Method for separating nucleic acid molecules

Abstract

The present invention provides a method (100) for separating a single-stranded nucleic acid molecule from one or more impurities, the method comprising: adding (110) a feed comprising the single-stranded nucleic acid molecule and one or more impurities to a chromatography medium comprising a matrix material functionalised with a hydrophobic ligand comprising an amine-derived aromatic group, and eluting (120) the single-stranded nucleic acid molecule from the chromatography medium. Further provided is the use of a chromatography medium for purification of a single-stranded nucleic acid molecule, wherein the chromatography medium comprises a matrix material functionalised with a hydrophobic ligand comprising an amine- derived aromatic group, as well as the use of a chromatography device for the purification of a single-stranded nucleic acid molecule, wherein the chromatography device comprises a chromatography medium for purification of a single-stranded nucleic acid molecule, wherein the chromatography medium comprises a matrix material functionalised with a hydrophobic ligand comprising an amine-derived aromatic group.

Description

[0001] METHOD FOR SEPARATING NUCLEIC ACID MOLECULES

[0002] Technical Field

[0003] The present invention relates to purification of biomolecules. More specifically, the present invention provides a method for separating single-stranded nucleic acid molecules from impurities, as well as use of a chromatography medium or a chromatography device for purification of single-stranded nucleic acid molecules.

[0004] Background

[0005] The growing understanding of functions of single-stranded nucleic acids (ss NAs) and their crucial roles in diseases promotes the application of various ss NAs to selectively function on proteins, transcripts, and genes, which previously have not been considered as druggable targets. Several short interfering RNAs (siRNAs), aptamers (i.e., short single-stranded oligonucleotides) and mRNA vaccines have been approved for clinical application (Zhu et al).

[0006] Purification of oligonucleotides synthesized by solid-phase synthesis is mainly performed by liquid chromatography, commonly by Anion Exchange (AIEX) chromatography, where the full-length oligonucleotide product is separated from process-related impurities under denaturing conditions, such as high pH and / or high salt concentration. RNA oligonucleotides with 2’ -OH are more sensitive to high pH than DNA oligonucleotides and may therefore be more challenging to purify by AIEX. When employing AIEX chromatography the oligonucleotides are separated based on the negative charge of the phosphate group of each nucleotide in the oligonucleotide. An advantage with AIEX chromatography is that the oligonucleotide can be purified in fully deprotected form. Reverse phase (RP) chromatography columns can also be used to purify oligonucleotides synthesized by solid-phase synthesis. An advantage with this technology is that one can achieve very good resolution between full-length oligonucleotide product and shorter un-desired oligonucleotides. This is achieved by not fully deprotecting the oligonucleotide after synthesis, but instead keeping the hydrophobic protection group for the 5’-OH of the oligonucleotide. Non-full-length oligonucleotides that have been capped (typically, acetylated) during the solid-phase synthesis do not have any protection group in the 5’-end. In essence, the hydrophobicity of the protection group for the 5 ’-OH provides retention in the RP chromatography column, while non-full-length oligonucleotides that lack the hydrophobic 5’ protection group are eluted earlier from the RP column. The disadvantage with RP chromatography to purify oligonucleotides is that it requires buffers comprising organic solvents, which is non-desirable from a sustainability perspective. In addition, RP chromatography often requires higher pressure than AIEX chromatography, and this is associated with higher cost for columns and chromatography equipment. Hydrophobic interaction chromatography (HIC) has also been used to purify short oligonucleotides, as described in WO2017 / 218454.

[0007] The production of mRNA could be carried out by use of host cells but is most commonly produced enzymatically, in a cell free system using in vitro transcription (IVT), where an RNA polymerase catalyses the synthesis of the target mRNA from nucleotide triphosphates (NTPs) substrates using a DNA template. Apart from the RNA polymerase (generally T7, SP6 or T3 RNA polymerases), the linear DNA template and the NTPs, the IVT 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.

[0008] For the mRNA to be translated into a protein or a polypeptide and thereby provide a therapeutic effect, the mRNA must cross the cell membrane to the cytosol where it is decoded by the ribosome. One of the most common strategies for mRNA delivery is by conjugation with lipid-based carriers, polymers, or peptides, such as liposomes or lipid nanoparticles (LNPs). Other approaches include direct injection of naked mRNA or via transfection of dendritic cells. 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. Following the IVT reaction, the DNA template is generally digested by addition of a DNA nuclease. Capping of the 5 'end is essential for both stability of the mRNA, by providing protection against exonuclease degradation, as well as improving protein translation. This can be performed during the IVT step by use of specific nucleotide such as the cap dinucleotide CleanCap® provided by TriLink or by using a separate post-IVT two-step enzymatic procedure based on the vaccinia capping enzyme (VCC). 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 digestion by deoxyribonuclease (DNase), precipitation, and steps including tangential flow filtration (TFF) and / or different modes of chromatography, including affinity, ion-pair, anion exchange and reversed phase (RP) chromatography.

[0009] Like for other biological drugs, the manufacturing of mRNA-based products is complex and robust testing is required to ensure the quality and safety of the final product. The main concern during purification of mRNA is to achieve efficient removal of the components used during the IVT and the following steps, such as enzymes, residual NTPs, DNA template and aberrant forms of RNA formed during the IVT such as double stranded RNA (dsRNA).

[0010] One of the strategies most commonly used for purification of mRNA involves affinity chromatography by use of poly-deoxythymidine (dT) oligonucleotide ligands, commonly denoted oligo dT ligands, that display a specific affinity for poly-adenylated mRNA constructs, commonly mRNA including a poly-A tail. However, in some cases it is not possible to use oligo dT ligands, for example for circular mRNA constructs lacking a poly-A tail and for mRNA constructs having a poly-A tail which is ‘hidden’ within the structure of the mRNA and thus is difficult to access for an oligo dT ligand.

[0011] There is thus a continued need in the art for improved and / or alternative methods for separating single-stranded nucleic acid molecules, such as mRNA, self-amplifying RNA, cyclic RNA, or antisense oligonucleotides, from impurities.

[0012] Summary

[0013] The above-mentioned object is achieved by providing a method for purification of single-stranded nucleic acid molecules by use of hydrophobic interaction chromatography (HIC).

[0014] More particularly, the present invention provides a method for separating a single-stranded nucleic acid molecule from one or more impurities, the method comprising: adding a feed comprising the single-stranded nucleic acid molecule and one or more impurities to a chromatography medium comprising a matrix material functionalised with a hydrophobic ligand comprising an aminederived aromatic group, and eluting the single-stranded nucleic acid molecule from the chromatography medium.

[0015] The hydrophobic ligand is as defined in claim 1.

[0016] The present invention also provides use of a chromatography medium for purification of a single-stranded nucleic acid molecule, wherein the chromatography medium comprises a matrix material functionalised with a hydrophobic ligand comprising an amine-derived aromatic group.

[0017] Further provided is use of a chromatography device for the purification of a single-stranded nucleic acid molecule, wherein the chromatography device comprises the chromatography medium as described in detail elsewhere herein.

[0018] Further features and advantages of the invention will become apparent from the following description of preferred embodiments of the invention, given by way of example only, which is made with reference to the accompanying drawings.

[0019] Brief Description of the Drawings

[0020] These and other aspects of the present disclosure will now be described in more detail, with reference to the appended drawings showing embodiment(s) of the invention, in which:

[0021] Fig. 1 is a flow chart outlining the steps of a method for separating a singlestranded nucleic acid molecule from one or more impurities according to the present invention.

[0022] Figs. 2 A-E show chromatograms for separation of mRNA, as described in Example 1 herein.

[0023] Figs. 3 A-B show chromatograms for separation of a DNA oligonucleotide, as described in Example 2 herein.

[0024] Detailed Description

[0025] The present invention solves or at least mitigates the problems associated with existing methods for purifying single-stranded nucleic acids by providing, as schematically illustrated in Fig. 1, a method 100 for separating a single-stranded nucleic acid molecule from one or more impurities, the method comprising: adding 110 a feed comprising the single-stranded nucleic acid molecule and one or more impurities to a chromatography medium comprising a matrix material functionalised with a hydrophobic ligand comprising an aminederived aromatic group, and eluting 120 the single-stranded nucleic acid molecule from the chromatography medium.

[0026] The single-stranded nucleic acid molecule may or may not comprise, at its 5'- end, a residual protective group originating from chemical synthesis of the nucleic acid molecule.

[0027] As shown in the experimental section below, the herein described method provides efficient binding of mRNA to the ligand at relatively low amounts of kosmotropic salt and allows for mild elution conditions at low salt concentration (i.e., at low conductivity) and without use of any organic solvent, which are favorable conditions for the mRNA. The result is a very sharp elution peak and a surprisingly high recovery of mRNA.

[0028] As further shown in the experimental section, the herein described method achieves binding of a DNA oligonucleotide, provided with a residual protective group originating from chemical synthesis of the DNA oligonucleotide, to the HIC ligand and elution thereof at mild elution conditions.

[0029] HIC applications for most other molecules, such as proteins and plasmid DNA (pDNA), are commonly associated with fronting and / or tailing of the elution peaks, i.e., broad elution peaks, which means either ending up with either a low recovery of the target molecule or a large eluate volume having a low concentration of the target molecule. This is also the case when using a hydrophobic ligand having an aminederived aromatic group as disclosed herein for purification of plasmid DNA.

[0030] The presently disclosed method also provides advantages compared to other types of chromatography. As mentioned further above, AIEX chromatography often requires denaturing elution conditions, e.g., including high pH and / or high salt concentration, which may damage the nucleic acid molecules to be purified and / or only provides low recovery of the target molecule, e.g., in relation to mRNA as the length of the mRNA construct increases. RP chromatography also involves several disadvantages, as described further above. Further, oligo dT affinity chromatography cannot be used for purification of all types of mRNA.

[0031] Accordingly, the present invention provides an improved and / or alternative method for purification of single-stranded nucleic acid molecules, such as RNA, e.g., mRNA, or oligonucleotides, such as RNA oligonucleotides or DNA oligonucleotides, including modified RNA / DNA oligonucleotides.

[0032] The term “chromatography medium” is used herein to denote a type of separation matrix.

[0033] The term "separation matrix" is used herein to denote a material comprising a matrix material to which one or more ligands comprising functional groups have been coupled. The functional groups of the ligand(s) bind compounds herein also called analytes, which are to be separated from a liquid sample and / or which are to be separated from other compounds present in the liquid sample.

[0034] In this context, "ligand" is a molecule that has a known or unknown affinity for a given analyte and includes any functional group, or capturing agent, immobilized on its surface, whereas "analyte" includes any specific binding partner to the ligand.

[0035] The analytes of particular interest according to the present invention are singlestranded nucleic acid molecules. The term “single-stranded nucleic acid molecule” encompasses ribonucleic acid (RNA) molecules as well as deoxyribonucleic acid (DNA) molecules, of various lengths ranging from oligonucleotides to longer RNA / DNA molecules, as specified in detail elsewhere herein. The single-stranded nucleic acid molecule may comprise one or more synthetic nucleotides.

[0036] The term "nucleotide" or “nucleoside” as used herein refer to individual nucleotides / nucleosides or varieties, analogues, or modifications of nucleotides / nucleosides, such as nucleotides / nucleosides having an alternative linking group, an analogous form of purine, an analogous form of pyrimidine, e.g., pseudouridine, a sugar analogue, and / or various natural posttranscriptional modifications, such as TV6-methyladenosine (m6A), TV6, 2'-O-dimethyladenosine (m6Am), 5-methylcytosine (m5C), 5-hydroxymethylcytosine (hm5C), inosine (I), pseudouridine (T), A1-methyladenosine (m'A), 2'-O-methylation (Nm), N4- acetylcytidine (ac4C), A7-m ethylguanosine (m7G) and dihydrouridine (D), etc. Nucleotides may have a modification to the natural phosphate group, for example phosphorothi oate .

[0037] The single-stranded nucleic acid molecule may be an RNA molecule.

[0038] The RNA molecule may be an in vitro transcribed RNA molecule or produced in a host cell, e.g., a yeast cell, such as Saccharomyces cerevisiae.

[0039] The RNA molecule may be an mRNA molecule.

[0040] The mRNA molecule may be an in vitro transcribed mRNA molecule or produced in a host cell, e.g., a yeast cell, such as Saccharomyces cerevisiae.

[0041] The single-stranded nucleic acid molecule may be a DNA molecule.

[0042] The single-stranded nucleic acid molecule may be an oligonucleotide.

[0043] The oligonucleotide may for example be DNA, RNA, non-natural versions thereof such as 2’-modified RNA including 2’- O[(CH2)nO]mCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, OCH2D(=0)N(H)CH3, and O(CH2)nON[CH2)nCH3]2, (where n and m are independently from 1 to about 10), or bicyclic RNA analogues such as locked nucleic acid (LNA), peptide nucleic acid (PNA), phosphorodiamidate morpholino oligonucleotide (PMO), or conjugated oligonucleotides, such as oligonucleotide-peptide conjugates (OPCs), which are synthetic constructs that combine peptides and nucleic acids in a single chimeric molecule.

[0044] The oligonucleotide may be an RNA oligonucleotide, including but not limited to a modified RNA oligonucleotide. Non-limiting examples of modifications have been described above. The RNA oligonucleotide preferably has a hydrophobic group attached to its 3’ or 5’ end.

[0045] The oligonucleotide may be a DNA oligonucleotide, including but not limited to a modified DNA oligonucleotide. Non-limiting examples of modifications have been described above.

[0046] The oligonucleotide may or may not comprise, at its 5'-end, a residual protective group originating from chemical synthesis of the oligonucleotide. Such protective group may be hydrophobic.

[0047] The single-stranded nucleic acid molecule may comprise from about 10 nucleotides to about 20,000 nucleotides, such as from about 20 nucleotides to about 10,000 nucleotides. More particularly, the single-stranded nucleic acid molecule may comprise: (i) from about 10 nucleotides to about 200 nucleotides, such as 10-100 nucleotides, 20-200 nucleotides, 20-100 nucleotides, or 50-200 nucleotides; or

[0048] (ii) from about 300 nucleotides to about 20,000 nucleotides, such as from about 500 nucleotides to about 10,000 nucleotides.

[0049] Single-stranded nucleic acid molecules having a size as defined in (i) above may advantageously be produced by chemical synthesis, also known as oligosynthesis.

[0050] Single-stranded nucleic acid molecules having a size as defined in (ii) above may advantageously be produced by biological synthesis, such as IVT.

[0051] The term “feed” is intended to mean a mobile phase, comprising the target compounds, i.e., single-stranded nucleic acid molecules. The terms “feed” and “liquid sample” may be used interchangeably herein. The target compounds are retained in the chromatography material by the ligand in preference to other components also present in the mobile phase. Such other components are normally referred to as impurities.

[0052] In case the feed comprises an in vitro transcribed RNA molecule as target compound, the one or more impurities may be template DNA, fragmented DNA, nucleotides, RNA polymerase, RNase inhibitor, and / or DNase. Remaining reagents, including non-incorporated nucleotides and peptides resulting from digestion of enzymes with proteinase K, as well as defect or incomplete target nucleic acid molecules, are also defined as impurities.

[0053] The matrix material of the chromatography medium may comprise porous particles, non-porous particles, or expanded bed media, or may be a convective flow material.

[0054] Porous particles may alternatively be called beads, and a chromatography material comprising such particles or beads may be called a resin.

[0055] Porous particles may for example be made of agarose, such as cross-linked agarose. Non-limiting examples of commercially available chromatography resins based on porous agarose particles include Sepharose resins (Cytiva, Sweden), comprising substantially spherical particles.

[0056] Another non-limiting example of a commercially available resin based on porous agarose particles is the PlasmidSelect Xtra chromatography resin (Cytiva, Sweden). Non-limiting examples of non-porous particles are magnetic beads and polystyrene beads, respectively. A non-limiting example of magnetic particles is Mag Sepharose (Cytiva, Sweden).

[0057] A non-limiting example of expanded bed media is STREAMLINE resins (Cytiva, Sweden).

[0058] The matrix material of the chromatography medium used may be a material having a mean flow pore size of 0.1-2.0 pm, wherein the matrix material is functionalised with a hydrophobic ligand comprising an amine-derived aromatic group to a ligand concentration up to 1500 pmol / g of matrix material, such as a ligand concentration of 10-1500 pmol / g matrix material.

[0059] The matrix material may be a convective flow matrix material. Such a material may for example be an adsorptive membrane where a flow through such materials is convective rather than diffusional. A convective matrix material includes any matrix in which application of a hydraulic pressure difference between the inflow and outflow of the matrix forces perfusion of the matrix, achieving substantially convective transport of the substance(s) into the matrix or out of the matrix, which can be affected very rapidly at a high flow rate.

[0060] Examples of convective matrix materials include porous adsorptive membranes, fibrous materials, and monolithic materials. The adsorptive membrane can for example be a polymeric membrane, such as a poly ether sulfone membrane (e.g., Mustang membrane chromatography capsules (Cytiva, Sweden). Another example of an adsorptive membrane is a polymer nanofiber membrane, such as for example cellulose, cellulose acetate and cellulose fibres, which have been treated for use as an adsorbent. Treatment may include one or more of cross-linking, derivatization, and coupling of a ligand. The matrix material may be a non-woven material comprising fibres, such as from cellulose. Such fibrous substrate may be based on electrospun polymeric fibres or cellulose fibres, and may e.g. have a cross-sectional diameter of 10-1000 nm, such as 200-800 nm, 200-400 nm or 300-400 nm. Convection-based chromatography and membrane adsorbers are described in for example US20140296464A1, US20160288089A1, US2019308169A1 and US2019234914A1, hereby incorporated by reference in their entireties. A polymer used for a polymeric membrane or polymer nanofiber membrane may be a natural or synthetic polymer, including a derivatized polymer. For example, a hydroxylated polymer may be used, such as to provide a hydroxylated polymeric membrane. A hydroxylated polymer may include a glycopolymer. In addition to cellulose and polyether sulfone as mentioned above, polymers useful in adsorptive membranes include polymers based on polytetrafluoroethylene (PTFE), polypropylene, polyamide, or polyacrylamide.

[0061] The adsorptive membrane could alternatively be a monolithic material, or a conventional membrane made by emulsification. Another alternative is a 3D-printed material.

[0062] Mean flow pore (MFP) size is an indicator of material flow characteristics, and is measured by capillary flow porometry, based on the displacement of a wetting liquid with a known surface tension from the sample pores by applying a gas at increasing pressure. The higher the MFP size, the larger the flow of liquid through the material at a given pressure. The mean flow pore size is calculated from the point at which 50 % of the flow goes through a sample. Mean flow pore size thus corresponds to the pore size calculated at the pressure where the wet curve and the half-dry curve meet.

[0063] A non-limiting example of a monolith is CIMmultus® (Sartorius, Germany).

[0064] The hydrophobic ligand comprising an aromatic group is an amine-derived aromatic ligand. The amine-derived aromatic ligand may comprise an aromatic ring having at least one nitrogen atom as member of the ring.

[0065] The hydrophobic ligand comprising an amine-derived aromatic group is a ligand comprising a thioether moiety connected to an aromatic ring, the aromatic ring preferably containing at least one nitrogen atom as a member of the ring.

[0066] The hydrophobic ligand comprising an amine-derived aromatic group may comprise an aryl group selected from pyridyl, phenyl, benzyl, toluyl, phenethyl, naphtyl, imidazolyl, pyrazolyl, pyrazinyl, pyrimidinyl, pyridazinyl, piperidinyl, morpholinyl, piperazinyl, indolyl, quinolinyl, purinyl groups and any substituted such group. Such substituted group may for example be a substituted phenyl group having one or more electron withdrawing groups. One example is nitrophenyl. The electron withdrawing group may be -CF3, or may be a group comprising nitrogen, such as -NO2 or -CN.

[0067] The aryl group may be pyridyl. The hydrophobic ligand comprising an amine-derived aromatic group, or a portion of said ligand, is described by formula I: wherein, independently

[0068] M represents a matrix material,

[0069] Z represents a linker, optionally including a spacer moiety,

[0070] X for each occurrence is NH, O or S,

[0071] Y is S or CH2, or is absent,

[0072] A is C or N,

[0073] B is H, a C1-C6 alkyl group such as a C1-C3 alkyl group, or an electron withdrawing group, or is absent, n is 0 or an integer 1-12, and m is 0 or an integer 1-12.

[0074] Preferably, one of X and Y is S.

[0075] The electron withdrawing group may be -CF3, or may be a group comprising nitrogen, such as -NO2 or -CN.

[0076] Preferably, at least one occurrence of A is N.

[0077] In embodiments where A is N, B may be absent.

[0078] The hydrophobic ligand may comprise a sulfur-containing moiety.

[0079] The hydrophobic ligand comprises a thioether moiety connected to an aromatic ring.

[0080] The hydrophobic ligand may be selected from 2-mercaptopyridine, 4- mercaptopyridine, vinyl pyridine, 2-mercaptoethanol, and 2-mercapto-ethyl-pyridine.

[0081] A currently preferred, non-limiting example of a hydrophobic ligand comprises a 2-mercaptopyridine group (e.g., trade names Capto PlasmidSelect and PlasmidSelect Xtra; Cytiva, Sweden). Such a ligand is previously known for separating open circular (OC) plasmid DNA (pDNA) from supercoiled (SC) pDNA. In such a process, pDNA is separated from RNA by first eluting OC pDNA, secondly eluting SC pDNA and subsequently eluting impurities, including RNA and host cell protein.

[0082] RNA is more hydrophobic than plasmid DNA, and thus generally binds stronger to a HIC ligand. RNA requires a lower amount of kosmotropic salt than pDNA to bind to a HIC ligand. It is known in the art that pDNA and RNA have different binding properties to ligands, so it is not a given that a ligand for plasmid purification would work for purification of RNA or other single-stranded nucleic acid molecules.

[0083] For the purpose of purifying single-stranded nucleic acid molecules, when the 2 -mercaptopyridine ligand is coupled to matrix material in the form of agarose beads, the ligand density may be from about 1.0 mg / ml to about 8.0 mg / ml, such as from about 2.0 to about 6.0 mg / ml. As a non-limiting example, the Capto PlasmidSelect chromatography medium (Cytiva, Sweden) may have a ligand density of about 3.0-5.6 mg / ml. Another non-limiting example is the PlasmidSelect Xtra chromatography medium (Cytiva, Sweden) which may have a ligand density of about 3.5 mg 2- mercaptopyridine / ml medium.

[0084] Another example of a hydrophobic ligand comprises a 1 -butylmercaptan group (e.g., trade name butyl-S; Cytiva, Sweden).

[0085] The chromatography medium may comprise a compound which couples the ligand(s) to the matrix material. The terms “linker”, “extender”, and “surface extender” may be used to describe such a compound, as further described below.

[0086] The linker may comprise a moiety resulting from the reaction of coupling the ligand to the matrix material. Well-known coupling chemistries are known to persons of skill in the art and may involve reaction with for example divinyl sulfone (DVS) or using epichlorohydrin (ECH) or allyl glycidyl ether (AGE). Thus, the linker may contain and be attached to the matrix material through, a moiety obtained by reaction of the matrix material, optionally activated or derivatised, with divinyl sulfone, by reaction with epichlorohydrin, or by reaction with allyl glycidyl ether. For example, the linker may comprise a vinyl sulfone moiety.

[0087] The hydrophobic ligand comprising an amine-derived aromatic group may be coupled to the matrix material via an “extender group”, or simply “extender”. The extender may be selected from polysaccharide structures and polymeric structures. The extender may be e.g. dextran, acrylamides or polyglycerol. If dextran is used as an extender, it may have a molecular weight in a range of from 5,000 to 2,000,000 Dalton. The extender group builds off of the substrate through polymerisation (undetermined length).

[0088] Optionally, the linker Z may also comprise a spacer moiety as described below.

[0089] In some embodiments, no extender group is used, and the ligand is immobilized / connected to the matrix material directly using for example a linker. The linker can be formed by the reagent used to activate the matrix material and that allows the coupling of the ligand. Well-established methods include for example the introduction of epoxy groups via the use of epichlorohydrin or 1,4-butanediol diglycidyl ether. Another example is the introduction of double bounds on the matrix material with the use of reagents such divinyl sulfone. The linker composed by these reagents can be further extended by a new functionalisation that would allow the coupling of the ligand.

[0090] Spacers, such as 2-12 carbon alkyls, linear or branched, or 2-12 carbon ethers, can be used between the hydrophobic ligand and a linker, such as vinyl sulfone. The linker may also react with an extender group and link it to the ligand or spacer. The spacer is the length of carbons between the linker and the ligand used to improve the base stability of the linkerligand bond.

[0091] The ligand may be connected via an oxygen atom to the matrix material.

[0092] In the herein disclosed method, in the step of adding 110 the feed comprising the single-stranded nucleic acid molecule to the chromatography medium comprising a matrix material functionalised with a hydrophobic ligand comprising an amine-derived aromatic group, the single-stranded nucleic acid molecule may be bound to the chromatography medium by applying a binding buffer comprising a kosmotropic salt.

[0093] Salts in water solvent are defined as kosmotropic (order-making) if they contribute to the stability and structure of water-water interactions. In contrast, chaotropic (disorder-making) salts have the opposite effect, disrupting water structure, increasing the solubility of nonpolar solvent particles, and destabilizing solute aggregates. Kosmotropes cause water molecules to favorably interact, which in effect stabilizes intramolecular interactions in macromolecules such as proteins (Moelbert S et al). A scale can be established for example by referring to the Hofmeister series, or lyotropic series, which is a classification of ions in order of their ability to salt out or salt in proteins (Hyde A et al). More particularly, the kosmotropic salt may comprise (i) an anion selected from a group consisting of CCh2', SCU2', S2O32', H2PO4', HPCU2' , acetate', citrate', and Cl', and (ii) a cation selected from a group consisting of NH , K+, Na+, and Lit

[0094] According to a currently preferred, non-limiting embodiment, the kosmotropic salt is ammonium sulfate, (NH^SCL.

[0095] Another non-limiting example of a kosmotropic salt is potassium chloride, KC1.

[0096] The concentration of the kosmotropic salt in the binding buffer may be from about 0.25 M to about 2.5 M, such as from about 0.5 M to about 1.5 M. This concentration range is applicable to ammonium sulfate, for example. For other kosmotropic salts, a higher concentration than 2.5 M, such as 3 M or 3.5 M, may be needed to make the single-stranded nucleic acid molecule bind to the chromatography medium. Such a concentration higher than 2.5 M may be applicable to KC1, for example. Different single-stranded nucleic acid molecules require different salt concentrations to bind to the hydrophobic ligand. Generally, a higher salt concentration is needed to bind DNA molecules (e.g., above 1.5 M ammonium sulfate) while RNA molecules bind at a lower salt concentration (e.g., below 1.5 M ammonium sulfate). The salt concentration to be selected also depends on the length of the nucleic acid molecule. Short single-stranded nucleic acid molecules, like oligonucleotides, require a higher salt concentration than longer single-stranded nucleic acid molecules.

[0097] Washing may be performed after the binding (adsorption) of the target molecule to the ligand but before elution, as is well known in the art, in order to remove retained undesired material.

[0098] In the herein disclosed method, in the step of eluting 120, the single-stranded nucleic acid molecule may be eluted from the chromatography medium by applying an elution buffer or water.

[0099] The elution buffer may be any buffer having a low salt concentration, provided that the stability of the nucleic acid molecule is maintained in the buffer selected. It is to be understood that a person skilled in the art can select a suitable elution buffer and a suitable concentration of salt. A non-limiting example of a suitable elution buffer is tris(hydroxymethyl)amino-methane (i.e., Tris), such as Tris 10 mM pH 7.5. Another non-limiting example of a suitable elution buffer is ethylene-diamine-tetraacetic acid (EDTA). Yet another example is to use an elution liquid containing no salt at all, i.e., water. If water is used to elute the single-stranded nucleic acid molecule from the chromatography medium, purified water is to be used. For example, Water For Injection (WFI) and similar grades of water may be suitable, provided that there is no ribonuclease (RNase) activity in the water selected.

[0100] It is to be understood that after the step of eluting 120 the single-stranded nucleic acid molecule from the chromatography medium, the method may further comprise a step of collecting the thus formed eluate containing the single-stranded nucleic acid molecule.

[0101] The residence time of the target compound on the chromatography medium may be selected depending on type of matrix material as well as type of target compound. As a non-limiting example, for purification of longer RNA molecules (approx. 1000- 2000 nucleotides), the residence time on Capto PlasmidSelect resin may be from about 0.5 min to about 6 min, such as from about 1 min to about 5 min, such as from about 2 min to about 4 min, or about 0.5 min, 1 min, 2 min, 3 min, 4 min, 5 min, or 6 min. For purification of shorter RNA / DNA molecules, i.e., oligonucleotides, the residence time on Capto PlasmidSelect resin may be from about 2 min to about 8 min, such as from about 4 min to about 7 min, such as 4 min, 5 min, 6 min, or 7 min.

[0102] The herein disclosed method may comprise a step comprising obtaining 102 (see Fig. 1) a feed comprising a single-stranded nucleic acid molecule.

[0103] Alternatively, the method may comprise a production step comprising obtaining 102 a single-stranded nucleic acid molecule by a suitable method of production.

[0104] As a non-limiting example, an RNA molecule may be obtained by in vitro transcription.

[0105] The method may further comprise subjecting an in vitro transcribed RNA molecule to one or more pre-purification steps 104 (see Fig. 1), such as digestion of template DNA by adding of DNase, before adding 110 the feed to the chromatography medium comprising a hydrophobic ligand.

[0106] According to another non-limiting example, an oligonucleotide may be obtained by solid-phase synthesis, in which the oligonucleotide is synthesized on a solid support material, such as a resin bead, using repetitive steps for each chain extension cycle. The 3’ end of a first nucleotide is attached to the solid support with a protective group, e.g., dimethoxy trityl, on the hydroxyl group where the oligonucleotide is to be synthesized. A nucleotide being provided with a protective group may be called a 5’ protected nucleotide. Before addition of the next nucleotide, the first nucleotide is de-protected, e.g., by using di chloroacetic acid in dichloromethane, or toluene, after which the next nucleotide is coupled to the free end of the first nucleotide. Steps of capping, deprotecting, and coupling of further nucleotides, including oxidation or sulfurisation, are repeated until a desired oligonucleotide sequence has been assembled.

[0107] Before adding 110 an oligonucleotide to a chromatography medium, the oligonucleotide must be cleaved from the solid-phase support material. However, the nucleotide added last may be kept in 5’ protected form, i.e., the protective group may be kept. The 5’ protective group may be a 5 ’-dimethoxytrityl (DMT) group. Where the protective group is hydrophobic, it can be utilized in hydrophobic interaction chromatography (HIC) to aid separation of the target oligonucleotides from impurities (e.g., unsuccessfully synthesized / unfinished oligonucleotide variants, which do not contain hydrophobic protective groups).

[0108] In general, the herein disclosed method may comprise one or more prepurification steps 104 before adding 110 the feed to the chromatography medium comprising a hydrophobic ligand. Such one or more pre-purifi cation steps may be selected from digestion of DNA, size-based separation such as filtration, affinity chromatography such as oligo-dT affinity chromatography, anion exchange chromatography, cation exchange chromatography, multimodal chromatography, and reversed phase chromatography.

[0109] A non-limiting example of a suitable pre-purifi cation step 104 is tangential flow filtration (TFF).

[0110] In case the target molecule is an RNA / DNA oligonucleotide, for which organic solvent(s) has been used to synthesize the target molecule, the feed needs to be conditioned by removing the organic solvent before purification by use of the hydrophobic ligand. In such a case, a suitable pre-purifi cation step 104 (more particularly, a conditioning step) is desalting and / or TFF.

[0111] The herein disclosed method may comprise one or more steps 130, subsequent to, i.e., being performed after, the step of eluting 120 the single-stranded nucleic acid molecule from the chromatography medium comprising a hydrophobic ligand. Such one or more steps 130 may be one or more purification or conditions steps, such as desalting or tangential flow filtration.

[0112] The herein disclosed method may further comprise a step of preparing 140 (see Fig. 1) a lipid nanoparticle comprising the purified single-stranded nucleic acid molecule. Lipid nanoparticles is a common form of administering nucleic acid molecules, such as mRNA, to a patient. However, as known by persons skilled in the art, other options exist, such as administering naked mRNA to the patient, thus obviating the need for a step of preparing 140 a lipid nanoparticle.

[0113] The RNA purified according to the disclosed method may be intended for use as an mRNA vaccine or for other therapeutic purposes for humans or animals. However, it is also envisaged that the purified mRNA or single-stranded nucleic acid referred to herein may be intended for other purposes where the mRNA or single-stranded nucleic acid as such is not to be used as a therapeutic.

[0114] According to a second aspect, the present invention provides use of a chromatography medium for purification of a single-stranded nucleic acid molecule, wherein the chromatography medium comprises a matrix material functionalised with a hydrophobic ligand comprising an amine-derived aromatic group.

[0115] The definitions of terms, examples, and general description of single-stranded nucleic acid molecules, matrix materials and hydrophobic ligands comprising an aminederived aromatic group, as given elsewhere herein, are applicable also in relation to this aspect of the present invention.

[0116] According to a third aspect, the present invention provides use of a chromatography device for the purification of a single-stranded nucleic acid molecule, wherein the chromatography device comprises the chromatography medium as described in detail elsewhere herein. The definitions of terms, examples, and general description of single-stranded nucleic acid molecules, matrix materials and hydrophobic ligands comprising an amine-derived aromatic group, as provided elsewhere herein, are applicable also in relation to this aspect of the present invention.

[0117] The chromatography device may be a chromatography column. The chromatography column may comprise chromatography medium having a matrix material comprising porous particles, a monolith, expanded bed media, or non-porous particles, such as polystyrene beads. Alternatively, the chromatography device may be a membrane adsorber unit, such as a capsule or a cassette. The membrane adsorber unit may comprise chromatography medium having a matrix material comprising a convective flow material, such as a porous adsorptive membrane or a fibrous material.

[0118] Alternatively, the chromatography device may be a container comprising non porous particles, such as magnetic beads.

[0119] It is to be understood that the present invention is not restricted to the below- described exemplifying embodiments thereof and that several conceivable modifications of the present invention are possible within the scope of the following claims. In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim. Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated. The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements.

[0120] EXPERIMENTAL SECTION

[0121] Example 1: Binding of mRNA on 2-mercaptopyridine resins

[0122] Materials and methods

[0123] Experiments aimed at binding / capture of two different mRNA constructs of respectively 2000 nt and 1000 nt, have been performed with two resins containing the ligand 2-mercaptopyridine (PlasmidSelect Xtra and Capto PlasmidSelect chromatography media; Cytiva, Sweden).

[0124] Binding has been tested in presence of different concentrations of ammonium sulfate (AS) ranging from 0.25 M to 2.5 M AS to promote binding of mRNA. Unless otherwise stated, the column has been pre-equilibrated with the chosen concentration of AS in either 20 mM Tris-EDTA (TE; 20 mM Tris, 2 mM EDTA) pH 8.0, or in 10 mM Tris pH 7.5.

[0125] The mRNA used originates from diluted IVT reactions where essentially all components, including dNTPs, proteins, and DNA fragments, except for mRNA, have been removed from the reaction mixture.

[0126] Loading of mRNA has been performed following conditioning of the material with a stock buffer of AS in either 20 mM TE pH 8.0, or in 10 mM Tris pH 7.5, to the chosen concentration of AS. Each loading was followed by a wash step of at least two column volumes (CV) using the same AS concentration and buffer as used as during loading.

[0127] Different loads of mRNA have been tested, ranging from about 0.1 mg of mRNA per mL of resin up to about 3.8 mg of mRNA per mL of resin. Similarly, different residence times during loading have also been tested, ranging from 1 to 5 minutes.

[0128] All experiments have been performed with 5 mL HiTrap PlasmidSelect Xtra (PSX) columns or 1 mL HiTrap Capto PlasmidSelect (CPS) columns.

[0129] Elution has been performed using a gradient to either 20 mM TE pH 8.0, or 10 mM Tris pH 7.5, or as a step elution using the same buffers. The UV cell path length used has been 2 mm or 0.5 mm.

[0130] A common starting point following IVT reactions is to perform diafiltration (DF) by tangential flow filtration (TFF). This step serves to remove most of the components from the IVT reaction into the permeate, except for mRNA which is retained in the retentate, as well as conditioning the material into the desired buffer environment. For comparison, an experiment was also performed with a sample containing all IVT components remaining after a completed IVT reaction, except for the mRNA construct which had been removed. This sample was loaded onto the Capto PlasmidSelect (CPS) medium at the concentration of AS that is sufficient for efficient binding of the 1000 nt construct. This sample is herein referred to as a ’’mock” diafiltration permeate. In addition, the behaviour of linear DNA template was tested at two relevant concentrations of AS, one with template only, and one with a mix of template and the corresponding mRNA product (2000 nt) from a purified IVT reaction.

[0131] Results

[0132] Loading of purified mRNA (2000 nt) at 2,5 M AS onto PSX

[0133] In the presence of 2.5 M AS in 20 mM TE pH 8.0 at 2.5 minutes residence time (RT), a complete binding of the approx. 2000 nt mRNA construct was achieved (data not shown). Loading of purified mRNA (2000 nt) at 1.0 M AS onto PSX

[0134] Complete binding of an approx. 2000 nt mRNA construct was achieved in presence of 1.0 M AS in 20 mM TE pH 8.0 at 2.5 minutes RT (data not shown).

[0135] Loading of purified mRNA (2000 nt) at 0.5 M AS onto PSX

[0136] At an AS concentration of 0.5 M in 20 mM TE pH 8.0, complete binding of the approx. 2000 nt mRNA construct was not achieved (data not shown).

[0137] Loading of purified mRNA (2000 nt) at 0.25 M AS onto PSX

[0138] An AS concentration of 0.25 in 20 mM TE pH 8.0 was not sufficient to promote binding of an approx. 2000 nt mRNA construct (data not shown).

[0139] Loading of purified mRNA (1000 nt) at 0.67 M AS onto CPS

[0140] As can be seen in Fig. 2A, minimal losses were detected in flow-through (FT) during binding of an approx. 1000 nt mRNA construct in the presence of 0.67 M AS in 10 mM Tris pH 7.5 with a load of approximately 3.0 mg mRNA per mL resin at 4 minutes RT. In Fig. 2A, the solid line represents UV 1 260 and the dotted line represents conductivity.

[0141] Loading of purified mRNA (1000 nt) at 0.67 M AS onto CPS

[0142] At a load of approximately 3.8 mg mRNA per mL resin, binding of an approx. 1000 nt mRNA construct, in the presence of 0.67 M AS in 10 mM Tris pH 7.5, was not complete due to significant occurrence of mRNA in the FT. Based on the results, shown in Fig. 2B , this indicated a dynamic binding capacity (DBC) of around 3 mg / mL at 0.67 M AS and 4 minutes RT.

[0143] Loading of mock IVT DF permeate fraction onto CPS at 0.67 M AS

[0144] The behaviour of the components removed from the IVT reaction, dNTPs, proteins, and DNA fragments, conditioned to 0.67 M AS in 10 mM Tris pH 7.5 and loaded onto CPS is shown in Fig. 2C. As can be seen, only a small fraction of these components bound to the resin while the bulk passed in the FT. In Fig. 2C, the solid line represents UV 1 260 and the dotted line represents conductivity. Loading of linear pDNA template onto PSX at 1.0 M AS

[0145] The behaviour of the template DNA that was used for the 2000 nt construct at 1.0 M AS in 20 mM TE pH 8.0 is shown in Fig. 2D. As can be seen, only a small fraction of the template material bound to the resin while the bulk passed in the FT. In Fig. 2D, the solid line represents UV 1 260 and the dotted line represents conductivity. The peak between 40 ml and 50 ml is an elution of some of the bound template material caused by an unintentional drop in conductivity, due to experimental method design issues in the software.

[0146] Loading of mix of linear pDNA template and mRNA (2000 nt) at 0.5 M AS The behaviour of a mix of the template DNA and the 2000 nt mRNA construct conditioned to 0.5 M AS in 20 mM TE pH 8.0 is shown in Fig. 2E. As can be seen, the template did not bind and passed in the FT while the mRNA was efficiently captured and eluted on the resin. In Fig. 2E, the solid line represents UV 1 260 and the dotted line represents conductivity. The peak between 40 ml and 50 ml is an elution caused by an unintentional drop in conductivity, due to experimental method design issues in the software.

[0147] Conclusion

[0148] As can be seen from the Figures 2A-E, elution of the mRNA is performed with a decreasing conductivity, i.e. low salt favours the elution This also means that the mRNA is successfully purified from not only DNA fragments and dNTPs, but also other components from the IVT reaction such as salts. Also Fig. 2E show salts occurring with the template DNA and FT, but not with the RNA elution peak. Thus, the method according to the present disclosure has been shown to successfully purify mRNA from essentially all other components from the IVT reaction. Example 2: Oligonucleotide purification

[0149] Materials and methods

[0150] Oligonucleotide purification was performed on AKTA Pure 25 (Cytiva) with a 0.5 mm UV-cell, fraction collector and samples were loaded using a Superloop.

[0151] A crude, single-stranded DNA 84-mer oligonucleotide with a 5 ’-dimethoxytrityl group (DMTr-ON) kept after synthesis was buffer exchanged to 50 mM Tris, pH 7.5 using a HiScreen desalting column (Cytiva) and then conditioned to 1.75 M (NEL^SC using a 3 M stock solution.

[0152] HiScreen Capto PlasmidSelect column (Cytiva) was equilibrated in binding buffer (1.75 M (NEL^SC , 50 mM Tris, pH 7.5) at 200 cm / h. The following steps were performed at 100 cm / h. Buffer exchanged and conditioned crude oligonucleotide was loaded using column load of 10 mg / mL resin. The column was washed with 14 column volumes (CV) binding buffer and full-length DMTr-ON oligonucleotide was eluted using a 0-100% gradient over 20 CV to 50 mM Tris pH 7.5. The residence time during elution was 6 minutes.

[0153] For analysis, 0.5 ml of the samples were buffer exchanged to 10 mM NaOH, pH 12 on a HiTrap desalting column. Full length oligonucleotide purity and concentration was analysed on a PA200 RS AIEX column, 4 pm, 50 x 4.6 mm (Thermo Fisher) on UltiMate 3000 High Performance Liquid Chromatography (HPLC) system with MWD, autosampler. The column temperature 60 °C and UV detection at 260 nm was used. The AIEX column is equilibrated in 10 mM Tris, 10 mM NaCKL at 1.0 mL / min, 1.5 pg sample was injected, then a 10-55% gradient to buffer B (10 mM Tris, 300 mM NaCICL) in 20 min for elution. The percentage of FLP (purity) was defined as peak area FLP / total UV trace area. A standard curve to calculate the amount in the injected sample was generated by injecting 0.25-1.75 pg of purified FLP. The obtained peak areas were plotted against the injected amounts to generate a linear regression to fit a standard curve. Results

[0154] The crude load had a full-length product (FLP) purity of 36%. In Fig. 3A, the chromatogram from Capto PlasmidSelect chromatography medium shows three UV- peaks. The first two peaks, FT (flow-through) and peak 2, contain minimal amounts of FLP according to HPLC analysis, as shown in Fig. 3B (where fractions 1-6 are shown as separate bars). The beginning of peak three, divided into fractions 1-5, and the shoulder after the main peak, fraction 6, contains more impurities. Fraction 2-5 contains the highest fraction purities. Fractions with above 70% purity were pooled and analysed again, and the received purity in the pool was 81% with 69% recovery. The highest purity in a collected fraction was 87%.

[0155] Example 3: Purification of mRNA on butyl-S resin

[0156] Materials and methods

[0157] Preparation of sample:

[0158] IVT uncapped mRNA (-2000 nt) material (DNase treated) in the concentration of 1.1 mg / mL was purified on a HiPrep 16 / 10 Sephadex 6FF by injecting 2 mL of IVT material onto the column at 5 mL / min flow of 50 mM TRIS pH 7.5 (RNase-free). The concentration of the eluate was determined by use of a UV-Vis-spectrophotometer (Nanodrop).

[0159] Resin material:

[0160] The experiment was performed on the following resin: Butyl-S (ligand having a 1- butylmercaptan group; Cytiva, Sweden).

[0161] Purification method:

[0162] 0.1 mg of mRNA was pre-conditioned to a final salt concentration of IM of (NH^SCL in 50 mM TRIS pH 7.5. The sample was injected via an injection loop onto a 1 mL Butyl-S FF HiTrap column with a residence time of 1 min. The column was equilibrated with 0.75M (NH^SCL in 50 mM TRIS pH 7.5 and elution was performed in stepgradient from 0.75M-0M of (NH^SCLin 50 mM TRIS pH 7.5 at a flow of 1 mL / min. Eluate fractions and flow-through fractions were collected and analysed by Nanodrop to determine concentration and by Fragment analyzer to check integrity. Results

[0163] The elution recovery for this experiment was 10% (purified mRNA, at 0.75M (NH4)2SO4). 35% of the mRNA could be detected in the flow-through (data not shown).

[0164] Example 4: Separation of mRNA under variable conditions

[0165] Experimental designs for separation of mRNA from impurities are performed with equipment and samples as in Example 1 above, with the following variations:

[0166] In terms of target compound: a) mRNA of various sizes, such as 1000 nt, 1500 nt, 2000 nt, 3000 nt, 4000 nt, 5000 nt, 6000 nt, 7500 nt, and 10000 nt; b) mRNA, which is uridine-depleted and / or including non-natural nucleosides; c) circular mRNA.

[0167] In terms of buffers and binding / elution conditions: a) Different concentrations of ammonium sulfate (AS), from about 0.2 M to about 1.4 M, such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4 M; b) Different AS elution gradient, or combination of a step elution and linear elution gradient; c) pH range 5.5-8.0 using any of the so-called Good’s buffers (including MES, ADA, PIPES, ACES, MOPSO, cholamine chloride, MOPS, BES, TES, HEPES, DIPSO, TAPSO, acetamidoglycine, POPSO, HEPPSO, HEPPS, tricine, Tris, glycinamide, glycylglycine, bicine, and TAPS); d) Acetate, citrate, or succinate at 10, 20, 50 or 100 mM; e) Phosphate pH 6.5-7.5 at 10, 20 , 50 , or 100 mM; f) KCl at l.0-2M; g) Addition of non-ionic kosmotropes, such as trehalose and glucose, to the binding buffer and / or to the elution buffer.

[0168] Example 5: Separation of oligonucleotides under variable conditions

[0169] Experimental designs for separation of oligonucleotides from impurities are performed with equipment as in Example 2 above, with the following variations: In terms of target compound: a) An RNA oligonucleotide instead of a DNA oligonucleotide; b) A mixture of RNA and DNA oligonucleotides; c) Different lengths of DNA / RNA oligonucleotide, from about 5 nt to about 200 nt; d) Oligonucleotides containing different hydrophobic groups on 3’ or 5’ end.

[0170] In terms of buffers and elution conditions: a) Different concentrations of ammonium sulfate (AS), 0.5-3 M; b) pH range 6.5-12 using any buffer concentration below 75 mM; c) Different salt, for example sodium sulfate, or citrate; d) Different residence time (lower than tested in Example 2); e) Shorter elution gradient or isocratic elution.

[0171] The above embodiments are to be understood as illustrative examples of the invention. Further embodiments of the invention are envisaged. It is to be understood that any feature described in relation to any one embodiment may be used alone, or in combination with other features described, and may also be used in combination with one or more features of any other of the embodiments, or any combination of any other of the embodiments. Furthermore, equivalents and modifications not described above may also be employed without departing from the scope of the invention, which is defined in the accompanying claims.

[0172] REFERENCES

[0173] Y Zhu et al, Cell Death and Disease, (2022) 13:644

[0174] WO20 17 / 218454

[0175] US20140296464A1

[0176] US20160288089A1 US2019308169A1

[0177] US2019234914A1

[0178] Moelbert Susanne et al, Kosmotropes and chaotropes: modelling preferential exclusion, binding and aggregate stability, Biophysical Chemistry, 2004 Dec, 112(1): 45-57 Hyde Adam M et al, General Principles and Strategies for Salting-Out Informed by the Hofmeister Series, Organic Process Research & Development, 2017, 21 (9): 1355— 1370.

Claims

CLAIMS1. A method (100) for separating a single-stranded nucleic acid molecule from one or more impurities, the method comprising: adding (110) a feed comprising the single-stranded nucleic acid molecule and one or more impurities to a chromatography medium comprising a matrix material functionalised with a hydrophobic ligand comprising an amine-derived aromatic group, and eluting (120) the single-stranded nucleic acid molecule from the chromatography medium, wherein the hydrophobic ligand or a portion thereof is described by formula I:wherein, independentlyM represents a matrix material,Z represents a linker, optionally including a spacer moiety,X for each occurrence is NH, O or S,Y is S or CH2, or is absent,A is C or N,B is H, a C1-C6 alkyl group such as a C1-C3 alkyl group, or an electron-withdrawing group, or is absent, n is 0 or an integer 1-12, and m is 0 or an integer 1-12, preferably wherein one of X and Y is S, and wherein the hydrophobic ligand comprises a thioether moiety connected to an aromatic ring2. The method according to claim 1, wherein the amine-derived aromatic group comprises an aryl group selected from pyridyl, phenyl, benzyl, toluyl, phenethyl, naphtyl, imidazolyl, pyrazolyl, pyrazinyl, pyrimidinyl, pyridazinyl, piperidinyl,morpholinyl, piperazinyl, indolyl, quinolinyl, purinyl groups and any substituted such group, optionally wherein the aryl group is pyridyl.

3. The method according to claim 1, wherein the hydrophobic ligand comprises a sulfur-containing moiety.

4. The method according to any one of claims 1-3, wherein the aromatic ligand is selected from 2-mercaptopyridine, 4-mercaptopyridine, vinyl pyridine, 2- mercaptoethanol and 2-mercapto-ethyl-pyridine, optionally wherein the ligand is 2- mercaptopyridine.

5. The method according to any one of claims 1-4, wherein the single-stranded nucleic acid molecule is bound to the chromatography medium by applying a binding buffer comprising a kosmotropic salt, optionally wherein the concentration of the kosmotropic salt is from about 0.25 M to about 2.5 M, such as from about 0.5 M to about 1.5 M, optionally wherein the kosmotropic salt is ammonium sulfate.

6. The method according to any one of claims 1-5, wherein the single-stranded nucleic acid molecule is eluted from the chromatography medium by applying an elution buffer or water.

7. The method according to claim 6, wherein the elution buffer has a lower concentration of a kosmotropic salt than the binding buffer.

8. The method according to any one of claims 1-7, wherein the single-stranded nucleic acid molecule is an RNA molecule.

9. The method according to any one of claims 1-8, wherein the single-stranded nucleic acid molecule is an mRNA molecule, optionally wherein the mRNA molecule is in vitro transcribed mRNA.

10. The method according to claim 8 or 9, wherein the feed comprises an in vitro transcribed RNA molecule and one or more impurities, optionally wherein at least one impurity is selected from template DNA, fragmented DNA, nucleotides, RNA polymerase, RNase inhibitor, and DNase.

11. The method according to any one of claims 8-10, further comprising a production step comprising obtaining (102) the RNA molecule by in vitro transcription, and optionally subjecting the in vitro transcribed RNA molecule to one or more prepurification steps (104), such as digestion of DNA by adding of DNase, before adding (110) the feed to the chromatography medium comprising a hydrophobic ligand.

12. The method according to any one of claims 1-8, wherein the single-stranded nucleic acid molecule at its 5 ’-end comprises a residual protective group originating from chemical synthesis of the single-stranded nucleic acid molecule.

13. The method according to any one of claims 1-12, further comprising one or more pre-purification steps (104) before adding (110) the feed to the chromatography medium comprising a hydrophobic ligand, optionally wherein the one or more prepurification steps are selected from digestion of DNA, size-based separation, affinity chromatography, anion exchange chromatography, cation exchange chromatography, multimodal chromatography, and reversed phase chromatography.

14. The method according to any one of claims 1-13, further comprising one or more purification steps (130) after eluting (120) the single-stranded nucleic acid molecule from the chromatography medium comprising a hydrophobic ligand, optionally wherein said one or more subsequent purification steps are selected from desalting and tangential flow filtration.

15. The method according to any one of claims 1-14, further comprising a step of preparing (140) a lipid nanoparticle comprising the purified single-stranded nucleic acid molecule.

16. The method according to any one of claims 1-15, wherein the single-stranded nucleic acid molecule comprises from about 10 nucleotides to about 20,000 nucleotides, such as from about 30 nucleotides to about 10,000 nucleotides, optionally wherein the single-stranded nucleic acid molecule comprises (i) from about 10 nucleotides to about 200 nucleotides, or (ii) from about 300 nucleotides to about 20,000 nucleotides, such as from about 500 nucleotides to about 10,000 nucleotides.

17. The method according to any one of claims 1-16, wherein the matrix material comprises porous particles, non-porous particles, or expanded bed media, or is a convective flow material, such as a porous adsorptive membrane, a fibrous material, or a monolithic material.

18. Use of a chromatography medium for purification of a single-stranded nucleic acid molecule, wherein the chromatography medium comprises a matrix material functionalised with a hydrophobic ligand comprising an amine-derived aromatic group, optionally wherein the single-stranded nucleic acid molecule at its 5 ’-end comprises a residual protective group originating from chemical synthesis of the single-stranded nucleic acid molecule, and wherein the hydrophobic ligand or a portion thereof is described by formula I:wherein, independentlyM represents a matrix material,Z represents a linker, optionally including a spacer moiety,X for each occurrence is NH, O or S,Y is S or CH2, or is absent,A is C or N,B is H, a C1-C6 alkyl group such as a C1-C3 alkyl group, or an electron-withdrawing group, or is absent,n is 0 or an integer 1-12, and m is 0 or an integer 1-12, preferably wherein one of X and Y is S, and wherein the hydrophobic ligand comprises a thioether moiety connected to an aromatic ring.

19. The use according to claim 18, wherein the amine-derived aromatic group comprises an aryl group selected from pyridyl, phenyl, benzyl, toluyl, phenethyl, naphtyl, imidazolyl, pyrazolyl, pyrazinyl, pyrimidinyl, pyridazinyl, piperidinyl, morpholinyl, piperazinyl, indolyl, quinolinyl, purinyl groups and any substituted such group, optionally wherein the aryl group is pyridyl.

20. The use according to claim 18, wherein the hydrophobic ligand comprises a sulfur-containing moiety.

21. The use according to any one of claims 18-20, wherein the aromatic ligand is selected from 2-mercaptopyridine, 4-mercaptopyridine, vinyl pyridine, 2- mercaptoethanol and 2-mercapto-ethyl-pyridine, optionally wherein the ligand is 2- mercaptopyridine.

22. The use according to any one of claims 18-21, wherein the matrix material comprises porous particles, non-porous particles, or expanded bed media, or is a convective flow material, such as a porous adsorptive membrane or a monolithic material.

23. The use according to any one of claims 18-22, wherein the single-stranded nucleic acid molecule is an mRNA molecule, and / or wherein the single-stranded nucleic acid molecule is an in vitro transcribed RNA molecule.

24. The use according to any one of claims 18-23, wherein the single-stranded nucleic acid molecule comprises from about 10 nucleotides to about 20,000 nucleotides, such as from about 30 nucleotides to about 10,000 nucleotides, optionally wherein the single-stranded nucleic acid molecule comprises:(i) from about 10 nucleotides to about 200 nucleotides, or(ii) from about 300 nucleotides to about 20,000 nucleotides, such as from about 500 nucleotides to about 10,000 nucleotides.

25. Use of a chromatography device for the purification of a single-stranded nucleic acid molecule, wherein the chromatography device comprises the chromatography medium as described in any one of claims 18-22, optionally wherein the chromatography device is selected from:(i) a chromatography column, optionally comprising chromatography medium having a matrix material comprising porous particles, a monolith, expanded bed media, or non-porous particles, such as polystyrene beads;(ii) a membrane adsorber unit, optionally comprising chromatography medium having a matrix material comprising a convective flow material, such as a porous adsorptive membrane or a fibrous material; and(iii) a container comprising non-porous particles, such as magnetic beads.

Images