A purification of RNA by flowthrough chromatography

Abstract

The present invention relates to the field of RNA purification and in particular to the purification of RNA using chromatography. More specifically, the present invention relates to a method of RNA purification using flowthrough column chromatography wherein a mobile phase comprising an RNA sample is applied to a stationary phase thereby being configured to have binding affinity for impurities other than target RNA, and wherein the mobile phase is free of organic solvents. The invention further relates to the use and a device for flowthrough purification of RNA wherein RNA flows through the stationary phase of the column thereby being free from impurities that remain in the stationary phase.

Description

[0001] A PURIFICATION OF RNA BY FLOWTHROUGH CHROMATOGRAPHY

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of RNA purification and in particular to the purification of RNA using chromatography. More specifically, the present invention relates to a method of RNA purification using flowthrough column chromatography wherein a mobile phase comprising an RNA sample is applied to a stationary phase thereby being configured to have binding affinity for impurities other than target RNA, and wherein the mobile phase is free of organic solvents. The invention further relates to the use and a device for flowthrough purification of RNA wherein RNA flows through the stationary phase of the column thereby being free from impurities that remain in the stationary phase.

[0004] BACKGROUND TO THE INVENTION

[0005] High-quality, pure, and intact RNA molecules are crucial for many downstream experiments, including RT-PCR, RT-qPCR, cloning, reverse transcription for cDNA synthesis, RNA- sequencing, but also for other applications such as delivering RNA therapeutics using nonviral vector-based delivery systems. There are various approaches to purify RNA including phenolchloroform extraction, ethanol precipitation, gel purification, spin column purification, chromatography, and the use of magnetic beads.

[0006] In vitro transcription is a simple and efficient process to synthesize RNA from a DNA template of any sequence using an RNA polymerase. While the reaction can yield large quantities of RNA, it contains impurities due to various unwanted activities of the polymerases. Chemical-, column- , gel-, and bead-based approaches as well as chromatography can be used - and are sometimes combined - to result in purified RNA free from unincorporated nucleotides, salts and proteins. Although (spin)column-based methods are an efficient and user-friendly way to remove impurities, loading capacity is limited and the recovery may be reduced. Another scalable approach to remove multiple process-related impurities such as proteins, unused reagent and buffer component from in vitro transcribed RNA is chromatography. Similar to proteins, RNA can be purified by chromatographic methods and is in particular capable of removing long or short RNA contaminants or contaminants bound to the RNA of interest. A number of chromatography techniques can be used including ion pair reverse-phase, ion exchange and affinity chromatography.

[0007] First of all, the essence of a typical chromatography-based process to purify RNA is based on the capturing or hybridization of RNA to a stationary phase, while impurities or contaminants are washed away. Afterwards, the ‘purified’ RNA is eluted and ready for down-stream processing. In most cases, salt needs to be added to the RNA to suppress the repulsive negative charges of RNA. Several disadvantages are associated with a typical chromatography-based process to purify RNA. One major disadvantage is that the yield of RNA obtained from chromatography can be relatively low due to insufficient binding of RNA to the column meaning that a large amount of starting material may be required. Alternatively, when the column is saturated with RNA, the remainder of RNA cannot or will have difficulty hybridizing and RNA is washed away leading to excessive loss of RNA. The capacity of a typical column in the bind-elute mode is low, so to scale up the process, bigger columns are required resulting in more (volume) of solvent (mobile phases) being used and bigger facilities. Secondly, RNA is a very fragile molecule that is easily degraded, and the capturing or hybridization to the column can cause damage to the RNA. A further drawback of conventional chromatography is that - after elution of RNA - the chromatography columns first need to be washed or sanitized before reusing it for another RNA purification batch. Even worse, in some cases columns cannot be recycled, and new columns need to be used in each batch leading to high costs. Lastly, the use of organic solvents during chromatography is disadvantageous since for example ethanol is a flammable and acetonitrile is a combustible and toxic substance.

[0008] Thus there remains a need for further and improved RNA purification methods, and in particular for those that allow cost- and time-efficient purification of RNAs at an industrial scale with high yield and pharmaceutical-grade purity while retaining the stability, biological potency and functionality of the RNA.

[0009] Accordingly, the inventors of the present invention have developed an innovative method for purifying RNA using flowthrough column chromatography wherein RNA flows through the column while contaminations or impurities other than target RNA bind to the column. The flowthrough column chromatography method of the invention involves the arrangement of a stationary phase and a mobile phase, wherein the stationary phase is configured to have binding affinity for impurities other than target RNA, and wherein the mobile phase is free of organic solvents.

[0010] The main advantage of purifying RNA with implementation of flowthrough chromatography is that the target RNA end sample has increased maximum amounts of RNA that can be processed per volume of stationary phase. In the method according to the invention, there is no issue of insufficient binding or RNA saturation of chromatography columns as RNA flows through the column while impurities and contaminants bind to the chromatography column. In this context, a multi-modal approach can be used wherein different types of stationary phases within one chromatography columns can be combined, or a serial approach wherein the same or different chromatography columns are sequentially connected to increase capacity and / or improve purity. A further advantage is that multiple types of impurities or contaminants can be captured by combining different types of stationary phases in one column or using different types of stationary phases in separate chromatography columns.

[0011] Since target RNA will not bind to the column, there is less or no chance of RNA degradation or shearing, which is particularly advantageous due to the fragile nature of RNA. Even more, due to the reduction in required stationary and (especially) mobile phase volumes, the present method can be performed using smaller equipment at significantly reduced footprint, which makes the procedure easy scalable, faster and less expensive. Lastly, the method according to the invention avoids the use of organic solvents in the mobile phase. This is particularly advantageous since these solvents may be toxic when administered to humans as part of a pharmaceutical composition and / or which may adversely impact on the stability of large RNAs. Further, the avoidance of flammable or toxic liquids and storage thereof, leads to an environmentally sustainable and operator safe procedure.

[0012] This flexible and modular RNA purification method can be adjusted to the specific need to purify an RNA sample. The method enables to purify RNA in such a way that it is extremely suitable to be encapsulated and formulated into a delivery system particle such as lipid or polymers, and to be used as a non-viral delivery system e.g. mRNA vaccine. To conclude, compared to conventional column chromatography, this flowthrough method for purifying RNA is comparatively simple, has a high yield, is scalable and rapid.

[0013] SUMMARY OF THE INVENTION

[0014] In a first aspect, the present invention provides a method of RNA purification using flowthrough column chromatography comprising one or more stationary phases and a mobile phase, wherein said method comprises applying a mobile phase (alternatively named mobile phase composition) comprising an RNA sample to a stationary phase thereby being configured to have binding affinity for impurities other than target RNA, and wherein the mobile phase is free of organic solvents.

[0015] In a further embodiment of the method of the present invention, said stationary phase comprises one or more stationary phases for a column chromatography method selected from the list comprising: affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, H-bond chromatography or any combinations thereof.

[0016] In a specific embodiment, said impurities are selected from the list comprising: protein impurities, nucleotides, enzymes, plasmid DNA, host cell DNA, host cell RNA, double stranded RNA (dsRNA), incomplete RNA fragments, and / or RNA aggregates. In particular, said method is particularly suitable as a method for in vitro transcribed RNA purification. In such specific embodiment, said impurities are in vitro transcription process-related impurities including but not limited to protein impurities, nucleotides, enzymes, plasmid DNA, while product-related impurities of the in vitro transcribed RNA include but are not limited to double stranded RNA (dsRNA), incomplete RNA fragments, and / or RNA aggregates. In yet a specific embodiment, said target RNA is single-stranded RNA, more in particular singlestranded mRNA, even more in particular full-length single-stranded mRNA.

[0017] In another embodiment of the method of the present invention, said stationary phase comprises at least one stationary phase for ion exchange chromatography, preferably at least one stationary phase for cation exchange chromatography.

[0018] In a particular embodiment, the present invention provides a method of RNA purification using flowthrough column chromatography comprising one or more stationary phases and a mobile phase, wherein said method comprises: applying a mobile phase composition comprising an RNA sample to a stationary phase configured to have binding affinity for impurities other than target RNA, wherein the mobile phase composition is free of organic solvents, and wherein said one or more stationary phases comprises a stationary phase for a cation exchange chromatography.

[0019] In a further specific embodiment of the method of the invention, said mobile phase is a buffer and / or salt solution having an ionic strength in the range of about and between 100 mM - 2 M, in particular about and between 500 mM - 1 .5 M, more in particular about 750 mM.

[0020] In yet a further embodiment, the present invention provides a method wherein the pH of the mobile phase is higher compared to the isoelectric point of said RNA sample, preferably wherein the pH of the mobile phase is at least about pH 4, more preferably in the range of about pH 4 to about pH 12.

[0021] In a very specific embodiment of the method of the present invention, the mobile phase is a salt solution with a concentration of about and between 0 M - 2 M. In a further embodiment, a method is provided wherein said stationary phase comprises a stationary phase for a cation exchange chromatography, and wherein said mobile phase is a salt solution with a concentration of about and between 0 M - 0.5 M having a pH in the range of about 4-8.

[0022] In still a further embodiment, a method is provided wherein said stationary phase for a column chromatography method is selected from the list comprising: affinity chromatography, anion exchange chromatography, hydrophobic interaction chromatography; and wherein said mobile phase is a salt solution with a concentration of about and between 0.1 M - 2 M having a pH in the range of about 6-8.

[0023] In still a further embodiment, a method is provided wherein said stationary phase for a column chromatography method comprises a stationary phase for a cation exchange chromatography and wherein said mobile phase is a salt solution with a concentration of about and between 0.1 M - 2 M having a pH in the range of about 6-8.

[0024] In another particular embodiment of the method of the present invention, target RNA flows through the stationary phase of the column thereby being free from impurities that remain bound to the stationary phase.

[0025] In another aspect, the present invention provides the use of a flowthrough chromatography column for RNA purification comprising one or more stationary phases and a mobile phase, wherein said stationary phase is configured to have binding affinity for impurities other than target RNA in the presence of a mobile phase which is free of organic solvents and comprises an RNA sample. In a particular embodiment, the stationary phase comprises two or more stationary phases.

[0026] In yet a further embodiment, the present invention provides the use of a flowthrough chromatography column for RNA purification comprising one or more stationary phases and a mobile phase, wherein said stationary phase is configured to have binding affinity for impurities other than target RNA in the presence of a mobile phase composition which is free of organic solvents and comprises an RNA sample, and wherein said stationary phase comprises a stationary phase for a cation exchange chromatography.

[0027] In a further aspect, the present invention provides a device for flowthrough purification of RNA comprising one or more chromatography columns comprising one or more stationary phases and a mobile phase, wherein said one or more stationary phases are configured to have binding affinity for impurities other than target RNA, and wherein said mobile phase is free of organic solvents.

[0028] In yet a further embodiment, the present invention provides a device for flowthrough purification of RNA comprising one or more chromatography columns comprising one or more stationary phases and a mobile phase, wherein said one or more stationary phases are configured to have binding affinity for impurities other than target RNA, and wherein said mobile phase comprises the use of a mobile phase composition which is free of organic solvents, and wherein said stationary phase comprises a stationary phase for a cation exchange chromatography.

[0029] BRIEF DESCRIPTION OF THE DRAWINGS

[0030] With specific reference now to the figures, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the different embodiments of the present invention only. They are presented in the cause of providing what is believed to be the most useful and readily description of the principles and conceptual aspects of the invention. In this regard no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention. The description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0031] FIG. 1 : Hydrophobic interaction chromatography comparative example.

[0032] (Panel A) RNA was loaded at high salt concentration (e.g. 50 mM Tris-HCI, 2 M NaCI). Salt concentration of the mobile phase is than decreased in stepwise manner. Fractions are collected and analyzed.

[0033] (Panel B) A 3-step method was performed consisting of a loading (2 M NaCI) step, a washing step (1 .2 M NaCI) and an elution step (500 mM NaCI).

[0034] (Panel C) Dynamic binding capacity of the column was determined by sequentially (4 times) loading 2mg fractions of RNA at loading conditions. During the third loading cycle (FT3), UV260 and UV280 are seen to increase. Clear breakthrough is observed during the fourth loading cycle (FT4) indicating that the binding capacity of the column is less than 6 mg of RNA per 1 ml of column volume.

[0035] FIG. 2: Hydrophobic interaction chromatography according to the invention.

[0036] (Panel A) Material was loaded at elution conditions (500 mM NaCI), forcing the RNA to remain in the mobile phase and only impurities to bind to the stationary phase. 9 fractions of 2 mg of RNA were loaded sequentially on 1 ml of column volume. Flowthrough fractions were collected and analyzed. The column was then washed with a mobile phase containing 0 mM NaCI, eluting all bound material from the column.

[0037] (Panel B) Protein content (pg / mg RNA) of the RNA present in the nine flowthrough fractions. (Panel C) dsRNA content (pg / mg) present in the nine flowthrough fractions.

[0038] FIG. 3: Multimodal chromatography wherein a stationary phase is used which combines characteristics of anion exchange and H-bond chromatography.

[0039] (Panel A) Comparative example wherein RNA is loaded at neutral pH (1 OOmM phosphate buffer pH 7) and eluted from the stationary phase at increased pH (e.g. 100mM phosphate buffer pH 10). Dynamic binding capacity (DBC) was assessed by sequentially loading fractions of 2.5mg RNA until breakthrough was observed.

[0040] (Panel B) Method according to the invention wherein RNA was loaded (9x) under elution conditions (pH10). Under these conditions (flowthrough mode), no RNA was observed to bind to the column.

[0041] (Panel C) Protein content (pg / mg RNA) of the RNA present in the nine flowthrough fractions.

[0042] FIG. 4: Anion exchange chromatography.

[0043] (Panel A) Anion exchange chromatography was performed on CIMmultus DEAE column. The column was sanitized using 1 M NaOH and equilibrated to 20 mM Tris-HCI, 1.223 M KCI, 0.2 mM EDTA. RNA was LiCI precipitated and resuspended in equilibration buffer. When the RNA was loaded on the equilibrated column, a large peak in absorbance at 260 nm was observed, corresponding to the RNA present in the flowthrough fraction. (Panel B) Residual protein content (pg / mg RNA) of the flowthrough fraction compared to the loaded material.

[0044] FIG. 5: Cation exchange chromatography.

[0045] (Panel A) Cation exchange chromatography was performed on CIMmultus SO3 column. The column was equilibrated under conditions that ensure that protein impurities but not the RNA bind to the stationary phase (here: 25 mM acetate buffer pH 4.0). 2mg of RNA was LiCI precipitated and resuspended in loading buffer (25 mM acetate buffer pH 4.0) and this material was loaded onto the column. After flushing the column with equilibration buffer, a clear flowthrough peak was observed. (Panel B) Residual protein content (pg / mg RNA) of the flowthrough fraction compared to the loaded material.

[0046] DETAILED DESCRIPTION OF THE INVENTION

[0047] The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0048] As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. By way of example, "a compound" means one compound or more than one compound.

[0049] The term "about" or "approximately" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of + / -10% or less, preferably + / -5% or less, more preferably + / - 1 % or less, and still more preferably + / -0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" or "approximately" refers is itself also specifically, and preferably, disclosed.

[0050] The present invention thus in particular relates to the purification of target RNA, and in particular to RNA purification method using flowthrough chromatography. The invention further relates to the use and a device for flowthrough purification of RNA wherein RNA flows through the stationary phase of the column, thereby being free from impurities that remain in the stationary phase.

[0051] The inventors of the present invention have found that the implementation of flowthrough-based chromatography in the purification of RNA (e.g. in vitro transcribed RNA) results in fast, scalable and highly pure target RNA with high yield compared to conventional chromatography-based techniques. The flowthrough column chromatography method of the invention involves the arrangement of a stationary phase and a mobile phase, wherein the stationary phase is configured to have binding affinity for impurities other than target RNA in the presence of a mobile phase, and wherein RNA flows through the column while contaminations or impurities other than target RNA remain bound to the column. Thus, the method of the present invention generally involves discarding materials which do not contain target RNA (or which do not contain the desired RNA species) while maintaining materials which contain target RNA (i.e. the desired RNA species).

[0052] The main advantage of purifying RNA with implementation of flowthrough chromatography is that the target RNA end sample has increased yield of RNA that can be processed per volume of stationary phase, requiring smaller equipment, lower mobile phase working volumes, and leading to an easy scalable method. There is no issue of insufficient binding or saturation of RNA to chromatography columns as target RNA flows through the column while multiple types of impurities and contaminants bind to the chromatography column. A further advantage is that there is less or no chance of RNA degradation or shearing, which is particularly advantageous due to the fragile nature of RNA.

[0053] Accordingly and as already detailed herein above, in a first aspect, the present invention provides a method of RNA purification using flowthrough column chromatography comprising one or more stationary phases and a mobile phase, wherein said method comprises applying a mobile phase comprising an RNA sample to a stationary phase thereby being configured to have binding affinity for impurities other than target RNA, and wherein the mobile phase is free of organic solvents.

[0054] In particular, the present invention provides a method of RNA purification using flowthrough column chromatography comprising one or more stationary phases and a mobile phase, wherein said method comprises applying a mobile phase composition comprising an RNA sample to a stationary phase configured to have binding affinity for impurities other than target RNA, wherein the mobile phase composition is free of organic solvents, and wherein said one or more stationary phases comprises a stationary phase for a cation exchange chromatography.

[0055] According to the present invention, the term "target RNA" is the desired RNA species that is obtained after the purification process. RNA refers to a molecule which comprises ribonucleotide residues and preferably being entirely or substantially composed of ribonucleotide residues. "Ribonucleotide" relates to a nucleotide with a hydroxyl group at the 2'-position of a 0- D- ribofuranosyl group. The term “target RNA” includes single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of a RNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in RNA molecules can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally- occurring RNA.

[0056] In a specific embodiment, said target RNA is single-stranded RNA, more in particular singlestranded mRNA, even more in particular full-length single-stranded mRNA. As used herein, "mRNA" means "messenger RNA" and relates to a "transcript" which may be produced using DNA as template and encodes a peptide or protein. mRNA typically comprises a cap at the 5’- end, 5' untranslated region (5’ -UTR), a protein or peptide coding region, a 3' untranslated region (3'-UTR) and a poly A tail at the 3’-end. mRNA has a limited halftime in cells and in vitro. Preferably, mRNA is produced by in vitro transcription using a DNA template. In one embodiment of the invention, the RNA is obtained by in vitro transcription. In particular, the method according to the invention is particularly suitable to purify in vitro transcribed RNA.

[0057] In the context of the present invention, the term "RNA sample" relates to a sample that comprises the target RNA as described herein (desired RNA), as well as undesired RNA and other impurities or contaminants which need to be removed or purified by the method according to the invention. For example, the RNA sample described herein can be obtained by for example, but not limited to, an in vitro transcription process. The in vitro transcription methodology is known to the skilled person and utilizes a nucleic acid production mixture comprising all necessary components that make up a reaction buffer that is essential to initiate a reaction. For example, such a mixture may comprise plasmid DNA, T7 RNA polymerase, bovine serum albumin (BSA), restriction enzymes (e.g. BfuA1 ), RNase inhibitor, RNA, Pyrophosphatase, anti-reverse cap analog (ARCA), guanosine triphosphate (GTP), adenosine triphosphate (ATP), uridine triphosphate (UTP), cytidine triphosphate (CTP), EDTA, HEPES, Spermidine, Pyrophospate, Dithiothreitol (DTT), Tris, MgClz, glycerol, sodium chloride, ethanol, lithium chloride, hydrogen chloride, and / or water. The exact conditions and consumables used in the transcription reaction depend on the amount of RNA needed for a specific application.

[0058] Accordingly, as used herein, the term “RNA sample” encompasses the desired “target RNA” as well as undesired RNA and unwanted impurities. As used herein, the term “impurities” is to be understood as RNA impurities encompassing any unwanted or extraneous RNA molecules, protein, nucleotide, enzymes, etc. that are present in the RNA sample that is loaded onto the chromatography column. These impurities can arise from various sources, such as incomplete removal of plasmid DNA, degradation of RNA during sample handling or storage, or contamination from other sources.

[0059] In a specific embodiment, said impurities are selected from the list comprising: protein impurities, nucleotides, enzymes, plasmid DNA, host cell DNA, host cell RNA, double stranded RNA (dsRNA), incomplete RNA fragments, and / or RNA aggregates. Examples of in vitro transcription process-related impurities include but are not limit to protein impurities, nucleotides, enzymes, plasmid DNA while of product-related impurities include but are not limited to double stranded RNA (dsRNA), incomplete RNA fragments, and / or RNA aggregates.

[0060] In other words, the term “target RNA” is used to make reference to the end product which is obtained after purification (desired RNA) while the term “RNA sample” is loaded onto the chromatography column and still comprises impurities. In a particular embodiment of the method of the present invention, the target RNA flows through the stationary phase of the chromatography column thereby being free from impurities that remain bound to the stationary phase.

[0061] As used herein, the term “RNA purification” is to be understood as a process of removing unwanted impurities or contaminants other than target RNA. The goal of RNA purification is to obtain highly pure and intact target RNA that can be suitably used for downstream applications, such as but not limited to gene expression analysis, RNA sequencing, reverse transcription- polymerase chain reaction (RT-PCR), as well as RNA that can be encapsulated and formulated into a delivery system particle such as lipid or polymers, and to be used as a non-viral delivery system e.g. mRNA vaccine. In particular, unintended byproducts of the in vitro production of mRNA may be responsible for immune responses. It has been shown that highly purified mRNA can avoid the immune response. The process of RNA purification is achieved by separating target RNA from other components, such as but not limited to plasmid DNA, dsRNA and proteins, using the flowthrough chromatography technique according to the invention. Thus, after purification the RNA is present in a purer form than before purification. This means that undesired components are present at lower amounts relative to the amount of desired target RNA than before purification.

[0062] In the context of the present invention, the term “flowthrough chromatography” refers to a chromatography technique utilized for the separation and purification of biomolecules including proteins, peptides, and nucleic acids. In the method according to the invention, the mobile phase which comprises an RNA sample is applied to (or loaded onto) a stationary phase packed within a column. As the mobile phase passes through the column by gravity or pressure, components of the RNA sample are separated based on their affinity and / or interaction strength with the stationary phase. Using the method according to the invention, the impurities and contaminants exhibit a stronger binding affinity to the stationary phase and are retained, while the target RNA remains unbound and flows through the column. Consequently, in the flowthrough chromatography method according to the invention, only the unbound target RNA is collected as the desired product, while the bound impurities and contaminants are eliminated.

[0063] The purification process is performed as a flowthrough process or continuous process with all the advantages it entails. The term “flowthrough” is to be understood as a process in which the RNA sample can be simultaneous applied or loaded and obtained from the process. For example, the purification of RNA can be performed in a continuous fashion through applying multiple batches of similar RNA samples until the chromatography column is saturated with impurities or until sufficient target RNA has been obtained. Optionally, a target sample that has been obtained in a first purification process can be loaded again to initiate the purification process again. Thus, the purified target RNA may be obtained in a continuous fashion, as long as RNA sample is loaded onto the column. In a particular embodiment, target RNA flows through the stationary phase of the column thereby being free from impurities that remain bound to the stationary phase. To avoid any misunderstanding, the wording as used herein means ‘essentially free from’ as it might be the case that a second purification step is needed to purify another unwanted impurity. In a specific embodiment, the target RNA according to the invention is essentially free, in particular 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% free from impurities. Typically, the purification of target RNA will be greater than 90% pure, often 95% to 100% pure as analyzed by HPLC.

[0064] As used herein, the term “stationary phase” refers to the solid or liquid material that is packed inside the chromatography column and used to separate and purify the RNA sample according to the invention. The stationary phase can be made of various materials, such as but not limited to silica, alumina, or polymers, and can be modified with different chemical groups to selectively bind to specific molecules or classes of molecules. The stationary phase is an important component of column chromatography, as it determines the selectivity, resolution, and efficiency of the separation. The choice of stationary phase depends on the properties of the RNA sample and the desired separation conditions, and can be optimized to achieve high purity and yield of the desired product.

[0065] In a specific embodiment, the stationary phase comprises one or more stationary phases for a column chromatography method selected from the list comprising: affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, H-bond chromatography or any combinations thereof. Examples of multimodal chromatography, which combines characteristics of different chromatography modalities are for example CIMmultus PrimaS, which combines characteristics of anion exchange and H-bond chromatography; Captocore 700, which combines size-exclusion with multimodal binding to capture impurities; or poros resins, which combines ion-exchange and hydrophobic interaction binding modalities.

[0066] In another embodiment, the chromatography column used in the method of the invention may comprise one or more stationary phases combined in one column (multi-modal approach), or the method can be performed by subsequently using the same or different types of stationary phases of separate chromatography columns (e.g. purifying an RNA sample to obtain the target RNA and then loading the target RNA again onto another type of chromatography column). In the case of a multi-modal approach wherein the characteristics of different chromatography modalities are combined in one stationary phase, it is advantageous that multiple impurities are removed within the same purification process. In the case of a serial approach wherein the same or different chromatography columns are sequentially placed or connected, it is advantageous that the capacity can be increased and / or multiple types of impurities are removed simultaneously. Where large-scale purification is performed, chromatography columns may be connected to each other in series for increased capacity.

[0067] In a specific embodiment, wherein an RNA samples are repeatably purified or before or after the purification process, the entire chromatography column may be sanitized with a solution to remove impurities that remained bound to the column and to prevent cross-contamination. A suitable sanitizing solution may be NaOH, in particular 1 M NaOH. In a further embodiment, the stationary phase is a stationary phase for cation exchange chromatography and is particularly suitable for binding protein impurities.

[0068] Where a sample comprises a desired target RNA and a non-desired RNA species of a different size, the one or more stationary phases comprise in particular a stationary phase for anion chromatography, affinity chromatography, or hydrophobic interaction chromatography.

[0069] In a further embodiment, the one or more stationary phases comprise a stationary phase for anion exchange chromatography and is particularly suitable for binding impurities including but not limited to NTPs, proteins, short RNA fragments, dsRNA, DNA template, or buffer components.

[0070] In a particular embodiment, the one or more stationary phases comprise a stationary phase for affinity chromatography and is particularly suitable for binding impurities including but not limited to NTPs, proteins, RNA fragments without poly A tails, DNA template, or buffer components.

[0071] In a further embodiment, the one or more stationary phases comprise a stationary phase for hydrophobic interaction chromatography and is particularly suitable for binding including but not limited to abortive RNA fragments, proteins, NTPs, DNA templates, buffer components, or dsRNA.

[0072] In a further embodiment, the one or more stationary phases comprise a stationary phase for Flbond chromatography and is particularly suitable for binding including but not limited to proteins, buffer components, NTPs (small molecules).

[0073] In another embodiment of the method of the present invention, said one or more stationary phases comprise at least one stationary phase for ion exchange chromatography, preferably at least one stationary phase for cation exchange chromatography.

[0074] In particular, the one or more stationary phases used in the invention comprise a stationary phase for ion exchange chromatography, more in particular for cation exchange chromatography.

[0075] Cation exchange chromatography is a chromatographic technique employed to separate and purify positively charged molecules based on their interaction with a negatively charged stationary phase. Since RNA molecules are typically negatively charged and many contaminants have a positive charge, typically anion exchange chromatography methods are used to capture the RNA molecules on the positively charged stationary phase, and to allow contaminant to flow- through. Herein, it was however found that cation exchange chromatography was also highly suitable for RNA purification, in particular in flow-through chromatography methods. As used herein, the term “mobile phase” or alternatively “mobile phase composition”, refers to a liquid or gas that flows through the chromatography column and carries the RNA sample through the stationary phase. The mobile phase can be composed of a single buffer or a mixture of buffers, and is chosen based on the properties of the RNA sample and the stationary phase. In a particular embodiment, the mobile phase that is used in chromatography columns according to the invention is free of organic solvents. Accordingly, the method of the invention does not require the use of organic solvents, and are ideally performed without the use of organic solvents, in particular without the use of organic solvents that may be toxic when administered to humans as part of a pharmaceutical composition and / or which may adversely impact on the stability of RNAs. Typical organic solvents used in conventional chromatography include for example Methanol (MeOH), Acetonitrile (ACN), Ethanol (EtOH), Isopropanol (IPA), Chloroform (CHC ), Dichloromethane (DCM), Hexane, Toluene. While organic solvents are widely used in chromatography, there are several disadvantages associated with their uses such as toxicity, environmental impact, cost, compatibility with detection methods, evaporation, flammability.

[0076] In a specific embodiment of the method of the invention, the mobile phase is a buffer solution, a salt solution or a combination thereof. As used herein, many buffer solutions are also salt solutions, but a buffer solution is not inherently a salt solution by definition. Although a buffer solution often contains one or more salts as part of its composition, a buffer solution is defined by its ability to resist changes in pH when small amounts of acid or base are added. On the other hand, some salts in solution fully dissociate and lack the weak acid or weak base pairs needed for buffering, so these types of salt solutions do not resist pH changes.

[0077] Typically, a buffer solution comprises: i) a weak acid and its conjugate base (often supplied as a salt) — for example, acetic acid (CH3COOH) and sodium acetate (CH3COONa). ii) a weak base and its conjugate acid (also often supplied as a salt) — for example, ammonia (NH3) and ammonium chloride (NH4CI).

[0078] In these cases, the "salt" in the buffer (like sodium acetate or ammonium chloride) serves as the conjugate form needed for the acid-base pair to function as a buffer.

[0079] On the other hand, for a buffer solutions as defined within the context of the present invention, a salt is not strictly required in the buffer solution as the buffers function can be maintained by weak acids and bases without added salts.

[0080] Suitable buffer solutions include but are not limited to phosphate buffers, Tris (Tris / HCI) buffers, sodium acetate buffers, HEPES buffers, MES buffers, Bis-Tris (Bis(2- hydroxyethyl)iminotris(hydroxymethyl)methane), Citrate Buffer. Particularly suitable buffer solutions are phosphate buffers or acetate buffers. Suitable salt solutions include but are not limited to sodium chloride (NaCI), potassium chloride (KCI), potassium phosphate (K2HPO4and KH2PO4), magnesium chloride (MgClz), calcium chloride (CaCIz), sodium acetate (NaOAc), sodium citrate (Na3C6HsO7), guanidine ghiocyanate (GuSCN), guanidine hydrochloride (GuHCI). Particularly suitable salt solutions are NaCI solutions. In particular, also combinations can be suitable applied for ex example Tris-buffered saline (TBS) and phosphate-buffered saline (PBS).

[0081] In a specific embodiment, the mobile phase can be optimized according to the different types of column chromatography, such as reverse-phase chromatography and ion-exchange chromatography, to achieve high separation efficiency, resolution, and purity of the desired product. For example, the ionic strength, pH, presence of salt, and specific concentrations of salt can be adjusted to optimize the separation conditions.

[0082] The inventors have found that for the purification of RNA using flowthrough column chromatography it is particularly useful to use a mobile phase which is free or organic solvents and wherein the ionic strength or pH of the mobile phase is adapted to the type of stationary phase thereby being configured to have binding affinity for impurities other than target RNA and to achieve a flowthrough of target RNA. For example, in the embodiments wherein a stationary phase is used suitable for anion exchange, affinity, or hydrophobic interaction chromatography, it is useful to use mobile phases wherein the buffer or salt concentration is modified, while in the embodiments wherein a stationary phase used suitable for anion exchange or cationic exchange chromatography, it is useful to use mobile phase wherein the pH is modified. Under these conditions (flowthrough mode), no, or substantially no, target RNA was observed to bind to the column.

[0083] In even a further embodiment, the temperature of the mobile phase may be increased compared to the temperature of the RNA sample resulting in a less sticky RNA sample.

[0084] In a further specific embodiment of the method of the invention, said mobile phase is a buffer and / or salt solution having an ionic strength in the range of about and between 100 mM - 2 M, in particular about and between 500 mM - 1 .5 M, more in particular about 750 mM.

[0085] In a specific embodiment, the mobile phase is a buffer solution, in particular a phosphate buffer or an acetate buffer, having an ionic strength in the range of about and between 100 mM - 2 M, in particular about and between 200 mM - 1 .9 M, more in particular about and between 300 mM - 1 .8 M, more in particular about and between 400 mM - 1 .7 M, more in particular about and between 500 mM - 1 .6 M, more in particular about and between 600 mM - 1 .5 M, more in particular about and between 700 mM - 1 .4 M such as for example about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, about 500 mM, about 550 mM, about 600 mM , about 650 mM, about 700 mM, about 750 mM, about 800 mM, about 850 mM; about 900 mM, about 950 mM, about 1000 mM, about 1 .1 M, about 1 .2 M, about 1 .3 M.

[0086] The buffer may further contain one or more salt(s), in addition to any buffering salts. Ideally, a salt type and concentration will be used such that RNA-protein interactions are weakened while maintaining the target RNA in solution. In a specific embodiment, the mobile phase is a salt solution, in particular NaCI, having an ionic strength in the range of about and between 0 mM - 2 M, in particular about and between 100 mM - 1 .9 M, more in particular about and between 200 mM - 1 .8 M, more in particular about and between 300 mM - 1 .7 M, more in particular about and between 400 mM - 1 .6 M, more in particular about and between 500 mM - 1 .5 M, more in particular about and 600 mM - 1 .4 M such as for example about 0 mM, about 50 mM, 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, about 500 mM, about 550 mM, about 600 mM , about 650 mM, about 700 mM, about 650 mM, about 700 mM, about 750 mM, about 800 mM, about 850 mM; about 900 mM, about 950 mM, about 1000 mM, about 1 .1 M, about 1 .2 M, about 1 .3 M.

[0087] In yet a further embodiment, the present invention provides a method wherein the pH of the mobile phase is higher compared to the isoelectric point of said RNA sample, in particular wherein the pH of the mobile phase is at least about pH 4, such as at least pH 4.5, at least pH 5, at least pH 5.5, at least pH 6, at least pH 6.5, at least pH 7, at least pH 7.5, at least pH 8, at least pH 8.5 at least pH 9, at least pH 9.5 at least pH 10, at least pH 10.5, at least pH 1 1 , at least pH 1 1 .5 but in particular a pH in the range of about pH 4 to about pH 12, more in particular a pH in the range of about pH 4 to about pH 8, even more in particular a pH in the range of about pH 6 to about pH 8.

[0088] The inventors have found that following embodiments enable a fast, industrial-scale purification process for obtaining pure RNA with high yield, and is particularly advantageous for removing protein and other contaminants from target RNA species (desired RNA) e.g. in an IVT reaction sample.

[0089] In very specific embodiment of the method of the present invention, when one or more of the stationary phases is a stationary phase for cation exchange chromatography, said mobile phase is a salt solution with a concentration of about and between 0 M - 0.5 M having a pH in the range of about pH 4 to about pH 8 and is particularly suitable for binding protein impurities. The inventors have found that a pH and a buffer or salt concentration in these concentrations are particularly advantageous, resulting in RNA purification with a high RNA yield and efficient protein removal.

[0090] In still a further embodiment, a method is provided wherein said one or more stationary phases for a column chromatography method is selected from the list comprising: affinity chromatography, anion exchange chromatography, hydrophobic interaction chromatography; and wherein said mobile phase is a salt solution with a concentration of about and between 0.1 M - 2 M having a pH in the range of about 6-8. The inventors have found that a pH and a buffer or salt concentration in these concentrations are particularly advantageous, resulting in RNA purification with a high RNA yield and efficient impurity removal.

[0091] In very specific embodiment of the method of the present invention, when one or more of the stationary phases is a stationary phase for anion exchange chromatography, said mobile phase is a salt solution with a concentration of about and between 0 M - 0.250 M having a pH at or above the isoelectric point of said RNA sample. In another embodiment of the method of the present invention, when the stationary phase is a stationary phase for anion exchange chromatography, said mobile phase is a salt solution with a concentration of about and between 0.250 M - 2 M having a pH of about and between pH 6 to about pH 8. These methods are particularly suitable for binding impurities including but not limited to NTPs, proteins, short RNA fragments, dsRNA, DNA template, or buffer components.

[0092] In another embodiment of the method of the present invention, when one or more of the stationary phases is a stationary phase for affinity exchange chromatography, said mobile phase is a salt solution with a concentration of about and between 0.1 M - 1 .5 M having a pH of about and between pH 6 to about pH 8 and is particularly suitable for binding impurities including but not limited to NTPs, proteins, RNA fragments without poly A tails, DNA template, or buffer components.

[0093] In another embodiment of the method of the present invention, when one or more of the stationary phases is a stationary phase for hydrophobic interaction chromatography, said mobile phase is a salt solution with a concentration of about and between 0.5 M - 1 .5 M having a pH of about and between pH 6 to about pH 8 and is particularly suitable for binding including but not limited to abortive RNA fragments, proteins, NTPs, DNA templates, buffer components, or dsRNA.

[0094] In a further embodiment, the purification process may further comprise an additional step of tangential flow filtration. The combination of both techniques is particularly suitable to increase purify cation efficiency even further without unduly compromising potency or stability and to provide compositions in which RNA is substantially cleared of contaminants.

[0095] As used herein, the term “Tangential Flow Filtration (TFF)”, also known as “crossflow filtration” is meant to be a filtration process wherein the feed flow travels tangentially across the surface of the filter (permeate), rather than into the filter. Everything else is retained on the feed side of the membrane as retentate. The principal advantage of this is that the filter cake (which can blind the filter) is substantially washed away during the filtration process, increasing the length of time that a filter unit can be operational. It can be a continuous process, unlike batch-wise dead-end filtration. Thus, operating in a TFF mode prevents the formation of a restrictive layer (concentrated biomolecule layer on the membrane surface that can foul or plug the membrane) by re-circulating the mixture allowing molecules smaller than the membrane pores to move toward and through the membrane.

[0096] In another aspect, the present invention provides the use of a flowthrough chromatography column for RNA purification comprising one or more stationary phases and a mobile phase, wherein said stationary phase is configured to have binding affinity for impurities other than target RNA in the presence of a mobile phase which is free of organic solvents and comprises an RNA sample. In a particular embodiment, the stationary phase comprises two or more stationary phases, in a very specific embodiment said stationary phase comprises a stationary phase for cation exchange chromatography.

[0097] In a further aspect, the present invention provides a device for flowthrough purification of RNA comprising one or more chromatography columns comprising one or more stationary phases and a mobile phase, wherein said one or more stationary phases are configured to have binding affinity for impurities other than target RNA, and wherein said mobile phase is free of organic solvents.

[0098] RNA purified according to this invention is useful as a component in pharmaceutical compositions, for example for use as a vaccine in immunizing subjects against various diseases. These compositions will typically include RNA and a pharmaceutically acceptable carrier. A pharmaceutical composition can also include one or more additional components but can also include a delivery system for the RNA e.g. an LNP, liposome, an oil-in-water emulsion, or a microparticle.

[0099] In another aspect, the present invention provides a method for preparing a pharmaceutical composition comprising the steps of:

[0100] (a) purifying RNA according to any embodiment of the method of the invention; and

[0101] (b) formulating the purified RNA as a pharmaceutical composition.

[0102] EXAMPLES

[0103] Example 1 : Hydrophobic interaction chromatography

[0104] In a comparative conventional chromatography method using a loading, wash and elution step, an RNA sample was loaded at high salt concentration (e.g. 50 mM Tris-HCI, 2 M NaCI). Salt concentration of the mobile phase is than decreased in stepwise manner. Fractions are collected and analyzed FIG. 1 A. It was observed that RNA was eluted completely as represented by the UV260 and UV280 peaks. Based on the data described above, a 3-step method was developed consisting of a loading (2 M NaCI) step, a washing step (1 .2 M NaCI) and an elution step (500 mM NaCI) FIG. 1B. Again, the UV260 and UV280 show RNA was eluted successfully from the column.

[0105] In a further comparative experiment, the dynamic binding capacity of the column was determined by sequentially loading 2 mg fractions of RNA at loading conditions. During the third loading cycle (FT3), UV260 and UV280 are seen to increase. Clear breakthrough is observed during the fourth loading cycle (FT4). This indicates that the binding capacity of the column is less than 6 mg of RNA per 1 ml of column volume FIG. 1C.

[0106] In the method according to the invention, an RNA sample was loaded at elution conditions (500 mM NaCI), forcing the RNA to remain in the mobile phase and only impurities to bind to the stationary phase. In this method, 9 fractions of 2 mg of RNA were loaded sequentially on 1 ml of column volume. Flowthrough fractions were collected and analyzed. The column was then washed with a mobile phase containing 0 mM NaCI, eluting all bound material (impurities) from the column FIG. 2A.

[0107] RNA recovery in the flowthrough fractions was very high and only 789.6 pg of RNA was found to be actually bound to the column (Table 1 ).

[0108] Table 1 When the protein content of the RNA present in the flowthrough fractions was analyzed, a clear reduction in residual protein levels was observed FIG. 2B. When the levels of double stranded RNA (dsRNA) are compared between the loaded material and the material in the flowthrough fractions, a downward trend also appears to be present FIG. 2C. These data clearly indicate that the flowthrough method is an effective way of RNA purification.

[0109] Given the fact that out of 18 mg of RNA, only 0.790 mg bound to the column, and the previously determined dynamic binding capacity (DBC) of 6 mg / ml, it can be concluded that using this modified method, a maximum amount of RNA of up to 136 mg could be processed per 1 ml of stationary phase.

[0110] Based on the data above, an estimate was made of the volume of mobile phase that would be required to process 1 g of RNA using bind-elute and flowthrough-based chromatography methods. Assuming a DBC of 6mg / ml, processing 1 g of RNA using bind-elute chromatography would require 167 ml of stationary phase. A wash and elution step, each consisting of 5 column volumes, bring the required mobile phase volume to 1 .670 L.

[0111] In the flowthrough mode, the required stationary phase volume is reduced to 56 ml (using the demonstrated capacity of 18 mg / ml). As no thorough washing is required in this method, a wash step of 1 CV is foreseen to flush the liquid from the column. As no target RNA is bound to the column, no elution step is required. The required mobile phase volume for this method is estimated as 56 ml. When the theoretical maximum capacity of 136 mg RNA per 1 ml column volume is taken into account, the required stationary and mobile phase volumes are further reduced to 8 ml (Table 2).

[0112] Table 2 Example 2: multimodal chromatography

[0113] Another method for RNA purification is using multimodal chromatography, which combines characteristics of different chromatography modalities. One stationary phase that may be used to perform multimodal chromatography is CIMmultus PrimaS, which combines characteristics of anion exchange and H-bond chromatography. In this method, RNA is loaded at neutral pH (100 mM phosphate buffer pH 7) and eluted from the stationary phase at increased pH (e.g. 100 mM phosphate buffer pH 10).

[0114] Dynamic binding capacity (DBC) was assessed by sequentially loading fractions of 2.5 mg RNA until breakthrough was observed. As shown below, UV260 and UV280 were stable during the first two loading cycles. During the third and fourth loading cycles, however, significant levels of breakthrough were observed, indicating that binding capacity had been exceeded FIG. 3A. Based in these experiments, it was concluded that binding capacity was <7.5 mg RNA per 1 ml of stationary phase and that up to 5 mg RNA per 1 ml stationary phase could safely be loaded.

[0115] As previously described in Example 1 , the method according to the invention was adapted so that the RNA was loaded under elution conditions (pH 10). Under these conditions (flowthrough mode), no RNA was observed to bind to the column FIG. 3B. As previously observed for hydrophobic interaction chromatography in Example 1 , running multimodal chromatography in flowthrough-mode results in a clear reduction of residual protein content in the purified RNA compared to the levels observed in the material prior to purification FIG. 3C.

[0116] Based on the demonstrated maximum process amounts for bind-elute and flowthrough chromatography, an estimate was made of the stationary and mobile phase volumes required for the purification of 1 g of RNA. For bind-elute chromatography, wash and elution volumes of 5 column volumes were taken into account, bringing the required mobile phase volume to 2 L (for a stationary phase of 200 ml). In flowthrough-mode, wash volume was reduced from 5 to 1 CV because in this method, the loaded volume only needs to be flushed from the column. As no binding to the column is expected, no elution step is required. This brings the required stationary and mobile phase volume to only 40ml (Table 3).

[0117] Table 3

[0118] Anion exchange chromatography was performed on CIMmultus DEAE column. The column was sanitized using 1 M NaOH and equilibrated to 20 mM Tris-HCI, 1 .223 M KCI, 0.2 mM EDTA. RNA was LiCI precipitated and resuspended in equilibration buffer. When the RNA was loaded on the equilibrated column, a large peak in absorbance at 260 nm was observed, corresponding to the RNA present in the flowthrough fraction FIG. 4A.

[0119] The flowthrough fraction was collected and the RNA was buffer exchanged to water for injection using Amicon Ultra-15 centrifugal filters. When the residual protein content of the purified material was determined, a clear reduction in protein content was observed compared to the loaded LiCI-precipicated mRNA FIG. 4B.

[0120] Cation exchange chromatography was performed on CIMmultus SO3 column. The column was equilibrated under conditions that ensure that protein impurities but not the RNA bind to the stationary phase (in the experiment shown below 25 mM acetate buffer pH 4.0). 2 mg of RNA was LiCI precipitated and resuspended in loading buffer (25 mM acetate buffer pH 4.0) and this material was loaded onto the column. After flushing the column with equilibration buffer, a clear flowthrough peak was observed FIG. 5A.

[0121] When the residual protein level of the flowthrough fraction was compared to that of the loaded material, a clear reduction was observed FIG. 5B. This demonstrates that cation exchange chromatography can be performed in a way that is compatible with the principles of flowthrough chromatography.

[0122] A common way of purifying RNA using affinity chromatography is oligo dT purification. This technique utilizes short, 2’-deoxythymidine (dT) oligonucleotides, usually 18- or 25-mers, attached to a stationary phase using a linker. Purification is performed by balancing electrostatic repulsion of the negatively charged phosphate groups and base pairing between the oligo dT sequence and the poly(A) tail of the in vitro transcribed mRNA. By adding a moderate concentration of salt, the RNA relaxes and electrostatic repulsion between the phosphate groups of the oligo dT sequence and the mRNA is significantly reduced. This allows base pairing to occur between the thymidine groups fixed on the stationary phase and the adenosines in the poly(A) tail of the mRNA. By reducing salt concentration of the mobile phase, the RNA can be eluted from the column.

[0123] Whereas oligo dT purification relies on an interaction between an oligo-sequence on the stationary phase and a corresponding oligo sequence on the target RNA, the oligo on the stationary phase can be easily adapted to correspond to an oligo sequence present on either non-target RNA or DNA impurities. By immobilizing an oligo sequence complementary to the DNA backbone, for example, it would be trivial to use affinity purification to bind DNA impurities while the target RNA remains unbound.

[0124] A specific embodiment of flowthrough-based affinity purification according to the invention is the removal of plasmid DNA using oligo dA or oligo dU chromatography. Such an application is entirely analogous to oligo dT based capture of polyadenylated RNA, with the notable change that in this application, deoxyadenine (dA) oligonucleotides would be immobilized rather than deoxythymidine (dT) oligonucleotides. Under moderate salt concentration, these dA oligonucleotides interact with the polythymidine stretch on the 3’ end of the linearized plasmid DNA, encoding the mRNA’s polyA tail. The target RNA, which does not contain sequences complementary to the sequences immobilized on the stationary phase, allowing effective removal of plasmid DNA from the in vitro transcribed mRNA.

[0125] Another possible embodiment of flowthrough-based affinity purification of RNA is based on the immobilization of affinity ligands targeting specific product- or process related impurities. A specific subset of such purification methods is immunoaffinity chromatography. This is a well- established process in which antibodies or antibody-related fragments are immobilized on a stationary phase. When a mobile phase comprising the antigen impurity is then passed over or through the stationary phase, this impurity is selectively captured onto the stationary phase An evident target for immunoaffinity purification is double stranded RNA, the detection and quantification of which is routinely performed using immunochemical assays utilizing the J2 antibody which specifically binds dsRNA and has in the past been successfully immobilized on solid carriers. If a solution of in vitro transcribed RNA comprising dsRNA impurities where to be passed over or through such a carrier carrying immobilized J2 antibody, the dsRNA in the antibody would be effectively captured by the stationary phase, while the RNA proper would remain unbound in the mobile phase, resulting in a depletion of dsRNA levels of the final RNA product.

Claims

-24-CLAIMS1 . A method of RNA purification using flowthrough column chromatography comprising one or more stationary phases and a mobile phase, wherein said method comprises applying a mobile phase composition comprising an RNA sample to a stationary phase configured to have binding affinity for impurities other than target RNA, wherein the mobile phase composition is free of organic solvents, and wherein said one or more stationary phases comprises a stationary phase for a cation exchange chromatography.

2. The method according to any one of the preceding claims, wherein said impurities are selected from the list comprising: protein impurities, nucleotides, enzymes, plasmid DNA, host cell DNA, host cell RNA, double stranded RNA (dsRNA), incomplete RNA fragments, and / or RNA aggregates.

3. The method according to any one of the preceding claims, said method is a method of in vitro transcribed RNA purification.

4. The method according to any one of the preceding claims, wherein said mobile phase composition is a buffer and / or salt solution having an ionic strength in the range of about and between 100 mM - 2 M, in particular about and between 500 mM - 1 .5 M, even more in particular about 750 mM.

5. The method according to any one of the preceding claims, wherein the pH of the mobile phase composition is higher compared to the isoelectric point of said RNA sample, in particular wherein the pH of the mobile phase composition is at least about pH 4, more preferably in the range of about pH 4 to about pH 12.

6. The method according to any one of the preceding claims, wherein the mobile phase composition is a salt solution with a concentration of about and between 0 M - 2 M.

7. The method according to any one of the preceding claims, wherein said mobile phase composition is a salt solution with a concentration of about and between 0 M - 0.5 M having a pH in the range of about 4-8.

8. The method according to any one of the preceding claims, wherein said mobile phase composition is a salt solution with a concentration of about and between 0.1 M - 2 M having a pH in the range of about 6-8.

9. The method according to any one of the preceding claims, wherein said target RNA flows through the stationary phase of the column thereby being free from impurities that remain bound to the stationary phase.

10. The method according to any one of the preceding claims, wherein said target RNA is single-stranded RNA, more in particular single-stranded mRNA, even more in particularfull-length single-stranded mRNA.1 1 . Use of a flowthrough chromatography column for RNA purification comprising one or more stationary phases and a mobile phase, wherein said stationary phase is configured to have binding affinity for impurities other than target RNA in the presence of a mobile phase composition which is free of organic solvents and comprises an RNA sample, and wherein said stationary phase comprises a stationary phase for a cation exchange chromatography.

12. Use according to claim 1 1 , wherein the stationary phase comprises two or more stationary phases.

13. A device for flowthrough purification of RNA comprising one or more chromatography columns comprising one or more stationary phases and a mobile phase, wherein said one or more stationary phases are configured to have binding affinity for impurities other than target RNA, and wherein said mobile phase comprises the use of a mobile phase composition which is free of organic solvents, and wherein said stationary phase comprises a stationary phase for a cation exchange chromatography.

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