Purification of ribonucleic acid oligonucleotides

Abstract

The presently claimed and described technology provides a chromatography method for purifying ribonucleic acid (RNA) comprising conditioning a sample comprising RNA with a binding liquid with a pH of between about 5.5 and 9.5 and optionally salt such as sodium or potassium chloride.

Description

PURIFICATION OF OLIGONUCLEOTIDESBACKGROUND

[0001] Oligonucleotides have emerged as an important class of therapeutics and vaccines in recent years and purification of ribonucleic acid (RNA) is a critical step that ensures the safety, efficacy, and quality of the final product. Purifying RNA is a challenging process due to its inherent instability and susceptibility to degradation. This intrinsic fragility necessitates stringent precautions during extraction and purification to preserve RNA integrity. Existing purification methods further complicate the process by exposing the RNA to high pH conditions. Balancing high yield with the need for high purity often involves trade-offs, requiring researchers to tailor methods to their specific samples and downstream needs. These challenges highlight the critical importance of using meticulous techniques, proper equipment, and RNase-free reagents to achieve successful RNA purification.

[0002] A need exists for a high yield RNA purification method that preserves RNA integrity without exposing the RNA to high pH conditions.BRIEF SUMMARY

[0003] One aspect of the disclosure is a method for purifying ribonucleic acid (RNA) comprising passing a sample with a pH of between about 5.5 and 9.5 and comprising RNA and optionally at least one salt through a chromatography adsorbent with a binding liquid, wherein the binding liquid has a pH of between about 5.5 and 9.5 and optionally comprises at least one salt, and wherein the RNA binds to the chromatography adsorbent; and eluting the RNA from the chromatography adsorbent with an elution liquid having a pH of between 5.5 and 9.5 and optionally comprising at least one salt.

[0004] In an aspect, the at least one salt is present in the binding and / or the elution liquid, independently of each other, in a concentration of between about 0 M and about 2 M; alternatively about 0.1 M; alternatively about 0.2 M; alternatively about 0.3 M; alternatively about 0.4 M; alternatively about 0.5 M; alternatively about 0.6 M; alternatively about 0.7 M; alternatively about 0.8 M; alternatively about 0.9 M; alternatively about 1.0 M; alternatively about 1.1 M; alternatively about 1.2 M; alternatively about 1.3 M; alternatively about 1.4 M; alternatively about 1.5 M;alternatively about 1.6 M; alternatively about 1.7 M; alternatively about 1.8 M; or alternatively about 1.9 M.

[0005] In an aspect, the at least one salt is selected from the group consisting of ammonium chloride, potassium chloride, sodium chloride, magnesium chloride, magnesium sulfate, magnesium nitrate, sodium citrate, ammonium sulfate, sodium sulfate, sodium acetate, potassium acetate, ammonium acetate, and combinations thereof.

[0006] In an aspect, the binding liquid and / or the elution liquid comprises at least a first salt and a second salt, wherein the first salt is selected from potassium chloride, sodium chloride, sodium acetate, potassium acetate, and ammonium acetate, and the second salt is selected from the group consisting of tris(hydroxymethyl)aminomethane, magnesium sulfate, magnesium nitrate, sodium citrate, ammonium sulfate, sodium sulfate, sodium acetate, potassium acetate, and ammonium acetate.

[0007] In an aspect, the elution liquid comprises at least one salt, wherein the at least one salt in the elution liquid and the at least one salt in the binding liquid are the same salt.

[0008] In an aspect, the pH of the binding liquid is about 6.0; alternatively about 5.5; alternatively about 6.5; alternatively about 7.0; alternatively about 7.5; alternatively about 8.0; alternatively about 8.5; alternatively about 9.

[0009] In an aspect, the eluted RNA has a purity of between about 70% and about 100%; alternatively about 71%, alternatively about 72%, alternatively about 73%, alternatively about 74%, alternatively about 75%, alternatively about 76%, alternatively about 77%, alternatively about 78%, alternatively about 79%, alternatively about 80%, alternatively about 81%, alternatively about 82%, alternatively about 83%, alternatively about 84%, alternatively about 85%, alternatively about 86%, alternatively about 87%, alternatively about 88%, alternatively about 89%, alternatively about 90%, alternatively about 91%, alternatively about 92%, alternatively about 93%, alternatively about 94%, alternatively about 95%, alternatively about 96%, alternatively about 97%, alternatively about 98%, or alternatively about 99%.

[0010] In an aspect, the binding liquid further comprises at least one buffering agent, chelating agent, chaotropic agent, alcohol, organic solvent, and / or polyol.

[0011] In an aspect, the buffering agent and / or chelating agent is selected from the group consisting of Tri s(hydroxymethyl)aminom ethane (TRIS), 2-(N-Morpholino)ethanesulfonic acid (MES), 4-(2-Hydroxyethyl)piperazine-l -ethanesulfonic acid (HEPES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-Tris(hydroxymethyl)methyl-3- aminopropanesulfonic acid (TAPSO), N-Bis(2 -hydroxy ethyl)-2-aminoethanesulfonic acid (BES), Piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES), Ethylenediaminetetraacetic acid (EDTA), Citric acid, Dimercaprol, Dithiothreitol, 3 Sodium 2, 3 -dimercaptopropane- 1 -sulfonate (DMPS), 2 Meso 2,3 -dimercaptosuccinic acid (DMSA), Pentetic acid or Diethylenetriaminepentaacetic acid (DTPA), Ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), N-(2- hydroxyethyl)ethylenediaminetriacetic acid (HEDTA), 5 Monoisoamyl DMSA (MIADMSA), nitrilotriacetic acid (NTA), Penicillamine, and combinations thereof.

[0012] In an aspect, the chaotropic agent is selected from the group consisting of urea, guanidine hydrochloride (GuHCl), guanidine thiocyanate (GuSCN), sodium iodide (Nal), sodium perchlorate (NaC104), potassium iodide (KI), lithium perchlorate (LiC104), thiourea, ammonium thiocyanate (NEESCN), 2-propanol, ethanol, and combinations thereof.

[0013] In an aspect, the polyol is selected from the group consisting of glycerol, propylene glycol, polyethylene glycol, ribitol, mannitol, and combinations thereof.

[0014] In an aspect, the sample is pretreated prior to the binding to the adsorbent.

[0015] In an aspect, the pretreatment comprises desalination, dilution, filtration, precipitation, denaturation, derivation, concentration, rehydration and / or decomplexation.

[0016] In an aspect, the chromatographic adsorbent is functionalized with a ligand that confers an ion exchange interaction, multimodal interaction (MMC) or hydrophobic interaction (HIC), and wherein a portion of the RNA binds to the chromatography adsorbent.

[0017] In an aspect, the chromatographic adsorbent comprises an ion exchange chromatography layer, resin or membrane, wherein a portion of the RNA binds to the ion exchange layer, resin, or membrane.

[0018] In an aspect, the ion exchange chromatography layer, resin, or membrane is an anion exchange chromatography layer, resin, or membrane.

[0019] In an aspect, the RNA is selected from the group consisting of transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), microRNA (miRNA), small interfering RNA (siRNA), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA) and small rDNA-derived RNA (srRNA).

[0020] In an aspect, the RNA comprises between about 10 nucleotides and about 30 nucleotides; alternatively about 11 nucleotides; alternatively about 12 nucleotides; alternatively about 13 nucleotides; alternatively about 14 nucleotides; alternatively about 15 nucleotides; alternatively about 16 nucleotides; alternatively about 17 nucleotides; alternatively about 18 nucleotides; alternatively about 19 nucleotides; alternatively about 20 nucleotides; alternatively about 21 nucleotides; alternatively about 22 nucleotides; alternatively about 23 nucleotides; alternatively about 24 nucleotides; alternatively about 25 nucleotides; alternatively about 26 nucleotides; alternatively about 27 nucleotides; alternatively about 28 nucleotides; alternatively about 29 nucleotides.

[0021] In an aspect, the RNA is derived from a natural source, such as a cell or a virus, by a chemical synthesis such as solid support-based oligo synthesis, in vitro transcription by an RNA polymerase, ligation-mediated synthesis, or a click-chemistry, such as azide or alkyne cycloadditions.

[0022] In an aspect, the RNA molecule is modified, at one or more positions, at the nucleobase, the sugar, the phosphodiester (PO) backbone or combinations thereof.

[0023] In an aspect, the backbone modification of the RNA is a boranophosphates (borano), phosphorothioates (PS), phosphorodithioates (PDS), phosphoramidates (PA), methylphosphonates (MP), amides (AM) or a triazole linkage (TL).

[0024] In an aspect, the sugar modification is a 2'-substituted sugar modification, a 2'-alkoxy sugar modification, a 2'-deoxy sugar modification, a 2 '-deoxy-2 '-fluoro sugar modification, a 2'-fluoro sugar modification, a 2'-methoxy sugar modification, a 2'-O-methyl sugar modification, a 2'-O- methoxyethyl sugar modification, a 2',4'-bridged nucleic acid (BNA) sugar modification, or a 2'- O,4'-C-ethylene-bridged nucleic acid (ENA) modification.

[0025] In an aspect, the nucleobase modification is a 2-thiouracil modification, a 2-thiocytosine modification, a 2-aminoadenine modification, a 2-aminopurine modification, a 2,6-diaminopurine modification, a 3 -nitropyrrole modification, a 4-thiouracil modification, a 5-substituted pyrimidines modification, a 5 -nitroindole modification, a 6-thioguanine modification, a N2- substituted purines modification, a N6-substituted purines modification, a O6-substituted purines modification, or a pseudouracil modification.

[0026] In an aspect, the chromatography adsorbent is washed with the binding liquid before eluting the RNA.

[0027] In an aspect, the elution is performed with a gradient elution, step-wise elution or isocratic elution.

[0028] A second aspect of the disclosure is a kit for purifying RNA comprising a chromatography adsorbent and a binding liquid, wherein the binding liquid has a pH of between about 5.5 and 9.5, and instructions for use.

[0029] These and other advantages, aspects, and novel features of the present disclosure, as well as details of illustrated embodiments thereof, will be more fully understood from the following description and drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Various aspects of the present disclosure will now be described, by way of example only, with reference to the attached Figures, wherein:

[0031] FIG. 1 is a chromatogram depicting the elution of RNA at pH 7.5 according to aspects of the disclosure with NaCl, 0.5 M Urea & NaCl, 1.0 M Urea & NaCl, or KC1 elution liquids.

[0032] FIG. 2 is a chromatogram depicting the elution of RNA at pH 9.0 according to aspects of the disclosure with NaCl, KC1, or KC1 and glycerol elution liquids.DETAILED DESCRIPTIONI. Introduction

[0033] Impurities found in RNA samples often include genomic DNA, proteins, organic solvents, salts, and phenols. Proteins, particularly ribonucleases, can also contaminate RNA preparations, and these enzymes, if not properly removed, can degrade the RNA. Residual organic solvents, such as phenol and chloroform may remain from earlier extraction steps. Salts or chemical byproducts from the extraction and / or precipitation of RNA can also be impurities in an RNA sample.

[0034] RNA is inherently unstable, with its 2’-hydroxyl group on the ribose sugar making it prone to hydrolysis. This instability can lead to degradation if the elution conditions — such as temperature, pH, and ionic strength — are not carefully optimized. Additionally, elution liquids must avoid harsh conditions that might compromise RNA integrity, while still ensuring efficient recovery from binding matrices, which can sometimes require fine-tuning for different RNA types or sizes. For example, purified mRNA must maintain its integrity, stability, and proper capping to ensure efficient delivery and translation in host cells. As such, eluting intact RNA duringpurification is particularly challenging due to its chemical properties and the delicate nature of the purification process.

[0035] Strong binding between RNA and a purification matrix can also make elution difficult, especially for larger or structured RNAs like ribosomal RNA or certain long non-coding RNAs. Ensuring efficient elution without degrading the RNA often necessitates liquids with specific compositions which might not be optimal for all downstream applications. Conversely, incomplete elution can lead to significant loss of RNA, particularly when working with small or low- abundance samples.

[0036] Furthermore, RNase contamination remains a persistent threat during the elution step. Even small amounts of RNase introduced through reagents, equipment, or the environment can degrade the RNA during or immediately after elution. Maintaining RNase-free conditions is especially critical because the elution liquid lacks the protective inhibitors typically present earlier in the extraction process.

[0037] A need exists for an RNA conditioning and purification method that preserves RNA integrity. In some aspects, the methods disclosed herein are used to condition and purify RNA in a manner where the RNA remains intact after elution. In some embodiments, the methods include conditioning the RNA with a liquid that has a pH of between about 5.5 and 9.5 and optionally one salt, passing a sample comprising RNA through a chromatography adsorbent, wherein at least a portion of the RNA binds to the chromatography adsorbent; and eluting the RNA from the chromatography adsorbent with an elution liquid.II. Definitions

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods described herein belong. Any reference to standard methods refers to the most recent available version of the method at the time of filing of this disclosure unless otherwise indicated.

[0039] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0040] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0041] The words “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments or aspects does not imply that other embodiments or aspects are not useful and is not intended to exclude other embodiments or aspects from the scope of the invention.

[0042] The term “comprises” and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0043] By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. “Consisting essentially of’ means including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0044] The singular form “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. These articles refer to one or to more than one (i.e., to at least one). As used herein, the term “or” is generally employed in its usual sense including “and / or” unless the content clearly dictates otherwise. The term “and / or” means any one or more of the items in the list joined by “and / or”. As an example, “x and / or y” means any element of the three-element set {(x), (y), (x, y)}. In other words, “x and / or y” means “one or both of x and y”. As another example, ”x, y, and / or z” means any element of the seven-element set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z)}. In other words, “x, y and / or z” means “one or more of x, y and z”.

[0045] Where ranges are given, endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Herein, “up to” a number (for example, up to 50)includes the number (for example, 50). The term “in the range” or “within a range” (and similar statements) includes the endpoints of the stated range.

[0046] Reference throughout this specification to “one aspect,” “an aspect,” “certain aspects,” or “some aspects,” “one embodiment,” “an embodiment,” “certain embodiment,” or “some embodiment,” etc., means that a particular feature, configuration, composition, or characteristic described in connection with the aspect is included in at least one aspect of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects.

[0047] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” As used herein in connection with a measured quantity, the term “about” refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The term “about” as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is + / -10%. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0048] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0049] The term “exemplary” means serving as a non-limiting example, instance, or illustration. As utilized herein, the terms “e.g.,” and “for example” set off lists of one or more non-limiting aspects, examples, instances, or illustrations.

[0050] As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. Biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, “substantially” may refer to being within at least about 20%, alternatively at least about 10%, alternatively at least about 5% of a characteristic or property of interest.

[0051] The invention is defined in the claims. However, below is a non-exhaustive listing of nonlimiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.III. Ribonucleic acid (RNA) purification processes

[0052] Ribonucleic acid (RNA) is typically single-stranded, allowing it to fold into complex three- dimensional shapes. This flexibility enables RNA to perform a variety of functions beyond genetic coding, such as catalysis and molecular signaling. Its presence in both the transcription of DNA into RNA and the translation of RNA into proteins makes it central to gene expression and cellular function.

[0053] RNA exists in several forms, each serving specific roles. Messenger RNA (mRNA) carries genetic instructions from DNA to ribosomes, where it guides protein synthesis. Self-amplifying RNA (saRNA) encodes both the target protein and replication machinery, enabling its amplification within cells to produce more protein from a smaller initial dose. Transfer RNA (tRNA) and ribosomal RNA (rRNA) play key roles in the translation process by delivering amino acids and forming the structural framework of ribosomes, respectively. Non-coding RNAs, such as small nuclear RNA (snRNA), microRNA (miRNA), small interfering RNA (siRNA), long noncoding RNA (IncRNA), piwi -interacting RNA (piRNA), tRNA-derived small RNA (tsRNA), and small rDNA-derived RNA (srRNA), regulate gene expression and participate in cellular processes like RNA splicing, regulating gene expression, gene silencing, and chromatin remodeling.

[0054] Hydrophobic Interaction Chromatography (HIC) is commonly used for RNA purification; however, this method suffers from several drawbacks. It may be challenging to differentiate between RNA species with similar hydrophobic properties, leading to co-purification of impurities or incomplete separation; HIC columns may have reduced binding capacity for large RNA molecules due to steric hindrance or insufficient hydrophobic regions; and the method mayexacerbate RNA degradation, especially if RNases are present or conditions are suboptimal for RNA stability.

[0055] While anion exchange chromatography is preferred, it is not employed due to the strong binding of RNA to the sorbent which can make elution challenging, even with high salt concentrations. This powerful interaction often requires harsh conditions, which risk compromising the integrity of the RNA. As a result, achieving efficient recovery while maintaining product quality can be a significant obstacle.

[0056] The inventors have developed a surprising and unexpected method that leverages a binding liquid with a pH of between about 5.5 and 9.5 with optionally at least one salt and a elution liquid which allows for high purification of intact RNA. The disclosed methods are also novel and inventive in that no high pH liquid is required, and a purity of RNA in excess of 95% is achievable.

[0057] In an aspect, the method includes passing a sample comprising RNA through a chromatography absorbent, wherein at least a portion of the RNA binds to the chromatography absorbent.

[0058] As used herein, “chromatography adsorbent” or “chromatographic adsorbent” refer to an affinity capture media used in chromatographic separation techniques to separate components of a mixture based on their varying interactions with the adsorbent. Chromatography adsorbents may vary in polarity, pore size, surface area, or surface chemistries depending on the components to be separated. The chromatography adsorbent may be in a variety of forms. For example, the chromatography adsorbent may be a layer, a resin, a monolith or a membrane, wherein at least a portion of the RNA binds to adsorbent layer, resin, monolith or membrane. The chromatography adsorbent may include characteristics that provide additional separation functionalities. For example, the chromatography adsorbent may include a magnetic or magnetically susceptible core that facilitates separation via a magnetic field. Examples of chromatography adsorbents include Sera-Mag Oligo(dT)-Coated Magnetic Particles (Cytiva®), Heparin Sepharose CL-6B (Cytiva®), SOURCE™ 30Q and SOURCE™ 30S high performance ion exchange chromatography resins (Cytiva®).

[0059] The chromatography adsorbent may be an ion exchange chromatography adsorbent. The chromatography adsorbent may include ion exchange resins that can either be cationic (negatively charged to attract and bind positively charged ions) or anionic (positively charged to attract and bind negatively charged ions).

[0060] The chromatography adsorbent may include an ion exchange chromatography layer, resin or membrane, wherein at least a portion of the RNA binds to the ion exchange chromatography layer, resin, or membrane. In another aspect, the ion exchange chromatography layer, resin, or membrane is an anion exchange chromatography layer, resin, or membrane. In this aspect, the negatively charged RNA molecules bind to the positively charged functional groups on the anion exchange chromatography layer, resin, or membrane. In certain embodiments, the chromatographic adsorbent is functionalized with a ligand that confer ion exchange interaction, multimodal interaction (MMC) or Hydrophobic interaction (HIC), and wherein a portion of the RNA binds to the chromatography adsorbent.

[0061] In an aspect, the bound RNA may be eluted from the chromatography adsorbent with an elution liquid. The elution liquid contains components that compete with the RNA for binding sites on the chromatography layer, resin, or membrane, causing the RNA to elute from the chromatography adsorbent. In some aspect, the elution is performed using a gradient elution, step- wise elution, or isocratic elution.

[0062] A gradient elution involves continuously varying the mobile phase composition (e.g., increasing solvent strength) to effect the separation of the RNA. A step-wise elution uses discrete changes in mobile phase composition at specific intervals, providing controlled separation of the RNA. In contrast, isocratic elution maintains a constant mobile phase composition throughout the elution process.

[0063] In an aspect, the chromatography adsorbent is contained within a chromatography device. In another aspect of the disclosure the chromatography adsorbent is any adsorbent usable in a chromatography device. As used herein, “chromatography device” refers to an apparatus used in the process of chromatography. Various chromatography devices are commercially available including the Mustang® E, Q and S chromatography membranes, or the Capto Q or Capto S resins (Cytiva®). The chromatography device may be part of a chromatography system. Various chromatography systems are commercially available including the AKTA® avant chromatography system (Cytiva®). Chromatography devices useful in the disclosed processes are described in U.S. Pub. No. 2023 / 0331773 and U.S. Pat. No. 7,094,347 each of which is incorporated by reference in its entirety.

[0064] In another aspect of the disclosure, the chromatography adsorbent is not used with a chromatography device. For example, the chromatography adsorbent may be mixed with a sample in a flask as a conditioning step.

[0065] The RNA in the RNA containing sample may also be derived from various sources depending on its intended use and production methods. In certain embodiments, the RNA is derived from a natural source, such as a cell or a virus. These include, but are not limited to, a bacterial culture, cells, cell culture, cell lysate, recombinant plasmids, genetically modified plasmids, and synthetic plasmids.

[0066] In an aspect, the RNA in the RNA containing sample is synthesized via cellular transcription.

[0067] Cellular transcription is a natural process occurring within cells, driven by RNA polymerase enzymes that transcribe DNA into RNA during gene expression. In eukaryotic cells, transcription occurs in the nucleus and involves multiple steps, including the addition of a 5’ cap, splicing to remove introns, and polyadenylation to produce mature RNA. Cellular transcription ensures precise regulation of RNA synthesis in response to environmental or developmental cues, producing diverse RNA species with highly specific roles. While this method is less suited for large-scale or modified RNA production due to its complexity and dependence on cellular machinery.

[0068] .

[0069] In some aspects, the RNA is derived from a chemical synthesis such as solid support-based oligo synthesis, in vitro transcription (IVT) by an RNA polymerase, , or a click-chemistry, such as azide or alkyne cycloadditions.

[0070] Solid support-based oligo synthesis involves synthesizing RNA using a resin a bead. This stepwise process, results in the sequential addition of nucleoside phosphoramidites facilitated by coupling agents. After the desired RNA sequence is assembled, the oligonucleotide is cleaved from the solid support and purified to remove byproducts and truncated sequences.

[0071] IVT is a cell-free method that uses purified enzymes, such as T7, SP6, or T3 RNA polymerases, to synthesize RNA from a DNA template containing the corresponding promoter. This highly controllable process allows for the production of custom RNA sequences with modifications, such as 5' caps or poly(A) tails, essential for stability and functionality inapplications like mRNA vaccines. IVT offers scalability and precision, enabling the rapid production of RNA for therapeutic and experimental purposes.

[0072] Ligation-mediated synthesis involves attaching specific RNA or DNA adapters to the ends of RNA molecules using RNA ligase, followed by reverse transcription to generate complementary DNA (cDNA). The ligation step allows for the precise addition of known sequences to the RNA, enabling efficient amplification and analysis.

[0073] Deriving RNA through click-chemistry involves employing bioorthogonal reactions to synthesize, label, or modify RNA molecules with high specificity and efficiency. For example, copper-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC), allows for the covalent attachment of functional groups or probes to RNA.

[0074] In other aspects the sample may be an environmental sample, clinical sample or food sample. The sample may be a fluid or liquid sample. In some aspects, the sample may be resuspended or reconstituted prior to passing through the chromatography adsorbent.

[0075] In certain embodiments, the RNA molecule is modified, at one or more positions, at the nucleobase, the sugar, the phosphodiester (PO) backbone or combinations thereof.

[0076] In some embodiments, the backbone modification is a boranophosphates (borano), phosphorothioates (PS), phosphorodithioates (PDS), phosphoramidates (PA), methylphosphonates (MP), amides (AM) or a triazole linkage (TL). In some embodiments, the sugar modifications is a 2'-substituted sugar modification, a 2'-alkoxy sugar modification, a 2'- deoxy sugar modification, a 2 '-deoxy-2 '-fluoro sugar modification, a 2'-fluoro sugar modification, a 2'-methoxy sugar modification, a 2'-O-methyl sugar modification, a 2'-O-methoxyethyl sugar modification, a 2',4'-bridged nucleic acid (BNA) sugar modification, or a 2'-O,4'-C-ethylene- bridged nucleic acid (ENA) modification. In some embodiments, the nucleobase modification is a 2-thiouracil modification, a 2-thiocytosine modification, a 2-aminoadenine modification, a 2- aminopurine modification, a 2,6-diaminopurine modification, a 3 -nitropyrrole modification, a 4- thiouracil modification, a 5-substituted pyrimidines modification, a 5-nitroindole modification, a 6-thioguanine modification, a N2-substituted purines modification, a N6-substituted purines modification, a ( -substituted purines modification, or a pseudouracil modification.

[0077] In certain embodiments, the RNA includes, but is not limited to, transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), microRNA (miRNA), small interferingRNA (siRNA), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA) and small rDNA-derived RNA (srRNA).

[0078] In certain embodiments, the RNA comprises between about 10 nucleotides and about 30 nucleotides; alternatively about 11 nucleotides; alternatively about 12 nucleotides; alternatively about 13 nucleotides; alternatively about 14 nucleotides; alternatively about 15 nucleotides; alternatively about 16 nucleotides; alternatively about 17 nucleotides; alternatively about 18 nucleotides; alternatively about 19 nucleotides; alternatively about 20 nucleotides; alternatively about 21 nucleotides; alternatively about 22 nucleotides; alternatively about 23 nucleotides; alternatively about 24 nucleotides; alternatively about 25 nucleotides; alternatively about 26 nucleotides; alternatively about 27 nucleotides; alternatively about 28 nucleotides; alternatively about 29 nucleotides.

[0079] In certain embodiments, the RNA containing sample has a pH of between about 5.5 and about 9.5, alternatively at about 5.5, alternatively at about 5.6, alternatively at about 5.7, alternatively at about 5.8, alternatively at about 5.9, alternatively at about 6.0, alternatively at about6.1, alternatively at about 6.2, alternatively at about 6.3, alternatively at about 6.4, alternatively at about 6.5, alternatively at about 6.6, alternatively at about 6.7, alternatively at about 6.8, alternatively at about 6.9, alternatively at about 7.0, alternatively at about 7.1, alternatively at about7.2, alternatively at about 7.3, alternatively at about 7.4, alternatively at about 7.5, alternatively at about 7.6, alternatively at about 7.7, alternatively at about 7.8, alternatively at about 7.9, alternatively at about 8.0, alternatively at about 8.1, alternatively at about 8.2, alternatively at about8.3, alternatively at about 8.4, alternatively at about 8.5, alternatively at about 8.6, alternatively at about 8.7, alternatively at about 8.8, alternatively at about 8.9, alternatively at about 9.0, alternatively at about 9.1, alternatively at about 9.2, alternatively at about 9.3, alternatively at about9.4, or alternatively at about 9.5.

[0080] In an aspect, the RNA containing sample additionally comprises at least one salt selected from the following group: ammonium chloride, potassium chloride, sodium chloride, magnesium chloride, magnesium sulfate, magnesium nitrate, sodium citrate, ammonium sulfate, sodium sulfate, sodium acetate, potassium acetate, ammonium acetate, and combinations thereof.

[0081] In an aspect, the salt in the RNA containing sample may be present in a concentration of about 0 M and about 2 M; alternatively about 0.01 M; alternatively about 0.02 M; alternatively about 0.03 M; alternatively about 0.04 M; alternatively about 0.05 M; alternatively about 0.06 M;alternatively about 0.07 M; alternatively about 0.08 M; alternatively about 0.09 M; alternatively about 0.1 M; alternatively about 0.2 M; alternatively about 0.3 M; alternatively about 0.4 M; alternatively about 0.5 M; alternatively about 0.6 M; alternatively about 0.7 M; alternatively about 0.8 M; alternatively about 0.9 M; alternatively about 1.0 M; alternatively about 1.1 M; alternatively about 1.2 M; alternatively about 1.3 M; alternatively about 1.4 M; alternatively about 1.5 M; alternatively about 1.6 M; alternatively about 1.7 M; alternatively about 1.8 M; or alternatively about 1.9 M.

[0082] In certain embodiments, the sample is pretreated prior to the RNA binding to the adsorbent. In certain embodiments, the pretreatment comprises desalination, dilution, filtration, precipitation, denaturation, derivation, concentration, rehydration and / or decomplexation.

[0083] In certain embodiments, the binding liquid has a pH of between about 5.5 and 9.5, alternatively at about 5.5, alternatively at about 5.6, alternatively at about 5.7, alternatively at about5.8, alternatively at about 5.9, alternatively at about 6.0, alternatively at about 6.1, alternatively at about 6.2, alternatively at about 6.3, alternatively at about 6.4, alternatively at about 6.5, alternatively at about 6.6, alternatively at about 6.7, alternatively at about 6.8, alternatively at about6.9, alternatively at about 7.0, alternatively at about 7.1, alternatively at about 7.2, alternatively at about 7.3, alternatively at about 7.4, alternatively at about 7.5, alternatively at about 7.6, alternatively at about 7.7, alternatively at about 7.8, alternatively at about 7.9, alternatively at about 8.0, alternatively at about 8.1, alternatively at about 8.2, alternatively at about 8.3, alternatively at about 8.4, alternatively at about 8.5, alternatively at about 8.6, alternatively at about 8.7, alternatively at about 8.8, alternatively at about 8.9, alternatively at about 9.0, alternatively at about 9.1, alternatively at about 9.2, alternatively at about 9.3, alternatively at about 9.4, or alternatively at about 9.5.

[0084] In certain embodiments, the binding liquid further comprises at least one salt including, but not limited to, ammonium chloride, potassium chloride, sodium chloride, magnesium chloride, magnesium sulfate, magnesium nitrate, sodium citrate, ammonium sulfate, sodium sulfate, sodium acetate, potassium acetate, ammonium acetate, and combinations thereof. The salt may be present in the binding liquid in a concentration of about 0 M and about 2 M; alternatively about 0.1 M; alternatively about 0.2 M; alternatively about 0.3 M; alternatively about 0.4 M; alternatively about 0.5 M; alternatively about 0.6 M; alternatively about 0.7 M; alternatively about 0.8 M; alternatively about 0.9 M; alternatively about 1.0 M; alternatively about 1.1 M; alternatively about 1.2 M;alternatively about 1.3 M; alternatively about 1.4 M; alternatively about 1.5 M; alternatively about 1.6 M; alternatively about 1.7 M; alternatively about 1.8 M; or alternatively about 1.9 M.

[0085] In certain embodiments, the binding liquid further comprises least one buffering agent, chelating agent, alcohol, organic solvent, chaotropic agent, and / or polyol. In certain embodiments, the buffering agent and / or chelating agent includes, but is not limited to, Tris(hydroxymethyl)aminomethane (TRIS), 2-(N-Morpholino)ethanesulfonic acid (MES), 4-(2- Hydroxyethyl)piperazine-1 -ethanesulfonic acid (HEPES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPSO), N-Bis(2- hydroxyethyl)-2-aminoethanesulfonic acid (BES), Piperazine-N,N' -bis(2-ethanesulfonic acid)(PIPES), N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES), Ethylenediaminetetraacetic acid (EDTA), Citric acid, Dimercaprol, Dithiothreitol, 3 Sodium 2,3 -dimercaptopropane- 1 -sulfonate (DMPS), 2 Meso 2,3 -dimercaptosuccinic acid (DMSA), Pentetic acid or Diethylenetriaminepentaacetic acid (DTPA), Ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), N-(2-hydroxy ethyl) ethylenediaminetriacetic acid (HEDTA), 5 Monoisoamyl DMSA (MIADMSA), nitrilotriacetic acid (NTA), Penicillamine, and combinations thereof.

[0086] In certain embodiments, the alcohol in the buffering agent includes, but is not limited to, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol and isobutanol. In certain embodiments, the organic solvent includes, but is not limited to, Acetic Acid, Acetone, Acetonitrile, Benzene, Butanol, Carbon Tetrachloride, Chloroform, Dichloromethane, Diethyl Ether, Dimethyl Sulfoxide (DMSO), Ethanol, Ethylenediamine (EDA), Ethyleneglycol, Ethyleneglycol Monomethyl Ether, Ethylpropyl Ether, Hexane, Isopropyl Alcohol, Methanol, Methyl Ethyl Ketone, Methylethyl Ether, Methylpropyl Ether, Methylpropyl Ether, N,N-Dimethylformamide (DMF), Naphtha, Neopentanol, Pentanol, Propanol, Toluene, and Xylene.

[0087] In certain embodiments, the buffering agent is TRIS and is present in a concentration of between about 5 mM and 100 mM, alternatively about 5 mM, alternatively about 10 mM, alternatively about 15 mM, alternatively about 20 mM, alternatively about 25 mM, alternatively about 30 mM, alternatively about 35 mM, alternatively about 40 mM, alternatively about 45 mM, alternatively about 50 mM, alternatively about 55 mM, alternatively about 60 mM, alternatively about 65 mM, alternatively about 70 mM, alternatively about 75 mM, alternatively about 80 mM, alternatively about 85 mM, alternatively about 90 mM, alternatively about 95 mM, or alternatively about 100 mM.

[0088] In certain embodiments, the chaotropic agent is selected from the group consisting of urea, guanidine hydrochloride (GuHCl), guanidine thiocyanate (GuSCN), sodium iodide (Nal), sodium perchlorate (NaC104), potassium iodide (KI), lithium perchlorate (LiC104), thiourea, ammonium thiocyanate (NH4SCN), 2-propanol, ethanol, and combinations thereof. In certain embodiments, the polyol is selected from the group consisting of glycerol, polyethylene glycol, propylene glycol, ribitol, mannitol, and combinations thereof.

[0089] In certain embodiments, the elution liquid has at least about 0.80 M of at least one salt. In some embodiments, the salt is present in a concentration of between about 0.80 M and 1.20 M, alternatively about 0.82 M, alternatively about 0.84 M, alternatively about 0.86 M, alternatively about 0.88 M, alternatively about 0.90 M, alternatively about 0.92 M, alternatively about 0.94 M, alternatively about 0.96 M, alternatively about 0.98 M, alternatively about 1.00 M, alternatively about 1.02 M, alternatively about 1.04 M, alternatively about 1.06 M, alternatively about 1.08 M, alternatively about 1.10 M, alternatively about 1.12 M, alternatively about 1.14 M, alternatively about 1.16 M, or alternatively about 1.18 M. In certain embodiments, the salt may be ammonium chloride, potassium chloride, sodium chloride, magnesium chloride, magnesium sulfate, magnesium nitrate, sodium citrate, ammonium sulfate, and combinations thereof.

[0090] In certain embodiments, the elution liquid has a pH of between about 5.5 and 9.5, alternatively at about 5.5, alternatively at about 5.6, alternatively at about 5.7, alternatively at about5.8, alternatively at about 5.9, alternatively at about 6.0, alternatively at about 6.1, alternatively at about 6.2, alternatively at about 6.3, alternatively at about 6.4, alternatively at about 6.5, alternatively at about 6.6, alternatively at about 6.7, alternatively at about 6.8, alternatively at about6.9, alternatively at about 7.0, alternatively at about 7.1, alternatively at about 7.2, alternatively at about 7.3, alternatively at about 7.4, alternatively at about 7.5, alternatively at about 7.6, alternatively at about 7.7, alternatively at about 7.8, alternatively at about 7.9, alternatively at about 8.0, alternatively at about 8.1, alternatively at about 8.2, alternatively at about 8.3, alternatively at about 8.4, alternatively at about 8.5, alternatively at about 8.6, alternatively at about 8.7, alternatively at about 8.8, alternatively at about 8.9, alternatively at about 9.0, alternatively at about 9.1, alternatively at about 9.2, alternatively at about 9.3, alternatively at about 9.4, or alternatively at about 9.5. In certain embodiments, the pH of the elution liquid is the same or about the same as the pH of the binding liquid. In other certain embodiments, the pH of the elution liquid is different than the pH of the binding liquid.

[0091] In certain embodiments, the salt of the elution liquid and the salt of the binding liquid are the same salt. In alternate embodiments, the salt of the binding liquid is different than the salt of the elution liquid. In certain embodiments, the chromatography adsorbent is washed with the binding liquid before eluting the RNA.

[0092] In an embodiment, the elution liquid may also include at least one basic amino acid. A basic amino acid is an amino acid that has a side chain (R group) that is positively charged at physiological pH (around 7.4). This positive charge is due to the presence of an extra amino group ( — NIL), which tends to accept a proton (H+). Non-limiting examples of basic amino acids include arginine, histidine, and lysine.

[0093] In an embodiment, the elution liquid comprises between about 0.09 M and about 1 M of at least one basic amino acid. In some embodiments, the elution liquid comprises more than 1 M of at least one basic amino acid. A basic amino acid is an amino acid that has a side chain (R group) that is positively charged at physiological pH (around 7.4). This positive charge is due to the presence of an extra amino group ( — NIL), which tends to accept a proton (H+). Non-limiting examples of basic amino acids include arginine, histidine, and lysine.

[0094] In some embodiments, the elution liquid comprises about 0.05 M and about 1 M of the at least one basic amino acid, alternatively about 0.075 M of at least one basic amino acid, alternatively about 0.1 M of at least one basic amino acid, alternatively about 0.125 M of at least one basic amino acid, alternatively about 0.15 M of at least one basic amino acid, alternatively about 0.2 M of at least one basic amino acid, alternatively about 0.25 M of at least one basic amino acid, alternatively about 0.3 M of at least one basic amino acid, alternatively about 0.35 M of at least one basic amino acid, alternatively about 0.4 M of at least one basic amino acid, alternatively about 0.45 M of at least one basic amino acid, alternatively about 0.5 M of at least one basic amino acid, alternatively about 0.55 M of at least one basic amino acid, alternatively about 0.6 M of at least one basic amino acid, alternatively about 0.65 M of at least one basic amino acid, alternatively about 0.7 M of at least one basic amino acid, alternatively about 0.75 M of at least one basic amino acid, alternatively about 0.8 M of at least one basic amino acid, alternatively about 0.85 M of at least one basic amino acid, alternatively about 0.9 M of at least one basic amino acid, or alternatively about 0.95 M of at least one basic amino acid.

[0095] In some aspects, the elution liquid comprises two or more basic amino acids or three or more basic amino acids.■

[0096] In an embodiment, the elution liquid comprises between about 0.05 M and about 1 M of at least one denaturant. In an embodiment, the elution liquid comprises more than about 1 M of at least one denaturant. A denaturant includes substances, such as, but not limited to urea, guanidine, and detergents (e.g., Sodium dodecyl sulfate), which interfere with non-covalent interactions - like hydrogen bonds, hydrophobic interactions, and ionic bonds.

[0097] In some embodiments, the elution liquid comprises about 0.075 M of at least one denaturant, alternatively about 0.1 M of at least one denaturant, alternatively about 0.125 M of at least one denaturant, alternatively about 0.15 M of at least one denaturant, alternatively about 0.2 M of at least one denaturant, alternatively about 0.25 M of at least one denaturant, alternatively about 0.3 M of at least one denaturant, alternatively about 0.35 M of at least one denaturant, alternatively about 0.4 M of at least one denaturant, alternatively about 0.45 M of at least one denaturant, alternatively about 0.5 M of at least one denaturant, alternatively about 0.55 M of at least one denaturant, alternatively about 0.6 M of at least one denaturant, alternatively about 0.65 M of at least one denaturant, alternatively about 0.7 M of at least one denaturant, alternatively about 0.75 M of at least one denaturant, alternatively about 0.8 M of at least one denaturant, alternatively about 0.85 M of at least one denaturant, alternatively about 0.9 M of at least one denaturant, or alternatively about 0.95 M of at least one denaturant.

[0098] In some aspects, the elution liquid comprises two or more denaturants or three or more denaturants.

[0099] In some aspects, the elution liquid comprises at least one basic amino acid and at least one denaturant. For example, in one embodiment, the elution liquid comprises about 0.1 M arginine and 0.1 M urea, alternatively about 0.2 M arginine and about 0.2 M urea, alternatively about 0.25 M arginine and about 0.5 M urea, or alternatively about 0.5 M arginine and about 1 M urea.

[0100] In another embodiment, the elution liquid comprises about 0.1 M arginine and 0.1 M guanidine, alternatively about 0.25 M arginine and about 0.25 M guanidine, or alternatively about 0.25 M arginine and about 0.5 M guanidine.

[0101] In another embodiment, the elution liquid comprises about 0.1 M histidine and 0.1 M urea, alternatively about 0.125 M histidine and about 0.25 M urea, alternatively about 0.25 M histidine and about 0.5 M urea, or alternatively about 0.5 M histidine and about 1 M urea.

[0102] The elution liquid may also comprise other suitable components including at least one buffering agent, chelating agent, alcohol, organic solvent, chaotropic agent, and / or polyol. In certain embodiments, the buffering agent and / or chelating agent includes, but is not limited to, Tris(hydroxymethyl)aminomethane (TRIS), 2-(N-Morpholino)ethanesulfonic acid (MES), 4-(2- Hydroxyethyl)piperazine-1 -ethanesulfonic acid (HEPES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPSO), N-Bis(2- hydroxyethyl)-2-aminoethanesulfonic acid (BES), Piperazine-N,N' -bis(2-ethanesulfonic acid)(PIPES), N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES), Ethylenediaminetetraacetic acid (EDTA), Citric acid, Dimercaprol, Dithiothreitol, 3 Sodium 2,3 -dimercaptopropane- 1 -sulfonate (DMPS), 2 Meso 2,3-dimercaptosuccinic acid (DMSA), Pentetic acid or Diethylenetriaminepentaacetic acid (DTPA), Ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), N-(2-hydroxy ethyl) ethylenediaminetriacetic acid (HEDTA), 5 Monoisoamyl DMSA (MIADMSA), nitrilotriacetic acid (NTA), Penicillamine, and combinations thereof.

[0103] In certain embodiments, the alcohol in the buffering agent includes, but is not limited to, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol and isobutanol. In certain embodiments, the organic solvent includes, but is not limited to, Acetic Acid, Acetone, Acetonitrile, Benzene, Butanol, Carbon Tetrachloride, Chloroform, Dichloromethane, Diethyl Ether, Dimethyl Sulfoxide (DMSO), Ethanol, Ethylenediamine (EDA), Ethyleneglycol, Ethyleneglycol Monomethyl Ether, Ethylpropyl Ether, Hexane, Isopropyl Alcohol, Methanol, Methyl Ethyl Ketone, Methylethyl Ether, Methylpropyl Ether, Methylpropyl Ether, N,N-Dimethylformamide (DMF), Naphtha, Neopentanol, Pentanol, Propanol, Toluene, and Xylene.

[0104] In certain embodiments, the chaotropic agent is selected from the group consisting of urea, guanidine hydrochloride (GuHCl), guanidine thiocyanate (GuSCN), sodium iodide (Nal), sodium perchlorate (NaCICri), potassium iodide (KI), lithium perchlorate (LiCICri), thiourea, ammonium thiocyanate (NH4SCN), 2-propanol, ethanol, and combinations thereof. In certain embodiments, the polyol is selected from the group consisting of glycerol, polyethylene glycol, propylene glycol, ribitol, mannitol, and combinations thereof.

[0105] In some aspects, the elution liquid may comprise a salt in a concentration of about 1 M or lower, alternatively in a concentration of about 750 mM or lower, alternatively in a concentration of about 500 mM or lower, alternatively in a concentration of about 250 mM or lower, alternativelyin a concentration of about 150 mM or lower, alternatively in a concentration of about 125 mM or lower, alternatively in a concentration of about 100 mM or lower, alternatively in a concentration of about 75 mM or lower, alternatively in a concentration of about 50 mM or lower, or alternatively in a concentration of about 25 mM or lower. In some aspects, the elution liquid may comprise a salt in a concentration of between about 25 mM and about 1000 mM. In some aspects, the salt may be ammonium chloride, potassium chloride, sodium chloride, magnesium chloride, magnesium sulfate, magnesium nitrate, sodium citrate, ammonium sulfate, or combinations thereof.

[0106] In certain embodiments, the eluted RNA has a purity of between about 70% and about 100%; alternatively about 71%, alternatively about 72%, alternatively about 73%, alternatively about 74%, alternatively about 75%, alternatively about 76%, alternatively about 77%, alternatively about 78%, alternatively about 79%, alternatively about 80%, alternatively about 81%, alternatively about 82%, alternatively about 83%, alternatively about 84%, alternatively about 85%, alternatively about 86%, alternatively about 87%, alternatively about 88%, alternatively about 89%, alternatively about 90%, alternatively about 91%, alternatively about 92%, alternatively about 93%, alternatively about 94%, alternatively about 95%, alternatively about 96%, alternatively about 97%, alternatively about 98%, or alternatively about 99%.

[0107] In certain aspects, the method results in a high yield or recovery of intact RNA. As used herein “% recovery” refers to the percentage of the initial amount of RNA that is successfully retrieved after a purification or processing step. A high % recovery indicates an efficient process with minimal loss of the RNA, while low % recovery suggests significant losses and potential areas for process improvement. In some aspects, % recovery may be referred to as % yield.

[0001] In some embodiments, the recovery of the purified intact RNA is between about 60% and about 100%, alternatively about 61%, alternatively about 62%, alternatively about 63%, alternatively about 64%, alternatively about 65%, alternatively about 66%, alternatively about 67%, alternatively about 68%, alternatively about 69%, alternatively about 70%, alternatively about 71%, alternatively about 72%, alternatively about 73%, alternatively about 74%, alternatively about 75%, alternatively at about 76%, alternatively at about 77%, alternatively at about 78%, alternatively at about 79%, alternatively at about 80%, alternatively at about 81%, alternatively at about 82%, alternatively at about 83%, alternatively at about 84%, alternatively at about 85%, alternatively at about 86%, alternatively at about 87%, alternatively at about 88%, alternatively at about 89%, alternatively at about 90%, alternatively at about 91%, alternatively atabout 92%, alternatively at about 93%, alternatively at about 94%, alternatively at about 95%, alternatively at about 96%, alternatively at about 97%, alternatively at about 98%, or alternatively at about 99%. In some embodiments, the recovery of the purified intact RNA is greater than 100%.

[0108] In certain embodiments, the method further comprises washing the chromatography device with a wash solution prior to eluting the RNA. In some aspects, the wash solution includes at least one salt selected from the group consisting of ammonium chloride, potassium chloride, sodium chloride, magnesium chloride, magnesium sulfate, magnesium nitrate, sodium citrate, ammonium sulfate, and combinations thereof. The salt may be present in a concentration of between about 0.80 M and 1.20 M, alternatively about 0.82 M, alternatively about 0.84 M, alternatively about 0.86 M, alternatively about 0.88 M, alternatively about 0.90 M, alternatively about 0.92 M, alternatively about 0.94 M, alternatively about 0.96 M, alternatively about 0.98 M, alternatively about 1.00 M, alternatively about 1.02 M, alternatively about 1.04 M, alternatively about 1.06 M, alternatively about 1.08 M, alternatively about 1.10 M, alternatively about 1.12 M, alternatively about 1.14 M, alternatively about 1.16 M, or alternatively about 1.18 M.

[0109] In an aspect, the method is carried out at temperature between about 20°C and about 30°C; alternatively at 21 °C, alternatively at 22°C, alternatively at 23 °C, alternatively at 24°C, alternatively at 25°C, alternatively at 26°C, alternatively at 27°C, alternatively at 28°C, or alternatively at 29°C.

[0110] One aspect of the disclosure includes a kit for purifying RNA comprising a chromatography adsorbent, a binding liquid with a pH of between about 5.5 and 9.5, and instructions for use.

[0111] The presently described technology and its advantages will be better understood by reference to the following examples. These examples are provided to describe specific implementations of the present technology. By providing these specific examples, it is not intended to limit the scope and spirit of the present technology. It will be understood by those skilled in the art that the full scope of the presently described technology encompasses the subject matter defined by the claims appending this specification, and any alterations, modifications, or equivalents of those claims.

[0112] EXAMPLES

[0113] Exemplary Purification of mRNA

[0114] Crude 21-mer RNA (trityl-off) was obtained for purification. Ten mL samples of the RNA were conditioned on a AKTA Pure 25 M, (Cytiva®) using a HiPrep 26 / 10 Desalting column (Cytiva®) with a binding liquid including either 20 mM Tris pH 7.5 pH or 25 mM Tris pH 9.0. Following the liquid exchange, additives urea, glycerol were added to some of the samples from stock solutions for further conditioning of the samples.

[0115] Table 1 lists exemplary binding liquids and elution liquids as described in the methods herein.

[0116] Table 1 : Binding liquids and elution liquids

[0117] The HiScreen Capto Q ImpRes column (Cytiva®) is equilibrated towards a binding liquid according to Table 1. Using a 10 mL Superloop® assembly (Cytiva®), 5 mL each of the corresponding conditioned samples (2.6 mg / mL) of Table 1 were loaded onto the HiScreen Capto Q ImpRes column at 155 cm / h and then washed with 5 column volumes (CV) of binding liquid. The RNA was eluted from the column using 15 CV 20% to 90% gradient towards the elution liquid using a flow rate of 100 cm / h. Two mL fractions of eluate were collected.

[0118] As shown in the conditions screening from pH 7.5 in Fig. 1, the full-length product (FLP) from 0.5 M Urea 05 and 1.0 M Urea 10 with NaCl gradient (Sample #2 and #3 respectively in Table 1) eluted earlier as compared to the NaCl gradient 15 (Sample #1 Table 1) and the KC1 gradient 20 (Sample #5 Table 1). The addition of urea did not significantly impact purity in the evaluated combinations as all were above 90% purity. In the pH 9.0 conditions screening, the full- length product (FLP) eluted earlier using a NaCl gradient 25 (Sample # 4 Table 1) as compared to KC1 gradient 30 (Sample #6 Table 1). The run with glycerol and KC1 gradient 35 (Sample #7 Table 1) at pH 9.0 resulted in the latest elution in the gradient as shown in Fig. 2. The sample with KC1 and glycerol at pH 9.0 also had the lowest conductivity in the elution liquid which may be partly attributed to the preparation of the sample where the volume increased when 85% glycerol was added to 1 M KC1 to make a 7.5% glycerol solution. However, comparing FLP elution peak at peak max, the conductivity is ~56 mS / cm in the glycerol and KC1 combination and ~73 mS / cm in the KC1 buffer.

[0119] The fractions of eluate correlating to the main peaks were analyzed by high performance liquid chromatography (HPLC). Samples (500 pl) were desalted using 5 mL HiTrap® Desalting column (Cytiva®) to 20 mM Tris pH 7.5 at a flow rate of 5 mL / min on a AKTA Pure 25 chromatography system (Cytiva®). The desalted eluate was analysed using an ion exchange DNAPac® PA200 RS AIEX column, 4 pm, 50x4.6 mm column (Thermo Fischer), to separate and quantify impurities from the FLP using 1.5 pg injections using an UltiMate 3000 HPLC system (Thermo Fisher).

[0120] Table 2 lists the purity, as measured by HPLC, of the eluate fractions from the HiScreen Capto Q ImpRes column with peak conductivity as described in the methods above.

[0121] Table 2 Purity of Main Peak Fractions

[0122] All features disclosed in the specification, including the claims, abstracts, and drawings, and all the steps in any method or process disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. Each feature disclosed in the specification, including the claims, abstract, and drawings, can be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0123] It will be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

CLAIMS1. A method for purifying ribonucleic acid (RNA) comprising: passing an RNA containing sample, wherein the sample has a pH of between about 5.5 and 9.5 and optionally comprises at least one salt through a chromatography adsorbent, with a binding liquid having a pH of between 5.5 and 9.5 and optionally comprising at least one salt, wherein at least a portion of the RNA binds to the chromatography adsorbent; and eluting at least a portion of the RNA from the chromatography adsorbent with an elution liquid having a pH of between 5.5 and 9.5 and optionally comprising at least one salt.

2. The method of claim 1, wherein the at least one salt is present in the binding liquid and / or elution liquid, independently of each other, in a concentration of between about 0 M and about 2 M; alternatively about 0.1 M; alternatively about 0.2 M; alternatively about 0.3 M; alternatively about 0.4 M; alternatively about 0.5 M; alternatively about 0.6 M; alternatively about 0.7 M; alternatively about 0.8 M; alternatively about 0.9 M; alternatively about 1.0 M; alternatively about 1.1 M; alternatively about 1.2 M; alternatively about 1.3 M; alternatively about 1.4 M; alternatively about 1.5 M; alternatively about 1.6 M; alternatively about 1.7 M; alternatively about 1.8 M; or alternatively about 1.9 M.

3. The method of claim 1 or claim 2, wherein the at least one salt is selected from the group consisting of ammonium chloride, potassium chloride, sodium chloride, magnesium chloride, magnesium sulfate, magnesium nitrate, sodium citrate, ammonium sulfate, sodium sulfate, sodium acetate, potassium acetate, ammonium acetate, and combinations thereof.

4. The method of claim 3, wherein the binding liquid and / or the elution liquid comprises at least a first salt and a second salt, wherein the first salt is selected from potassium chloride, sodium chloride, sodium acetate, potassium acetate, or ammonium acetate, and the second salt is selected from the group consisting of magnesium sulfate, magnesium nitrate, sodium citrate, ammonium sulfate, sodium sulfate, sodium acetate, potassium acetate, or ammonium acetate.

5. The method of any one of claims 1 to 4, wherein the elution liquid comprises at least one salt, wherein the at least one salt in the elution liquid and the at least one salt in the binding liquid are the same.

6. The method of any one of claims 1 to 5, wherein the pH of the binding liquid is about 6.0, alternatively about 6.5, alternatively 7.5; alternatively about 8.0; alternatively about 8.5; or alternatively about 9.

7. The method of any one of claims 1 to 6, wherein the eluted RNA has a purity of between about 70% and about 100%; alternatively about 71%, alternatively about 72%, alternatively about 73%, alternatively about 74%, alternatively about 75%, alternatively about 76%, alternatively about 77%, alternatively about 78%, alternatively about 79%, alternatively about 80%, alternatively about 81%, alternatively about 82%, alternatively about 83%, alternatively about 84%, alternatively about 85%, alternatively about 86%, alternatively about 87%, alternatively about 88%, alternatively about 89%, alternatively about 90%, alternatively about 91%, alternatively about 92%, alternatively about 93%, alternatively about 94%, alternatively about 95%, alternatively about 96%, alternatively about 97%, alternatively about 98%, or alternatively about 99%.

8. The method of any one of claims 1 to 7, wherein the binding liquid further comprises least one buffering agent, chelating agent, chaotropic agent, alcohol, organic solvent and / or polyol.

9. The method of claim 8, wherein the buffering agent and / or chelating agent is selected from the group consisting of Tri s(hydroxymethyl)aminom ethane (TRIS), 2-(N- Morpholino)ethanesulfonic acid (MES), 4-(2-Hydroxyethyl)piperazine-l -ethanesulfonic acid (HEPES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-Tris(hydroxymethyl)methyl-3- aminopropanesulfonic acid (TAPSO), N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), Piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), N-(2-Acetamido)-2- aminoethanesulfonic acid (ACES), Ethylenediaminetetraacetic acid (EDTA), Citric acid, Dimercaprol, Dithiothreitol, 3 Sodium 2,3 -dimercaptopropane- 1 -sulfonate (DMPS),2 Meso 2,3 -dimercaptosuccinic acid (DMSA), Pentetic acid or Diethylenetriaminepentaacetic acid (DTP A), Ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), N-(2- hydroxyethyl)ethylenediaminetriacetic acid (HEDTA), 5 Monoisoamyl DMSA (MIADMSA), nitrilotriacetic acid (NTA), Penicillamine, and combinations thereof.

10. The method of claim 8, wherein the chaotropic agent is selected from the group consisting of urea, guanidine hydrochloride (GuHCl), guanidine thiocyanate (GuSCN), sodium iodide (Nal), sodium perchlorate (NaC104), potassium iodide (KI), lithium perchlorate (LiC104), thiourea, ammonium thiocyanate (NEkSCN), 2-propanol, ethanol, and combinations thereof.

11. The method of claim 8, wherein the polyol is selected from the group consisting of glycerol, polyethylene glycol, propylene glycol, ribitol, mannitol, and combinations thereof.

12. The method of any one of claim 1 to 11, wherein the sample is pretreated prior to the RNA binding to the adsorbent.

13. The method of claim 12, wherein the pretreatment comprises desalination, dilution, filtration, precipitation, denaturation, derivation, concentration, rehydration and / or decomplexation.

14. The method of any one of claims 1 to 13, wherein the chromatographic adsorbent is functionalized with a ligand that confer ion exchange interaction, multimodal interaction (MMC) or Hydrophobic interaction (HIC), and wherein a portion of the RNA binds to the chromatography adsorbent.

15. The method of claim 14, wherein the chromatography adsorbent comprises an ion exchange chromatography layer, resin or membrane, wherein a portion of the RNA binds to the ion exchange layer, resin, or membrane.

16. The method of claim 14 or 15, wherein the ion exchange chromatography layer, resin, or membrane is an anion exchange chromatography layer, resin, or membrane.

17. The method of any one of claims 1 to 16, wherein the RNA is selected from the group consisting of transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), microRNA (miRNA), small interfering RNA (siRNA), Piwi-interacting RNA (piRNA), tRNA- derived small RNA (tsRNA) and small rDNA-derived RNA (srRNA).

18. The method of any one of claims 1 to 17, wherein the RNA comprises between about 10 nucleotides and about 30 nucleotides; alternatively about 11 nucleotides; alternatively about 12 nucleotides; alternatively about 13 nucleotides; alternatively about 14 nucleotides; alternatively about 15 nucleotides; alternatively about 16 nucleotides; alternatively about 17 nucleotides;alternatively about 18 nucleotides; alternatively about 19 nucleotides; alternatively about 20 nucleotides; alternatively about 21 nucleotides; alternatively about 22 nucleotides; alternatively about 23 nucleotides; alternatively about 24 nucleotides; alternatively about 25 nucleotides; alternatively about 26 nucleotides; alternatively about 27 nucleotides; alternatively about 28 nucleotides; or alternatively about 29 nucleotides.

19. The method of any one of claims 1 to 18, wherein the RNA is derived from a natural source, such as a cell or a virus, by a chemical synthesis such as solid support-based oligo synthesis, in vitro transcription by an RNA polymerase, ligase-mediated conjugation, or a Clickchemistry, such as azide or alkyne cycloadditions or by chemical conjugation .

20. The method of any one of claims 1 to 19 wherein the RNA is modified, at one or more positions, at the nucleobase, the sugar, the phosphodiester (PO) backbone or combinations thereof.

21. The method of claim 20, wherein the backbone modification is a boranophosphates (borano), phosphorothioates (PS), phosphorodithioates (PDS), phosphoramidates (PA), methylphosphonates (MP), amides (AM) or a triazole linkage (TL).

22. The method of claim 20, wherein the sugar modification is a 2 '-substituted sugar modification, a 2'-alkoxy sugar modification, a 2'-deoxy sugar modification, a 2 '-deoxy-2 '-fluoro sugar modification, a 2'-fluoro sugar modification, a 2'-methoxy sugar modification, a 2'-O-methyl sugar modification, a 2'-O-methoxyethyl sugar modification, a 2',4'-bridged nucleic acid (BNA) sugar modification, ora 2'-O,4'-C-ethylene-bridged nucleic acid (ENA) modification.

23. The method of claim 20, wherein the nucleobase modification is a 2-thiouracil modification, a 2-thiocytosine modification, a 2-aminoadenine modification, a 2-aminopurine modification, a 2,6-diaminopurine modification, a 3 -nitropyrrole modification, a 4-thiouracil modification, a 5-substituted pyrimidines modification, a 5-nitroindole modification, a 6-thioguanine modification, aN2-substituted purines modification, aN6-substituted purines modification, a O6-substituted purines modification, or a pseudouracil modification.

24. The method of any one of claims 1 to 23, further comprising the step of washing the adsorbent with the binding liquid before eluting the RNA.

25. The method of any one of claims 1 to 24, wherein the elution is performed with a gradient elution, step-wise elution, or isocratic elution.

26. A kit for purifying RNA comprising a chromatography device comprising a chromatographic adsorbent and a conditioning liquid, wherein the conditioning liquid has a pH of between about 5.5 and 9.5, and instructions for use.

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