Purification and analysis of RNA

Abstract

The disclosure relates to methods for purifying RNA from a sample using size exclusion chromatography (SEC). Uses of a SEC medium for purifying RNA from a sample are also provided.

Description

[0001] I

[0002] PURIFICATION AND ANALYSIS OF RNA

[0003] FIELD

[0004] The present invention relates to methods for purifying RNA from a sample and, in particular from a sample obtainable by in vitro transcription of RNA. The invention also relates to method of determining the concentration of RNA in a sample.

[0005] BACKGROUND

[0006] Reliable systems for purifying and separating biomolecules in a solution is a critical component of pharmaceutical and biotechnical industries. These industries put high demands on high quality products that are produced in a safe way.

[0007] The use of mRNA for pharmaceutical applications is a fast-emerging field and the development of effective vaccines against COVID-19 has clearly demonstrated the great potential of mRNA technology. This has paved the way for a range of future novel vaccines and therapies, which is, to a large extent, due to that the development and manufacture of vaccines and therapeutics based on mRNA have been proven to be faster than other platforms. mRNA technology enables rapid tailoring of the mRNA sequence in response to seasonal variations, which makes mRNA-based vaccines an attractive therapeutic strategy for future pandemics, as well as endemics and other infectious diseases such as rabies, Zika, and cytomegalovirus infection. In addition to this, there are several clinical studies ongoing which are based on mRNA treatment of diseases, such as HIV, various types of cancers, cystic fibrosis and diabetes.

[0008] There are several other benefits of the mRNA technology compared to traditional vaccines and DNA vaccines. In contrast to vaccines based on attenuated or inactivated viruses, mRNA is specific and will only express a defined antigen which will induce a directed immune response. Construct of mRNA vaccines is based on insertion of the encoded antigen into a DNA template which is then used for in vitro transcription (IVT) of mRNA. As such, any desired sequence can be designed, produced in vitro and delivered to different types of cells. Compared with DNA-based vaccines, mRNA vaccines are both safer, as the probability of random genome integration is virtually zero, and do not require nuclear entry for expression. The mRNA only needs to reach the cytosol, where it is translated into the antigen using the cellular translation machinery. Furthermore, as the expression of the coded antigens is transient, the mRNA is generally quickly degraded within a few days at most by native cellular processes involving RNases. In addition, since the production of mRNA is based on a cell free system using IVT, safety concerns regarding the presence of cell-derived impurities and viral contaminants are virtually eliminated compared to the production of biologicals found in most other platforms as the mRNA cannot create any protein other than the protein for which it is coded. Additionally, due to the short reaction time required for IVT, the actual processing time is also significantly lower compared to other vaccine manufacturing processes, which often are both complex and where the upstream production commonly extends for up to a week or even more. mRNA technology has also shown to be a good candidate for use within personalized medicine. Personalized medicine is an approach to healthcare that considers an individual's genetic makeup, lifestyle and environmental factors to develop personalized treatment plans. The use of mRNA vaccines in personalized medicine involves the development of vaccines tailored to an individual's unique genetic profile. For example, cancer vaccines can be designed to target specific mutations or neoantigens found in a patient's tumor, providing a personalized treatment option. Similarly, vaccines for autoimmune diseases can be tailored to an individual's specific immune profile, improving their efficacy and reducing the risk of adverse effect.

[0009] The production of mRNA by IVT is an enzyme-based reaction, where an RNA polymerase (generally T7, SP6 or T3 RNA polymerases) catalyses the synthesis of the target mRNA from nucleotide triphosphates (NTPs) substrates using a DNA template. Apart from these, the IVT reaction requires other components such as the polymerase cofactor Mg2+and optimized buffer conditions at a suitable pH. Additional components and reagents may also be used, depending on the process design. The template must be produced in advance, usually by enzymatic linearization of purified plasmid DNA, but it can also be produced by amplification of the region of interest using PCR. Similarly to eukaryotic mRNA, capping of the 5'end increases stability of the mRNA by providing protection against exonuclease degradation, as well as improves protein translation. This can be performed for example during the IVT step by use of specific nucleotide such as the cap dinucleotide CleanCap® (TriLink BioTechnologies) or by using a separate post-IVT two-step enzymatic procedure based on the vaccinia capping enzyme (VCC). Although the use of cap analogues is faster and does not require the set-up of a second enzymatic reaction, VCC capping has a higher efficiency at a considerably lower cost, which may have a significant impact at larger scales. The IVT reaction can be performed within a few hours and generally produce milligram quantities of mRNA per millilitre, depending on scale and design. Compared to the timeconsuming processes of conventional vaccines, this provides an additional benefit as this significantly lowers the probability for contamination to occur. From a manufacturing perspective the flexible nature of the mRNA platform is highly valuable since the encoded antigen can be changed without affecting the physical-chemical characteristics of the mRNA backbone and as the performance of the IVT reaction is not dependent on the antigen encoded in the template this allows for standardized procedures. Thus, the combination of speed and flexibility associated with their development and production, the low costs and relatively simple manufacturing, as well as the ease of scalability makes the mRNA technology a very efficient platform to attack pandemics. The exact composition of the IVT reaction components may vary significantly depending on application, construct, enzymes used as well as other factors. Likewise, the exact conditions for template digestion can vary significantly and is not restricted to the conditions shown in the examples hereinbelow, since the IVT material can have been processed / exposed to other additional steps e.g. capping or other chromatographic steps.

[0010] Lab scale production of mRNA is typically performed as a one-step synthesis IVT reaction followed by the precipitation of the RNA. There are several commercial kits available to produce mRNA for preclinical studies at laboratory scale. However, their costs are high and not suitable for large-scale production. Rather, large-scale production of mRNA vaccines generally consists of a one or two step in vitro reaction, followed by purification involving multiple steps. This can include precipitation and steps including tangential flow filtration (TFF) and / or different modes of chromatography. The mRNA manufacturing process can generally be divided into upstream processing, which consists of the enzymatic generation of mRNA by IVT, and downstream processing which includes the unit operations required for purifying the mRNA product. The choice of unit operations may have a significant impact on the process economy as well as the cost per dose, which in turn is directly related to production titers and scale. While still being an area in rapid development, there are currently no established production process platforms similar to those for other common biopharmaceuticals such as used in mAb processes. In addition, only very limited information is still yet publicly available for existing large-scale processes, including those used in COVID vaccine production. Consequently, mRNA processing and purification strategies can be expected to vary considerably from process-to-process and will likely also change over time. An important aspect of mRNA processes, in comparison to other bioprocesses, is the scale, where volumes for the IVT reactions rarely are expected to be larger than 50 L.

[0011] As indicated above, there is a variety of preparative techniques available today for use in the separation of mRNA from residual components following IVT reactions, such as TFF or different modes of chromatography, including affinity chromatography, ion-pair chromatography and anion exchange chromatography.

[0012] TFF, also known as crossflow filtration, is a filter system, relying on the utilization of microfiltration or ultrafiltration membranes, for purification, separation, concentration and purification of biomolecules and particles in a solution. Microfiltration membranes typically have a pore size between 0.1 pm and 10 pm, while ultrafiltration membranes have a pore size between 0.001 pm and 0.1 pm and are therefore often used for concentrating and desalting dissolved proteins, peptides, nucleic acids, carbohydrates and other molecules.

[0013] There are some drawbacks with using TFF for purification of RNA following an IVT reaction. Firstly, mRNA may be purified using TFF with a limited purity. The purity is mainly dependent on the membrane used as well as the number of diafiltration volumes applies. Assuming that an appropriate filter membrane and surface are used, a relatively high purity can be accomplished after a single run of TFF. However, this comes at the cost of longer process times and reduced recoveries. Thus, the time required to obtain a purified mRNA sample is expected to be significantly prolonged. Although TFF may be used in diafiltration mode for removal of residual IVT components in the permeate, this relies on the use of units with discrete surface areas, which will limit scalability and has a great impact on the process time. In addition to this, most small-scale TFF membranes are not designed for use with sample volumes at mL scale or lower. This becomes a problem since, as discussed earlier, mRNA-based therapies have been shown to have a great potential for use in personalized medicine.

[0014] Affinity chromatography, ion-pair chromatography and anion exchange chromatography have been used in the separation of mRNA from residual components following IVT reactions. In particular, WO 2014 / 140211 teaches the use of core bead flow- through chromatography for RNA purification alone or in combination with TFF and / or hydroxyapatite chromatography. In these prior art methods for purification of IVT reaction mixtures, the initial sample has to be diluted prior to loading, and the chromatography method requires a frequent harsh cleaning step (cleaning-in-place, CIP), e.g. using a composition comprising 0.5-1 M NaOH for core beads, or a mixture of 30% isopropanol and 1 M NaOH, to remove components of the initial sample which are retained by the chromatography matrix. Such cleaning step and subsequent column regeneration as prerequisites for column reusability entail several disadvantages. As such, the time needed for RNA purification is significantly prolonged. Moreover, the number of cycles in which such column can be used for RNA purification (i.e. the life-time of the column) is limited, at least due to the frequent harsh cleaning step which irreversibly compromises column performance for RNA purification. Additionally, mRNA may be purified from IVT reaction mixtures using core bead flow-through chromatography, resulting in limited purity, such as about 90 % purity or less, and therefore core bead flow-through chromatography typically is required to be supplemented with additional methods for achieving pharmaceutical grade purity of mRNA. Core bead chromatography often suffers from low capacity (or processability) when used in combination with complex start materials, i.e. when there is a lot of or a diversity in type of contaminants that need to be removed.

[0015] Taken together, efficient purification of in vitro produced mRNA is continuing to be a challenge. Despite the obvious benefits and advantages with mRNA, the quality control is as rigid as for any other biopharmaceutical drug, thus safety, efficacy and quality are key in the manufacturing of mRNA-based products. mRNA following a IVT reaction must be free from the components used during the IVT, such as enzymes, residual NTPs and DNA templates.

[0016] Therefore, there is an unmet need for RNA purification methods, wherein said method is cost-, time- and resource-efficient, but also allow for purification of very small sample volumes without compromising the pharmaceutical-grade purity and yield of the purified RNA. Said method should be suitable for use when RNA is purified from an IVT reaction sample, and thus the sample is comprising several different biological and non- biological components.

[0017] DESCRIPTION OF THE INVENTION

[0018] One object of the invention is to provide methods and uses having unique and advantageous characteristics for purifying RNA from a sample.

[0019] Another object of the invention is to provide methods and uses for purifying RNA from a sample with a high recovery. Another object of the invention is to provide methods and uses for purifying a sample containing RNA, such as an IVT reaction mixture, to yield RNA of high purity.

[0020] Another object of the invention is to provide methods and uses for purifying RNA from a sample, wherein said method has a short process time.

[0021] Another object of the invention is to provide methods and uses for purifying RNA from a sample, wherein said methods and uses are suitable for small-scale as well as for large-scale RNA purification.

[0022] Another object of the invention is to provide methods and uses for purifying mRNA from an IVT reaction sample.

[0023] Another object of the invention is to provide methods for purifying RNA from a sample, wherein said methods and uses comprise determining the concentration of the purified RNA.

[0024] Another object of the invention is to provide methods for purifying RNA from a sample, wherein said methods and uses comprise conditioning of said RNA.

[0025] Another object of the invention is to provide methods and uses for purifying RNA from a sample, wherein said methods and uses comprise conditioning of said RNA and determining the concentration of the purified RNA.

[0026] One or more of these objects, and other objects that are apparent to the skilled person from reading the entire disclosure, are met by the various aspects disclosed.

[0027] Thus, in a first aspect, the present disclosure provides a method for purifying RNA from a sample comprising the RNA, DNA, protein and optionally further components, wherein said method comprises a) enzymatically digesting DNA in said sample to obtain a DNase-treated sample, and b) separating RNA of said DNase-treated sample from proteins and residual DNA fragments present in said DNase-treated sample by size exclusion chromatography (SEC) using a chromatography medium.

[0028] It is understood that step b) also separates the RNA of said DNase-treated sample from optional further components, including NTPs, buffer components, salts, detergents and additional reagents.

[0029] SEC, also known as gel filtration, is the mildest of all the chromatography techniques known in the art, which separates molecules by differences in size as they pass through a resin packed in a column. SEC is widely used to partition proteins based on size. The present inventors have found that SEC is surprisingly beneficial for use in a method for purifying

[0030] RNA.

[0031] As demonstrated by the appended Examples, the use of SEC in a method for purifying RNA is contemplated to offer advantages in terms of avoiding the drawbacks associated with existing methods known for RNA purification. As such, the method as disclosed herein provides a significantly reduced process time for RNA purification in comparison to that of known techniques. In order to achieve the degree of purity offered by the present invention using only TFF, many diafiltration volumes (e.g. at least 5-7) would be needed. Accordingly, the inventors envision that the process time when using the method as disclosed herein may be at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90% shorter in comparison to the process time when using TFF for RNA purification. As an example, where TFF would take 30 minutes or more, the method according to the present invention can achieve the same purification in less than 2 minutes. Moreover, the present method offers a higher recovery yield of the RNA in comparison to that obtainable by existing methods, such as using TFF. Additionally, the method as disclosed herein is beneficial for purifying RNA to achieve RNA with high purity. In particular, it is contemplated that the present method is superior to known techniques as it is suitable for purifying RNA within a short period of time, while maintaining a high recovery yield and high purity of the RNA. Without wishing to be bound by theory, a single step of SEC in the method as disclosed herein for separating RNA of a DNase-treated sample from other components, such as proteins and residual DNA fragments, present in said sample is envisioned to be sufficient to achieve the above advantageous properties of the disclosed method. The present method could be used to replace a TFF step. As such, the present method can be used without a TFF step, i.e., the method does not comprise a TFF step. In embodiments, the method at least does not comprise a TFF step before step b). However, it is envisaged that the method can also be used in conjunction with a TFF step, either before and / or after the TFF step.

[0032] As illustrated in the appended Examples, the method is easily scalable to different sample volumes, such as to sample volumes of the sub-mL range and above. Therefore, the method is suitable for both small- and large-scale RNA purification. The inventors envision that the method as disclosed herein is fully scalable according to the volume of the sample and thus, is suitable for all possible applications in RNA technology. In certain embodiments, the method is thus provided for small- and large-scale RNA purification, wherein the volume of the sample is in the sub-mL range or higher. In one embodiment, the method is provided for large-scale RNA purification, wherein the volume of the sample is about 500 mL or higher. In fact, the method could be used for sample volumes of 10 L or higher. The upper scalability limit will only depend on the availability of column formats suitable for these volumes. In one embodiment, the method is provided for small-scale RNA purification, wherein the volume of the sample is about 500 mL or less, such as about 100 mL or less, such as about 50 mL or less, such as about 10 mL or less, such as about 5 mL or less, such as about 4 mL or less, such as about 3 mL or less, such as about 2 mL or less, such as about 1 mL or less. The method is particularly advantageous for small-scale RNA purification since the currently used preferred method for such purpose, wherein RNA is precipitated with lithium-chloride (LiCI) or TRIzol to remove components other than RNA present in a sample. However, both methods have safety concerns and none of the stated methods are really scalable. Precipitation with LiCI furthermore requires a prolonged process time as typically occurs overnight. In precipitation with LiCI, concentrations above 1 mg / ml are desirable due to the low recovery when having too low concentrations. Likewise, precipitation of RNA with other methods such as ammonium acetate and phenol / chloroform should preferably also be avoided. Most, if not all, precipitation methods, not limited to the methods mentioned above, are time-consuming and involve potentially toxic components. Furthermore, they require limited working volumes and are consequently difficult to scale-up for a large-scale production process. Thus, the present method is advantageous in that any precipitation can be excluded from the process. Another non-limiting reason for this particular advantage is that the present method is suitable for purifying RNA from sample volumes applicable for example in precision (also known as personalized) medicine. By excluding a precipitation step, the process may furthermore be GMP (Good Manufacturing Practice) compliant.

[0033] In addition, the method is suitable for use in existing chromatography systems, such as AKTA Pure™ chromatography system (Cytiva®), thereby enabling its effortless introduction with wide applicability in established laboratories and industrial settings for RNA purification. Furthermore, the method is also suitable for, and fully scalable to, bioprocess scale systems that are GMP compliant.

[0034] Without being bound by theory, the present inventors have found that the method as disclosed herein is suitable for conditioning said RNA of the sample. Conditioning of a RNA sample means that the sample is provided in a desired controlled environment e.g. with regard to buffer system, salt content, pH, etc. Unlike techniques such as ion exchange chromatography ( I EX) or affinity chromatography (AC), molecules do not bind to the chromatography resin, which means that buffer composition does not directly affect resolution (the degree of separation between peaks in a resulting chromatogram). Consequently, a significant advantage of SEC for use in RNA purification is that conditions can be varied to suit the type of sample or the requirements for further purification, analysis, or storage without altering the separation. Moreover, the present inventors have found that the method as disclosed herein is suitable for determining concentration of the RNA separated in step b) of the method as disclosed herein. It is also to be understood that in the method as disclosed herein, separation of RNA of a DNase-treated sample from proteins and residual DNA fragments present in said DNase-treated sample using SEC may occur simultaneously with conditioning of said RNA and / or determining the concentration of the RNA separated in step b) of the method as disclosed herein.

[0035] As it is demonstrated in the appended examples, the present method for purifying RNA, which may further comprise conditioning of said RNA and / or determination of the concentration of the RNA separated in step b) of the method as disclosed herein, may be achieved by subjecting a partially purified and / or a non-purified RNA sample to SEC. The term "partially purified RNA sample" as used herein refers to a sample comprising RNA that has been subjected to at least one RNA purification step but still comprises at least one nondesired component, in particular protein and / or residual nucleic acid contaminants, which may be present in a sample comprising RNA prior to any RNA purification step. The term "non-purified RNA sample" as used herein, refers to a sample comprising RNA, which has not been subjected to any RNA purification step and comprises at least one non-desired component, in particular protein and / or residual nucleic acid contaminants, which may be present in a sample comprising RNA prior to any purification step. The term may also be referred to as a "IVT reaction mixture". It is to be understood that the term "at least one non-desired component" refers to components which are not desired in the purified RNA sample obtained by the method as disclosed herein. Besides protein and / or residual nucleic acid contaminants, non-desired components may include buffer components, such as salts. Non-desired components may be dependent on one or more subsequent processing steps of the purified RNA sample obtained by the method as disclosed herein. The term "purified RNA sample obtained by the method as disclosed herein" refers to a sample comprising RNA that is essentially free of non-desired components, in particular any protein and residual nucleic acid contaminants, which may be present in a sample comprising RNA prior to any purification step.

[0036] As demonstrated in the appended Examples, the inventors have found that the method as disclosed herein is beneficial for separating RNA of the sample from components other than RNA present in said sample. Non-limiting examples of these components include, beside proteins and residual DNA fragments, other components required for an IVT reaction, such as cofactors, salts and NTPs. Thus, in one embodiment said step b) of the method as disclosed herein comprises separating RNA of said DNase-treated sample from proteins, residual DNA fragments and any additional components other than said RNA, said proteins and said residual DNA fragments present in said DNase-treated sample, such as salts and other buffer components and / or NTPs.

[0037] The term "DNase-treated sample" as used herein, refers to a sample comprising RNA which has been subjected to DNase treatment. The method as disclosed herein involves DNase-treatment of the sample according to step a) of the method. It is to be understood that said treatment results in the fragmentation of the DNA to an extent by which the size of the residual DNA fragments is substantially smaller than that of the RNA present in said sample, thus RNA and residual DNA fragments are separated from each other during SEC according to step b) of the method.

[0038] The term "chromatography medium" as used herein refers to the core of chromatography technology and is a critical raw material which serves as a stationary phase, through which the components of a mixture (such as a sample) is carried through. Typical chromatography media are beaded resins, which are well known to those skilled in the art and are media used for example to capture and / or polish monoclonal antibodies, antibody fragments, vaccines and other biomolecules. Hence, the chromatography medium may comprise beads, such as a matrix in the form of a plurality of beads, e.g. formed of polymeric material as detailed elsewhere herein. The beads are typically porous.

[0039] In one embodiment, said chromatography medium comprises a homogeneous porous matrix. It is to be understood that the term "homogeneous matrix" as used herein refers to a matrix which is uniform. In particular, the beads of the matrix may be homogeneous in that they are formed of a uniform material, a single layer of material, and / or have a uniform porosity. In other words, homogeneous porous matrix refers to a matrix which can be characterized with one type of property, structure and material. For the sake of clarity, beads which comprise more than one layer, such as a core and a shell (also referred to as "core beads"), are not to be understood as being homogeneous beads or as forming a homogeneous porous matrix. On the other hand, SEC resins are typically chromatography media formed of a homogeneous porous matrix. Thus, in one embodiment of the present invention, said chromatography medium is a SEC resin comprising a homogeneous porous matrix. The skilled person is aware that biomolecules which are larger than the pores are unable to diffuse into the beads, while biomolecules that range in size between the smallest and largest pores of the beads can penetrate the pores to varying degrees based on their size. Molecules smaller than the smallest pore (such as salts) can enter the total pore volume. Accordingly, when a chromatography medium comprising porous beads is packed in a column and is used in a chromatography purification, all liquids will enter the pores, and the largest molecules that are excluded from the pores will elute in the void volume (i.e., the elution volume of molecules that do not enter the pores and elute first), while the smallest molecules, such as salts, are eluted last. The molecules in the void volume are larger than the largest pores. It is thus appreciated that the resolution of separation of RNA from proteins and residual DNA fragments present in a DNase-treated sample according to the method as disclosed herein may be affected by the pore size of the chromatography medium used in the method. In other cases, it may also be relevant to define the resolution as molecules ability to diffuse in and out of the pores of the chromatography medium. In this definition of resolution, the resolution comprises three parts, i.e. selectivity, retention capacity and efficiency. Each of these terms is affected by the specific components of the method. A column's particle size, in particular, affects the efficiency term of the resolution equation. Efficiency is ultimately derived from the theoretical plate model of chromatography. Conceptually, a plate refers to one complete equilibrated transfer (or partition) of a solute between the mobile and stationary phases. Efficiency is a qualitative term used to measure the number of theoretical plates in a given column, or the degree to which an analyte partitions between the mobile and stationary phases. In relation to particle size, efficiency is inversely proportional. As particle size decreases, efficiency increases, and more resolution is achieved. In contrast, efficiency is directly proportional to the column length. Shortening the analysis time with maintained resolution can be achieved by and decreasing the length of the column by the same factor as the particle size. It is also beneficial that efficiency is inversely proportional to the square of the peak width— higher efficiencies produce narrower peak widths. Narrow peak widths enhance resolution by lengthening the baseline between two adjacent peaks. The present inventors demonstrate several types of chromatography media comprising a homogeneous porous matrix suitable for the method as disclosed herein and illustrate in the appended examples that depending on the size of the RNA, the most optimal chromatography medium may be selected.

[0040] The matrix typically comprises porous beads which may comprise a polymeric material, such as a polysaccharide and derivates thereof, e.g. agarose such as cross-linked agarose, or dextran, such as cross-linked dextran and combinations thereof.

[0041] In one embodiment, said matrix may comprise a polymeric material selected from the group consisting of crosslinked agarose, crosslinked dextran and crosslinked copolymer of allyl dextran and N,N'-methylene bisacrylamide. In one embodiment, said matrix is or comprises crosslinked agarose or crosslinked dextran. In one embodiment, said matrix is crosslinked agarose or crosslinked copolymer of allyl dextran and N,N'-methylene bisacrylamide. In one embodiment, said matrix is crosslinked dextran or crosslinked copolymer of allyl dextran and N,N'-methylene bisacrylamide. In one embodiment, said matrix is crosslinked dextran. In one embodiment, said matrix is crosslinked copolymer of allyl dextran and N,N'-methylene bisacrylamide. In one particular embodiment, said matrix is crosslinked agarose. Cross-linking increases the mechanical strength of the resin. In particular, crosslinked agarose can endure a higher flow pressure compared to other polysaccharides such as dextran or copolymers comprising dextran, as well as compared to for instance polyacrylamide-based matrices. Additionally, dextran-based matrices may swell or shrink depending on which solvent is used, whereas crosslinked agarose remains stable. Thus, in a preferred embodiment, said matrix is crosslinked agarose.

[0042] Polysaccharide beads, such as agarose beads, useful in the present invention can be produced by conventional means well known in the art and can be produced e.g. as described in US6602990 Bl, US7396467B2 or US8309709 B2, which are incorporated herein. The higher the initial agarose concentration, the smaller the pore sizes that form after crosslinking.

[0043] The agarose beads may comprise 0.5-20 wt.% agarose. The agarose content may be 0.5-20 wt.%, 1.5-20 wt.%, 2-20 wt.%, >2-20 wt.%, 2.5-20 wt.%, 3-20 wt.%, 3.5-20 wt.%, 4-20 wt.%, 4.5-20 wt.%, 5-20 wt.%, 5.5-20 wt.%, 6-20 wt.%, 2-10 wt.%, 4-10 wt.%.

[0044] In embodiments, said beads have a median particle size D50v in the range of 5-200 pm. D50v is the median particle size of the cumulative volume distribution, and can be measured by an electronic particle counter, such as Multisizer™3 Coulter Counter (Beckman Coulter) using the electrical sensing zone (ESZ) method. Where particles are porous, to correct for porosity of the particles, a narrow-sieved fraction of the sample is analyzed by both the ESZ method and by image analysis (B. Gbransson, E. Dagerus: Image analysis as a tool for calibration of Electrical Sensing Zone instruments in the size measurement of porous spherical particles, in P. J. Lloyd (ed.): Particle Size Analysis 1988. 1988 John Wiley & Sons Ltd, pp. 159-166) using a microscope or image analyzer system such as Zeiss Axioplan 1 microscope (Carl Zeiss) or equivalent together with image processing software containing a macro-programming function working for different morphologies and with the ability to display, process, measure and plot statistical results including volume distributed particle size distributions. The correlation between the median results from the two methods is determined used as the basis for calculating a factor, called the response factor, needed to correct the ESZ-instrument result to be equal to the microscope result.

[0045] In embodiments, the bead size D50v may be 5-200 pm, 10-200 pm, 20-200 pm, 40- 200 pm, 60-200 pm, 80-200 pm, 100-200 pm, 120-200 pm, 140-200 pm, 160-200 pm, 180- 200 pm, 5-180 pm, 5-160 pm, 5-140 pm, 5-120 pm, 5-100 pm, 5-80 pm, 5-60 pm, 5-40 pm, 5-20 pm, or 10-50 pm. Particles smaller than D50v of 5 pm may result in high pack-pressure and column-nets, i.e. leakage of small beads through the column filter.

[0046] The inventors have found that the method as disclosed herein is beneficial for purifying RNA, wherein said chromatography medium comprises a homogeneous porous matrix that does not retain RNA. Thus, in one particular embodiment, the matrix is nonfunctionalized. In the present context, a non-functionalized matrix lacks reactivity and adsorptive properties. Accordingly, a non-functionalized matrix does not bind biomolecules and therefore, upon contacting a sample comprising a biomolecule with a chromatography medium comprising non-functionalized matrix beads, the chromatography beads do not bind any biomolecules of the sample (although some biomolecules, depending on their size, may be retained to varying degrees). In other words, said non-functionalized matrix is inactive and / or inert with regards to any biomolecules of the sample. Typical SEC resins are composed of such non-functionalized matrices. Thus, in one embodiment, said chromatography medium is a SEC resin comprising a homogeneous porous matrix that is non-functionalized.

[0047] In light of the above, the skilled person will appreciate that functionalized matrices that are functionalized with a ligand which does not bind RNA and / or is inactive and / or inert with regards to RNA, are not expected to retain RNA in a chromatography method. The present inventors envision that RNA may be purified according to the herein disclosed method, wherein a chromatography medium comprises a homogeneous porous matrix functionalized with any such ligand. Non-limiting examples of such ligands include affinity ligands directed to target molecules other than RNA, such as protein A based affinity ligands for affinity capture of IgG (for example in case of MabSelect SuRe™ resins (Cytiva®) and MabSelect PrismA™ resins (Cytiva®)), albumin-binding affinity ligands (for example in case of Blue Sepharose FF resins (Cytiva®)) and affinity ligands for viral vectors such as AAV and AVB Sepharose High Performance resins (Cytiva®). Other non-limiting examples of such ligands are cation exchange (CIEX) ligands, such as sulfonate ("S") ligands and sulphopropyl ("SP") ligands (for example in case of SP Sepharose FF resins (Cytiva®) and SP Sepharose HP resins (Cytiva®)), as well as metal affinity ligands for IMAC, such as in case of HisTrap FF resins (Cytiva®). The Blue Sepharose FF, SP Sepharose FF and HisTrap FF resins (Cytiva®), all use a typical SEC matrix, Sepharose 6 FF (Cytiva®), as the base matrix to which the respective ligands are coupled. AVB Sepharose High Performance and SP Sepharose HP resins (Cytiva®) have the ligands coupled onto a base matrix which is another typical SEC matrix, Sepharose HP (Cytiva®). Those skilled in the art appreciate that since these ligands do not bind RNA and since the base matrices are typical SEC matrices, these chromatography media are expected to be applicable in the method as disclosed herein. This is further shown in Example 13 below. Thus, in some embodiments of the method, the matrix is non-functionalized or is functionalized with a ligand that does not bind RNA. In one embodiment, the matrix is functionalized with a ligand that does not bind RNA. A functionalized matrix may potentially provide a selective purification of the RNA in that it may bind a specific component other than RNA that needs to be removed from the RNA sample.

[0048] In one embodiment, the chromatography medium is packed in a column. As demonstrated in the appended examples, various column formats are suitable for the method as disclosed herein. Moreover, the inventors envision that said chromatography medium may be packed in any column format and size. In one embodiment, said column has a diameter of between about 25 mm and about 50 mm and a bed height of between about 100 mm and about 250 mm. In one specific embodiment, which has been used in an experimental set-up, the height / diameter ratio is 3.85. However, higher ratios as well as lower ratios, e.g. in the range of 0.5-30, such as 0.5-10, such as 2-5, may be suitable as long as the load volume (% of sample volume loaded in relation to the column volume) is adjusted accordingly. In one embodiment, said column has a column volume and a volume loading capacity, wherein said volume loading capacity is less than said column volume. Column volume (CV) is known as the geometrical volume of a column interior (also known as the chromatography bed). The term "volume loading capacity", as referred to herein, corresponds to the maximum sample volume that can be loaded onto a column without compromising the purity and yield of the purified product, in this case the purified RNA. The volume loading capacity may be expressed in percentage of the CV and the sample-to- column volume ratio influences resolution. In fact, the skilled person is aware that sample volume is one important parameter in SEC which influences high-resolution separation. The inventors have found that a sample loading volume that is lower than the CV is beneficial in the method as disclosed herein. In some embodiments, the volume loading capacity is equal to or less than about 50% of the CV, such as equal to or less than about 40% of the CV, such as equal to or less than about 35% of the CV, such as equal to or less than about 30% of the CV, such as equal to or less than about 25% of the CV, such as equal to or less than about 20% of the CV, such as equal to or less than about 15% of the CV, such as equal to or less than about 10% of the CV. As demonstrated in the appended Examples, a sample loading volume that is about 20% or less of the CV is particularly advantageous for the method as disclosed herein. Thus, in some embodiments, the volume loading capacity is from about 1% to about 25% of the CV, such as from about 1% to about 20% of the CV, such as from about 2% to about 20% of the CV, such as from about 2% to about 15% of the CV. In one particular embodiment, the volume loading capacity is about 20% of the CV. In yet another particular embodiment, the volume loading capacity is about 10% of the CV.

[0049] The method may comprise a chromatography medium which is packed in a column to provide a column volume, wherein the method may comprise a step of loading a volume of the sample onto the column, wherein said volume of the sample is equal to or less than 50% of the column volume, such as 1 - 25 % of the column volume as detailed above.

[0050] The sample does not need to be diluted. A range of different sample volumes can be used depending on the initial concentration. The same is valid for flow velocities, which will be limited mainly by (but not exclusively) column format and system used.

[0051] As discussed above, the inventors have found that RNA may be purified from a sample in high purity according to the method as disclosed herein. As demonstrated in the Examples below (e.g. in Example 1) by a re-injection of an RNA sample purified according to the disclosed method, near 100% purity may be achieved. Thus, in one embodiment, RNA is purified from said sample in a purity of at least about 90%, such as at least about 91%, such as at least about 92%, such as at least about 93%, such as at least about 94%, such as at least about 95%, such as at least about 96%, such as at least about 97%, such as at least about 98%, such as at least about 99%, such as about 100%. In one embodiment, RNA is purified from said sample in a purity of at least about 95%, such as at least about 96%, such as at least about 97%, such as at least about 98%, such as at least about 99%, such as about 100%. In one embodiment, RNA is purified from said sample in a purity of from at least about 95% to about 100%. In one embodiment, RNA is purified from said sample in a purity of at least about 95%. In one embodiment, RNA is purified from said sample in a purity of about 100%. As illustrated in the appended Examples, the inventors have surprisingly found that said purity may be achieved by a single step of SEC according to the disclosed method, since by reinjection of an RNA sample purified according to the method disclosed herein, said purity may be confirmed. This is considered advantageous since such purification efficiency may significantly accelerate standard procedures for RNA purification. Thus, in one particular embodiment, said purity is achieved by step b) of the method as disclosed herein without further steps of separating RNA of said DNase-treated sample from proteins and residual DNA fragments present in said DNase-treated sample. The terms used herein relating to RNA purity are to be understood in relation to all other non-desired components present in said sample, in particular to residual DNA fragments and residual proteins present in said sample, which has been subjected to the method as disclosed herein.

[0052] The RNA concentration of the sample is dependent on the volume loaded on the column. The RNA concentration of the sample may be 10 mg / mL or less, such as 5 mg / mL or less. In certain embodiments, the sample concentration of the RNA is 1.0-1.5 mg / mL using a 10% column volume load. In certain embodiments, the RNA concentration of the sample is 4-6 mg / mL using a 5% column volume load. Accordingly, sample loading volumes may be adapted according to the concentration of the RNA in the sample.

[0053] As indicated above, the inventors have found that mRNA may be purified from a sample in a high recovery yield according to the method as disclosed herein. As demonstrated in the Examples by re-injection of RNA samples purified according to the disclosed method, near 100% recovery yield may be achieved. Thus, in one embodiment, RNA is purified from said sample in a recovery yield of at least 90%, such as at least about 91%, such as at least about 92%, such as at least about 93%, such as at least about 94%, such as at least about 95%, such as at least about 96%, such as at least about 97%, such as at least about 98%, such as at least about 99%, such as about 100%. In one embodiment, RNA is purified from said sample in a recovery yield of at least about 95%, such as at least about 96%, such as at least about 97%, such as at least about 98%, such as at least about 99%, such as about 100%. In one embodiment, RNA is purified from said sample in a recovery yield of from at least about 95% to about 100%. In one embodiment, RNA is purified from said sample in a recovery yield of at least about 95%. In one embodiment, RNA is purified from said sample in a recovery yield of about 100%. The terms used herein relating to RNA recovery yield are to be understood in relation to the initial amount of RNA present in said sample, which has been subjected to the method as disclosed herein.

[0054] The inventors envision that the invention allows for packing of the column to be more optimal for higher flow rates.

[0055] As explained above, said RNA, said proteins and said residual DNA fragments are not bound to said matrix that is not functionalized and / or functionalized with a ligand that does not bind and / or is inactive and / or inert with regards to these sample components. In such cases, as it is demonstrated by the inventors in the appended Examples, said RNA, said proteins and said residual DNA fragments are eluted from the chromatography medium using a single buffer composition, such as a single buffer composition of at least about 1.3 CV, such as at least about 1.5 CV. Accordingly, a column packed with such chromatography medium may be reused for a new run, without implementing any method steps, such as elution, cleaning, regeneration and / or re-equilibration, for removing retained components of the sample. Thus, in one embodiment, said RNA, said proteins and said residual DNA fragments are eluted from the chromatography medium used in step b) using the same buffer. The volume of said buffer may be at least about 1.3 CV, such as at least about 1.5 CV. In particular, the RNA may be purified from the sample in a volume of said buffer corresponding to 0.5 CV or less. In some particular embodiments, the method as disclosed herein allows collection of the purified RNA in less than 10% of the time required for TFF. Examples of such buffers include 10m M Tris-HCI pH 7.5-8 and compositions thereof such as in the range of 10-100 mM Tris pH 7.0-8.0, 10-100 mM Phosphate pH 6.5 -8.0, with or without the presence of salts such as NaCI or KCI or other types of additives such as, but not restricted to, detergents, EDTA, reducing agents, and poloxamers. Optionally, a chaotropic agent such as urea or guanidine hydrochloride may also be present, in such cases typically added after IVT but before or during SEC. Virtually any pH and buffer composition could be used, as long as the stability of RNA is maintained. This is further shown in Example 9, showing that resolution for the separation is not affected by different buffer compositions and / or pH of said compositions. Thus, the RNA is stable and the elution profile the same regardless of pH and composition, within the normal working range for RNA. Accordingly, the inventors have found that the present method is surprisingly beneficial for RNA purification, as the chromatography medium used in SEC according to step b) of the method may be reused sequentially in the method as disclosed herein without a need for removing retained components of the sample. The inventors illustrate in Example 7 that the chromatography medium used in SEC according to step b) of the method may be reused at least ten times without any compromise on the performance of said medium for separating RNA from said proteins and said residual DNA fragments. The inventors in fact envision that the chromatography medium used in SEC according to step b) of the method may be reused at least about 50 to about 100 times, or more, without any compromise on the performance of said medium for separating RNA from said proteins and said residual DNA fragments. By avoiding or at least reducing the frequency of cleaning-in-place (CIP) procedures, substantial amount of processing time, as well as consumption of chemicals, may be saved. Moreover, the inventors have found that omitting such cleaning step is also beneficial for increasing the lifetime of the chromatography medium used in SEC according to step b). Still, the inventors envision that it may be advantageous to apply an occasional cleaning-in-place (CIP) step with the purpose of removing unwanted and / or nonspecifically bound material, for example using 0.1-1M NaOH or any other bioprocess acceptable CIP solution and GMP, for example to avoid any unwanted contamination, such as RNase activity. Such CIP of the chromatography medium used in SEC according to step b) of the method may thus be introduced in between every 5 such use or less frequently, such as between every 10 such use or less frequently. In other words, step b) can be repeated at least 5 times, such as at least 10 times, consecutively without an intervening CIP step.

[0056] The single buffer may comprise a reducing agent. This will improve the separation of T7 RNA polymerase from the mRNA during the chromatography step b) of the method disclosed above. Suitable reducing agents are known to the skilled person, and one example of reducing agents is for instance TCEP. The improved separation of T7 RNA polymerase from mRNA when including a reducing agent is shown in Example 8. In the present disclosure, the single buffer, being both an equilibration buffer as well as an elution buffer, is sometimes referred to as a running buffer. The two terms "single buffer" and "running buffer" may be used interchangeably. As appreciated by those skilled in the art, in order for a chromatography medium to be used and / or reused in a chromatography method, it is advised to equilibrate said chromatography medium prior to sample loading. Thus, the method as disclosed herein may further comprise a step prior to step b), wherein said chromatography medium is equilibrated. Such equilibration may be performed by using a buffer used in said SEC in step b) of the method in a volume of at least about 1.3 CV, such as at least about 1.5 CV. Moreover, said chromatography medium used in said SEC in step b) of the method may be equilibrated using a buffer used in said SEC in step b) of the method in a volume of at least about 1.3 CV, such as at least about 1.5 CV, prior to subjecting said DNase-treated sample to said SEC.

[0057] As discussed above, the present method is advantageous for conditioning the RNA present in said sample. Thus, in one embodiment, said method comprises conditioning of said RNA. In some embodiments, said method comprises conditioning of said RNA during said SEC as defined in step b) of the method as disclosed herein; and / or said method comprises a further step of SEC, wherein said conditioning of said RNA occurs during said further step of SEC. In one embodiment, said conditioning occurs during said SEC as defined in step b) of the method as disclosed herein. In one embodiment, the method as disclosed herein comprises a further step of SEC, wherein said conditioning occurs during said further step of SEC. In certain embodiments, a chromatography medium as defined above in relation to said SEC in step b) is used in said further step of SEC.

[0058] As discussed above and demonstrated in appended Example 5, the present inventors have surprisingly found that the method as disclosed herein is suitable for determining concentration of the RNA separated in step b) of the method. Thus, in some embodiments, the concentration of the RNA separated in step b) is determined using the elution profile obtained by said SEC in step b); and / or the method as disclosed herein comprises a further step of SEC, wherein the concentration of the RNA separated in step b) is determined using the elution profile obtained by said further step of SEC. In one embodiment, the concentration of the RNA separated in step b) is determined using the elution profile obtained by said SEC in step b). In another embodiment, the method as disclosed herein comprises a further step of SEC, wherein the concentration of the RNA separated in step b) is determined using the elution profile obtained by said further step of SEC. In certain embodiments, a chromatography medium as defined above in relation to said SEC in step b) is used in said further step of SEC. Said further step of SEC for determining said concentration of the RNA may be an analytical SEC, as further explained below.

[0059] As discussed earlier, said conditioning of the RNA and said determination of the concentration of the RNA separated in step b) may occur simultaneously. Thus, in some embodiments, said RNA is conditioned during step b) and the concentration of the RNA separated in step b) is determined using the elution profile obtained by said SEC in step b). In some embodiments, said method comprises a further step of SEC, wherein said RNA is conditioned during said further step of SEC and the concentration of the RNA separated in step b) is determined using the elution profile obtained by said further step of SEC. In one embodiment, said RNA is conditioned during step b) and the concentration of the RNA separated in step b) is determined using the elution profile obtained by said further step of SEC. It will be appreciated that said further step of SEC is not performed with the purpose of separating RNA of said DNase-treated sample from proteins and residual DNA fragments present in said DNase-treated sample in the embodiments discussed immediately above.

[0060] As demonstrated in the appended Examples, in certain embodiments said RNA is a single stranded RNA, such as an mRNA. Said RNA may be a synthetic RNA. Accordingly, in one embodiment of the method, said sample is obtained by an RNA manufacturing method, such as by in vitro transcription (IVT). In some embodiments, the RNA comprises a sequence of at least about 500 nucleotides, such as at least about 750 nucleotides, such as at least about 1000 nucleotides, such as at least about 1500 nucleotides, such as at least about 2000 nucleotides, such as at least about 3000 nucleotides, such as at least about 4000 nucleotides. In some embodiments, said RNA comprises a sequence of at least about 750 nucleotides, such as at least about 800 nucleotides, such as at least about 850 nucleotides, such as at least about 900 nucleotides, such as at least about 950 nucleotides, such as at least about 1000 nucleotides, such as at least about 1500 nucleotides, such as at least about 2000 nucleotides, such as at least about 3000 nucleotides, such as at least about 4000 nucleotides. In some embodiments, said RNA comprises a sequence of at least about 750 nucleotides, such as at least about 800 nucleotides, such as at least about 850 nucleotides, such as at least about 900 nucleotides, such as at least about 950 nucleotides, such as at least about 1000 nucleotides or more. As demonstrated in the appended examples, in certain embodiments, said RNA comprises a sequence of from about 1000 nucleotides to about 5000 nucleotides, such as from about 1000 nucleotides to about 4500 nucleotides. In some embodiments, the RNA comprises a sequence of at least about 20000 nucleotides, or more. According to some embodiments, the RNA is a linear sequence. In another embodiments, the RNA is a circular RNA. As discussed above, capping of the 5' end may be important for both stability of an mRNA as well as improving protein translation. Such capping may be performed during IVT. Thus, in one embodiment, said mRNA is capped. Furthermore, modified nucleotides may be applied for mRNA production and may be useful in nucleic acid therapeutics. These are known to alter subsequent usability of mRNA, for example by affecting translation, stability and / or splicing. Accordingly, in one embodiment, said mRNA comprises natural nucleotides and / or modified nucleotides. In one embodiment, said mRNA comprises natural nucleotides. In one embodiment, said mRNA comprises modified nucleotides.

[0061] As shown in Examples 11 and 12 below, RNA with sequences smaller than 400 nucleotides will enter the beads of the SEC resin. However, this may be dependent on the flow rate. A slower flow rate leads to interaction of the smaller RNA molecules with the resin, whereas larger RNA molecules are less prone to enter the resin, regardless of flow rate. Thus, with the herein disclosed method a separation of mRNA larger than 400 nucleotides is achieved, regardless of flow rate. For smaller mRNA, it is more dependent on flow rate and there is a risk that mRNA may be lost to the second peak comprising proteins, residual DNA fragments present in said DNase-treated sample, as well as salts and other buffer components and / or NTPs, resulting in a lower recovery for the mRNA.

[0062] The RNA may include a poly(A) sequence, e.g. at the 3' end of a linear mRNA.

[0063] In some embodiments, the method as disclosed herein further comprises at least one additional method step. This at least one additional method step could be included prior to or subsequent to the method as disclosed herein. The step can be a chromatographic capture step, i.e. a chromatography step that binds the target RNA, which is subsequently eluted. In some embodiments, said additional method step is selected from the group consisting of affinity chromatography, hydrophobic interaction chromatography, desalting and buffer exchange, anion exchange, chromatography with multimodal / mixed chromatography resins, and any combination thereof. According to some embodiments, the method as disclosed herein does not comprise an additional method, such as TFF, before and / or after the SEC step. As discussed above, SEC according to step b) of the method as disclosed herein may be used to simultaneously condition RNA in an appropriate buffer system as well as to remove unwanted residual reagents from the sample, which conditioning allows direct load onto a second column in an additional method step, such as affinity capture by use of a oligo dT ligand (e.g. in the form of membrane chromatography for example as described in WO 2022 / 162018 or CIMmultus® Oligo dT Monolithic Column (Sartorius)), and / or any other mode of capture, such as hydrophobic interaction chromatography (HIC) and / or anion exchange (Al EX) purification, aimed at capturing RNA. Said additional method step may be a further step of SEC for conditioning of said RNA and / or for removal of buffer and salt components. Said additional method step may be preceding and / or subsequent to step b) of the method as disclosed herein. In some embodiments, said additional method step is subsequent to step b).

[0064] As explained above, the inventors have found the present method particularly useful for purifying RNA, wherein a single step of SEC according to step b) of the method may be sufficient to obtain a RNA sample with high purity. Accordingly, in some embodiments, said method does not comprise an additional method step preceding and / or subsequent to step a) and / or step b) for separating RNA of said DNase-treated sample from proteins and / or residual DNA fragments present in said DNase-treated sample.

[0065] In certain embodiments of the method as disclosed herein and as supported by the appended Examples, said RNA is purified on a preparative scale, semi-preparative scale or on an analytical scale. In one embodiment, said RNA is purified on a preparative scale. In another embodiment, said RNA is purified on a semi-preparative scale. In yet another embodiment, said RNA is purified on an analytical scale. Additionally, said SEC in step b) may be preparative SEC or analytical SEC. As appreciated by those skilled in the art, preparative SEC typically refers to a high-resolution size-based separation of biomolecules with fractionation and analytical SEC refers to a high-resolution size-based separation without fractionation. The two types are not mutually exclusive. Preparative SEC is normally performed to isolate one or more components of a sample and the separated components obtained in preparative SEC can directly be transferred to a suitable buffer for assay or storage. Analytical SEC is typically performed to check the quality of the sample, to establish the presence or study the properties of a biomolecule or to measure the relative proportions of analytes in a mixture. As such, analytical SEC may be applied for determining concentration of the RNA separated in step b) of the method as disclosed herein. For the sake of clarity, it is to be understood that preparative SEC may be used for purifying RNA on a preparative scale (i.e. large-scale purification) or on a semi-preparative scale (i.e. small-scale purification), depending on the type of sample subjected to the method as disclosed herein. For example, small-scale purification may be beneficial for samples having a small sample volume, e.g. as applicable in precision medicine. Large-scale purification may be on the other hand beneficial for purifying mass components often utilized for manufacturing purposes, such as for example in preparation for mRNA vaccines. As discussed above, the inventors surprisingly found that the method as disclosed herein is beneficial in both applications. This is shown in Example 10, where different formats of chromatography columns are compared.

[0066] The skilled person will appreciate that the embodiments discussed above in relation to the first aspect of the present disclosure, are equally relevant and applicable to the second aspect disclosed below. This particularly applies to embodiments relating to the properties of the size exclusion chromatography medium. For the sake of brevity these will not be repeated in relation to the below aspects or will only be briefly mentioned.

[0067] As evident based on the above, the inventors have found that a size exclusion chromatography medium is unexpectedly advantageous in the method for purifying RNA according to the present disclosure.

[0068] Thus, in a second aspect of the invention there is provided a use of a size exclusion chromatography medium for separating RNA present in a DNase-treated sample from proteins and residual DNA fragments present in said DNase-treated sample. In one embodiment, said use further comprises determining the concentration of the RNA separated from said proteins and said residual DNA fragments. In one embodiment, said use further comprises conditioning of said RNA. In one particular embodiment, said use further comprises conditioning of said RNA and determining the concentration the RNA separated from said proteins and said residual DNA fragments.

[0069] It should be understood that said RNA is separated from said proteins and said residual DNA fragments simultaneously. In one embodiment, said size exclusion chromatography medium is used simultaneously for separating said RNA from said proteins and said residual DNA fragments and for determining concentration of the RNA separated from said proteins and said residual DNA fragments. In one embodiment, said size exclusion chromatography medium is used simultaneously for separating said RNA from said proteins and said residual DNA fragments, for determining concentration of the RNA separated from said proteins and said residual DNA fragments and for conditioning said RNA. In one embodiment, said size exclusion chromatography medium is used simultaneously for separating said RNA from said proteins and said residual DNA fragments and for conditioning said RNA. In one embodiment, said matrix is non-functionalized or functionalized with a ligand that does not retain RNA. In one particular embodiment, the matrix is nonfunctionalized. In one embodiment, said matrix selected from the group consisting of crosslinked agarose, crosslinked dextran and crosslinked copolymer of allyl dextran and N,N'-methylene bisacrylamide. In one embodiment, said matrix is crosslinked agarose or crosslinked dextran. In one embodiment, said matrix is crosslinked agarose or crosslinked copolymer of allyl dextran and N,N'-methylene bisacrylamide. In one embodiment, said matrix is crosslinked dextran or crosslinked copolymer of allyl dextran and N,N'-methylene bisacrylamide. In one embodiment, said matrix is crosslinked dextran. In one embodiment, said matrix is crosslinked copolymer of allyl dextran and N,N'-methylene bisacrylamide. In a preferred embodiment, said matrix is crosslinked agarose.

[0070] In one embodiment, said size exclusion chromatography medium is packed in a column. In one embodiment, said column has a diameter of from about 25 mm to about 50 mm and a bed height of from about 100 mm to about 250 mm.

[0071] In one embodiment, said column has a column volume and a volume loading capacity, wherein said volume loading capacity is less than said column volume. In one embodiment, the volume loading capacity is equal to or less than about 50% of the column volume, such as equal to or less than about 40% of the column volume, such as equal to or less than about 35% of the column volume, such as equal to or less than about 30% of the column volume, such as equal to or less than about 25% of the column volume, such as equal to or less than about 20% of the column volume, such as equal to or less than about 15% of the column volume, such as equal to or less than about 10% of the column volume. In one embodiment, the volume loading capacity is from about 1% to about 25% of the column volume, such as from about 1% to about 20% of the column volume, such as from about 2% to about 20% of the column volume, such as from about 2% to about 15% of the column volume. In one embodiment, the volume loading capacity is about 20% of the column volume, preferably about 10% of the column volume.

[0072] In yet another embodiment, said RNA is purified from said DNase-treated sample in a purity of at least about 90%, such as at least about 91%, such as at least about 92%, such as at least about 93%, such as at least about 94%, such as at least about 95%, during said use. In some embodiments, the RNA is purified from said DNase-treated sample in a purity of at least about 96%, such as at least about 97%, such as at least about 98%, such as at least about 99%, during said use. In one embodiment, said RNA is purified from said DNase-treated sample in a recovery yield of at least 90%, such as at least about 91%, such as at least about 92%, such as at least about 93%, such as at least about 94%, such as at least about 95%, during said use. In some embodiments, the RNA is purified from said DNase-treated sample in a recovery yield of at least about 96%, such as at least about 97%, such as at least about 98%, such as at least about 99%, during said use.

[0073] In one embodiment, the RNA is a single stranded RNA, such as an mRNA. In one embodiment, said sample is obtained by in vitro transcription. In one embodiment, the RNA comprises a linear sequence of at least about 500 nucleotides, such as at least about 750 nucleotides, such as at least about 1000 nucleotides, such as at least about 1500 nucleotides, such as at least about 2000 nucleotides, such as at least about 3000 nucleotides, such as at least about 4000 nucleotides.

[0074] As discussed above relating to the first aspect of the disclosure, the inventors illustrate in Example 7 that the chromatography medium used in SEC according to the present disclosure may be reused at least ten times without any compromise on the performance of said medium for separating RNA from said proteins and said residual DNA fragments. The inventors in fact envision that the use of the chromatography medium as disclosed herein may be repeated at least about 50 to about 100 times, or more, without any compromise on the performance of said medium for separating RNA from said proteins and said residual DNA fragments. Thus, in one embodiment, said use of the chromatography medium as disclosed herein is a repeated use, such as wherein said chromatography medium is used according to the present disclosure at least about 10 times, such as at least about 50 to about 100 times, or more. It is to be understood that said repeated use of the chromatography medium does not require a harsh cleaning step in between uses according to the present disclosure. The inventors envision that occasional CIP, for example using a solution comprising 0.5-1.0 M NaOH, of the chromatography medium for the use as disclosed herein may be advantageous, for example to avoid any unwanted contamination, such as RNase activity. Thus, said repeated use may comprise a standard CIP of the chromatography medium in between every 5 uses or less frequently, such as between every 10 uses or less frequently, according to the present disclosure.

[0075] It is noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" also include plural referents unless the context clearly dictates otherwise. As used herein, when the term "about" or "approximately" is used in relation to a numerical value, it is to be interpreted as a range of ± 10 %, such as ± 9 %, such as ± 8 %, such as ± 7 %, such as ± 6 %, such as ± 5 %, such as ± 4 %, such as ± 3 %, such as ± 2 %, such as ± 1 %. For example, when the value is stated to be about 10, this means that the value is in fact in the range of from 9 to 11, such as in the range of from 9.9 to 10.9, such as in the range of from 9.8 to 10.8, such as in the range of from 9.7 to 10.7, such as in the range of from 9.6 to 10.6, such as in the range of from 9.5 to 10.5, such as in the range of from 9.4 to 10.4, such as in the range of from 9.3 to 10.3, such as in the range of from 9.2 to 10.2, such as in the range of from 9.1 to 10.1.

[0076] The skilled person knows that numerical values relating to measurements are subject to measurement errors which place limits on their accuracy. For this reason, the general convention in the scientific and technical literature is applied: the last decimal place of a numerical value indicates its degree of accuracy. Where no other error margins are given, the maximum margin is ascertained by applying the rounding-off convention to the last decimal place e.g. for a measurement of 3.5 cm, the error margin is 3.45-3.54. When interpreting ranges of values in patent specifications, the skilled person proceeds on the same basis.

[0077] BRIEF DESCRIPTION OF THE FIGURES

[0078] Figure 1 shows a chromatogram from the experiment wherein mRNA is purified from a DNase-treated crude IVT sample using SEC (Example 1). Curve (A) corresponds to the elution profile of the first run recorded at 260 nm. Curve (B) corresponds to the elution profile of the second run recorded at 260 nm. Curve (C) corresponds to the conductivity measurement during the first run. Curve (D) corresponds to the conductivity measurement during the second run.

[0079] Figure 2 shows chromatograms from the experiments wherein mRNAs of various lengths are purified from DNase-treated crude IVT reaction samples using SEC (Example 2). Figure 2A shows separation of a sample comprising 1042 nucleotide-long mRNA product from residual components. Figure 2B shows separation of a sample comprising 1975 nucleotide-long mRNA product from residual components. Figure 2C shows separation of a sample comprising 4525 nucleotide-long mRNA product from residual components. In all figures, curve (E) represents measurement of absorbance at 260 nm. Curve (F) represents measurement of absorbance at 280 nm. Curve (G) represents measurement of conductivity. Figure 3 shows elution volumes in SEC of individual nucleotides used in IVT reaction samples and of reference proteins (Example 3). Figure 3A shows a chromatogram with the relative elution position and absorbance maximum for all nucleotides used in IVT reaction samples: ATP (H), GTP (I), UTP (J) and CTP (K). Absorbance is measured at 260 nm. Figure 3B shows a chromatogram with elution profiles of various reference proteins thyroglobulin (L), ferritin (M) and blue dextran (N) obtained in SEC and superimposed to the elution profile (O) and conductivity curve (P) of a DNase-treated crude IVT sample comprising mRNA (4525 nucleotide-long) obtained in SEC under the same conditions as used for the reference proteins. Absorbance was measured at 260 nm.

[0080] Figure 4 shows the chromatograms recorded at 280 nm from experiments wherein the volume loading capacity for purifying mRNA using SEC is investigated (Example 4). Figure 4 shows superimposed chromatograms of four separate runs with different sample loading volumes. Elution profiles using a loading volume of 21.2% of CV (S), a loading volume of 10.6% of CV (Q), a loading volume of 5.3% of CV (R) and a loading volume of 2.1% of CV (T) are shown.

[0081] Figure 5 shows the results from the experiments wherein the determination of mRNA concentration using SEC is investigated (Example 5). Figure 5A shows superimposed chromatograms recorder at 260 nm according to Example 5, wherein eluted mRNA from a first injection (U) was re-injected. Re-injection was repeated four times. The corresponding elution profiles for each injection in descending order are: first injection (U), second injection (V), third injection (W), fourth injection (X) and fifth injection (Y). Figure 5B shows a calibration curve of recorded absorbance data for both peak heights and peak areas for each re-injection obtained in figure 5A.

[0082] Figure 6 shows the chromatograms from the experiments wherein different base matrices are tested for use in SEC for purifying mRNA of various lengths from a IVT reaction samples (Example 6). Figure 6A shows the elution profiles recorded at 260 nm in the first run after injection of a DNase-treated crude IVT reaction sample comprising a 1975 nucleotide-long mRNA product to Sepharose 6FF, Sepharose 4FF and Sepharose HP, respectively. Figure 6B shows the elution profiles recorded at 260 nm in the second run, wherein the two eluted peaks from the first run were collected, pooled and thereafter run on the same columns and base matrices. Figure 6C shows chromatograms recorded at 260 nm, wherein DNase-treated crude IVT reaction samples comprising mRNA of different lengths (1042, 1975 and 4525 nucleotide-long mRNA, respectively) according to Example 6 were injected to three different base matrices (Sepharose 6FF, Sepharose 4FF and Sepharose HP, respectively). Figure 6D shows chromatograms recorded at 260 nm, wherein pooled elution peaks (2nd run) and purified mRNA (3rd run) obtained from purifying DNase- treated IVT reaction samples comprising mRNA of different lengths (1042, 1975 and 4525 nucleotide-long mRNA, respectively) according to Example 6 were injected to two different base matrices.

[0083] Figure 7 shows superimposed chromatograms of 10 consecutive SEC runs of a sample containing purified mRNA according to Example 7. Absorbance was measured at 260 nm.

[0084] Figure 8 shows elution profiles of purified T7 RNA polymerase (abbreviated as T7 RNAP in figure) on Sepharose 6 FF. Figure 8A shows the SDS-PAGE of purified T7-RNA polymerase, indicating 99% purity. Curve (AA) in Figure 8B shows the chromatogram of purified T7 RNAP recorded at 280 nm (left y-axis) where the Sepharose 6FF column is equilibrated and eluted with Tris 10 mM, EDTA ImM, tris(2-carboxyethyl) phosphine (TCEP) 5 mM, pH 7.5. Curve (AB) corresponds to identical injection with Tris 10 mM, EDTA ImM pH 7.5 as running buffer. Curve (AC) shows a chromatogram at 260 nm (right y-axis) of purified 1975 nucleotide-long mRNA on identical Sepharose 6 FF column with Tris 10 mM, EDTA ImM, TCEP 5mM, pH 7.5 as running buffer, to illustrate the elution volumes of mRNA in relation to T7 RNA polymerase.

[0085] Figure 9 shows a comparison of using different running buffers in the method according to the invention. Fig. 9A shows the superimposed UV260 traces of Sepharose 6FF purification of crude IVT comprising of 1975-nucleotides mRNA in various of buffer pH. Curve (AD) corresponds to running buffer of Tris 10 mM, EDTA ImM, pH 7.5 (the default condition). Curve (AE) corresponds to running buffer of sodium acetate 50 mM, pH 4.5. Curve (AF) corresponds to running buffer of sodium acetate 50 mM, pH 5.5. Curve (AG) corresponds to running buffer of sodium citrate 50 mM, pH 6.5. Curve (AH) corresponds to running buffer of HEPES 50 mM, pH 7.5. Curve (Al) corresponds to running buffer of HEPES 50 mM, pH 8.5. Both mRNA fractions (Peak 1, abbreviated Pl in Figure 9B) and impurity fractions (Peak2, abbreviated P2 in Figure 9B) are analyzed by Fragment Analyzer to visualize presence of mRNA and mRNA integrity in respective buffer pH.

[0086] Figure 10 shows the results from the experiments illustrating different column formats and scalability. Fig. 10A shows the superimposed UV 260 curves of preparative purification of DNase-treated crude IVT comprising of 1975-nucleotides mRNA. All three columns were with 20 cm bed height, crude IVT sample of 15% CV was injected to equilibrated columns. Curve (AJ) corresponds to the run performed on 2xHiScreen column. Curve (AK) corresponds to the run performed on 2xHiPrep column. Curve (AL) corresponds to the run performed on XK50 / 30 column. In Figure 10B, Peak 1 (abbreviated for Pl) for mRNA and peak 2 (abbreviated for P2) for impurities were analysed on Fragment Analyzer for mRNA integrity analysis.

[0087] Figure 11 shows a superimposed chromatogram of UV260 of crude IVT comprising of RNA in size range of 100 to 400 nucleotides on Sepharose 6FF. In Figure 11A, a linear flow rate of 67 cm / h is applied whereas in Figure 11B the flow rate is 200 cm / h. Curve (AS) in Fig. 11A and curve (AV) in Fig. 11B correspond to crude IVT comprising of 100-nucleotides RNA. Curve (AT) in Fig. 11A and curve (AW) in Fig. 11B correspond to crude IVT comprising of 200- nucleotides RNA. Curve (AU) in Fig. 11A and curve (AX) in Fig. 11B correspond to crude IVT comprising of 400-nucleotides RNA.

[0088] Figure 12 illustrate the UV260 traces on Sepharose 6FF runs with crude IVT comprising of either 200 or 400 nucleotides mRNA together with their respective conductivity curve and fractionation information. In Figure 12A, curve (AW) shows UV260 trace of crude IVT comprising of 200 nucleotides in overlay with its conductivity curve (AZ) and the reference line (BA) for middle of conductivity peak. Likewise, curve (AX) shows UV260 trace of crude IVT comprising of 400 nt nucleotides in overlay with its conductivity curve (BB) and the reference line (BC) for middle of conductivity peak. Using the reference line (BA and BC, respectively) from conductivity, P2 fractions (labelled with white arrows) were selected for further analysis, together with Pl fractions (labelled with black arrow) on CGE by Fragment Analyzer. In Figure 12B, Pl fractions from Figure 12A are re-injected at 5.37% CV load on to Sepharose 6FF 2xHiScreen column to illustrate the elution profile of pooled peak 1 fractions. Curve (BD) corresponds to Peak 1 fraction pooled from curve (AW) in Fig. 12A (200 nucleotides mRNA) reinjected on Sepharose 6FF. Curve (BD) corresponds to Peak 1 fraction pooled from curve (AX) in Fig. 12A (400 nucleotides mRNA) reinjected on Sepharose 6FF. The peak 1 fractions isolated for both 200 and 400 nucleotides contain only mRNA, free from impurities such as free NTPs. In Figure 12C, CGE analysis of fractions collected from P2 in Figure 12A (white arrows) and 12B (grey arrows) are performed by Fragment Analyzer, indicating 400 nucleotides mRNA does not enter the pore of Sepharose 6FF whereas 200 nucleotides mRNA does. Figure 13 shows chromatograms of the respective elution profiles of the analysed samples using SEC base matrices coupled with a sulphopropyl CIEX ligand (Fig. 13A) and with a butyl HIC ligand (Fig. 13B). As shown in the chromatograms in Fig. 13A and Fig. 13B a first elution peak, with a peak maximum approximately between 1.7 and 2.1 mL, was recorded at 260 nm which corresponds to the elution of mRNA. Thereafter, a second elution peak, with a peak maximum approximately between 3.3 and 4.1 mL, was recorded. The second peak corresponds to residual components of the analysed crude IVT samples, such as fragmented DNA, enzymes and free NTPs.

[0089] EXAMPLES

[0090] While the invention has been described with reference to various exemplary aspects and embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or method to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention is not to be limited to any particular embodiment, but that the invention will include all embodiments falling within the scope of the appended claims.

[0091] The invention will be further illustrated by the following non-limiting Examples. They are offered for illustrative purposes only and are not intended to limit the invention in any manner. Those of skilled in the art will recognize a variety of non-critical parameters which can be changed or adapted to yield essentially the same results. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, volumes, etc.), but some experimental error and deviations may be present. Unless otherwise indicated, the practice of the invention employs conventional methods of biochemistry, biophysics, molecular biology, pharmacology and standard laboratory techniques, within the skill of the art. Such techniques are explained fully in the existing literature. Additionally, it will be apparent to one skilled in the art that the methods for RNA purification applied herein can also be applied to RNA molecules not exemplified below and contemplated by the present inventors to fall within the scope of the disclosure.

[0092] In summary, methods for purifying RNA according to the present disclosure is exemplified below on both preparative and analytical scales, wherein RNA of a DNase- treated sample is separated from proteins and residual DNA fragments present in said sample using SEC. The Examples showcase obtaining purified RNA samples in a recovery yield of at least 95% or higher, and / or with a purity of at least 95% or higher. Moreover, the Examples demonstrate measuring concentration of RNA in a sample using SEC as well as applicability of various base matrices for the method as disclosed herein. Robustness of the method and remarkable reusability of a SEC chromatography medium for the disclosed method are also illustrated.

[0093] Example 1: Purification of mRNA from DNase treated crude IVT samples using SEC

[0094] This Example demonstrates the method as disclosed herein, wherein RNA of a DNase-treated sample is separated from proteins and residual DNA fragments present in said sample using SEC. In particular, a DNase-treated crude IVT reaction sample comprising a 1975 nucleotide-long mRNA product was subjected to SEC, by which mRNA was separated from residual components, such as nucleotides, enzymes, proteins, salts and DNA fragments present in said sample following nuclease digestion.

[0095] Material and methods

[0096] A crude IVT reaction sample comprising a 1975 nucleotide-long mRNA product, free NTPs, DNA template for said mRNA and RNA polymerase (T7 RNA polymerase) was obtained from a reaction mixture containing 40 mM Tris-HCI pH 8, 50 mM MgAc, 10 mM DTT, 0.02% TritonX-100 and 7.5 mM of respectively, ATP, CTP, GTP, UTP, 1 U / pL Murine RNase inhibitor, 0.002 U / pL Inorganic Pyrophosphatase, 4 U / pL T7 RNA Polymerase and 150 pg / mL DNA template. For the sake of clarity, the term "mRNA product" as used in the Examples presented herein refers to mRNA transcripts obtained in an IVT reaction based on a DNA template.

[0097] To fragment the DNA template present in the crude IVT reaction sample, DNase digestion of the template was performed according to standard laboratory procedures. Briefly, template digestion was performed on the IVT reaction by the addition of CaCI2 to 2.5 mM and DNase I followed by incubation at 37.7°C.

[0098] Following incubation the DNase I was inactivated by addition of EDTA to a final concentration of 6 mM. Following all steps, the material was diluted to a final dilution factor of 5x, i.e. the mRNA concentration is 5x lower than directly after the IVT reaction.

[0099] In order to ensure reliable UV monitoring in this particular Example, the DNase- treated crude IVT reaction sample was diluted 5x prior to loading onto a Prepacked Prep 26 / 10 Sepharose 6 FF (Fast Flow) column (26 mm x 100 mm) with a CV of 53 ml (Cytiva), as described above. Sepharose 6FF is formed of spherical beads of cross-linked 6% agarose and has a particle size d50v of approximately 90 pm, and the exclusion limits [M] for globular proteins and dextran is ~ 4 x 106respectively ~ 2 x 106.

[0100] Specifications of the SEC method: sample loading volume: 4 mL buffer: 10 mM Tris pH 7.5, 1 mM EDTA flow velocity: 5 mL / min (equal to 57 cm / h). running time: approximately 20 minutes

[0101] Recordings: Abs: UVjgo, conductivity

[0102] The initial sample concentration of crude diluted IVT was 0.25 mg / mL of which the eluted fractions with purified RNA with the highest absorbance were selected and pooled, resulting in a concentration of 0.15 mg / mL which was injected in the following experiment. The buffer used for equilibration and elution was 10 mM Tris pH 7.5, 1 mM EDTA.

[0103] In other experiments using the same column, sample volumes up to 5 mL, approximately 10% of the CV, has been used, as well as higher flow velocities, up to 15 mL / min, equivalent of 170 cm / h.

[0104] Results and conclusion

[0105] The experiment was performed by an initial injection of 4 mL of the crude DNase- treated IVT reaction sample to the SEC column and run as described above. As shown by curve (A) in the chromatogram in Fig. 1, two elution peaks were recorded. The first elution peak approximately between 17 and 25 mL corresponds to the elution of mRNA and a second elution peak starting at about 35 mL corresponds to the elution of residual components present in the crude DNase-treated IVT reaction sample, respectively. Salts present in the sample buffer are eluted last, as can be seen by the increase in conductivity with a peak maximum between 53-54 mL (curve (C) in Fig. 1).

[0106] Thereafter, the eluted mRNA was collected and used for a second injection. 4 mL of the collected mRNA sample was injected to the SEC column and run as described above. As shown by curve (B) in the chromatogram in Fig. 1, only one elution peak approximately between 17 and 25 mL was recorded, corresponding to the first elution peak in the first run. Conductivity was also measured for this run (curve (D) in Fig. 1). It is apparent from the results of the second run, that the second elution profile lacks a second elution peak corresponding to the second elution peak in the first run. Moreover, as apparent from the conductivity curves, the amount of salts in the collected mRNA sample used in the second injection is significantly less, near zero, in comparison to that in the sample of the first run. This is indicative of that components of the crude DNase-treated IVT reaction sample other than mRNA are successfully separated from the mRNA, and the collected mRNA sample after the first run is a purified mRNA sample. As apparent from the comparison of the elution profiles of the first and the second injections, mRNA has been purified at least with about 95% purity, in fact close to about 100% purity.

[0107] In conclusion, mRNA of the crude DNase-treated IVT reaction sample was successfully separated from other components of the sample (such as proteins, DNA fragments, salts and other buffer components) in a single run of SEC, as apparent from the comparison of the chromatograms obtained in the first and second runs. Thus, the method as described herein is surprisingly beneficial in purifying mRNA from a sample.

[0108] Example 2: Purification of mRNAs of various lengths from DNase treated crude IVT reaction samples

[0109] In this Example, purification of mRNA constructs of various lengths is demonstrated according to the method as disclosed herein. In particular, the Example presents three experiments, wherein three crude IVT reaction samples comprising either a 1042 nucleotide-long mRNA product, a 1975 nucleotide-long mRNA product or a 4525 nucleotide- long mRNA product are subjected to SEC, by which mRNA was separated from residual components, such as nucleotides, enzymes, proteins, salts and DNA fragments present in said sample following nuclease digestion.

[0110] Material and methods

[0111] The Experiment was prepared and performed as described in Example 1 but in the present case three different samples were obtained, comprising an mRNA product of one of the three different sizes (1042 nucleotide-long, 1975 nucleotide-long or 4525 nucleotide- long), free NTPs, DNA template for said mRNAs respectively, and RNA polymerase (T7 RNA polymerase). DNase treatment of the samples was performed prior to loading as described above.

[0112] The samples were diluted 5x and thereafter injected and run individually on a

[0113] Prepacked Prep 26 / 10 Sepharose 6 FF column 26 mm x 100 mm (Cytiva) with a CV of 53 ml as described in Example 1. Absorbance in the present experiments was measured at two different wavelengths, at 260 and 280 nm.

[0114] Results and conclusion

[0115] The chromatograms presented in Fig. 2 correspond to the respective elution profiles of the analysed samples: the sample comprising the 1042 base-long mRNA product (2A), the sample comprising the 1975 base-long mRNA product (2B) and the sample comprising the 4525 nucleotide-long mRNA product (2C). For each case, curve (E) represents the absorbance at 260 nm, curve (F) represents the absorbance at 280 nm, and curve (G) represents the conductivity.

[0116] As shown in all chromatograms in Fig. 2A, 2B and 2C, a first elution peak, approximately between 13 and 24 mL, was recorded at both 260 and 280 nm which corresponds to the elution of mRNA. Thereafter, a second elution peak, approximately between 33 and 60 mL, was also recorded at both wavelengths. This second peak corresponds to residual components of the analysed crude IVT samples, such as fragmented DNA, enzymes and free NTPs. As demonstrated by monitoring conductivity, salts present in the sample buffer were eluted last.

[0117] It is apparent from the results that SEC can efficiently be used to separate mRNA of various lengths from residual components present in a crude IVT reaction sample. As can be readily understood from the chromatograms presented in Fig. 2A-C, this separation occurs at a resolution which enables obtaining a purified mRNA sample. In fact, the entire volume of the first peak containing the mRNA can be collected without incorporating any eluted volume which contains residual components of the initial sample. Thus, mRNA can be purified from the tested samples in a high recovery yield and also with high purity independently of the size of the tested mRNAs.

[0118] Thus, the method as disclosed herein is suitable for purifying a wide range of different RNAs, such as single-stranded RNAs having a length of at least about 1000 nucleotides, since mRNA products of various lengths within an extended range could be efficiently separated from remaining components of a crude DNase-treated IVT reaction sample in a single run of SEC. Based on the presented results for individual mRNA products, the inventors also envision that mRNA products which comprise a mix of mRNA transcripts of various lengths, such as mRNA transcripts having a length of at least about 1000 nucleotides, can also be efficiently purified from a sample using SEC. Example 3: Elution volumes of individual nucleotides and reference proteins used in IVT reactions

[0119] This Example describes control experiments which demonstrate the elution profile of free NTPs as well as reference proteins in SEC. The presented results are useful in evaluating relative elution volumes corresponding to proteins and free NTPs present in a RNA-containing sample subjected to SEC, for Example a DNase-treated crude IVT reaction sample, and demonstrate the performance of the herein disclosed method for the removal of these from said sample.

[0120] Material and methods

[0121] Free NTPs: Four samples, each comprising one of the nucleotides , ATP, GTP, UTP or CTP were prepared to evaluate elution profile of free NTPs in SEC. The samples were individually tested using a Prepacked Prep 26 / 10 Sepharose 6 FF column (26 mm x 100 mm) with a CV of 53 ml (Cytiva). Equilibration was performed with 10 mM Tris pH 7.5, 1 mM EDTA. SEC method was performed as described in Example 1, with a flow rate of 15 mL / min and totally 75 pl of 100 nM NTP was injected. Absorbance was measured at 260 nm.

[0122] Reference proteins: Samples comprising individual model proteins of different sizes were prepared using reference protein stock solutions of the Gel Filtration HMW Calibration Kit (Cytiva). In particular, samples comprising Thyroglobulin (669 kDa), Ferritin (440 kDa) or Blue Dextran (2000 kDa) were prepared and analysed in SEC using a Prepacked Prep 26 / 10 Sepharose 6FF column (26 mm x 100 mm) with a CV of 53 ml (Cytiva). SEC was performed as described in Example 1, and absorbance was measured at 260 nm. Elution profiles of the tested reference proteins obtained in the individual runs were superimposed to the elution profile of a DNase-treated crude IVT sample comprising mRNA (4525 nucleotide-long) obtained in SEC under the same conditions as used for the reference proteins, as described in Example 2 (see Fig. 3B).

[0123] Results and conclusion Free NTPs:

[0124] The results from the different runs are shown in Fig. 3A. The absorbance at 260 nm of each nucleotide, in descending order, is ATP (H), GTP (I), UTP (J) and CTP (K). As can be seen in the chromatogram, the relative elution position and absorbance maximum for all nucleotide occurs around 42-44 mL.

[0125] Accordingly, free NTPs present in a sample, such as a crude IVT reaction sample, are expected to have an elution volume with an absorbance maximum at 42-43 mL when a Prepacked Prep 26 / 10 Sepharose 6 FF column (26 mm x 100 mm) with a CV of 53 ml is used for SEC. As apparent from the above Examples as well as Fig. 1 and 2, wherein the same column was used for purifying mRNA according to the method disclosed herein, the second peak corresponding to residual components of the analysed samples has an elution volume with an absorbance maximum at 42-43 mL. Thus, it is concluded that residual components of the sample which correspond the second elution peak shown in Fig. 1 and 2 encompass free NTPs present in the analysed sample. In other words, mRNA of the analysed samples which has been separated from the residual components, and which corresponds to the first peak of the presented chromatograms in Examples 1 and 2 is free from any free NTPs.

[0126] In conclusion, mRNA of crude DNase-treated IVT reaction samples as demonstrated in Examples 1 and 2 is successfully separated from each type of residual free nucleotides present in said sample using the method as disclosed herein. Moreover, the present data support that free NTPs are expected to elute subsequently to mRNA present in a sample using a SEC matrix for purifying mRNA when relative elution volumes of these are compared, thus the herein demonstrated separation of these occurs independently of the column dimensions. Consequently, it is concluded that in each of the herein described Examples, free NTPs are eluted separately (in the corresponding second peaks) from the RNA present in the analysed samples, when RNA is purified according to the disclosed method.

[0127] Reference proteins:

[0128] The largest expected entity that needs to be removed following an IVT reaction is the T7 RNA polymerase at 99 kDa. As expected, and shown on Fig. 3B, the largest molecule in the kit, Blue Dextran 2000, elutes at a similar position as the 4525 nucleotide-long mRNA construct (N). The largest proteins Thyroglobulin (669 kDa) and Ferritin (440 kDa) also elute at a similar elution volume ((L) and (M), respectively). T7 RNA polymerase has a significantly lower molecular weight than that of these reference proteins and is thus expected to elute at a higher elution volume than the elution volume observed for Blue Dextran 2000, Thyroglobulin and Ferritin. Accordingly, T7 RNA polymerase is expected to be eluted separately from the elution peak corresponding to the mRNA construct present in the IVT reaction sample (the first elution peak (O) shown in Fig. 3B). Thereby, mRNA free from residual proteins may be purified according to the method as disclosed herein. These results together support that the second elution peak of the elution profile (O) of a DNase-treated crude IVT sample comprising the 4525 nucleotide-long mRNA corresponds to the elution volume of T7 RNA polymerase present in said sample.

[0129] In summary, mRNA of crude DNase-treated IVT reaction samples as demonstrated in for example, Examples 1 and 2 is successfully separated from each type of residual free nucleotides as well as residual proteins present in said sample using the method as disclosed herein.

[0130] Example 4: Volume loading capacity for purifying mRNA

[0131] In this Example, different sample loading volumes were tested in the method as disclosed herein using a DNase-treated IVT reaction sample comprising mRNA for loading in different volumes. In particular, sample loading volume in relation to the CV was evaluated for achieving sufficient separation of mRNA from residual components present in a DNase- treated IVT reaction sample using SEC.

[0132] Materials and methods

[0133] A crude IVT reaction sample comprising a 1927 nucleotide-long mRNA product, free NTPs, DNA template for said mRNA and RNA polymerase (T7 RNA polymerase) was obtained. DNase treatment of the sample was performed prior to loading, as described in Example 1.

[0134] The thereby obtained sample having an mRNA concentration of 5.5 mg / mL was injected at a flow rate of 0.5 mL / min in four different sample loading volumes as delineated below and run individually on a HiScreen column (Cytiva) packed with Sepharose 6 FF (Cytiva) and having a CV of 4.7 mL, a bed height of 100 mm and a diameter of 7.7 mm:

[0135] 100 pL (equals to 2.1% of CV)

[0136] 250 pL (equals to 5.3% of CV) 500 pL (equals to 10.6% of CV) 1000 pL (equals to 21, .2% of CV) Results and conclusion

[0137] Fig. 4 shows chromatograms recorded for the tested sample loading volumes, namely 21.2% CV load (S), 10.6% CV load (Q), 5.3% CV load (R) and 2.1% CV load (T), using the HiScreen column packed with Sepharose 6 FF and having a CV of 4.7 mL and a bed height of 100 mm and a diameter of 7.7 mm. Two elution peaks were recorded for each sample, wherein, as demonstrated before, the first elution peak corresponds to the elution of mRNA and the second peak corresponds to the elution of residual components present in the crude DNase-treated IVT reaction sample. It is noted that that in the present case the absolute elution volumes differ from those described in the above Examples due to the difference in the column dimensions, however, the relative elution volumes of the peaks are indicative of the separation of the mRNA from the residual components.

[0138] As can be seen in Fig. 4, all of the tested sample loading volumes enable a separation between the peaks, i.e. are applicable for the separation of mRNA from residual components present in the initial DNase-treated crude IVT reaction sample using SEC. Accordingly, the use of a sample loading volume that is less than the CV is demonstrated for RNA purification according to the present method. In particular, it is demonstrated that using a sample loading volume that is about 20% of the CV or less can be used in the method disclosed herein. Thus, a SEC column having a volume loading capacity of about 20% or below can be efficiently used.

[0139] When looking at the chromatograms in Fig. 4 in comparison, it is apparent that a sample loading volume of about 10% of the CV or less is advantageous for obtaining a better resolution of the peaks, and thus, of the separation of mRNA from residual components present in the sample. This may be considered beneficial in obtaining a higher recovery yield of a purified RNA sample with a higher purity. The inventors envision that a sample loading volume which is less than the CV, and preferably is about 20% of the CV or less, enables separation of RNA from residual components present in the DNase-treated sample using SEC.

[0140] In conclusion, it is demonstrated herein that the disclosed method is beneficial in purifying RNA from a DNase treated sample using SEC, wherein the volume loading capacity of the column used in the method is about 20% of the CV or less, such is about 10% of the CV or less. Example 5: Determination of mRNA concentration using SEC

[0141] This Example demonstrates the method as disclosed herein, wherein the concentration of RNA present in a sample is obtained with high accuracy using SEC. In particular, the present Example demonstrates that the concentration of RNA may be obtained during purifying RNA from a sample using SEC as well as by analysing a purified RNA sample using SEC.

[0142] Material and methods

[0143] A crude IVT reaction sample comprising a 1975 nucleotide-long mRNA product, free NTPs, DNA template for said mRNA and RNA polymerase (T7 RNA polymerase) was obtained. As described in Example 1, the sample was subjected to a DNase-treatment and was diluted 5x, and an additional 5x dilution with 10 mM Tris pH 7.5 and 1 mM EDTA, prior to loading onto and run on a Prepacked Prep 26 / 10 Sepharose 6 FF column (26 mm x 100 mm) with a CV of 53 ml (Cytiva).

[0144] The experiment was performed by an initial injection of 4 mL of the crude DNase- treated IVT reaction sample to the SEC column. 4 mL of the resulting mRNA eluate was thereafter re-injected. 4 mL sequential re-injection of each eluate was performed for a total of four times in addition to the first injection with the crude DNase-treated IVT material. Absorbance was recorded at 260 nm during the runs.

[0145] The concentration in the injected samples was determined from the measured absorbance values at 260 nm in the chromatogram both by the peak height and the peak area. The corresponding mRNA mass (in mg) in the injected volume was calculated using the mass extinction coefficient of single-stranded RNA (mg-1 mL cm-1) with a 0.2 cm path length UV cell. The data from the peak height and peak area, respectively, was used to construct calibration curves, which was compared to separate analysis with an UV spectrometer (NanoDrop) according to standard protocols.

[0146] Results and conclusion

[0147] The initial injection of 4 mL of the crude DNase-treated IVT reaction sample to the SEC column is represented by curve (U) (first injection) in the chromatogram in Fig. 5A. The curve (U) shows two elution peaks, wherein the first elution peak, approximately between 17 and 25 mL, corresponds to the elution of mRNA and the second elution peak, starting at about 35 mL corresponds, to the elution of residual components present in the crude DNase-treated IVT sample. As described above, the mRNA eluate, i.e. the first peak elute, was then re-injected to the column (curve (V); second injection). As can be seen from curve (V), there is only one elution peak between 17 and 25 mL, indicating that the mRNA was successfully separated from the residual components during the first run. As apparent from the lack of the second elution peak, mRNA has been purified with near 100% purity, such as at least about 95% purity. The eluate from the second injection was thereafter re-injected again ((W), third injection). In the same way, the fourth injection is represented by curve (X), and the fifth injection is represented by the curve (Y).

[0148] The measured absorbance maximums and peak areas of the mRNA elution peaks together with the calculated mRNA concentrations are presented in Table 1 below. The concentration of each sample was also measured using Nanodrop and the corresponding values are also indicated in Table 1. As apparent from the comparison of the values presented in Table 1, RNA concentration measured by the method as described herein using SEC corresponds to the concentrations measured by the conventional method using Nanodrop with high accuracy.

[0149] Table 1. mRNA concentrations obtained using SEC and Nanodrop

[0150] Run Sample origin NanoDrop SEC SEC area height

[0151] Injection 1 Crude ND* 250.5 250.2

[0152] Injection 2 Eluate 1 154.5 153.0 152.9

[0153] Injection s Eluate 2 82.1 80.0 81.6

[0154] Injection 4 Eluate S 47.0 48.0 48.1

[0155] Injection s Eluate 4 31.4 32.0 30.7

[0156] * Not possible to analyze

[0157] As it can also be seen from comparing the chromatograms in Fig. 5A, each reinjection corresponds to a lower absorbance maximum of the elution peak between 17 and 25 mL, which indicates that the mRNA concentration of the injected sample was gradually decreased by re-injection of the same volume (4 mL) of the eluates collected in the previous SEC runs, which corresponds to a gradual dilution of the samples during these consecutive runs. The recorded absorbance data for both peak heights and peak areas for each re- injection (obtained from the second to the fifth injections) of this serial dilution were thus presented as calibration curves as shown in Fig. 5B. As apparent from this figure, linear regression analysis of these is indicative of RNA concentration measurement with high accuracy (R2>0.9995). Accordingly, both type of data, i.e. peak hight and peak area, obtained from the elution profile during SEC of a sample comprising RNA are suitable for determining the concentration of said RNA.

[0158] In conclusion, the present Example demonstrates that the concentration of RNA in a sample, such as a DNase-treated crude IVT reaction sample and / or a purified RNA sample, can be measured with high accuracy using SEC according to the method as disclosed herein, based on the elution profile obtained during the run of said sample, such as by recording absorbance at 260 nm. The present Example demonstrates concentration determination of single-stranded RNA in a sample, but it is to be appreciated the herein disclosed method also enables concentration determination of double-stranded RNA by using the corresponding extinction coefficient for the above calculation.

[0159] Example 6: Testing of different base matrices used in SEC for purifying mRNA of various lengths from DNase treated crude IVT reaction samples

[0160] In this Example, purification of RNA of various lengths is demonstrated according to the method as disclosed herein using different SEC matrix alternatives, wherein RNA of a DNase-treated sample is separated from proteins and residual DNA fragments present in said sample using SEC.

[0161] Material and methods a) In a first set of experiments, a crude IVT reaction sample comprising free NTPs, RNA polymerase (T7 RNA polymerase) and a 1975 nucleotide-long mRNA product together with the corresponding DNA template was obtained. The sample was subjected to DNase treatment, as described in Example 1. 100 pl of the sample was injected at 0.5 ml / min flow rate and run on different HiScreen columns (7.7 mm x 100 mm) having a CV of 4.7 mL packed with the following base cross-linked agarose matrices: 1) Sepharose 6 FF (Cytiva), 2) Sepharose 4 FF (Cytiva), and 3) Sepharose High Performance (Cytiva). Sepharose 6FF is based on spherical cross-linked 6% agarose and has a particle size d5ov of approximately 90 pm, and the exclusion limits [M ] for globular proteins and dextran is ~ 4 x 106Da and ~ 2 x 106Da, respectively. Sepharose 4FF is based on spherical cross-linked 4% agarose and has a particle size d5ov of approximately 90 pm, and the exclusion limits [M] for globular proteins and dextran is ~ 3 x 107Da and ~ 6 x 106Da, respectively. Regarding Sepharose High Performance, it is also based on cross-linked 6% agarose with a mean bead size of 34 pm, and an exclusion limit for globular proteins of ~ 4 x 106Da. Further product specifications can be found on cytivalifesciences.com. Chromatograms recorded during these runs are presented in Fig. 6A. b) In a second set of experiments, eluted peaks (both the first and second peaks corresponding to the mRNA eluate and the eluate of the remaining components of a crude IVT reaction sample) from runs as described in a) were collected and pooled for a second run using the same columns and base matrices as indicated above. 100 pl of the pooled samples was injected at 0.5 ml / min flow rate and run using the above specified SEC method. Chromatograms recorded during these runs are presented in Fig. 6B. c) In a third set of experiments, eluted mRNA peaks (corresponding to the first peak during mRNA purification using SEC according to the method as disclosed herein) from runs as described in a) were collected for a second run using the same columns and base matrices as indicated above. In this case, three different types of crude IVT reaction samples were used in the first run to purify mRNA of various lengths using the indicated columns and base matrices. Each sample comprised free NTPs, RNA polymerase (T7 RNA polymerase) and one of 1) a 1042 nucleotide-long mRNA product, 2) a 1975 nucleotide-long mRNA product or 3) a 4525 nucleotide-long mRNA product together with the corresponding DNA template for the respective mRNAs. The samples were subjected to DNase treatment, as described in Example 1 prior to the first run. 100 pl of the collected mRNA peak samples (having an mRNA concentration of 0.1 pg / pl) was injected at 0.5 ml / min flow rate and run using the above specified SEC method as in Example 1. Chromatograms of runs from the third set of experiments are presented in Fig. 6C. d) For a fourth set of experiments, three different types of IVT reaction samples were prepared to purify mRNA of various lengths using different HiScreen columns (7.7 mm x 100 mm) having a CV of 4.7 mL packed with the following base matrices:

[0162] Sepharose CL-2B (Cytiva),

[0163] Sephacryl S-500 (Cytiva).

[0164] Each sample comprised free NTPs, RNA polymerase (T7 RNA polymerase) and one of 1) a 1042 nucleotide-long mRNA product, 2) a 1975 nucleotide-long mRNA product or 3) a 4525 nucleotide-long mRNA product together with the corresponding DNA template for the respective mRNAs. The samples were subjected to DNase treatment, as described in Example 1 prior to the first run. Eluted peaks (both the first and second peaks corresponding to the mRNA eluate and the eluate of the remaining components of a crude IVT reaction sample) from the first runs as described immediately above were collected and pooled for a second run using the same columns and base matrices as indicated above. In this second run, 100 pl of the pooled samples was injected at 0.5 ml / min flow rate and run using the above specified SEC method (described in a)). Eluted mRNA peaks (corresponding to the first peak eluted in the first runs) were collected for a third injection using the same columns and base matrices as indicated above. In the third run, 100 pl of the collected mRNA peak samples was injected at 0.5 ml / min flow rate and run using the above specified SEC method. Chromatograms of the second and third runs are presented in Fig. 6D.

[0165] During the above tests, absorbance was measured at 260 nm. The columns were equilibrated between each run using a buffer containing 10 mM Tris (pH 7.5) and 1 mM EDTA with a volume corresponding to at least one column volume.

[0166] Results and conclusion

[0167] The results of experiments a) and b) as depicted in Fig. 6A and B, are indicative that each tested further SEC matrix is suitable for purifying RNA according to the method as disclosed herein. In particular, as demonstrated by two elution peaks in each chromatogram in Fig. 6A, mRNA of the DNase-treated crude IVT samples was separated from residual components present in said samples. Resolution (Res.) of the two elution peaks enables obtaining a purified mRNA sample in high recovery yield with high purity. Moreover, as apparent from Fig. 6B and the presented values of resolution and peak volume at half height (PV), separation of mRNA from residual components occurs with reproducible resolution and PV when a pooled sample is tested, as described in experiment b) above. Accordingly, separation between the first and the second peaks was achieved with both direct injection of the DNase-treated crude IVT reaction sample as well as a pooled sample of the first and the second peaks, as described above.

[0168] The results of experiment c) as depicted in Fig. 6C, are indicative that each tested alternative SEC matrix is suitable for obtaining a purified RNA sample with high purity according to the method as disclosed herein. Importantly, purified mRNA with high purity was obtained, independently of the matrix used and of the length of the tested mRNA. Since the presented chromatograms contain no detectable second peaks which would be indicative of residual components other than RNA present in the initial sample which has been purified according to the present method, it is concluded that mRNA has been purified at least with 95% purity, such as with about 100% purity, in each case.

[0169] The obtained chromatograms are also informative on the shape of the mRNA elution peaks which may be characterized by the asymmetry value thereof, as known in the art. The asymmetry value is dependent on the matrix used as well as on the nature of the sample analyzed. It is ideally near 1, which means that the peak has a sharp symmetrical shape. The skilled person appreciates that a value near 1 may be beneficial in the separation of components of a sample using SEC, since a better resolution may be obtained between components having different elution volumes. A peak having an asymmetry value greater than 1 is indicative of a "tailing effect" which means that the elution of the component is prolonged. When this happens, the purity and / or recovery yield of the purified component may be compromised. As demonstrated in Fig. 6C, the asymmetry value obtained using the same matrix varies depending on the size of the purified mRNA, moreover varies between different matrices when purified mRNA of the same length is analyzed. Consequently, while each of the tested SEC matrices is suitable for purifying RNA according to the method as disclosed herein, it is envisioned that depending on the size of the RNA, the most suitable SEC matrix may be chosen accordingly. Such selection may be beneficial for optimizing purity and / or RNA recovery yield. Based on the values of asymmetry presented in Fig. 6C, the Sepharose 6 FF resin (Cytiva), appears to perform particularly well among the tested alternatives, independently of the size of the mRNA.

[0170] The results of experiment d) as depicted in Fig. 6D, are indicative that each tested alternative SEC matrix is suitable for purifying RNA according to the method as disclosed herein. In particular, as demonstrated by two elution peaks in each chromatogram of the second runs (corresponding to the mRNA eluate and the eluate of the remaining components of a crude IVT reaction sample, respectively) and the lack of a second elution peak in each chromatogram of the third runs (corresponding to the eluate of the remaining components of a crude IVT reaction sample, respectively), mRNA of the DNase-treated crude IVT samples was separated from residual components present in said samples using each matrix. It is thereby concluded that a purified mRNA sample is obtained using each tested matrix and independently of the size of the mRNA present in the initial sample. Since the second elution peaks are absent in chromatograms of the third runs, it is also concluded that the mRNA sample obtained by collecting the first elution peak from the second runs can be characterized with near 100%, such as at least 95% purity.

[0171] In conclusion, the present Example demonstrates that various SEC base matrices are suitable for use in a method as disclosed herein for purifying RNA of various lengths.

[0172] Example 7: Testing of reusability of a SEC column in a method as disclosed herein

[0173] This Example demonstrates reusability capacity of a column packed with a SEC matrix in a method as disclosed herein. In particular, sequential injection and run of a purified RNA sample comprising a 1975 nucleotide-long mRNA product has been carried out according to the present method without performing any cleaning steps in between the runs. The below presented results indicate that a column packed with a SEC matrix in a method as disclosed herein can be reused and / or recycled for the method rapidly and without any decline in its performance.

[0174] Material and methods

[0175] A purified mRNA sample comprising a 1975 nucleotide-long mRNA product has been obtained. 100 pl of the sample (having an mRNA concentration of 0.125 mg / mL) was injected and run consecutively 10 times at a flow rate of 0.5 mL / min on a HiScreen column (7.7 mm x 100 mm) having a CV of 4.7 mL packed with Sepharose 6 FF (Cytiva). No cleaning steps in between the runs have been performed. No separate equilibration was performed between the runs, since the buffer volumes used before and after sample injection is sufficient for equilibration between the injections. Absorbance at 260 nm was measured.

[0176] Results and conclusion

[0177] Fig. 7 shows superimposed chromatograms of the 10 consecutive SEC runs, wherein one elution peak corresponding to the elution of mRNA present in the purified mRNA sample appears near identical between each individual run. The peak area of these elution peaks is presented in Table 2. Accordingly, measured peak areas between the superimposed elution peaks are near identical with a standard deviation of 0.6214 (and 0.260965 with excluding the peak area of the first run). Table 2. Peak area of the elution peak ofmRNA in 10 consecutive SEC analyses of a purified mRNA sample

[0178] In conclusion, this Example shows that a column packed with a SEC matrix can be used with reliable performance and high accuracy in consecutive runs of samples comprising RNA without implementing a cleaning step of the column in between the runs. While the present Example concerns re-injection of a purified mRNA sample, it is to be understood that the herein demonstrated accuracy, reproducibility and reusability apply to consecutive runs of any samples comprising RNA according to the method as disclosed herein. Thus, the disclosed method is expected to efficiently separate mRNA from a sample with high accuracy and steady performance also after a repeated number of runs. Thus, the method as disclosed herein enables a repeated number of SEC runs for purifying mRNA, wherein a harsh cleaning step between individual runs is omitted. This is advantageous for numerous reasons. As such, the time needed for obtaining a purified RNA sample is significantly reduced in comparison to other methods known in the art. Moreover, due to the absence of a harsh cleaning step, which is normally needed in conventional methods for mRNA purification using chromatography, such as core bead flow-through chromatography, and which exhibit the column to very harsh conditions, the lifetime of the column is significantly increased without compromising the recovery yield and purity of the purified mRNA. The inventors envision, that a chromatography medium, such as a column, packed with a SEC matrix may be reused at least 50-100 times, such as 100 times or more in a method as disclosed herein, without implementing any harsh cleaning step in between the runs.

[0179] Example 8: T7 RNAP alone elutes in peak 2 when reducing agent is included in running buffer

[0180] T7 RNA polymerase is the key component for mRNA synthesis during IVT but also a major impurity to be separated from mRNA during downstream purification. In order to illustrate the elution profile of T7 RNAP alone on Sepharose 6 FF in size-exclusion mode as described for mRNA purification, T7 RNAP was purified to high purity and injected to Sepharose 6FF under similar running conditions as used for mRNA purification. This Example demonstrates that when including reducing agent in running buffer, pure T7 RNA polymerase (T7 RNAP) likely is preserved in its monomeric state and elutes at retention volume similar or later than peak 2 as seen for free NTPs and example proteins shown in Example 1 and 3, thereby separated from the mRNA peak (peak 1). When reducing agent is not included in running buffer, pure T7 RNA polymerase elutes partially in peak 1, likely due to formation of higher oligomer or aggregates.

[0181] The simultaneous detection of mRNA and T7 RNAP by UV from a crude IVT run on Sepharose 6FF is regarded impossible due to the low amount of residual T7 RNAP in a crude IVT reaction, as well as the significantly stronger interfering UV signal originated from mRNA.

[0182] Material and methods

[0183] Sepharose 6FF packed in 2xHiScreen format with 20 cm bed height, 9.3 mL column volume was coupled to an AKTA Pure 25 system coupled with a 10 mm path length UV cell. 10% CV of sample volume was injected in each run and linear flow rate was set to 200 cm / h. T7 RNAP with molecular weight of 99 KDa was purified to 99% purity verified by SDS-PAGE. Purified T7 RNAP was dissolved in 200 mM NaCI, Tris-HCI 100 mM, EDTA 2mM, Triton X-100, TCEP 5mM pH 8 before injection. T7 RNAP was detected using a UV cell with 10 mm path length. During 2 consecutive Sepharose 6FF runs, the column was equilibrated and ran with respective buffer of Tris 10 mM, EDTA ImM, pH 7.5 with or without TCEP 5mM.

[0184] In a control run, purified 1975 nucleotide-long mRNA of 0.17 mg was injected on the same Sepharose 6FF column as above, equilibrated and ran with Tris 10 mM, EDTA ImM, TCEP 5mM, pH 7.5. Results and conclusion

[0185] When purified 1975 nucleotide-long mRNA of 10% CV was injected onto Sepharose 6 FF packed in 2xHiScreen format with 20 cm bed height and equilibrated in buffer comprising of Tris 10 mM, EDTA ImM, TCEP 5mM pH 7.5, one elution peak was recorded for UV 260 nm, as seen in curve (AC) in Fig. 8B ranging from approximately 0.375 to 0.625 CV in retention volume.

[0186] When purified T7 RNAP of at least 10-fold concentrated as in IVT reaction was injected onto Sepharose 6 FF, it was clear that the inclusion of reducing agent (in this case TCEP 5mM) in running buffer was important to maintain T7 RNAP eluting as a single peak starting at around 0.875 CV, as seen in curve (AA) in Fig 8B. When reducing agent was not included in running buffer, T7 RNAP shows elution profile consisting of 2 peaks (curve (AB) in Fig. 8B). The first peak ranging from approximately 0.31 to 0.625 CV therefore overlaps with that of pure mRNA, whereas the majority of T7 RNAP elutes at a second peak with retention volume starting at 0.875 CV.

[0187] In conclusion, a buffer condition where pure T7 RNAP is retained to elute later than that of mRNA on Sepharose 6FF is identified.

[0188] Example 9: S6FF separation of mRNA and impurities works across pH range of 4.5 to 8.5 Material and methods

[0189] DNase-treated crude IVT comprising of 1975-nucleotides long mRNA with concentration of 3.3 mg / ml was injected at 15% CV as load to 2xHiScreen Sepharose 6 FF column (bed height 20 cm, 9.3 mL CV) . Before each run, the column was equilibrated for 2 CV with respective running buffer and ran with the same buffer at linear flow rate of 180 cm / h. The buffers used include Tris 10 mM, EDTA ImM, pH 7.5; sodium acetate 50 mM, pH 4.5, sodium acetate 50 mM, pH 5.5, sodium citrate 50 mM, pH 6.5; HEPES 50 mM, pH 7.5 and HEPES 50 mM, pH 8.5.

[0190] Peak 1 for mRNA and peak 2 for impurities were analyzed on Fragment Analyzer: mRNA peak 1 fractions were diluted to final concentration of 0.2 mg / ml before analysis and peak 2 fractions were kept undiluted.

[0191] Results and conclusions Equilibration and running buffer of Tris 10 mM, EDTA ImM, pH 7.5 (the default condition) corresponds to curve (AD) in Fig. 9A and TE pH 7.5 in Fig. 9B; sodium acetate 50 mM, pH 4.5 corresponds to curve (AE) in Fig. 9A and NaAc 4.5 in Fig. 9B; sodium acetate 50 mM, pH 5.5, corresponds to curve (AF) in Fig. 9A and NaAc 5.5 in Fig 9B; sodium citrate 50 mM, pH 6.5 corresponds to curve (AG) in Fig. 9A and NaCit 6.5 in Fig. 9B; HEPES 50 mM, pH 7.5 corresponds to curve (AH) in Fig 9A and HEPES 7.5 in Fig 9B; HEPES 50 mM, pH 8.5, corresponds curve (Al) in Fig. 9A and HEPES 8.5 in Fig. 9B.

[0192] As shown in Fig. 9A,the separation offered by Sepharose 6FF for mRNA from other impurities is functional across the tested buffer pH range, with two clear and distinct peaks around 0.5 CV (Pl) and 1 CV (P2), respectively. Fig. 9B shows that mRNA of 1975-nucleotides size is found only in Pl fractions, while P2 is free from mRNA, regardless of tested buffer. The mRNA integrity was regarded unchanged in all the buffer pH tested. pH range lower than 4.5 or higher than 8.5 were not tested in this experiment, out of concern of the mRNA stability in those conditions.

[0193] Example 10: Sepharose 6FF offers scalable, preparative purification of mRNA from crude IVT

[0194] Material and method

[0195] A crude IVT reaction sample comprising free NTPs, RNA polymerase (T7 RNA polymerase) and a 1975 nucleotide-long mRNA product together with the corresponding DNA template was obtained as described in Example 1. The sample was subjected to DNase treatment, also as described in Example 1. The crude IVT originating from the same batch was applied to three sizes of Sepharose 6FF columns including 2xHiScreen (diameter 0.77 cm), 2xHiPrep (diameter 2.6 cm ) and XK50 / 30 (diameter 5 cm). A UV cell with 0.5 mm path length was used for all three runs. Column format information is listed in Table 3 below. A linear flow rate of 180 cm / h was applied on 2XHiScreen and 2xHiPrep column, whereas a lower linear flow rate of 60 cm / h was used on the XK50 / 30 column, out of respect of the delta-pressure limit of the particular column unit used in the experiment. Normally, the XK50 / 30 column can be run with a higher flow rate. Larger column sizes listed in Table 3, such as AxiChrom 50, AxiChrom 70 and RTP (80 / 250) were not tested in this experiment, but we are able to project the performance and productivity of these formats in Table 3 by using very moderate linear flow rate with consideration for both the tolerance of column formats as well as the flow-pressure property of Sepharose 6FF resin. Table 3. Scale and productivity

[0196] Column Bed Column Linear flow Delta Load Sample Elution Cycle Productivity format height volume velocity pressure volume cone volume time units cm mL cm / h mPa CV mg / mL CV h mg / h

[0197] 2xHiScreen 20 9.3 180 0.3 0.15 3.3 2 0.22 21

[0198] 2xHiPrep 20 106 180 0.3 0.15 3.3 2 0.22 236

[0199] XK50 / 30* 20 373 60 0.03 0.15 3.3 2 0.67 284

[0200] AxiChrom 50 20 392 180 0.15 0.15 3.3 2 0.22 873

[0201] AxiChrom 70 20 769 180 0.15 0.15 3.3 2 0.22 1713

[0202] RTP (80 / 250) 25 1300 180 0.12 0.15 3.3 2 0.28 2317

[0203] * A lower linear flow velocity was used for this column due to the selected packing conditions but according to Sepharose 6 FF instruction manual the flow / pressure profile is 250 to 400 cm / h with delta column pressure of 0.1 MPa in the same column format

[0204] Peak 1 (Pl) for mRNA and peak 2 (P2) for impurities were analysed on Fragment Analyzer for mRNA integrity analysis, as described in Example 9.

[0205] Results and conclusions

[0206] Sepharose 6FF could be used for preparative purification of mRNA with scalable processable sample volume and productivity when appropriate column size is chosen for the input scale. As shown in Fig. 10B, three Sepharose 6FF columns of increasing sizes exhibit similar elution profiles when purifying DNase-treated crude IVT sample comprising of 1975- nucleotide mRNA, with the mRNA successfully extracted in Peak 1 only whereas Peak 2 is free from mRNA.

[0207] As shown in Table 3, Sepharose 6FF packed in column formats starting from 2xHiPrep upwards offers a productivity of at least 236 mg of mRNA per hour, estimated by amount of mRNA purified within 2CV of elution. The separation of mRNA from DNAse- treated crude IVT is similar for different column formats with a common bed height of 20 cm and a common sample load of 15% CV (see Fig. 10A). Peak 1 (Pl) is at approx. 0.4-0.7 CV, and peak 2 (P2) is at approx.1-1.3 CV. Due to the chemical and structural characteristics of mRNA, especially long constructs, the crude IVT sample as well as the eluted mRNA exhibits high viscosity. Special attention should be paid on delta-column pressure during mRNA purification by Sepharose 6FF, since high pressure contributed by mRNA is expected during sample application and mRNA elution in Peak 1. The delta-column pressure is influenced by many factors, including but not limited to mRNA concentration in sample, percent of CV loaded and size of the mRNA. The actual reachable productivity by Sepharose 6FF is subject to the practical tolerance by the specific mRNA of interest.

[0208] Example 11: Impact of flow rate on the separation profile of smaller RNA by Sepharose 6FF

[0209] Material and method

[0210] Crude IVT reactions comprising free NTPs, RNA polymerase (T7 RNA polymerase) and either as 100, 200 or 400 nucleotide-long mRNA product together with the corresponding DNA template was obtained as described in Example 1. The crude IVT samples were subjected to DNase treatment, as described in Example 1 before being injected individually on Sepharose 6FF in 2xHiScreen column format (CV 9.3 mL). The sample load was 4.3% of CV for all runs. The chromatography was performed with 67 cm / h or 200 cm / h linear flow rate on an AKTA Pure 25 system with an UV cell of 2 mm path length. Unified running buffer of Tris 10 mM, EDTA ImM pH 7.5 was used for all runs.

[0211] Results and conclusions

[0212] Smaller mRNA such as 100 and 200 nucleotides sizes are likely able to enter the pore of Sepharose 6FF resin. When slower linear flow rate was applied, UV260 traces of both 100 (curve (AS) in Fig. 11A) and 200 (curve (AT) in Fig. 11A) nucleotides mRNA show a split peak and delayed peak top within the expected mRNA elution range, as compared to a faster flow rate (curve (AV) for 100 nucleotides mRNA and curve (AW) for 200 nucleotides mRNA in Fig. 11B). This indicates that at slower flow rate, both mRNA interact more with the Sepharose 6FF resin, causing delay in their elution. In contrast, mRNA of 400 nucleotides size (curve (AU) in Fig. 11A, curve (AX) in Fig. 11B) has similar elution profile regardless of linear flow rate applied. Performing Sepharose 6FF purification on crude IVT comprising of mRNA that are smaller than 400 nucleotides likely require faster linear flow rate to maximize the separation from the impurities. Example 12: mRNA size cut-off for successful separation from crude IVT by Sepharose 6FF

[0213] Material and method

[0214] DNase treated crude IVTs comprising of either 200 or 400 nucleotides mRNA are individually injected to Sepharose 6FF packed in 2xHiScreen column format (20 cm bed height). Chromatography was performed using Tris 10 mM, EDTA ImM, pH 7.5 as running buffer on an AKTA Pure 25 system coupled with an UV cell of 2 mm path length. The linear flow rate was set to be 200 cm / h for both runs. The fractions from Sepharose 6FF crude mRNA purification were pooled for peak 1 for re-injection onto the same column. In addition, peak 2 fractions of crude purification as well as new fractions obtained from reinjecting peak 1 pool were further analysed for presence of mRNA on CGE by Fragment Analyzer.

[0215] Results and conclusion mRNA of 400 nucleotides (curve (AX) in Fig. 12A) does not enter the pore of Sepharose 6FF resin as verified by absence in Peak 2 fractions, whereas mRNA of 200 nucleotides (curve (AW) in Fig. 12A) does. In practice, Sepharose 6FF may not provide complete extraction of mRNA content from crude IVT comprising of mRNA with sizes smaller than 400 due to the interaction of smaller mRNA to the resin and the partial partition of smaller mRNA into peak 2 (as shown in Fig. 12C), together with smaller impurities such as NTP. However, peak 1 extraction of both 200 nucleotides (curve (BD)) and 400 nucleotides mRNA (curve (BE)) is confirmed to be mRNA (See Fig. 12B) and are found to eluate in peak 1 position from re-run analysis, at similar retention volumes as in their crude purification. These results indicate Sepharose 6FF could still provide partial extraction of mRNA with sizes smaller than 400 nucleotides, although with reduced recovery due to partition of these smaller mRNA into peak 2 (See fig. 12C).

[0216] Example 13: Testing of different functionalized base matrices

[0217] In this Example, purification of RNA is demonstrated according to the method as disclosed herein using two SEC base matrix alternatives coupled with two different ligands, one being a cation exchange (CIEX) ligand and one being a hydrophobic interaction (HIC) ligand. None of the ligands bind RNA under conditions used for SEC with mRNA from DNase treated crude IVT reaction samples Material and methods

[0218] A crude IVT reaction sample comprising free NTPs, RNA polymerase (T7 RNA polymerase) and a 1975 nucleotide-long mRNA product together with the corresponding DNA template was obtained as described in Example 1. The sample was subjected to DNase treatment, also as described in Example 1.

[0219] 100 pl of the DNase-treated crude IVT reaction sample was diluted 50x prior to loading at 0.5 ml / min flow rate on two different HiTrap columns (16 mm x 25 mm) having a CV of 5.0 mL packed with the following base cross-linked agarose resins:

[0220] 1. HiTrap™ SP FF (Cytiva®),

[0221] 2. HiTrap™ Butyl HP (Cytiva®)

[0222] Resin 1 has the same base matrix as Sepharose 6 FF functionalized with a sulphopropyl cation exchange (CIEX) ligand and resin 2 same base matrix as Sepharose HP functionalized with a butyl ligand.

[0223] During the above tests, performed on a AKTA Pure 25 system, absorbance was measured at 260 nm with a 10 mm path length UV cell.

[0224] . The columns were equilibrated between each run using a buffer containing 10 mM Tris (pH 7.5) and 1 mM EDTA with a volume corresponding to at least one column volume.

[0225] Results and conclusion

[0226] Chromatograms from the CIEX testing is presented in Fig. 13A and from the HIC testing in Fig. 13B. For both resins tested, the elution profile is substantially the same as for a non-functionalized resin as in the Examples above. Thus, the presented results indicate that a column packed with a SEC base matrix coupled with a ligand that does not bind RNA when using SEC conditions can be used for separation of mRNA from proteins and residual DNA fragments present in crude DNase-treated IVT samples.

[0227] ITEMIZED LIST OF EMBODIMENTS

[0228] 1. A method for purifying RNA from a sample comprising the RNA, DNA, protein and optionally further components, wherein said method comprises a) enzymatically digesting DNA in said sample to obtain a DNase-treated sample, and b) separating RNA of said DNase-treated sample from proteins and residual DNA fragments present in said DNase-treated sample by size exclusion chromatography (SEC) using a chromatography medium. 2. The method according to item 1, wherein said chromatography medium comprises a homogeneous porous matrix.

[0229] 3. The method according to item 2, wherein the matrix is non-functionalized or functionalized with a ligand that does not retain RNA, such as wherein the matrix is nonfunctionalized.

[0230] 4. The method according to any preceding item, wherein the chromatography medium is packed in a column.

[0231] 5. The method according to item 4, wherein said column has a column volume (CV) and a volume loading capacity and wherein said volume loading capacity is less than said CV.

[0232] 6. The method according to any proceeding item, wherein the chromatography medium is packed in a column to provide a column volume (CV), wherein the method comprises a step of loading a volume of the sample onto the column, wherein said volume of the sample is equal to or less than 50% of the CV, such as 1-25 % of the CV.

[0233] 7. The method according to item 5, wherein the volume loading capacity is equal to or less than about 50% of the column volume (CV), such as equal to or less than about 40% of the CV, equal to or less than about 35% of the CV, equal to or less than about 30% of the CV, equal to or less than about 25% of the CV, equal to or less than about 20% of the CV, equal to or less than about 15% of the CV, or equal to or less than about 10% of the CV.

[0234] 8. The method according to item 5 or 7, wherein the volume loading capacity is about 1% to about 25% of the CV, such as about 1% to about 20% of the CV, such as about 2% to about 20% of the CV, such as about 2% to about 15% of the CV.

[0235] 9. The method according to any one of items 5 to 8, wherein the volume loading capacity is about 20% of the column volume, preferably about 10% of the column volume.

[0236] 10. The method according to any preceding item, wherein RNA is purified from said sample in a purity of at least about 90%, such as at least about 91%, such as at least about 92%, such as at least about 93%, such as at least about 94%, such as at least about 95%.

[0237] 11. The method according to item 10, wherein said purity is achieved by step b) without further steps of separating RNA of said DNase-treated sample from proteins and residual DNA fragments present in said DNase-treated sample.

[0238] 12. The method according to any preceding item, wherein RNA is purified from said sample in a recovery yield of at least 90%, such as at least about 91%, such as at least about 92%, such as at least about 93%, such as at least about 94%, such as at least about 95%. 13. The method according to any preceding item, wherein said RNA, said proteins and said residual DNA fragments are eluted from the chromatography medium used in step b) using the same buffer.

[0239] 14. The method according to any preceding item, wherein said method comprises conditioning of said RNA.

[0240] 15. The method according to any preceding item, wherein said method further comprises, subsequent to step b, one or more chromatography steps, such as an ion exchange chromatography, an affinity chromatography step, a hydrophobic interaction chromatography step or a multimodal chromatography step.

[0241] 16. The method according to item 14, wherein said conditioning occurs during said SEC as defined in step b) in item 1; and / or wherein said method comprises a further step of SEC wherein said conditioning of said RNA occurs during said further step of SEC.

[0242] 17. The method according to any preceding item, wherein said method comprises determining the concentration of the RNA separated from said proteins and said residual DNA fragments.

[0243] 18. The method according to item 17, wherein the concentration of the RNA separated from said proteins and said residual DNA fragments is determined using the elution profile obtained by said SEC as defined in step b) in item 1; and / or wherein the method comprises a further step of SEC wherein the concentration of the RNA separated from said proteins and said residual DNA fragments is determined using the elution profile obtained by said further step of SEC.

[0244] 19. The method according to item 16 or 18, wherein a chromatography medium as defined in any one of items 1 to 9 is used in said further step of SEC.

[0245] 20. The method according to any preceding item, wherein the RNA is a single stranded RNA, such as an mRNA.

[0246] 21. The method according to any preceding item, wherein said sample is obtained by an RNA manufacturing method, such as by in vitro transcription.

[0247] 22. The method according to any preceding item, wherein the RNA comprises a linear sequence of at least about 500 nucleotides (nt), such as at least about 750 nt, such as at least about 1000 nt, such as at least about 1500 nt, such as at least about 2000 nt, such as at least about 3000 nt, such as at least about 4000 nt.

[0248] 23. The method according to any preceding item, wherein the method further comprises at least one additional method step. 24. The method according to item 23, wherein said additional method step is subsequent to step b).

[0249] 25. The method according to item 23 or 24, wherein said additional method step is selected from the group consisting of affinity chromatography, hydrophobic interaction chromatography, desalting and buffer exchange, anion exchange, chromatography with multimodal / mixed chromatography resins and any combination thereof.

[0250] 26. The method according to any preceding item, wherein said method does not comprise an additional method step preceding and / or subsequent to step a) and / or step b) for separating RNA of said DNase-treated sample from proteins and / or residual DNA fragments present in said DNase-treated sample.

[0251] 27. The method according to any preceding item, wherein said RNA is purified on a preparative scale, semi-preparative scale or on an analytical scale, such as wherein said RNA is purified on a preparative scale.

[0252] 28. Use of a size exclusion chromatography medium for separating RNA present in a DNase-treated sample from proteins and residual DNA fragments present in said DNase- treated sample.

[0253] 29. The use according to item 28, wherein said use further comprises determining concentration of the RNA separated from said proteins and said residual DNA fragments.

[0254] 30. The use according to item 28 or 29, wherein said use further comprises conditioning of said RNA.

[0255] 31. The use according to any one of items 28 to 30, wherein said use comprises simultaneously separating said RNA from said proteins and said residual DNA fragments and determining concentration of the RNA separated from said proteins and said residual DNA fragments.

[0256] 32. The use according to any one of items 28 to 31, wherein said use comprises simultaneously separating said RNA from said proteins and said residual DNA fragments and conditioning of said RNA.

[0257] 33. The use according to any one of items 28 to 32, wherein said use comprises simultaneously separating said RNA from said proteins and said residual DNA fragments, determining concentration of the RNA separated from said proteins and said residual DNA fragments and conditioning of said RNA.

[0258] 34. The use according to any one of items 28 to 33, wherein said size exclusion chromatography medium comprises a homogeneous porous matrix. 35. The use according to item 34, wherein the matrix is non-functionalized or functionalized with a ligand that does not retain RNA, such as wherein the matrix is nonfunctionalized.

[0259] 36. The use according to any one of items 28 to 35, wherein said size exclusion chromatography medium is packed in a column.

[0260] 37. The use according to item 36, wherein said column has a column volume (CV) and a volume loading capacity, and wherein said volume loading capacity is less than said CV.

[0261] 38. The use according to item 37, wherein the volume loading capacity is equal to or less than about 50% of the CV, such as equal to or less than about 40% of the CV, such as equal to or less than about 35% of the CV, such as equal to or less than about 30% of the CV, such as equal to or less than about 25% of the CV, such as equal to or less than about 20% of the CV, such as equal to or less than about 15% of the CV, such as equal to or less than about 10% of the CV.

[0262] 39. The use according to item 37 or 38, wherein the volume loading capacity is from about 1% to about 25% of the CV, such as from about 1% to about 20% of the CV, about 2% to about 20% of the CV, or about 2% to about 15% of the CV.

[0263] 40. The use according to any one of items 37 to 39, wherein the volume loading capacity is about 20% of the column volume, preferably about 10% of the column volume.

[0264] 41. The use according to any one of items 28 to 40, wherein said RNA is purified from said DNase-treated sample in a purity of at least about 90%, such as at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95%, during said use.

[0265] 42. The use according to any one of items 28 to 41, wherein said RNA is purified from said DNase-treated sample in a recovery yield of at least 90%, such as at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95%, during said use.

[0266] 43. The use according to any one of items 28 to 42, wherein the RNA is a single stranded RNA, such as an mRNA.

[0267] 44. The use according to any one of items 28 to 43, wherein said sample is obtained by in vitro transcription.

[0268] 45. The use according to any one of items 28 to 44, wherein the RNA comprises a linear sequence of at least about 500 nucleotides (nt), such as at least about 750 nt, at least about 1000 nt, at least about 1500 nt, at least about 2000 nt, at least about 3000 nt, or at least about 4000 nt.

[0269] 46. The method according to any one of items 1-T1 or the use according to any one of items 34-45, wherein said matrix comprises at least one material selected from the group consisting of crosslinked agarose, crosslinked dextran and crosslinked copolymer of allyl dextran and N,N'-methylene bisacrylamide.

[0270] 47. The method according to any one of items 1-T1 or the use according to any one of items 34-45, wherein said matrix is selected from the group consisting of crosslinked agarose, crosslinked dextran and crosslinked copolymer of allyl dextran and N,N'-methylene bisacrylamide, and combinations thereof.

[0271] 48. The method or the use according to item 46 or 47, wherein said material or matrix is crosslinked agarose.

[0272] 49. The method according to item 2 or the use according to item 34, wherein said matrix comprises beads having a median particle size D50v, such as a bead size D50v of 5- 200 pm, 10-200 pm, 20-200 pm, 40-200 pm, 60-200 pm, 80-200 pm, 100-200 pm, 120-200 pm, 140-200 pm, 160-200 pm, 180-200 pm, 5-180 pm, 5-160 pm, 5-140 pm, 5-120 pm, 5- 100 pm, 5-80 pm, 5-60 pm, 5-40 pm, 5-20 pm, or 10-50 pm.

[0273] 50. The method according to any one of items 4-9 or the use according to any one of items 36-40, wherein the column has a diameter and a bed height with a height / diameter ratio of 0.5-30, such as 0.5-10, such as 2-5, such as 3.85.

[0274] 51. The method according to any of the items 1-27, wherein no precipitation is performed before step b).

[0275] 52. The method according to any of the items 1-27 or 51, wherein step b) is performed using a single buffer, said single buffer comprising a reducing agent.

[0276] REFERENCES

[0277] WO 2014 / 140211

[0278] WO 2022 / 162018

[0279] US6602990 Bl

[0280] US7396467B2

[0281] US8309709 B2 B. Gbransson, E. Dagerus: Image analysis as a tool for calibration of Electrical Sensing Zone instruments in the size measurement of porous spherical particles, in P. J. Lloyd (ed.): Particle Size Analysis 1988. 1988 John Wiley & Sons Ltd, pp. 159-166

Claims

CLAIMS1. A method for purifying RNA from a sample comprising the RNA, DNA, protein and optionally further components, wherein said method comprises a) enzymatically digesting DNA in said sample to obtain a DNase-treated sample, and b) separating RNA of said DNase-treated sample from proteins and residual DNA fragments present in said DNase-treated sample by size exclusion chromatography (SEC) using a chromatography medium.

2. The method according to claim 1, wherein said chromatography medium comprises a homogeneous porous matrix.

3. The method according to claim 2, wherein the matrix is non-functionalized or functionalized with a ligand that does not retain RNA, such as wherein the matrix is nonfunctionalized.

4. The method according to any preceding claim, wherein the chromatography medium is packed in a column, wherein said column has a column volume and a volume loading capacity and wherein said volume loading capacity is less than said column volume.

5. The method according to any proceeding claim, wherein the chromatography medium is packed in a column to provide a column volume, wherein the method comprises a step of loading a volume of the sample onto the column, wherein said volume of the sample is equal to or less than 50% of the column volume, such as 1 - 25 % of the column volume.

6. The method according to claim 4, wherein the volume loading capacity is from about 1% to about 25% of the column volume (CV), such as about 1% to about 20% of the CV, such as about 2% to about 20% of the CV, such as about 2% to about 15% of the CV.

7. The method according to any preceding claim, wherein RNA is purified from said sample in a purity of at least about 90%, such as at least about 91%, such as at least about 92%, such as at least about 93%, such as at least about 94%, such as at least about 95%.

8. The method according to any preceding claim, wherein RNA is purified from said sample in a recovery yield of at least 90%, such as at least about 91%, such as at least about 92%, such as at least about 93%, such as at least about 94%, such as at least about 95%.

9. The method according to any preceding claim, wherein said method comprises conditioning of said RNA and / or determining the concentration of the RNA separated from said proteins and said residual DNA fragments.

10. The method according to any preceding claim, wherein said method further comprises, subsequent to step b, one or more chromatography steps, such as an ion exchange chromatography, an affinity chromatography step, a hydrophobic interaction chromatography step or a multimodal chromatography step.

11. The method according to any preceding claim, wherein said sample is obtained by an RNA manufacturing method, such as by in vitro transcription.

12. Use of a size exclusion chromatography medium for separating RNA present in a DNase-treated sample from proteins and residual DNA fragments present in said DNase- treated sample.

13. The use according to claim 12, wherein said use further comprises determining the concentration of the RNA separated from said proteins and said residual DNA fragments and / or conditioning of said RNA.

14. The use according to claim 12 or 13, wherein said size exclusion chromatography medium comprises a homogeneous porous matrix.

15. The use according to claim 14, wherein the matrix is non-functionalized or functionalized with a ligand that does not retain RNA, such as wherein the matrix is nonfunctionalized.

16. The use according to any one of claims 12 to 15, wherein said size exclusion chromatography medium is packed in a column, wherein said column has a column volume (CV) and a volume loading capacity, and wherein said volume loading capacity is less than said column volume.

17. The use according to claim 16, wherein the volume loading capacity is from about 1% to about 25% of the CV, such as from about 1% to about 20% of the CV, such as from about 2% to about 20% of the CV, such as from about 2% to about 15% of the CV.

18. The method or use according to any of claims 2-11 or 14-17, wherein said material or matrix is crosslinked agarose.

19. The method or use according to any one of the preceding claims, wherein no precipitation is performed before the size exclusion chromatography step b), or before the use of the size exclusion chromatography medium.

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