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

Abstract

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

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202080067535.4, filed on July 17, 2020, entitled “Method for determining at least one parameter of a sample composition comprising nucleic acids such as RNA and optionally present particles”. Technical Field

[0002] This disclosure generally relates to the field of analyzing nucleic acids such as RNA, and in particular to determining at least one parameter of a sample composition comprising nucleic acids (especially RNA) and optionally present particles. Background Technology

[0003] The use of recombinant nucleic acids (such as DNA or RNA) to deliver exogenous genetic information into target cells is well-known. Advantages of using RNA include transient expression and non-transformation characteristics. RNA does not need to enter the cell nucleus for expression and cannot integrate into the host genome, thus eliminating various risks such as tumorigenesis.

[0004] Recombinant nucleic acids can be administered to subjects in need in their naked form; however, recombinant nucleic acids are typically administered using pharmaceutical compositions. For example, RNA can be delivered via so-called nanoparticle formulations containing RNA and a medium for forming the nanoparticles, such as cationic lipids, mixtures of cationic lipids and cofactor lipids, or cationic polymers.

[0005] The fate of these nanoparticle formulations is controlled by a variety of key factors (e.g., the integrity and concentration of nucleic acids within the nanoparticles; the amount of free nucleic acids; the size, size distribution, quantitative size distribution, and morphology of the nanoparticles; etc.). For example, these factors were identified as specific attributes that should be analyzed and described in the 2018 FDA "Liposome Drug Products Guidance." A current limitation in the clinical application of nanoparticle formulations may lie in the lack of homogeneous, pure, and well-characterized nanoparticle formulations. This is also due to the shortcomings of all existing techniques used to determine these factors.

[0006] For example, current techniques used to determine the integrity and / or concentration of nucleic acids in nanoparticles (such as dye-based, gel electrophoresis, microchannel electrophoresis, or capillary electrophoresis (CE)) are labor-intensive, expensive, prone to artifacts due to sample preparation steps, and unable to provide sufficient information and / or analyze large volumes of samples. In one technique that currently uses dyes (such as fluorescent dyes), the dye itself can introduce variability, which can affect the reliability of the measurement results. Furthermore, most gel electrophoresis-based techniques require multiple washing steps, the use of special run buffers that increase program length, and special precautions due to the use of toxic reagents. For example, agarose gel electrophoresis is unreliable due to several parameters (e.g., the quality of the agarose, gel casting, dye / sensitivity (requiring larger sample volumes), exposure time, processing of raw data, and standardization of densitometric software (28S / 18S methods)). Compared to agarose gel electrophoresis, microchannel-based, chip-based, or capillary electrophoresis techniques offer faster run times and improved data quality, but require manual handling to start and load the gel, labels, and samples onto the system. CE instruments lack the sensitivity, dynamic range, and separation quality required for adequate RNA quality / quantity analysis.

[0007] Furthermore, a key challenge in characterizing nanoparticle formulations lies in the quantitative determination of the size distribution of particles contained within the formulation. This is particularly true for particles with diameters smaller than approximately 500 nm (i.e., the range associated with most pharmaceutical products). Another unmet need is to determine the size distribution of nanoparticle formulations, where the 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 about size, or they only measure small, optionally unrepresentative subsets of samples, or their sensitivity (or other parameters, such as the refractive index gradient relative to the bulk phase) varies considerably for particles of different sizes, which strongly affects the obtained size distribution.

[0008] Several techniques exist for measuring particle size in the lower submicron range (approximately 100 nm), such as dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), electron microscopy (EM), and size exclusion chromatography (SEC)-UV.

[0009] DLS provides information about the diffusion constant of nanoparticles, from which the hydrodynamic radius R can be calculated using the Stokes-Einstein equation. hHowever, DLS only provides average data, thus requiring the use of certain algorithms to numerically calculate particle size. For quantitatively reliable figures, nanoparticle formulations should be unimodal and monodisperse, which is not the case for many products, including nanoparticle pharmaceutical formulations. The most widely used algorithm in DLS is the so-called cumulative analysis (DE Koppel, J. Chem. Phys. 57 (1972) 4814-4820), which, by default, only assumes a unimodal size distribution and provides physically meaningful figures only when the polydispersity is below a certain threshold. Other DLS algorithms (see, for example, Provencher, SW, Comput. Phys. Commun. 1982, 27, 229–242) provide size curves that depend largely on the fitting parameters, and several very different spectra can correspond to the same dataset. This analysis is hampered by the fact that the light scattering intensity of large particles is much higher than that of smaller particles, making it difficult to determine the fraction of smaller particles in the presence of much larger particles.

[0010] Non-precipitated scattering (NTA) is a method for determining particle size from their diffusion constants by observing the scattered light from a very small (diluted) subset of a sample over time using a microscope. In principle, NTA can provide a quantitative size distribution spectrum; however, it can only measure very diluted samples, and the particles must be present in a relatively small size range—that is, very small particles cannot be identified against a much larger background due to the much higher scattering intensity. Therefore, NTA is not suitable as a routine method for determining the quantitative size distribution of pharmaceutical formulations. Furthermore, NTA has a higher statistical standard deviation compared to other techniques (e.g., DLS). This is a direct result of the one to three orders of magnitude lower particle counts analyzed by NTA. In particular, trace particles with biological effects (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 determine a suitable setup for accurate measurements. Typically, samples used for NTA measurements must be diluted 10–1000 times, which can cause problems, particularly concentration-dependent aggregation or disintegration of particles. Due to all these drawbacks, it is difficult to establish NTA as a quality control method.

[0011] EM provides quantitative information about the size, shape, and morphology of individual particles, but the number of particles that can be analyzed is even lower than NTA. Therefore, EM has similar or identical drawbacks to NTA in that the measured particles may not be representative of the entire sample. Other major disadvantages of this technique are high cost, complex sample preparation, and long turnaround time for analyzed samples. This is why EM is not commonly used as a GMP method. Another problem with EM is that sample fixation can lead to artifacts (e.g., shrinkage, aggregation, etc.). If the sample is not fixed (e.g., in Cryo-EM), the sample may have low contrast and be unanalyzable.

[0012] Other separation techniques such as SEC-UV are unsuitable because interactions with the column matrix can lead to problems (e.g., adsorption or elution delays). Due to the limited size range of SEC columns, nanoparticles may not be sufficiently dispersed, or they 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 related to particle size. Other dispersion methods, such as analytical ultracentrifugation (AUC), only allow indirect size measurements, such as based on sedimentation coefficients, where several assumptions must be made to calculate the size distribution. Moreover, AUC is both expensive and time-consuming, and it is not a commonly used method in routine quality control. Therefore, AUC is also unsuitable as a routine quality control method for determining quantitative size profiles.

[0013] In view of the above, to ensure the reproducible quality of nanoparticle formulations, advanced analytical methods for in-depth particle characterization are needed. In particular, an improved method for analyzing nanoparticle formulations containing nucleic acids (especially RNA) is needed, wherein the method preferably (i) provides information on formulation characteristics (e.g., the quantitative size distribution of particles contained in the formulation (especially for particles with a diameter less than 500 nm); (ii) provides information on particle composition characteristics (e.g., the amount of nucleic acids (especially RNA) contained in the particles, especially as a function of particle size, such as the ratio of the amount of nucleic acids (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), especially as a function of particle size); (iii) is GMP-compatible; (iv) is independent of the use of dyes; (v) is semi-automatic; and / or (vi) can be used to analyze changes in one or more reactions during the preparation and / or storage of the composition. The composition comprises nucleic acids (such as RNA) and optionally present particles, subject to the influence of conditions (e.g., salt concentration; temperature; pH or buffer concentration; light / radiation; oxygen; shear force; pressure; freeze / thaw cycles; dry / restore cycles; addition of excipients (e.g., stabilizers and / or chelating agents); type and / or source of particle-forming compounds (especially lipids and / or polymers); charge ratio; and / or ratio of nucleic acids (such as RNA) to particle-forming compounds (especially lipids and / or polymers). Preferably, the method provides data on one or more of the following parameters: nucleic acid (such as RNA) integrity; total amount of nucleic acids (such as RNA); amount of free nucleic acids (such as RNA); amount of nucleic acids (such as RNA) bound to particles; size of particles containing nucleic acids (such as RNA) (e.g., based on the radius of gyration (R) of particles containing nucleic acids (such as RNA)). g The hydrodynamic radius (R) of particles containing nucleic acids (such as RNA) and / or particles containing nucleic acids (such as RNA). h Size distribution of particles containing nucleic acids (such as RNA) (e.g., based on R...) g or R h Value); quantitative size distribution of particles containing nucleic acids (such as RNA) (e.g., based on R). g or R h Value); molecular weight of nucleic acid (such as RNA); and / or shape of the particle containing nucleic acid (such as RNA) (e.g., shape and / or form factor). Optionally, other parameters may include one or more of the following: amount of surface nucleic acid (such as amount of surface RNA), amount of encapsulated nucleic acid (such as amount of encapsulated RNA), amount of accessible nucleic acid (such as amount of accessible RNA), size of nucleic acid (especially RNA) (e.g., based on R). g or R h Value), the size distribution of nucleic acids (especially RNA) (e.g., based on R).g or R h Value), quantitative size distribution of nucleic acids (especially RNA) (e.g., based on R). g or R h Value), nucleic acid (especially RNA) encapsulation efficiency, the ratio of the amount of nucleic acid (e.g., RNA) bound to the particle to the total amount of particle-forming compounds (especially lipids and / or polymers) in the particle, the ratio of the amount of positively charged portions of particle-forming compounds (especially 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 portions of particle-forming compounds (especially lipids and / or polymers) in the particle to the amount of negatively charged portions of nucleic acid (e.g., RNA) bound to the particle.

[0014] The inventors were surprised to find that the methods and uses described herein meet the above requirements. Summary of the Invention

[0015] In a first aspect, this disclosure provides a method for determining one or more parameters of a sample composition, wherein the sample composition comprises nucleic acids (such as RNA) and optionally present particles, the method comprising:

[0016] (a) Perform field-flow fractionation on at least a portion of the sample composition to fractionate the components contained in the sample composition by size to produce one or more sample fractions;

[0017] (b) Measure at least one signal selected from UV, fluorescence, and refractive index (RI) signals from at least one of the one or more sample fractions obtained in step (a), and optionally measure a light scattering (LS) signal; and

[0018] (c) Calculate one or more parameters from at least one signal selected from UV signals, fluorescence signals, and RI signals, and optionally from the LS signal, 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 the particle, and size of the particle containing nucleic acid (e.g., RNA) (particularly based on the radius of gyration (R) of the particle containing nucleic acid (e.g., RNA). g The hydrodynamic radius (R) of particles containing nucleic acids (especially RNA) and / or particles containing nucleic acids (especially RNA). h ), and the size distribution of particles containing nucleic acids (such as RNA) (e.g., based on R of particles containing nucleic acids (especially RNA) g or R h The quantitative size distribution of particles containing nucleic acids (such as RNA) and the R value of particles containing nucleic acids (especially RNA). g or Rh Value). Generally, the size distribution and / or quantitative size distribution of particles containing nucleic acids (such as RNA) can be given as the number of particles containing nucleic acids (such as RNA), the molar amount of particles containing nucleic acids (such as RNA), or the mass of particles containing nucleic acids (such as RNA), each as a function of their size. Additional optional parameters include the molecular weight of the nucleic acid (especially RNA), the amount of surface nucleic acid (such as the amount of surface RNA), the amount of encapsulated nucleic acid (such as the amount of encapsulated RNA), the amount of accessible nucleic acid (such as the amount of accessible RNA), and the size of the nucleic acid (especially RNA) (particularly, based on the R value of the nucleic acid (especially RNA)). g and / or R h Value), size distribution of nucleic acids (especially RNA) (e.g., R based on nucleic acids (especially RNA) g or R h Value), quantitative size distribution of nucleic acids (especially RNA) (e.g., R based on nucleic acids (especially RNA) g or R h Value), shape factor, form factor, and nucleic acid (especially RNA) encapsulation efficiency. Generally, the size distribution and / or quantitative size distribution of nucleic acids (especially RNA) can be given as the number of nucleic acid (especially RNA) molecules, the molar amount of nucleic acids (especially RNA), or the mass of nucleic acids (especially RNA), each as a function of their size. Other additional optional parameters include the ratio of the amount of nucleic acid (e.g., RNA) bound to the particle to the total amount of particle-forming compounds (especially lipids and / or polymers) in the particle, wherein said ratio can be given as a function of particle size; the ratio of the amount of positively charged portions of particle-forming compounds (especially lipids and / or polymers) in the particle to the amount of nucleic acid (e.g., RNA) bound to the particle, wherein said ratio can be given as a function of particle size; and the charge ratio of the amount of positively charged portions of particle-forming compounds (especially lipids and / or polymers) in the particle to the amount of negatively charged portions of nucleic acid (e.g., RNA) bound to the particle, wherein said charge ratio is typically expressed as an N / P ratio and can be given as a function of particle size.

[0019] In the first subgroup of the first aspect, the method includes:

[0020] (a) Perform field-flow fractionation on at least a portion of the sample composition to fractionate the components contained in the sample composition by size to produce one or more sample fractions;

[0021] (b) Measure at least the UV signal and optionally the light scattering (LS) signal of at least one of the one or more sample fractions obtained in step (a); and

[0022] (c) Calculate one or more parameters from the UV signal and optionally from the LS signal.

[0023] In a second and preferred subgroup of the first aspect, the method is used to determine one or more parameters of a sample composition, wherein the sample composition comprises RNA and optionally present particles, the method comprising:

[0024] (a) Perform field-flow fractionation on at least a portion of the sample composition to fractionate the components contained in the sample composition by size to produce one or more sample fractions;

[0025] (b) Measure at least one signal selected from UV, fluorescence, and refractive index (RI) signals from at least one of the one or more sample fractions obtained in step (a), and optionally measure a light scattering (LS) signal; and

[0026] (c) Calculate the one or more parameters from at least one signal selected from UV signals, fluorescence signals and RI signals, and optionally from the LS signal.

[0027] In a third and more preferred subgroup of the first aspect, the method is used to determine one or more parameters of a sample composition, wherein the sample composition comprises RNA and optionally present particles, the method comprising:

[0028] (a) Perform field-flow fractionation on at least a portion of the sample composition to fractionate the components contained in the sample composition by size to produce one or more sample fractions;

[0029] (b) Measure at least the UV signal and optionally the light scattering (LS) signal of at least one of the one or more sample fractions obtained in step (a); and

[0030] (c) Calculate one or more parameters from the UV signal and optionally from the LS signal.

[0031] In one embodiment of the first aspect (particularly, in one embodiment of the first, second, or third subgroup of the first aspect), R based on particles containing nucleic acids (such as RNA) g The size, size distribution, and / or quantitative size distribution of the particles containing nucleic acids (such as RNA) are calculated. In another embodiment of the first aspect (particularly, in another embodiment of the first, second, or third subgroup of the first aspect), the R value is based on the particles containing nucleic acids (such as RNA). hThe size, size distribution, and / or quantitative size distribution of the particles containing nucleic acids (such as RNA) are calculated. In another embodiment of the first aspect (particularly, in another embodiment of the first, second, or third subgroup of the first aspect), the R value is based on the particles containing nucleic acids (such as RNA). g The values ​​are based on R values ​​of particles containing nucleic acids (such as RNA). h The value is calculated to determine the size, size distribution, and / or quantitative size distribution of the particles containing nucleic acids (such as RNA). That is, this implementation generates two datasets: one based on R... g Value, a value based on R h value).

[0032] In one embodiment of the first aspect (particularly, in one embodiment of the first, second, or third subgroup of the first aspect), said one or more parameters include the size, size distribution, and / or quantitative size distribution of the nucleic acid (such as RNA), based on the R of the nucleic acid (such as RNA). g The value is calculated to determine the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA). In another embodiment of the first aspect (particularly, in another embodiment of the first, second, or third subgroup of the first aspect), wherein said one or more parameters include the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA), based on the R... h The value is calculated to determine the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA). In another embodiment of the first aspect (particularly, in another embodiment of the first, second, or third subgroup of the first aspect), wherein said one or more parameters include the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA), based on the R... g Values ​​and respectively based on the R of nucleic acids (such as RNA) h The value is calculated to determine the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA). That is, this implementation generates two datasets, one based on R... g Value, a value based on R h value).

[0033] 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 classification is preferably a flow field-flow classification, such as asymmetric flow field-flow classification (AF4) or hollow fiber flow field-flow classification (HF5).

[0034] In one embodiment of the first aspect (particularly in one embodiment of the first, second or third subgroup of the first aspect), step (a) is performed using a membrane having a molecular weight cutoff (MW) suitable for preventing nucleic acid (especially RNA) from permeating the membrane, preferably a membrane with a MW cutoff in the range of 2kDa-30kDa, such as a membrane with a MW cutoff of 10kDa.

[0035] In one embodiment of the first aspect (particularly in one embodiment of the first, second or third subgroup of the first aspect), step (a) is performed using polyethersulfone (PES) or a regenerated cellulose membrane.

[0036] In one embodiment of the first aspect (particularly, in one embodiment of the first, second, or third subgroup of the first aspect), step (a) is performed using: (I) a cross-flow rate up to 8 mL / min, preferably up to 4 mL / min, more preferably up to 2 mL / min, for example, a cross-flow rate profile; and / or (II) an injection flow rate in the range of 0.05-0.35 mL / min, preferably in the range of 0.10-0.30 mL / min, more preferably in the range of 0.15-0.25 mL / min; and / or (III) a detector flow rate in the range of 0.30-0.70 mL / min, preferably in the range of 0.40-0.60 mL / min, more preferably in the range of 0.45-0.55 mL / min.

[0037] 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 spectrum preferably includes a fractionation phase, which allows the components contained in the control or sample composition to be fractionated / separated according to their size to produce one or more sample fractions. Preferably, the cross-flow rate changes during this fractionation phase (e.g., starting from a value (e.g., from about 1 to about 4 mL / min) and then decreasing to a lower value (e.g., from about 0 to about 0.1 mL / min), or starting from a value (e.g., from about 0 to about 0.1 mL / min) and then increasing to a higher value (e.g., from about 1 to about 4 mL / min)), wherein the change can be in any manner, e.g., continuous (e.g., linear or exponential) or gradual. Preferably, the cross-flow rate spectrum includes a fractionation phase, wherein the cross-flow rate changes continuously (preferably exponentially), starting from a value (e.g., from about 1 to about 4 mL / min) and then decreasing to a lower value (e.g., from about 0 to about 0.1 mL / min). The fractionation stage can have any length suitable for fractionating / separating components contained in the sample composition according to their size, such as about 5 min to about 60 min, about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 30 min. The cross-flow rate spectrum can contain additional stages (e.g., 1, 2, 3, or 4 stages) which can be before and / or after the fractionation stage (e.g., 1 before the fractionation stage and 1, 2, or 3 after the fractionation stage) and can be used to separate non-nucleic acid (especially non-RNA) components (e.g., proteins, peptides, mononucleotides, etc.) contained in the sample composition from nucleic acids (especially RNA) contained in the sample composition to concentrate nucleic acids (especially RNA) contained in the sample composition and / or regenerate the field-flow fractionation device (e.g., remove all components bound to the membrane of the device). Preferably, the crossflow velocity of these additional stages is constant for each additional stage, and the length of each of the additional stages is independently in the range of about 5 min to about 60 min (e.g., about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 30 min).For example, the cross velocity spectrum may include (i) a first additional stage prior to the grading stage, wherein the cross velocity of the first additional stage is constant and the same as the cross low rate at the beginning of the grading stage (the length of the first additional stage may be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 10 min, about 20 min, or about 30 min); and (ii) a second additional stage after the grading stage, wherein the cross velocity of the second additional stage is constant and the same as the cross low rate at the end of the grading stage (the length of the second additional stage may be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min). (approximately 5 min, approximately 20 min to approximately 40 min, or approximately 25 min to approximately 35 min, or approximately 10 min, approximately 20 min, or approximately 30 min); and optionally (iii) a third additional stage following the second additional stage, wherein the crossflow rate of the third additional stage is constant and different from that of the second additional stage (the length of the third additional stage may be in the range of approximately 5 min to approximately 60 min, such as approximately 10 min to approximately 50 min, approximately 15 min to approximately 45 min, approximately 20 min to approximately 40 min, or approximately 25 min to approximately 35 min, or approximately 10 min, approximately 20 min, or approximately 30 min).In embodiments where the cross-flow rate spectrum includes a grading phase, the cross-flow rate varies continuously (preferably exponentially), starting from a value (e.g., about 1 to about 4 mL / min) and then decreasing to a lower value (e.g., about 0 to about 0.1 mL / min). Preferably, the cross-flow rate spectrum further includes (i) a first additional phase preceding the grading phase, wherein the cross-flow rate of the first additional phase is constant and the same as the low cross-flow rate at the start of the grading phase (e.g., about 1 to about 4 mL / min) (the length of the first additional phase may be in the range of about 5 min to about 30 min, such as about 6 min to about 25 min, about 7 min to about 20 min, or about 8 min to about 15 min, or about 10 min to about 12 min, or about 5 min, about 10 min, or about 12 min); (ii) a second additional phase following the grading phase, wherein the cross-flow rate of the second additional phase is constant and the same as the low cross-flow rate at the start of the grading phase. The cross-flow rate at the end of the first stage is the same (e.g., about 0.01 to 0.1 mL / min) (the length of the second additional stage may be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 30 min); and optionally (iii) a third additional stage after the second additional stage, wherein the cross-flow rate of the third additional stage is constant and lower than that of the second additional stage (e.g., the cross-flow rate of the third additional stage is 0) (the length of the third additional stage may be in the range of about 5 min to about 30 min, such as about 6 min to about 25 min, about 7 min to about 20 min, or about 8 min to about 15 min, or about 10 min to about 12 min, or about 5 min, about 10 min, or about 12 min). Preferred examples of such cross-flow rate spectra are as follows: 1.0-2.0 mL / min for 10 min, an exponential gradient from 1.0-2.0 mL / min to 0.01-0.07 mL / min over 30 min; 0.01-0.07 mL / min for 30 min; and 0 mL / min for 10 min.

[0038] 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 acids (especially RNA) contained in the sample composition is calculated using the integrity of a control nucleic acid (especially RNA).

[0039] In a first specific instance of this implementation scheme of the first aspect, the integrity of the control nucleic acid (especially RNA) is determined by the following steps:

[0040] (a') Field-flow fractionation, particularly AF4 or HF5, is performed on at least a portion of a control composition containing control nucleic acid (especially RNA) to fractionate the components contained in the control composition by size to produce one or more control fractions.

[0041] (b') Measure at least one signal selected from UV signal, fluorescence signal and refractive index (RI) signal from at least one of one or more control fractions obtained in step (a');

[0042] (c'1) Calculate the area from the maximum height of a UV, fluorescence, or RI peak to the end of the UV, fluorescence, or RI peak obtained in step (b'), thereby obtaining A. 50％ (Comparison);

[0043] (c'2) Calculate the total area of ​​a peak used in step (c'1) from at least one signal selected from UV, fluorescence, and RI signals obtained in step (b') to obtain A. 100％ (Comparison); and

[0044] (c'3) Determine A 50％ (Comparison) and A 100％ The ratio between (control) and (control) is used to obtain the integrity of the control nucleic acid (especially RNA) (I (control)).

[0045] In this first example, the integrity of nucleic acids (especially RNA) contained in the sample composition can be calculated through the following steps:

[0046] (c1) Calculate the area from the maximum height of the sample UV, fluorescence, or RI peak corresponding to the control UV, fluorescence, or RI peak used in step (c'1) to the end of the sample UV, fluorescence, or RI peak from at least one signal selected from UV, fluorescence, and RI obtained in step (b), thereby obtaining A. 50％ (sample);

[0047] (c2) Calculate the total area of ​​the sample UV, fluorescence, or RI peaks used in step (c1) from the sample UV, fluorescence, or RI signals obtained in step (b), thereby obtaining A. 100％ (sample);

[0048] (c3) Determine A 50％ (sample) and A 100％ The ratio between (samples) is used to obtain I (sample); and

[0049] (c4) Determine the ratio between I (sample) and I (control) to obtain the integrity of the nucleic acids (especially RNA) contained in the sample composition.

[0050] In a second specific instance of this implementation scheme of the first aspect, the integrity of the control nucleic acid (especially RNA) is determined by the following steps:

[0051] (a”) Field-flow fractionation, particularly AF4 or HF5, is performed on at least a portion of a control composition containing control nucleic acid (especially RNA) to fractionate the components contained in the control composition by size to produce one or more control fractions.

[0052] (b”) Measure at least one signal selected from UV, fluorescence, and RI signals from at least one of the one or more control fractions obtained in step (a”); and

[0053] (c”) The height of a UV, fluorescence, or RI peak (H(control)) is determined from at least one signal selected from UV, fluorescence, and RI signals obtained in step (b”) to obtain the integrity of the control nucleic acid (especially RNA).

[0054] In this second example, the integrity of nucleic acids (especially RNA) contained in the sample composition can be calculated through the following steps:

[0055] (c1') Determine 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 UV, fluorescence, and RI signals obtained in step (b); and

[0056] (c2') Determine the ratio between H (sample) and H (control) to obtain the integrity of the nucleic acids (especially RNA) contained in the sample composition.

[0057] In a third specific instance of this implementation scheme of the first aspect (with regard to the first subgroup of the first aspect), the integrity of the control nucleic acid (especially RNA) is determined by the following steps:

[0058] (a') Field-flow fractionation, particularly AF4 or HF5, is performed on at least a portion of a control composition containing control nucleic acid (especially RNA) to fractionate the components contained in the control composition by size to produce one or more control fractions.

[0059] (b') Measure the UV signal of at least one of the one or more control fractions obtained from step (a');

[0060] (c'1) Calculate the area from the maximum height of a UV peak to the end of the UV peak using the UV signal obtained in step (b'), thereby obtaining A. 50％ (Comparison);

[0061] (c'2) Calculate the total area of ​​one peak used in step (c'1) from the UV signal obtained in step (b') to obtain A. 100％ (Comparison); and

[0062] (c'3) Determine A 50％ (Comparison) and A 100％ The ratio between (control) and (control) is used to obtain the integrity of the control nucleic acid (especially RNA) (I (control)).

[0063] In this third example, the integrity of nucleic acids (especially RNA) contained in the sample composition can be calculated through the following steps:

[0064] (c1) 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 from the UV signal obtained in step (b), thereby obtaining A. 50％ (sample);

[0065] (c2) Calculate the total area of ​​the sample UV peaks used in step (c1) from the sample UV signal obtained in step (b), thereby obtaining A. 100％ (sample);

[0066] (c3) Determine A 50％ (sample) and A 100％ The ratio between (samples) is used to obtain I (sample); and

[0067] (c4) Determine the ratio between I (sample) and I (control) to obtain the integrity of the nucleic acids (especially RNA) contained in the sample composition.

[0068] In a fourth specific instance of this implementation scheme of the first aspect (with regard to the first subgroup of the first aspect), the integrity of the control nucleic acid (especially RNA) is determined by the following steps:

[0069] (a”) Field-flow fractionation, particularly AF4 or HF5, is performed on at least a portion of a control composition containing control nucleic acid (especially RNA) to fractionate the components contained in the control composition by size to produce one or more control fractions.

[0070] (b”) Measure the UV signal of at least one of the one or more control fractions obtained from step (a”); and

[0071] (c”) Determine the height of a UV peak (H(control)) from the UV signal obtained in step (b”) to obtain the integrity of the control nucleic acid (especially RNA).

[0072] In this fourth example, the integrity of nucleic acids (especially RNA) contained in the sample composition can be calculated through the following steps:

[0073] (c1') Determine 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); and

[0074] (c2') Determine the ratio between H (sample) and H (control) to obtain the integrity of the nucleic acids (especially RNA) contained in the sample composition.

[0075] In the fifth specific instance of this implementation scheme of the first aspect (regarding the second subgroup of the first aspect), the integrity of the control RNA is determined by the following steps:

[0076] (a') Field-flow fractionation, particularly AF4 or HF5, is performed on at least a portion of a control composition containing control RNA to fractionate the components contained in the control composition by size to produce one or more control fractions.

[0077] (b') Measure at least one signal selected from UV signal, fluorescence signal and RI signal from at least one of one or more control fractions obtained in step (a');

[0078] (c'1) Calculate the area from the maximum height of a UV, fluorescence, or RI peak to the end of the UV, fluorescence, or RI peak obtained in step (b'), thereby obtaining A. 50％ (Comparison);

[0079] (c'2) Calculate the total area of ​​a peak used in step (c'1) from at least one signal selected from UV, fluorescence, and RI signals obtained in step (b') to obtain A. 100％ (Comparison); and

[0080] (c'3) Determine A 50％ (Comparison) and A 100％ The ratio between (control) and (control) is used to obtain the integrity of the control RNA (I (control)).

[0081] In this fifth example, the integrity of the RNA contained in the sample composition can be calculated through the following steps:

[0082] (c1) Calculate the area from the maximum height of the sample UV, fluorescence, or RI peak corresponding to the control UV, fluorescence, or RI peak used in step (c'1) to the end of the sample UV, fluorescence, or RI peak from at least one signal selected from UV, fluorescence, and RI obtained in step (b), thereby obtaining A. 50％ (sample);

[0083] (c2) Calculate the total area of ​​the sample UV, fluorescence, or RI peaks used in step (c1) from the sample UV, fluorescence, or RI signals obtained in step (b), thereby obtaining A. 100％ (sample);

[0084] (c3) Determine A 50％ (sample) and A 100％ The ratio between (samples) is used to obtain I (sample); and

[0085] (c4) Determine the ratio between I (sample) and I (control) to obtain the integrity of the RNA contained in the sample composition.

[0086] In the sixth specific instance of this implementation scheme of the first aspect (regarding the second subgroup of the first aspect), the integrity of the control RNA is determined by the following steps:

[0087] (a”) Perform field-flow fractionation, particularly AF4 or HF5, on at least a portion of a control composition containing control RNA, thereby fractionating the components contained in the control composition by size to produce one or more control fractions.

[0088] (b”) Measure at least one signal selected from UV, fluorescence, and RI signals from at least one of the one or more control fractions obtained in step (a”); and

[0089] (c”) The height of a UV, fluorescence, or RI peak (H(control)) is determined from at least one signal selected from UV, fluorescence, and RI signals obtained in step (b”) to obtain the integrity of the control RNA.

[0090] In this sixth example, the integrity of the RNA contained in the sample composition can be calculated through the following steps:

[0091] (c1') Determine 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 UV, fluorescence, and RI signals obtained in step (b); and

[0092] (c2') Determine the ratio between H (sample) and H (control) to obtain the integrity of the RNA contained in the sample composition.

[0093] In the seventh specific instance of this implementation scheme of the first aspect (with regard to the third subgroup of the first aspect), the integrity of the control RNA is determined by the following steps:

[0094] (a') Field-flow fractionation, particularly AF4 or HF5, is performed on at least a portion of a control composition containing control RNA to fractionate the components contained in the control composition by size to produce one or more control fractions.

[0095] (b') Measure the UV signal of at least one of the one or more control fractions obtained from step (a');

[0096] (c'1) Calculate the area from the maximum height of a UV peak to the end of the UV peak using the UV signal obtained in step (b'), thereby obtaining A. 50％ (Comparison);

[0097] (c'2) Calculate the total area of ​​one peak used in step (c'1) from the UV signal obtained in step (b') to obtain A. 100％ (Comparison); and

[0098] (c'3) Determine A 50％ (Comparison) and A 100％ The ratio between (control) and (control) is used to obtain the integrity of the control RNA (I (control)).

[0099] In this seventh example, the integrity of the RNA contained in the sample composition can be calculated through the following steps:

[0100] (c1) 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 from the UV signal obtained in step (b), thereby obtaining A. 50％ (sample);

[0101] (c2) Calculate the total area of ​​the sample UV peaks used in step (c1) from the sample UV signal obtained in step (b), thereby obtaining A. 100％ (sample);

[0102] (c3) Determine A 50％ (sample) and A 100％ The ratio between (samples) is used to obtain I (sample); and

[0103] (c4) Determine the ratio between I (sample) and I (control) to obtain the integrity of the RNA contained in the sample composition.

[0104] In the eighth specific instance of this implementation scheme of the first aspect (with regard to the third subgroup of the first aspect), the integrity of the control RNA is determined by the following steps:

[0105] (a”) Perform field-flow fractionation, particularly AF4 or HF5, on at least a portion of a control composition containing control RNA, thereby fractionating the components contained in the control composition by size to produce one or more control fractions.

[0106] (b”) Measure the UV signal of at least one of the one or more control fractions obtained from step (a”); and

[0107] (c”) Determine the height of a UV peak (H(control)) from the UV signal obtained in step (b”) to obtain the integrity of the control RNA.

[0108] In this eighth example, the integrity of the RNA contained in the sample composition can be calculated through the following steps:

[0109] (c1') Determine 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); and

[0110] (c2') Determine the ratio between H (sample) and H (control) to obtain the integrity of the RNA contained in the sample composition.

[0111] 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) the nucleic acid extinction coefficient (especially the RNA extinction coefficient) or (ii) the nucleic acid calibration curve (especially the RNA calibration curve).

[0112] 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 acids (especially RNA) and particles, such as lipid complex particles and / or lipid nanoparticles and / or polyplex particles and / or lipid polycomplex particles and / or virus-like particles, wherein the nucleic acids (especially RNA) are bound to the particles.

[0113] 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 (especially the amount of total RNA) is determined by: (i) treating at least a portion of the sample composition with a releasing agent; (ii) performing steps (a)-(c) with at least the portion obtained from step (i); and (iii) determining the amount of nucleic acid (especially RNA) as described herein (e.g., by using (i) a nucleic acid extinction coefficient (especially an RNA extinction coefficient) or (ii) a nucleic acid calibration curve (especially an RNA calibration curve)). 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.

[0114] In one embodiment of the first aspect (particularly, in one embodiment of the first, second, or third subgroup of the first aspect), the releasing agent is (i) a surfactant, such as anionic surfactants (e.g., sodium dodecyl sulfate), zwitterionic surfactants (e.g., tetradecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate), etc. (3-14)) cationic surfactants, nonionic surfactants or mixtures thereof; (ii) alcohols, such as fatty alcohols (e.g., ethanol), or mixtures of alcohols; or (iii) a combination of (i) and (ii).

[0115] 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: performing steps (a)-(c) without the addition of a releasing agent, particularly in the absence of any releasing agent; and determining the amount of nucleic acid (especially RNA) as described herein (e.g., using (i) the nucleic acid extinction coefficient (especially the RNA extinction coefficient) or (ii) the nucleic acid calibration curve (especially the RNA calibration curve)).

[0116] 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 particle is determined by: subtracting the amount of free nucleic acid (especially RNA) as determined herein from the amount of total nucleic acid (especially RNA) as determined herein (e.g., by treating at least a portion of the sample composition with a releasing agent; (ii) performing steps (a)-(c) with at least the portion obtained from step (i); and (iii) determining the amount of nucleic acid (especially RNA) as described herein (e.g., using (i) a nucleic acid extinction coefficient (especially RNA extinction coefficient) or (ii) a nucleic acid calibration curve (especially RNA calibration curve))).

[0117] 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 an LS signal, such as a dynamic light scattering (DLS) signal and / or a static light scattering (SLS) signal, such as a multi-angle light scattering (MALS) signal, of at least one of the sample fractions obtained from step (a).

[0118] In one embodiment of the first aspect (particularly, in one embodiment of the first, second, or third subgroup of the first aspect), the radius of gyration (R) is calculated from the LS signal obtained from step (b). g ) value and / or hydrodynamic radius (R h The value is used to determine the size of particles containing nucleic acids (especially RNA). In one embodiment of the first aspect (particularly, in one embodiment of the first, second, or third subgroup of the first aspect), step (b) includes measuring the dynamic light scattering (DLS) signal of at least one of the one or more sample fractions obtained from step (a), and step (c) includes calculating R from the DLS signal. h Value. In one embodiment of the first aspect (particularly, in one embodiment of the first, second, or third subgroup of the first aspect), step (b) includes measuring a static light scattering (SLS) signal, such as a MALS signal, of at least one of the one or more sample fractions obtained in step (a), and step (c) includes calculating R from the SLS signal. gValue. In one embodiment of the first aspect (particularly, in one embodiment of the first, second, or third subgroup of the first aspect), step (b) includes measuring a dynamic light scattering (DLS) signal and a static light scattering (SLS) signal, such as a MALS signal, of at least one of the one or more sample fractions obtained from step (a), and step (c) includes calculating R. g and R h Value. This latter implementation produces two datasets of particle size containing nucleic acids (such as RNA), namely, one based on R. g Value, a value based on R h value.

[0119] In one embodiment of the first aspect (particularly, in one embodiment of the first, second, or third subgroup of the first aspect), by applying at least one signal selected from UV signals, fluorescence signals, and RI signals obtained from step (b) to the R signal determined as described herein. g or R h Value (e.g., calculated from the SLS signal obtained in step (b)). g The value or R is calculated from the DLS signal obtained in step (b). h The size distribution of particles containing nucleic acids (especially RNA) is determined by plotting the UV signal obtained from step (b) against the R values ​​as described herein. In a first example of this implementation (a first subgroup of the first aspect), the size distribution of particles containing nucleic acids (especially RNA) is determined by plotting the UV signal obtained from step (b) against the R values ​​as described herein. g or R h Value (e.g., calculated from the SLS signal obtained in step (b)). g The value or R is calculated from the DLS signal obtained in step (b). h The size distribution of particles containing nucleic acids (especially RNA) is determined by plotting the values. In a second example of this embodiment (a second subgroup of the first aspect), the size distribution of particles containing nucleic acids (especially RNA) is determined by plotting at least one signal selected from UV signals, fluorescence signals, and RI signals obtained from step (b) against the R values ​​determined herein. g or R h Value (e.g., calculated from the SLS signal obtained in step (b)). g The value or R is calculated from the DLS signal obtained in step (b). h The size distribution of RNA-containing particles was determined by plotting the UV signal obtained from step (b) against the R values ​​as described herein. In a third example of this implementation (a third subgroup concerning the first aspect), the size distribution of RNA-containing particles was determined by plotting the UV signal obtained from step (b) against the R values ​​as described herein. g or R h Value (e.g., calculated from the SLS signal obtained in step (b)). g The value or R is calculated from the DLS signal obtained in step (b). hThe size distribution of RNA-containing particles can be determined by plotting the values. In each of the first, second, and third examples above, the size distribution of RNA-containing particles can be determined by plotting the values. g value, R h The size distribution of particles containing nucleic acids (especially RNA) is determined based on the value or both. If in R g Value and R h Determining the size distribution of particles containing nucleic acids (especially RNA) based on the values ​​will generate two datasets: one based on R... g The magnitude distribution of values ​​and an R-based h Distribution of values.

[0120] In one embodiment of the first aspect (particularly, in one embodiment of the first, second, or third subgroup of the first aspect), by converting UV, fluorescence, or RI signals into cumulative weight fractions and comparing the cumulative weight fractions with R... g or R h Plot the values ​​as R. g or R h The quantitative size distribution of particles containing nucleic acids (especially RNA) is calculated by plotting UV, fluorescence, or RI signals as a function of their values. In a first example of this embodiment (a first subgroup of the first aspect), the UV signal is converted to a cumulative weight fraction and the cumulative weight fraction is plotted against the RI signal. g or R h Plot the values ​​as R. g or R h The quantitative size distribution of particles containing nucleic acids (especially RNA) is calculated by plotting the UV signal as a function of its value. In a second example of this embodiment (a second subgroup of the first aspect), the UV, fluorescence, or RI signal is converted into a cumulative weight fraction and the cumulative weight fraction is plotted against the RI signal. g or R h Plot the values ​​as R. g or R h The quantitative size distribution of RNA-containing particles is calculated by plotting UV, fluorescence, or RI signals as a function of their values. In a third example of this embodiment (a third subgroup concerning the first aspect), the UV signal is converted to a cumulative weight fraction and the cumulative weight fraction is plotted against RI. g or R h Plot the values ​​as R. g or R h The quantitative size distribution of RNA-containing particles is calculated from a graph of the UV signal as a function of the value. In each of the first, second, and third examples above, the quantitative size distribution of RNA-containing particles can be calculated from the graph of the UV signal as a function of the value. g value, R h The quantitative size distribution of particles containing nucleic acids (especially RNA) is determined based on the value or both. If in R gValue and R h Determining the quantitative size distribution of particles containing nucleic acids (especially RNA) based on the values ​​will generate two datasets: one based on R... g A quantitative distribution of values ​​and an R-based distribution h The quantitative distribution of values.

[0121] 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 includes D10, D50, and / or D90 values ​​(e.g., based on R...). g or R h (Value). If in R g Value and R h Determining the quantitative size distribution of particles containing nucleic acids (especially RNA) based on the values ​​will generate two datasets: one based on R... g The values ​​of D10, D50 and / or D90, and a set of R-based values. h The D10, D50, and / or D90 values.

[0122] 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 include (or) at least two, preferably at least three, parameters as specified herein (including additional optional parameters), particularly at least two, preferably at least three, selected from the following parameters: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to the particle, and the size distribution of the particles containing nucleic acid (especially RNA) (particularly based on the radius of gyration (R) of the particles containing nucleic acid (especially RNA)). g The hydrodynamic radius (R) of particles containing nucleic acids (especially RNA) and / or particles containing nucleic acids (especially RNA). h And the quantitative size distribution of particles containing nucleic acids (especially RNA) (e.g., based on R...). g or R h (Value). If in R g Value and R h Determining the size distribution of particles containing nucleic acids (especially RNA) based on the values ​​will generate two datasets: one based on R... g Value, a value based on R h However, according to the present invention, the two datasets of particle size distributions containing nucleic acids (especially RNA) are considered as only one parameter (instead of two parameters). Furthermore, if the fractogram obtained by field-flow fractionation shows more than one particle peak, the determination of the size distribution of each particle peak is considered as only one parameter (instead of a parameter for each particle peak). This also applies to R...g Value and R h Based on the values, the quantitative size distribution of particles containing nucleic acids (especially RNA) is determined.

[0123] In one embodiment of the first aspect (particularly, in one embodiment of the first, second, or third subgroup of the first aspect, particularly in a preferred embodiment of the third subgroup of the first aspect), the one or more parameters include a quantitative size distribution of particles containing nucleic acids (especially RNA) (e.g., based on the radius of gyration (R) of particles containing nucleic acids (especially RNA)). g The hydrodynamic radius (R) of particles containing nucleic acids (especially RNA) and / or particles containing nucleic acids (especially RNA). h And at least one of the remaining parameters (including additional optional parameters) specified herein, such as at least two parameters; preferably these remaining parameters are selected from: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to the particle, and the size distribution of the particles containing nucleic acid (especially RNA) (e.g., based on R). g or R h In one embodiment of the first aspect (particularly, in one embodiment of the first, second, or third subgroup of the first aspect, particularly in a preferred embodiment of the third subgroup of the first aspect), the one or more parameters include a quantitative size distribution of particles containing nucleic acids (especially RNA) (e.g., based on R...). g or R h The value) and at least one parameter selected from the following, such as at least two parameters: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to the particle, and the size distribution of the particles containing nucleic acid (especially RNA) (e.g., based on R). g or R h In one embodiment of the first aspect (particularly, in one embodiment of the first, second, or third subgroup of the first aspect, particularly in a preferred embodiment of the third subgroup of the first aspect), the one or more parameters include a quantitative size distribution of particles containing nucleic acids (especially RNA) (e.g., based on R...). g or R h The values ​​of free nucleic acids (especially RNA) and the amount of nucleic acids (especially RNA) bound to particles. If in R... g Value and R h Determining the quantitative size distribution of particles containing nucleic acids (especially RNA) based on the values ​​will generate two datasets: one based on R... g Value, a value based on R hValues. However, according to the present invention, the two datasets of quantitative size distribution of particles containing nucleic acids (especially RNA) are considered as only one parameter (instead of two parameters). Furthermore, if the classification pattern obtained by field-flow classification shows more than one particle peak, the determination of the quantitative size distribution of each particle peak is considered as only one parameter (instead of one parameter per particle peak). This also applies to R... g Value and R h Based on the values, the size distribution of particles containing nucleic acids (especially RNA) is determined.

[0124] 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), particularly free nucleic acid (especially RNA), is determined by measuring a UV signal, for example, at a wavelength in the range of 260 nm to 280 nm, such as at a wavelength of 260 nm or 280 nm, and using the extinction coefficient of nucleic acid (especially RNA) at the corresponding wavelength (e.g., 260 nm or 280 nm).

[0125] In one embodiment of the first aspect (particularly, in one embodiment of the first, second, or third subgroup of the first aspect, especially in a preferred embodiment of the third subgroup of the first aspect), the size distribution of particles containing nucleic acids (especially RNA) (e.g., based on R...) g or R h The quantitative size distribution of particles containing nucleic acids (especially RNA) and / or R-values ​​(e.g., based on R-values). g or R h The size distribution of the RNA-containing particles is in the range of 10-2000 nm, preferably in the range of 20-1500 nm, such as 30-1200 nm, 40-1100 nm, 50-1000 nm, 60-900 nm, 70-800 nm, 80-700 nm, 90-600 nm, or 100-500 nm, or for example in the range of 10-1000 nm, 15-500 nm, 20-450 nm, 25-400 nm, 30-350 nm, 40-300 nm, or 50-250 nm. In a preferred embodiment of the third subgroup of the first aspect, the (quantitative) size distribution of the RNA-containing particles (e.g., based on R...) g or R h The value is in the range of 10-1000nm, such as in the range of 15-500nm, 20-450nm, 25-400nm, 30-350nm, 40-300nm or 50-250nm.

[0126] In one embodiment of the first aspect (particularly in one embodiment of the first, second or third subgroup of the first aspect), the nucleic acid (especially RNA) is 10-15,000 nucleotides in length, such as 40-15,000 nucleotides, 100-12,000 nucleotides or 200-10,000 nucleotides.

[0127] 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.

[0128] In one embodiment of the first aspect (particularly in one embodiment of the first, second, or third subgroup of the first aspect), the measurement of at least one signal selected from UV signals, fluorescence signals, and RI signals, optionally LS signals such as SLS, for example MALS signals, and / or DLS signals, is performed online, and / or step (c) is performed online.

[0129] 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 in a cycle of steps (a)-(c).

[0130] In one embodiment of the first aspect (particularly, in one embodiment of the first, second, or third subgroup of the first aspect), the at least portion of the sample composition is diluted with a solvent or solvent mixture before field-flow fractionation is performed on the sample composition, the solvent or solvent mixture being capable of preventing the formation of particulate aggregates. In one embodiment, the solvent mixture is a mixture of water and an organic solvent such as formamide.

[0131] In one embodiment of the first aspect (particularly in one embodiment of the first, second, or third subgroup of the first aspect), the UV signal is measured by using circular dichroism (CD) spectroscopy.

[0132] In a second aspect, this disclosure provides a method for analyzing the effect of changing one or more reaction conditions when providing a composition comprising nucleic acids (such as RNA) and optionally present particles, the method comprising:

[0133] (A) Provides a first composition comprising nucleic acids (such as RNA) and optionally present particles;

[0134] (B) Provide a second composition comprising nucleic acid (such as RNA) and optionally present particles, wherein the provision of the second composition differs from the provision of the first composition only in one or more reaction conditions;

[0135] (C) A method of first aspect is used to subject a portion of the first composition to determine one or more parameters of the first composition;

[0136] (D) subjecting a corresponding portion of the second composition to the method used in step (C) to determine one or more parameters of the second composition; and

[0137] (E) Compare one or more parameters of the first composition obtained in step (C) with one or more corresponding parameters of the second composition obtained in step (D).

[0138] 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 the particle, and size of the particle containing nucleic acid (e.g., RNA) (particularly based on the radius of gyration (R) of the particle containing nucleic acid (e.g., RNA). g The hydrodynamic radius (R) of particles containing nucleic acids (such as RNA) and / or particles containing nucleic acids (such as RNA). h ), and the size distribution of particles containing nucleic acids (such as RNA) (e.g., based on R of particles containing nucleic acids (such as RNA) g or R h The quantitative size distribution of particles containing nucleic acids (such as RNA) and the R value of particles containing nucleic acids (such as RNA). g or R h Values). Generally, the size distribution and / or quantitative size distribution of particles containing nucleic acids (such as RNA) can be given as the number of particles containing nucleic acids (such as RNA), the molar amount of particles containing nucleic acids (such as RNA), or the mass of particles containing nucleic acids (such as RNA), each as a function of their size. Additional optional parameters include the molecular weight of the nucleic acid (especially RNA), the amount of surface nucleic acid (such as the amount of surface RNA), the amount of encapsulated nucleic acid (such as the amount of encapsulated RNA), the amount of accessible nucleic acid (such as the amount of accessible RNA), and the size of the nucleic acid (especially RNA) (particularly, based on the R value of the nucleic acid (such as RNA)). g and / or R h Value), size distribution of nucleic acids (especially RNA) (e.g., R based on nucleic acids (such as RNA) g or R h Value), quantitative size distribution of nucleic acids (especially RNA) (e.g., R based on nucleic acids (such as RNA) gor R h Value), shape factor, form factor, and nucleic acid (especially RNA) encapsulation efficiency. Generally, the size distribution and / or quantitative size distribution of nucleic acids (especially RNA) can be given as the number of nucleic acid (especially RNA) molecules, the molar amount of nucleic acids (especially RNA), or the mass of nucleic acids (especially RNA), each as a function of their size. Other additional optional parameters include the ratio of the amount of nucleic acid (e.g., RNA) bound to the particle to the total amount of particle-forming compounds (especially lipids and / or polymers) in the particle, wherein said ratio can be given as a function of particle size; the ratio of the amount of positively charged portions of particle-forming compounds (especially lipids and / or polymers) in the particle to the amount of nucleic acid (e.g., RNA) bound to the particle, wherein said ratio can be given as a function of particle size; and the charge ratio of the amount of positively charged portions of particle-forming compounds (especially lipids and / or polymers) in the particle to the amount of negatively charged portions of nucleic acid (e.g., RNA) bound to the particle, wherein said charge ratio is typically expressed as an N / P ratio and can be given as a function of particle size.

[0139] In one embodiment of the second aspect, the one or more parameters include (or) at least two, preferably at least three, parameters as specified herein (including additional optional parameters), particularly at least two, preferably at least three, selected from the following parameters: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to the particle, and the size distribution of the particles containing nucleic acid (especially RNA) (e.g., based on the R of the particles containing nucleic acid (such as RNA)). g or R h The quantitative size distribution of particles containing nucleic acids (especially RNA), based on R values ​​(e.g., R values ​​of particles containing nucleic acids such as RNA). g or R h The parameters include the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to the particle, and the molecular weight of the nucleic acid (especially RNA). In one embodiment of the second aspect, the one or more parameters include (or) at least two, preferably at least three, selected from the following parameters: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to the particle, and the size distribution of the particles containing nucleic acid (especially RNA) (e.g., based on the R value of nucleic acid (especially RNA)). g or R h Values) and quantitative size distribution of particles containing nucleic acids (especially RNA) (e.g., R based on nucleic acid (especially RNA) g or R h value).

[0140] In one embodiment of the second aspect, the method of the first aspect used in steps (C) and (D) is a method comprising:

[0141] (a) At least a portion of the composition (e.g., the first composition for step (C) or the second composition for step (D)) is subjected to field-flow classification, thereby classifying the components contained in the composition by their size to produce one or more composition fractions;

[0142] (b) Measure at least one signal selected from UV signal, fluorescence signal, and refractive index (RI) signal from at least one of the one or more composition fractions obtained in step (a), and optionally measure light scattering (LS) signal; and

[0143] (c) Calculate the one or more parameters from at least one signal selected from UV signals, fluorescence signals and RI signals, and optionally from the LS signal.

[0144] In the first subgroup of the second aspect, the method of the first aspect used in steps (C) and (D) is a method comprising:

[0145] (a) At least a portion of the composition (e.g., the first composition for step (C) or the second composition for step (D)) is subjected to field-flow classification, thereby classifying the components contained in the composition by their size to produce one or more fractions;

[0146] (b) Measure at least the UV signal and optionally the light scattering (LS) signal of at least one of the one or more fractions obtained from step (a); and

[0147] (c) Calculate one or more parameters from the UV signal and optionally from the LS signal.

[0148] In the second and preferred subgroup of the second aspect, the method of the first aspect used in steps (C) and (D) is a method for determining one or more parameters of a sample composition (e.g., the first composition for step (C) or the second composition for step (D)), wherein the sample composition comprises RNA and optionally present particles, the method comprising:

[0149] (a) Field-flow fractionation of at least a portion of the sample composition to classify the components contained in the sample composition by size to produce one or more sample fractions;

[0150] (b) Measure at least one signal selected from UV, fluorescence, and refractive index (RI) signals from at least one of the one or more sample fractions obtained in step (a), and optionally measure a light scattering (LS) signal; and

[0151] (c) Calculate the one or more parameters from at least one signal selected from UV signals, fluorescence signals and RI signals, and optionally from the LS signal.

[0152] In the third and more preferred subgroup of the second aspect, the method of the first aspect used in steps (C) and (D) is a method for determining one or more parameters of a sample composition (e.g., the first composition for step (C) or the second composition for step (D)), wherein the sample composition comprises RNA and optionally present particles, the method comprising:

[0153] (a) Field-flow fractionation of at least a portion of the sample composition to classify the components contained in the sample composition by size to produce one or more sample fractions;

[0154] (b) Measure at least the UV signal and optionally the light scattering (LS) signal of at least one of the one or more sample fractions obtained in step (a); and

[0155] (c) Calculate one or more parameters from the UV signal and optionally from the LS signal.

[0156] In one embodiment of the second aspect (particularly, in one embodiment of the first, second, or third subgroup of the second aspect), R based on particles containing nucleic acids (such as RNA) g The size, size distribution, and / or quantitative size distribution of particles containing nucleic acids (such as RNA) are calculated. In another embodiment of the second aspect (particularly, in another embodiment of the first, second, or third subgroup of the second aspect), the R value is based on the particles containing nucleic acids (such as RNA). h The size, size distribution, and / or quantitative size distribution of particles containing nucleic acids (such as RNA) are calculated. In another embodiment of the second aspect (particularly, in another embodiment of the first, second, or third subgroup of the second aspect), the R value is based on the particles containing nucleic acids (such as RNA). g The values ​​are based on R values ​​of particles containing nucleic acids (such as RNA). h The value is calculated for the size, size distribution, and / or quantitative size distribution of particles containing nucleic acids (such as RNA). That is, this implementation generates two datasets: one based on R... g Value, a value based on R h value).

[0157] In one embodiment of the second aspect (particularly, in one embodiment of the first, second, or third subgroup of the second aspect), said one or more parameters include the size, size distribution, and / or quantitative size distribution of the nucleic acid (such as RNA), based on the R of the nucleic acid (such as RNA). g The value is calculated to determine the size, size distribution, and / or quantitative size distribution of the nucleic acid (such as RNA). In another embodiment of the second aspect (particularly, in another embodiment of the first, second, or third subgroup of the second aspect), wherein said one or more parameters include the size, size distribution, and / or quantitative size distribution of the nucleic acid (such as RNA), based on the R of the nucleic acid (such as RNA). h The value is calculated to determine the size, size distribution, and / or quantitative size distribution of the nucleic acid (such as RNA). In another embodiment of the second aspect (particularly, in another embodiment of the first, second, or third subgroup of the second aspect), wherein said one or more parameters include the size, size distribution, and / or quantitative size distribution of the nucleic acid (such as RNA), based on the R of the nucleic acid (such as RNA). g Values ​​and respectively based on the R of nucleic acids (such as RNA) h This implementation calculates the size, size distribution, and / or quantitative size distribution of nucleic acids (such as RNA). Specifically, it generates two datasets: one based on R... g Value, a value based on R h value).

[0158] 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, and size of the particle containing nucleic acid (especially RNA) (particularly, based on the radius of gyration (R) of the particle containing nucleic acid (especially RNA)). g The hydrodynamic radius (R) of particles containing nucleic acids (especially RNA) and / or particles containing nucleic acids (especially RNA). h ), and the size distribution of particles containing nucleic acids (especially RNA) (e.g., based on R g or R h Values) and quantitative size distribution of particles containing nucleic acids (especially RNA) (e.g., based on R). g or R h The molecular weight of the nucleic acid (especially RNA) that is present, and the molecular weight of the nucleic acid that is present.

[0159] 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 (or) at least two, preferably at least three, parameters selected from: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to the particle, and the size distribution of the particles containing nucleic acid (especially RNA) (e.g., based on the R of the particles containing nucleic acid (especially RNA)). g or R h The quantitative size distribution of particles containing nucleic acids (especially RNA), based on R values. g or R h The parameters include the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to the particle, and the molecular weight of the nucleic acid (especially RNA). In one embodiment of the first, second, or third subgroup of the second aspect, the one or more parameters include (or) at least two, preferably at least three, selected from the following parameters: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to the particle, and the size distribution of the particles containing nucleic acid (especially RNA) (e.g., based on the R value of the particles containing nucleic acid (especially RNA)). g or R h The quantitative size distribution of particles containing nucleic acids (especially RNA) and the R value of particles containing nucleic acids (especially RNA). g or R h (Value). If in R g Value and R h Determining the size distribution of particles containing nucleic acids (especially RNA) based on the values ​​will generate two datasets: one based on R... g Value, a value based on R h Values. However, according to the present invention, the two datasets of particle size distributions containing nucleic acids (especially RNA) are considered as only one parameter (instead of two parameters). Furthermore, if the classification pattern obtained by field-flow classification shows more than one particle peak, the determination of the size distribution of each particle peak is considered as only one parameter (instead of a parameter for each particle peak). This also applies to R... g Value and R h Based on the values, the quantitative size distribution of particles containing nucleic acids (especially RNA) is determined.

[0160] In one embodiment of the second aspect (particularly, in one embodiment of the first, second, or third subgroup of the second aspect, particularly in a preferred embodiment of the third subgroup of the second aspect), the one or more parameters include a quantitative size distribution of particles containing nucleic acids (especially RNA) (e.g., based on the radius of gyration (R) of particles containing nucleic acids (especially RNA)). gThe hydrodynamic radius (R) of particles containing nucleic acids (especially RNA) and / or particles containing nucleic acids (especially RNA). h And at least one of the remaining parameters (including additional optional parameters) specified herein, such as at least two parameters; preferably these remaining parameters are selected from: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to the particle, and the size distribution of the particles containing nucleic acid (especially RNA) (e.g., based on R). g or R h In one embodiment of the second aspect (particularly, in one embodiment of the first, second, or third subgroup of the second aspect, particularly in a preferred embodiment of the third subgroup of the second aspect), the one or more parameters include a quantitative size distribution of particles containing nucleic acids (especially RNA) (e.g., based on R...). g or R h The value) and at least one parameter selected from the following, such as at least two parameters: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to the particle, and the size distribution of the particles containing nucleic acid (especially RNA) (e.g., based on R). g or R h In one embodiment of the second aspect (particularly, in one embodiment of the first, second, or third subgroup of the second aspect, particularly in a preferred embodiment of the third subgroup of the second aspect), the one or more parameters include a quantitative size distribution of particles containing nucleic acids (especially RNA) (e.g., based on R...). g or R h The values ​​of free nucleic acids (especially RNA) and the amount of nucleic acids (especially RNA) bound to particles. If in R... g Value and R h Determining the quantitative size distribution of particles containing nucleic acids (especially RNA) based on the values ​​will generate two datasets: one based on R... g Value, a value based on R h Values. However, according to the present invention, the two datasets of quantitative size distribution of particles containing nucleic acids (especially RNA) are considered as only one parameter (instead of two parameters). Furthermore, if the classification pattern obtained by field-flow classification shows more than one particle peak, the determination of the quantitative size distribution of each particle peak is considered as only one parameter (instead of one parameter per particle peak). This also applies to R... g Value and R h Based on the values, the size distribution of particles containing nucleic acids (especially RNA) is determined.

[0161] In one embodiment of the second aspect (particularly in one embodiment of the first, second, or third subgroup of the second aspect), the one or more parameters are determined in a cycle of steps (a)-(c).

[0162] In one embodiment of the second aspect (particularly in one embodiment of the first, second, or third subgroup of the second aspect), the one or more reaction conditions include any one of the following: salt concentration / ionic strength; temperature; pH or buffer concentration; light / radiation; oxygen; shear force; pressure; freeze / thaw cycle; dry / restore cycle; addition of excipients (e.g., stabilizers and / or chelating agents); type and / or source of particle-forming compounds (particularly lipids and / or polymers); charge ratio; physical state; and the ratio of nucleic acids (particularly RNA) to particle-forming compounds (particularly lipids and / or polymers constituting particles). Exemplary salt concentrations include salts of 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, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 mM NaCl. Exemplary temperature conditions include low temperatures (e.g., -20°C), ambient or room temperature, moderate temperatures (e.g., 30°C), or high temperatures (e.g., 50°C). Exemplary conditions regarding the type and / or source of the particulate-forming compound are cationic lipids against cationic polymers, cationic lipids against zwitterionic lipids, or polyethylene glycol-modified lipids against non-polyethylene glycol-modified lipids. Exemplary charge ratios of positive to negative charges in nucleic acid (especially RNA) particles are from about 6:1 to about 1:2, such as from about 5:1 to about 1.2:2, from about 4:1 to about 1.4:2, from about 3:1 to about 1.6:2, from about 2:1 to about 1.8:2, or from about 1.6:1 to about 1:1. Exemplary ratios of nucleic acids (especially RNA) to particle-forming compounds (especially lipids and / or polymers constituting the particles) include ratios of nucleic acids (especially RNA) to total lipids in the range of from about 1:100 to about 10:1 (w / w).

[0163] 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 classification is preferably a flow field-flow classification, such as asymmetric flow field-flow classification (AF4) or hollow fiber flow field-flow classification (HF5).

[0164] In one embodiment of the second aspect (particularly in one embodiment of the first, second or third subgroup of the second aspect), step (a) is performed using a membrane having a molecular weight cutoff (MW) suitable for preventing nucleic acid (especially RNA) from permeating the membrane, preferably a membrane with a MW cutoff in the range of 2kDa-30kDa, such as a membrane with a MW cutoff of 10kDa.

[0165] In one embodiment of the second aspect (particularly in one embodiment of the first, second or third subgroup of the second aspect), step (a) is performed using polyethersulfone (PES) or a regenerated cellulose membrane.

[0166] In one embodiment of the second aspect (particularly, in one embodiment of the first, second, or third subgroup of the second aspect), step (a) is performed using: (I) a cross-flow rate up to 8 mL / min, preferably up to 4 mL / min, more preferably up to 2 mL / min, for example, a cross-flow rate profile; and / or (II) an injection flow rate in the range of 0.05-0.35 mL / min, preferably in the range of 0.10-0.30 mL / min, more preferably in the range of 0.15-0.25 mL / min; and / or (III) a detector flow rate in the range of 0.30-0.70 mL / min, preferably in the range of 0.40-0.60 mL / min, more preferably in the range of 0.45-0.55 mL / min.

[0167] 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 spectrum preferably includes a fractionation phase that allows the components contained in the composition (such as a control or sample composition, particularly the first composition for step (C) or the second composition for step (D)) to be fractionated / separated by their size to produce one or more composition fractions. Preferably, the cross-flow rate changes during this fractionation phase (e.g., starting from a value (e.g., from about 1 to about 4 mL / min) and then decreasing to a lower value (e.g., from about 0 to about 0.1 mL / min), or starting from a value (e.g., from about 0 to about 0.1 mL / min) and then increasing to a higher value (e.g., from about 1 to about 4 mL / min)), which can be varied in any way, e.g., continuously (e.g., linearly or exponentially) or gradually. Preferably, the cross-flow rate spectrum includes a fractionation phase in which the cross-flow rate changes continuously (preferably exponentially), starting from a value (e.g., from about 1 to about 4 mL / min) and then decreasing to a lower value (e.g., from about 0 to about 0.1 mL / min). The fractionation stage can have any length suitable for fractionating / separating the components contained in the composition according to their size, such as about 5 min to about 60 min, about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 30 min. The cross-flow rate spectrum can contain additional stages (e.g., 1, 2, 3 or 4 stages) which can be before and / or after the fractionation stage (e.g., 1 before the fractionation stage and 1, 2 or 3 after the fractionation stage) and can be used to separate non-nucleic acid (especially non-RNA) components (e.g., proteins, peptides, mononucleotides, etc.) contained in the composition from nucleic acids (especially RNA) contained in the composition to concentrate the nucleic acids (especially RNA) contained in the composition and / or regeneration field-flow fractionation device (e.g., removing all components bound to the membrane of the device). Preferably, the crossflow velocity of these additional stages is constant for each additional stage, and the length of each of the additional stages is independently in the range of about 5 min to about 60 min (e.g., about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 30 min).For example, the cross velocity spectrum may include (i) a first additional stage prior to the grading stage, wherein the cross velocity of the first additional stage is constant and the same as the low cross velocity at the beginning of the grading stage (the length of the first additional stage may be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 10 min, about 20 min, or about 30 min); and (ii) a second additional stage after the grading stage, wherein the cross velocity of the second additional stage is constant and the same as the low cross velocity at the end of the grading stage (the length of the second additional stage may be about 5 min to about 60 min). Within the range, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 10 min, about 20 min, or about 30 min); and optionally (iii) a third additional stage after the second additional stage, wherein the crossflow rate of the third additional stage is constant and different from that of the second additional stage (the length of the third additional stage may be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 10 min, about 20 min, or about 30 min).In embodiments where the cross-flow rate spectrum includes a grading phase, the cross-flow rate varies continuously (preferably exponentially), starting from a value (e.g., about 1 to about 4 mL / min) and then decreasing to a lower value (e.g., about 0 to about 0.1 mL / min). Preferably, the cross-flow rate spectrum further includes (i) a first additional phase preceding the grading phase, wherein the cross-flow rate of the first additional phase is constant and the same as the low cross-flow rate at the start of the grading phase (e.g., about 1 to about 4 mL / min) (the length of the first additional phase may be in the range of about 5 min to about 30 min, such as about 6 min to about 25 min, about 7 min to about 20 min, or about 8 min to about 15 min, or about 10 min to about 12 min, or about 5 min, about 10 min, or about 12 min); (ii) a second additional phase following the grading phase, wherein the cross-flow rate of the second additional phase is constant and the same as the low cross-flow rate at the start of the grading phase. The cross-flow rate at the end of the first stage is the same (e.g., about 0.01 to 0.1 mL / min) (the length of the second additional stage may be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 30 min); and optionally (iii) a third additional stage after the second additional stage, wherein the cross-flow rate of the third additional stage is constant and lower than that of the second additional stage (e.g., the cross-flow rate of the third additional stage is 0) (the length of the third additional stage may be in the range of about 5 min to about 30 min, such as about 6 min to about 25 min, about 7 min to about 20 min, or about 8 min to about 15 min, or about 10 min to about 12 min, or about 5 min, about 10 min, or about 12 min). Preferred examples of such cross-flow rate spectra are as follows: 1.0-2.0 mL / min for 10 min, an exponential gradient from 1.0-2.0 mL / min to 0.01-0.07 mL / min over 30 min; 0.01-0.07 mL / min for 30 min; and 0 mL / min for 10 min.

[0168] 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 acids (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).

[0169] In a first specific instance of this implementation scheme in the second aspect, the integrity of the control nucleic acid (especially RNA) is determined through the following steps:

[0170] (a') Perform field-flow fractionation, particularly AF4 or HF5, on at least a portion of a control composition containing control nucleic acid (especially RNA) to fractionate the components contained in the control composition by size to produce one or more control fractions.

[0171] (b') Measure at least one signal selected from UV signal, fluorescence signal and refractive index (RI) signal from at least one of one or more control fractions obtained in step (a');

[0172] (c'1) Calculate the area from the maximum height of a UV, fluorescence, or RI peak to the end of the UV, fluorescence, or RI peak obtained in step (b'), thereby obtaining A. 50％ (Comparison);

[0173] (c'2) Calculate the total area of ​​a peak used in step (c'1) from at least one signal selected from UV, fluorescence, and RI signals obtained in step (b') to obtain A. 100％ (Comparison); and

[0174] (c'3) Determine A 50％ (Comparison) and A 100％ The ratio between (control) and (control) is used to obtain the integrity of the control nucleic acid (especially RNA) (I (control)).

[0175] In this first example, the integrity of nucleic acids (especially RNA) contained in the sample composition (e.g., the first composition for step (C) or the second composition for step (D)) can be calculated by the following steps:

[0176] (c1) Calculate the area from the maximum height of the sample UV, fluorescence, or RI peak corresponding to the control UV, fluorescence, or RI peak used in step (c'1) to the end of the sample UV, fluorescence, or RI peak from at least one signal selected from UV, fluorescence, and RI obtained in step (b), thereby obtaining A. 50％ (sample);

[0177] (c2) Calculate the total area of ​​the sample UV, fluorescence, or RI peaks used in step (c1) from the sample UV, fluorescence, or RI signals obtained in step (b), thereby obtaining A. 100％ (sample);

[0178] (c3) Determine A 50％ (sample) and A 100％ The ratio between (samples) is used to obtain I (sample); and

[0179] (c4) Determine the ratio between I (sample) and I (control) to obtain the integrity of the nucleic acids (especially RNA) contained in the sample composition.

[0180] In a second specific instance of this implementation scheme in the second aspect, the integrity of the control nucleic acid (especially RNA) is determined by the following steps:

[0181] (a”) Field-flow fractionation, particularly AF4 or HF5, is performed on at least a portion of a control composition containing control nucleic acid (especially RNA) to fractionate the components contained in the control composition by size to produce one or more control fractions.

[0182] (b”) Measure at least one signal selected from UV, fluorescence, and RI signals from at least one of the one or more control fractions obtained in step (a”); and

[0183] (c”) The height of a UV, fluorescence, or RI peak (H(control)) is determined from at least one signal selected from UV, fluorescence, and RI signals obtained in step (b”) to obtain the integrity of the control nucleic acid (especially RNA).

[0184] In this second example, the integrity of nucleic acids (especially RNA) contained in the sample composition (e.g., the first composition for step (C) or the second composition for step (D)) can be calculated by the following steps:

[0185] (c1') Determine 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 UV, fluorescence, and RI signals obtained in step (b); and

[0186] (c2') Determine the ratio between H (sample) and H (control) to obtain the integrity of the nucleic acids (especially RNA) contained in the sample composition.

[0187] In a third specific instance of this implementation scheme of the second aspect (regarding the first subgroup of the second aspect), the integrity of the control nucleic acid (especially RNA) is determined by the following steps:

[0188] (a') Field-flow fractionation, particularly AF4 or HF5, is performed on at least a portion of a control composition containing control nucleic acid (especially RNA) to fractionate the components contained in the control composition by size to produce one or more control fractions.

[0189] (b') Measure the UV signal of at least one of the one or more control fractions obtained from step (a');

[0190] (c'1) Calculate the area from the maximum height of a UV peak to the end of the UV peak using the UV signal obtained in step (b'), thereby obtaining A. 50％ (Comparison);

[0191] (c'2) Calculate the total area of ​​one peak used in step (c'1) from the UV signal obtained in step (b') to obtain A. 100％ (Comparison); and

[0192] (c'3) Determine A 50％ (Comparison) and A 100％ The ratio between (control) and (control) is used to obtain the integrity of the control nucleic acid (especially RNA) (I (control)).

[0193] In this third example, the integrity of nucleic acids (especially RNA) contained in the sample composition (e.g., the first composition for step (C) or the second composition for step (D)) can be calculated by the following steps:

[0194] (c1) 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 from the UV signal obtained in step (b), thereby obtaining A. 50％ (sample);

[0195] (c2) Calculate the total area of ​​the sample UV peaks used in step (c1) from the sample UV signal obtained in step (b), thereby obtaining A. 100％ (sample);

[0196] (c3) Determine A 50％ (sample) and A 100％ The ratio between (samples) is used to obtain I (sample); and

[0197] (c4) Determine the ratio between I (sample) and I (control) to obtain the integrity of the nucleic acids (especially RNA) contained in the sample composition.

[0198] In a fourth specific instance of this implementation scheme of the second aspect (regarding the first subgroup of the second aspect), the integrity of the control nucleic acid (especially RNA) is determined by the following steps:

[0199] (a”) Field-flow fractionation, particularly AF4 or HF5, is performed on at least a portion of a control composition containing control nucleic acid (especially RNA) to fractionate the components contained in the control composition by size to produce one or more control fractions.

[0200] (b”) Measure the UV signal of at least one of the one or more control fractions obtained from step (a”); and

[0201] (c”) Determine the height of a UV peak (H(control)) from the UV signal obtained in step (b”) to obtain the integrity of the control nucleic acid (especially RNA).

[0202] In this fourth example, the integrity of nucleic acids (especially RNA) contained in the sample composition (e.g., the first composition for step (C) or the second composition for step (D)) can be calculated by the following steps:

[0203] (c1') Determine 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); and

[0204] (c2') Determine the ratio between H (sample) and H (control) to obtain the integrity of the nucleic acids (especially RNA) contained in the sample composition.

[0205] In the fifth specific instance of this implementation scheme of the second aspect (regarding the second subgroup of the second aspect), the integrity of the control RNA is determined by the following steps:

[0206] (a') Field-flow fractionation, particularly AF4 or HF5, is performed on at least a portion of a control composition containing control RNA to fractionate the components contained in the control composition by size to produce one or more control fractions.

[0207] (b') Measure at least one signal selected from UV signal, fluorescence signal and RI signal from at least one of one or more control fractions obtained in step (a');

[0208] (c'1) Calculate the area from the maximum height of a UV, fluorescence, or RI peak to the end of the UV, fluorescence, or RI peak obtained in step (b'), thereby obtaining A. 50％ (Comparison);

[0209] (c'2) Calculate the total area of ​​a peak used in step (c'1) from at least one signal selected from UV, fluorescence, and RI signals obtained in step (b') to obtain A. 100％ (Comparison); and

[0210] (c'3) Determine A 50％ (Comparison) and A 100％ The ratio between (control) and (control) is used to obtain the integrity of the control RNA (I (control)).

[0211] 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)) can be calculated by the following steps:

[0212] (c1) Calculate the area from the maximum height of the sample UV, fluorescence, or RI peak corresponding to the control UV, fluorescence, or RI peak used in step (c'1) to the end of the sample UV, fluorescence, or RI peak from at least one signal selected from UV, fluorescence, and RI obtained in step (b), thereby obtaining A. 50％ (sample);

[0213] (c2) Calculate the total area of ​​the sample UV, fluorescence, or RI peaks used in step (c1) from the sample UV, fluorescence, or RI signals obtained in step (b), thereby obtaining A. 100％ (sample);

[0214] (c3) Determine A 50％ (sample) and A 100％ The ratio between (samples) is used to obtain I (sample); and

[0215] (c4) Determine the ratio between I (sample) and I (control) to obtain the integrity of the RNA contained in the sample composition.

[0216] In the sixth specific instance of this implementation scheme of the second aspect (regarding the second subgroup of the second aspect), the integrity of the control RNA is determined by the following steps:

[0217] (a”) Perform field-flow fractionation, particularly AF4 or HF5, on at least a portion of a control composition containing control RNA, thereby fractionating the components contained in the control composition by size to produce one or more control fractions.

[0218] (b”) Measure at least one signal selected from UV, fluorescence, and RI signals from at least one of the one or more control fractions obtained in step (a”); and

[0219] (c”) The height of a UV, fluorescence, or RI peak (H(control)) is determined from at least one signal selected from UV, fluorescence, and RI signals obtained in step (b”) to obtain the integrity of the control RNA.

[0220] 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)) can be calculated by the following steps:

[0221] (c1') Determine 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 UV, fluorescence, and RI signals obtained in step (b); and

[0222] (c2') Determine the ratio between H (sample) and H (control) to obtain the integrity of the RNA contained in the sample composition.

[0223] In the seventh specific instance of this implementation scheme of the second aspect (regarding the third subgroup of the second aspect), the integrity of the control RNA is determined by the following steps:

[0224] (a') Field-flow fractionation, particularly AF4 or HF5, is performed on at least a portion of a control composition containing control RNA to fractionate the components contained in the control composition by size to produce one or more control fractions.

[0225] (b') Measure the UV signal of at least one of the one or more control fractions obtained from step (a');

[0226] (c'1) Calculate the area from the maximum height of a UV peak to the end of the UV peak using the UV signal obtained in step (b'), thereby obtaining A. 50％ (Comparison);

[0227] (c'2) Calculate the total area of ​​one peak used in step (c'1) from the UV signal obtained in step (b') to obtain A. 100％ (Comparison); and

[0228] (c'3) Determine A 50％ (Comparison) and A 100％ The ratio between (control) and (control) is used to obtain the integrity of the control RNA (I (control)).

[0229] 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)) can be calculated by the following steps:

[0230] (c1) 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 from the UV signal obtained in step (b), thereby obtaining A. 50％ (sample);

[0231] (c2) Calculate the total area of ​​the sample UV peaks used in step (c1) from the sample UV signal obtained in step (b), thereby obtaining A. 100％ (sample);

[0232] (c3) Determine A 50％ (sample) and A 100％ The ratio between (samples) is used to obtain I (sample); and

[0233] (c4) Determine the ratio between I (sample) and I (control) to obtain the integrity of the RNA contained in the sample composition.

[0234] In the eighth specific instance of this implementation scheme of the second aspect (regarding the third subgroup of the second aspect), the integrity of the control RNA is determined by the following steps:

[0235] (a”) Perform field-flow fractionation, particularly AF4 or HF5, on at least a portion of a control composition containing control RNA, thereby fractionating the components contained in the control composition by size to produce one or more control fractions.

[0236] (b”) Measure the UV signal of at least one of the one or more control fractions obtained from step (a”); and

[0237] (c”) Determine the height of a UV peak (H(control)) from the UV signal obtained in step (b”) to obtain the integrity of the control RNA.

[0238] 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)) can be calculated by the following steps:

[0239] (c1') Determine 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); and

[0240] (c2') Determine the ratio between H (sample) and H (control) to obtain the integrity of the RNA contained in the sample composition.

[0241] In one embodiment of the second aspect (particularly in one embodiment of the first, second or third subgroup of the second aspect), the amount of nucleic acid (especially RNA) is determined by using (i) the extinction coefficient of nucleic acid (especially the extinction coefficient of RNA) or (ii) the nucleic acid calibration curve (especially the RNA calibration curve).

[0242] 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 acids (especially RNA) and particles, such as lipid complex particles and / or lipid nanoparticles and / or polyplex particles and / or lipid polycomplex particles and / or virus-like particles, with the nucleic acids (especially RNA) bound to the particles.

[0243] 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 (especially 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)-(c) with at least the portion obtained from step (i); and (iii) determining the amount of nucleic acid (especially RNA) as described herein (e.g., using (i) a nucleic acid extinction coefficient (especially an RNA extinction coefficient) or (ii) a nucleic acid calibration curve (especially 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.

[0244] In one embodiment of the second aspect (particularly, in one embodiment of the first, second, or third subgroup of the second aspect), the releasing agent is (i) a surfactant, such as anionic surfactants (e.g., sodium dodecyl sulfate), zwitterionic surfactants (e.g., tetradecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate), etc. (3-14)) cationic surfactants, nonionic surfactants or mixtures thereof; (ii) alcohols, such as fatty alcohols (e.g., ethanol), or mixtures of alcohols; or (iii) a combination of (i) and (ii).

[0245] 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: performing steps (a)-(c) without the addition of a releasing agent, particularly in the absence of any releasing agent; and determining the amount of nucleic acid (especially RNA) as described herein (e.g., using (i) the nucleic acid extinction coefficient (especially the RNA extinction coefficient) or (ii) the nucleic acid calibration curve (especially the RNA calibration curve)).

[0246] 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 particle is determined by: subtracting the amount of free nucleic acid (especially RNA) as determined herein from the amount of total nucleic acid (especially RNA) as determined herein (e.g., by 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)-(c) with at least the portion obtained from step (i); and (iii) subtracting the amount of free nucleic acid (especially RNA) as determined herein (e.g., by performing steps (a)-(c) without the addition of a releasing agent, particularly in the absence of any releasing agent; and subtracting the amount of free nucleic acid (especially RNA) as determined herein (e.g., by using (i) a nucleic acid extinction coefficient (especially RNA extinction coefficient) or (ii) a nucleic acid calibration curve (especially an RNA calibration curve)) as determined herein).

[0247] In one embodiment of the second aspect (particularly in one embodiment of the second or third subgroup of the second aspect), step (b) further includes measuring an LS signal, such as dynamic light scattering (DLS) and / or static light scattering (SLS), for example, a multi-angle light scattering (MALS) signal, of at least one of the sample fractions obtained from step (a).

[0248] In one embodiment of the second aspect (particularly, in one embodiment of the second or third subgroup of the second aspect), the radius of gyration (R) is calculated from the LS signal obtained from step (b). g ) value and / or hydrodynamic radius (R h The value is used to determine the size of particles containing nucleic acids (especially RNA). In one embodiment of the second aspect (particularly, in one embodiment of the first, second, or third subgroup of the second aspect), step (b) includes measuring the dynamic light scattering (DLS) signal of at least one of the sample fractions obtained from step (a), and step (c) includes calculating R from the DLS signal. h Value. In one embodiment of the second aspect (particularly, in one embodiment of the first, second, or third subgroup of the second aspect), step (b) includes measuring a static light scattering (SLS) signal, such as a MALS signal, of at least one of the one or more sample fractions obtained from step (a), and step (c) includes calculating R from the SLS signal. gValue. In one embodiment of the second aspect (particularly, in one embodiment of the first, second, or third subgroup of the second aspect), step (b) includes measuring a dynamic light scattering (DLS) signal and a static light scattering (SLS) signal, such as a MALS signal, of at least one of the one or more sample fractions obtained from step (a), and step (c) includes calculating R. g and R h Value. This latter implementation produces two datasets of particle size containing nucleic acids (such as RNA), namely, one based on R. g Value, a value based on R h value.

[0249] In one embodiment of the second aspect (particularly, in one embodiment of the first, second, or third subgroup of the second aspect), by applying at least one signal selected from UV signals, fluorescence signals, and RI signals obtained from step (b) to the R signal determined as described herein. g or R h Value (e.g., calculated from the SLS signal obtained in step (b)). g The value or R is calculated from the DLS signal obtained in step (b). h The size distribution of particles containing nucleic acids (especially RNA) is determined by plotting the UV signal obtained from step (b) against the R values ​​as described herein. In a first example of this implementation (a first subgroup of the second aspect), the size distribution of particles containing nucleic acids (especially RNA) is determined by plotting the UV signal obtained from step (b) against the R values ​​as described herein. g or R h Value (e.g., calculated from the SLS signal obtained in step (b)). g The value or R is calculated from the DLS signal obtained in step (b). h The size distribution of particles containing nucleic acids (especially RNA) is determined by plotting the values. In a second example of this embodiment (a second subgroup of the second aspect), the size distribution of particles containing nucleic acids (especially RNA) is determined by plotting at least one signal selected from UV signals, fluorescence signals, and RI signals obtained from step (b) against the R values ​​as described herein. g or R h Value (e.g., calculated from the SLS signal obtained in step (b)). g The value or R is calculated from the DLS signal obtained in step (b). h The size distribution of RNA-containing particles was determined by plotting the UV signal obtained from step (b) against the R values ​​as described herein. In a third example of this implementation (a third subgroup concerning the second aspect), the size distribution of RNA-containing particles was determined by plotting the UV signal obtained from step (b) against the R values ​​as described herein. g or R h Value (e.g., calculated from the SLS signal obtained in step (b)). g The value or R is calculated from the DLS signal obtained in step (b). hThe size distribution of RNA-containing particles can be determined by plotting the values. In each of the first, second, and third examples above, the size distribution of RNA-containing particles can be determined by plotting the values. g value, R h The size distribution of particles containing nucleic acids (especially RNA) is determined based on the value or both. If in R g Value and R h Determining the size distribution of particles containing nucleic acids (especially RNA) based on the values ​​will generate two datasets: one based on R... g The magnitude distribution of values ​​and an R-based h Distribution of values.

[0250] In one embodiment of the second aspect (particularly, in one embodiment of the first, second, or third subgroup of the second aspect), by converting UV, fluorescence, or RI signals into cumulative weight fractions and comparing the cumulative weight fractions with R... g or R h Plot the values ​​as R. g or R h The quantitative size distribution of particles containing nucleic acids (especially RNA) is calculated by plotting UV, fluorescence, or RI signals as a function of their values. In a first example of this embodiment (a first subgroup of the second aspect), the UV signal is converted to a cumulative weight fraction and the cumulative weight fraction is plotted against the RI signal. g or R h Plot the values ​​as R. g or R h The quantitative size distribution of particles containing nucleic acids (especially RNA) is calculated by plotting the UV signal as a function of its value. In a second example of this implementation (a second subgroup of the second aspect), the UV, fluorescence, or RI signal is converted into a cumulative weight fraction and the cumulative weight fraction is plotted against the RI signal. g or R h Plot the values ​​as R. g or R h The quantitative size distribution of RNA-containing particles is calculated by plotting UV, fluorescence, or RI signals as a function of their values. In a third example of this embodiment (a third subgroup concerning the second aspect), the UV signal is converted to a cumulative weight fraction and the cumulative weight fraction is plotted against RI. g or R h Plot the values ​​as R. g or R h The quantitative size distribution of RNA-containing particles is calculated from a graph of the UV signal as a function of the value. In each of the first, second, and third examples above, the quantitative size distribution of RNA-containing particles can be calculated from the graph of the UV signal as a function of the value. g value, R h The quantitative size distribution of particles containing nucleic acids (especially RNA) is determined based on the value or both. If in R gValue and R h Determining the quantitative size distribution of particles containing nucleic acids (especially RNA) based on the values ​​will generate two datasets: one based on R... g A quantitative distribution of values ​​and an R-based distribution h The quantitative distribution of values.

[0251] 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 includes D10, D50, and / or D90 values. If in R... g Value and R h Determining the quantitative size distribution of particles containing nucleic acids (especially RNA) based on the values ​​will generate two datasets: one based on R... g The values ​​of D10, D50 and / or D90, and a set of R-based values. h The D10, D50, and / or D90 values.

[0252] 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), particularly free nucleic acid (especially RNA), is determined by measuring a UV signal, for example, at a wavelength in the range of 260 nm to 280 nm, such as at a wavelength of 260 nm or 280 nm, and using the extinction coefficient of nucleic acid (especially RNA) at the corresponding wavelength (e.g., 260 nm or 280 nm).

[0253] In one embodiment of the second aspect (particularly, in one embodiment of the first, second, or third subgroup of the second aspect, especially in a preferred embodiment of the third subgroup of the second aspect), the size distribution of particles containing nucleic acids (especially RNA) (e.g., based on R...) g or R h The quantitative size distribution of particles containing nucleic acids (especially RNA) and / or R-values ​​(e.g., based on R-values). g or R h The size distribution of the RNA-containing particles is in the range of 10-2000 nm, preferably in the range of 20-1500 nm, such as 30-1200 nm, 40-1100 nm, 50-1000 nm, 60-900 nm, 70-800 nm, 80-700 nm, 90-600 nm, or 100-500 nm, or for example in the range of 10-1000 nm, 15-500 nm, 20-450 nm, 25-400 nm, 30-350 nm, 40-300 nm, or 50-250 nm. In a preferred embodiment of the third subgroup of the second aspect, the (quantitative) size distribution of the RNA-containing particles (e.g., based on R...) g or R hThe value is in the range of 10-1000nm, for example, in the range of 15-500nm, 20-450nm, 25-400nm, 30-350nm, 40-300nm or 50-250nm.

[0254] In one embodiment of the second aspect (particularly in one embodiment of the first, second, or third subgroup of the second aspect), the nucleic acid (especially RNA) is 10-15,000 nucleotides in length, such as 40-15,000 nucleotides, 100-12,000 nucleotides, or 200-10,000 nucleotides.

[0255] 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.

[0256] In one embodiment of the second aspect (particularly in one embodiment of the first, second, or third subgroup of the second aspect), the measurement of at least one signal selected from UV signals, fluorescence signals, and RI signals, optionally LS signals such as SLS, for example MALS signals, and / or DLS signals, is performed online, and / or step (c) is performed online.

[0257] In one embodiment of the second aspect (particularly, in one embodiment of the first, second, or third subgroup of the second aspect), before field-flow fractionation of at least a portion of the sample composition (e.g., the first composition for step (C) or the second composition for step (D)), the at least portion of the sample composition is diluted with a solvent or solvent mixture capable of preventing the formation of particulate aggregates. In one embodiment, the solvent mixture is a mixture of water and an organic solvent such as formamide.

[0258] In one embodiment of the second aspect (particularly in one embodiment of the first, second, or third subgroup of the second aspect), the UV signal is measured by using circular dichroism (CD) spectroscopy.

[0259] It should be understood that any implementation described in the context of the first aspect can also be applied to any implementation of the second aspect.

[0260] In a third aspect, this disclosure provides the use of field-flow fractionation in determining one or more parameters of a sample composition comprising nucleic acids (such as RNA) and optionally present particles, wherein the one or more parameters include nucleic acid (such as RNA) integrity, total amount of nucleic acids (such as RNA), amount of free nucleic acids (such as RNA), amount of nucleic acids (such as RNA) bound to particles, and size of particles containing nucleic acids (such as RNA) (particularly based on the radius of gyration (R) of particles containing nucleic acids (such as RNA)). g The hydrodynamic radius (R) of particles containing nucleic acids (such as RNA) and / or particles containing nucleic acids (such as RNA). h ), and the size distribution of particles containing nucleic acids (such as RNA) (e.g., based on R of particles containing nucleic acids (such as RNA) g or R h The quantitative size distribution of particles containing nucleic acids (such as RNA) and the R value of particles containing nucleic acids (such as RNA). g or R h Value). Generally, the size distribution and / or quantitative size distribution of particles containing nucleic acids (such as RNA) can be given as the number of particles containing nucleic acids (such as RNA), the molar amount of particles containing nucleic acids (such as RNA), or the mass of particles containing nucleic acids (such as RNA), each as a function of their size. Additional optional parameters include the molecular weight of the nucleic acid (such as RNA), the amount of surface nucleic acid (such as the amount of surface RNA), the amount of encapsulated nucleic acid (such as the amount of encapsulated RNA), the amount of accessible nucleic acid (such as the amount of accessible RNA), and the size of the nucleic acid (especially RNA) (particularly based on the R value of the particles containing nucleic acids (such as RNA)). g and / or R h Value), size distribution of nucleic acids (especially RNA) (e.g., R based on nucleic acids (such as RNA) g or R h Value), quantitative size distribution of nucleic acids (especially RNA) (e.g., R based on nucleic acids (such as RNA) g or R hValue), shape factor, form factor, and nucleic acid (especially RNA) encapsulation efficiency. Generally, the size distribution and / or quantitative size distribution of nucleic acids (especially RNA) can be given as the number of nucleic acid (especially RNA) molecules, the molar amount of nucleic acids (especially RNA), or the mass of nucleic acids (especially RNA), each as a function of their size. Other additional optional parameters include the ratio of the amount of nucleic acid (e.g., RNA) bound to the particle to the total amount of particle-forming compounds (especially lipids and / or polymers) in the particle, wherein said ratio can be given as a function of particle size; the ratio of the amount of positively charged portions of particle-forming compounds (especially lipids and / or polymers) in the particle to the amount of nucleic acid (e.g., RNA) bound to the particle, wherein said ratio can be given as a function of particle size; and the charge ratio of the amount of positively charged portions of particle-forming compounds (especially lipids and / or polymers) in the particle to the amount of negatively charged portions of nucleic acid (e.g., RNA) bound to the particle, wherein said charge ratio is typically expressed as an N / P ratio and can be given as a function of particle size.

[0261] In one embodiment of the third aspect, the field-flow classification includes:

[0262] (a) Perform field-flow fractionation on at least a portion of the sample composition to fractionate the components contained in the sample composition by size to produce one or more sample fractions;

[0263] (b) Measure at least one signal selected from UV, fluorescence, and refractive index (RI) signals from at least one of the one or more sample fractions obtained in step (a), and optionally measure a light scattering (LS) signal; and

[0264] (c) Calculate the one or more parameters from at least one signal selected from UV signals, fluorescence signals and RI signals, and optionally from the LS signal.

[0265] In the first subgroup of the third aspect, the field-flow classification includes:

[0266] (a) Perform field-flow fractionation on at least a portion of the sample composition to fractionate the components contained in the sample composition by size to produce one or more sample fractions;

[0267] (b) Measure at least the UV signal and optionally the light scattering (LS) signal of at least one of the one or more sample fractions obtained in step (a); and

[0268] (c) Calculate one or more parameters from the UV signal and optionally from the LS signal.

[0269] In the second and preferred subgroup of the third aspect, the use is for determining one or more parameters of a sample composition, wherein the sample composition comprises RNA and optionally present particles, and the field-flow fractionation includes:

[0270] (a) Perform field-flow fractionation on at least a portion of the sample composition to fractionate the components contained in the sample composition by size to produce one or more sample fractions;

[0271] (b) Measure at least one signal selected from UV, fluorescence, and refractive index (RI) signals from at least one of the one or more sample fractions obtained in step (a), and optionally measure a light scattering (LS) signal; and

[0272] (c) Calculate the one or more parameters from at least one signal selected from UV signals, fluorescence signals and RI signals, and optionally from the LS signal.

[0273] In the third and more preferred subgroup of the third aspect, the use is for determining one or more parameters of a sample composition, wherein the sample composition comprises RNA and optionally present particles, and the field-flow fractionation includes:

[0274] (a) Perform field-flow fractionation on at least a portion of the sample composition to fractionate the components contained in the sample composition by size to produce one or more sample fractions;

[0275] (b) Measure at least the UV signal and optionally the light scattering (LS) signal of at least one of the one or more sample fractions obtained in step (a); and

[0276] (c) Calculate one or more parameters from the UV signal and optionally from the LS signal.

[0277] In one embodiment of the third aspect (particularly, in one embodiment of the first, second, or third subgroup of the third aspect), R based on particles containing nucleic acids (such as RNA) g The size, size distribution, and / or quantitative size distribution of particles containing nucleic acids (such as RNA) are calculated. In another embodiment of the third aspect (particularly, in another embodiment of the first, second, or third subgroup of the third aspect), the R value is based on the particles containing nucleic acids (such as RNA). h The size, size distribution, and / or quantitative size distribution of particles containing nucleic acids (such as RNA) are calculated. In another embodiment of the third aspect (particularly, in another embodiment of the first, second, or third subgroup of the third aspect), the R value is based on the particles containing nucleic acids (such as RNA). g The values ​​are based on R values ​​of particles containing nucleic acids (such as RNA).h The value is calculated for the size, size distribution, and / or quantitative size distribution of particles containing nucleic acids (such as RNA). That is, this implementation generates two datasets: one based on R... g Value, a value based on R h value).

[0278] In one embodiment of the third aspect (particularly, in one embodiment of the first, second, or third subgroup of the third aspect), said one or more parameters include the size, size distribution, and / or quantitative size distribution of the nucleic acid (such as RNA), based on the R of the nucleic acid (such as RNA). g The value is calculated to determine the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA). In another embodiment of the third aspect (particularly, in another embodiment of the first, second, or third subgroup of the third aspect), said one or more parameters include the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA), based on the R... h The value is calculated to determine the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA). In another embodiment of the third aspect (particularly, in another embodiment of the first, second, or third subgroup of the third aspect), said one or more parameters include the size, size distribution, and / or quantitative size distribution of the nucleic acid (e.g., RNA), based on the R... g Values ​​and respectively based on the R of nucleic acids (such as RNA) h This implementation calculates the size, size distribution, and / or quantitative size distribution of nucleic acids (such as RNA). Specifically, it generates two datasets: one based on R... g Value, a value based on R h value).

[0279] 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 classification is a flow field-flow classification, such as asymmetric flow field-flow classification (AF4) or hollow fiber flow field-flow classification (HF5).

[0280] In one embodiment of the third aspect (particularly, in one embodiment of the first, second, or third subgroup of the third aspect), the field-flow fractionation uses a membrane with a molecular weight cutoff (MW) suitable for preventing nucleic acid (especially RNA) from permeating the membrane, preferably a membrane with a MW cutoff in the range of 2kDa-30kDa, such as a membrane with a MW cutoff of 10kDa.

[0281] 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 classification uses a polyethersulfone (PES) or regenerated cellulose membrane.

[0282] In one embodiment of the third aspect (particularly, in one embodiment of the first, second, or third subgroup of the third aspect), step (a) is performed using: (I) a cross-flow rate up to 8 mL / min, preferably up to 4 mL / min, more preferably up to 2 mL / min, for example, a cross-flow rate profile; and / or (II) an injection flow rate in the range of 0.05-0.35 mL / min, preferably in the range of 0.10-0.30 mL / min, more preferably in the range of 0.15-0.25 mL / min; and / or (III) a detector flow rate in the range of 0.30-0.70 mL / min, preferably in the range of 0.40-0.60 mL / min, more preferably in the range of 0.45-0.55 mL / min.

[0283] 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 spectrum preferably includes a fractionation phase, which allows the components contained in the control or sample composition to be fractionated / separated according to their size to produce one or more sample fractions. Preferably, the cross-flow rate changes during this fractionation phase (e.g., starting from a value (e.g., from about 1 to about 4 mL / min) and then decreasing to a lower value (e.g., from about 0 to about 0.1 mL / min), or starting from a value (e.g., from about 0 to about 0.1 mL / min) and then increasing to a higher value (e.g., from about 1 to about 4 mL / min)), wherein the change can be in any manner, e.g., continuous (e.g., linear or exponential) or gradual. Preferably, the cross-flow rate spectrum includes a fractionation phase, wherein the cross-flow rate changes continuously (preferably exponentially), starting from a value (e.g., from about 1 to about 4 mL / min) and then decreasing to a lower value (e.g., from about 0 to about 0.1 mL / min). The fractionation stage can have any length suitable for fractionating / separating components contained in the sample composition according to their size, such as about 5 min to about 60 min, about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 30 min. The cross-flow rate spectrum can contain additional stages (e.g., 1, 2, 3, or 4 stages) which can be before and / or after the fractionation stage (e.g., 1 before fractionation and 1, 2, or 3 after fractionation) and can be used to separate non-nucleic acid (especially non-RNA) components (e.g., proteins, peptides, mononucleotides, etc.) contained in the sample composition from nucleic acids (especially RNA) contained in the sample composition to concentrate nucleic acids (especially RNA) contained in the sample composition and / or regenerate the field-flow fractionation device (e.g., remove all components bound to the membrane of the device). Preferably, the crossflow velocity of these additional stages is constant for each additional stage, and the length of each of the additional stages is independently in the range of about 5 min to about 60 min (e.g., about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 30 min).For example, the cross velocity spectrum may include (i) a first additional stage prior to the grading stage, wherein the cross velocity of the first additional stage is constant and the same as the low cross velocity at the beginning of the grading stage (the length of the first additional stage may be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 10 min, about 20 min, or about 30 min); and (ii) a second additional stage after the grading stage, wherein the cross velocity of the second additional stage is constant and the same as the low cross velocity at the end of the grading stage (the length of the second additional stage may be about 5 min to about 60 min). Within the range, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 10 min, about 20 min, or about 30 min); and optionally (iii) a third additional stage after the second additional stage, wherein the crossflow rate of the third additional stage is constant and different from that of the second additional stage (the length of the third additional stage may be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 10 min, about 20 min, or about 30 min).In embodiments where the cross-flow rate spectrum includes a grading phase, the cross-flow rate varies continuously (preferably exponentially), starting from a value (e.g., about 1 to about 4 mL / min) and then decreasing to a lower value (e.g., about 0 to about 0.1 mL / min). Preferably, the cross-flow rate spectrum further includes (i) a first additional phase preceding the grading phase, wherein the cross-flow rate of the first additional phase is constant and the same as the low cross-flow rate at the start of the grading phase (e.g., about 1 to about 4 mL / min) (the length of the first additional phase may be in the range of about 5 min to about 30 min, such as about 6 min to about 25 min, about 7 min to about 20 min, or about 8 min to about 15 min, or about 10 min to about 12 min, or about 5 min, about 10 min, or about 12 min); (ii) a second additional phase following the grading phase, wherein the cross-flow rate of the second additional phase is constant and the same as the low cross-flow rate at the start of the grading phase. The cross-flow rate at the end of the first stage is the same (e.g., about 0.01 to 0.1 mL / min) (the length of the second additional stage may be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 30 min); and optionally (iii) a third additional stage after the second additional stage, wherein the cross-flow rate of the third additional stage is constant and lower than that of the second additional stage (e.g., the cross-flow rate of the third additional stage is 0) (the length of the third additional stage may be in the range of about 5 min to about 30 min, such as about 6 min to about 25 min, about 7 min to about 20 min, or about 8 min to about 15 min, or about 10 min to about 12 min, or about 5 min, about 10 min, or about 12 min). Preferred examples of such cross-flow rate spectra are as follows: 1.0-2.0 mL / min for 10 min, an exponential gradient from 1.0-2.0 mL / min to 0.01-0.07 mL / min over 30 min; 0.01-0.07 mL / min for 30 min; and 0 mL / min for 10 min.

[0284] 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 acids (especially RNA) contained in the sample composition is determined by using the integrity of a control nucleic acid (especially RNA).

[0285] In a first specific instance of this implementation scheme in the third aspect, the integrity of the control nucleic acid (especially RNA) is determined through the following steps:

[0286] (a') Field-flow fractionation, particularly AF4 or HF5, is performed on at least a portion of a control composition containing control nucleic acid (especially RNA) to fractionate the components contained in the control composition by size to produce one or more control fractions.

[0287] (b') Measure at least one signal selected from UV signal, fluorescence signal and refractive index (RI) signal from at least one of one or more control fractions obtained in step (a');

[0288] (c'1) Calculate the area from the maximum height of a UV, fluorescence, or RI peak to the end of the UV, fluorescence, or RI peak obtained in step (b'), thereby obtaining A. 50％ (Comparison);

[0289] (c'2) Calculate the total area of ​​a peak used in step (c'1) from at least one signal selected from UV, fluorescence, and RI signals obtained in step (b') to obtain A. 100％ (Comparison); and

[0290] (c'3) Determine A 50％ (Comparison) and A 100％ The ratio between (control) and (control) is used to obtain the integrity of the control nucleic acid (especially RNA) (I (control)).

[0291] In this first example, the integrity of nucleic acids (especially RNA) contained in the sample composition can be calculated through the following steps:

[0292] (c1) Calculate the area from the maximum height of the sample UV, fluorescence, or RI peak corresponding to the control UV, fluorescence, or RI peak used in step (c'1) to the end of the sample UV, fluorescence, or RI peak from at least one signal selected from UV, fluorescence, and RI obtained in step (b), thereby obtaining A. 50％ (sample);

[0293] (c2) Calculate the total area of ​​the sample UV, fluorescence, or RI peaks used in step (c1) from the sample UV, fluorescence, or RI signals obtained in step (b), thereby obtaining A. 100％ (sample);

[0294] (c3) Determine A 50％ (sample) and A 100％ The ratio between (samples) is used to obtain I (sample); and

[0295] (c4) Determine the ratio between I (sample) and I (control) to obtain the integrity of the nucleic acids (especially RNA) contained in the sample composition.

[0296] In a second specific instance of this implementation scheme in the third aspect, the integrity of the control nucleic acid (especially RNA) is determined through the following steps:

[0297] (a”) Field-flow fractionation, particularly AF4 or HF5, is performed on at least a portion of a control composition containing control nucleic acid (especially RNA) to fractionate the components contained in the control composition by size to produce one or more control fractions.

[0298] (b”) Measure at least one signal selected from UV, fluorescence, and RI signals from at least one of the one or more control fractions obtained in step (a”); and

[0299] (c”) The height of a UV, fluorescence, or RI peak (H(control)) is determined from at least one signal selected from UV, fluorescence, and RI signals obtained in step (b”) to obtain the integrity of the control nucleic acid (especially RNA).

[0300] In this second example, the integrity of nucleic acids (especially RNA) contained in the sample composition can be calculated through the following steps:

[0301] (c1') Determine 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 UV, fluorescence, and RI signals obtained in step (b); and

[0302] (c2') Determine the ratio between H (sample) and H (control) to obtain the integrity of the nucleic acids (especially RNA) contained in the sample composition.

[0303] In a third specific instance of this implementation scheme of the third aspect (regarding the first subgroup of the third aspect), the integrity of the control nucleic acid (especially RNA) is determined by the following steps:

[0304] (a') Field-flow fractionation, particularly AF4 or HF5, is performed on at least a portion of a control composition containing control nucleic acid (especially RNA) to fractionate the components contained in the control composition by size to produce one or more control fractions.

[0305] (b') Measure the UV signal of at least one of the one or more control fractions obtained from step (a');

[0306] (c'1) Calculate the area from the maximum height of a UV peak to the end of the UV peak using the UV signal obtained in step (b'), thereby obtaining A. 50％ (Comparison);

[0307] (c'2) Calculate the total area of ​​one peak used in step (c'1) from the UV signal obtained in step (b') to obtain A. 100％ (Comparison); and

[0308] (c'3) Determine A 50％ (Comparison) and A 100％ The ratio between (control) and (control) is used to obtain the integrity of the control nucleic acid (especially RNA) (I (control)).

[0309] In this third example, the integrity of nucleic acids (especially RNA) contained in the sample composition can be calculated through the following steps:

[0310] (c1) 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 from the UV signal obtained in step (b), thereby obtaining A. 50％ (sample);

[0311] (c2) Calculate the total area of ​​the sample UV peaks used in step (c1) from the sample UV signal obtained in step (b), thereby obtaining A. 100％ (sample);

[0312] (c3) Determine A 50％ (sample) and A 100％ The ratio between (samples) is used to obtain I (sample); and

[0313] (c4) Determine the ratio between I (sample) and I (control) to obtain the integrity of the nucleic acids (especially RNA) contained in the sample composition.

[0314] In a fourth specific instance of this implementation scheme of the third aspect (regarding the first subgroup of the third aspect), the integrity of the control nucleic acid (especially RNA) is determined by the following steps:

[0315] (a”) Field-flow fractionation, particularly AF4 or HF5, is performed on at least a portion of a control composition containing control nucleic acid (especially RNA) to fractionate the components contained in the control composition by size to produce one or more control fractions.

[0316] (b”) Measure the UV signal of at least one of the one or more control fractions obtained from step (a”); and

[0317] (c”) Determine the height of a UV peak (H(control)) from the UV signal obtained in step (b”) to obtain the integrity of the control nucleic acid (especially RNA).

[0318] In this fourth example, the integrity of nucleic acids (especially RNA) contained in the sample composition can be calculated through the following steps:

[0319] (c1') Determine 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); and

[0320] (c2') Determine the ratio between H (sample) and H (control) to obtain the integrity of the nucleic acids (especially RNA) contained in the sample composition.

[0321] In the fifth specific instance of this implementation scheme of the third aspect (regarding the second subgroup of the third aspect), the integrity of the control RNA is determined by the following steps:

[0322] (a') Field-flow fractionation, particularly AF4 or HF5, is performed on at least a portion of a control composition containing control RNA to fractionate the components contained in the control composition by size to produce one or more control fractions.

[0323] (b') Measure at least one signal selected from UV signal, fluorescence signal and RI signal from at least one of one or more control fractions obtained in step (a');

[0324] (c'1) Calculate the area from the maximum height of a UV, fluorescence, or RI peak to the end of the UV, fluorescence, or RI peak obtained in step (b'), thereby obtaining A. 50％ (Comparison);

[0325] (c'2) Calculate the total area of ​​a peak used in step (c'1) from at least one signal selected from UV, fluorescence, and RI signals obtained in step (b') to obtain A. 100％ (Comparison); and

[0326] (c'3) Determine A 50％ (Comparison) and A 100％ The ratio between (control) and (control) is used to obtain the integrity of the control RNA (I (control)).

[0327] In this fifth example, the integrity of the RNA contained in the sample composition can be calculated through the following steps:

[0328] (c1) Calculate the area from the maximum height of the sample UV, fluorescence, or RI peak corresponding to the control UV, fluorescence, or RI peak used in step (c'1) to the end of the sample UV, fluorescence, or RI peak from at least one signal selected from UV, fluorescence, and RI obtained in step (b), thereby obtaining A. 50％ (sample);

[0329] (c2) Calculate the total area of ​​the sample UV, fluorescence, or RI peaks used in step (c1) from the sample UV, fluorescence, or RI signals obtained in step (b), thereby obtaining A. 100％ (sample);

[0330] (c3) Determine A 50％ (sample) and A 100％ The ratio between (samples) is used to obtain I (sample); and

[0331] (c4) Determine the ratio between I (sample) and I (control) to obtain the integrity of the RNA contained in the sample composition.

[0332] In the sixth specific instance of this implementation scheme of the third aspect (regarding the second subgroup of the third aspect), the integrity of the control RNA is determined by the following steps:

[0333] (a”) Perform field-flow fractionation, particularly AF4 or HF5, on at least a portion of a control composition containing control RNA, thereby fractionating the components contained in the control composition by size to produce one or more control fractions.

[0334] (b”) Measure at least one signal selected from UV, fluorescence, and RI signals from at least one of the one or more control fractions obtained in step (a”); and

[0335] (c”) The height of a UV, fluorescence, or RI peak (H(control)) is determined from at least one signal selected from UV, fluorescence, and RI signals obtained in step (b”) to obtain the integrity of the control RNA.

[0336] In this sixth example, the integrity of the RNA contained in the sample composition can be calculated through the following steps:

[0337] (c1') Determine 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 UV, fluorescence, and RI signals obtained in step (b); and

[0338] (c2') Determine the ratio between H (sample) and H (control) to obtain the integrity of the RNA contained in the sample composition.

[0339] In the seventh specific instance of this implementation scheme of the third aspect (regarding the third subgroup of the third aspect), the integrity of the control RNA is determined by the following steps:

[0340] (a') Field-flow fractionation, particularly AF4 or HF5, is performed on at least a portion of a control composition containing control RNA to fractionate the components contained in the control composition by size to produce one or more control fractions.

[0341] (b') Measure the UV signal of at least one of the one or more control fractions obtained from step (a');

[0342] (c'1) Calculate the area from the maximum height of a UV peak to the end of the UV peak using the UV signal obtained in step (b'), thereby obtaining A. 50％ (Comparison);

[0343] (c'2) Calculate the total area of ​​one peak used in step (c'1) from the UV signal obtained in step (b') to obtain A. 100％ (Comparison); and

[0344] (c'3) Determine A 50％ (Comparison) and A 100％ The ratio between (control) and (control) is used to obtain the integrity of the control RNA (I (control)).

[0345] In this seventh example, the integrity of the RNA contained in the sample composition can be calculated through the following steps:

[0346] (c1) 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 from the UV signal obtained in step (b), thereby obtaining A. 50％ (sample);

[0347] (c2) Calculate the total area of ​​the sample UV peaks used in step (c1) from the sample UV signal obtained in step (b), thereby obtaining A. 100％ (sample);

[0348] (c3) Determine A 50％ (sample) and A 100％ The ratio between (samples) is used to obtain I (sample); and

[0349] (c4) Determine the ratio between I (sample) and I (control) to obtain the integrity of the RNA contained in the sample composition.

[0350] In the eighth specific instance of this implementation scheme of the third aspect (regarding the third subgroup of the third aspect), the integrity of the control RNA is determined by the following steps:

[0351] (a”) Perform field-flow fractionation, particularly AF4 or HF5, on at least a portion of a control composition containing control RNA, thereby fractionating the components contained in the control composition by size to produce one or more control fractions.

[0352] (b”) Measure the UV signal of at least one of the one or more control fractions obtained from step (a”); and

[0353] (c”) Determine the height of a UV peak (H(control)) from the UV signal obtained in step (b”) to obtain the integrity of the control RNA.

[0354] In this eighth example, the integrity of the RNA contained in the sample composition can be calculated through the following steps:

[0355] (c1') Determine 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); and

[0356] (c2') Determine the ratio between H (sample) and H (control) to obtain the integrity of the RNA contained in the sample composition.

[0357] 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) the nucleic acid extinction coefficient (especially the RNA extinction coefficient) or (ii) the nucleic acid calibration curve (especially the RNA calibration curve).

[0358] 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 acids (especially RNA) and particles, such as lipid complex particles and / or lipid nanoparticles and / or polyplex particles and / or lipid polycomplex particles and / or virus-like particles, wherein the nucleic acids (especially RNA) are bound to the particles.

[0359] 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)-(c) with at least the portion obtained from step (i); and (iii) determining the amount of nucleic acid (especially RNA) as described herein (e.g., by using (i) a nucleic acid extinction coefficient (especially an RNA extinction coefficient) or (ii) a nucleic acid calibration curve (especially an RNA calibration curve)). 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.

[0360] In one embodiment of the third aspect (particularly, in one embodiment of the first, second, or third subgroup of the third aspect), the releasing agent is (i) a surfactant, such as anionic surfactants (e.g., sodium dodecyl sulfate), zwitterionic surfactants (e.g., tetradecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate), etc. (3-14)) cationic surfactants, nonionic surfactants or mixtures thereof; (ii) alcohols, such as fatty alcohols (e.g., ethanol), or mixtures of alcohols; or (iii) a combination of (i) and (ii).

[0361] 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: performing steps (a)-(c) without the addition of a releasing agent, particularly in the absence of any releasing agent; and determining the amount of nucleic acid (especially RNA) as described herein (e.g., using (i) the nucleic acid extinction coefficient (especially the RNA extinction coefficient) or (ii) the nucleic acid calibration curve (especially the RNA calibration curve)).

[0362] 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 particle is determined by: subtracting the amount of free nucleic acid (especially RNA) as determined herein from the amount of total nucleic acid (especially RNA) as determined herein (e.g., by treating at least a portion of the sample composition with a releasing agent; (ii) performing steps (a)-(c) with at least the portion obtained from step (i); and (iii) determining the amount of nucleic acid (especially RNA) as described herein (e.g., using (i) a nucleic acid extinction coefficient (especially RNA extinction coefficient) or (ii) a nucleic acid calibration curve (especially RNA calibration curve))).

[0363] 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 an LS signal, such as a dynamic light scattering (DLS) signal and / or a static light scattering (SLS) signal, such as a multi-angle light scattering (MALS) signal, of at least one of the sample fractions obtained from step (a).

[0364] In one embodiment of the third aspect (particularly, in one embodiment of the first, second, or third subgroup of the third aspect), the radius of gyration (R) is calculated from the LS signal obtained from step (b). g ) value and / or hydrodynamic radius (R h The value is used to determine the size of particles containing nucleic acids (especially RNA). In one embodiment of the third aspect (particularly, in one embodiment of the first, second, or third subgroup of the third aspect), step (b) includes measuring the dynamic light scattering (DLS) signal of at least one of the one or more sample fractions obtained from step (a), and step (c) includes calculating R from the DLS signal. h Value. In one embodiment of the third aspect (particularly, in one embodiment of the first, second, or third subgroup of the third aspect), step (b) includes measuring a static light scattering (SLS) signal, such as a MALS signal, of at least one of the one or more sample fractions obtained from step (a), and step (c) includes calculating R from the SLS signal. gValue. In one embodiment of the third aspect (particularly, in one embodiment of the first, second, or third subgroup of the third aspect), step (b) includes measuring a dynamic light scattering (DLS) signal and a static light scattering (SLS) signal, such as a MALS signal, of at least one of the one or more sample fractions obtained from step (a), and step (c) includes calculating R. g and R h Value. This latter implementation produces two datasets of particle size containing nucleic acids (such as RNA), namely, one based on R. g Value, a value based on R h value.

[0365] In one embodiment of the third aspect (particularly, in one embodiment of the first, second, or third subgroup of the third aspect), by applying at least one signal selected from UV signals, fluorescence signals, and RI signals obtained from step (b) to the R signal determined as described herein. g or R h Value (e.g., calculated from the SLS signal obtained in step (b)). g The value or R is calculated from the DLS signal obtained in step (b). h The size distribution of particles containing nucleic acids (especially RNA) is determined by plotting the UV signal obtained from step (b) against the R values ​​as described herein. In a first example of this implementation (the first subgroup of the third aspect), the size distribution of particles containing nucleic acids (especially RNA) is determined by plotting the UV signal obtained from step (b) against the R values ​​as described herein. g or R h Value (e.g., calculated from the SLS signal obtained in step (b)). g The value or R is calculated from the DLS signal obtained in step (b). h The size distribution of particles containing nucleic acids (especially RNA) is determined by plotting the values. In a second example of this embodiment (a second subgroup of the third aspect), the size distribution of particles containing nucleic acids (especially RNA) is determined by plotting the values ​​obtained from step (b) against the values ​​of at least one signal selected from UV signals, fluorescence signals, and RI signals as described herein. g or R h Value (e.g., calculated from the SLS signal obtained in step (b)). g The value or R is calculated from the DLS signal obtained in step (b). h The size distribution of RNA-containing particles was determined by plotting the UV signal obtained from step (b) against the R values ​​as described herein. In a third example of this implementation (a third subgroup concerning the third aspect), the size distribution of RNA-containing particles was determined by plotting the UV signal obtained from step (b) against the R values ​​as described herein. g or R h Value (e.g., calculated from the SLS signal obtained in step (b)). g The value or R is calculated from the DLS signal obtained in step (b). hThe size distribution of RNA-containing particles can be determined by plotting the values. In each of the first, second, and third examples above, the size distribution of RNA-containing particles can be determined by plotting the values. g value, R h The size distribution of particles containing nucleic acids (especially RNA) is determined based on the value or both. If in R g Value and R h Determining the size distribution of particles containing nucleic acids (especially RNA) based on the values ​​will generate two datasets: one based on R... g The magnitude distribution of values ​​and an R-based h Distribution of values.

[0366] In one embodiment of the third aspect (particularly, in one embodiment of the first, second, or third subgroup of the third aspect), by converting UV, fluorescence, or RI signals into cumulative weight fractions and comparing the cumulative weight fractions with R... g or R h Plot the values ​​as R. g or R h The quantitative size distribution of particles containing nucleic acids (especially RNA) is calculated by plotting UV, fluorescence, or RI signals as a function of their values. In a first example of this embodiment (the first subgroup of the third aspect), the UV signal is converted to a cumulative weight fraction and the cumulative weight fraction is plotted against the RI signal. g or R h Plot the values ​​as R. g or R h The quantitative size distribution of particles containing nucleic acids (especially RNA) is calculated by plotting the UV signal as a function of its value. In a second example of this implementation (a second subgroup of the third aspect), the UV, fluorescence, or RI signal is converted into a cumulative weight fraction and the cumulative weight fraction is plotted against the RI signal. g or R h Plot the values ​​as R. g or R h The quantitative size distribution of RNA-containing particles is calculated by plotting UV, fluorescence, or RI signals as a function of their values. In a third example of this embodiment (a third subgroup concerning the third aspect), the UV signal is converted to a cumulative weight fraction and the cumulative weight fraction is plotted against RI. g or R h Plot the values ​​as R. g or R h The quantitative size distribution of RNA-containing particles is calculated from a graph of the UV signal as a function of the value. In each of the first, second, and third examples above, the quantitative size distribution of RNA-containing particles can be calculated from the graph of the UV signal as a function of the value. g value, R h The quantitative size distribution of particles containing nucleic acids (especially RNA) is determined based on the value or both. If in R gValue and R h Determining the quantitative size distribution of particles containing nucleic acids (especially RNA) based on the values ​​will generate two datasets: one based on R... g A quantitative distribution of values ​​and an R-based distribution h The quantitative distribution of values.

[0367] 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 includes D10, D50, and / or D90 values. If in R g Value and R h Determining the quantitative size distribution of particles containing nucleic acids (especially RNA) based on the values ​​will generate two datasets: one based on R... g The values ​​of D10, D50 and / or D90, and a set of R-based values. h The D10, D50, and / or D90 values.

[0368] 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 include (or) at least two, preferably at least three, parameters as specified herein (including additional optional parameters), particularly at least two, preferably at least three, selected from the following parameters: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to the particle, and the size distribution of the particles containing nucleic acid (especially RNA) (particularly based on the radius of gyration (R) of the particles containing nucleic acid (especially RNA)). g The hydrodynamic radius (R) of particles containing nucleic acids (especially RNA) and / or particles containing nucleic acids (especially RNA). h And the quantitative size distribution of particles containing nucleic acids (especially RNA) (e.g., based on R...). g or R h (Value). If in R g Value and R h Determining the size distribution of particles containing nucleic acids (especially RNA) based on the values ​​will generate two datasets: one based on R... g Value, a value based on R h Values. However, according to the present invention, the two datasets of particle size distributions containing nucleic acids (especially RNA) are considered as only one parameter (instead of two parameters). Furthermore, if the classification pattern obtained by field-flow classification shows more than one particle peak, the determination of the size distribution of each particle peak is considered as only one parameter (instead of a parameter for each particle peak). This also applies to R... g Value and R h Based on the values, the quantitative size distribution of particles containing nucleic acids (especially RNA) is determined.

[0369] In one embodiment of the third aspect (particularly, in one embodiment of the first, second, or third subgroup of the third aspect, particularly in a preferred embodiment of the third subgroup of the third aspect), the one or more parameters include a quantitative size distribution of particles containing nucleic acids (especially RNA) (e.g., based on the radius of gyration (R) of particles containing nucleic acids (especially RNA)). g The hydrodynamic radius (R) of particles containing nucleic acids (especially RNA) and / or particles containing nucleic acids (especially RNA). h And at least one of the remaining parameters (including additional optional parameters) specified herein, such as at least two parameters; preferably these remaining parameters are selected from: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to the particle, and the size distribution of the particles containing nucleic acid (especially RNA) (e.g., based on R). g or R h Value). In one embodiment of the third aspect (particularly, in one embodiment of the first, second, or third subgroup of the third aspect, especially in a preferred embodiment of the third subgroup of the third aspect), the one or more parameters include a quantitative size distribution of particles containing nucleic acids (especially RNA) (e.g., based on R). g or R h The value) and at least one parameter selected from the following, such as at least two parameters: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to the particle, and the size distribution of the particles containing nucleic acid (especially RNA) (e.g., based on R). g or R h Value). In one embodiment of the third aspect (particularly, in one embodiment of the first, second, or third subgroup of the third aspect, especially in a preferred embodiment of the third subgroup of the third aspect), the one or more parameters include a quantitative size distribution of particles containing nucleic acids (especially RNA) (e.g., based on R). g or R h The values ​​of free nucleic acids (especially RNA) and the amount of nucleic acids (especially RNA) bound to particles. If in R... g Value and R h Determining the quantitative size distribution of particles containing nucleic acids (especially RNA) based on the values ​​will generate two datasets: one based on R... g Value, a value based on R hValues. However, according to the present invention, the two datasets of quantitative size distribution of particles containing nucleic acids (especially RNA) are considered as only one parameter (instead of two parameters). Furthermore, if the classification pattern obtained by field-flow classification shows more than one particle peak, the determination of the quantitative size distribution of each particle peak is considered as only one parameter (instead of one parameter per particle peak). This also applies to R... g Value and R h Based on the values, the size distribution of particles containing nucleic acids (especially RNA) is determined.

[0370] 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 in a cycle of steps (a)-(c).

[0371] 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), particularly free nucleic acid (especially RNA), is determined by measuring a UV signal, for example, at a wavelength in the range of 260 nm to 280 nm, such as at a wavelength of 260 nm or 280 nm, and using the extinction coefficient of nucleic acid (especially RNA) at the corresponding wavelength (e.g., 260 nm or 280 nm).

[0372] In one embodiment of the third aspect (particularly, in one embodiment of the first, second, or third subgroup of the third aspect, especially in a preferred embodiment of the third subgroup of the third aspect), the size distribution of particles containing nucleic acids (especially RNA) (e.g., based on R...) g or R h The quantitative size distribution of particles containing nucleic acids (especially RNA) and / or R-values ​​(e.g., based on R-values). g or R h The size distribution of the RNA-containing particles is in the range of 10-2000 nm, preferably in the range of 20-1500 nm, such as 30-1200 nm, 40-1100 nm, 50-1000 nm, 60-900 nm, 70-800 nm, 80-700 nm, 90-600 nm, or 100-500 nm, or for example in the range of 10-1000 nm, 15-500 nm, 20-450 nm, 25-400 nm, 30-350 nm, 40-300 nm, or 50-250 nm. In a preferred embodiment of the third subgroup of the third aspect, the (quantitative) size distribution of the RNA-containing particles (e.g., based on R...) g or R hThe value is in the range of 10-1000nm, for example, in the range of 15-500nm, 20-450nm, 25-400nm, 30-350nm, 40-300nm or 50-250nm.

[0373] In one embodiment of the third aspect (particularly, in one embodiment of the first, second, or third subgroup of the third aspect), the nucleic acid (especially RNA) is 10-15,000 nucleotides in length, such as 40-15,000 nucleotides, 100-12,000 nucleotides, or 200-10,000 nucleotides.

[0374] In one embodiment of the third aspect (particularly, in one embodiment of the first subgroup of the third aspect), the nucleic acid is RNA. In this embodiment and in the 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.

[0375] In one embodiment of the third aspect (particularly, in one embodiment of the first, second, or third subgroup of the third aspect), the measurement of at least one signal selected from UV signals, fluorescence signals, and RI signals, optionally LS signals such as SLS, for example, MALS signals, and / or DLS signals, is performed online, and / or step (c) is performed online.

[0376] In one embodiment of the third aspect (particularly, in one embodiment of the first, second, or third subgroup of the third aspect), the at least portion of the sample composition is diluted with a solvent or solvent mixture before field-flow fractionation is performed on the sample composition, the solvent or solvent mixture being capable of preventing the formation of particulate aggregates. In one embodiment, the solvent mixture is a mixture of water and an organic solvent such as formamide.

[0377] In one embodiment of the third aspect (particularly, in one embodiment of the first, second, or third subgroup of the third aspect), the UV signal is measured by using circular dichroism (CD) spectroscopy.

[0378] It should be understood that any implementation described in the context of the first or second aspect may also be applied to any implementation of the third aspect.

[0379] The further implementation plan is as follows:

[0380] 1. A method for determining one or more parameters of a sample composition, wherein the sample composition comprises RNA and optionally present particles, the method comprising:

[0381] (a) Perform field-flow fractionation on at least a portion of the sample composition to fractionate the components contained in the sample composition by size to produce one or more sample fractions;

[0382] (b) Measure at least the UV signal and optionally the light scattering (LS) signal of at least one of the one or more sample fractions obtained in step (a); and

[0383] (c) Calculate one or more parameters from the UV signal and optionally from the LS signal.

[0384] The parameters mentioned above include RNA integrity, total RNA amount, 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.

[0385] 2. The method of Project 1, wherein the field-flow classification is a flow field-flow classification, such as asymmetric flow field-flow classification (AF4) or hollow fiber flow field-flow classification (HF5).

[0386] 3. The method of Project 1 or 2, wherein step (a) is performed using a membrane having a molecular weight cutoff (MW) suitable for preventing RNA permeation, preferably a membrane with a MW cutoff in the range of 2kDa-30kDa, such as a membrane with a MW cutoff of 10kDa.

[0387] 4. The method of any one of items 1-3, wherein step (a) is performed using polyethersulfone (PES) or a regenerated cellulose membrane.

[0388] 5. The method of any one of items 1-4, wherein step (a) is performed using a crossflow rate of up to 8 mL / min, preferably up to 4 mL / min, more preferably up to 2 mL / min.

[0389] 6. The method of any one of items 1-5, wherein step (a) is performed using the following cross-flow rate spectrum: 1.0-2.0 mL / min for 10 min, an exponential gradient from 1.0-2.0 mL / min to 0.01-0.07 mL / min over 30 min; 0.01-0.07 mL / min for 30 min; and 0 mL / min for 10 min.

[0390] 7. The method of any one of items 1-6, wherein step (a) is performed using an injection flow rate in the range of 0.05-0.35 mL / min, preferably in the range of 0.10-0.30 mL / min, and more preferably in the range of 0.15-0.25 mL / min.

[0391] 8. The method of any one of items 1-7, wherein step (a) is performed using a detector flow rate in the range of 0.30-0.70 mL / min, preferably in the range of 0.40-0.60 mL / min, more preferably in the range of 0.45-0.55 mL / min.

[0392] 9. The method of any one of items 1-8, wherein the integrity of the RNA contained in the sample composition is calculated using the integrity of control RNA.

[0393] 10. The method of Project 9, wherein the integrity of the control RNA is determined by the following steps:

[0394] (a') Field-flow fractionation, particularly AF4 or HF5, is performed on at least a portion of a control composition containing control RNA to fractionate the components contained in the control composition by size to produce one or more control fractions.

[0395] (b') Measure the UV signal of at least one of the one or more control fractions obtained from step (a');

[0396] (c'1) Calculate the area from the maximum height of a UV peak to the end of the UV peak using the UV signal obtained in step (b'), thereby obtaining A. 50％ (Comparison);

[0397] (c'2) Calculate the total area of ​​one peak used in step (c'1) from the UV signal obtained in step (b') to obtain A. 100％ (Comparison); and

[0398] (c'3) Determine A 50％ (Comparison) and A 100％ The ratio between (control) and (control) is used to obtain the integrity of the control RNA (I (control)).

[0399] 11. The method of Item 10, wherein the integrity of RNA contained in the sample composition is calculated by the following steps:

[0400] (c1) 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 from the sample UV signal obtained in step (b), thereby obtaining A. 50％ (sample);

[0401] (c2) Calculate the total area of ​​the sample UV peaks used in step (c1) from the sample UV signal obtained in step (b), thereby obtaining A. 100％ (sample);

[0402] (c3) Determine A 50％(sample) and A 100％ The ratio between (samples) is used to obtain I (sample); and

[0403] (c4) Determine the ratio between I (sample) and I (control) to obtain the integrity of the RNA contained in the sample composition.

[0404] 12. The method of Project 9, wherein the integrity of the calculated control RNA is determined by the following steps:

[0405] (a”) Perform field-flow fractionation, particularly AF4 or HF5, on at least a portion of a control composition containing control RNA, thereby fractionating the components contained in the control composition by size to produce one or more control fractions.

[0406] (b”) Measure the UV signal of at least one of the one or more control fractions obtained from step (a”); and

[0407] (c”) Determine the height of a UV peak (H(control)) from the UV signal obtained in step (b”) to obtain the integrity of the control RNA.

[0408] 13. The method of Item 12, wherein the integrity of the RNA contained in the sample composition is calculated by the following steps:

[0409] (c1') Determine 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); and

[0410] (c2') Determine the ratio between H (sample) and H (control) to obtain the integrity of the RNA contained in the sample composition.

[0411] 14. The method of any one of items 1-13, wherein the amount of RNA is determined by using (i) RNA extinction coefficient or (ii) RNA calibration curve.

[0412] 15. The method of any one of items 1-14, wherein the sample composition comprises RNA and particles, such as lipid complex particles and / or lipid nanoparticles and / or polyplex particles and / or lipid polycomplex particles and / or virus-like particles, wherein the RNA binds to the particles.

[0413] 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)-(c) with at least the portion obtained from step (i); and (iii) determining the amount of RNA as described in Item 14.

[0414] 17. The method of Item 16, wherein in step (a) a field-flow classification is performed using a liquid phase containing a releasing agent.

[0415] 18. The method of item 16 or 17, wherein the releasing agent is (i) a surfactant, such as anionic surfactant (e.g., sodium dodecyl sulfate), amphoteric surfactant (e.g., tetradecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate), or (…). (3-14)) cationic surfactants, nonionic surfactants or mixtures thereof; (ii) alcohols, such as fatty alcohols (e.g., ethanol), or mixtures of alcohols; or (iii) a combination of (i) and (ii).

[0416] 19. The method of any one of items 15-18, wherein the amount of free RNA is determined by: performing steps (a)-(c) without the addition of a releasing agent, particularly in the absence of any releasing agent; and determining the amount of RNA as described in item 14.

[0417] 20. The method of any one of items 15-19, wherein the amount of RNA bound to the particle is determined by subtracting the amount of free RNA as determined in item 19 from the amount of total RNA as determined in any one of items 16-18.

[0418] 21. The method of any one of items 15-20, wherein step (b) further comprises measuring an LS signal, such as a dynamic light scattering (DLS) signal and / or a static light scattering (SLS) signal, such as a multi-angle light scattering (MALS) signal, of at least one of the sample fractions obtained from step (a).

[0419] 22. The method of Project 21, wherein the radius of gyration (R) is calculated from the LS signal obtained from step (b). g ) value and / or hydrodynamic radius (R h The value is used to determine the size of particles containing RNA.

[0420] 23. The method of Project 21, wherein the experimentally determined R g and / or R h Value smoothing, preferably by experimentally determined or calculated R... g or R h The values ​​are fitted to a polynomial or linear function, and R is recalculated based on the polynomial or linear fit. g or R h value.

[0421] 24. The method of any one of items 21-23, wherein the UV signal obtained from step (b) is compared with the R determined as described in item 22. g or R hPlot the values ​​to determine the size distribution of RNA-containing particles.

[0422] 25. The method of any one of items 21-24, wherein the UV signal is converted into a cumulative weight fraction and the cumulative weight fraction is plotted against R. g or R h Plot the values ​​as R. g The quantitative size distribution of RNA-containing particles was calculated from the UV signal plot as a function of the value.

[0423] 26. The method of Item 25, wherein the quantitative size distribution includes D10, D50 and / or D90 values.

[0424] 27. The method of any one of items 22-26, wherein step (b) comprises measuring a dynamic light scattering (DLS) signal of at least one of one or more sample fractions obtained in step (a), and step (c) comprises calculating R from said DLS signal. h value.

[0425] 28. The method of any one of items 15-27, wherein the one or more parameters comprise (or) at least two, preferably at least three, selected from the following parameters: the amount of free RNA, the amount of RNA bound to the particle, the size distribution of the RNA-containing particle, and the quantitative size distribution of the RNA-containing particle.

[0426] 29. The method of any one of items 15-28, wherein the amount of RNA, particularly free RNA, is determined by measuring a UV signal at 260 nm and using an RNA extinction coefficient at 260 nm or by measuring a UV signal at 280 nm and using an RNA extinction coefficient at 280 nm.

[0427] 30. The method of any one of items 1-29, wherein the size distribution of the RNA-containing particles and / or the quantitative size distribution of the RNA-containing particles are in the range of 20-1500 nm, such as 30-1200 nm, 40-1100 nm, 50-1000 nm, 60-900 nm, 70-800 nm, 80-700 nm, 90-600 nm or 100-500 nm, such as in the range of 10-1000 nm, 15-500 nm, 20-450 nm, 25-400 nm, 30-350 nm, 40-300 nm or 50-250 nm.

[0428] 31. The method of any one of items 1-30, wherein the length of said RNA is 10-15,000 nucleotides, such as 40-15,000 nucleotides, 100-12,000 nucleotides or 200-10,000 nucleotides.

[0429] 32. The method of any one of items 1-31, wherein the RNA is in vitro transcribed RNA, particularly in vitro transcribed mRNA.

[0430] 33. The method of any one of items 1-32, wherein the measurement of the UV signal, optionally the LS signal, such as SLS, for example, the MALS signal and / or the DLS signal, is performed online, and / or step (c) is performed online.

[0431] 34. The method of any one of items 15-33, wherein, prior to performing field-flow classification on at least a portion of the sample composition, the at least a portion of the sample composition is diluted with a solvent or solvent mixture capable of preventing the formation of particulate aggregates.

[0432] 35. The method of item 34, wherein the solvent mixture is a mixture of water and an organic solvent such as formamide.

[0433] 35a. The method of any one of items 1-35, wherein the UV signal is measured by using circular dichroism (CD) spectroscopy.

[0434] 36. A method for analyzing the effect of changing one or more reaction conditions when providing a composition comprising RNA and optionally present particles, the method comprising:

[0435] (A) Provides a first composition comprising RNA and optionally present particles;

[0436] (B) Provide a second composition comprising RNA and optionally present particles, wherein the provision of the second composition differs from the provision of the first composition only in one or more reaction conditions;

[0437] (C) Performing a portion of the first composition on any one of items 1-35 and 35a to determine one or more parameters of the first composition;

[0438] (D) subjecting a corresponding portion of the second composition to the method used in step (C) to determine one or more parameters of the second composition; and

[0439] (E) Compare one or more parameters of the first composition obtained in step (C) with one or more corresponding parameters of the second composition obtained in step (D).

[0440] 37. The method of Item 36, wherein said one or more reaction conditions include any one 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 cycle; dry / recovery cycle; addition of excipients (e.g., stabilizers and / or chelating agents); type and / or source of particle-forming compounds (particularly lipids and / or polymers, e.g., cationic lipids to cationic polymers, cationic lipids to zwitterionic lipids, or polyethylene glycol-modified lipids to non-polyethylene glycol-modified lipids); charge ratio; physical state; and the ratio of RNA to particle-forming compounds (particularly lipids and / or polymers).

[0441] 38. Use of field-fluid fractionation in determining one or more parameters of a sample composition comprising RNA and optionally present 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.

[0442] 39. Application of Item 38, wherein the field-flow classification includes:

[0443] (a) Perform field-flow fractionation on at least a portion of the sample composition to fractionate the components contained in the sample composition by size to produce one or more sample fractions;

[0444] (b) Measure at least the UV signal and optionally the light scattering (LS) signal of at least one of the one or more sample fractions obtained in step (a); and

[0445] (c) Calculate one or more parameters from the UV signal and optionally from the LS signal.

[0446] 40. Applications of items 38 or 39, wherein the field-flow classification is a flow field-flow classification, such as asymmetric flow field-flow classification (AF4) or hollow fiber flow field-flow classification (HF5).

[0447] 41. Use of any one of items 38-40, wherein the field-flow fractionation uses a membrane having a molecular weight cutoff (MW) suitable for preventing RNA permeation, preferably a membrane with a MW cutoff in the range of 2 kDa to 30 kDa, such as a membrane with a MW cutoff of 10 kDa.

[0448] 42. Use of any of items 38-41, wherein the field-flow classification uses a polyethersulfone (PES) or regenerated cellulose membrane.

[0449] 43. The use of any one of items 39-42, wherein step (a) is performed using the following:

[0450] (I) Cross-flow rates up to 8 mL / min, preferably up to 4 mL / min, more preferably up to 2 mL / min, with the following cross-flow rate profiles: 1.0–2.0 mL / min for 10 min, followed by an exponential gradient from 1.0–2.0 mL / min to 0.01–0.07 mL / min over 30 min; 0.01–0.07 mL / min for 30 min; and 0 mL / min for 10 min; and / or

[0451] (II) An injection flow rate in the range of 0.05-0.35 mL / min, preferably in the range of 0.10-0.30 mL / min, and more preferably in the range of 0.15-0.25 mL / min; and / or

[0452] (III) The detector flow rate is in the range of 0.30-0.70 mL / min, preferably in the range of 0.40-0.60 mL / min, and more preferably in the range of 0.45-0.55 mL / min.

[0453] 44. The use of any one of items 38-43, wherein the integrity of RNA contained in the sample composition is determined by using the integrity of control RNA.

[0454] 45. The purpose of item 44, which involves determining the integrity of control RNA through the following steps:

[0455] (a') Field-flow fractionation, particularly AF4 or HF5, is performed on at least a portion of a control composition containing control RNA to fractionate the components contained in the control composition by size to produce one or more control fractions.

[0456] (b') Measure the UV signal of at least one of the one or more control fractions obtained from step (a');

[0457] (c'1) Calculate the area from the maximum height of a UV peak to the end of the UV peak using the UV signal obtained in step (b'), thereby obtaining A. 50％ (Comparison);

[0458] (c'2) Calculate the total area of ​​one peak used in step (c'1) from the UV signal obtained in step (b') to obtain A. 100％ (Comparison); and

[0459] (c'3) Determine A 50％ (Comparison) and A 100％ The ratio between (control) and (control) is used to obtain the integrity of the control RNA (I (control)).

[0460] 46. ​​The use of item 45, wherein the integrity of the RNA contained in the sample composition is calculated by the following steps:

[0461] (c1) 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 from the sample UV signal obtained in step (b), thereby obtaining A. 50％ (sample);

[0462] (c2) Calculate the total area of ​​the sample UV peaks used in step (c1) from the sample UV signal obtained in step (b), thereby obtaining A. 100％ (sample);

[0463] (c3) Determine A 50％ (sample) and A 100％ The ratio between (samples) is used to obtain I (sample); and

[0464] (c4) Determine the ratio between I (sample) and I (control) to obtain the integrity of the RNA contained in the sample composition.

[0465] 47. The purpose of item 44, in which the integrity of the calculated control RNA is determined through the following steps:

[0466] (a”) Perform field-flow fractionation, particularly AF4 or HF5, on at least a portion of a control composition containing control RNA, thereby fractionating the components contained in the control composition by size to produce one or more control fractions.

[0467] (b”) Measure the UV signal of at least one of the one or more control fractions obtained from step (a”); and

[0468] (c”) Determine the height of a UV peak (H(control)) from the UV signal obtained in step (b”) to obtain the integrity of the control RNA.

[0469] 48. The use of item 47, wherein the integrity of the RNA contained in the sample composition is calculated by the following steps:

[0470] (c1') Determine 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); and

[0471] (c2') Determine the ratio between H (sample) and H (control) to obtain the integrity of the RNA contained in the sample composition.

[0472] 49. The use of any one of items 38-48, wherein the amount of RNA is determined by using (i) RNA extinction coefficient or (ii) RNA calibration curve.

[0473] 50. Use of any one of items 39-49, wherein the sample composition comprises RNA and particles, such as lipid complex particles and / or lipid nanoparticles and / or polyplex particles and / or lipid polycomplex particles and / or virus-like particles, wherein RNA is bound to the particles and / or the particles contain RNA.

[0474] 51. The use of item 50, wherein the amount of total RNA is determined by: (i) treating at least a portion of the sample composition with a releasing agent; (ii) performing steps (a)-(c) with at least the portion obtained from step (i); and (iii) determining the amount of RNA as described in item 49.

[0475] 52. The use of item 51, wherein a liquid phase containing a releasing agent is used in step (a) for field-flow classification.

[0476] 53. Use of item 51 or 52, wherein the releasing agent is (i) a surfactant, such as anionic surfactant (e.g., sodium dodecyl sulfate), amphoteric surfactant (e.g., tetradecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate), or (…). (3-14)) cationic surfactants, nonionic surfactants or mixtures thereof; (ii) alcohols, such as fatty alcohols (e.g., ethanol), or mixtures of alcohols; or (iii) a combination of (i) and (ii).

[0477] 54. The use of any one of items 50-53, wherein the amount of free RNA is determined by: performing steps (a)-(c) without the addition of a releasing agent, particularly in the absence of any releasing agent; and determining the amount of RNA as described in item 49.

[0478] 55. The use of any of items 50-54, wherein the amount of RNA bound to the particle is determined by subtracting the amount of free RNA as determined in item 53 from the amount of total RNA as determined in any of items 51-53.

[0479] 56. The use of any one of items 50-55, wherein step (b) further comprises measuring an LS signal, such as a dynamic light scattering (DLS) signal and / or a static light scattering (SLS) signal, such as a multi-angle light scattering (MALS) signal, of at least one of the sample fractions obtained from step (a).

[0480] 57. The purpose of item 56, in which the radius of gyration (R) is calculated from the LS signal obtained from step (b). g ) value and / or hydrodynamic radius (R h The value is used to determine the size of particles containing RNA.

[0481] 58. The purpose of item 57, in which R is determined experimentally. g and / or R h Value smoothing, preferably by experimentally determined or calculated R... g or R h The values ​​are fitted to a polynomial or linear function, and R is recalculated based on the polynomial or linear fit. g or R h value.

[0482] 59. Use of any one of items 56-58, wherein the UV signal obtained from step (b) is compared with the R determined as described in item 57. g or R h Plot the values ​​to determine the size distribution of RNA-containing particles.

[0483] 60. The use of any one of items 56-59, wherein the UV signal is converted into a cumulative weight fraction and the cumulative weight fraction is compared with R. g or R h Plot the values ​​as R. g or R h The quantitative size distribution of RNA-containing particles was calculated from the UV signal plot as a function of the value.

[0484] 61. Use of Item 60, wherein the quantitative size distribution includes D10, D50 and / or D90 values.

[0485] 62. The use of any one of items 57-61, wherein step (b) further comprises measuring a dynamic light scattering (DLS) signal of at least one of one or more sample fractions obtained from step (a), and step (c) comprises calculating R from said DLS signal. h value.

[0486] 63. Use of any one of items 50-62, wherein the one or more parameters comprise (or) at least two, preferably at least three, selected from the following parameters: the amount of free RNA, the amount of RNA bound to the particle, the size distribution of the RNA-containing particle, and the quantitative size distribution of the RNA-containing particle.

[0487] 64. The use of any one of items 50-63, wherein the amount of RNA, particularly free RNA, is determined by measuring a UV signal at 260 nm and using an RNA extinction coefficient at 260 nm or by measuring a UV signal at 280 nm and using an RNA extinction coefficient at 280 nm.

[0488] 65. The use of any one of items 38-64, wherein the size distribution of RNA-containing particles and / or the quantitative size distribution of RNA-containing particles are in the range of 20-1500 nm, such as 30-1200 nm, 40-1100 nm, 50-1000 nm, 60-900 nm, 70-800 nm, 80-700 nm, 90-600 nm or 100-500 nm, such as in the range of 10-1000 nm, 15-500 nm, 20-450 nm, 25-400 nm, 30-350 nm, 40-300 nm or 50-250 nm.

[0489] 66. Use of any of items 38-64, wherein the length of said RNA is 10-15,000 nucleotides, such as 40-15,000 nucleotides, 100-12,000 nucleotides or 200-10,000 nucleotides.

[0490] 67. Use of any one of items 38-65, wherein the RNA is in vitro transcribed RNA, particularly in vitro transcribed mRNA.

[0491] 68. The use of any of items 39-67, wherein the measurement of the UV signal, optionally the LS signal, such as SLS, for example, the MALS signal and / or the DLS signal, is performed online, and / or step (c) is performed online.

[0492] 69. The use of any one of items 39-68, wherein, prior to field-flow fractionation of at least a portion of the sample composition, the at least portion of the sample composition is diluted with a solvent or solvent mixture capable of preventing the formation of particulate aggregates.

[0493] 70. Use of item 69, wherein the solvent mixture is a mixture of water and an organic solvent such as formamide.

[0494] 70a. The use of any of items 39-70, wherein the UV signal is measured by means of circular dichroism (CD) spectroscopy.

[0495] In a fourth aspect, this disclosure provides a data processing apparatus / system comprising means for performing any method of this disclosure, particularly the methods of the first aspect (e.g., the methods defined in any of items 1-35 and 35a) and / or the methods of the second aspect (e.g., the methods defined in items 36 or 37).

[0496] In a fifth aspect, this disclosure provides a computer program suitable for performing any method of this disclosure, particularly the method of the first aspect (e.g., the method as defined in any of items 1-35 and 35a) and / or the method of the second aspect (e.g., the method as defined in item 36 or 37).

[0497] In a sixth aspect, this disclosure provides a computer-readable storage medium or data carrier that includes the program of the fifth aspect of this disclosure.

[0498] Other aspects of this disclosure are disclosed herein. Attached Figure Description

[0499] Figure 1 The time-flow spectrum of asymmetric flow field-flow classification (AF4) separation is shown, where the detector flow rate (Vd) is 0.5 mL / min, the crossflow (Vx) starts at 1.5 mL / min and decreases exponentially to 0.04 mL / min.

[0500] Figure 2 This diagram illustrates an overview of a preferred computational procedure for estimating relative RNA integrity. An example of an AF4 gradation pattern displayed using a UV signal at 260 nm after RNA isolation is shown. A) Limits for calculations of control RNA. B) Limits for calculations of the total RNA peak area. C) Limits for calculations of slightly degraded RNA. D) Limits for calculations of the peak area of ​​total slightly degraded RNA.

[0501] Figure 3 The following describes RNA quantification without the use of standards: A) Analysis of RNA stock solutions with different injection volumes using AF4-UV-RI. Plot the peak area under the curve (solid UV line; dashed RI line) against the injection volume and fit a linear regression. B) Measure successive dilutions of RNA using the same injection volume using AF4-UV-RI and analyze as in A).

[0502] Figure 4 shows the quantification of degraded RNA using AF4-UV-RI without standards: A) Representative AF4 grading maps of different thermally degraded RNAs and untreated RNA are shown (curves represent UV signals at 260 nm). B) Applying Lambert-Beer's law, the UV signals of different degraded RNAs are directly correlated with concentration.

[0503] Figure 5 Representative grading patterns are shown for sample particle compositions (containing lipids and RNA in a molar ratio of 1.3 / 2) separated by the AF4 method disclosed herein. Solid lines represent light scattering (LS) signals at a 90° angle and indicate particle peaks (t = ~35 min), while dashed lines represent UV signals (recorded at 260 nm) and reflect bound (t = ~38 min) and unbound RNA (t = ~20 min).

[0504] Figure 6 illustrates quantitative RNA integrity measurements of unprepared RNA: A) Representative AF4 grading profiles of different thermally degraded RNAs (n=3; curves represent UV signals at 260 nm). B) Summary of the average calculated relative integrity of four thermally degraded RNAs of different lengths (RNA#1-4; size: 986-1688 nt). Error bars represent standard deviations (n=3). C) Representative AF4 grading profiles obtained from RNA#2 using different proportions (untreated, fully thermally degraded, and a 50:50 mixture of untreated and fully thermally degraded RNA). D) Validation experiment: different RNAs (RNA#1-3, 986-1688 nt) were thermally degraded and mixed in a prescribed manner (for the AF4 grading profile of mixture 4, see...). Figure 6C The results were analyzed using AF4-UV measurements. The bar graph represents the relative RNA integrity determined using the AF4 method disclosed herein (dark, medium, and light gray bars) compared to theoretically calculated values ​​(black bars).

[0505] Figure 7 demonstrates the applicability of UV signaling for RNA quantification compared to using fluorescent dyes (proof of concept). A) The UV peak and fluorescence (FS) integral of the sample composition (containing RNA and fluorescently labeled particles) are correlated with the corresponding total RNA amount in the sample composition. B) It was found that the calculated ratio of UV to FS peak areas of the sample composition is constant over a wide mass range (1–15 μg total RNA in the sample composition).

[0506] Figure 8 shows the UV ratio as a parameter of the RNA sample composition. A) The UV peak integrals of free RNA and particle-bound RNA are related to the corresponding nominal total RNA content in the sample composition. B) Calculation of the UV ratio of free RNA (peak) and bound RNA (peak).

[0507] Figure 9 illustrates a proof-of-concept for quantifying particle size distribution using AF4-UV-MALS. A) Representative AF4 gradation plots of the sample composition (RNA and Atto594-labeled particles): Dashed lines represent UV traces recorded at 260 nm, and solid lines represent fluorescence signals (FS) emitted at 624 nm. B) Calculation of the UV / FS ratio (dashed line) and its modulus against the radius of gyration (R). g Plot the UV signal (highlighted gray peak) from the recorded particle peak fraction (elution time: 22–60 min) and the UV signal from the recorded particle peak fraction (elution time: 22–60 min). Plot the R value at a change of less than 50% of the UV / FS ratio. g Area highlighted (box). R between 50 and 300 nm g Within the range, the UV / FS ratio varies little and provides reliable magnitude values. Smaller R... g The value is affected by RNA signaling. A larger R value... g The value is affected by scattering. In general, these affected R values... g The value is less than 10% of the total signal. C) Quantitative quality parameters (D10, D50, D90) are calculated based on cumulative weight fraction analysis using fluorescence emission at 624 nm and UV signal at 260 nm.

[0508] Figure 10 Representative AF4 gradation spectra of the sample composition (RNA and particles) are shown, detected using LS signal at 90° and UV at 260 nm. The calculated radius of gyration (Ry) is also shown. g The values ​​(gray squares) are derived from multi-angle light scattering (MALS) using a Berry plot, while the hydrodynamic radius (R) is derived from the hydrodynamic radius. h The value is derived from online dynamic light scattering (DLS; gray circle).

[0509] Figure 11 This illustrates the quantification of particle size distribution in a complex sample composition using AF4-UV-MALS. A) shows the AF4-UV-MALS elution spectrum of the sample composition (RNA and particles), the UV signal at 260 nm for RNA detection (dashed line), and the light scattering signal at 90° (solid line). The corresponding radius of gyration (R) from the MALS signal is also shown. g A) Values ​​are displayed as black dots. B) The experimentally determined RMS values ​​of the particle peaks (elution time: 26-55 min) are fitted to a polynomial equation (light gray line). C) The UV signal (solid line) is plotted as the RMS value of the polynomial fit. g Functions of value (see) Figure 11 B), and plot the corresponding cumulative weight fraction as a function of the UV signal (dashed line).

[0510] Figure 12 illustrates the separation and qualitative analysis of different sample compositions (prepared by mixing lipids and RNA at different lipid / RNA ratios (0.1–0.9) with 100 mM NaCl) using the AF4 method disclosed herein. A) For each different sample composition, the UV signal (at 260 nm), light scattering signal (at 90°), and the corresponding radius of gyration (R) calculated using the Berry plot are superimposed and displayed. g B) The R value calculated from the MALS signal. g Plot the values ​​against the appropriate cumulative weight fraction analysis, and then calculate the corresponding D90 values. C) Plot the R values ​​derived from the cumulative weight fraction analysis. g The (D90) value is plotted as a function of the lipid / RNA ratio with 100 mM NaCl (black dots) or no NaCl (hollow dots).

[0511] Figure 13 This shows how the hydrodynamic radius (R) is used to illustrate the process. h ) value and R g Values ​​are correlated to estimate the "shape factor". These values ​​are suitable for linear regression, and the resulting slope provides information about the particle shape.

[0512] Figure 14 This paper illustrates the separation and characterization of different particle compositions (LPX, LNP, polyplex particles (PLX), liposomes, VLPs+LPX) using the AF4 method disclosed herein. The separation / detection is shown as AF4-UV-MALS-DLS. LS at a 90° angle is displayed as a solid line, representing the particle peak. The dashed line represents the UV signal recorded at 260 nm (for RNA detection). Radius gyration (R) g The values ​​(black dots) are derived from the multi-angle light scattering (MALS) signal using a Zimm plot. Dynamic light scattering (DLS; gray dots) provides the hydrodynamic radius (R). hIndividual particle peak fractions are highlighted by gray bars. A) Representative fractionation map of an LPX sample containing lipids and RNA in a molar ratio of 1.3 / 2 after AF4-UV-MALS-DLS separation / detection. B) Representative fractionation map of a composition containing two types of particles (short RNA-LPX:VLP, 1:1 mixture). C) Representative fractionation map of a liposome sample (positively charged liposomes composed of DOTMA and DOPE in a molar ratio of 2 / 1). D) Representative fractionation map of an LPX sample (positively charged LPX containing DOTMA and cholesterol, and RNA in a molar ratio of 4 / 1). E) Representative fractionation map of a lipid nanoparticle (LNP) sample composed of DODMA, cholesterol, DOPE, PEG (molar ratio of 1.2 / 1.44 / 0.3 / 0.06) and RNA in a molar ratio of 3 / 1. F) A representative hierarchical map of particles containing JetPEI polymer and IVT-RNA or saRNA, with a particle-to-RNA ratio of 12 / 1.

[0513] Figure 15 Analysis of RNA behavior in the presence of ions (sodium chloride) is shown. Exemplary AF4 gradation maps of unprepared RNA from different sodium chloride concentrations (0-50 mM) are shown (showing light scattering signals at 90°). Radius of gyration (R) g The value is derived from multi-angle light scattering (MALS) using the Zimm plot.

[0514] Figure 16 shows the characterization of RNA after treatment with sodium chloride. A) shows the RNA from cumulative weight fraction analysis at different sodium chloride concentrations (0-50 mM). g (D50) value. B) RNAR g (D50) value (from) Figure 16A Plot the sodium chloride concentration against the ratio (mM sodium chloride to nm R) and calculate the proportion. g The linear fit of the proportions of NaCl values ​​from 0 to 10 mM is represented by a thick line, while the dotted line represents the fit from 10 to 50 mM NaCl. Gray and black lines represent instances measured with two different RNA concentrations.

[0515] Figure 17This paper illustrates 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 in particle-free compositions by UV absorption at 260 nm. RNA amounts were plotted against their respective UV peak areas (AUC*min) to generate linear calibration curves. B) Different amounts of particulate compositions (containing 1–15 μg of total RNA) were analyzed using the AF4 method. The superimposed AF4 hierarchical plots show the UV signal at 260 nm. The first peak (elution time: ~20 min) corresponds to free RNA, while the second peak (elution time: ~38 min) corresponds to particles (bound RNA). The amounts of free and unbound RNA in the particulate compositions can be calculated relative to reference RNA (=100%) (see [link to AF4 method]). Figure 17 A). C) To demonstrate the linearity of this method, free RNA (see Figure 17 B) and reference, naked RNA (see Figure 17 A) The UV peak integral plot is a function of different RNA amounts (1-15 μg). D) As a second, preferred procedure (direct method) for quantifying free RNA, it defines the unbound RNA peak and can directly calculate the RNA amount using the specific extinction coefficient of RNA.

[0516] Figure 18 The analysis of free RNA amounts in sample compositions with different physicochemical behaviors is shown. A) AF4-UV grading plots of particulate compositions without NaCl ((DOTMA / DOPE 2 / 1) / RNA complexes mixed in variable charge ratios (0.1–0.9)) or B) particulate compositions with 100 mM NaCl are shown. C) A plot showing the calculated percentage (mol / mol) of unbound RNA with 100 mM NaCl (black circles) and without NaCl (hollow circles) using AF4-UV detection at 260 nm. All mixtures were prepared in duplicate and measured at least twice. Error bars represent standard deviations. D) A plot showing the concentration (μg / mL) of unbound RNA with 100 mM NaCl (black circles) and without NaCl (hollow circles) calculated using the extinction coefficient of RNA at 260 nm.

[0517] Figure 19The quantification of total RNA in the particle composition is shown. A) AF4 grading map of Zwittergent-treated naked RNA isolated 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 grading map of the particle composition with UV detection (solid line), free RNA (highlighted in gray) and bound RNA (second peak), and LS signal at 90° (dashed line). C) Corresponding AF4 grading map of the RNA composition, in which the particles have been dissolved with a releasing agent (liquid phase containing 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 the releasing agent (Zwittergent).

[0518] Figure 20 shows the integrity of free RNA and total RNA in a sample composition containing RNA and particles. A) UV traces of particles separated using the AF4 method disclosed herein, where the particles have varying RNA integrity (untreated RNA: solid black line; partially thermally degraded RNA: dotted line; mixture (50% untreated and 50% fully degraded in a defined manner): dashed line; fully degraded RNA in particles: solid gray line). B) Quantification of intact free RNA (dark gray) and total free RNA (black) and fully degraded (light gray) free RNA in particles. C) UV traces of particles dissolved after AF4 separation (using a releasing agent in liquid phase). D) Determined integrity of free and total RNA in particles as measured by AF4-UV. The bar graph represents the relative RNA integrity (gray bars) of free RNA compared to the determined integrity (black bars) of total RNA in particles.

[0519] Figure 21 This document illustrates a scheme for determining different fractions of RNA (total, bound, encapsulated, accessible, surface, and unbound RNA) using the AF4 method disclosed herein. For example, the AF4 method can be used to quantify accessible and / or surface RNA using the fluorescence emission signal of an intercalating dye (e.g., GelRED). A quantitative combination of free (unbound), total, and accessible RNA can be used to calculate encapsulated, bound, and surface RNA. GelRED fluorescence emission at 600 nm is enhanced by intercalating RNA.

[0520] Figure 22 shows (A) linearity of fluorescence detection using the AF4 method disclosed herein; (B) a bar graph showing the relative amounts of accessible (black bars) and encapsulated (gray bars) RNA; and (C) a comparison of the relative amounts of free RNA in the particulate composition, wherein the amounts have been determined using different RNA detection methods: UV absorption at 260 nm (black bars) and fluorescence emission signal (FS) at 600 nm (gray bars).

[0521] Figure 23 This illustrates the analysis of RNA integrity using the AF4 method disclosed herein without the use of a reference RNA. A) Shows an exemplary AF4 grading map of a long saRNA with an LS signal at 90° (dotted line) and a UV signal at 260 nm (solid line). The thick black line represents the molecular weight curve derived from the MALS signal. B) For a better overview, in Figure 23 In the upper plot of B, only the molecular weight curve from (A) is shown as a solid line. The limit for the total RNA peak (peak 1) is set based on the total UV peak signal (i.e., from t = 10 min to t = 40 min). Here, the limit for the "complete" RNA peak (peak 2) is set by the first derivative of the molecular weight curve (derived from MALS). The first derivative of the molecular weight curve is calculated (…). Figure 23 (B. Dotted line in the figure below). The more horizontal portion of the molecular weight curve reflects retention time, where the fraction of undegraded RNA exists. Based on this, the integration limit can be selected, and the amount of undegraded RNA in the sample can be calculated.

[0522] Figure 24 illustrates the quantitative analysis of free and bound RNA, using UV to determine particle size distribution, particularly the cumulative RNA weight fraction, RNA mass in RNA-lipid complex (LPX) fractions, and RNA copy number per LPX fraction. A) shows a representative AF4 fractionation pattern of the RNALPX sample composition with LS signal at 90° (solid line) and UV signal at 260 nm (dashed line). The UV signal shows two peaks, where the first peak represents the amount of free, unbound RNA, and the second peak originates from the RNA-containing LPX nanoparticles. The UV signal directly represents the amount of RNA in different fractions as a function of elution time. Radius of gyration (R) g (B) The thick line originates from the MALS signal. (B) shows the signal from... Figure 24A The UV signal at 260 nm (dashed line) and the solid line show the cumulative weight fraction based on the area under the UV signal. C) shows the weight fraction obtained by varying the R values ​​of particles of a specific size. g The amount of RNA bound in the RNALPX sample composition was determined using the absorbance at 260 nm during fractionation (Δt = 1 min). This was done to calculate the different R... g The amount of RNA fractionated was determined using only the LPX peak (i.e., Figure 24A and 24B The second peak begins at t = ~24 min and ends at t = ~60 min). D) shows the peak from... Figure 24C The results shown in the figure are calculated for each R g RNA copy number is calculated for each fraction (bars, left y-axis). g The number of particles in the fraction is represented by the corresponding dotted line curve (the second right y-axis).

[0523] Figure 25 illustrates the feasibility of using circular dichroism (CD) spectroscopy in the AF4 method disclosed herein. A) Shows a representative AF4 gradation map of an RNA-lipid complex (LPX) formulation with an LS signal (solid line) at a 90° angle and a CD signal (dotted line) recorded at 260 nm, where the latter represents unbound RNA (first peak; t = 18 min) and bound RNA (second peak; t = 35 min). B) Calibration curves of naked RNA generated using UV detection at 260 nm and CD detection at 260 nm in parallel. The peak areas under the curve (CD: solid squares and solid lines; UV: solid triangles and dotted lines) are plotted against the amount of injected RNA. The ratio of the peak areas of the CD and UV signals is shown as dots (second right y-axis). C) Different amounts of RNALPX samples (2–15 μg) are analyzed using the AF4 method. The area under the curve (AUC) of the CD signal from the appropriate naked RNA is correlated with the appropriate total AUC of the CD signal, where plotting the respective CD peak AUC values ​​against the amount of RNA results in a linear fit (R²). 2 =0.998). The relative amounts (%) of unbound RNA (hollow squares) and bound RNA (hollow circles) in the RNALPX sample composition were determined by correlating the amounts of unbound RNA and bound RNA with the total amount of RNA. Invention Details

[0525] While this disclosure is described in further detail below, it should be understood that this disclosure is not limited to the specific methods, schemes, and reagents described herein, as these can 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 this disclosure, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0526] The elements of this disclosure will be described in more detail below. These elements are listed with specific embodiments; however, it should be understood that they can be combined in any way and in any number to produce additional embodiments. The various examples and preferred embodiments described should not be construed as limiting this disclosure to the explicitly described embodiments. Such description should be understood to support and cover embodiments combining the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, unless the context otherwise requires, any permutation and combination of all elements described in this application should be considered as disclosed by the description of this application. For example, if AF4 is used as field-fluid fractionation in a preferred embodiment of the method of this disclosure, and the nucleic acid (e.g., RNA) is in vitro transcribed RNA in another preferred embodiment of the method of this disclosure, then in another preferred embodiment of the method of this disclosure, AF4 is used as field-fluid fractionation, and the nucleic acid (e.g., RNA) is in vitro transcribed RNA.

[0527] Preferably, the terminology used herein is such as "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G. Leuenberger, B. Nagel, and H. The definition is given in Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0528] Unless otherwise stated, the practice of this disclosure will employ conventional chemical, biochemical, cell biological, immunological, and recombinant DNA techniques, as explained in the literature in this field (see, for example, Organikum, Deutscher Verlag der Wissenschaften, Berlin 1990; Streitwieser / Heathcook, "Organische Chemie", VCH, 1990; Beyer / Walter, "Lehrbuch der Organischen Chemie", S. Hirzel Verlag Stuttgart, 1988; Carey / Sundberg, "Organische Chemie", VCH, 1995; March, "Advanced Organic Chemistry", John Wiley & Sons, 1985; Chemie Lexikon, Falbe / Regitz (Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0529] Throughout the specification and appended claims, unless the context otherwise requires, the word “comprise” and variations such as “comprises” and “comprising” shall be understood to mean including the stated members, integers or steps, or groups of members, integers or steps, but not excluding any other members, integers or steps, or groups of members, integers or steps. The term “substantially constitutes…” excludes other members, integers or steps of any substantial significance. The term “comprise” encompasses the term “substantially constitutes…”, which in turn encompasses the term “consistent with…”. Therefore, whenever the term “comprises” appears in this application, it may be replaced by the term “substantially constitutes…” or “consistent with…”. Similarly, whenever the term “substantially constitutes…” appears in this application, it may be replaced by the term “consistent with…”.

[0530] The terms “a,” “an,” and “this,” as well as similar designations, used in the context of describing this disclosure (particularly in the context of the claims) should be understood to cover both singular and plural forms, unless otherwise specified herein or clearly contradicted by the context. The enumeration of ranges of values ​​herein is solely for shorthand purposes to individually refer to each distinct value falling within that range. Each individual value is incorporated into this specification as it is individually stated herein, unless otherwise stated herein or clearly contradicted by the context. All methods described herein may be performed in any suitable order. The use of any and all instances or exemplary language (e.g., “such”) provided herein is solely for the purpose of better illustrating this disclosure and does not constitute a limitation on the scope of any additional claims made herein. No language in this specification should be construed as implying any unclaimed element essential to the practice of this disclosure.

[0531] When used herein, “and / or” is considered as a specific disclosure of each of two specified features or components, which may or may not contain the other. For example, “X and / or Y” is considered as a specific disclosure of (i) X, (ii) Y, and (iii) each of X and Y, as if each were listed separately herein.

[0532] In the context of this disclosure, the term "about" indicates an interval of accuracy, which, as will be understood by those skilled in the art, still ensures the technical effect of the feature under consideration. This 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 understood by those skilled in the art, such specific deviation from the numerical value of a given technical effect will depend on the nature of the technical effect. For example, natural or biotechnological effects can generally have a larger such deviation than man-made or engineered effects.

[0533] The list of ranges of values ​​in this document is provided solely as a shorthand method for individually referring to each distinct value falling within the range. Unless otherwise stated herein, each individual value is incorporated into this specification as it is individually stated herein.

[0534] The use of any and all instances or exemplary language (e.g., “such”) provided herein is for the purpose of better illustrating the invention and does not constitute a limitation on the scope of the otherwise claimed invention. No language in this specification should be construed as implying any unclaimed element essential to the practice of the invention.

[0535] Several documents are cited throughout the main text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturers' specifications, instructions, etc.), whether mentioned above or below, is incorporated herein in its entirety. Nothing herein shall be construed as an admission that the invention does not enjoy priority of any prior application.

[0536] definition

[0537] Definitions applicable to all aspects of this disclosure are provided below. Unless otherwise stated, the following terms have the following meanings. Any undefined term has its generally accepted meaning.

[0538] As used herein, terms such as “reduction” or “inhibition” refer to the ability to cause a reduction in the overall level, for example, a reduction of about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, or about 75% or more. The term “inhibition” or similar phrases include complete or substantially complete inhibition, i.e., reduction to 0 or substantially to 0.

[0539] In one embodiment, terms such as “increase” or “enhance” refer 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%.

[0540] As used in this article, "physiological pH" refers to a pH of approximately 7.5.

[0541] As used in this disclosure, "%w / v" refers to weight-volume percentage, which is a unit of concentration that measures the amount of solute in grams (g) and is expressed as a percentage of the total volume of the solution in milliliters (mL).

[0542] The term "ionic strength" refers to the mathematical relationship between the quantity of different types of ions and their respective charges in a given solution. Therefore, ionic strength I... S In mathematics, it is represented by the following formula:

[0543]

[0544] Where c is the molar concentration of a specific ion class, and z is the absolute value of its charge. The sum Σ is taken from all the different types of ions (i) in the solution.

[0545] According to this disclosure, in one embodiment, the term "ionic strength" refers to the presence of monovalent ions. Regarding the presence of divalent ions, particularly divalent cations, their concentration or effective concentration (the presence of free ions) is sufficiently low in one embodiment to prevent RNA degradation due to the presence of chelating agents. In one embodiment, the concentration or effective concentration of divalent ions is below the catalytic level 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, there are no or substantially no free divalent ions.

[0546] "Osmotic pressure" refers to the concentration of a specific solute, expressed as the number of solute osmoles per kilogram of solvent.

[0547] The term "freezing" refers to the solidification of a liquid, usually accompanied by the removal of heat.

[0548] The term "lyophilizing" or "lyophilization" refers to the freeze-drying of a substance by freezing it and then reducing the surrounding pressure to allow the freezing medium in the substance to sublimate directly from the solid phase to the liquid phase.

[0549] The term "spray drying" refers to the process of drying a substance by spraying a mixture of (heated) gas and a fluid atomized (sprayed) inside a container (spray dryer), where the solvent from the formed droplets evaporates, resulting in a dried powder.

[0550] The term "restore" refers to adding a solvent, such as water, to a dried product to restore it to a liquid state, such as its original liquid state.

[0551] In the context of this disclosure, the term "recombinant" means "prepared by genetic engineering." In one embodiment, the "recombinant object" in the context of this disclosure is not naturally occurring.

[0552] As used herein, the term "naturally occurring" means the fact that an object can be found in nature. For example, peptides or nucleic acids that exist in organisms (including viruses), can be isolated from natural sources, and have not been intentionally modified by humans in a laboratory are naturally occurring. The term "found in nature" means "existing in nature" and includes both known objects and objects that have not yet been found and / or isolated from nature but may be found and / or isolated from natural sources in the future.

[0553] As used herein, the terms “room temperature” and “ambient temperature” are used interchangeably and refer to a temperature of at least about 15°C, preferably about 15°C to about 35°C, about 15°C to about 30°C, about 15°C to about 25°C, or about 17°C to about 22°C. Such temperatures include 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, and 22°C.

[0554] The term "ethanol injection technique" refers to a process in which an ethanol solution containing lipids is rapidly injected into an aqueous solution via a needle. This action disperses the lipids throughout the solution and promotes the formation of lipid structures, such as lipid vesicles or liposomes. Generally, the nucleic acid (especially RNA) lipid complex particles described herein can be obtained by adding nucleic acids (especially RNA) to colloidal liposome dispersions. In one embodiment, such colloidal liposome dispersions are formed using the ethanol injection technique as follows: an ethanol solution containing lipids such as cationic lipids (e.g., DOTMA) and additional lipids is injected into an aqueous solution under stirring. In one embodiment, the nucleic acid (especially RNA) lipid complex particles described herein are obtained without an extrusion step.

[0555] The term EDTA refers to disodium ethylenediaminetetraacetate. All concentrations of disodium EDTA are provided.

[0556] The term "alkyl" refers to a monovalent group of a saturated straight-chain or branched hydrocarbon. Preferably, the alkyl group comprises 1-12 (e.g., 1-10) carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 1-8 carbon atoms, such as 1-6 or 1-4 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, isopropyl (also known as 2-propyl or 1-methylethyl), butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, secondary pentyl, neopentyl, 1,2-dimethyl-propyl, isopentyl, n-hexyl, isohexyl, secondary hexyl, n-heptyl, isoheptyl, n-octyl, 2-ethylhexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, etc.

[0557] According to 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 linked together by peptide bonds. The term "protein" refers to a large peptide, particularly a peptide having at least about 151 amino acids; however, the terms "peptide" and "protein" are generally used synonymously herein.

[0558] According to this disclosure, preferably, once taken up or introduced, i.e., transfected or transduced into cells, a nucleic acid such as RNA (preferably mRNA) that encodes a peptide or protein leads to the expression of said peptide or protein, said cells may be present in vitro or in a subject. The cells 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 their surface.

[0559] According to this disclosure, terms such as “nucleic acid expression” and “nucleic acid encoding” or similar terms are used interchangeably herein, and with respect to a particular peptide or polypeptide, it means that, if present in a suitable environment, preferably within a cell, nucleic acid can be expressed to produce said peptide or polypeptide.

[0560] According to this disclosure, a portion or fragment of a peptide or protein preferably has 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 to nucleic acids. For example, a pharmacologically active fragment of a peptide or protein has at least one pharmacological activity of the peptide or protein from which that fragment 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, up to 12, up to 15, up to 20, up to 30, or up to 55 consecutive amino acids of the peptide or protein.

[0561] According to this disclosure, a peptide or protein analog is a modified form of the peptide or protein from which it is derived and has at least one functional property of the peptide or protein. For example, a pharmacologically active peptide or protein analog has at least one pharmacological activity of the peptide or protein from which the analog is derived. Such modifications include any chemical modifications and include single or multiple substitutions, deletions, and / or additions of any molecule associated with the protein or peptide (such as carbohydrates, lipids, and / or proteins or peptides). In one embodiment, a “protein or peptide analog” includes those modified forms resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristylation, isopreneation, esterification, alkylation, derivatization, introduction of a protecting / blocking group, protease cleavage, or binding to an antibody or another cellular ligand. The term “analog” also extends to all functional chemical equivalents of the protein and peptide.

[0562] According to this disclosure, "antigen" encompasses any substance that can elicit an immune response and / or any substance targeted by an immune response or immune mechanism such as a cellular response. This also includes cases where an antigen is processed into an antigenic peptide and an immune response or immune mechanism targets one or more antigenic peptides, particularly if presented in the context of MHC molecules. In particular, "antigen" refers to any substance, preferably a peptide or protein, that reacts specifically with antibodies or T lymphocytes (T cells). According to the invention, the term "antigen" includes any molecule containing at least one epitope, such as a T-cell epitope. Preferably, in the context of this disclosure, an antigen is a molecule that, optionally after processing, induces an immune response, preferably antigen- (including cells expressing the antigen) specific. In one embodiment, the antigen is a disease-associated antigen, such as a tumor antigen, viral antigen, or bacterial antigen, or an epitope derived from such an antigen.

[0563] According to this disclosure, any suitable antigen can be used as a candidate for an immune response, which can be both humoral and cellular immune responses. In the context of some embodiments of this disclosure, the antigen is preferably presented by cells, preferably by antigen-presenting cells, in the context of MHC molecules, which results in an immune response against the antigen. The antigen is preferably a product corresponding to or derived from a naturally occurring antigen. Such naturally occurring antigens can include or may be derived from allergens, viruses, bacteria, fungi, parasites, and other infectious agents and pathogens, or the antigen may also be a tumor antigen. According to the invention, the antigen may correspond to a naturally occurring product, such as a viral protein or a portion thereof.

[0564] In a preferred embodiment, the antigen is a tumor antigen, i.e., part of tumor cells, particularly those that are predominantly found within cells or serve as tumor cell surface antigens. In another embodiment, the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, such as a virus, bacteria, single-celled organism, or parasite, such as viral antigens like viral ribonucleoproteins or capsid proteins. In particular, the antigen should be presented by MHC molecules, which leads to regulation, particularly activation of immune system cells (preferably CD4+ and CD8+ lymphocytes), especially through the regulation of T cell receptor activity.

[0565] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule containing an epitope that stimulates the host's immune system to produce a cellular antigen-specific immune response and / or humoral antibody response against the disease. Disease-associated antigens include pathogen-associated antigens, i.e., antigens associated with microbial infections, typically microbial antigens (such as bacterial or viral antigens), or cancer-associated antigens, typically tumor antigens (such as tumor antigens).

[0566] The term "tumor antigen" refers to components of cancer cells, which can originate from the cytoplasm, cell surface, or cell nucleus. Specifically, it refers to antigens that are produced intracellularly or act as surface antigens on tumor cells. For example, tumor antigens include carcinoembryonic antigen, α1-fetoprotein, isoferrin and fetal sulfoglycoprotein, α2-H-ferritin and γ-fetoprotein, as well as various viral tumor antigens. According to this disclosure, tumor antigens preferably include any antigen that is characteristic of a tumor or cancer and of tumor cells or cancer cells in terms of type and / or expression level.

[0567] The term "viral antigen" refers to any viral component that possesses antigenic properties, meaning it can elicit an immune response in an individual. Viral antigens can be viral ribonucleoproteins or envelope proteins.

[0568] The term "bacterial antigen" refers to any bacterial component that possesses antigenic properties, meaning it can elicit an immune response in an individual. Bacterial antigens can originate from the cell wall or cytoplasmic membrane of bacteria.

[0569] The term "epitope" refers to an antigenic determinant in a molecule such as an antigen, that is, a portion or fragment of a molecule recognized by the immune system, for example, the one recognized by antibody T cells or B cells, particularly when presented in the context of MHC molecules. Epitopes of proteins preferably comprise continuous or discontinuous portions of the protein, and are preferably about 5 to about 100 amino acids in length, more preferably about 5 to about 50, more preferably about 8 to about 0, and most preferably about 10 to about 25 amino acids. For example, epitopes may preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. Particularly preferred epitopes in the context of this invention are T-cell epitopes.

[0570] Terms such as “epitope,” “antigen fragment,” “immunogenic peptide,” and “antigen peptide” are used interchangeably herein and preferably refer to an incomplete representation of an antigen that is preferably capable of evoking an immune response against the antigen or a cell expressing, containing, or preferably presenting the antigen. Preferably, the term refers to the immunogenic portion of an antigen. Preferably, it is a portion of the antigen that is recognized (i.e., specifically bound) by a T-cell receptor, particularly if presented in the context of MHC molecules. Certain preferred immunogenic portions bind to MHC class I or II molecules.

[0571] The term "T-cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when presented in the context of MHC molecules. The term "major histocompatibility complex" and the abbreviation "MHC" encompasses MHC class I and MHC class II molecules and refers to gene complexes present in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen-presenting or diseased cells in immune responses, where MHC proteins or molecules bind peptide epitopes and present them for recognition by T-cell receptors on T cells. MHC-encoded proteins are expressed on the cell surface and present T cells with both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments from invading microorganisms). In the case of class I MHC / peptide complexes, the binding peptide is typically about 8–about 10 amino acids long, although longer or shorter peptides may be effective. In the case of class II MHC / peptide complexes, the binding peptide is typically about 10–about 25 amino acids long, particularly about 13–about 18 amino acids long, while longer or shorter peptides may be effective.

[0572] The term "target" should refer to a substance, such as a cell or tissue, that is a target of an immune response, such as a cellular immune response. Targets include cells that present antigens or antigenic epitopes (i.e., peptide fragments derived from antigens). In one embodiment, the target cell is a cell that expresses an antigen and preferably presents said antigen using a class I MHC.

[0573] The term "portion" refers to a fraction. Regarding a specific structure such as an amino acid sequence or a protein, the term "portion" can refer to a continuous or discontinuous portion of that structure.

[0574] The terms "part" and "fragment" are used interchangeably herein and refer to a continuous element. For example, a part of a structure, such as an amino acid sequence or a protein, refers to a continuous element of that structure. When used in the context of a composition, the term "part" means a portion of the composition. For example, a part of the composition can be any portion ranging from 0.1% to 99.9% (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%) of the composition.

[0575] "Antigen processing" refers to the degradation of an antigen into processed products, which are fragments of the antigen (e.g., protein degradation into peptides), and one or more of these fragments are associated (e.g., by binding) with MHC molecules for presentation by cells (preferably antigen-presenting cells of specific T cells).

[0576] "Antigen-reactive CTL" indicates that CD8+ cells respond to an antigen or a peptide derived from said antigen. + T cells, along with type I MHC, are presented on the surface of antigen-presenting cells.

[0577] According to the present invention, CTL responsiveness may include sustained calcium flux, cell division, production of cytokines such as IFN-γ and TNF-α, upregulation of activating markers such as CD44 and CD69, and specific cytolytic killing of target cells expressing tumor antigens. CTL responsiveness can also be determined using artificial reporter molecules that accurately indicate CTL responsiveness.

[0578] The terms “immune response” and “immune reaction” are used interchangeably in their conventional sense herein, referring to a comprehensive bodily response to an antigen, and preferably to a cellular immune response, a humoral immune response, or both. According to the invention, the terms “immune response to…” or “immune response against…” relating to a substance (such as an antigen, cell, or tissue) refer to an immune response (such as a cellular response) against said substance. An immune response may include one or more reactions selected from: the production of antibodies against one or more antigens, and antigen-specific T lymphocytes (preferably CD4+). + and CD8 +T lymphocytes, preferably CD8 + The expansion of T lymphocytes can be detected in various in vitro proliferation or cytokine production tests.

[0579] In the context of this invention, the terms "inducing an immune response" and "initiating 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. An immune response can be protective / defensive / prophylactic and / or therapeutic. An immune response can be against any immunogen or antigen or antigenic peptide, preferably against tumor-associated antigens or pathogen-associated antigens (e.g., antigens of viruses such as influenza viruses (A, B, or C), CMV, or RSV)). In this context, "inducing" can mean that there was no immune response against a specific antigen or pathogen prior to induction, but it can also mean that there was a certain level of immune response against a specific antigen or pathogen prior to induction, and that the immune response was enhanced after induction. Therefore, 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 cancer, or the disease condition is improved by inducing an immune response.

[0580] The terms "cellular immune response," "cellular response," "cell-mediated immunity," or similar terms refer to cellular responses involving cells characterized by expressing antigens and / or presenting antigens using class I or II MHC. Cellular responses involve cells called T cells or T lymphocytes, which act as "helpers" or "killers." Helper T cells (also known as CD4+) + T cells play a central role in regulating the immune response, while killer cells (also known as cytotoxic T cells, cytolytic T cells, CD8+ cells)... + T cells (or CTLs) kill cells such as diseased cells.

[0581] The term "humoral immune response" refers to the process by which a living organism produces antibodies in response to substances and organisms, which ultimately neutralize and / or eliminate said substances and organisms. The specificity of the antibody response is mediated by T and / or B cells through the binding of membrane-associated receptors to a single specific antigen. After binding the appropriate antigen and receiving various other activation signals, B lymphocytes divide, producing memory B cells and antibody-secreting plasma cell clones, each clone producing antibodies that recognize the same antigenic epitopes recognized by its antigen receptor. Memory B lymphocytes remain dormant until they are subsequently activated by their specific antigen. These lymphocytes provide the cellular basis of memory and lead to an escalation of the antibody response upon re-exposure to the specific antigen.

[0582] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an epitope on an antigen. Specifically, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, and any combination thereof. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain comprises a light chain variable region (VL) and a light chain constant region (CL). The variable and constant regions are also referred to herein as variable domains and constant domains, respectively. The VH and VL regions can be further subdivided into highly denatured regions called complementarity-determining regions (CDRs), scattered within more conserved regions called framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of VH are called HCDR1, HCDR2, and HCDR3, and the CDRs of VL are called LCDR1, LCDR2, and LCDR3. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of an antibody include a heavy chain constant region (CH) and a light chain constant region (CL), where CH can be further subdivided into constant domain CH1, a hinge region, and constant domains CH2 and CH3 (arranged in the following order from the amino terminus to the carboxyl terminus: CH1, CH2, CH3). The constant regions of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Antibodies can be complete immunoglobulins derived from natural or recombinant sources and can be the immunologically active portion of a complete immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies and humanized antibodies.

[0583] The term "immunoglobulin" refers to proteins of the immunoglobulin superfamily, preferably antigen receptors such as antibodies or B cell receptors (BCRs). Immunoglobulins are characterized by their domains, specifically immunoglobulin domains that exhibit the characteristic immunoglobulin (Ig) fold. The term encompasses both membrane-bound immunoglobulins and soluble immunoglobulins. Membrane-bound immunoglobulins, also known as surface immunoglobulins or membrane immunoglobulins, are generally part of the BCR. Soluble immunoglobulins are generally called antibodies. Immunoglobulins typically consist of several chains, usually two identical heavy chains and two identical light chains linked by disulfide bonds. These chains are primarily composed of immunoglobulin domains, such as V... L (Variable light chain) domain, C L (Constant light chain) structural domain, V H(variable heavy chain) domain and C H (Constant Heavy Chain) Domain C H 1. C H 2. C H 3 and C H 4. There are five types of mammalian immunoglobulin heavy chains: α, δ, ε, γ, and μ, which result in different 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 carboxyl terminals. There are two types of light chains in mammals: λ and κ. Immunoglobulin chains contain variable and constant regions. The constant region is largely conserved across different isotypes of immunoglobulins, while the variable portion is highly diverse and results in antigen recognition.

[0584] The terms “vaccination” and “immunization” describe the process of treating or preventing a cause of treatment in an individual and involve the following procedures: administering to an individual one or more immunogens or antigens or derivatives thereof, particularly in the form of RNA encoding them, as described herein, and stimulating an immune response against said one or more immunogens or antigens or cells characterized by presenting said one or more immunogens or antigens.

[0585] "Cells characterized by antigen presentation" or "antigen-presenting cells" or "MHC molecules that present antigens on the surface of antigen-presenting cells" or similar expressions refer to cells such as diseased cells, especially tumor cells, or antigen-presenting cells that present antigens or antigenic peptides directly or after processing in the presence of MHC molecules (preferably MHC class I and / or MHC class II molecules, most preferably MHC class I molecules).

[0586] In the context of this disclosure, the term "transcription" refers to a process in which the genetic code in a DNA sequence is transcribed into RNA. The RNA can then be translated into peptides or proteins.

[0587] Regarding RNA, the terms "expression" or "translation" refer to a process within the cell's ribosomes through which the mRNA chain directs the assembly of amino acid sequences to produce peptides or proteins.

[0588] As used herein, the terms “optional” or “optionally” mean that an event, situation, or condition described below may or may not occur, and the description includes both the possibility that the event, situation, or condition may occur and the possibility that it may not occur.

[0589] The "radius of gyration" of the particle about its axis of rotation (abbreviated as R in this article) gR is the radial distance from a point to the axis of rotation. If we assume that the entire mass of the particle is concentrated at that point, then its moment of inertia about a given axis will be the same as its actual mass distribution. Mathematically, R... g This is the root-mean-square distance between a particle's component and its center of mass or a given axis. For example, for a macromolecule composed of n mass elements, with mass m... i (i = 1, 2, 3, ..., n), located at a fixed distance s from the center of mass. i R g It is the s of all mass elements i 2 The square root of the average mass value can be calculated as follows:

[0590]

[0591] The radius of gyration can be determined experimentally or calculated, for example, by using light scattering. In particular, for small scattering vectors... The structure function S is defined as follows:

[0592]

[0593] Where N is the number of components (Guinier's Law).

[0594] The "D10 value," specifically related to the quantitative size distribution of particles, is the diameter of 10% of the particles smaller than this value. The D10 value is a method for describing the proportion of the smallest particles within a particle swarm (such as within a particle peak obtained from a field-flow classification).

[0595] The "D50 value," specifically related to the quantitative size distribution of particles, is the diameter of 50% of the particles smaller than this value. The D50 value is a method for describing the average particle size of a particle group (such as within a particle peak obtained from a field-flow classification).

[0596] The "D90 value," particularly concerning the quantitative size distribution of particles, represents the diameter of 90% of the particles smaller than this value. The "D95," "D99," and "D100" values ​​have corresponding meanings. The D90, D95, D99, and D100 values ​​are methods for describing the proportion of the largest particles within a particle swarm (e.g., within a particle peak obtained from a field-flow classification).

[0597] The "hydrodynamic radius" of a particle (sometimes called the "Stokes radius" or "Stokes-Einstein radius") is the radius of a hypothetical hard sphere that diffuses at the same velocity as the particle. The hydrodynamic radius relates to the particle's mobility and takes into account not only its size but also the effects of the solvent. For example, a smaller charged particle with stronger hydration may have a larger hydrodynamic radius than a larger charged particle with weaker hydration. This is because the smaller particle drags more water molecules as it moves through the solution. Since the actual size of the particle in the solvent cannot be directly measured, the hydrodynamic radius can be defined by the Stokes-Einstein equations:

[0598]

[0599] Where k B η is the Boltzmann constant; T is the temperature; η is the viscosity of the solvent; and D is the diffusion coefficient. The diffusion coefficient can be determined experimentally, for example, by using dynamic light scattering (DLS). Therefore, one method for determining the hydrodynamic radius of a particle or particle group (such as the hydrodynamic radius of particles contained in the sample or control compositions disclosed herein, or the hydrodynamic radius of particle peaks obtained by field-flow fractionation of such sample or control compositions) is to measure the DLS signal of said particle or particle group (such as the DLS signal of particles contained in the sample or control compositions disclosed herein, or the DLS signal of particle peaks obtained by field-flow fractionation of such sample or control compositions).

[0600] As used in this article, the term "shape factor" refers to R. g Values ​​(such as recalculated R) g Value) and hydrodynamic radius (R) h The proportion of the value. It can be determined by R. g Values ​​(such as recalculated R) g Value) for hydrodynamic radius (R) h The values ​​are plotted and the data points are fitted to a function (e.g., a linear function) to determine or calculate.

[0601] As used in this article, the term "form factor" refers to the hydrodynamic radius (R). h ) value and R g Values ​​(such as recalculated R) g The ratio of the value. It can be expressed by the proportion of the hydrodynamic radius (R). h ) value for R g Values ​​(such as recalculated R) g The values ​​are plotted and the data points are fitted to a function (e.g., a linear function) to determine or calculate.

[0602] As used herein, “nucleic acid encapsulation efficiency” refers to the ratio of the amount of encapsulated nucleic acid contained in a sample or control composition to the total amount of nucleic acid contained in the sample or control composition, which comprises nucleic acid and particles. For example, in the case where the nucleic acid is RNA, as used herein, “RNA encapsulation efficiency” refers to the ratio of the amount of encapsulated RNA contained in a sample or control composition comprising RNA and particles to the total amount of RNA contained in the sample or control composition.

[0603] As used herein, the term "membrane" refers to a size-selective barrier that allows molecules of a certain size (referred to as "retention" or "molecular weight cutoff (MW)") to pass through but prevents molecules of a certain size (i.e., retention, such as MW cutoff) from passing through. Preferably, the membrane is synthetic. Examples of membranes suitable for the methods and / or uses of this disclosure include ultrafiltration membranes, polyethersulfone (PES) membranes, regenerated cellulose membranes, polyvinylidene fluoride (PVDF) membranes, and other ultrafiltration membranes.

[0604] As used herein, the term “aggregate” refers to a cluster of particles in which the particles are identical or very similar and adhere to each other in a non-covalent manner (e.g., through ionic interactions, H-bridge interactions, dipole interactions and / or van der Waals interactions).

[0605] As used in this article, "light scattering" refers to a physical process in which light is forced to deviate from a straight trajectory by one or more paths due to local inhomogeneities in the medium through which it passes.

[0606] The term "UV" stands for ultraviolet light and specifies the electromagnetic spectrum band with wavelengths from 10 nm to 400 nm, which is shorter than visible light but longer than X-rays.

[0607] As used herein, the term "circular dichroism spectrum" or "CD spectrum" refers to a spectrum using circularly polarized light. Preferably, the CD spectrum involves the differential absorption of left-handed and right-handed light.

[0608] The term "UV CD light" or "UV CD signal" refers to circularly polarized light with wavelengths ranging from 10 nm to 400 nm, which is shorter than visible light but longer than X-rays.

[0609] As used herein, the terms "multi-angle light scattering" or "MALS" refer to techniques for measuring light scattered from a sample at multiple angles. "Multi-angle" in this context means that the scattered light can be detected at different discrete angles during measurement, for example, by a single detector moving within a range including selected specific angles 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 Scattering). Based on the MALS signal of the composition containing particles and by using an appropriate form (e.g., Zimm plot, Berry plot, or Debye plot), the radius of gyration (R) can be determined. g This allows for the determination of the particle size. Preferably, the Zimm diagram is a graphical representation using the following equation:

[0610]

[0611] Where c is the mass concentration of particles in the solvent (g / mL); A2 is the second virial coefficient (mol·mL / g). 2 P(θ) is a formal factor related to the dependence of scattered light intensity on angle; R θ It is the excess Rayleigh ratio (cm) -1 K* is an optical constant equal to 4π. 2 η o (dn / dc) 2 λ0 -4 N A -1 , where η o λ is the refractive index of the solvent at the incident radiation (vacuum) wavelength, λ0 is the incident radiation (vacuum) wavelength (nm), and N A It is Avogadro's number (mol) -1 ), dn / dc is the differential refractive index increment (mL / g) (see, for example, Buchholz et al. (Electrophoresis 22(2001), 4118-4128); BHZimm (J. Chem. Phys. 13(1945), 141; P. Debye (J. Appl. Phys. 15(1944): 338; and W. Burchard (Anal. Chem. 75(2003), 4279-4291). Preferably, the Berry plot is calculated according to the following:

[0612]

[0613] Among them, c and R θ And K* as defined above. Preferably, the Debye diagram is calculated according to the following terms:

[0614]

[0615] Among them, c and R θ And K* as defined above. Although nucleic acids (especially RNA) are not particles in the sense defined above, the size of nucleic acids (especially RNA) can also be determined using any of the above forms (e.g., Zimm diagrams, Berry diagrams, or Debye diagrams), assuming that nucleic acids (especially RNA) are in a random coil form. Therefore, in one embodiment of the method and / or use of this disclosure, R based on nucleic acid (such as RNA) g Values ​​are used to calculate the size, size distribution, and / or quantify the size distribution of nucleic acids (such as RNA). In another embodiment of the methods and / or uses of this disclosure, the R value is based on nucleic acids (such as RNA). h Values ​​are used to calculate the size, size distribution, and / or quantify the size distribution of nucleic acids (such as RNA). In another embodiment of the methods and / or uses of this disclosure, the R value is based on nucleic acids (such as RNA). g Values ​​and respectively based on the R of nucleic acids (such as RNA) h This implementation calculates the size, size distribution, and / or quantitative size distribution of nucleic acids (such as RNA). Specifically, it generates two datasets: one based on R... g Value, a value based on R h value).

[0616] As used herein, “dynamic light scattering” or “DLS” refers to a technique for determining the size and size distribution spectrum of particles (particularly regarding the hydrodynamic radius of the particles). A monochromatic light source, typically a laser, is incident on the sample through a polarizer. The scattered light then passes through a second polarizer, where it is detected and the resulting image is projected onto a screen. Particles in the solution are struck by the light and diffract it in all directions. The diffracted light from the particles can be constructive interference (bright areas) or destructive interference (dark areas). This process is repeated over short time intervals, and the resulting speckle pattern set is analyzed by comparing the autocorrelator of the light intensity at each point over time.

[0617] As used herein, "static light scattering" or "SLS" refers to a technique for determining the size and size distribution spectrum of particles (particularly concerning the particle's radius of gyration and / or molar mass). High-intensity monochromatic light, typically a laser, is emitted into a solution containing the particles. One or more detectors are used to measure the scattering intensity at one or more angles. Angle dependence is required to obtain accurate measurements of the molar mass and radius of all macromolecules. Therefore, simultaneous measurements at several angles relative to the direction of incident light are called multi-angle light scattering (MALS) or multi-angle laser scattering (MALLS), and are generally considered the standard implementation of static light scattering.

[0618] The terms “elution time” and “retention time” are used interchangeably in this document and refer to the time it takes for a particular analyte to pass through the system (e.g., from the injection point of a field-flow fractionation device to the detector) under set conditions.

[0619] The expression "continuous change" means that the change from one value to different values ​​is steady, that is, without any jumps. Examples of continuous change are linear change or exponential change (such as linear gradient or exponential gradient).

[0620] The expression "gradual change" means that the change from one value to a different value is not discontinuous, but rather jumps from a first specific value to a second specific value, thus omitting at least one of the values ​​between the first and second values. An example of gradual change is a flow rate spectrum that starts with a first value (e.g., 10 mL / min) and ends with a second value (e.g., 0 mL / min), where the flow rate during this spectrum can only be integers (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 mL / min), thus omitting values ​​between these integers.

[0621] As used herein, the phrase “providing a composition comprising nucleic acids (such as RNA) and optionally present particles” means that such a composition is provided by any means, for example, it may be prepared, processed (such as purified and / or dried) and / or stored.

[0622] Nucleic acid

[0623] According to this disclosure, the term "nucleic acid" includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term includes genomic DNA, cDNA, mRNA, recombinant-produced, and chemically synthesized molecules. According to this disclosure, nucleic acids can exist as single-stranded or double-stranded, and as linear or covalently circular closed molecules. According to this disclosure, nucleic acids can be isolated. According to this disclosure, the term "isolated nucleic acid" means that the nucleic acid is: (i) amplified in vitro, for example by polymerase chain reaction (PCR) for DNA or in vitro transcription for RNA (using, for example, RNA polymerase), (ii) produced by clonal recombination, (iii) purified, for example by cleavage and separation by gel electrophoresis, or (iv) synthesized, for example by chemical synthesis.

[0624] The term "nucleoside" (abbreviated as "N" in this document) refers to compounds that can be considered as nucleotides without a phosphate group. A nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose), while a nucleotide consists of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.

[0625] The five standard nucleosides that typically constitute naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine, and guanosine. These five nucleosides are usually abbreviated by their single-letter codes U, A, T, C, and G. However, thymidine is more commonly written as "dT" ("d" stands for "deoxy") because it contains a 2'-deoxyfuranose moiety instead of the furanose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) rather than ribonucleic acid (RNA). Conversely, uridine is found in RNA rather than DNA. The other three nucleosides can be found in both RNA and DNA. In RNA, they are represented as A, C, and G, while in DNA, they are represented as dA, dC, and dG.

[0626] The modified purine (A or G) or pyrimidine (C, T or U) base moiety is preferably modified with one or more alkyl groups, more preferably with one or more C groups. 1-4 Alkyl groups, or more preferably one or more methyl groups. Specific examples of modified purine or pyrimidine base moieties include N... 7 -alkyl-guanine, N 6 -alkyl-adenine, 5-alkyl-cytosine, 5-alkyl-uracil and N(1)-alkyl-uracil, such as N 7 -C 1-4 Alkyl-guanine, N 6 -C 1-4 Alkyl-adenine, 5-C 1-4 Alkyl-cytosine, 5-C 1-4 Alkyl-uracil and N(1)-C 1-4Alkyl-uracil, preferably N 7 -Methyl-guanine, N 6 -Methyl-adenine, 5-methyl-cytosine, 5-methyl-uracil and N(1)-methyl-uracil.

[0627] In this disclosure, the term "DNA" refers to a nucleic acid molecule comprising deoxyribonucleotide residues. In a preferred embodiment, the DNA contains all or most of the deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" means a nucleotide lacking a hydroxyl group at the 2'-position of β-D-ribofuranosyl. DNA encompasses, but is not limited to, double-stranded DNA, single-stranded DNA, isolated DNA such as partially purified DNA, substantially pure DNA, synthetic DNA, recombinant 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 substances to internal DNA nucleotides or the ends of DNA. Nucleotides in DNA are also considered herein to be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the purposes of this disclosure, these altered DNAs are considered analogs of naturally occurring DNA. If, based on the total number of nucleotide residues in a molecule, the content of deoxyribonucleotide residues exceeds 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%), then the molecule contains "a majority of deoxyribonucleotide residues". The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (regardless of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or their analogues).

[0628] In one embodiment, the DNA is recombinant DNA and can be obtained by cloning nucleic acids, particularly cDNA. cDNA can be obtained by reverse transcription of RNA.

[0629] In this disclosure, the term "RNA" refers to a nucleic acid molecule comprising ribonucleotide residues. In a preferred embodiment, the RNA contains all or most of the ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2'-position of the β-D-furanose group. RNA encompasses, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinant 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 a non-nucleotide substance to an internal RNA nucleotide or the terminus 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 the purposes of this disclosure, these 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. If, based on the total number of nucleotide residues in the molecule, the content of ribonucleotide residues exceeds 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%), then the molecule contains "major ribonucleotide residues". The total number of nucleotide residues in the molecule is the sum of all nucleotide residues (regardless of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or their analogues).

[0630] According to this disclosure, "RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), single-stranded RNA (ssRNA), dsRNA, repressive RNA (such as antisense ssRNA, small interfering RNA (siRNA) or microRNA (miRNA)), activating RNA (such as small activating RNA) and immune-stimulating RNA (isRNA).

[0631] As used herein, the term "in vitro transcription" or "IVT" refers to transcription performed in a cell-free manner (i.e., RNA production). That is, IVT does not use living cells / cultured cells, but rather uses transcription mechanisms extracted from cells (e.g., cell lysates or their isolated components, including RNA polymerases (preferably T7, T3, or SP6 polymerases)).

[0632] According to this disclosure, the term "mRNA" means "messenger RNA" and refers to a "transcription" that can be produced 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 this disclosure, mRNA is preferably produced from a DNA template via in vitro transcription (IVT). As shown above, in vitro transcription methods are known to those skilled in the art, and various in vitro transcription kits are commercially available.

[0633] mRNA is single-stranded, but it can contain self-complementary sequences that allow portions of the mRNA to fold and pair with themselves to form a double helix.

[0634] According to this disclosure, "dsRNA" means double-stranded RNA and is RNA having two partially or completely complementary strands.

[0635] The length of RNA can vary from 10 nucleotides to 15,000 nucleotides, such as 40-15,000, 100-12,000, or 200-10,000 nucleotides. In one embodiment, the RNA is repressive RNA and has a length of 10-100 nucleotides (such as up to 90 nucleotides, up to 80 nucleotides, up to 70 nucleotides, up to 60 nucleotides, up to 50 nucleotides, up to 45 nucleotides, up to 35 nucleotides, up to 25 nucleotides, or up to 20 nucleotides). In one embodiment, the RNA encodes a peptide or protein and has a length of at least 45 nucleotides (e.g., at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides), preferably up to 15,000, such as up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides, or up to 10,000 nucleotides.

[0636] In some embodiments of this disclosure, the RNA is mRNA associated with an RNA transcript encoding a peptide or protein. As established in the art, mRNA generally contains a 5' untranslated region (5'-UTR), a peptide-coding region, and a 3' untranslated region (3'-UTR). In some embodiments, the RNA is produced by in vitro transcription or chemical synthesis. In one embodiment, mRNA is produced by in vitro transcription using a DNA template. In vitro transcription methods are known to those skilled in the art; see, for example, Molecular Cloning: A Laboratory Manual, 2 nd Edition, J. Sambrook et al. eds., ColdSpring Harbor Laboratory Press, Cold Spring Harbor 1989. In addition, various in vitro transcription kits are commercially available, for example, from Thermo Fisher Scientific (such as TranscriptAid). TM T7 reagent kit T7 reagent kit ), New England BioLabs Inc. (such as HiScribe TM T7 reagent kit, HiScribe TM T7 ARCA mRNA kit), Promega (such as RiboMAX) TM , Systems), Jena Bioscience (such as SP6 or T7 transcription kits), and Epicentre (such as AmpliScribe) TM To provide modified RNA, corresponding modified nucleotides, such as modified naturally occurring nucleotides, non-naturally occurring nucleotides, and / or modified non-naturally occurring nucleotides, may be incorporated during synthesis (preferably in vitro transcription), or the modification may be achieved and / or added to the RNA after transcription.

[0637] In one embodiment, the RNA is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter used to control transcription can be any promoter of any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly cDNA, and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0638] In the context of this disclosure, the RNA, preferably mRNA, contains one or more modifications, for example, to increase its stability and / or increase translation efficiency and / or reduce immunogenicity and / or reduce cytotoxicity. For example, to increase the expression of RNA (especially mRNA), it may be modified within the coding region, i.e., the sequence encoding the expressed peptide or protein, preferably without altering the sequence of the expressed peptide or protein. Such modifications are described, for example, in WO 2007 / 036366 and PCT / EP2019 / 056502, and include the following: 5'-cap structure; elongation or truncation of a naturally occurring poly(A) tail; alteration of the 5'- and / or 3'-untranslated region (UTR), such as the introduction of a UTR unrelated to the coding region of the RNA; replacement of one or more naturally occurring nucleotides with synthetic nucleotides; and codon optimization (e.g., alteration, preferably an increase in the GC content of the RNA). According to this disclosure, the term "modification" in the context of modified RNA (preferably mRNA) preferably refers to any modification of RNA (preferably mRNA) that is not naturally present in said RNA.

[0639] In some embodiments, the RNA (preferably mRNA) according to this disclosure comprises a 5'-cap structure. In one embodiment, the RNA (preferably mRNA) does not have a capless 5'-triphosphate. In one embodiment, the RNA (preferably mRNA) may comprise a conventional 5'-cap and / or a 5'-cap analogue. The term "conventional 5'-cap" refers to a cap structure found at the 5'-end of an mRNA molecule, typically composed of guanosine 5'-triphosphate (Gppp), which is linked to the 5'-end of the next nucleotide of the mRNA via its triphosphate portion (i.e., guanosine is linked to the remainder of the mRNA via a 5'-to-5' triphosphate bond). Guanosine may be located at position N. 7 Methylation (leading to cap structure m) 7 (Gppp). The term "5'-cap analogue" refers to a 5'-cap that is based on the conventional 5'-cap, but has been modified in m 7 The 2'- or 3'-position of the guanosine structure is modified to prevent the 5'-cap analogue from integrating in the reverse direction (these 5'-cap analogues are also called anti-reverse cap analogues (ARCA)). Particularly preferred 5'-cap analogues are those with one or more substitutions at the bridging and non-bridging oxygen sites of the phosphate bridge, such as 5'-cap analogues modified with thiophosphate at the β-phosphate (e.g., m2). 7,2'OG(5')ppSp(5')G (referred to as beta-S-ARCA or β-S-ARCA), as described in PCT / EP2019 / 056502, the entire disclosure of which is incorporated herein by reference. Providing 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 suitable 5'-cap compound, wherein the 5'-cap structure is co-transcribed into the resulting RNA strand, or RNA (preferably mRNA) can be generated, for example, by in vitro transcription, and the 5'-cap structure can be ligated to RNA post-transcribed using a capping enzyme, such as a capping enzyme of vaccinia virus.

[0640] In some embodiments, the RNA (preferably mRNA) according to this disclosure comprises 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's 5'-cap structure.

[0641] In

[0642] In some embodiments, the RNA (preferably mRNA) comprises cap0, cap1, or cap2, preferably cap1 or cap2. According to this disclosure, the term "cap0" indicates the structure "m..." 7 "GpppN", where N is any nucleoside with an OH moiety at position 2'. According to this disclosure, the term "cap1" indicates the structure "m". 7 "GpppNm", where Nm is any nucleotide with an OCH3 moiety at position 2'. According to this disclosure, the term "cap2" represents the structure "m". 7 "GpppNmNm", where each Nm is independently any nucleoside with an OCH3 moiety at position 2'.

[0643] The D1 diastereomer of beta-S-ARCA (β-S-ARCA) has the following structure:

[0644]

[0645] "β-S-ARCA D1 diastereomer" or "β-S-ARCA(D1)" is a diastereomer of β-S-ARCA that elutes first on the HPLC column compared to the D2 diastereomer of β-S-ARCA (β-S-ARCA(D2)), thus exhibiting a shorter retention time. The HPLC is preferably analytical HPLC. In one embodiment, a Supelcosil LC-18-T RP column, preferably 5 μm, 4.6 x 250 mm, is used for separation, allowing a flow rate of 1.3 ml / min to be applied. In one embodiment, a methanol gradient in ammonium acetate is used, for example, a 0-25% linear gradient of methanol in 0.05 M ammonium acetate at pH 5.9 over 15 min. UV detection (VWD) can be performed at 260 nm, and fluorescence detection (FLD) can be performed with excitation at 280 nm and detection at 337 nm.

[0646] Component of cap1, 5'-cap analogue m2 7,3'-O Gppp(m1 2'-O ApG (also known as m2) 7,3'O G(5')ppp(5')m 2'-O ApG has the following structure:

[0647]

[0648] An exemplary cap0 RNA containing β-S-ARCA and RNA has the following structure:

[0649]

[0650] Includes m2 7,3'O An exemplary cap0 of G(5')ppp(5')G and RNA has the following structure:

[0651] Includes m2 7,3'-O Gppp(m1 2'-O An exemplary cap1 RNA of ApG and RNA has the following structure:

[0652]

[0653] As used herein, the term "poly(A) tail" or "poly-A sequence" refers to a continuous or discontinuous sequence of adenosine 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 RNA described herein. A continuous poly-A tail is characterized by a series of adenosine residues. Continuous poly-A tails are typical in nature. The RNAs disclosed herein may have a poly-A tail that is ligated to a free 3' end of the RNA after transcription by a template-independent RNA polymerase, or a poly-A tail encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0654] The poly-A tail of approximately 120 A nucleotides has been shown to have a beneficial effect on RNA levels in transfected eukaryotic cells as well as on protein levels translated from an open reading frame located upstream (5') of the poly-A tail (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0655] The poly-A tail can have any length. In some embodiments, the poly-A tail comprises, is substantially composed 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, "substantially composed of" means that the majority of the nucleotides in the poly-A tail are A nucleotides, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the nucleotides in the poly-A tail are A nucleotides, but the remaining nucleotides are allowed to be nucleotides other than A nucleotides, such as U nucleotides (uridine monophosphate), G nucleotides (guanosine monophosphate), or C nucleotides (cytidine monophosphate). In this context, "composed of" means that all the nucleotides in the poly-A tail, i.e., 100% of the nucleotides in the poly-A tail are A nucleotides. The term "A nucleotide" or "A" refers to adenosine monophosphate.

[0656] In some implementations, a poly-A tail is attached during RNA transcription, for example, during the preparation of in vitro transcribed RNA, based on a DNA template containing repeating dT nucleotides (deoxythymidines) in a strand complementary to the coding strand. The DNA sequence (coding strand) encoding the poly-A tail is called a poly(A) box.

[0657] In some embodiments, the poly(A) box present in the DNA coding strand is essentially composed of dA nucleotides, but interrupted by a random sequence of four nucleotides (dA, dC, dG, and dT). The length of such a random sequence can be 5-50, 10-30, or 10-20 nucleotides. Such a box is disclosed in WO 2016 / 005324 A1, which is incorporated herein by reference. Any poly(A) box disclosed in WO 2016 / 005324 A1 can be used in this invention. A poly(A) box encompassing such a box, which is essentially composed of dA nucleotides but interrupted by a random sequence of four nucleotides (dA, dC, dG, and dT) having an evenly distributed length of, for example, 5-50 nucleotides, exhibits constant proliferation of plasmid DNA at the DNA level in *E. coli* and remains associated with beneficial properties supporting RNA stability and translation efficiency at the RNA level. Therefore, in some embodiments, the poly-A tail in the RNA molecule described herein consists primarily of A nucleotides, but is interrupted by a random sequence of four nucleotides (A, C, G, U). Such a random sequence can be 5-50, 10-30, or 10-20 nucleotides in length.

[0658] In some implementations, there are no nucleotides other than A on the 3'-terminal flanking of the poly-A tail; that is, the poly-A tail is not covered or followed by nucleotides other than A at its 3'-terminus.

[0659] In some embodiments, the RNA according to this disclosure comprises a 5'-UTR and / or a 3'-UTR. The terms "untranslated region" or "UTR" refer to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or to a corresponding region in an RNA molecule such as mRNA. Untranslated regions (UTRs) may be present at the 5' (upstream) (5'-UTR) and / or the 3' (downstream) (3'-UTR) of an open reading frame. If present, the 5'-UTR is located at the 5'-terminus, upstream of the start codon of the protein-coding region. The 5'-UTR is located downstream of the 5'-cap (if present), for example, directly adjacent to the 5'-cap. If present, the 3'-UTR is located at the 3'-terminus, downstream of the stop codon of the protein-coding region, but the term "3'-UTR" preferably does not include a poly-A sequence. Thus, the 3'-UTR is located upstream of the poly-A sequence (if present), for example, directly adjacent to the poly(A) sequence. Incorporating a 3'-UTR into the 3'-untranslated region of an RNA (preferably mRNA) molecule can lead to improved translation efficiency. A synergistic effect can be achieved by incorporating two or more such 3'-UTRs (preferably arranged head-to-tail; see, for example, Holtkamp et al., Blood 108, 4009-4017 (2006)). The 3'-UTRs can be autologous or heterologous to the RNA (preferably mRNA) to which they are incorporated. In one specific embodiment, the 3'-UTR is derived from a globin gene or mRNA, such as α2-globin, α1-globin, or β-globin, preferably β-globin, more preferably the gene or mRNA of human β-globin. For example, RNA (preferably mRNA) can be modified by replacing an existing 3'-UTR with one or more, preferably two, copies of a 3'-UTR, or by inserting one or more, preferably two, copies of a 3'-UTR derived from a globin gene, such as α2-globin, α1-globin, or β-globin, preferably β-globin, more preferably human β-globin.

[0660] The RNA (preferably mRNA) may have modified ribonucleotides to increase its stability and / or reduce its immunogenicity and / or cytotoxicity. For example, in one embodiment, uridine in the RNA described herein is replaced (partially or completely, preferably completely) with a modified nucleoside. In some embodiments, the modified nucleoside is modified uridine.

[0661] In some embodiments, the uridine used to replace the modified uridine is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), 5-methyl-uridine (m5U) and combinations thereof.

[0662] In some embodiments, the nucleoside that replaces (partially or completely, preferably completely) the uridine modification in the RNA can be any one or more of the following: 3-methyluridine (m 3 U), 5-methoxyuridine (mo) 5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-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 (mchm) 5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5- Prolynyl-uridine, 1-propynyl-pseudouridine, 5-taurate methyl-uridine (τm5U), 1-taurate methyl-pseudouridine, 5-taurate methyl-2-thio-uridine (τm5s2U), 1-taurate methyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deazo-pseudouridine, 2-thio-1-methyl-1-deazo-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine 5-Methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2′-O-methyl-uridine (Um), 5,2′-O-dimethyluridine (m5Um), 2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyluridine (s2Um), 5-methoxycarbonylmethyl-2′-O-methyluridine (mcm5Um), 5-carbamoylmethyl-2′-O-methyluridine (ncm5Um), 5-carboxymethylaminomethyl-2′-O-methyluridine (cmnm5Um), 3 2′-O-dimethyluridine (m3Um), 5-(isopentenylaminomethyl)-2′-O-methyluridine (inm5Um), 1-thiouridine, deoxythymidine, 2′-F-arasu-uridine, 2′-F-uridine, 2′-OH-arasu-uridine, 5-(2-methoxycarbonylvinyl)uridine, 5-[3-(1-E-propenylamino)uridine or any other modified uridine known in the art.

[0663] RNA (preferably mRNA) modified with pseudouridine (partially or completely, preferably completely replacing uridine) is referred to herein as “Ψ-modified”, and the term “m1Ψ-modified” indicates that the RNA (preferably mRNA) contains N(1)-methylpseudouridine (partially or completely, preferably completely replacing uridine). Furthermore, the term “m5U-modified” indicates that the RNA (preferably mRNA) contains 5-methyluridine (partially or completely, preferably completely replacing uridine). Such Ψ-, m1Ψ-, or m5U-modified RNAs exhibit reduced immunogenicity compared to their unmodified forms and are therefore preferred in applications where induction of an immune response is avoided or minimized.

[0664] The codons of the RNA (preferably mRNA) disclosed herein can be further optimized, for example, to increase the GC content of the RNA and / or replace rare codons in the cell (or subject), wherein the peptide or protein of interest is expressed by codons that are synonymous frequent codons in the cell (or subject).

[0665] The combination of the above modifications, namely, incorporation of a 5'-cap structure, incorporation of a poly-A sequence, exposure of a poly-A sequence, alteration of the 5'- and / or 3'-UTR (e.g., incorporation of one or more 3'-UTRs), replacement of one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine and / or pseudouridine (Ψ) or N(1)-methylpseudouridine (m1Ψ) or 5-methyluridine (m5U) for uridine), and codon optimization, has a synergistic effect on improving the stability and translation efficiency of RNA (preferably mRNA). Therefore, in a preferred embodiment, the RNA (preferably mRNA) according to this disclosure contains 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 poly-A sequence to expose the poly-A sequence, (iii) alteration of the 5'- and / or 3'-UTR (e.g., incorporation of one or more 3'-UTRs), (iv) replacement of one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine and / or pseudouridine (Ψ) or N(1)-methylpseudouridine (m1Ψ) or 5-methyluridine (m5U) for uridine), and (v) codon optimization.

[0666] In one embodiment, the RNA according to this disclosure comprises a nucleic acid sequence encoding a peptide or protein, preferably a pharmaceutically active peptide or protein.

[0667] In a preferred embodiment, the RNA according to this disclosure comprises a nucleic acid sequence encoding a peptide or protein, preferably a pharmaceutically active peptide or protein, and is capable of expressing said peptide or protein, particularly if transferred to cells or a subject. Therefore, the RNA according to the invention preferably contains a coding region (open reading frame (ORF)) encoding a peptide or protein, preferably a pharmaceutically active peptide or protein. In this respect, an "open reading frame" or "ORF" is a continuous sequence of codons, beginning with a start codon and ending with a stop codon.

[0668] According to this disclosure, the term "pharmaceutically active peptide or protein" refers to a peptide or protein that can be used to treat an individual, wherein the expression of the peptide or protein would be beneficial, for example, in improving symptoms of a disease or condition. Preferably, the pharmaceutically active peptide or protein has therapeutic or palliative properties and can be administered to improve, alleviate, relieve, reverse, delay the onset of, or reduce the severity of one or more symptoms of a disease or condition. Preferably, when administered to an individual in a therapeutically effective amount, the pharmaceutically active peptide or protein has a positive or beneficial effect on the individual's condition or disease state. Pharmacologically active peptides or proteins may have preventative properties and can be used to delay the onset of a disease or condition or reduce its severity. The term "pharmaceutically active peptide or protein" includes complete proteins or polypeptides and may also refer to their pharmaceutically active fragments. It may also include pharmaceutically active analogs of peptides or proteins.

[0669] 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-engineered proteins, and blood proteins.

[0670] The term "cytokine" refers to proteins with a molecular weight of approximately 5-20 kDa that participate in cell signaling (e.g., paracrine, endocrine, and / or autocrine signaling). Specifically, upon release, cytokines influence cellular behavior around their release site. Examples of cytokines include lymphokines, interleukins, chemokines, interferons, and tumor necrosis factor (TNF). According to this disclosure, cytokines do not include hormones or growth factors. Cytokines differ from hormones in that (i) they generally function at 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 generally characterized by antiviral, antiproliferative, and immunomodulatory activities. Interferons are proteins that alter and regulate intracellular gene transcription by binding to interferon receptors on the surface of regulated cells, thereby preventing intracellular viral replication. Interferons can be classified into two types. IFN-γ is the only type II interferon; the others are type I interferons. Specific examples of cytokines include erythropoietin (EPO), colony-stimulating factor (CSF), granulocyte colony-stimulating ...

Claims

1. A method for determining one or more parameters of a sample composition, wherein the sample composition comprises RNA and particles bound to the RNA, the method comprising: (a) subjecting at least a portion of the sample composition to field-flow fractionation, thereby fractionating components contained in the sample composition according to their size to produce one or more sample fractions; (b) measuring at least a UV signal and optionally a light scattering (LS) signal of at least one of the one or more sample fractions obtained from step (a); and (c) calculating said one or more parameters from said UV signal and optionally from said LS signal, wherein the one or more parameters include RNA integrity, the total amount of RNA, the amount of free RNA, the amount of RNA associated with particles, the size of particles containing RNA, the size distribution of particles containing RNA, and the quantitative size distribution of particles containing RNA, wherein at least two parameters are determined selected from the group consisting of the amount of free RNA, the amount of RNA associated with particles, the size distribution of particles containing RNA, and the quantitative size distribution of particles containing RNA, wherein the amount of RNA is determined by using (i) an RNA extinction coefficient or (ii) an RNA calibration curve, wherein the amount of RNA bound to particles is determined by subtracting the amount of free RNA from the amount of total RNA, wherein the amount of free RNA is determined by: performing steps (a)-(c) without adding a releasing agent, and determining the amount of RNA; and the amount of total RNA is determined by: (i) treating a portion of the sample composition with a releasing agent; (ii) performing steps (a)-(c) with the portion obtained from step (i); and (iii) determining the amount of RNA.

2. Use of field-flow fractionation in determining one or more parameters of a sample composition, wherein the sample composition comprises RNA and particles bound to the RNA, wherein the one or more parameters include RNA integrity, the total amount of RNA, the amount of free RNA, the amount of RNA bound to particles, the size of particles containing RNA (such as the hydrodynamic radius of particles containing RNA), the size distribution of particles containing RNA, and the quantitative size distribution of particles containing RNA, wherein at least two parameters selected from the following are determined: the amount of free RNA, the amount of RNA bound to particles, the size distribution of particles containing RNA, and the quantitative size distribution of particles containing RNA.

3. The method of claim 2, wherein the field-flow fractionation comprises: (a) subjecting at least a portion of the sample composition to field-flow fractionation, thereby fractionating components contained in the sample composition according to their size to produce one or more sample fractions; (b) measuring at least a UV signal and optionally a light scattering (LS) signal of at least one of the one or more sample fractions obtained from step (a); as well as (c) calculating said one or more parameters from said UV signal and optionally from said LS signal, wherein the amount of RNA is determined by using (i) an RNA extinction coefficient or (ii) an RNA calibration curve, wherein the amount of RNA bound to particles is determined by subtracting the amount of free RNA from the amount of total RNA, wherein the amount of free RNA is determined by: performing steps (a)-(c) without adding a releasing agent, and determining the amount of RNA; and the amount of total RNA is determined by: (i) treating a portion of the sample composition with a releasing agent; (ii) performing steps (a)-(c) with the portion obtained from step (i); and (iii) determining the amount of RNA.

4. The method of claim 1 or the use of claim 2 or 3, 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).

5. The method of claim 1 or 4 or the use of claim 3 or 4, wherein step (a) is performed using: - a membrane having a molecular weight cut-off (MW) suitable for preventing RNA from penetrating the membrane, preferably a membrane having a MW cut-off in the range of 2 kDa-30 kDa, such as a membrane having a MW cut-off of 10 kDa; - polyethersulfone (PES) or regenerated cellulose membrane; - a cross flow rate of up to 8 mL / min, preferably up to 4 mL / min, more preferably up to 2 mL / min; - The following cross-flow profile: 1.0-2.0 mL / min for 10 min, exponential gradient from 1.0-2.0 mL / min to 0.01-0.07 mL / min in 30 min; 0.01-0.07 mL / min for 30 min; and 0 mL / min for 10 min; - an injection rate in the range of 0.05-0.35 mL / min, preferably in the range of 0.10-0.30 mL / min, more preferably in the range of 0.15-0.25 mL / min; and / or - a detector flow rate in the range of 0.30-0.70 mL / min, preferably in the range of 0.40-0.60 mL / min, more preferably in the range of 0.45-0.55 mL / min.

6. The method of any one of claims 1, 4 and 5 or the use of any one of claims 2-5, wherein the integrity of the RNA contained in the sample composition is calculated or determined using the integrity of the control RNA; preferably, wherein (i) Determine the integrity of the control RNA by the following steps: (a') subjecting at least part of a control composition containing a control RNA to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions; (b') measuring the UV signal of at least one of the one or more control fractions obtained from step (a'); (c'1) calculating the area from the maximum height of a UV peak to the end of the UV peak from the UV signal obtained in step (b') to obtain A 50％ (control); (c'2) Calculating the total area of ​​one peak used in step (c'1) from the UV signal obtained in step (b') to obtain A 100％ (control); and (c'3) Determine A 50％ (control) and A 100％ (control), thereby obtaining the integrity of the control RNA (I(control)), Wherein, preferably, the integrity of the RNA contained in the sample composition is calculated by the following steps: (c1) calculating the area from the maximum height of the sample UV peak corresponding to the control UV peak used in step (c'1) to the end of the sample UV peak from the sample UV signal obtained in step (b), thereby obtaining A 50％ (sample); (c2) calculating the total area of ​​the sample UV peak used in step (c1) from the sample UV signal obtained in step (b), Thus obtaining A 100％ (sample); (c3) Determine A 50％ (Sample) and A 100％ (samples), thereby obtaining I(sample); and (c4) determining the ratio between I(sample) and I(control) to obtain the integrity of the RNA contained in the sample composition; or (ii) wherein the integrity of the control RNA is determined by the following steps: (a") subjecting at least part of a control composition containing a control RNA to field-flow fractionation, in particular AF4 or HF5, thereby fractionating the components contained in the control composition according to their size to produce one or more control fractions; (b") measuring the UV signal of at least one of the one or more control fractions obtained from step (a"); and (c") determining the height of a UV peak (H(control)) from the UV signal obtained in step (b"), thereby obtaining the integrity of the control RNA, Wherein, preferably, the integrity of the RNA contained in the sample composition is calculated by the following steps: (c1′) determining the height of the sample UV peak (H(sample)) corresponding to the reference UV peak used in step (c″) from the UV signal obtained in step (b); and (c2') Determine the ratio between H(sample) and H(control) to obtain the integrity of the RNA contained in the sample composition.

7. The method according to any one of claims 1 and 4 to 6 or the use according to any one of claims 2 to 6, wherein the particle bound to the RNA is a lipid complex particle and / or a lipid nanoparticle and / or a polyplex particle and / or a lipid polymer complex particle and / or a virus-like particle.

8. The method according to any one of claims 1 and 4 to 7 or the use according to any one of claims 3 to 7, wherein in step (a), field-flow fractionation is performed using a liquid phase containing a releasing agent; and / or 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 ( 3-14)), a cationic surfactant, a nonionic surfactant or a mixture thereof; (ii) an alcohol, such as a fatty alcohol (e.g., ethanol), or a mixture of alcohols; or (iii) a combination of (i) and (ii).

9. The method of any one of claims 1 and 4 to 8 or the use of any one of claims 3 to 8, wherein step (b) further comprises measuring an LS signal, such as a dynamic light scattering (DLS) signal and / or a static light scattering (SLS), e.g., a multi-angle light scattering (MALS) signal, of at least one of the one or more sample fractions obtained in step (a), wherein Preferably, - Calculate the radius of gyration (R) by the LS signal obtained from step (b) g ) value and / or hydrodynamic radius (R h ) values ​​to determine the size of the RNA-containing particles; or - Make the experimentally determined R g and / or R h The values ​​are smoothed, preferably by dividing the experimentally determined or calculated R g or R h The values ​​are fitted to a polynomial or linear function and R is recalculated based on the polynomial or linear fit g or R h value; and / or - R determined by dividing the UV signal obtained from step (b) by g or R h The values ​​are plotted to determine the size distribution of particles containing the RNA; and / or - by converting the UV signal into a cumulative weight fraction and comparing the cumulative weight fraction to the determined R g or R h The values ​​are plotted and displayed as R g or R h Calculate the quantitative size distribution of the RNA-containing particles from a plot of the UV signal as a function of the UV value, wherein more preferably, the quantitative size distribution comprises D10, D50 and / or D90 values; and / or - step (b) comprises measuring a dynamic light scattering (DLS) signal of at least one of the one or more sample fractions obtained from step (a), and step (c) comprises calculating R from said DLS signal h value.

10. The method according to any one of claims 1 and 4 to 9 or the use according to any one of claims 2 to 9, wherein the one or more parameters are two parameters selected from the group consisting of: the amount of free RNA, the amount of RNA bound to particles, the size distribution of particles containing RNA, and the quantitative size distribution of particles containing RNA, or the one or more parameters include at least three or are three parameters selected from the group consisting of: the amount of free RNA, the amount of RNA bound to particles, the size distribution of particles containing RNA, and the quantitative size distribution of particles containing RNA; and / or The amount of RNA, in particular free RNA, is determined by measuring the UV signal at 260 nm and using the RNA extinction coefficient at 260 nm, or by measuring the UV signal at 280 nm and using the RNA extinction coefficient at 280 nm.

11. The method of any one of claims 1 and 4 to 10 or the use of any one of claims 2 to 10, 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 10-2000 nm, preferably in the range of 20-1500 nm, such as 30-1200 nm, 40-1100 nm, 50-1000, 60-900 nm, 70-800 nm, 80-700 nm, 90-600 nm or 100-500 nm, for example in the range of 10-1000 nm, 15-500 nm, 20-450 nm, 25-400 nm, 30-350 nm, 40-300 nm or 50-250 nm; - The length of the RNA is 10-15,000 nucleotides, such as 40-15,000 nucleotides, 100-12,000 nucleotides or 200-10,000 nucleotides; - the RNA is in vitro transcribed RNA, in particular in vitro transcribed mRNA; and / or - measuring a UV signal, optionally a LS signal, such as a SLS, e.g. a MALS signal and / or a DLS signal number, is performed online, and / or step (c) is performed online.

12. The method of any one of claims 1 and 4 to 11 or the use of any one of claims 2 to 11, wherein before subjecting at least part of the sample composition to field-flow fractionation, said at least part of the sample composition is diluted with a solvent or solvent mixture which is capable of preventing the formation of particle aggregates, wherein, Preferably, the solvent mixture is a mixture of water and an organic solvent such as formamide.

13. The method of any one of claims 1 and 4 to 12 or the use of any one of claims 2 to 12, wherein measuring the UV signal is performed by using circular dichroism (CD) spectroscopy.

14. A method of analyzing the effect of changing one or more reaction conditions in providing a composition comprising RNA and particles that bind to the RNA, the method comprising: (A) providing a first composition comprising RNA and particles; (B) providing a second composition comprising RNA and particles, wherein providing the second composition differs from providing the first composition only in one or more reaction conditions; (C) subjecting a portion of the first composition to the method of any one of claims 1 to 13, thereby determining one or more parameters of the first composition; (D) subjecting a corresponding portion of the second composition to the method used in step (C), thereby determining one or more parameters of the second composition; as well as (E) comparing one or more parameters of the first composition obtained in step (C) with the corresponding one or more parameters of the second composition obtained in step (D).

15. The method of claim 14, 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 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; dry / reconstitute cycles; addition of excipients (e.g., stabilizers and / or chelators); type and / or source of particle-forming compounds (particularly lipids and / or polymers, e.g., cationic lipids versus zwitterionic lipids or PEGylated lipids versus non-PEGylated lipids); charge ratio; physical state; and the ratio of RNA to particle-forming compounds (particularly lipids and / or polymers).

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