METHOD FOR DETERMINING AT LEAST ONE PARAMETER OF A SAMPLE COMPOSITION COMPRISING NUCLEIC ACID, SUCH AS RNA, AND OPTIONALLY PARTICLES

TECHNICAL FIELD

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

BACKGROUND

The use of a recombinant nucleic acid (such as DNA or RNA) for delivery of foreign genetic information into target cells is well known. The advantages of using RNA include transient expression and a non-transforming character. RNA does not need to enter the nucleus in order to be expressed and moreover cannot integrate into the host genome, thereby eliminating diverse risks such as oncogenesis.

A recombinant nucleic acid may be administered in naked form to a subject in need thereof; however, usually a recombinant nucleic acid is administered using a pharmaceutical composition. For example, RNA may be delivered by so-called nanoparticle formulations containing RNA and a nanoparticle forming vehicle, e.g., a cationic lipid, a mixture of a cationic lipid and helper lipid, or a cationic polymer.

The fate of such nanoparticle formulations is controlled by diverse key-factors (e.g., integrity and concentration of the nucleic acid in the nanoparticles; amount of free nucleic acid; size, size distribution, quantitative size distribution, and morphology of the nanoparticles; etc.). These factors are, e.g., referred to in the FDA “Liposome Drug Products Guidance” from 2018 as specific attributes which should be analyzed and specified. The limitations to the clinical application of current nanoparticle formulations may lie in the lack of homogeneous, pure and well-characterized nanoparticle formulations. This is also due to the fact that all the existing techniques for determining these factors have some drawbacks.

For example, the current techniques for the determination of integrity and/or concentration of nucleic acid in nanoparticles (such as methods based on a dye, gel electrophoresis, microchannel electrophoresis, or capillary electrophoresis (CE)) are labor-intensive, costly, utilize sample preparation steps causing artefacts, cannot provide adequate information and/or cannot analyze high numbers of samples. In one current technique using a dye (such as a fluorescent dye), the dye by itself can causes differences, which can affect reliability of the measured results. In addition, most of the techniques based on gel electrophoresis require multiple washing steps, the use of special running buffers that increase the length of the procedure, and special precautions due to the use of toxic reagents. For example, the agarose gel technique is affected by multiple parameters (e.g., quality of the agarose, cast of the gel, dye/sensitivity (higher amount of sample is needed), exposure time, processing the raw data and standardized evaluation by densitometry software (28S/18S method)) which make this technique unreliable. The techniques based on microchannel, chip-based electrophoresis or capillary electrophoresis provide faster run times and improved data quality compared to agarose gel electrophoresis, but require hands-on processing for priming and loading of gel, markers, and samples onto the system. CE instruments lack the sensitivity, dynamic range, and separation quality required for adequate RNA quality/quantity analysis.

Furthermore, one key challenge for characterizing nanoparticle formulations lies in the quantitative determination of the size distribution of the particles contained in the formulations. This is particularly the case for particles with a diameter smaller than about 500 nm, i.e., the range which is relevant for most pharmaceutical products. A further unmet need lies in the determination of the size distribution of nanoparticle formulations, where the size distribution is broad or complex (in particular asymmetric). All existing techniques which are available for characterization of nanoparticle formulations have certain drawbacks: e.g., they do not provide direct quantitative information on size or they measure only small, optionally not representative, subsets of the samples, or the sensitivity for particles with different sizes (or other parameters, e.g., refractive index gradients with respect to the bulk phase) is very different, which strongly affects the obtained size distributions.

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

DLS provides information on the diffusion constant of nanoparticles, from which the hydrodynamic radius, R_h, is calculated using the Stokes-Einstein equation. However, DLS provides only average data, from which particle sizes are numerically calculated using certain algorithms. In order to obtain quantitatively reliable numbers, nanoparticle formulations should be monomodal and monodisperse, which is not the case for many products, including nanoparticulate pharmaceutical formulations. The algorithm which is most widely used in DLS is the so-called cumulative analysis (D. E. Koppel, J. Chem. Phys. 57 (1972) 4814-4820) which as a premise only assumes monomodal size distributions and provides physically meaningful numbers only if the polydispersity is below a certain threshold. Other algorithms for DLS (cf., e.g., Provencher, S. W., Comput. Phys. Commun. 1982, 27, 229-242) provide size curves which heavily depend on the fitting parameters, and several, very different profiles can correspond to the same data set. Such analysis is hampered by the fact that the light scattering intensity for large particles is much higher than for smaller particles, which makes it difficult to determine fractions of smaller particles in the presence of much larger ones.

NTA is a method which determines the sizes of particles from their diffusion constant by microscopically observing scattered light from very small (diluted) subsets of the samples over time. NTA, in principle, is able to provide quantitative size distribution profiles; however, only very diluted samples can be measured, and the particles must be present in a relatively small size range, i.e., very small particles cannot be determined on a background of much larger ones, due to their much higher scattering intensity. Therefore, NTA is not suitable as a regularly applicable method for determining quantitative size distributions for pharmaceutical formulations. Furthermore, the statistical standard deviation of NTA is high compared to other techniques (e.g., DLS). This is a direct consequence of one to three orders of magnitude lower amount of particles analyzed by NTA. In particular, marginal amounts of particles (e.g., aggregates), which have biological impact, can be underestimated or cannot even be detected by NTA. NTA requires several time-consuming optimization steps (e.g., video capture setting, different sample dilutions, etc.) to identify suitable settings for an accurate measurement. Typically, the samples for the NTA measurement have to be diluted by a factor of 10-1000-fold which can cause problems, especially with concentration depending aggregation or disassembly of particles. Due to all these disadvantages, it is difficult to establish NTA as a quality control method.

EM provides quantitative information on size, shape and morphology of individual particles, but the number of particles which can be analyzed is even lower than for NTA. Therefore, EM has disadvantages similar or identical to those of NTA in the sense that the measured particles may not be representative for the total sample. Additional major drawbacks of this technique are the high costs, complex sample preparation and long turn-around time for analyzing the samples. This is why EM is not commonly used as a GMP method. Another problem of EM is that fixation of the samples can causes artifacts (e.g., shrinking, aggregation, etc.). If samples are not fixed (e.g., in Cryo-EM), the samples may have low contrast and cannot be analyzed.

Other separation techniques like SEC-UV are not applicable, because interaction with the column matrix can causes problems (e.g., absorption or delay of the elution). The nanoparticles cannot adequately dispersed because of the limited size range of the SEC column, or the nanoparticles cannot separated from aggregates. Furthermore, SEC-UV does not provide a quantitative size distribution in the sense that mass or particle numbers are directly correlated to the to the particle size. Other dispersive methods, such as Analytical Ultracentrifugation (AUC), allow only indirect size measurements, for example as based on the sedimentation coefficients, where several assumptions have to be made in order to calculate size profiles. In addition, AUC is costly and time consuming and it is not a common method in regular quality control. Therefore, also AUC is not appropriate for determining quantitative size profiles as a regular quality control method.

In view of the above, to assure reproducible quality of nanoparticle formulations, advanced analytical methods for in-depth particle characterization are needed. In particular, there is a need for an improved method of analyzing nanoparticle formulations containing a nucleic acid (especially RNA), wherein said method preferably (i) provides information on characteristics of the formulation (such as quantitative size distribution of the particles contained in the formulation (in particular with respect to particles having a diameter of less than 500 nm); (ii) provides information on characteristics of the particle composition (e.g., the amount of nucleic acid (especially RNA) contained in the particles, in particular as a function of the particle size, such as the ratio of the amount of nucleic acid (especially RNA) contained in the particles to the amount of particle forming compounds (in particular lipids and/or polymers, e.g., cationic lipid vs. cationic polymer), in particular as a function of the particle size); (iii) is GMP-compatible; (iv) does not depend on the use of a dye; (v) is semi-automatic; and/or (vi) can be used to analyze the effect of altering one or more reaction conditions (e.g., salt concentration; temperature; pH or buffer concentration; light/radiation; oxygen; shear force; pressure; freezing/thawing cycle; drying/reconstitution cycle; addition of excipient(s) (e.g., stabilizer and/or chelating agent); type and/or source of particle forming compounds (in particular lipids and/or polymers); charge ratio; and/or ratio of nucleic acid (such as RNA) to particle forming compounds (in particular lipids and/or polymers)) when preparing and/or storing a composition comprising a nucleic acid (such as RNA) and optionally particles. Preferably, said method provides data regarding one or more of the following parameters: nucleic acid (such as RNA) integrity; the total amount of nucleic acid (such as RNA); the amount of free nucleic acid (such as RNA); the amount of nucleic acid (such as RNA) bound to particles; the size of nucleic acid (such as RNA) containing particles (e.g., based on the radius of gyration (R_g) of nucleic acid (such as RNA) containing particles and/or the hydrodynamic radius (R_h) of nucleic acid (such as RNA) containing particles); the size distribution of nucleic acid (such as RNA) containing particles (e.g., based on R_gor R_hvalues); the quantitative size distribution of nucleic acid (such as RNA) containing particles (e.g., based on R_gor R_hvalues); the molecular weight of nucleic acid (such as RNA); and/or the shape (e.g., the shape and/or form factor) of nucleic acid (such as RNA) containing particles. Optionally, additional parameters may include one or more of the following: 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), the size of the nucleic acid (especially RNA) (e.g., based on R_gor R_hvalues), the size distribution of the nucleic acid (especially RNA) (e.g., based on R_gor R_hvalues), the quantitative size distribution of the nucleic acid (especially RNA) (e.g., based on R_gor R_hvalues), the nucleic acid (especially RNA) encapsulation efficiency, the ratio of the amount of nucleic acid (such as RNA) bound to particles to the total amount of particle forming compounds (in particular lipids and/or polymers) in the particles, the ratio of the amount of positively charged moieties of particle forming compounds (in particular lipids and/or polymers) in the particles to the amount of nucleic acid (such as RNA) bound to particles, and the charge ratio of the amount of positively charged moieties of particle forming compounds (in particular lipids and/or polymers) in the particles to the amount of negatively charged moieties of nucleic acid (such as RNA) bound to particles (N/P ratio)

The inventors surprisingly found that the methods and uses described herein fulfill the above mentioned requirements.

SUMMARY

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

(a) subjecting at least a part of the sample composition to field-flow fractionation, thereby fractioning the components contained in the sample composition by their size so as to produce one or more sample fractions;

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

(c) calculating from the at least one signal selected from the group consisting of the UV signal, the fluorescence signal, and the RI signal, and optionally from the LS signal, the one or more parameters, wherein the one or more parameters comprise the nucleic acid (such as RNA) integrity, the total amount of nucleic acid (such as RNA), the amount of free nucleic acid (such as RNA), the amount of nucleic acid (such as RNA) bound to particles, the size of nucleic acid (such as RNA) containing particles (in particular, based on the radius of gyration (R_g) of nucleic acid (such as RNA) containing particles and/or the hydrodynamic radius (R_h) of nucleic acid (especially RNA) containing particles), the size distribution of nucleic acid (such as RNA) containing particles (e.g., based on R_gor R_hvalues of nucleic acid (especially RNA) containing particles), and the quantitative size distribution of nucleic acid (such as RNA) containing particles (e.g., based on R_gor R_hvalues of nucleic acid (especially RNA) containing particles). Generally, the size distribution and/or quantitative size distribution of nucleic acid (such as RNA) containing particles can be given as the number of the nucleic acid (such as RNA) containing particles, the molar amount of the nucleic acid (such as RNA) containing particles, or the mass of the nucleic acid (such as RNA) containing particles each as a function of their size. Additional optional parameters include the molecular weight of 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), the size of nucleic acid (especially RNA) (in particular, based on R_gand/or R_hvalues of nucleic acid (especially RNA)), the size distribution of nucleic acid (especially RNA) (e.g., based on R_gor R_hvalues of nucleic acid (especially RNA)), the quantitative size distribution of nucleic acid (especially RNA) (e.g., based on R_gor R_hvalues) of nucleic acid (especially RNA)), the shape factor, the form factor, and the nucleic acid (especially RNA) encapsulation efficiency. Generally, the size distribution and/or quantitative size distribution of nucleic acid (especially RNA) can be given as the number of the nucleic acid (especially RNA) molecules, the molar amount of the nucleic acid (especially RNA), or the mass of the nucleic acid (especially RNA) each as a function of their size. Further additional optional parameters include the ratio of the amount of nucleic acid (such as RNA) bound to particles to the total amount of particle forming compounds (in particular lipids and/or polymers) in the particles, wherein said ratio may be given as a function of the particle size; the ratio of the amount of positively charged moieties of particle forming compounds (in particular lipids and/or polymers) in the particles to the amount of nucleic acid (such as RNA) bound to particles, wherein said ratio may be given as a function of the particle size; and the charge ratio of the amount of positively charged moieties of particle forming compounds (in particular lipids and/or polymers) in the particles to the amount of negatively charged moieties of nucleic acid (such as RNA) bound to particles, wherein said charge ratio is usually denoted as N/P ratio and may be given as a function of the particle size.

In a first subgroup of the first aspect, the method comprises:

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

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

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

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

In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA) containing particles is/are calculated based on the R_gvalues of the nucleic acid (such as RNA) containing particles. In another embodiment of the first aspect (in particular, in another embodiment of the first, second or third subgroup of the first aspect), the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA) containing particles is/are calculated based on the R_hvalues of the nucleic acid (such as RNA) containing particles. In another embodiment of the first aspect (in particular, in another embodiment of the first, second or third subgroup of the first aspect), the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA) containing particles is/are calculated based on the R_gvalues of the nucleic acid (such as RNA) containing particles and separately based on the R_hvalues of nucleic acid (such as RNA) containing particles (i.e., this embodiment results in two data sets for the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA) containing particles, one based on the R_gvalues and one based on the R_hvalues).

In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), where the one or more parameters comprise the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA), the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA) is/are calculated based on the R_gvalues of the nucleic acid (such as RNA). In another embodiment of the first aspect (in particular, in another embodiment of the first, second or third subgroup of the first aspect), where the one or more parameters comprise the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA), the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA) is/are calculated based on the R_hvalues of the nucleic acid (such as RNA). In another embodiment of the first aspect (in particular, in another embodiment of the first, second or third subgroup of the first aspect), where the one or more parameters comprise the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA), the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA) is/are calculated based on the R_gvalues of the nucleic acid (such as RNA) and separately based on the R_hvalues of nucleic acid (such as RNA) (i.e., this embodiment results in two data sets for the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA), one based on the R_gvalues and one based on the R_hvalues).

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

In one embodiment of the first aspect (in particular, 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 (MW) cut-off suitable to prevent the nucleic acid (especially RNA) from permeating the membrane, preferably a membrane having a MW cut-off in the range of from 2 kDa to 30 kDa, such as a MW cut-off of 10 kDa.

In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), step (a) is performed using a polyethersulfon (PES) or regenerated cellulose membrane.

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

In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), the cross flow rate profile preferably contains a fractioning phase which allows the components contained in the control or sample composition to fraction/separate by their size so as to produce one or more sample fractions. It is preferred that the cross flow rate changes during this fractioning phase (e.g., starting from one value (such as about 1 to about 4 mL/min) and then decreasing to a lower value (such as about 0 to about 0.1 mL/min) or starting from one value (such as about 0 to about 0.1 mL/min) and then increasing to a higher value (such as about 1 to about 4 mL/min), wherein the change can be by any means, e.g., a continuous (such as linear or exponential) change or a stepwise change. Preferably, the cross flow rate profile contains a fractioning phase, wherein the cross flow rate changes continuously (preferably exponentially) starting from one value (such as about 1 to about 4 mL/min) and then decreasing to a lower value (such as about 0 to about 0.1 mL/min). The fractioning phase may have any length suitable to fraction/separate the components contained in the sample composition by their size, e.g., 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. The cross flow rate profile may contain additional phases (e.g., 1, 2, 3, or 4 phases) which may be before and/or after the fractioning phase (e.g., one before and 1, 2, or 3 after the fractioning phase) and which may serve to separate non-nucleic acid (especially non RNA) components contained in the sample composition (e.g., proteins, polypeptides, mononucleotides, etc.) from the nucleic acid (especially RNA) contained in the sample composition, to focus the nucleic acid (especially RNA) contained in the sample composition and/or to regenerate the field-flow fractionation device (e.g., to remove all components bound to the membrane of the device). Preferably, the cross flow rate of these additional phases is constant for each additional phase and the length of each of the additional phases is independently for each of the additional phases in the range of about 5 min to about 60 min (such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 30 min). For example, the cross flow rate profile may contain (i) a first additional phase which is before the fractioning phase, wherein the cross flow rate of said first additional phase is constant and is the same cross low rate with which the fractioning phase starts (the length of the first additional phase may be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 10 min or about 20 min or about 30 min); (ii) a second additional phase which is after the fractioning phase, wherein the cross flow rate of said second additional phase is constant and is the same cross low rate with which the fractioning phase ends (the length of the second additional phase may be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 10 min or about 20 min or about 30 min); and optionally (iii) a third additional phase which is after the second additional phase, wherein the cross flow rate of said third additional phase is constant and different from that of the second additional phase (the length of the third additional phase may be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 10 min or about 20 min or about 30 min). In the embodiment, where the cross flow rate profile contains a fractioning phase, wherein the cross flow rate changes continuously (preferably exponentially) starting from one value (such as about 1 to about 4 mL/min) and then decreasing to a lower value (such as about 0 to about 0.1 mL/min), it is preferred that the cross flow rate profile further contains (i) a first additional phase which is before the fractioning phase, wherein the cross flow rate of said first additional phase is constant and is the same cross low rate with which the fractioning phase starts (such as 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 or about 10 min or about 12 min); (ii) a second additional phase which is after the fractioning phase, wherein the cross flow rate of said second additional phase is constant and is the same cross low rate with which the fractioning phase ends (such as about 0.01 to 0.1 mL/min) (the length of the second additional phase 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 phase which is after the second additional phase, wherein the cross flow rate of said third additional phase is constant and lower than that of the second additional phase (e.g., the cross flow rate of said third additional phase is 0) (the length of the third 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 or about 10 min or about 12 min). A preferred example of such a cross flow rate profile is the following: 1.0 to 2.0 mL/min for 10 min, an exponential gradient from 1.0 to 2.0 mL/min to 0.01 to 0.07 mL/min within 30 min; 0.01 to 0.07 mL/min for 30 min; and 0 mL/min for 10 min.

In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), the integrity of the nucleic acid (especially RNA) contained in the sample composition is calculated using the integrity of a control nucleic acid (especially RNA).

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

(a′) subjecting at least a part of a control composition containing control nucleic acid (especially RNA) to field-flow fractionation, in particular AF4 or HF5, thereby fractioning the components contained in the control composition by their size so as to produce one or more control fractions;

(b′) measuring at least one signal selected from the group consisting of the UV signal, the fluorescence signal, and the refractory index (RI) signal of least one of the one or more control fractions obtained from step (a′);

(c′1) calculating from the at least one signal selected from the group consisting of the UV signal, the fluorescence signal, and the RI signal obtained in step (b′) the area from the maximum height of one UV, fluorescence, or RI peak to the end of the UV, fluorescence, or RI peak, thereby obtaining A_50%(control);

(c′2) calculating from the at least one signal selected from the group consisting of the UV signal, the fluorescence signal, and the RI signal obtained in step (b′) the total area of the one peak used in step

(c′1), thereby obtaining A_100%(control); and

(c′3) determining the ratio between A_50%(control) and A_100%(control), thereby obtaining the integrity of the control nucleic acid (especially RNA) (I(control)).

In this first example, the integrity of the nucleic acid (especially RNA) contained in the sample composition may be calculated by the following steps:

(c1) calculating from the at least one signal selected from the group consisting of the UV signal, the fluorescence signal, and the RI signal obtained from step (b) 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, thereby obtaining A_50%(sample);

(c2) calculating from the sample UV, fluorescence, or RI signal obtained from step (b) the total area of the sample UV, fluorescence, or RI peak used in step (c1), thereby obtaining A_100%(sample);

(c3) determining the ratio between A_50%(sample) and A_100%(sample), thereby obtaining I(sample); and

(c4) determining the ratio between I(sample) and I(control), thereby obtaining the integrity of the nucleic acid (especially RNA) contained in the sample composition.

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

(a″) subjecting at least a part of a control composition containing control nucleic acid (especially RNA) to field-flow fractionation, in particular AF4 or HF5, thereby fractioning the components contained in the control composition by their size so as to produce one or more control fractions;

(b″) measuring at least one signal selected from the group consisting of the UV signal, the fluorescence signal, and the RI signal of least one of the one or more control fractions obtained from step (a″); and

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

In this second example, the integrity of the nucleic acid (especially RNA) contained in the sample composition may be calculated by the following steps:

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

(c2′) determining the ratio between H(sample) and H(control), thereby obtaining the integrity of the nucleic acid (especially RNA) contained in the sample composition.

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

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

(c′1) calculating from the UV signal obtained in step (b′) the area from the maximum height of one UV peak to the end of the UV peak, thereby obtaining A_50%(control);

(c′2) calculating from the UV signal obtained in step (b′) the total area of the one peak used in step (c′1), thereby obtaining A_100%(control); and

(c′3) determining the ratio between A_50%(control) and A_100%(control), thereby obtaining the integrity of the control nucleic acid (especially RNA) (I(control)).

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

(c1) calculating from the UV signal obtained from step (b) the area from the maximum height of the sample UV peak corresponding to the control UV peak used in step (c′1) to the end of the sample UV peak, thereby obtaining A_50%(sample);

(c2) calculating from the sample UV signal obtained from step (b) the total area of the sample UV peak used in step (c1), thereby obtaining A_100%(sample);

(c3) determining the ratio between A_50%(sample) and A_100%(sample), thereby obtaining I(sample); and

(c4) determining the ratio between I(sample) and I(control), thereby obtaining the integrity of the nucleic acid (especially RNA) contained in the sample composition.

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

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

(c″) determining from the UV signal obtained in step (b″) the height of one UV peak (H(control)), thereby obtaining the integrity of the control nucleic acid (especially RNA).

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

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

(c2′) determining the ratio between H(sample) and H(control), thereby obtaining the integrity of the nucleic acid (especially RNA) contained in the sample composition.

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

(a′) subjecting at least a part of a control composition containing control RNA to field-flow fractionation, in particular AF4 or HF5, thereby fractioning the components contained in the control composition by their size so as to produce one or more control fractions;

(b′) measuring at least one signal selected from the group consisting of the UV signal, the fluorescence signal, and the RI signal of least one of the one or more control fractions obtained from step (a′);

(c′3) determining the ratio between A_50%(control) and A_100%(control), thereby obtaining the integrity of the control RNA (I(control)).

In this fifth example, the integrity of the RNA contained in the sample composition may be calculated by the following steps:

(c1) calculating from the at least one signal selected from the group consisting of the UV signal, the fluorescence signal, and the RI signal obtained from step (b) 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 (el) to the end of the sample UV, fluorescence, or RI peak, thereby obtaining A_50%(sample);

(c2) calculating from the sample UV, fluorescence, or RI signal obtained from step (b) the total area of the sample UV, fluorescence, or RI peak used in step (c1), thereby obtaining A_100%(sample);

(c3) determining the ratio between A_50%(sample) and A_100%(sample), thereby obtaining I(sample); and

(c4) determining the ratio between I(sample) and I(control), thereby obtaining the integrity of the RNA contained in the sample composition.

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

(a″) subjecting at least a part of a control composition containing control RNA to field-flow fractionation, in particular AF4 or HF5, thereby fractioning the components contained in the control composition by their size so as to produce one or more control fractions;

In this sixth example, the integrity of the RNA contained in the sample composition may be calculated by the following steps:

(c2′) determining the ratio between H(sample) and H(control), thereby obtaining the integrity of the RNA contained in the sample composition.

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

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

(c′1) calculating from the UV signal obtained in step (b′) the area from the maximum height of one UV peak to the end of the UV peak, thereby obtaining A_50%(control);

(c′2) calculating from the UV signal obtained in step (b′) the total area of the one peak used in step (c′1), thereby obtaining A_100%(control); and

(c′3) determining the ratio between A_50%(control) and A_100%(control), thereby obtaining the integrity of the control RNA (I(control)).

In this seventh example, the integrity of the RNA contained in the sample composition may be calculated by the following steps:

(c2) calculating from the sample UV signal obtained from step (b) the total area of the sample UV peak used in step (c1), thereby obtaining A_100%(sample);

(c3) determining the ratio between A_50%(sample) and A_100%(sample), thereby obtaining I(sample); and

(c4) determining the ratio between I(sample) and I(control), thereby obtaining the integrity of the RNA contained in the sample composition.

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

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

(c″) determining from the UV signal obtained in step (b″) the height of one UV peak (H(control)), thereby obtaining the integrity of the control RNA.

In this eighth example, the integrity of the RNA contained in the sample composition may be calculated by the following steps:

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

(c2′) determining the ratio between H(sample) and H(control), thereby obtaining the integrity of the RNA contained in the sample composition.

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

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

In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), the amount of total nucleic acid (especially the amount of total RNA) is determined by (i) treating at least a part of the sample composition with a release agent; (ii) performing steps (a) to (c) with at least the part obtained from step (i); and (iii) determining the amount of nucleic acid (especially RNA) as specified 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, the field-flow-fractionation is preferably performed using a liquid phase containing the release agent.

In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), the release agent is (i) a surfactant, such as an anionic surfactant (e.g., sodium dodecylsulfate), a zwitterionic surfactant (e.g., n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent® 3-14)), a cationic surfactant, a non-ionic surfactant, or a mixture thereof; (ii) an alcohol, such as an aliphatic alcohol (e.g., ethanol), or a mixture of alcohols; or (iii) a combination of (i) and (ii).

In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), the amount of free nucleic acid (especially RNA) is determined by performing steps (a) to (c) without the addition of a release agent, in particular in the absence of any release agent; and determining the amount of nucleic acid (especially RNA) as specified 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 one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), the amount of nucleic acid (especially RNA) bound to particles is determined by subtracting the amount of free nucleic acid (especially RNA) as determined herein (e.g., by performing steps (a) to (c) without the addition of a release agent, in particular in the absence of any release agent; and determining the amount of nucleic acid (especially RNA) as specified 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))) from the amount of total nucleic acid (especially RNA) as determined herein (e.g., by (i) treating at least a part of the sample composition with a release agent; (ii) performing steps (a) to (c) with at least the part obtained from step (i); and (iii) determining the amount of nucleic acid (especially RNA) as specified 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 one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), step (b) further comprises measuring the LS signal, such as the dynamic light scattering (DLS) signal and/or the static light scattering (SLS), e.g., multi-angle light scattering (MALS), signal, of least one of the one or more sample fractions obtained from step (a).

In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), the size of nucleic acid (especially RNA) containing particles is determined by calculating from the LS signal obtained from step (b) the radius of gyration (R_g) values and/or the hydrodynamic radius (R_h) values. In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), step (b) comprises measuring the dynamic light scattering (DLS) signal of least one of the one or more sample fractions obtained from step (a), and step (c) comprises calculating the R_hvalues from the DLS signal. In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), step (b) comprises measuring the static light scattering (SLS), e.g., MALS, signal of least one of the one or more sample fractions obtained from step (a), and step (c) comprises calculating the R_gvalues from the SLS signal. In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), step (b) comprises measuring the dynamic light scattering (DLS) signal and the static light scattering (SLS), e.g., MALS, signal of least one of the one or more sample fractions obtained from step (a), and step (c) comprises calculating the R_gand R_hvalues. This latter embodiment results in two data sets for the size of nucleic acid (such as RNA) containing particles, i.e., one based on the R_gvalues and one based on the R_hvalues.

In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), the size distribution of nucleic acid (especially RNA) containing particles is determined by plotting the at least one signal selected from the group consisting of the UV signal, the fluorescence signal, and the RI signal obtained from step (b) against the R_gor R_hvalues determined as specified herein (e.g., by calculating the R_gvalues from the SLS signal obtained from step (b) or by calculating the R_hvalues from the DLS signal obtained from step (b)). In a first example of this embodiment (relating to the first subgroup of the first aspect), the size distribution of nucleic acid (especially RNA) containing particles is determined by plotting the UV signal obtained from step (b) against the R_gor R_hvalues determined as specified herein (e.g., by calculating the R_gvalues from the SLS signal obtained from step (b) or by calculating the R_hvalues from the DLS signal obtained from step (b)). In a second example of this embodiment (relating to the second subgroup of the first aspect), the size distribution of RNA containing particles is determined by plotting the at least one signal selected from the group consisting of the UV signal, the fluorescence signal, and the RI signal obtained from step (b) against the R_gor R_hvalues determined as specified herein (e.g., by calculating the R_gvalues from the SLS signal obtained from step (b) or by calculating the R_hvalues from the DLS signal obtained from step (b)). In a third example of this embodiment (relating to the third subgroup of the first aspect), the size distribution of RNA containing particles is determined by plotting the UV signal obtained from step (b) against the R_gor R_hvalues determined as specified herein (e.g., by calculating the R_gvalues from the SLS signal obtained from step (b) or by calculating the R_hvalues from the DLS signal obtained from step (b)). In each of the above first, second and third examples, the size distribution of nucleic acid (especially RNA) containing particles can be determined on the basis of the R_gvalues, the R_hvalues or both. If the size distribution of nucleic acid (especially RNA) containing particles is determined on the basis of the R_gvalues and the R_hvalues, this results in two data sets, i.e., one size distribution based on the R_gvalues and one size distribution based on the R_hvalues.

In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), the quantitative size distribution of nucleic acid (especially RNA) containing particles is calculated from the plot showing the UV, fluorescence, or RI signal as function of the R_gor R_hvalues by transforming the UV, fluorescence, or RI signal into a cumulative weight fraction and plotting the cumulative weight fraction against the R_gor R_hvalues. In a first example of this embodiment (relating to the first subgroup of the first aspect), the quantitative size distribution of nucleic acid (especially RNA) containing particles is calculated from the plot showing the UV signal as function of the R_gor R_hvalues by transforming the UV signal into a cumulative weight fraction and plotting the cumulative weight fraction against the R_gor R_hvalues. In a second example of this embodiment (relating to the second subgroup of the first aspect), the quantitative size distribution of RNA containing particles is calculated from the plot showing the UV, fluorescence, or RI signal as function of the R_gor R_hvalues by transforming the UV, fluorescence, or RI signal into a cumulative weight fraction and plotting the cumulative weight fraction against the R_gor R_hvalues. In a third example of this embodiment (relating to the third subgroup of the first aspect), the quantitative size distribution of RNA containing particles is calculated from the plot showing the UV signal as function of the R_gor R_hvalues by transforming the UV signal into a cumulative weight fraction and plotting the cumulative weight fraction against the R_gor R_hvalues. In each of the above first, second and third examples, the quantitative size distribution of nucleic acid (especially RNA) containing particles can be determined on the basis of the R_gvalues, the R_hvalues or both. If the quantitative size distribution of nucleic acid (especially RNA) containing particles is determined on the basis of the R_gvalues and the R_hvalues, this results in two data sets, i.e., one quantitative size distribution based on the R_gvalues and one quantitative size distribution based on the R_hvalues.

In one embodiment of the first aspect (in particular, 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_gor R_hvalues). If the quantitative size distribution of nucleic acid (especially RNA) containing particles is determined on the basis of the R_gvalues and the R_hvalues, this results in two data sets, i.e., one set of D10, D50, and/or D90 values based on the R_gvalues and one set of D10, D50, and/or D90 values based on the R_hvalues.

In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), the one or more parameters comprise (or are) at least two, preferably at least three, parameters as specified herein (including the additional optional parameters), in particular at least two, preferably at least three, parameters selected from the group consisting of: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to particles, the size distribution of nucleic acid (especially RNA) containing particles (in particular, based on the radius of gyration (R_g) of nucleic acid (especially RNA) containing particles and/or the hydrodynamic radius (R_h) of nucleic acid (especially RNA) containing particles), and the quantitative size distribution of nucleic acid (especially RNA) containing particles (e.g., based on R_gor R_hvalues). If the size distribution of nucleic acid (especially RNA) containing particles is determined on the basis of the R_gvalues and the R_hvalues, this results in two data sets, i.e., one based on the R_gvalues and one based on the R_hvalues. However, according to the present invention, these two data sets for the size distribution of nucleic acid (especially RNA) containing particles are only considered as one parameter (and not as two parameters). In addition, in case the fractogram obtained by the field-flow fractionation shows more than one particle peak, the determination of the size distribution for each of the particle peaks is only considered as one parameter (and not as one parameter for each of the particle peaks). The same applies to the situation where the quantitative size distribution of nucleic acid (especially RNA) containing particles is determined on the basis of the R_gvalues and the R_hvalues.

In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect, in particular in a preferred embodiment of the third subgroup of the first aspect), the one or more parameters comprise the quantitative size distribution of nucleic acid (especially RNA) containing particles (e.g., based on the radius of gyration (R_g) of nucleic acid (especially RNA) containing particles and/or the hydrodynamic radius (R_h) of nucleic acid (especially RNA) containing particles) and optionally at least one parameter, such as at least two parameters, of the remaining parameters specified herein (including the additional optional parameters); preferably these remaining parameters are selected from the group consisting of: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to particles, and the size distribution of nucleic acid (especially RNA) containing particles (e.g., based on R_gor R_hvalues). In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect, in particular in a preferred embodiment of the third subgroup of the first aspect), the one or more parameters comprise the quantitative size distribution of nucleic acid (especially RNA) containing particles (e.g., based on R_gor R_hvalues) and at least one parameter, such as at least two parameters, selected from the group consisting of: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to particles, and the size distribution of nucleic acid (especially RNA) containing particles (e.g., based on R_gor R_hvalues). In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect, in particular in a preferred embodiment of the third subgroup of the first aspect), the one or more parameters comprise the quantitative size distribution of nucleic acid (especially RNA) containing particles (e.g., based on R_gor R_hvalues), the amount of free nucleic acid (especially RNA), and the amount of nucleic acid (especially RNA) bound to particles. If the quantitative size distribution of nucleic acid (especially RNA) containing particles is determined on the basis of the R_gvalues and the R_hvalues, this results in two data sets, i.e., one based on the R_gvalues and one based on the R_hvalues. However, according to the present invention, these two data sets for the quantitative size distribution of nucleic acid (especially RNA) containing particles are only considered as one parameter (and not as two parameters). In addition, in case the fractogram obtained by the field-flow fractionation shows more than one particle peak, the determination of the quantitative size distribution for each of the particle peaks is only considered as one parameter (and not as one parameter for each of the particle peaks). The same applies to the situation where the size distribution of nucleic acid (especially RNA) containing particles is determined on the basis of the R_gvalues and the R_hvalues.

In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), the amount of nucleic acid (especially RNA), in particular free nucleic acid (especially RNA), is determined by measuring the UV signal, e.g., 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 nucleic acid (especially RNA) extinction coefficient at the corresponding wavelength (e.g., 260 nm or 280 nm).

In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect, in particular in a preferred embodiment of the third subgroup of the first aspect), the size distribution of nucleic acid (especially RNA) containing particles (e.g., based on R_gor R_hvalues) and/or the quantitative size distribution of nucleic acid (especially RNA) containing particles (e.g., based on R_gor R_hvalues) is/are within the range of 10 to 2000 nm, preferably within the range of 20 to 1500 nm, such as 30 to 1200 nm, 40 to 1100 nm, 50 to 1000, 60 to 900 nm, 70 to 800 nm, 80 to 700 nm, 90 to 600 nm, or 100 to 500 nm, or such as within the range of 10 to 1000 nm, 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm, or 50 to 250 nm. In a preferred embodiment of the third subgroup of the first aspect, the (quantitative) size distribution of RNA containing particles (e.g., based on R_gor R_hvalues) is within the range of 10 to 1000 nm, such as within the range of 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm, or 50 to 250 nm.

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

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

In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), measuring the at least one signal selected from the group consisting of the UV signal, the fluorescence signal, and the RI signal, optionally the LS signal, such as the SLS, e.g., MALS, signal and/or the DLS signal, is performed on-line and/or step (c) is performed on-line.

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

In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), before subjecting at least a part of the sample composition to field-flow fractionation, the at least part of the sample composition is diluted with a solvent or solvent mixture, said solvent or solvent mixture being able to prevent the formation of aggregates of the particles. In one embodiment, the solvent mixture is a mixture of water and an organic solvent, e.g., formamide.

In one embodiment of the first aspect (in particular, in one embodiment of the first, second or third subgroup of the first aspect), measuring the UV signal is performed by using circular dichroism (CD) spectroscopy.

In a second aspect, the present disclosure provides a method of analyzing the effect of altering one or more reaction conditions when providing a composition comprising a nucleic acid (such as RNA) and optionally particles, the method comprising:

(A) providing a first composition comprising nucleic acid (such as RNA) and optionally particles;

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

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

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

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

In one embodiment of the second aspect, the one or more parameters comprise the nucleic acid (such as RNA) integrity, the total amount of nucleic acid (such as RNA), the amount of free nucleic acid (such as RNA), the amount of nucleic acid (such as RNA) bound to particles, the size of nucleic acid (such as RNA) containing particles (in particular, based on the radius of gyration (R_g) of nucleic acid (such as RNA) containing particles and/or the hydrodynamic radius (R_h) of nucleic acid (such as RNA) containing particles), the size distribution of nucleic acid (such as RNA) containing particles (e.g., based on R_gor R_hvalues of nucleic acid (such as RNA) containing particles), and the quantitative size distribution of nucleic acid (such as RNA) containing particles (e.g., based on R_gor R_hvalues of nucleic acid (such as RNA) containing particles). Generally, the size distribution and/or quantitative size distribution of nucleic acid (such as RNA) containing particles can be given as the number of the nucleic acid (such as RNA) containing particles, the molar amount of the nucleic acid (such as RNA) containing particles, or the mass of the nucleic acid (such as RNA) containing particles each as a function of their size. Additional optional parameters include the molecular weight of 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), the size of nucleic acid (especially RNA) (in particular, based on R_gand/or R_hvalues of nucleic acid (such as RNA)), the size distribution of nucleic acid (especially RNA) (e.g., based on R_gor R_hvalues of nucleic acid (such as RNA)), the quantitative size distribution of nucleic acid (especially RNA) (e.g., based on R_gor R_hvalues of nucleic acid (such as RNA)), the shape factor, the form factor, and the nucleic acid (especially RNA) encapsulation efficiency. Generally, the size distribution and/or quantitative size distribution of nucleic acid (especially RNA) can be given as the number of the nucleic acid (especially RNA) molecules, the molar amount of the nucleic acid (especially RNA), or the mass of the nucleic acid (especially RNA) each as a function of their size. Further additional optional parameters include the ratio of the amount of nucleic acid (such as RNA) bound to particles to the total amount of particle forming compounds (in particular lipids and/or polymers) in the particles, wherein said ratio may be given as a function of the particle size; the ratio of the amount of positively charged moieties of particle forming compounds (in particular lipids and/or polymers) in the particles to the amount of nucleic acid (such as RNA) bound to particles, wherein said ratio may be given as a function of the particle size; and the charge ratio of the amount of positively charged moieties of particle forming compounds (in particular lipids and/or polymers) in the particles to the amount of negatively charged moieties of nucleic acid (such as RNA) bound to particles, wherein said charge ratio is usually denoted as N/P ratio and may be given as a function of the particle size.

In one embodiment of the second aspect, the one or more parameters comprise (or are) at least two, preferably at least three, parameters as specified herein (including the additional optional parameters), in particular at least two, preferably at least three, parameters selected from the group consisting of: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to particles, the size distribution of nucleic acid (especially RNA) containing particles (e.g., based on R_gor R_hvalues of nucleic acid (such as RNA) containing particles), the quantitative size distribution of nucleic acid (especially RNA) containing particles (e.g., based on R_gor R_hvalues of nucleic acid (such as RNA) containing particles), and the molecular weight of nucleic acid (especially RNA). In one embodiment of the second aspect, the one or more parameters comprise (or are) at least two, preferably at least three, parameters selected from the group consisting of: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to particles, the size distribution of nucleic acid (especially RNA) containing particles (e.g., based on R_gor R_hvalues of nucleic acid (especially RNA)), and the quantitative size distribution of nucleic acid (especially RNA) containing particles (e.g., based on R_gor R_hvalues of nucleic acid (especially RNA)).

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

(a) subjecting at least a part of the composition (e.g., the first composition for step (C) or the second composition for step (D)) to field-flow fractionation, thereby fractioning the components contained in the composition by their size so as to produce one or more composition fractions;

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

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

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

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

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

In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect), the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA) containing particles is/are calculated based on the R_gvalues of the nucleic acid (such as RNA) containing particles. In another embodiment of the second aspect (in particular, in another embodiment of the first, second or third subgroup of the second aspect), the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA) containing particles is/are calculated based on the R_hvalues of the nucleic acid (such as RNA) containing particles. In another embodiment of the second aspect (in particular, in another embodiment of the first, second or third subgroup of the second aspect), the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA) containing particles is/are calculated based on the R_gvalues of the nucleic acid (such as RNA) containing particles and separately based on the R_hvalues of nucleic acid (such as RNA) containing particles (i.e., this embodiment results in two data sets for the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA) containing particles, one based on the R_gvalues and one based on the R_hvalues).

In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect), where the one or more parameters comprise the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA), the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA) is/are calculated based on the R_gvalues of the nucleic acid (such as RNA). In another embodiment of the second aspect (in particular, in another embodiment of the first, second or third subgroup of the second aspect), where the one or more parameters comprise the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA), the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA) is/are calculated based on the R_hvalues of the nucleic acid (such as RNA). In another embodiment of the second aspect (in particular, in another embodiment of the first, second or third subgroup of the second aspect), where the one or more parameters comprise the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA), the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA) is/are calculated based on the R_gvalues of the nucleic acid (such as RNA) and separately based on the R_hvalues of nucleic acid (such as RNA) (i.e., this embodiment results in two data sets for the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA), one based on the R_gvalues and one based on the R_hvalues).

In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect), the one or more parameters comprise the nucleic acid (especially RNA) integrity, the total amount of nucleic acid (especially RNA), the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to particles, the size of nucleic acid (especially RNA) containing particles (in particular, based on the radius of gyration (R_g) of nucleic acid (especially RNA) containing particles and/or the hydrodynamic radius (R_h) of nucleic acid (especially RNA) containing particles), the size distribution of nucleic acid (especially RNA) containing particles (e.g., based on R_gor R_hvalues), and the quantitative size distribution of nucleic acid (especially RNA) containing particles (e.g., based on R_gor R_hvalues), and optionally the molecular weight of nucleic acid (especially RNA).

In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect), the one or more parameters comprise (or are) at least two, preferably at least three, parameters selected from the group consisting of: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to particles, the size distribution of nucleic acid (especially RNA) containing particles (e.g., based on R_gor R_hvalues of nucleic acid (especially RNA) containing particles), the quantitative size distribution of nucleic acid (especially RNA) containing particles (e.g., based on R_gor R_hvalues of nucleic acid (especially RNA) containing particles), and the molecular weight of nucleic acid (especially RNA). In one embodiment of the first, second or third subgroup of the second aspect, the one or more parameters comprise (or are) at least two, preferably at least three, parameters selected from the group consisting of: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to particles, the size distribution of nucleic acid (especially RNA) containing particles (e.g., based on R_gor R_hvalues of nucleic acid (especially RNA) containing particles), and the quantitative size distribution of nucleic acid (especially RNA) containing particles (e.g., based on R_gor R_hvalues of nucleic acid (especially RNA) containing particles). If the size distribution of nucleic acid (especially RNA) containing particles is determined on the basis of the R_gvalues and the R_hvalues, this results in two data sets, i.e., one based on the R_gvalues and one based on the R_hvalues. However, according to the present invention, these two data sets for the size distribution of nucleic acid (especially RNA) containing particles are only considered as one parameter (and not as two parameters). In addition, in case the fractogram obtained by the field-flow fractionation shows more than one particle peak, the determination of the size distribution for each of the particle peaks is only considered as one parameter (and not as one parameter for each of the particle peaks). The same applies to the situation where the quantitative size distribution of nucleic acid (especially RNA) containing particles is determined on the basis of the R_gvalues and the R_hvalues.

In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect, in particular in a preferred embodiment of the third subgroup of the second aspect), the one or more parameters comprise the quantitative size distribution of nucleic acid (especially RNA) containing particles (e.g., based on the radius of gyration (R_g) of nucleic acid (especially RNA) containing particles and/or the hydrodynamic radius (R_h) of nucleic acid (especially RNA) containing particles) and optionally at least one parameter, such as at least two parameters, of the remaining parameters specified herein (including the additional optional parameters); preferably these remaining parameters are selected from the group consisting of: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to particles, and the size distribution of nucleic acid (especially RNA) containing particles (e.g., based on R_gor R_hvalues). In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect, in particular in a preferred embodiment of the third subgroup of the second aspect), the one or more parameters comprise the quantitative size distribution of nucleic acid (especially RNA) containing particles (e.g., based on R_gor R_hvalues) and at least one parameter, such as at least two parameters, selected from the group consisting of: the amount of free nucleic acid (especially RNA), the amount of nucleic acid (especially RNA) bound to particles, and the size distribution of nucleic acid (especially RNA) containing particles (e.g., based on R_gor R_hvalues). In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect, in particular in a preferred embodiment of the third subgroup of the second aspect), the one or more parameters comprise the quantitative size distribution of nucleic acid (especially RNA) containing particles (e.g., based on R_gor R_hvalues), the amount of free nucleic acid (especially RNA), and the amount of nucleic acid (especially RNA) bound to particles. If the quantitative size distribution of nucleic acid (especially RNA) containing particles is determined on the basis of the R_gvalues and the R_hvalues, this results in two data sets, i.e., one based on the R_gvalues and one based on the R_hvalues. However, according to the present invention, these two data sets for the quantitative size distribution of nucleic acid (especially RNA) containing particles are only considered as one parameter (and not as two parameters). In addition, in case the fractogram obtained by the field-flow fractionation shows more than one particle peak, the determination of the quantitative size distribution for each of the particle peaks is only considered as one parameter (and not as one parameter for each of the particle peaks). The same applies to the situation where the size distribution of nucleic acid (especially RNA) containing particles is determined on the basis of the R_gvalues and the R_hvalues.

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

In one embodiment of the second aspect (in particular, in one embodiment of the first, second, or third subgroup of the second aspect), the one or more reaction conditions comprise any of the following: salt concentration/ionic strength; temperature; pH or buffer concentration; light/radiation; oxygen; shear force; pressure; freezing/thawing cycle; drying/reconstitution cycle; addition of excipient(s) (e.g., stabilizer and/or chelating agent); type and/or source of particle forming compounds (in particular lipids and/or polymers); charge ratio; physical state; and ratio of nucleic acid (especially RNA) to particle forming compounds (in particular lipids and/or polymers constituting the particles). Exemplary salt concentrations include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 mM of a salt, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 mM NaCl. Exemplary temperature conditions include low temperature (such as −20° C.), ambient or room temperature, middle temperature (such as 30° C.) or high temperature (such as 50° C.). Exemplary conditions regarding type and/or source of particle forming compounds are cationic lipid vs. cationic polymer, cationic lipid vs. zwitterionic lipid, or pegylated lipid vs. unpegylated lipid. An exemplary charge ratio of positive charges to negative charges in the nucleic acid (especially RNA) particles is from about 6:1 to about 1:2, such as about 5:1 to about 1.2:2, about 4:1 to about 1.4:2, about 3:1 to about 1.6:2, about 2:1 to about 1.8:2, or about 1.6:1 to about 1:1. An exemplary ratio of nucleic acid (especially RNA) to particle forming compounds (in particular the lipids and/or polymers constituting the particles) include ratios of nucleic acid (especially RNA) to total lipids in the range of from about 1:100 to about 10:1 (w/w).

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

In one embodiment of the second aspect (in particular, 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 (MW) cut-off suitable to prevent the nucleic acid (especially RNA) from permeating the membrane, preferably a membrane having a MW cut-off in the range of from 2 kDa to 30 kDa, such as a MW cut-off of 10 kDa.

In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect), step (a) is performed using a polyethersulfon (PES) or regenerated cellulose membrane.

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

In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect), the cross flow rate profile preferably contains a fractioning phase which allows the components contained in the composition (such as control or sample composition, in particular the first composition for step (C) or the second composition for step (D)) to fraction/separate by their size so as to produce one or more composition fractions. It is preferred that the cross flow rate changes during this fractioning phase (e.g., starting from one value (such as about 1 to about 4 mL/min) and then decreasing to a lower value (such as about 0 to about 0.1 mL/min) or starting from one value (such as about 0 to about 0.1 mL/min) and then increasing to a higher value (such as about 1 to about 4 mL/min), wherein the change can be by any means, e.g., a continuous (such as linear or exponential) change or a stepwise change. Preferably, the cross flow rate profile contains a fractioning phase, wherein the cross flow rate changes continuously (preferably exponentially) starting from one value (such as about 1 to about 4 mL/min) and then decreasing to a lower value (such as about 0 to about 0.1 mL/min). The fractioning phase may have any length suitable to fraction/separate the components contained in the composition by their size, e.g., 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. The cross flow rate profile may contain additional phases (e.g., 1, 2, 3, or 4 phases) which may be before and/or after the fractioning phase (e.g., one before and 1, 2, or 3 after the fractioning phase) and which may serve to separate non-nucleic acid (especially non RNA) components contained in the composition (e.g., proteins, polypeptides, mononucleotides, etc.) from the nucleic acid (especially RNA) contained in the composition, to focus the nucleic acid (especially RNA) contained in the composition and/or to regenerate the field-flow fractionation device (e.g., to remove all components bound to the membrane of the device). Preferably, the cross flow rate of these additional phases is constant for each additional phase and the length of each of the additional phases is independently for each of the additional phases in the range of about 5 min to about 60 min (such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 30 min). For example, the cross flow rate profile may contain (i) a first additional phase which is before the fractioning phase, wherein the cross flow rate of said first additional phase is constant and is the same cross low rate with which the fractioning phase starts (the length of the first additional phase may be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 10 min or about 20 min or about 30 min); (ii) a second additional phase which is after the fractioning phase, wherein the cross flow rate of said second additional phase is constant and is the same cross low rate with which the fractioning phase ends (the length of the second additional phase may be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 10 min or about 20 min or about 30 min); and optionally (iii) a third additional phase which is after the second additional phase, wherein the cross flow rate of said third additional phase is constant and different from that of the second additional phase (the length of the third additional phase may be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 10 min or about 20 min or about 30 min). In the embodiment, where the cross flow rate profile contains a fractioning phase, wherein the cross flow rate changes continuously (preferably exponentially) starting from one value (such as about 1 to about 4 mL/min) and then decreasing to a lower value (such as about 0 to about 0.1 mL/min), it is preferred that the cross flow rate profile further contains (i) a first additional phase which is before the fractioning phase, wherein the cross flow rate of said first additional phase is constant and is the same cross low rate with which the fractioning phase starts (such as 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 or about 10 min or about 12 min); (ii) a second additional phase which is after the fractioning phase, wherein the cross flow rate of said second additional phase is constant and is the same cross low rate with which the fractioning phase ends (such as about 0.01 to 0.1 mL/min) (the length of the second additional phase 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 phase which is after the second additional phase, wherein the cross flow rate of said third additional phase is constant and lower than that of the second additional phase (e.g., the cross flow rate of said third additional phase is 0) (the length of the third 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 or about 10 min or about 12 min). A preferred example of such a cross flow rate profile is the following: 1.0 to 2.0 mL/min for 10 min, an exponential gradient from 1.0 to 2.0 mL/min to 0.01 to 0.07 mL/min within 30 min; 0.01 to 0.07 mL/min for 30 min; and 0 mL/min for 10 min.

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

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

(c′3) determining the ratio between A_50%(control) and A_100%(control), thereby obtaining the integrity of the control nucleic acid (especially RNA) (I(control)).

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

(c2) calculating from the sample UV, fluorescence, or RI signal obtained from step (b) the total area of the sample UV, fluorescence, or RI peak used in step (c1), thereby obtaining A_100%(sample);

(c3) determining the ratio between A_50%(sample) and A_100%(sample), thereby obtaining I(sample); and

(c4) determining the ratio between I(sample) and I(control), thereby obtaining the integrity of the nucleic acid (especially RNA) contained in the sample composition.

Ina second particular example of this embodiment of the second aspect, the integrity of a control nucleic acid (especially RNA) is determined by the following steps:

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

(c2′) determining the ratio between H(sample) and H(control), thereby obtaining the integrity of the nucleic acid (especially RNA) contained in the sample composition.

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

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

(c′1) calculating from the UV signal obtained in step (1:0 the area from the maximum height of one UV peak to the end of the UV peak, thereby obtaining A_50%(control);

(c′2) calculating from the UV signal obtained in step (b) the total area of the one peak used in step (c′1), thereby obtaining A_100%(control); and

(c′3) determining the ratio between A_50%(control) and A_100%(control), thereby obtaining the integrity of the control nucleic acid (especially RNA) (I(control)).

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

(c2) calculating from the sample UV signal obtained from step (b) the total area of the sample UV peak used in step (c1), thereby obtaining A_100%(sample);

(c3) determining the ratio between A_50%(sample) and A_100%(sample), thereby obtaining I(sample); and

(c4) determining the ratio between I(sample) and I(control), thereby obtaining the integrity of the nucleic acid (especially RNA) contained in the sample composition.

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

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

(c″) determining from the UV signal obtained in step (b″) the height of one UV peak (H(control)), thereby obtaining the integrity of the control nucleic acid (especially RNA).

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

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

(c2′) determining the ratio between H(sample) and H(control), thereby obtaining the integrity of the nucleic acid (especially RNA) contained in the sample composition.

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

(c′3) determining the ratio between A_50%(control) and A_100%(control), thereby obtaining the integrity of the control RNA (I(control)).

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)) may be calculated by the following steps:

(c2) calculating from the sample UV, fluorescence, or RI signal obtained from step (b) the total area of the sample UV, fluorescence, or RI peak used in step (c1), thereby obtaining A_100%(sample);

(c3) determining the ratio between A_50%(sample) and A_100%(sample), thereby obtaining I(sample); and

(c4) determining the ratio between I(sample) and I(control), thereby obtaining the integrity of the RNA contained in the sample composition.

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

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)) may be calculated by the following steps:

(c2′) determining the ratio between H(sample) and H(control), thereby obtaining the integrity of the RNA contained in the sample composition.

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

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

(c′1) calculating from the UV signal obtained in step (b′) the area from the maximum height of one UV peak to the end of the UV peak, thereby obtaining A_50%(control);

(c′2) calculating from the UV signal obtained in step (b′) the total area of the one peak used in step (c′1), thereby obtaining A_100%(control); and

(c′3) determining the ratio between A_50%(control) and A_100%(control), thereby obtaining the integrity of the control RNA (I(control)).

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)) may be calculated by the following steps:

(c2) calculating from the sample UV signal obtained from step (b) the total area of the sample UV peak used in step (c1), thereby obtaining A_100%(sample);

(c3) determining the ratio between A_50%(sample) and A_100%(sample), thereby obtaining I(sample); and

(c4) determining the ratio between I(sample) and I(control), thereby obtaining the integrity of the RNA contained in the sample composition.

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

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

(c″) determining from the UV signal obtained in step (b″) the height of one UV peak (H(control)), thereby obtaining the integrity of the control RNA.

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)) may be calculated by the following steps:

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

(c2′) determining the ratio between H(sample) and H(control), thereby obtaining the integrity of the RNA contained in the sample composition.

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

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

In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect), the amount of total nucleic acid (especially the amount of total RNA) is determined by (i) treating at least a part of the sample composition (e.g., the first composition for step (C) or the second composition for step (D)) with a release agent; (ii) performing steps (a) to (c) with at least the part obtained from step (i); and (iii) determining the amount of nucleic acid (especially RNA) as specified 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, the field-flow-fractionation is preferably performed using a liquid phase containing the release agent.

In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect), the release agent is (i) a surfactant, such as an anionic surfactant (e.g., sodium dodecylsulfate), a zwitterionic surfactant (e.g., n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent® 3-14)), a cationic surfactant, a non-ionic surfactant, or a mixture thereof; (ii) an alcohol, such as an aliphatic alcohol (e.g., ethanol), or a mixture of alcohols; or (iii) a combination of (i) and (ii).

In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect), the amount of free nucleic acid (especially RNA) is determined by performing steps (a) to (c) without the addition of a release agent, in particular in the absence of any release agent; and determining the amount of nucleic acid (especially RNA) as specified 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 one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect), the amount of nucleic acid (especially RNA) bound to particles is determined by subtracting the amount of free nucleic acid (especially RNA) as determined herein (e.g., by performing steps (a) to (c) without the addition of a release agent, in particular in the absence of any release agent; and determining the amount of nucleic acid (especially RNA) as specified 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))) from the amount of total nucleic acid (especially RNA) as determined herein (e.g., by (i) treating at least a part of the sample composition (e.g., the first composition for step (C) or the second composition for step (D)) with a release agent; (ii) performing steps (a) to (c) with at least the part obtained from step (i); and (iii) determining the amount of nucleic acid (especially RNA) as specified 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 one embodiment of the second aspect (in particular, in one embodiment of the second or third subgroup of the second aspect), step (b) further comprises measuring the LS signal, such as the dynamic light scattering (DLS) and/or the static light scattering (SLS), e.g., multi-angle light scattering (MALS), signal, of least one of the one or more sample fractions obtained from step (a).

In one embodiment of the second aspect (in particular, in one embodiment of the second or third subgroup of the second aspect), the size of nucleic acid (especially RNA) containing particles is determined by calculating from the LS signal obtained from step (b) the radius of gyration (R_g) values and/or the hydrodynamic radius (R_h) values. In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect), step (b) comprises measuring the dynamic light scattering (DLS) signal of least one of the one or more sample fractions obtained from step (a) and step (c) comprises calculating the R_hvalues from the DLS signal. In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect), step (b) comprises measuring the static light scattering (SLS), e.g., MALS, signal of least one of the one or more sample fractions obtained from step (a), and step (c) comprises calculating the R_gvalues from the SLS signal. In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect), step (b) comprises measuring the dynamic light scattering (DLS) signal and the static light scattering (SLS), e.g., MALS, signal of least one of the one or more sample fractions obtained from step (a) and step (c) comprises calculating the R_gand R_hvalues. This latter embodiment results in two data sets for the size of nucleic acid (such as RNA) containing particles, i.e., one based on the R_gvalues and one based on the R_hvalues.

In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect), the size distribution of nucleic acid (especially RNA) containing particles is determined by plotting the at least one signal selected from the group consisting of the UV signal, the fluorescence signal, and the RI signal obtained from step (b) against the R_gor R_hvalues determined as specified herein (e.g., by calculating the R_gvalues from the SLS signal obtained from step (b) or by calculating the R_hvalues from the DLS signal obtained from step (b)). In a first example of this embodiment (relating to the first subgroup of the second aspect), the size distribution of nucleic acid (especially RNA) containing particles is determined by plotting the UV signal obtained from step (b) against the R_gor R_hvalues determined as specified herein (e.g., by calculating the R_gvalues from the SLS signal obtained from step (b) or by calculating the R_hvalues from the DLS signal obtained from step (b)). In a second example of this embodiment (relating to the second subgroup of the second aspect), the size distribution of RNA containing particles is determined by plotting the at least one signal selected from the group consisting of the UV signal, the fluorescence signal, and the RI signal obtained from step (b) against the R_gor R_hvalues determined as specified herein (e.g., by calculating the R_gvalues from the SLS signal obtained from step (b) or by calculating the R_hvalues from the DLS signal obtained from step (b)). In a third example of this embodiment (relating to the third subgroup of the second aspect), the size distribution of RNA containing particles is determined by plotting the UV signal obtained from step (b) against the R_gor R_hvalues determined as specified herein (e.g., by calculating the R_gvalues from the SLS signal obtained from step (b) or by calculating the R_hvalues from the DLS signal obtained from step (b)). In each of the above first, second and third examples, the size distribution of nucleic acid (especially RNA) containing particles can be determined on the basis of the R_gvalues, the R_hvalues or both. If the size distribution of nucleic acid (especially RNA) containing particles is determined on the basis of the R_gvalues and the R_hvalues, this results in two data sets, i.e., one size distribution based on the R_gvalues and one size distribution based on the R_hvalues.

In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect), the quantitative size distribution of nucleic acid (especially RNA) containing particles is calculated from the plot showing the UV, fluorescence, or RI signal as function of the R_gor R_hvalues by transforming the UV, fluorescence, or RI signal into a cumulative weight fraction and plotting the cumulative weight fraction against the R_gor R_hvalues. In a first example of this embodiment (relating to the first subgroup of the second aspect), the quantitative size distribution of nucleic acid (especially RNA) containing particles is calculated from the plot showing the UV signal as function of the R_gor R_hvalues by transforming the UV signal into a cumulative weight fraction and plotting the cumulative weight fraction against the R_gor R_hvalues. In a second example of this embodiment (relating to the second subgroup of the second aspect), the quantitative size distribution of RNA containing particles is calculated from the plot showing the UV, fluorescence, or RI signal as function of the R_gor R_hvalues by transforming the UV, fluorescence, or RI signal into a cumulative weight fraction and plotting the cumulative weight fraction against the R_gor R_hvalues. In a third example of this embodiment (relating to the third subgroup of the second aspect), the quantitative size distribution of RNA containing particles is calculated from the plot showing the UV signal as function of the R_gor R_hvalues by transforming the UV signal into a cumulative weight fraction and plotting the cumulative weight fraction against the R_gor R_hvalues. In each of the above first, second and third examples, the quantitative size distribution of nucleic acid (especially RNA) containing particles can be determined on the basis of the R_gvalues, the R_hvalues or both. If the quantitative size distribution of nucleic acid (especially RNA) containing particles is determined on the basis of the R_gvalues and the R_hvalues, this results in two data sets, i.e., one quantitative size distribution based on the R_gvalues and one quantitative size distribution based on the R_hvalues.

In one embodiment of the second aspect (in particular, 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 the quantitative size distribution of nucleic acid (especially RNA) containing particles is determined on the basis of the R_gvalues and the R_hvalues, this results in two data sets, i.e., one set of D10, D50, and/or D90 values based on the R_gvalues and one set of D10, D50, and/or D90 values based on the R_hvalues.

In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect), the amount of nucleic acid (especially RNA), in particular free nucleic acid (especially RNA), is determined by measuring the UV signal, e.g., 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 nucleic acid (especially RNA) extinction coefficient at the corresponding wavelength (e.g., 260 nm or 280 nm).

In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect, in particular in a preferred embodiment of the third subgroup of the second aspect), the size distribution of nucleic acid (especially RNA) containing particles (e.g., based on the R_gor R_hvalues) and/or the quantitative size distribution of nucleic acid (especially RNA) containing particles (e.g., based on the R_gor R_hvalues) is/are within the range of 10 to 2000 nm, preferably within the range of 20 to 1500 nm, such as 30 to 1200 nm, 40 to 1100 nm, 50 to 1000, 60 to 900 nm, 70 to 800 nm, 80 to 700 nm, 90 to 600 nm, or 100 to 500 nm, or such as within the range of 10 to 1000 nm, 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm, or 50 to 250 nm. In a preferred embodiment of the third subgroup of the second aspect, the (quantitative) size distribution of RNA containing particles (e.g., based on R_gor R_hvalues) is within the range of 10 to 1000 nm, such as within the range of 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm, or 50 to 250 nm.

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

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

In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect), measuring the at least one signal selected from the group consisting of the UV signal, the fluorescence signal, and the RI signal, optionally the LS signal, such as the SLS, e.g., MALS, signal and/or the DLS signal, is performed on-line and/or step (c) is performed on-line.

In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect), before subjecting at least a part of the sample composition (e.g., the first composition for step (C) or the second composition for step (D)) to field-flow fractionation, the at least part of the sample composition is diluted with a solvent or solvent mixture, said solvent or solvent mixture being able to prevent the formation of aggregates of the particles. In one embodiment, the solvent mixture is a mixture of water and an organic solvent, e.g., formamide.

In one embodiment of the second aspect (in particular, in one embodiment of the first, second or third subgroup of the second aspect), measuring the UV signal is performed by using circular dichroism (CD) spectroscopy.

It is understood that any embodiment described herein in the context of the first aspect may also apply to any embodiment of the second aspect.

In a third aspect, the present disclosure provides the use of field-flow-fractionation for determining one or more parameters of a sample composition comprising nucleic acid (such as RNA) and optionally particles, wherein the one or more parameters comprise the nucleic acid (such as RNA) integrity, the total amount of nucleic acid (such as RNA), the amount of free nucleic acid (such as RNA), the amount of nucleic acid (such as RNA) bound to particles, the size of nucleic acid (such as RNA) containing particles (in particular, based on the radius of gyration (R_g) of nucleic acid (such as RNA) containing particles and/or the hydrodynamic radius (R_h) of nucleic acid (such as RNA) containing particles), the size distribution of nucleic acid (such as RNA) containing particles (e.g., based on R_gor R_hvalues of nucleic acid (such as RNA) containing particles), and the quantitative size distribution of nucleic acid (such as RNA) containing particles (e.g., based on R_gor R_hvalues of nucleic acid (such as RNA) containing particles). Generally, the size distribution and/or quantitative size distribution of nucleic acid (such as RNA) containing particles can be given as the number of the nucleic acid (such as RNA) containing particles, the molar amount of the nucleic acid (such as RNA) containing particles, or the mass of the nucleic acid (such as RNA) containing particles each as a function of their size. Additional optional parameters include the molecular weight of 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), the size of nucleic acid (especially RNA) (in particular, based on R_gand/or R_hvalues of nucleic acid (such as RNA) containing particles), the size distribution of nucleic acid (especially RNA) (e.g., based on R_gor R_hvalues of nucleic acid (such as RNA)), the quantitative size distribution of nucleic acid (especially RNA) (e.g., based on R_gor R_hvalues of nucleic acid (such as RNA)), the shape factor, the form factor, and the nucleic acid (especially RNA) encapsulation efficiency. Generally, the size distribution and/or quantitative size distribution of nucleic acid (especially RNA) can be given as the number of the nucleic acid (especially RNA) molecules, the molar amount of the nucleic acid (especially RNA), or the mass of the nucleic acid (especially RNA) each as a function of their size. Further additional optional parameters include the ratio of the amount of nucleic acid (such as RNA) bound to particles to the total amount of particle forming compounds (in particular lipids and/or polymers) in the particles, wherein said ratio may be given as a function of the particle size; the ratio of the amount of positively charged moieties of particle forming compounds (in particular lipids and/or polymers) in the particles to the amount of nucleic acid (such as RNA) bound to particles, wherein said ratio may be given as a function of the particle size; and the charge ratio of the amount of positively charged moieties of particle forming compounds (in particular lipids and/or polymers) in the particles to the amount of negatively charged moieties of nucleic acid (such as RNA) bound to particles, wherein said charge ratio is usually denoted as N/P ratio and may be given as a function of the particle size.

In one embodiment of the third aspect, the field-flow fractionation comprises:

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

In a first subgroup of the third aspect, the field-flow fractionation comprises:

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

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

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

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

In one embodiment of the third aspect (in particular, in one embodiment of the first, second or third subgroup of the third aspect), the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA) containing particles is/are calculated based on the R_gvalues of the nucleic acid (such as RNA) containing particles. In another embodiment of the third aspect (in particular, in another embodiment of the first, second or third subgroup of the third aspect), the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA) containing particles is/are calculated based on the R_hvalues of the nucleic acid (such as RNA) containing particles. In another embodiment of the third aspect (in particular, in another embodiment of the first, second or third subgroup of the third aspect), the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA) containing particles is/are calculated based on the R_gvalues of the nucleic acid (such as RNA) containing particles and separately based on the R_hvalues of nucleic acid (such as RNA) containing particles (i.e., this embodiment results in two data sets for the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA) containing particles, one based on the R_gvalues and one based on the R_hvalues).

In one embodiment of the third aspect (in particular, in one embodiment of the first, second or third subgroup of the third aspect), where the one or more parameters comprise the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA), the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA) is/are calculated based on the R_gvalues of the nucleic acid (such as RNA). In another embodiment of the third aspect (in particular, in another embodiment of the first, second or third subgroup of the third aspect), where the one or more parameters comprise the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA), the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA) is/are calculated based on the R_hvalues of the nucleic acid (such as RNA). In another embodiment of the third aspect (in particular, in another embodiment of the first, second or third subgroup of the third aspect), where the one or more parameters comprise the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA), the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA) is/are calculated based on the R_gvalues of the nucleic acid (such as RNA) and separately based on the R_hvalues of nucleic acid (such as RNA) (i.e., this embodiment results in two data sets for the size, size distribution, and/or quantitative size distribution of nucleic acid (such as RNA), one based on the R_gvalues and one based on the R_hvalues).

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

In one embodiment of the third aspect (in particular, in one embodiment of the first, second or third subgroup of the third aspect), the field-flow-fractionation uses a membrane having a molecular weight (MW) cut-off suitable to prevent nucleic acid (especially RNA) from permeating the membrane, preferably a membrane having a MW cut-off in the range of from 2 kDa to 30 kDa, such as a MW cut-off of 10 kDa.

In one embodiment of the third aspect (in particular, in one embodiment of the first, second or third subgroup of the third aspect), the field-flow-fractionation uses a polyethersulfon (PES) or regenerated cellulose membrane.

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

In one embodiment of the third aspect (in particular, in one embodiment of the first, second or third subgroup of the third aspect), the cross flow rate profile preferably contains a fractioning phase which allows the components contained in the control or sample composition to fraction/separate by their size so as to produce one or more sample fractions. It is preferred that the cross flow rate changes during this fractioning phase (e.g., starting from one value (such as about 1 to about 4 mL/min) and then decreasing to a lower value (such as about 0 to about 0.1 mL/min) or starting from one value (such as about 0 to about 0.1 mL/min) and then increasing to a higher value (such as about 1 to about 4 mL/min), wherein the change can be by any means, e.g., a continuous (such as linear or exponential) change or a stepwise change. Preferably, the cross flow rate profile contains a fractioning phase, wherein the cross flow rate changes continuously (preferably exponentially) starting from one value (such as about 1 to about 4 mL/min) and then decreasing to a lower value (such as about 0 to about 0.1 mL/min). The fractioning phase may have any length suitable to fraction/separate the components contained in the sample composition by their size, e.g., 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. The cross flow rate profile may contain additional phases (e.g., 1, 2, 3, or 4 phases) which may be before and/or after the fractioning phase (e.g., one before and 1, 2, or 3 after the fractioning phase) and which may serve to separate non-nucleic acid (especially non RNA) components contained in the sample composition (e.g., proteins, polypeptides, mononucleotides, etc.) from the nucleic acid (especially RNA) contained in the sample composition, to focus the nucleic acid (especially RNA) contained in the sample composition and/or to regenerate the field-flow fractionation device (e.g., to remove all components bound to the membrane of the device). Preferably, the cross flow rate of these additional phases is constant for each additional phase and the length of each of the additional phases is independently for each of the additional phases in the range of about 5 min to about 60 min (such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 30 min). For example, the cross flow rate profile may contain (i) a first additional phase which is before the fractioning phase, wherein the cross flow rate of said first additional phase is constant and is the same cross low rate with which the fractioning phase starts (the length of the first additional phase may be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 10 min or about 20 min or about 30 min); (ii) a second additional phase which is after the fractioning phase, wherein the cross flow rate of said second additional phase is constant and is the same cross low rate with which the fractioning phase ends (the length of the second additional phase may be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 10 min or about 20 min or about 30 min); and optionally (iii) a third additional phase which is after the second additional phase, wherein the cross flow rate of said third additional phase is constant and different from that of the second additional phase (the length of the third additional phase may be in the range of about 5 min to about 60 min, such as about 10 min to about 50 min, about 15 min to about 45 min, about 20 min to about 40 min, or about 25 min to about 35 min, or about 10 min or about 20 min or about 30 min). In the embodiment, where the cross flow rate profile contains a fractioning phase, wherein the cross flow rate changes continuously (preferably exponentially) starting from one value (such as about 1 to about 4 mL/min) and then decreasing to a lower value (such as about 0 to about 0.1 mL/min), it is preferred that the cross flow rate profile further contains (i) a first additional phase which is before the fractioning phase, wherein the cross flow rate of said first additional phase is constant and is the same cross low rate with which the fractioning phase starts (such as 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 or about 10 min or about 12 min); (ii) a second additional phase which is after the fractioning phase, wherein the cross flow rate of said second additional phase is constant and is the same cross low rate with which the fractioning phase ends (such as about 0.01 to 0.1 mL/min) (the length of the second additional phase 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 phase which is after the second additional phase, wherein the cross flow rate of said third additional phase is constant and lower than that of the second additional phase (e.g., the cross flow rate of said third additional phase is 0) (the length of the third 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 or about 10 min or about 12 min). A preferred example of such a cross flow rate profile is the following: 1.0 to 2.0 mL/min for 10 min, an exponential gradient from 1.0 to 2.0 mL/min to 0.01 to 0.07 mL/min within 30 min; 0.01 to 0.07 mL/min for 30 min; and 0 mL/min for 10 min.

In one embodiment of the third aspect (in particular, in one embodiment of the first, second or third subgroup of the third aspect), the integrity of the nucleic acid (especially RNA) contained in the sample composition is determined using the integrity of a control nucleic acid (especially RNA).

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

(c′3) determining the ratio between A_50%(control) and A_100%(control), thereby obtaining the integrity of the control nucleic acid (especially RNA) (I(control)).

In this first example, the integrity of the nucleic acid (especially RNA) contained in the sample composition may be calculated by the following steps:

(c1) calculating from the at least one signal selected from the group consisting of the UV signal, the fluorescence signal, and the RI signal obtained from step (b) 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 (el) to the end of the sample UV, fluorescence, or RI peak, thereby obtaining A_50%(sample);

(c2) calculating from the sample UV, fluorescence, or RI signal obtained from step (b) the total area of the sample UV, fluorescence, or RI peak used in step (c1), thereby obtaining A_100%(sample);

(c3) determining the ratio between A_50%(sample) and A_100%(sample), thereby obtaining I(sample); and

(c4) determining the ratio between I(sample) and I(control), thereby obtaining the integrity of the nucleic acid (especially RNA) contained in the sample composition.

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

In this second example, the integrity of the nucleic acid (especially RNA) contained in the sample composition may be calculated by the following steps:

(c2′) determining the ratio between H(sample) and H(control), thereby obtaining the integrity of the nucleic acid (especially RNA) contained in the sample composition.

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