WO2024231285A1

WO2024231285A1 - Method of analysing contaminants in rna products by ion-pair chromatography

Info

Publication number: WO2024231285A1
Application number: PCT/EP2024/062285
Authority: WO
Inventors: Svenja GROBE; Simon KLENK; Anja BESCHNITT; Nicole M. HARTUNG
Original assignee: Biontech SE
Current assignee: Biontech SE
Priority date: 2023-05-05
Filing date: 2024-05-03
Publication date: 2024-11-14
Anticipated expiration: 2025-11-05

Abstract

A method of analysis of substances which are contaminants in a sample comprising target RNA, typically prepared by in vitro transcription, the method comprising the steps of: a) preparing a sample comprising the target RNA; b) separating the target RNA from the substances by liquid chromatography, wherein the liquid chromatography is ion-pair reversed-phase chromatography, wherein the ion pair comprises a primary (C3-8)alkylamine or a salt thereof; and c) analysis of the substances by one or both of: i) ultraviolet-visible spectroscopy and ii) mass spectroscopy is provided.

Description

METHOD OF ANALYSING CONTAMINANTS IN RNA PRODUCTS BY ION-PAIR CHROMATOGRAPHY

Field of the Invention

The present invention relates to the analysis (quantification and /or characterization) of substances which form contaminants in a sample of RNA, particularly although not exclusively a sample of RNA produced by in vitro transcription. It also relates to the analysis (quantification and /or characterization) of these substances in the absence of the target RNA. This method can be used for the quality control of RNA, in particular RNA produced by in vitro transcription.

Background to the Invention

As is well known to the person skilled in the art, transcription is the process of copying a segment of DNA into RNA. The segments of DNA transcribed into RNA molecules that can encode proteins are said to produce messenger RNA (mRNA). A number of mRNA therapeutics and vaccines are under development and have been approved for marketing.

However, the production of RNA by in vitro transcription is not a clean process, and typically results in by-products, which means the target RNA contains a number of contaminants. Examples of such contaminants include short-chain oligonucleotides (such as up to 120 nucleotides), nucleotide contaminants, and RNA cap analogs. A sample of RNA may also include proteins. The formation route of these contaminants as well as their biological effects are unclear. Different formation mechanisms, e.g., T7 polymerase drop off or self-priming reactions, have been discussed in the literature.

A number of methods have been developed in the art in order to analyse the contaminants in a sample of RNA. In particular, WO2017/140345 describes a method for detecting by-products of in vitro transcription in a sample comprising an in vitro transcribed target RNA, the method comprising the steps of a) preparing a sample comprising a target RNA by in vitro transcription; b) purifying the target RNA, thereby providing a purified target RNA sample; and c) detecting the by-products in the purified target RNA sample by HPLC.

In particular, WO2017/140345 describes a method wherein the HPLC method is ionpair reversed phase chromatography. In this method, the ion-pairing reagent is triethylammonium acetate.

WO 2019/036683 Al relates to a method of separating a nucleic acid from a mixture comprising one or more additional nucleic acids or impurities. This document discloses an IP -RP -HPLC method using both a size selective ion pairing agent and a composition selective ion pairing agent. WO 2019/036683 Al does not disclose a method wherein the mobile phase used in the IP -RP -HPLC comprises between 5 and 350 mM 1,1,1,3,3,3-hexafluoroisopropanol.

US 2022/325309 Al relates to a method for producing a single-stranded RNA, said method including purifying the reaction product by reverse-phase column chromatography using a mobile phase comprising at least one ammonium salt(s). US 2022/325309 Al does not disclose the analysis of a sample comprising target RNA prepared by in vitro transcription and does not employ a IP-RP-HPLC method wherein the mobile phase used in the IP-RP-HPLC comprises between 5 and 350 mM 1,1,1 ,3 ,3 ,3 -hexafluoroisopropanol .

Donegan Michael et al. (Effect of ion-pairing reagent hydrophobicity on liquid chromatography and mass spectrometry analysis of oligonucleotides, Journal of Chromatography A, vol. 1666, 2022, 462860) relates to a study looking into the effect of ion-pairing reagent hydrophobicity on liquid chromatography and mass spectrometry analysis of oligonucleotides. This document discloses IP-RP-LC for the analysis of oligonucleotides having a length between 15 and 60 nucleotides. This document does not disclose the analysis of a sample comprising target RNA prepared by in vitro transcription.

WO 2023/055879 Al relates to a method of separating molecular species of a guanine-rich oligonucleotide from a mixture of molecular species. The method is disclosed as being used for the separation of siRNAs and involves IP-RP-HPLC in the presence of hexyl ammonium acetate and detection by UV at 260 nm. WO 2023/055879 Al does not disclose the analysis of a sample comprising target RNA prepared by in vitro transcription.

Finally, Marreel Kris et al. (Diving into the Structural Details of In Vitro Transcribed mRNA Using Liquid Chromatography-Mass Spectrometry Based Oligonucleotide Profiling, LCGC Europe, 2022, pp 220-236) relates to the use of LCMS oligonucleotide profiling for elucidating structural details of in vitro transcribed mRNA. This document does not disclose a IP-RP-HPLC method wherein the mobile phase used in the IP-RP-HPLC comprises between 5 and 350 mM 1, 1,1, 3,3,3- hexafluoroisopropanol.

Summary of the Invention

According to a first aspect of the invention, there is provided a method of analysis of substances which are contaminants in a sample comprising target RNA prepared by in vitro transcription, the method comprising the steps of: a) preparing a sample comprising the target RNA; b) separating the target RNA from the substances by liquid chromatography, wherein the liquid chromatography is ion-pair reversed-phase chromatography, wherein the ion pair comprises a primary (C3-8)alkylamine or a salt thereof and the mobile phase used in the ion-pair reversed-phase chromatography comprises between 5 and 350 mM 1,1,1,3,3,3-hexafluoroisopropanol; and c) analysis of the substances by one or both of: i) ultraviolet-visible spectroscopy and ii) mass spectroscopy.

According to a second aspect of the invention, there is provided a method of analysis of substances which are contaminants in a sample comprising target RNA, the method comprising the steps of: a) preparing a sample comprising the target RNA; b) separating the target RNA from the substances by liquid chromatography, wherein the liquid chromatography is ion-pair reversed-phase chromatography, wherein the ion pair comprises a primary (C3-8)alkylamine or a salt thereof; and c) analysis of the substances by one or both of: i) ultraviolet-visible spectroscopy and ii) mass spectroscopy.

According to a third aspect of the invention, there is provided a method of analysis of substances in a sample, the substances being selected from the group consisting of oligonucleotides, nucleotides, nucleosides and cap analogs, the method comprising the steps of: a) preparing a sample comprising the substances; b) separating the substances by liquid chromatography, wherein the liquid chromatography is ion-pair reversed-phase chromatography, wherein the ion pair comprises a primary (C3-8)alkylamine or a salt thereof; and c) analysis of the substances by one or both of: i) ultraviolet-visible spectroscopy and ii) mass spectroscopy.

In one embodiment, the analysis by ultraviolet-visible spectroscopy comprises quantification of the substances. The quantification may be relative quantification or absolute quantification, as defined below.

In one embodiment, the analysis by ultraviolet-visible spectroscopy comprises characterization of the substances.

In one embodiment, the analysis by mass spectroscopy comprises characterization of the substances.

Advantages and Surprising Findings

It has been unexpectedly found by the present inventors that carrying out the ion-pair reversed phase chromatography using a primary (C3-8)alkylamine or a salt thereof as ion pairing reagent results in an improved separation of the substances than was possible using ion-pairing reagents used in the art, in particular triethylammonium salts as used in WO2017/140345. This is because the more hydrophobic nature of the unbranched mid chain alkylamines supports a more length-based separation. Enhanced analyte retention and better resolution of longer oligonucleotides are the result.

In addition, while the chromatographic method as used in WO2017/140345 comprises stepped gradient elution, the stepped gradient used in the method of the invention, when used together with the primary (C3-8)alkylamine or salt thereof as ion pair, enables a chromatographic separation over a wide length range starting from nucleotides over oligonucleotides (e.g., 5 to 120 nucleotides) to the full length RNA (e.g. > 2000 nucleotides) in a reasonable analysis time.

In addition, the combination of the primary (C3-8) alkylamine and 1, 1,1, 3,3,3- hexafluoroisopropanol (HFIP) as the ion pairing reagents instead of the respective salt (e.g. acetate) improves the chromatographic separation.

Furthermore, when mass spectrometry is used for characterization (either in the first second or third aspects of the invention), the primary (C3-8) alkylamine/HFIP combination is more suitable because of its reduced ion suppression and thus, enhanced sensitivity in mass spectrometric detection.

Brief Description of the Figures

Figure 1 shows the UV absorbance spectrum of standards and in vitro transcribed (IVT) RNA with the specified measure at 260 nm of the components when separated according to the ion pair reversed phase chromatography - coupled to UV absorbance spectroscopy (IPRP-UV) method of the invention - (A) being the chromatogram of the complete analytical run and (B) zoomed at 2 - 32 min;

Figure 2 shows the UV absorbance spectrum at 260 nm of a collected fraction obtained with the specified IPRP-UV method of the invention; (A) showing the chromatogram of the complete analytical run and (B) zoomed at 3 - 31 min;

Figure 3 (A) shows chromatograms resulting from analysis of a collected fraction separated with the specified IPRP -UV-MS method; 3(A) showing the chromatograms of the complete analytical run as total ion chromatogram (TIC) and extracted ion chromatogram (EIC) of the m/z corresponding to the capped 5’ end after enzymatic cleavage in the digested contaminants fraction and digested RNA as well as the MS spectra over the detected peak; and

Figure 3(B) shows the chromatograms of the complete analytical run as TIC and EIC of the expected 3’ end after enzymatic cleavage in the digested contaminants fraction and digested RNA as well as the MS spectra over the detected peak.

Definitions

In the following, definitions will be provided which apply to all aspects of the present disclosure. The following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps. The term "consisting essentially of means excluding other members, integers or steps of any essential significance. The term "comprising" encompasses the term "consisting essentially of' which, in turn, encompasses the term "consisting of. Thus, at each occurrence in the present application, the term "comprising" may be replaced with the term "consisting essentially of or "consisting of. Likewise, at each occurrence in the present application, the term "consisting essentially of may be replaced with the term "consisting of .

The terms "a", "an" and "the" and similar references used in the context of describing the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context.

Where used herein, "and/or" is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "X and/or Y" is to be taken as specific disclosure of each of (i) X, (ii) Y, and (iii) X and Y, just as if each is set out individually herein. The term "alkyl" refers to a monoradical of a saturated straight or branched hydrocarbon. Preferably, the alkyl group comprises from 1 to 40, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, carbon atoms, such as 1 to 30, such as 1 to 20 carbon atoms, such as 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, such as 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. The term “Cl-n alkyl” denotes an alkyl group containing 1 to n carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, iso-propyl (also called 2-propyl or 1 -methylethyl), butyl, iso-butyl, tert-butyl, n-pentyl, iso-pentyl, sec-pentyl, neo-pentyl, 1,2- dimethylpropyl, iso-amyl, n-hexyl, iso-hexyl, and sec-hexyl.

The term "nucleoside", as used herein, refers to a nucleobase, which may be adenine ("A"), guanine ("G"), cytosine ("C"), uracil ("U"), thymine ("T") linked to a carbohydrate, for example D-ribose (in RNA - the unit being termed a “ribonucleoside”) or 2'-deoxy -D-ribose (in DNA - the unit being termed a “deoxyribonucleoside”), through a glycosidic bond between the anomeric carbon of the carbohydrate (F -carbon atom of the carbohydrate) and the nucleobase. When the nucleobase is purine, e.g., A or G, the ribose sugar is generally attached to the Imposition of the heterocyclic ring of the purine. When the nucleobase is pyrimidine, e.g., C, T or U, the sugar is generally attached to the N1 -position of the heterocyclic ring. The nucleobase may be a modified nucleobase, such as N⁶-methyladenosine, 5- methyl-cytosine, 5-methyl-uridine (m5U), pseudouridine (y) or N(l)-m ethylpseudouridine (mly).

The carbohydrate portion of the nucleoside may be substituted or unsubstituted. Substituted ribose sugars include, but are not limited to, those in which one or more of the carbon atoms, for example the 2'-carbon atom, is substituted with one or more of the same or different Cl, F, R, OR, NR2 or halogen groups, where each R is independently H, Ci-Ce alkyl or C5-C14 aryl. Ribose examples include ribose, 2'- deoxyribose, 2',3'-dideoxy-ribose, 2'-haloribose, 2'-fluororibose, 2'-chlororibose, and 2'-alkylribose, e.g., 2'-O-methyl, 4'-alpha-anomeric nucleotides, F -alpha-anomeric nucleotides (Asseline et al, Nucl. Acids Res., 1991, 19, 4067-74) 2'-O-[2-(N- methylcarbamoyl)ethyl]ribose (Yamada et al., J. Org. Chem. 2011, 76, 3042-53). The term “nucleoside analogue”, as used herein, is intended to encompass compounds in which the carbohydrate portion of the nucleoside is replaced with a non-natural group. In one embodiment, the 2’-0 and 4’-C or the 3’0- and 4’C positions of the ribose group are linked by a covalent bond or linker (typically a methylene or ethylene group) - such groups are termed "locked nucleic acids" or "LNA" The structures of LNAs can be found, for example, in Wengel, et al., Chemical Communications (1998) 455; Koshkin et al., Tetrahedron (1998) 54:3607; Jesper Wengel, Accounts of Chem. Research (1999) 32:301; Obika, et al., Tetrahedron Letters (1997) 38:8735; Obika, et al., Tetrahedron Letters (1998) 39:5401; and Obika, et al., Bioorganic Medicinal Chemistry (2008) 16:9230, and in WO 98/22489; WO 98/39352 and WO 99/14226).

In other embodiments, the carbohydrate moiety of the nucleotide is replaced with an N-(2-aminoethyl) glycine unit - such groups are termed “peptide nucleic acids” or “PNA”. PNAs can be produced synthetically using any technique known in the art. See, e.g., U.S. Pat. Nos.: 6,969,766; 7,211,668; 7,022,851; 7,125,994; 7,145,006; and 7,179,896. See also U.S. Pat. Nos.: 5,539,082; 5,714,331; and 5,719,262 for the preparation of PNAs. Further teaching of PNA compounds can be found in Nielsen et al., Science, 254: 1497-1500, 1991.

In other embodiments, the C2'-C3' bond of the carbohydrate moiety has been cleaved - such groups are termed “unlocked nucleic acid” or “UNA” moieties. UNAs are disclosed, for example, in WO 2016/070166.

In other embodiments, the carbohydrate moiety of the nucleotide is replaced with a morpholino group, the nucleobase being present at the 3 -position of the morpholino group and the 6-position of the adjacent morpholino group linked (via a -CH2-O- linkage) to the phosphorus of the intersubunit linkage, which is in turn linked to the nitrogen of the adjacent morpholino group. Typically, in such compounds, the negatively charged oxygen of the phosphate intersubunit linkage is replaced by an amide or substituted amide group - such compounds having both the morpholino backbone and phosphorodiamidate inter-subunit linkage are termed “phosphorodiamidate morpholino” (or simply “morpholino” groups). Their general structure is as described in Figure 2 of Summerton, J., et al., Antisense & Nucleic Acid Drug Development, 7: 187-195 (1997) and their synthesis, structures, and binding characteristics of morpholino oligomers are detailed in U.S. Patent Nos.: 5,698,685; 5,217,866; 5,142,047; 5,034,506; 5,166,315; 5,521,063; 5,506,337; 8,076,476; and 8,299,206.

The term "nucleotide" as used herein means a nucleoside (or nucleoside analogue) in a phosphorylated form (a phosphate ester of a nucleoside or nucleoside analogue), as a monomer unit or within a polynucleotide polymer. The phosphate group may be present at any oxygen on the sugar portion of the nucleotide. Typically, the phosphate group is present on the 3’-position or the 5’-position, preferably the 5’-position. The phosphate group may comprise any number of phosphate units, typically 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate units. Preferably, the phosphate group is a monophosphate (1 phosphate unit), diphosphate (2 phosphate units) or triphosphate (3 phosphate units). Sulfur may substitute for oxygen in any or all of the phosphate groups to form a thiophosphate group. "Nucleotide 5'-triphosphate" refers to a nucleotide with a triphosphate ester group at the 5' position, sometimes denoted as "NTP", or "dNTP" and "ddNTP" to particularly point out the structural features of the ribose sugar. The triphosphate ester group may include sulfur substitutions for the various oxygen moieties, e.g., alpha-thio-nucleotide 5'- triphosphates. Nucleotides can exist in the mono-, di-, or tri-phosphorylated forms. The carbon atoms of the ribose present in nucleotides are designated with a prime character (') to distinguish them from the backbone numbering in the bases. For a review of polynucleotide and nucleic acid chemistry see Shabarova, Z. and Bogdanov, A. Advanced Organic Chemistry of Nucleic Acids, VCH, New York, 1994.

RNA

The nucleic acid which undergoes the method of the present invention to quantify (and optionally identify) the contaminants present therein is RNA. According to the present disclosure, the term "RNA" means a nucleic acid molecule which includes ribonucleotide residues. RNA typically comprises the naturally occurring nucleic acids adenosine (A), uridine (U), cytidine (C) and guanosine (G). In preferred embodiments, the RNA contains all or a majority of ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2'-position of a P-D-ribofuranosyl group. RNA encompasses without limitation, double stranded RNA, 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 may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered/modified nucleotides (or modified nucleosides) can be referred to as analogs of naturally occurring nucleotides (nucleosides), and the corresponding RNAs containing such altered/modified nucleotides or nucleosides (i.e., altered/modified RNAs) can be referred to as analogs of naturally occurring RNAs. A molecule contains "a majority of ribonucleotide residues" if the content of ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof). "RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), trans-amplifying RNA (taRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA) and immunostimulatory RNA (isRNA). In some embodiments, "RNA" refers to mRNA. The active ingredient may be mRNA, saRNA, taRNA, or mixtures thereof. The active ingredient is preferably mRNA. In some instances, the active ingredient is not siRNA.

In a preferred embodiment, the RNA comprises an open reading frame (ORF) encoding a peptide, polypeptide or protein. Said RNA may be capable of or configured to express the encoded peptide, polypeptide, or protein. For example, said RNA may be RNA encoding and capable of or configured for expressing a pharmaceutically active peptide or protein. In some embodiments, RNA is able to interact with the cellular translation machinery allowing translation of the peptide or protein. A cell may produce the encoded peptide or protein intracellularly (e.g. in the cytoplasm), may secrete the encoded peptide or protein, or may produce it on the surface. Alternatively, the RNA can be non-coding RNA such as antisense-RNA, micro RNA (miRNA) or siRNA. mRNA

In preferred embodiments of all aspects of the disclosure, the nucleic acid is mRNA. According to the present disclosure, the term "mRNA" means "messenger-RNA" and includes a "transcript" which may be generated by using a DNA template. Generally, mRNA encodes a peptide, polypeptide or protein. As established in the art, the RNA (such as mRNA) generally contains a 5' untranslated region (5'-UTR), a peptide/polypeptide/protein coding region and a 3' untranslated region (3'-UTR). mRNA is single-stranded but may contain self-complementary sequences that allow parts of the mRNA to fold and pair with itself to form double helices.

According to the present disclosure, "dsRNA" means double-stranded RNA and is RNA with two partially or completely complementary strands.

In preferred embodiments of the present disclosure, the mRNA relates to an RNA transcript which encodes a peptide, polypeptide or protein.

In some embodiments, the RNA which preferably encodes a peptide, polypeptide or protein has a length of at least at least 45 nucleotides, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides, up to 10,000, such up to 11,000, up to 12,000, up to 13,000 nucleotides, up to 14,000 nucleotides or up to 15,000 nucleotides. In some embodiments, the RNA (such as mRNA) is produced by in vitro transcription or chemical synthesis. Preferably, the RNA (such as mRNA) is produced by in vitro transcription using a DNA template. The term "in vitro transcription" or "IVT" as used herein means that the transcription (/.< ., the generation of RNA) is conducted in a cell-free manner. I.e., IVT does not use living/cultured cells but rather the transcription machinery extracted from cells (e.g, cell lysates or the isolated components thereof, including an RNA polymerase (preferably T7, T3 or SP6 polymerase)). The in vitro transcription methodology is known to the skilled person; cf., e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989. Furthermore, a variety of in vitro transcription kits is commercially available, e.g., from Thermo Fisher Scientific (such as TranscriptAid™ T7 kit, MEGAscript® T7 kit, MAXIscript®), New England BioLabs Inc. (such as HiScribe™ T7 kit, HiScribe™ T7 ARCA mRNA kit), Promega (such as RiboMAX™, HeLaScribe®, Riboprobe® systems), Jena Bioscience (such as SP6 or T7 transcription kits), and Epicentre (such as AmpliScribe™).

For providing modified RNA (such as mRNA), correspondingly modified nucleotides, such as modified naturally occurring nucleotides, non-naturally occurring nucleotides and/or modified non-naturally occurring nucleotides, can be incorporated during synthesis (preferably in vitro transcription), or modifications can be effected in and/or added to the mRNA after transcription. The RNA (such as mRNA) may be modified. The RNA (such as mRNA) may comprise modified nucleotides or nucleosides, such as N⁶-methyladenosine, 5-methyl-cytosine, 5-methyl-uridine (m5U), pseudouridine (y) or N(l)-methyl-pseudouridine (mly). One or more uridine in the RNA described herein may be replaced by a modified nucleoside. The modified nucleoside may be a modified uridine. The RNA may comprise a modified nucleoside in place of at least one uridine. Preferably, the RNA may comprise a modified nucleoside in place of each uridine (e.g., all of the uridines in the RNA are replaced with a modified nucleoside). The modified nucleoside may be independently selected from N⁶-methyladenosine, pseudouridine (y), Nl-methyl-pseudouridine (mly), and 5-methyl-uridine (m5U). The modified nucleoside is preferably pseudouridine (y) or Nl-methyl-pseudouridine (ml\|/). In some embodiments, RNA (such as mRNA) is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. Particular examples of RNA polymerases are the T7, T3, and SP6 RNA polymerases. Preferably, the in vitro transcription is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA.

In some embodiments of the present disclosure, the RNA (such as mRNA) is "replicon RNA" (such as "replicon mRNA") or simply a "replicon", in particular "self-replicating RNA" (such as "self-replicating mRNA") or "self-amplifying RNA" (or "self-amplifying mRNA"). The lipid particles containing RNA as described herein may contain mRNA, saRNA, taRNA, or mixtures thereof. The lipid particles containing RNA as described herein may contain an mRNA encoding a replicase protein, and one or more RNA molecules capable of being replicated or amplified by the replicase.

Inhibitory RNA

In some embodiments of all aspects of the disclosure, the nucleic acid is an inhibitory RNA.

The term "inhibitory RNA" as used herein means RNA which selectively hybridizes to and/or is specific for a target mRNA, thereby inhibiting (e.g., reducing) transcription and/or translation thereof. Inhibitory RNA includes RNA molecules having sequences in the antisense orientation relative to the target mRNA. Suitable inhibitory oligonucleotides typically vary in length from five to several hundred nucleotides, more typically about 20 to 70 nucleotides in length or shorter, even more typically about 10 to 30 nucleotides in length. Examples of inhibitory RNA include antisense RNA, ribozyme, iRNA, siRNA and miRNA. In some embodiments of all aspects of the disclosure, the inhibitory RNA is siRNA. The term "antisense RNA" as used herein refers to an RNA which hybridizes under physiological conditions to DNA comprising a particular gene or to mRNA of said gene, thereby inhibiting transcription of said gene and/or translation of said mRNA. The size of the antisense RNA may vary from 15 nucleotides to 15,000, preferably 20 to 12,000, in particular 100 to 10,000, 150 to 8,000, 200 to 7,000, 250 to 6,000, 300 to 5,000 nucleotides, such as 15 to 2,000, 20 to 1,000, 25 to 800, 30 to 600, 35 to 500, 40 to 400, 45 to 300, 50 to 250, 55 to 200, 60 to 150, or 65 to 100 nucleotides.

By "small interfering RNA" or "siRNA" as used herein is meant an RNA molecule, preferably greater than 10 nucleotides in length, more preferably greater than 15 nucleotides in length, and most preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length that is capable of binding specifically to a portion of a target mRNA. This binding induces a process, in which said portion of the target mRNA is cut or degraded and thereby the gene expression of said target mRNA inhibited. A range of 19 to 25 nucleotides is the most preferred size for siRNAs. Typically siRNAs comprise a single molecule in which two complementary portions are base-paired and are covalently linked by a single-stranded "hairpin" area. Without wishing to be bound by any theory, it is believed that the hairpin area of the siRNA molecule is cleaved intracellularly by the "Dicer" protein (or its equivalent) to form an siRNA of two individual base-paired RNA molecules.

As used herein, "downregulation target mRNA" refers to an RNA molecule that is a target for downregulation. In some embodiments, the downregulation target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide as specified herein. In some embodiments, the pharmaceutically active peptide or polypeptide is one whose expression (in particular increased expression, e.g., compared to the expression in a healthy subject) is associated with a disease. In some embodiments, the downregulation target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide whose expression (in particular increased expression, e.g., compared to the expression in a healthy subject) is associated with cancer.

According to the present disclosure, siRNA can be targeted to any stretch of approximately 19 to 25 contiguous nucleotides in any of the target mRNA sequences (the "target sequence"). Techniques for selecting target sequences for siRNA are given, for example, in Tuschl T. et al., "The siRNA User Guide", revised Oct. 11, 2002, the entire disclosure of which is herein incorporated by reference. Further guidance with respect to the selection of target sequences and/or the design of siRNA can be found on the webpages of Protocol Online (www.protocol-online.com) using the keyword "siRNA". Thus, in some embodiments, the sense strand of the siRNA used in the present disclosure comprises a nucleotide sequence substantially identical to any contiguous stretch of about 19 to about 25 nucleotides in the target mRNA. siRNA can be obtained using a number of techniques known to those of skill in the art. For example, siRNA can be chemically synthesized or recombinantly produced. Preferably, siRNA is transcribed from recombinant circular or linear DNA plasmids using any suitable promoter. Selection of other suitable promoters is within the skill in the art. Selection of plasmids suitable for transcribing siRNA, methods for inserting nucleic acid sequences for expressing the siRNA into the plasmid, and IVT methods of in vitro transcription of said siRNA are within the skill in the art.

The term "miRNA" (microRNA) as used herein relates to non-coding RNAs which have a length of 21 to 25 (such as 21 to 23, preferably 22) nucleotides and which induce degradation and/or prevent translation of target mRNAs. miRNAs are typically found in plants, animals and some viruses, wherein they are encoded by eukaryotic nuclear DNA in plants and animals and by viral DNA (in viruses whose genome is based on DNA), respectively. miRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNAs), usually resulting in translational repression or target degradation and gene silencing. miRNA can be obtained using a number of techniques known to those of skill in the art. For example, miRNA can be chemically synthesized or recombinantly produced using methods known in the art (e.g., by using commercially available kits such as the miRNA cDNA Synthesis Kit sold by Applied Biological Materials Inc.). Preferably, miRNA is transcribed from recombinant circular or linear DNA plasmids using any suitable promoter. In this specification the term “target RNA” means the RNA which is to be separated from the contaminants (as defined below). Typically, the target RNA is produced by an in vitro transcription process. The length and the sequence of the target RNA is determined by the sequence of the nucleic acid template which is subjected to the RNA in vitro transcription reaction. Hence, the target RNA is the full-length RNA transcript. In contrast, the contaminants typically are either longer or shorter than the target RNA.

The target RNA may further comprise a cap structure on its 5' terminus. In one embodiment, the cap analog is added to the RNA in vitro transcription reaction. In another embodiment, the cap analog is added via an enzymatic reaction. The target RNA may also comprise modified nucleotides, if these modified nucleotides had been added to the RNA in vitro transcription reaction mixture. In contrast, RNA containing modified nucleotides which had not been added to the RNA in vitro transcription reaction mixture is considered as a by-product. If the target RNA is mRNA, it will preferably code for proteins, in particular those which have an antigen character, and for example all recombinantly produced or naturally occurring proteins, which are known to a person skilled in the art from the prior art and are used for therapeutic, diagnostic or research purposes. In particular, the antigens may be tumour antigens or antigens of pathogens, for example of viral, bacterial or protozoal organisms.

Capped RNA

In one embodiment, the RNA is a capped RNA. The capped RNA may be any capped RNA, either natural or synthetic. Typically, the RNA comprises nucleotides in which a ribose sugar has a base attached to the T position, and a phosphate group which may be attached at the 5 ’-position or the 3 ’-position. The base may be adenine (A), cytosine (C), guanine (G) or uracil (U). Typically, the RNA is capped mRNA.

The cap may have any structure, natural or synthetic, which is capable of performing the function of binding to the cap-binding complex and EIF4E enabling the RNA to undergo translation during protein synthesis and/or protecting the RNA from degradation via 5'-3' exonucleases. In one embodiment, the cap has a structure of formula (I):

PM is a mono-or polyphosphate moiety containing 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphate units, the oxo (=0) groups in any of the phosphate units being optionally replaced with a thio (=S) group;

R is an end-cap moiety;

B is a nucleobase, optionally alkylated on a nitrogen atom by a Ci-4 alkyl group;

R’ is selected from OH, O(Ci-4 alkyl), and halogen; and the squiggly line represents the rest of the RNA molecule.

In formula (I), R may represent any group capable of allowing the cap to perform the above-mentioned function of binding to the cap-binding complex and EIF4E enabling the RNA to undergo translation during protein synthesis and/or protecting the RNA from degradation via 5'-3' exonucleases.

In one embodiment, the cap has a structure of formula (la):

Nuc is a nucleoside or nucleoside analogue;

In formulae (I) and (la), PM is preferably a monophosphate (1 phosphate unit), diphosphate (2 phosphate units), triphosphate (3 phosphate units) or tetraphosphate (4 phosphate units). Any one or all of the oxo (=0) groups in the phosphate units (preferably only 1 oxo group in 1 phosphate unit) may be replaced with a thio (=S) group.

In one embodiment of formula (la), Nuc is a nucleoside, which may be a ribonucleoside or deoxyribonucleoside (as defined above).

In another embodiment of formula (la), Nuc is a nucleoside analogue, as defined above. The nucleoside analogue may comprise a locked nucleic acid (LNA) moiety, a peptide nucleic acid (PNA) moiety, an unlocked nucleic acid (UNA) moiety or a morpholino moiety, as defined above.

In one embodiment of either formula (I) or (la), R’ is OH or OCH3.

In one embodiment, the cap has a structure of formula (lb):

or a salt thereof, wherein:

Ri is selected from OH, O(Ci-4 alkyl), and halogen;

R2 is selected from H, OH, and O(Ci-4 alkyl), and halogen;

R3 is selected from OH, O(Ci-4 alkyl), and halogen;

R4 is H, OH, O(Ci-4 alkyl), halogen, or a nucleobase, optionally alkylated on a nitrogen atom by a Ci-4 alkyl group; n is 1, 2 or 3;

Xi, each X2, and X3, are each independently O or S; and the squiggly line represents the rest of the RNA molecule.

In one embodiment of formula (lb), R4 is OH. In one embodiment of formula (lb), R4 is a nucleobase, optionally alkylated on a nitrogen atom by a Ci-4 alkyl group.

In one embodiment, the cap has a structure of formula (Ic):

or a salt thereof, wherein:

B and B’ are each independently nucleobases, each optionally alkylated on a nitrogen atom by a Ci-4 alkyl group;

Ri is selected from OH, O(Ci-4 alkyl), and halogen;

R2 is selected from H, OH, and O(Ci-4 alkyl), and halogen;

R3 is selected from OH, O(Ci-4 alkyl), and halogen; n is 1, 2 or 3;

In one embodiment of formula (Ic), B is selected from adenine ("A"), guanine ("G"), cytosine ("C"), or uracil ("U"), each optionally alkylated on a nitrogen atom by a Ci-4 alkyl group, such as by a methyl group. In one embodiment of formula (Ic), B is G, optionally methylated on the nitrogen at the 7’ -position.

In one embodiment of either formula (lb) or (Ic), B’ is selected from adenine ("A"), guanine ("G"), cytosine ("C"), or uracil ("U"), each optionally alkylated on a nitrogen atom by a Ci-4 alkyl group, such as by a methyl group. In one embodiment of either formula (lb) or (Ic), B’ is G, optionally methylated on the nitrogen at the 7’-position.

In one embodiment of either formula (lb) or (Ic), Ri is OH or OCH3. In one embodiment of either formula (lb) or (Ic), R2 is H, OH, or OCH3.

In one embodiment of either formula (lb) or (Ic), R3 is OH or OCH3.

In one embodiment of either formula (lb) or (Ic), n is 1.

In one embodiment of either formula (lb) or (Ic), Xi is O. In one embodiment of either formula (lb) or (Ic), X3 is O. In one embodiment of either formula (lb) or (Ic), Xi is S. In one embodiment of either formula (lb) or (Ic), X3 is S. In one embodiment of either formula (lb) or (Ic), each X2 is O. In one embodiment of either formula (lb) or (Ic), each X2 is S.

In one embodiment of formula (Ic), B and B’ are both G, each optionally methylated on the nitrogen at the 7’-position. In one embodiment of formula (Ic), B is G, and B’ is 7’-methyl-G.

In one embodiment, of either formula (lb) or (Ic), n is 1, Xi and X3 are O, and X2 is O. In one embodiment of either formula (lb) or (Ic), n is 1, Xi and X3 are O, and X2 is S.

In one embodiment, the cap is a naturally occurring cap structure. One example of a naturally occurring cap structure is a 7-methyl guanosine that is linked via a triphosphate bridge to the 5 '-end of the first transcribed nucleotide, resulting in a dinucleotide cap of m⁷ G(5')ppp(5')N, where N is any nucleoside. This cap is a structure of formula (Ic) in which B’ is 7-methyl-G; n is 1, each X is O, X’ is O; and Ri, R2 and R3 are all OH.

In vivo, the cap is added enzymatically. The cap is added in the nucleus and is catalyzed by the enzyme guanylyl transferase. The addition of the cap to the 5' terminal end of RNA occurs immediately after initiation of transcription. The terminal nucleoside is typically a guanosine, and is in the reverse orientation to all the other nucleotides, i.e., G(5')ppp(5')GpNpNp. A common cap for mRNA produced by in vitro transcription is m⁷G(5')ppp(5')G, which has been used as the dinucleotide cap in transcription with T7 or SP6 RNA polymerase in vitro to obtain RNAs having a cap structure in their 5'-termini. The prevailing method for the in vitro synthesis of capped mRNA employs a pre-formed dinucleotide of the form m⁷ G(5')ppp(5')G ("m7 GpppG") as an initiator of transcription.

In one embodiment, the cap structure is a synthetic occurring cap structure. One example of a synthetic dinucleotide cap used in in vitro translation experiments is the Anti-Reverse Cap Analogue ("ARCA"), which is generally a modified cap analogue in which the 2' or 3' OH group is replaced with -OCH3. ARCA and triple-methylated cap analogues are incorporated in the forward orientation. Chemical modification of m⁷ G at either the 2' or 3' OH group of the ribose ring results in the cap being incorporated solely in the forward orientation, even though the 2' OH group does not participate in the phosphodiester bond. (Jemielity, J. et al., RNA, 2003, 9: 1108-1122). The selective procedure for methylation of guanosine at N7 and 3' O-methylation and 5' diphosphate synthesis has been established (Kore, A. and Parmar, G. Nucleosides, Nucleotides, and Nucleic Acids, 2006, 25, 337-340, and Kore, A. R., et al.

Nucleosides, Nucleotides, and Nucleic Acids 2006, 25, 307-14).

In one embodiment, the cap structure is that of P-S-ARCA, which is a structure of formula (Ic) in which B is G, B’ is 7’-methyl-G; n is 1, Xi and X3 is O, X2 is S; Ri is OH; R₂ is OCH3; and R3 is OH.

In one embodiment, the cap structure is that of CleanCap® 413, which is a structure of formula (Ic) in which B is A, B’ is 7’-methyl-G; n is 1, Xi, X2 and X3 are all O; Ri is OH; R2 is OH; R3 is OCH3, and the structure is connected at the squiggly line to G via a monophosphate intersubunit linkage. CleanCap® 413 is commercially available from TriLink Biotechnologies.

In one embodiment, the cap structure is that of CleanCap® AU, which is a structure of formula (Ic) in which B is A, B’ is 7’-methyl-G; n is 1, Xi, X2 and X3 are all O; Ri is OH; R2 is OH; R3 is OCH3, and the structure is connected at the squiggly line to U via a monophosphate intersubunit linkage. CleanCap® AU is commercially available from TriLink Biotechnologies.

Substances for analysis (quantification / characterization

The method of the first aspect of the present invention separates the target RNA from substances which are contaminants in the target RNA. In this specification, the term “contaminant” in its broadest sense refers to any chemical species in the sample other than the target RNA. According to the invention, the substances may be analysed using the method of the invention. Following separation from the target RNA by the ion-pair reverse phase chromatography method of step b), these substances may be analysed, using either or both of the methods used in step c) of this aspect of the invention. In one embodiment, the substances may be analysed (quantified and or characterized) using the ultraviolet-visible spectroscopy method described in step c)i). In one embodiment, the substances may be analysed (in one embodiment, characterized) using the method of the invention, and in particular the mass spectroscopy method described in step c)ii). In one embodiment, the substances may be analysed (in one embodiment, characterized) using both the ultraviolet-visible spectroscopy method described in step c) i), and the mass spectroscopy method described in step c)ii).

The method of the third aspect of the present invention also enables these substances to be analysed (quantified and/or characterized) even in the absence of a target RNA. Following separation by the ion-pair reversed phase chromatography method of step b), these substances may be analysed using either or both of the methods used in step c) of this aspect of the invention. In one embodiment, the substances may be analysed (quantified and/or characterized) using the ultraviolet-visible spectroscopy method described in step c)i). In one embodiment, the substances may be characterized using the mass spectroscopy method described in step c)ii). In one embodiment, the substances may be analysed (in one embodiment, characterized) using both the ultraviolet-visible spectroscopy method described in step c) i), and the mass spectroscopy method described in step c)ii).

In one embodiment, the substances to be analysed comprise a degraded or treated RNA. In one embodiment, the substances to be analysed comprise a heat degraded or heat treated RNA. In one embodiment, the substances to be analysed comprise an enzyme-degraded or enzyme-treated RNA.

In one embodiment, the substances to be quantified and/or characterized are nucleotides. In this aspect, the term “nucleotide” is as defined generally above, and encompasses both natural and synthetic nucleotides as described and exemplified above.

In one embodiment, the substances to be quantified and/or characterized are nucleosides. In this aspect, the term “nucleoside” is as defined generally above, and encompasses both natural and synthetic nucleosides as described and exemplified above.

In one embodiment, the substances, to be quantified and/or characterized are oligonucleotides. In this aspect, the term “oligonucleotide” encompasses both natural and synthetic oligonucleotides, and both oligonucleotides having a natural ribosephosphate backbone (as described above in relation to RNA) and oligonucleotides having other chemical backbones (such as peptide oligonucleotides, morpholinos and locked oligonucleotides), as described above in relation to nucleoside analogues. In one embodiment, the oligonucleotides to be quantified and/or characterized have at least 2 nucleotides, such as at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 nucleotides. In one embodiment, the oligonucleotides to be quantified and/or characterized have about 2 to 120 nucleotides in length, even more typically about 2 to 50 nucleotides in length.

In one embodiment, the substances to be quantified and/or characterized are capping analogs. In this specification, the term “capping analogs” is defined and exemplified above in relation to capped RNA.

In one embodiment, the substances to be quantified and/or characterized may contain poly-A tails. As is known to the person skilled in the art, the poly-A tail is a long chain of adenine nucleotides that is added to an RNA, typically a messenger RNA (mRNA) molecule during RNA processing to increase the stability of the molecule. The poly-A tail, if present, is at the 3 ’-end of the molecule. In one embodiment, the poly-A tail comprises 10-200 adenine nucleotides. In one embodiment, the poly-A tail comprises 30-120 adenine nucleotides.

In one embodiment, proteins may also be detected using the methods of the invention. In one embodiment, the substances to be detected using the methods of the invention do not include proteins. In one embodiment, the substances to be quantified and/or characterized using the methods of the invention do not include proteins.

In one embodiment, the contaminants are by-products of the method used to form the RNA. A by-product is a secondary product of a manufacturing process or a chemical reaction, which differs from the target product of said process or reaction in its size and/or chemical structure. In one embodiment, the by-product is produced by the RNA polymerase during the RNA in vitro transcription process.

Additionally or alternatively, the by-product may comprise short RNAs which have a lower number of nucleotides than the target RNA, but have part of the sequence of the target RNA and may therefore also be considered as fragments of the target RNA. These short RNAs may, for example, be produced by premature termination of transcription, i.e. the transcription stops before the end of the sequence to be transcribed is reached. Hence, these short RNAs typically comprise the 5' sequence of the target RNA. Also additionally or alternatively, the by-product may comprise long RNAs which have a higher number of nucleotides than the target RNA and comprise the complete sequence of the target RNA and additional nucleotides. These long RNAs may, for example, be produced by incomplete termination of the transcription or by incomplete linearization of the plasmid providing the template DNA.

Also additionally or alternatively, the by-products may comprise double-stranded RNA or DNA/RNA hybrids which are produced by RNA-dependent polymerization catalyzed by the RNA polymerase. In the method of the present invention the detection of antisense RNA or DNA molecules may be indicative for these by-products. The by-product may also be an RNA having the same or a shorter or longer length as the target RNA in which one or more modified nucleotides are present, if the target RNA does not comprise modified nucleotides.

Step a) - Preparing Sample

The first step of the method of the present invention is the preparation of a sample. In the first and second aspects of the invention, the sample includes the target RNA. In the third aspect of the invention, the sample does not comprise the target RNA.

The RNA may be produced by any means known in the art. In one embodiment, the RNA is produced by transcription of a corresponding DNA sequence. In one embodiment, the RNA is produced by in vitro transcription of a corresponding DNA sequence.

In some embodiments, RNA is produced by in vitro transcription, originally developed by Krieg and Melton (Methods Enzymol., 1987, 155 397-415) for the synthesis of RNA using an RNA phage polymerase. Typically, these reactions include at least a phage RNA polymerase (for example, T7, T3 or SP6), a DNA template containing a phage polymerase promoter, nucleotides (in particular nucleoside triphosphates, such as ATP, CTP, GTP and UTP or modified nucleotides like Nl-Me- Pseudo-UTP), and a buffer containing a salt (in particular a magnesium salt).

RNA synthesis yields may be optimized by increasing nucleotide concentrations, adjusting magnesium concentrations and by including inorganic pyrophosphatase (US 5,256,555; Gurevich, et al., Anal. Biochem. 1991, 195, 207-213; Sampson, J.R. and Uhlenbeck, O.C., Proc. Natl. Acad. Sci. USA. 1988, 85, 1033-1037; Wyatt, J.R., et al., Biotechniques, 1991, 11, 764-769). Some embodiments utilize commercial kits for the large-scale synthesis of in vitro transcripts (e.g., MEGAscript®, Ambion). The RNA synthesized in these reactions is usually characterized by a 5' terminal nucleotide that has a triphosphate at the 5' position of the ribose. Typically, depending on the RNA polymerase and promoter combination used, this nucleotide is a guanosine, although it can be an adenosine (see e.g., Coleman, T. M., et al., Nucleic Acids Res., 2004, 32, el4). In the embodiments wherein the RNA is a capped RNA, to synthesize a capped RNA by in vitro transcription, a cap analogue is included in the transcription reaction. The cap analogue may be any compound capable of forming an RNA having the desired cap. In some embodiments, the RNA polymerase will incorporate the cap analogue as readily as any of the other nucleotides; that is, there is no bias for the cap analogue. In some embodiments, the cap analogue will be incorporated at the 5' terminus by the enzyme guanylyl transferase.

In one embodiment, the contaminants and / or substances to be analysed may be subjected to hydrolysis to produce hydrolysis products. When the substance is RNA the hydrolysis products may comprise shorter-chain oligonucleotides, nucleotides, and/or nucleosides.

The RNA may be hydrolysed by contacting it with an enzyme, such as an RNAase enzyme. The precise nature of the enzyme is not limited, provided that it is capable of hydrolysing the RNA to produce hydrolysis products. Examples of suitable RNA- specific RNAses A and T1 are RNAse A, which cleaves at the 3’ end at C and U, and RNAse Tl, which cleaves at G, respectively, leaving a 3’ phosphate group.

Step a’) - Purification

In one embodiment of the invention, the method comprises the step of purifying the sample (in the first and second aspects, the sample containing the target RNA; in the third aspect, not containing the target RNA). This step is carried out after the sample preparation step a) but before the separation step b). Certain aspects of the purification step, where carried out, may be incorporated into the sample preparation step a).

In this specification the terms "purification", "purified" or "purifying", takes its general meaning in the art as referring to removal of undesired substances from the sample, such that the sample which enters the ion-pair reversed-phase liquid chromatography step b) contains a lower amount of these undesired substances. In one embodiment, purification results in the undesired substances being eliminated from the sample.

In one embodiment, the purifying step a’) removes components of the in vitro transcription method from the sample. In one embodiment, the components comprise products that are introduced into the in vitro transcription method. In one embodiment, the components comprise by-products of the in vitro transcription method. Typical such components include proteins, DNA, and salts.

In relation to the first and second aspects of the invention, purification may result in the target RNA being separated and/or isolated from the components of the RNA in vitro transcription reaction present in the sample comprising the target RNA after the RNA in vitro transcription reaction.

Thus, in one aspect after purification the purified target RNA sample has a higher purity than the target RNA-containing sample prior to purification, i.e. the amount of contaminants is lower than in the sample after transcription, but before purification. Undesired constituents of RNA- containing samples which therefore need to be separated may in particular be by-products of the RNA in vitro transcription reaction, or also excessively long transcripts if plasmids are not completely linearised. In addition, components of the RNA in vitro transcription reaction mixture, such as proteins, in particular enzymes, for example RNases and polymerases, and nucleotides may be separated from the target RNA in the purification step.

In one embodiment, the purifying step a’) removes proteins from the sample.

After the purification step, the target RNA has a higher purity than before the purification step, but may still contain contaminants which may be detected by the method of the present invention. The degree of purity after the purification step may be more than 70% or 75%, in particular more than 80% or 85%, very particularly more than 90% or 95% and most favourably 99% or more. The degree of purity may for example be determined by an analytical HPLC as described herein, wherein the percentage provided above is determined in a similar manner to the quantification step c) below. Step b) - Separation by ion-pair reversed phase chromatography

In step b) of the method of the present invention, the target RNA is separated from the substances to be analysed by ion-pair reversed phase chromatography.

As is well understood by the person skilled in the art, the term "chromatography" in its broadest sense refers to a technique for separation of mixtures in which, typically, the mixture is dissolved in a fluid called the "mobile phase" or “eluent”, which carries it through a structure holding another material called the "stationary phase."

Chromatography may be carried out according to a wide range of possible techniques, which are generally well known to those skilled in the art. The chromatography technique may be classified by the physical state of the mobile phase.

In the present invention, the chromatography method used in step b) is liquid chromatography (i.e. wherein the mobile phase is a liquid). Liquid chromatography methods in which the stationary phase is more polar than the mobile phase (e.g., toluene as the mobile phase, silica as the stationary phase) are termed normal phase liquid chromatography (NPLC) and the opposite (e.g., water-methanol mixture as the mobile phase and C18 (octadecyl silyl) as the stationary phase) is termed reversed phase liquid chromatography (RPLC).

The ion-pair reversed phase chromatography used in the present invention is a form of high performance liquid chromatography (HPLC). In the HPLC process, a pressurized liquid solvent containing the sample mixture is passed through a column filled with a solid adsorbent material leading to the interaction of components of the sample with the adsorbent material. Since different components interact differently with the adsorbent material, this leads to the separation of the components as they flow out of the column. The operational pressure in HPLC process is typically between 50 and 350 bar. Generally, the term HPLC includes reversed phase HPLC (RP-HPLC), size exclusion chromatography, gel filtration, affinity chromatography, hydrophobic interaction chromatography or ion pair chromatography. In one embodiment, the chromatography used in step (c) is ultra high performance liquid chromatography (UHPLC). Typically, this is carried out at a pressure of around 400-1200 bar.

Reversed phase HPLC uses a non-polar stationary phase and a moderately polar mobile phase and therefore works with hydrophobic interactions which result from repulsive forces between a relatively polar solvent, the relatively non-polar analyte, and the non-polar stationary phase (reversed phase principle). The retention time on the column is therefore longer for molecules which are more non-polar in nature, allowing polar molecules to elute more readily. The retention time is increased by the addition of polar solvent to the mobile phase and decreased by the addition of more hydrophobic solvent. The characteristics of the specific RNA molecule as an analyte may play an important role in its retention characteristics. In general, an analyte having more apolar functional groups results in a longer retention time because it increases the molecule's hydrophobicity and therefore the interaction with the nonpolar stationary phase. Very large molecules, however, can result in incomplete interaction between the large analyte surface and the alkyl chain. Retention time increases with hydrophobic surface area, which is roughly inversely proportional to solute size. Branched chain compounds elute more rapidly than their corresponding isomers because the overall surface area is decreased.

The separation of step b) of the method of the present invention is therefore preferably performed at an analytical scale. In an analytical scale method, a quantity of RNA such as 1 ng to 1000 ng, preferably 1 to 100 pg, may be introduced for a single run. If a plurality of runs is performed, the quantity increases in direct proportion to the number of runs. The remainder of the purified target RNA sample can be further processed to the final RNA product, such as an RNA product for administration to a patient, if the analysis according to the method of the invention indicates that the amount of contaminants is within a range which is acceptable for a final RNA product.

Stationary phases for use in the HPLC analysis are known in the art. Preferably, the stationary phase is selected from the group consisting of a porous polystyrene, a porous non-alkylated polystyrene, a polystyrene-divinylbenzene, a porous non- alkylated polystyrene-divinylbenzene, a porous silica gel, a porous silica gel modified with non-polar residues, a porous silica gel modified with carbon chains, carbon chains, a porous silica gel modified with phenylic residues, and a porous polymethacrylate.

The stationary phase used for step b) of the method of the present invention is preferably a porous silica gel modified with carbon chains, typically Cl -40 alkyl chains, preferably C4-30 alkyl chains, more preferably from butyl-, octyl and/or octadecyl, and most preferably an octadecyl carbon chain. An example of such a silica gel is that used on the column ACQUITY Premier BEH Cl 8 Column with VanGuard FIT from Waters.

The silica gel may have a particle size of 0.5 to 5 pm, preferably of 0.7 to 4 pm, more preferably of 1 to 3 pm, even more preferably of 1.5 to 2 pm and most preferably of 1.7 pm.

The pore size of the porous silica gel may be 50 to 300 A, preferably 70 to 250 A, more preferably 100 to 200 A, even more preferably 120 to 170 A and most preferably it is 130 A.

The stationary phase is conventionally located in a column. V2A steel is conventionally used as the material for the column, but other materials may also be used for the column provided they are suitable for the conditions prevailing during HPLC. Conventionally the column is straight.

In one embodiment, the HPLC column has a length of 5 cm to 100 cm. In one embodiment, the HPLC column has a length of 10 cm to 50 cm. In one particularly preferred embodiment, the HPLC column has a length of 15 cm.

In one embodiment, the HPLC column has an inner diameter of 0.5 mm to 10 mm. In one embodiment, the HPLC column has an inner diameter of 1 mm to 5 mm. In one particularly preferred embodiment, the HPLC column has an inner diameter of 2.1 mm. In one embodiment, the method of step b) is carried out at a column temperature of between 40 to 80 °C. In one embodiment, the method of step b) is carried out at a column temperature of between 50 - 70°C.

The flow rate of the mobile phase is selected such that good separation of the substances can be achieved. In one embodiment, the method of step b) is carried out at a flow rate of 0.1 to 1 mL/min. In one embodiment, the method of step b) is carried out at a flow rate of 0.1 to 0.5 mL/min. In one embodiment, the method of step b) is carried out at a flow rate of 0.2 mL/min.

According to the present invention, the HPLC is performed as ion-pair, reversed phase HPLC. Ion-pair reversed-phase HPLC is a specific form of reversed-phase HPLC in which an ion with a lipophilic residue and positive charge is added to the mobile phase as counter-ion for the negatively charged RNA. When used with common hydrophobic HPLC phases in the reversed-phase mode, ion pair reagents can be used to selectively increase the retention of the RNA. The formation of ion pairs between the negatively charged analytes and the positively charged ion pairing reagent molecules with hydrophobic residues results in a generally length-based separation mechanism where the retention time increases with oligonucleotide length. This allows a length approximation of the contaminants in RNA samples.

According to the present invention, the ion used in the ion-pair reversed phase chromatography is a C3-8 primary alkylamine or a salt thereof. The term "primary alkylamine" refers to a compound of the formula R-NH2, wherein R is an alkyl group, as defined above, either in its broadest aspect or any preferred aspect. The alkyl group has from 3 to 8 carbon atoms. Exemplary primary alkylamines include 1- propylamine, 2-propylamine, 1 -butylamine, 2-butylamine, tert-butylamine, 1- pentylamine, 2-pentylamine, 3 -pentylamine, neopentylamine, l,2-dimethylpropyl-2- amine, 1 -hexylamine, 2-hexylamine, 3 -hexylamine, 2,2-dimethylbutylamine, 1- heptylamine, 2-heptylamine, 3 -heptylamine, 4-heptylamine, 1 -octylamine, 2- octylamine, 3-octylamine, 4-octylamine. Preferably the C3-8 primary alkylamine is an unbranched C3-8 primary alkylamine, such as 1 -propylamine, 1 -butylamine, 1- pentylamine, 1 -hexylamine, 1 -heptylamine, or 1 -octylamine. C4-6 primary amines (preferably unbranched C4-6 primary amines) are preferred, C5-6 primary amines (preferably unbranched C5-6 primary amines) are more preferred, and C6 primary amines are most preferred, especially 1 -hexylamine.

The C3-8 primary alkylamine may be present either as the free base, a salt or a mixture thereof. In one embodiment, the C3-8 primary alkylamine may be present in the form of a free base. In one embodiment, the C3-8 primary alkylamine may be present in the form of a salt, typically an acid addition salt. In one embodiment, the C3-8 primary alkylamine may be present in the form of a mixture of the free base and a salt, typically an acid addition salt. As C3-8 primary alkylamines are weak bases, it will be readily understood by the person skilled in the art that, depending on the pH, the C3-8 primary alkylamine will typically be present in the form of an equilibrium mixture of the free base and an acid addition salt, the nature of the counter-ion depending on the nature of the other components present in the sample.

Examples of suitable acid addition salts include the acetate, formate and hydrochloride/chloride salts.

In contrast to the triethylammonium salts used to carry out the ion-pair reversed phase chromatography as described in WO2017/140345, it has been found that carrying out the ion-pair reversed phase chromatography using a primary (C3-8)alkylamine or a salt thereof as ion pairing reagent results in an improved separation of the substances to be analysed than was possible using ion-pairing reagents used in the art.

In one embodiment, the concentration of the C3-8 primary alkylamine is between 1 and 200 mM. In one embodiment, the concentration of the C3-8 primary alkylamine is between 2 and 50 mM. In one embodiment, the concentration of the C3-8 primary alkylamine is between 5 and 20 mM. In one particularly preferred embodiment, the concentration of the C3-8 primary alkylamine is 15 mM.

When the liquid chromatography method used in step b) uses a first mobile phase and a second mobile phase, in one embodiment, the concentration of the C3-8 primary alkylamine in the first (aqueous) mobile phase is between 1 and 200 mM. In one embodiment, the concentration of the C3-8 primary alkylamine in the first mobile phase is between 2 and 50 mM. In one particularly preferred embodiment, the concentration of the C3-8 primary alkylamine in the first mobile phase is 15 mM.

When the liquid chromatography method used in step b) uses a first mobile phase and a second mobile phase, in one embodiment, the concentration of the C3-8 primary alkylamine in the second mobile phase (containing organic solvent) is between 0.2 and 20 mM. In one embodiment, the concentration of the C3-8 primary alkylamine in the second mobile phase is between 1 and 4 mM. In one particularly preferred embodiment, the concentration of the C3-8 primary alkylamine in the second mobile phase is 3 mM.

The liquid chromatography method used in step b) may use any suitable solvent capable of eluting the substances from the column, to enable the contaminants to be quantified and/or characterized according to the subsequent method steps. In one embodiment, the liquid chromatography method used in step b) uses an aqueous solvent as mobile phase. In one embodiment, the liquid chromatography method used in step b) uses an organic solvent as mobile phase. In one embodiment, the liquid chromatography method used in step b) uses a mixture of an aqueous solvent and an organic solvent as mobile phase. Typically, elution is carried out by increasing the percentage of mobile phase containing organic solvent from the beginning to the end of the elution.

When the liquid chromatography method used in step b) uses an organic solvent as mobile phase (either alone or as a mixture with an aqueous solvent), in one embodiment, the organic solvent is a Cl-4 alcohol, a C2-4 nitrile, a C3-4 ketone, or a mixture thereof. In one embodiment, the organic solvent is acetonitrile, methanol or isopropanol or a mixture thereof.

When the liquid chromatography method used in step b) uses a mixture of an aqueous solvent and an organic solvent, in one embodiment, the mixture comprises acetonitrile and water. In one embodiment, the mixture comprises methanol and water.

When the liquid chromatography method used in step b) uses a mixture of an aqueous solvent and an organic solvent as the mobile phase, the aqueous solvent and the organic solvent may be present in any proportions capable of performing the elution. In one embodiment, the mixture contains 1 to 99% by volume of organic solvent and 1 to 99% by volume of aqueous solvent. In one embodiment, the mixture contains 10 to 90% by volume of organic solvent and 10 to 90% by volume of aqueous solvent. In one embodiment, the mixture contains 70 to 90% by volume of organic solvent and 10 to 30% by volume of aqueous solvent.

In one embodiment, the liquid chromatography method used in step b) uses a first mobile phase and a second mobile phase, the first mobile phase comprising an aqueous solvent and the second mobile phase comprising a mixture of an aqueous solvent and an organic solvent. In one embodiment, the second mobile phase contains 1 to 99% by volume of organic solvent and 1 to 99% by volume of aqueous solvent. In one embodiment, the second mobile phase contains 10 to 90% by volume of organic solvent and 10 to 90% by volume of aqueous solvent. In one embodiment, the second mobile phase contains 70 to 90% by volume of organic solvent and 10 to 30% by volume of aqueous solvent.

When the liquid chromatography method used in step b) uses a first mobile phase and a second mobile phase, in one embodiment, the first and second mobile phases are present in a volume ratio of 1 : 19 to 1 :2. In one embodiment, the first and second mobile phases are present in a volume ratio of 1 :9 to 1 :3. In one especially preferred embodiment, the first and second mobile phases are present in a volume ratio of 1 :4.

In one preferred aspect of the invention, the mobile phase used in the ion-pair reversed-phase chromatography comprises 1,1,1,3,3,3-hexafluoroisopropanol. In contrast to the methods of the prior art, incorporating 1, 1,1, 3,3,3 -hexafluoroisopropanol into the mobile phase used in the ion-pair reversed-phase chromatography results in stronger retention of the analytes and improved chromatographic resolution. It further increases sensitivity in mass spectrometric detection compared to the use of alkylamine salts alone.

While it is possible for the 1,1,1,3,3,3-hexafluoroisopropanol to be introduced undiluted into the liquid chromatography column, it is more typically provided in the form of a solution. In this solution, the solvent may be an aqueous solvent, an organic solvent, or a mixture thereof, as defined and exemplified above. In one embodiment, the organic solvent in which the 1,1,1,3,3,3-hexafhioroisopropanol is dissolved is a Cl -4 alcohol, a C2-4 nitrile, a C3-4 ketone, or a mixture of any thereof, optionally mixed with an aqueous solvent, preferably water. In one embodiment, the organic solvent in which the 1,1,1,3,3,3-hexafluoroisopropanol is dissolved is acetonitrile, methanol or isopropanol or a mixture thereof, optionally mixed with an aqueous solvent, preferably water.

It is particularly preferred that the mobile phase in which the 1, 1,1, 3,3,3- hexafluoroisopropanol is dissolved in an aqueous mobile phase, preferably water, and in second mobile phase, preferably a mixture of methanol and water. Typically, the mixture contains 1 to 99% by volume of methanol and 1 to 99% by volume of water. Preferably, the mixture contains 50 to 95% by volume of methanol and 5 to 50% by volume of water. More preferably, the mixture contains 70 to 90% by volume of methanol and 10 to 30% by volume of water. Most preferably, the mixture contains 75 to 85% by volume of methanol and 15 to 25% by volume of water. In one particularly preferred example, the mixture contains 80% by volume of methanol and 20 % by volume of water.

In one embodiment, the concentration of the 1,1,1,3,3,3-hexafluoroisopropanol is between 5 and 400 mM. In one embodiment, the concentration of the 1, 1,1, 3,3,3- hexafluoroisopropanol is between 5 and 350 mM. In one embodiment, the concentration of the 1,1,1,3,3,3-hexafluoroisopropanol is between 5 and 300 mM. In one embodiment, the concentration of the 1,1,1,3,3,3-hexafluoroisopropanol is between 5 and 250 mM. In one embodiment, the concentration of the 1, 1,1, 3,3,3- hexafluoroisopropanol is between 5 and 200 mM. In one embodiment, the concentration of the 1,1,1,3,3,3-hexafhioroisopropanol is between 5 and 150 mM. In one embodiment, the concentration of the 1,1,1,3,3,3-hexafluoroisopropanol is between 5 and 100 mM. In one embodiment, the concentration of the 1, 1,1, 3,3,3- hexafluoroisopropanol is between 10 and 100 mM. In one embodiment, the concentration of the 1,1,1,3,3,3-hexafhioroisopropanol is between 50 and 100 mM. In one embodiment, the concentration of the 1,1,1,3,3,3-hexafluoroisopropanol is between 60 and 100 mM. In one embodiment, the concentration of the 1, 1,1, 3,3,3- hexafluoroisopropanol is between 70 and 100 mM. In one embodiment, the concentration of the 1,1,1,3,3,3-hexafluoroisopropanol is between 80 and 100 mM. In one embodiment, the concentration of the 1,1,1,3,3,3-hexafluoroisopropanol is between 30 and 70 mM. In one embodiment, the concentration of the 1, 1,1, 3,3,3- hexafluoroisopropanol is between 40 and 60 mM.

When the liquid chromatography method used in step b) uses a first mobile phase and a second mobile phase, in one embodiment, the concentration of the 1, 1,1, 3,3,3- hexafluoroisopropanol in the first (aqueous) mobile phase is between 5 and 400 mM. In one embodiment, the concentration of the 1,1,1,3,3,3-hexafluoroisopropanol in the first mobile phase is between 10 and 100 mM. In one embodiment, the concentration of the 1,1,1,3,3,3-hexafluoroisopropanol in the first mobile phase is between 40 and 60 mM.

When the liquid chromatography method used in step b) uses a first mobile phase and a second mobile phase, in one embodiment, the concentration of the 1, 1,1, 3,3,3- hexafluoroisopropanol in the second mobile phase containing organic solvent is between 1 and 80 mM. In one embodiment, the concentration of the 1, 1,1, 3,3,3- hexafluoroisopropanol in the second mobile phase is between 2 and 20 mM. In one embodiment, the concentration of the 1,1,1,3,3,3-hexafluoroisopropanol in the second mobile phase is between 8 and 12 mM.

In one embodiment, the liquid chromatography used in step b) comprises elution from a liquid chromatography column by means of gradient separation. Elution may proceed isocratically or by means of gradient separation. In isocratic separation, elution of the RNA proceeds with a single eluent or a constant mixture of a plurality of eluents, wherein the solvents described above in detail may be used as eluent. In a preferred embodiment, gradient separation is performed wherein the composition of the eluent is varied by means of a gradient program. The equipment necessary for gradient separation is known to a person skilled in the art. Gradient elution may here proceed either on the low pressure side by mixing chambers or on the high pressure side by further pumps.

The following gradient program has proven particularly useful in the ion-pair reversed phase chromatography method of the present invention: mobile phase A: 15 mM 1-hexylamine (HA) / 50 mM 1 J , 3, 3, 3 -hexafluoroisopropanol (HFIP) in H2O; mobile phase B: mobile phase A in a mixture with methanol (20:80, v/v); with the following gradient: start with 85% A and 15% B, change to 40 % A and 60% B in 5 minutes, then hold at 40 % A and 60% B for 5 minutes, change to 20% A and 80% B in 20 minutes, change to 10 % A and 90% B in 0.1 minutes, then hold at 10 % A and 90% B for 4.9 minutes, change to 90% A and 15 % B in 0.1 minutes, then rinse with initial conditions for 6.9 minutes.

The ion-pair reverse phase chromatography used in step b) of the present invention enables the substances, in particular the short oligonucleotides (typically having a chain length up to and including 120 nucleotides), cap analogs, and nucleotides, to elute before the target RNA and thereby separate them from the target RNA and allows their quantification and/or characterization in the subsequent steps described below.

Step c), option i)- analysis by UV-Visible spectroscopy

In one embodiment, step c) of the method according to the invention comprises analysis of the substances by ultraviolet-visible (UV/Vis) spectroscopy. In one embodiment of the method according to the invention, step c) comprises quantification of the substances by ultraviolet-visible (UV/Vis) spectroscopy. In one embodiment of the method according to the invention, step c) comprises characterization of the substances by ultraviolet-visible (UV/Vis) spectroscopy.

As is known to the person skilled in the art, (UV/Vis) spectroscopy is an absorption spectroscopic method comprising passing ultraviolet and/or visible light through a sample and measuring the absorbance of the ultraviolet and/or visible light after it has passed through to determine the concentration of the substances. Typically, this method involves use of a UV/Vis spectrophotometer for detection. This is typically coupled to the apparatus used to perform the liquid chromatography. The presence of an analyte gives a response which is proportional to the concentration of the analyte. The method is most often used in a quantitative way to determine concentrations of an absorbing species in solution, using the Beer-Lambert law:

A = logio (7o/7) = zcL where A is the measured absorbance, Io is the intensity of the incident light at a given wavelength, I is the transmitted intensity, L the path length through the sample, and c the concentration of the absorbing species. For each species and wavelength, a is a constant known as the molar absorptivity or extinction coefficient. This constant is a fundamental molecular property in a given solvent, at a particular temperature and pressure, and has units of MM* cm.

Typically, the wavelength of the ultraviolet and/or visible light to which the sample is subjected may be between 100 nm and 750 nm. Preferably, the wavelength of the ultraviolet light to which the sample is subjected is between 200 nm and 300 nm.

More preferably, the wavelength of the ultraviolet light to which the sample is subjected is between 240 nm and 280 nm. A particularly preferred wavelength for nucleic acid detection is 260 nm.

In one embodiment, the instrument's response to the analyte in the unknown is compared with the response to a reference standard. This is very similar to the use of calibration curves. The response (e.g., peak area) for a particular concentration is known as the response factor.

Typically, the wavelength of the ultraviolet and/or visible light to which the reference sample is subjected may be between 100 nm and 750 nm. Preferably, the wavelength of the visible light to which the reference sample is subjected is between 500 nm and 600 nm. More preferably, the wavelength of the visible light to which the reference sample is subjected is between 550 nm and 560 nm. A particularly preferred wavelength for reference samples is 600 nm. In the first and second aspects of the invention, the method may be used for quantification of the substances which are contaminants in the RNA sample. Quantification may be defined as concentration or amount.

In one embodiment, the quantification is relative quantification (i.e. measuring the quantities of the substances relative to one another, and to the target RNA). The relative concentration can be determined via the relative peak areas of the contaminants (after blank subtraction) and the summed peak areas of the contaminants and RNA.

In one embodiment, the quantification is absolute quantification (i.e. measuring the absolute quantities of the substances in the sample). The absolute concentration can be calculated using the total peak area of the contaminants (after blank subtraction) and the molar extinction coefficient corresponding to an oligonucleotide with the mean substances’ length. The relative concentration can then here be determined by the ratio of the absolute contaminant concentration and the total RNA concentration of the sample. The total RNA concentration of the sample is determined with a spectrophotometer using ultraviolet absorption spectroscopy at 260 nm.

In one embodiment of the third aspect of the invention, the method may be used for characterization of the substances which were present in the sample.

Step c), option ii)-analysis by mass spectrometry

In one embodiment, the method of the invention comprises the step c) of analysis of the substances by mass spectroscopy. In one embodiment, the method of the invention comprises the step of characterization of the substances by mass spectroscopy.

In one embodiment of the invention, step c ii) may be carried out after step c i) of analysis (quantification / characterization) of the substances by ultraviolet-visible (UV/Vis) spectroscopy. This step of the method of the present invention, when carried out, comprises characterization by mass spectrometry. As is known to the person skilled in the art, a mass spectrometer typically consists of three components: an ion source, a mass analyzer, and a detector. The ionizer converts a portion of the sample into ions. As detailed below, there are a wide variety of ionization techniques, depending on the phase (solid, liquid, gas) of the sample and the efficiency of various ionization mechanisms for the unknown species. The mass spectrometer also typically comprises an extraction system which removes ions from the sample, which are then targeted through the mass analyzer and onto the detector. The difference in mass-to-charge (m/z) of the ions or ion fragments thereof allows the mass analyzer to sort the ions by their mass-to-charge ratio. Finally, the detector measures the value of an indicator quantity and thus provides data for calculating the abundances of each ion present.

For the purpose of characterization the mass spectrometry method should typically possess the following features: high resolution, quadrupole for ion selection, and enabling fragmentation of the ions formed.

In a typical mass spectrometry procedure, the first step comprises ionization of a sample. In another embodiment, the ionization comprises Atmospheric Pressure Chemical Ionisation (APCI). In another embodiment, the ionization comprises Atmospheric Pressure Photon Ionization (APPI).

In another embodiment, the ionization comprises electrospray ionization (ESI), in which the liquid containing the analyte(s) of interest is dispersed by electrospray into a fine aerosol. These ionization techniques are well known to the person skilled in the art. Ionization, in particular electron ionization, may cause some of the sample's molecules to break into charged fragments.

Following ionization, the ions produced in the first step are then separated according to their mass-to-charge (m/z) ratio in the mass analyzer. This is typically carried out by one or more of the following mass to charge separation techniques: by quadrupole electric fields as used in quadrupole mass spectrometers, by ion trap quadrupole electric fields as used by ion trap mass spectrometers, by longitudinal ion travelling time as used by time of flight mass spectrometers and by electric and/or magnetic field deflection as traditionally used by electric and magnetic sector mass spectrometers.

Following separation, the ions are detected. Typically, the detector records either the charge induced or the current produced when an ion passes by or hits a surface. In a scanning instrument, the signal produced in the detector during the course of the scan versus where the instrument is in the scan will produce a mass spectrum, a record of ions as a function of m/z.

According to one embodiment of the present invention, the mass spectrometry step c)ii) is used in tandem with the chromatographic separation technique used in step b) and optionally the analysis of the substances by ultraviolet-visible (UV/Vis) spectroscopy in step c)i). As the chromatographic technique is liquid chromatography, the combination technique being known as liquid chromatographymass spectrometry (LC/MS, LCMS or LC-MS). As described in relation to step b) above, and is generally known to the person skilled in the art, this technique separates compounds chromatographically using a liquid mobile phase. Typically, the liquid phase is a mixture of water and organic solvents. The stream of separated compounds is then fed into the mass spectrometer for ionization, mass analysis and detection as described above.

In one embodiment, the mass spectrometry used in step c)ii) is tandem mass spectrometry. Tandem mass spectrometry, also known as MS/MS, MS² or MSⁿ (where n is at least 2, preferably 2 to 10, more preferably 2 to 5, even more preferably 2 or 3, most preferably 2) involves multiple steps of mass spectrometry selection, with some form of fragmentation occurring in between the steps. Tandem mass spectrometry is especially preferred as the mass spectrometry method of the present invention when coupled to liquid chromatography, the analytes can be determined with high selectivity, flexibility and sensitivity.

Typically, tandem mass spectrometry involves the following steps:

(i) Ionization of a sample to produce ions. The ionization may be carried out using any of the ionization techniques generally described above, in particular Electrospray Ionization (ESI), Secondary Electrospray Ionization (SESI), Extractive Electrospray Ionization (EESI), Neutral Desorption Electrospray Ionization (ND-ESI), Atmospheric Pressure Chemical Ionization (APCI), Atmospheric Pressure Photon Ionization (APPI), Direct Analysis in Real Time (DART).

(ii) Separating the ions according to their mass-to-charge ratio to produce one or more precursor ions. The separation is carried out as generally described above.

(iii) Fragmentation of the one or more separated precursor ions to yield product ions. There are many methods used to fragment the ions and these can result in different types of fragmentation and thus different information about the structure and composition of the molecule. In one embodiment, the fragmentation method comprises collision-induced dissociation. Typically, this method involves the collision of an ion with a neutral atom or molecule in the gas phase and subsequent dissociation of the ion. In another embodiment, the fragmentation technique comprises in-source fragmentation (i.e. fragmentation in the ionization chamber) in which the ionization process is sufficiently violent to leave the resulting ions with sufficient internal energy to fragment within the mass spectrometer (e.g. by electron impact, Chemical Ionization or "accelerated ion dissociation"). All of these techniques are well known to the person skilled in the art.

(iv) Separating the product ions obtained from the fragmentation process according to their mass-to-charge ratio. The separation is typically carried out as generally described above.

(v) Detection of the separated ions. The detection is typically carried out as generally described above.

In one embodiment, the tandem mass spectrometry is quadrupole ion trap mass spectrometry. As is known to the person skilled in the art, a quadrupole ion trap is a type of ion trap that uses dynamic electric fields to trap charged particles.

In one embodiment, the tandem mass spectrometry is quadrupole time of flight mass spectrometry. As is known to the person skilled in the art, a quadrupole time-of-flight mass spectrometer is a triple quadrupole mass spectrometer, as described above, with the final quadrupole replaced by a time-of-flight device. As is known to the person skilled in the art, time-of-flight mass spectrometry (TOFMS) is a method of mass spectrometry in which the mass-to-charge ratio (m/z) of an ion is determined via a time measurement. The technique involves acceleration of the ions by an electric field of known strength. This acceleration results in an ion having the same kinetic energy as any other ion that has the same charge. The velocity of the ion depends on the mass-to-charge ratio. The time that it subsequently takes for the particle to reach a detector at a known distance is measured. This time will depend on the mass-to- charge ratio of the particle, heavier particles reaching lower speeds. From this time and the known experimental parameters, the user can determine the mass-to-charge ratio of the ion.

In one embodiment, the tandem mass spectrometry is Quadrupole Ion Trap mass spectrometry. In one embodiment, the tandem mass spectrometry is Quadrupole-Time of Flight mass spectrometry. In one embodiment, the tandem mass spectrometry is Ion Mobility-Quadrupole Ion Trap-Time of Flight mass spectrometry. In one embodiment, the tandem mass spectrometry is Quadrupole-Orbitrap mass spectrometry. In one embodiment, the tandem mass spectrometry is Quadrupole Ion Trap mass spectrometry. In one embodiment, the tandem mass spectrometry is Ion Mobility Spectrometer-Quadrupole Ion Trap mass spectrometry. In one embodiment, the tandem mass spectrometry is Quadrupole-Orbitrap Mass spectrometry. In one embodiment, the tandem mass spectrometry is Quadrupole Ion Trap-Orbitrap mass spectrometry. In one embodiment, the tandem mass spectrometry is Time of Flight, Ion Trap-Fourier Transform mass spectrometry. Details of these techniques are known to the person skilled in the art.

In one embodiment, the mass spectrometry is carried out in full scan monitoring mode. As is known to the person skilled in the art, full scan monitoring involves scanning the mass range from the smallest the highest mass of ions expected (compared with selected ion monitoring mode in which data is only collected on the selected masses of interest).

In one embodiment, the mass spectrometry is carried out in full scan monitoring mode with additional data-dependent or independent fragmentation. As is known to the person skilled in the art, the selected ions are further fragmented and the fragmentation products are scanned in a selected mass range. This information can be used for more detailed characterization of the analytes. Examples

Example 1 - Analysis of standards and in vitro transcribed (IVT) RNA with the specified HPLC-UV method

A composition containing RNA and the further substances referred to below was separated using the ion pair reversed phase chromatography method of the present invention and analysed by UV spectroscopy.

15 mM 1-hexylamine (HA) / 50 mM 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) in H2O was used as mobile phase A, and a mixture of mobile phase A with methanol (20:80, v/v) was used as mobile phase B (see Gong 2014 Rapid Commun. Mass Spectrom. DOI: 10.1002/rcm.6773).

The components were separated on an ACQUITY Premier BEH C18 column with VanGuard FIT, 1.7 pm, 2.1 x 150 mm, pore size 130 A, from Waters, at a flow rate of 0.2 mL/min at 60°C with the following gradient: 15% - 60% B in 5 min, hold for 5 min, 60% - 80% B in 20 min, 80% - 90% B in 0.1 min, hold for 4.9 min, 90% - 15 % B in 0.1 min, rinse with initial conditions for 6.9 min. 10 pL of each sample were injected.

Shown in Figure 1 are the UV absorbance spectra at 260 nm from (A) the chromatogram of the complete analytical run and (B) a zoomed at 2 - 32 min. The following are noted: i) Standards mix with 1 pM uncapped oligonucleotides (5, 10, 15, 20, 40, 60, 80, 100, 120 nucleotides), 10 pM nucleotides (ATP, CTP, GTP, UTP, mlyTP), 10 pM cap analogs (CleanCap®413, P-S-ARCA DI cap) and 0.25 mg/mL RNA (2135 nucleotides) (grey) ii) 1 mg/mL luciferase-RNA (2135 nucleotides) produced with mutant T7 polymerase and purified with magnetic beads (dotted) iii) 1 mg/mL luciferase RNA (2135 nucleotides) produced with regular T7 polymerase and purified with magnetic beads (black) iv) Injection blank (H2O) (light grey) v) Gradient (dashed) The chromatogram illustrates the differences between the same RNA that was produced under different IVT conditions. The signals between approximately 10 and 22 min corresponding to the contaminants are more pronounced in the RNA produced with the T7 polymerase mutant. Based on the retention times of the oligonucleotide standards corresponding to the same sequence, the estimated length of the oligonucleotide contaminants in the RNAs range mainly between 10 and 20 nucleotides.

Example 2 - Analysis of a collected HPLC fraction with the specified HPLC-UV method of the invention

The fraction analysed in this example contains the contaminants that were separated from the luciferase-RNA (2135 nucleotides) produced with mutant T7 polymerase, collected and concentrated. It was separated using the ion pair reversed phase chromatography method and analysed by UV spectroscopy according to the method of the present invention. The contaminants fraction was subjected to enzymatic digestion for 1 h at 37°C with RNA-specific RNAses A and Tl. The RNAse A cleaves at the 3’ end at C and U, and RNAse Tl at G, respectively, leaving a 3’ phosphate group.

The components were separated under the same ion pair reversed phase chromatography conditions as in Example 1.

The volume corresponding to 5.4 pmol each of undigested and digested contaminant fraction samples were injected. 25 pL of the reagent blank and 10 pL of the injection blank were injected.

Shown in Figure 2 is the UV trace at 260 nm of the ion pair reversed phase chromatography. Shown are (A) the chromatogram of the complete analytical run and (B) a zoomed at 3 - 31 min. The following are noted: i) Collected HPLC fraction containing the oligonucleotide contaminants - undigested (black) ii) Collected HPLC fraction containing the oligonucleotide contaminants - after enzymatic digestion (grey) iii) Reagent blank of enzymatic digestion (light grey) iv) Injection blank (H2O) (dashed) The chromatogram illustrates that the collected contaminant fraction consists of RNA oligonucleotides. These are digested by the RNA-specific RNAses to smaller oligonucleotides or nucleotides which shifts their retention time to the beginning of the chromatogram.

Example 3 - Analysis of a collected HPLC fraction with the HPLC-UV-MS method of the invention

The fraction analysed in this example contains the contaminants that were separated from the luciferase-RNA (2135 nucleotides) produced with mutant T7 polymerase by the same HPLC-UV method.

The contaminants fraction and the luciferase-RNA (2135 nucleotides) produced with mutant T7 polymerase were subjected to enzymatic digestion for 1 h at 37°C with RNA-specific RNAses A and Tl. The RNAse A cleaves at the 3’ end at C and U, and RNAse Tl at G, respectively, leaving a 3’ phosphate group. The components were then separated via ion pair reversed chromatography with the same conditions used in Example 1. The volume corresponding to 5.4 pmol of the samples was injected. Figure 3 (A) shows the chromatograms of the complete analytical run as total ion chromatogram (TIC) and extracted ion chromatogram (EIC) of m/z=1238.1196 — 1238.1444 in i) the digested contaminants fraction and iii) the digested luciferase- RNA produced with mutant T7 polymerase. This m/z corresponds the expected capped 5’ end after enzymatic cleavage: m7(3'OMeG)(5')ppp(5')(2'OMeA)pGp. This was confirmed with the MS spectra over the 12.90 min peaks of ii) the digested contaminants fraction and iv) the digested the luciferase-RNA produced with mutant T7 polymerase.

Shown in (B) are the chromatograms of the complete analytical run as TIC and EIC of m/z= 929.3211 - 929.5069 in i) the digested contaminants fraction and iii) the digested luciferase-RNA produced with mutant T7 polymerase. This m/z corresponds the expected 3’ end after enzymatic cleavage: AsoGp, which was only detected in the luciferase RNA at 22.96 min as also shown in the iii) MS spectrum.

With the RNA-specific digestion and the analysis via the HPLC-MS method, the contaminants in the collected fraction were further characterized as capped on the 5’ end, but lacking a polyA sequence at the 3’ end. Example 4 - Sequence Mapping

Analysis of the luciferase-RNA (2135 nucleotides) produced with mutant T7 polymerase with the specified HPLC-UV-MS method.

The components were separated via ion pair reversed chromatography with the same conditions as used in Example 1.

Using a commercial software (BioPharma Finder 5.1™ from Thermo Fisher Scientific), the MS data of the early eluting contaminants was compared to the 5’ sequence of the luciferase-RNA and the calculated expected MS data.

All theoretical oligonucleotides from the first 1-30 nucleotides (n+1) were mapped to the MS data of the full scan with a mass error < 10 ppm and MS area > 25,000. 5’ end was set as variable with X = CleanCap®413, triphosphate, diphosphate, monophosphate or hydroxy group. Partially, the identification was confirmed by fragment ion spectra that matched with the calculated expected fragment ions, as shown in Table 1 below.

X- AGACGAACUAGUAUUCUUCUGGUCCCCACA (SEQ ID NO: 1).

20-30 AGACGAACUAGUAUUCUUCUGGUCCCCACA 14.10

21-27 AGACGAACUAGUAUUCUUCUGGUCCCCACA 12.23

21-28 AGACGAACUAGUAUUCUUCUGGUCCCCACA 12.72

Table 1 c = CleanCap®413, ppp = Triphosphate

Only few of the abundant peaks could be matched to the expected 5’ T7 polymerase drop-off products comprising a CleanCap® 413 at the 5’ end. The others were mapped to randomly to the sequence. Therefore, other mechanisms for the formation of these oligonucleotide contaminants are likely.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, biochemistry, molecular biology, biotechnology or related fields are intended to be within the scope of the following claims.

Claims

1. A method of analysis of substances which are contaminants in a sample comprising target RNA prepared by in vitro transcription, the method comprising the steps of: a) preparing a sample comprising the target RNA; b) separating the target RNA from the substances by liquid chromatography, wherein the liquid chromatography is ion-pair reversed-phase chromatography, wherein the ion pair comprises a primary (C3-8)alkylamine or a salt thereof and the mobile phase used in the ion-pair reversed-phase chromatography comprises between 5 and 350 mM 1,1,1,3,3,3-hexafhioroisopropanol; and c) analysis of the substances by one or both of: i) ultraviolet-visible spectroscopy and ii) mass spectroscopy.

2. A method according to claim 1, comprising the following additional step after step a) but before step b): a’) purifying the target RNA.

3. A method according to claim 2, wherein the purifying step a’) removes components of the in vitro transcription method from the sample.

4. A method according to claim 3, wherein the components comprise (i) products that are introduced into the in vitro transcription method or (ii) by-products of the in vitro transcription method.

5. A method according to claim 3 or 4, wherein the products or components comprise proteins, DNA and salts.

6. A method according to any preceding claim, wherein the substances to be determined are selected from the group consisting of oligonucleotides, nucleotides, nucleosides, and cap analogs.

7. A method according to any preceding claim, wherein the analysis by ultraviolet- visible spectroscopy comprises quantification of the substances.

8. A method according to claim 7, wherein the quantification is selected from relative quantification and absolute quantification.

9. A method according to any preceding claim, wherein the analysis by ultraviolet- visible spectroscopy comprises characterization of the substances.

10. A method according to any preceding claim, wherein the analysis by mass spectroscopy comprises characterization of the substances.

11. A method according to any one of claims 1 to 10, wherein the liquid chromatography used in step b) uses a mixture of an aqueous solvent and an organic solvent as mobile phase.

12. A method according to claim 11, wherein the organic solvent is a Cl-4 alcohol, a C2-4 nitrile, a C3-4 ketone, or a mixture thereof.

13. A method according to claim 12, wherein the organic solvent is acetonitrile, methanol or isopropanol or a mixture thereof.

14. A method according to any one of claims 1 to 13, where the liquid chromatography used in step b) comprises elution from a liquid chromatography column by means of gradient separation.