattering (SLS), such as a multi-angle light scattering (MALS) signal, of the one or more sample fractions obtained from step (a).

58. The size of the RNA-containing particles can be determined from the LS signal obtained from step (b) by calculating the radius of gyration (R ｇ ) value and / or hydrodynamic radius (R ｈ 58. The use according to claim 57, wherein the value is determined by calculating the

59. The experimentally determined R ｇ value and / or said R ｈ The value is preferably experimentally determined or calculated. ｇ value or the R ｈ The values are fitted to a polynomial or linear function to obtain an R based on the polynomial or linear fit. ｇ Value or R ｈ 59. Use according to claim 58, wherein the values are smoothed by recalculating them.

60. The size distribution of the RNA-containing particles is determined by dividing the UV signal from step (b) by the R determined as defined in claim 58. ｇ value or the R ｈ 60. The use of any one of claims 57 to 59, wherein the value is determined by plotting the value of

61. The quantitative size distribution of RNA-containing particles is ｇ value or the R ｈ From a plot showing the UV signal as a function of the value, the UV signal is converted to a cumulative weight fraction, and the cumulative weight fraction is converted to the R ｇ value or the R ｈ 61. The use according to any one of claims 57 to 60, wherein the value is calculated by plotting the value against the

62. 62. The use of claim 61, wherein the quantitative size distribution comprises a D10 value, a D50 value, and / or a D90 value.

63. Step (b) comprises measuring at least one dynamic light scattering (DLS) signal of the one or more sample fractions obtained from step (a), and step (c) comprises determining R from the DLS signal. ｈ 63. The use of any one of claims 58 to 62, comprising calculating a value.

64. 64. The use of any one of claims 51 to 63, wherein 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 RNA, the amount of RNA bound to particles, the size distribution of RNA-containing particles, and the quantitative size distribution of RNA-containing particles.

65. 65. Use according to any one of claims 51 to 64, wherein the amount of RNA, in particular the amount of free RNA, is determined by measuring the UV signal at 260 nm and using the RNA attenuation coefficient at 260 nm or by measuring the UV signal at 280 nm and using the RNA attenuation coefficient at 280 nm.

66. 66. The use according to any one of claims 39 to 65, wherein the size distribution of RNA-containing particles and / or the quantitative size distribution of RNA-containing particles is in the range of 10 to 2000 nm, preferably 20 to 1500 nm, such as 30 to 1200 nm, 40 to 1100 nm, 50 to 1000, 60 to 900 nm, 70 to 800 nm, 80 to 700 nm, 90 to 600 nm, or 100 to 500 nm, such as 10 to 1000 nm, 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm, or 50 to 250 nm.

67. 67. The use of any one of claims 39 to 66, wherein the RNA has a length of 10 to 15,000 nucleotides, such as 40 to 15,000 nucleotides, 100 to 12,000 nucleotides, or 200 to 10,000 nucleotides.

68. 68. The use according to any one of claims 39 to 67, wherein the RNA is in vitro transcribed RNA, in particular in vitro transcribed mRNA.

69. 69. Use according to any one of claims 40 to 68, wherein the step of measuring the UV signal, optionally the LS signal, such as SLS, such as MALS signal and / or the DLS signal, is carried out online and / or step (c) is carried out online.

70. 70. The use of any one of claims 40 to 69, wherein 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, the solvent or solvent mixture being capable of preventing the formation of particle aggregates.

71. 71. The use according to claim 70, wherein the solvent mixture is a mixture of water and an organic solvent, such as formamide.

72. 72. The use according to any one of claims 40 to 71, wherein the step of measuring the UV signal is carried out by using CD spectroscopy.

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