Single-stranded RNA purification method

Abstract

applying a sample containing single-stranded RNA to a solid phase carrying predominantly or exclusively primary amino groups on its surface at a pH sufficient to at least predominantly bind said single-stranded RNA; eluting the adsorbed single-stranded RNA from the surface of the solid phase by exposing the surface of the solid phase to an increasing pH; A method for purifying single-stranded RNA, comprising:

Description

Technical Field

[0001] The present invention relates to a method for removing double-stranded RNA from an essentially aqueous mixture of double-stranded RNA and single-stranded RNA.

Background Art

[0002] The synthesis of messenger RNA (mRNA) for gene therapy applications results in preparations that contain an unwanted subpopulation of double-stranded (ds) RNA in addition to the desired single-stranded (ss) RNA. These dsRNA species form post-synthesis by intrastrand interactions of complementary sequences within the base strands. The formation of dsRNA sequences can also occur by pairing with complementary sequences between adjacent ssRNA molecules, thereby creating non-specific intermolecular dimers and higher-order multimers that can also contain intrastrand ds sequences. Double-stranded RNA, when injected into a subject, induces an unwanted potentially lethal immune response, and removing this is a particular goal of purification.

[0003] Methods for reducing the dsRNA content in mRNA preparations are known. The level of dsRNA contamination can be reduced by affinity adsorption chromatography using a cellulose-based chromatography medium [1-3]. The exact mechanism of adsorption is not certain, but dsRNA binds under certain conditions while ssRNA passes through. This method is simple and effective on a laboratory scale, but is burdensome due to its low capacity. The low capacity is associated with large columns that, on a manufacturing scale, reduce the productivity of manufacturing facilities, require large amounts of buffer, large amounts of manufacturing area, and extended process times. This method also causes dilution of the processed ssRNA, which is associated with an increase in the volume of the product that burdens subsequent purification steps.

[0004] Alternatively, dsRNA levels can be reduced by reversed-phase chromatography (RPC) using styrene-divinylbenzene (SDVB) solid phase [4-7]. Because RPC uses toxic, flammable organic solvents, it requires specialized equipment that is extremely expensive at industrial scale to mitigate the risk of fire and explosion. RPC also imposes further burdens on safety issues related to the toxicity of organic solvents in the work environment and the handling of hazardous waste. In addition to the solvent issues, RPC separation often imposes the further burden of requiring high temperatures to obtain the best results.

[0005] Anion exchange chromatography has proven useful in the purification of low-molecular-weight mRNA (less than 1000 nucleotides) [8]. Anion exchange media that have been evaluated to date include so-called strong anion exchangers, such as quaternary amine (QA) anion exchangers. So-called weak anion exchangers, particularly diethylaminoethyl (DEAE) anion exchangers using tertiary amine ligands, have also been evaluated.

[0006] Anion exchange chromatography has limited utility in removing DNA and protein contaminants from large mRNA (1,000–10,000 bases), and only at elevated operating temperatures [9]. Raising the temperature to 65°C allows for the elution of large mRNA in a sodium chloride gradient. However, high-temperature operations impose complex logistical burdens, as buffers, samples, and columns must all be pre-equilibriumized and precisely maintained at a specific operating temperature throughout the entire process, potentially for several years, across all batches, for the entire lifecycle of the product.

[0007] Proteins become more electrically positive and less electronegative as the pH decreases, which in turn stops their association with anion exchangers. Therefore, it is known that proteins are eluted from anion exchangers by a decreasing pH gradient. This approach does not work for RNA because its charge characteristics remain constant between approximately pH 2.6 and pH 13.0. Increasing the pH is known to cause RNA to bind more strongly, and therefore this method does not work for eluting biomolecules from anion exchangers. While proteins can be eluted from cation exchangers by increasing the pH gradient, ssRNA and dsRNA do not bind to cation exchangers, so this method is not useful for separating dsRNA from ssRNA. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] A novel method has been developed for removing dsRNA from mixed preparations with ssRNA, representing an improvement over known methods. This method is relevant to mRNA of all sizes, but particularly to large or gigantic mRNAs, such as those in the 1,000–25,000 base range. The method enables the separation of ssRNA and dsRNA bound to a primary amino solid phase by an increasing pH gradient. ssRNA elutes at a higher pH than dsRNA. This method also allows for the fractionation of ssRNA according to its size. [Means for solving the problem]

[0009] According to the present invention, a method for purifying single-stranded RNA is claimed, the method being: A step of applying a sample containing single-stranded RNA to a solid phase that primarily supports or exclusively supports primary amino groups on its surface, at a pH at least sufficient to bind the single-stranded RNA; A step of exposing the surface of the solid phase to an increasing pH, thereby eluting the adsorbed single-stranded RNA from the surface of the solid phase. Includes.

[0010] In one embodiment of the method of the present invention, at least one step may be provided in which, after applying the sample and before eluting the single-stranded RNA, the solid phase is washed with a washing buffer having a higher ionic strength than the elution buffer used for eluting the single-stranded RNA.

[0011] In another embodiment of the method of the present invention, the at least one washing step may be provided to reduce the higher ionic strength.

[0012] In a further embodiment of the method of the present invention, at least one washing step may be provided after applying the sample and before eluting the single-stranded RNA, using a washing buffer having an elevated pH to maintain the single-stranded RNA adsorbed to the solid phase and desorb any remaining double-stranded RNA.

[0013] The washing steps can be combined as two subsequent washing steps after the sample has been applied to the solid phase.

[0014] In yet another embodiment of the method of the present invention, the ionic strength of the washing buffer can be selected to be in a range of 0.5 M to 12 M, 1.0 M to 10 M, 2.0 M to 8.0 M, or 4.0 M to 6.0 M higher than the ionic strength of the buffer required to elute the single-stranded RNA. When NaCl is used, the ionic strength corresponds to the molar concentration of the solution. For example, if the corresponding ionic strength at which ssRNA elutes is less than 2.0 M, the ionic strength of the washing buffer may be 2.5 M. In another example, if the ionic strength at which ssRNA elutes is 0.5 M, the ionic strength of the washing buffer may be 1.0 M.

[0015] Typically, the molar concentration of the washing buffer is in the range of 0.51 M to 12.0 M, while the molar concentration at which ssRNA elutes is in the range of 0.01 M to 0.5 M.

[0016] In yet another embodiment of the method of the present invention, the ionic strength of the washing buffer can be adjusted by the concentration of a chaotropic salt, particularly a chaotropic salt selected from the group consisting of guanidinium salts, thiocyanates, perchlorates, and combinations thereof.

[0017] In a further embodiment of the method of the present invention, the elution of the single-stranded RNA from the surface of the solid phase can be performed with an elution buffer having a pH in the range of pH 7.5 to pH 12.0, pH 8.0 to pH 11.5, pH 8.5 to pH 11, or pH 9.0 to pH 10.5.

[0018] In a further embodiment of the method of the present invention, the application of the sample to the solid phase may occur at a pH value of less than approximately 8.5.

[0019] In further embodiments of the method of the present invention, a chelating agent may be present in the essentially aqueous mixture, in the environment of the solid phase surface prior to contact with the aqueous mixture, in the buffer for eluting the single-stranded RNA from the solid phase surface, and / or in a separate buffer used between the step of applying the sample to the solid phase and / or the step of eluting the single-stranded RNA from the solid phase surface.

[0020] In another embodiment of the present invention, the chelating agent can be independently selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), citrate, phosphoric acid, or ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), tris(2-aminoethyl)amine (TREN), and mixtures thereof.

[0021] Typically, the single-stranded RNA is in the size range of 1,000 to 25,000 base pairs.

[0022] The subject of the present invention is also the use of a solid phase containing predominantly primary amino groups or only primary amino groups on its surface for single-stranded RNA purification. In particular, single-stranded RNA can be purified or separated from double-stranded RNA by increasing the pH.

[0023] The surprising nature of this method is emphasized by the fact that it goes against the teachings of the prior art. In the published matter of this technical field, it is taught that it is impossible to elute large ssRNA from large dsRNA using strong anion exchangers (QA) and weak anion exchangers (DEAE) at ambient temperature.

[0024] The fact that it was considered impossible to elute any biomolecular species from any anion exchanger by increasing the pH emphasizes the second surprising feature of the present invention. It goes against the fundamental principle known in this technical field for decades. This principle is that acidic solutes (negatively charged net) bind more strongly to strong anion exchangers with an increase in pH. In the rare cases where anion exchangers are eluted with a pH gradient, contrary to the method of the present invention, the anion exchangers are exclusively used for elution with a descending pH gradient.

[0025] The present invention embodies a third surprising feature without precedent in this technical field. Preliminary data shows that ssRNA species are sorted according to size in the elution of ssRNA from a primary amino solid phase by a pH gradient, and higher pH values are required to elute larger ssRNA species. Notably, the size discrimination between dsRNA species is relatively poor.

[0026] The present invention also embodies a fourth surprising feature without precedent in the art. Experimental data indicate that dsRNA does not interact with the primary amino solid phase in the same manner as ssRNA behaves on the same surface. This is unexpected since ssRNA and dsRNA are compositionally identical in a given in vitro transcription mixture and have the same number of nucleotide bases in the same sequence. Notably, a wide variety of salts can displace most of the dsRNA from the primary amino solid phase without eluting the desired ssRNA. Further notably, ssRNA is not even eluted by extremely high concentrations of known invasive chaotropic salts.

[0027] A theory explaining why the paradoxical behavior of the primary amino solid phase eluted with an ascending pH gradient contravenes predictions based on traditional anion exchangers, how to separate dsRNA from ssRNA, or how to achieve size fractionation of ssRNA has not yet been developed.

[0028] Not only does high-salt washing simply displace dsRNA prior to elution of ssRNA, but it also provides a unique opportunity to dissociate complexes between ssRNA, which would otherwise be stable, and contaminating substances such as proteins and DNA. This provides a solution to a major problem that has only recently been recognized in the art. Nucleic acids often exist in stable complexes with contaminating substances. Some of these complexes elute under the same or nearly the same conditions as pure nucleic acids. Thus, these complexes are Trojan horses that bring the contaminating substances into what should be the pure ssRNA elution fraction, even though the possibility should have been excluded based on the properties when the contaminating substances are independent. Dissociating high-salt washing offers the possibility of stopping that contamination route. The complex dissociation potential of this approach is enhanced with chaotropic salts such as guanidine, which embody a stronger dissociation potential than neutral salts such as NaCl. The complex dissociation ability can be further enhanced by the presence of chelating agents.

[0029] The inability of contaminating dsRNA to bind to the primary amino solid phase in a high-salt chemical environment also enables a workflow simplification comparable to that of affinity chromatography, a technique known in the art. Affinity chromatography is a technique for covalently binding biospecific ligands, such as antibodies, to a solid phase. When a contaminated sample carrying the antibody's target is applied to the solid phase, only the target molecule is captured, while the contaminants are removed by flowing through the column. After washing the column to remove trace levels of unwanted species, the target molecule is eluted into a single, highly purified fraction. In this case, the sample containing dsRNA and ssRNA is loaded into a high-salt environment at neutral pH. The majority of the dsRNA flows through the solid phase. The highly purified ssRNA is eluted into a concentrated fraction by increasing the pH.

[0030] This simplification of the workflow is a slightly modified process using the same principle as that used in the basic configuration of the present invention, the principle of which is that dsRNA is not bound by the primary amino solid phase in the presence of high concentrations of salt, as will be recognized by those experienced in the art.

[0031] The ultimate surprising feature of this invention is that the elution behavior of contaminants with a different composition from RNA is similar to that of dsRNA, but different from that of ssRNA. Such contaminants include proteins and DNA, both of which elute faster than ssRNA in a pH gradient. The majority of these are also removed by salts applied to the solid phase before their trace residues are separated from the ssRNA by an increasing pH gradient.

[0032] General description of the invention In a general embodiment, the present invention is a solid-phase extraction method for removing dsRNA from a preparation containing a mixture of double-stranded messenger RNA (dsRNA) and single-stranded messenger RNA (ssRNA). The present invention further relates to a method for separating ssRNA species according to their size.

[0033] In certain embodiments, the present invention relates to the separation of dsRNA from ssRNA and / or the separation of ssRNA species according to their size, wherein the dsRNA and ssRNA are bound to a primary amino solid phase by non-covalent interactions.

[0034] In another specific embodiment, the present invention relates to the separation of dsRNA from ssRNA using an ascending pH gradient in which dsRNA elutes at a lower pH value than ssRNA. Smaller ssRNA species elute at a lower pH value than larger ssRNA species.

[0035] In more specific embodiments, the present invention relates to a pH gradient spanning pH 3.5 to 11.5, or pH 4.5 to 11.5, pH 5.5 to 11.5, pH 6.5 to 11.5, or pH 7.5 to 11.5, or pH 8.5 to 11.5, or pH 8.5 to 11.0, or pH 8.5 to 10.5, or pH 8.5 to 10.0, or pH 8.5 to 9.5, or pH 8.5 to 9.0, or a higher pH range, a lower pH range, or an intermediate range. The pH range is specified because the values ​​required to separate dsRNA from a particular ssRNA or to fractionate ssRNA species of different sizes depend on their respective sizes.

[0036] In one embodiment, the pH value can be continuously increased over a specific range that forms a so-called linear gradient. In a related embodiment, the pH can be increased discontinuously in separate steps, forming a so-called stepped gradient. In another related embodiment, the gradient may consist of a single step.

[0037] In another embodiment, elution by increasing pH may be performed in the presence of a salt for the purpose of increasing ssRNA recovery. In such embodiments, the salt species may be sodium chloride at concentrations ranging from 10 mM to 250 mM, or 20 mM to 200 mM, or 50 mM to 100 mM. In another such embodiment, the salt species may be potassium chloride at concentrations within a similar range, or guanidine-HCl at concentrations within a similar range, or guanidine thiocyanate at concentrations within a similar range. The presence of a salt during elution also allows ssRNA to be eluted at lower pH values.

[0038] In another aspect, the present invention relates to a method for washing ssRNA bound to a primary amino solid phase with a chelating agent.

[0039] In some embodiments, the chelating agent may be ethylenediaminetetraacetic acid (EDTA) at concentrations of 2 mM to 200 mM, or 5 mM to 100 mM, or 10 mM to 50 mM, or 20 mM to 25 mM, or lower, or in an intermediate range, or higher up to maximum complete saturation. In closely related embodiments, the chelating agent may be citric acid, phosphoric acid, a salt of ethylene glycol bis(2-aminoethyl ether)-N,N',N'-tetraacetic acid (EGTA), or tris(2-aminoethyl)amine (TREN), or a salt of another chelating agent, or a mixture of chelating agents at concentrations over the same range described for EDTA.

[0040] In some embodiments of the present invention, treatment with a chelating agent may include any of the following to carry out the steps of the method, including a pH elution: adding a chelating agent to the sample; replacing the sample buffer with a metal ion-free buffer; replacing the sample buffer with a chelating buffer; and using either a metal ion-free or chelating buffer. In some of these embodiments, the type and / or concentration of the chelating agent may vary in each step of the method.

[0041] In one embodiment, the present invention relates to the separation of dsRNA and ssRNA by washing the primary amino solid phase with salt to remove a subset of dsRNA before performing the final separation of dsRNA and ssRNA by increasing the pH. In all such embodiments, the concentration of salt during washing is higher than the concentration of salt present during elution.

[0042] In some embodiments, the salt used to wash the solid phase before elution by increasing the pH can be any type of salt at any concentration, up to the maximum complete saturation concentration. In one such embodiment, where the salt is sodium chloride saturated at about 5.0 M, the salt concentration may be in the range of 10 mM to 5 M, or 50 mM to 5 M, or 100 mM to 5 M, or 500 mM to 5 M, or 1 M to 5 M, or 2 M to 5 M, or 3 M to 5 M, or 4 M to 5 M, or an intermediate range, but preferably in the range of 1 M to 5 M. In closely related embodiments, the salt may be potassium chloride. It will be recognized that RNA can precipitate with other high concentrations of neutral salts such as lithium chloride, as can RNA precipitate with sodium chloride or potassium chloride, etc. It will also be recognized that RNA precipitation during sample application or elution is highly undesirable because it can interfere with the flow of buffer through the solid-phase chromatography apparatus. Strictly speaking, this is not a problem if salt washing is performed after loading the sample to remove excess salt before elution, but it is often preferable to completely avoid the problem by using a non-RNA precipitating salt instead.

[0043] In one embodiment, where the RNA precipitate salt is replaced with a non-RNA precipitate salt, the salt is a chaotropic salt such as guanidinium hydrochloride that saturates at about 6 M, and the concentration may be in the range of 10 mM to 6 M, or 50 mM to 6 M, or 100 mM to 6 M, or 500 mM to 6 M, or 1 M to 6 M, or 2 M to 6 M, or 3 M to 6 M, or 4 M to 6 M, or 5 M to 6 M, or an intermediate range, preferably in the range of 3 M to 4 M. In closely related embodiments where the salt is a chaotropic salt such as guanidine thiocyanate that saturates at approximately 12 M, the concentration may be 10 mM to 12 M, or 50 mM to 12 M, or 100 mM to 12 M, or 500 mM to 12 M, or 1 M to 12 M, or 2 M to 12 M, or 3 M to 12 M, or 4 M to 12 M, or 5 M to 12 M, or 6 M to 12 M, or 7 M to 12 M, or 8 M to 12 M, or 9 M to 12 M, or 10 M to 12 M, or 11 M to 12 M, or in an intermediate range, preferably in the range of 1.5 M to 3.0 M. From these two examples of salts, it will be clear that other salts can be used similarly, up to but not exceeding their saturation point.

[0044] In some embodiments, a chelating agent can be used in combination with a salt. In various embodiments of the present invention, treatment with a salt and / or chelating agent may include any of the following: adding a salt and chelating agent to a sample; replacing the sample buffer with a buffer containing a salt and / or chelating agent; replacing the sample buffer with a buffer containing a salt and / or chelating agent; and using a salt-containing buffer and / or chelating agent-containing buffer to carry out the steps of the method, including elution of ssRNA. In some such embodiments, the type of salt, the chelating agent, and their respective concentrations may vary in each step of the method.

[0045] In some embodiments, dsRNA can be separated from ssRNA by equilibrating the sample with a high concentration of non-RNA precipitate salt, such as guanidine thiocyanate, plus a chelating agent such as EDTA. The solid phase can be equilibrated with a buffer containing the same concentrations of guanidine thiocyanate and EDTA. While the sample is applied to the solid phase, most of the dsRNA does not bind to the solid phase. By the end of the washing step with the same concentrations of guanidine thiocyanate and EDTA, the dsRNA has been reduced to trace levels. The guanidine thiocyanate and EDTA are then washed away from the system with a buffer that does not contain them. The remaining dsRNA is separated from the ssRNA by an ascending pH gradient.

[0046] In preliminary embodiments where the objective is to isolate a certain amount of purified ssRNA for some purpose, it is most desirable to neutralize the pH of the ssRNA immediately after pH elution to minimize exposure to pH values ​​close to pH 9 or higher. Experimental data have shown that ssRNA exposed to alkaline conditions only for the duration required to carry out the method of the present invention retains its native composition and remains stable indefinitely. Neutralization, which is essentially instantaneous, can be carried out by recovering the fraction in a neutralizing solution. Alternatively, rapid neutralization may be performed by adding the neutralizing solution immediately after fraction recovery. Neutralization can also be carried out after fraction recovery by buffer exchange methods including chromatography or diafiltration. All of these methods are commonly practiced and well known in the art, for example, in the field of affinity chromatography, where pH neutralization after sample elution is routine.

[0047] The method of the present invention can be carried out using any apparatus commonly used in the art, for example, the primary amino solid phase can be placed in a chromatographic apparatus. Typically, the solid phase surface may be in the form of a monolith, a packed particle column, a packed nanofiber column, a membrane adsorbent, or a hydrogel, among other chromatographic formats.

[0048] The method of the present invention can be used for analytical or preparative purposes. While applicable to all mRNAs, it is particularly useful for mRNAs in the size range of 1,000 to 25,000 base pairs. Specific chromatographic conditions may vary depending on the size of the RNA and the distribution of contaminants in the sample applied. Adjusting specific conditions to achieve the best analytical or preparative results for any particular ssRNA species uses the same experimental skills known to practitioners of chromatography techniques for decades.

[0049] To purify ssRNA to a greater extent than can be achieved by any single processing method alone, the method of the present invention may include one or more additional processing methods, either preceding, succeeding, or preceding and succeeding. [Brief explanation of the drawing]

[0050] [Figure 1] This demonstrates the separation of dsRNA from ssRNA using a primary amino solid phase, where the dsRNA is removed by a sodium chloride step before eluting the ssRNA using a pH gradient at ambient temperature. [Figure 2] This demonstrates the separation of dsRNA from ssRNA using a primary amino solid phase, where the dsRNA is removed by a 6M guanidine step before eluting the ssRNA using a pH gradient at ambient temperature. [Figure 3] This demonstrates that elution of ssRNA from strongly and weakly anion-exchanged materials fails under a pH gradient at ambient temperature. [Figure 4] This paper demonstrates the separation of plasmid DNA from ssRNA using a pH gradient at ambient temperature with a primary amino acid solid phase. [Figure 5] This demonstration shows how plasmid DNA can be separated from ssRNA using a primary amino solid phase, with the DNA being removed by a salt step before eluting the ssRNA using a pH gradient at ambient temperature. [Figure 6]This study demonstrates the effectiveness of combining salt and pH gradient elution at ambient temperature for the separation of DNA and ssRNA. [Figure 7] This study demonstrates the separation of large DNA and large dsRNA from small ssRNA on a primary amino solid phase using a pH gradient at ambient temperature. [Figure 8] This demonstrates the separation of dsRNA from ssRNA using a primary amino solid phase, where DNA is removed by a chelating agent-chaotrope combination step before eluting ssRNA using a pH gradient at ambient temperature. [Figure 9] The inclusion of chelating salts reduces the ssRNA elution pH in a pH gradient. [Figure 10] This shows monitoring of in vitro transcription reactions at different time points using a primary amine solid phase eluted under a pH gradient in the presence of salt. [Modes for carrying out the invention]

[0051] The term "primary amino solid phase" refers to a solid phase suitable for chromatography that primarily or exclusively supports primary amino ligands on its surface. The terms "primary amino solid phase," "solid phase bearing primary amino groups," "primary amine-bearing solid phase," "primary amino-bearing solid phase," or "primary amine solid phase" are synonymous and interchangeable. Secondary amines should be absent or present in small quantities on the solid phase surface. Tertiary and quaternary amines should be absent or present in small quantities. Negatively charged residues should be absent. Uncharged hydrophobic or hydrogen-bonding residues may be present.

[0052] The term “primary amino group” refers to a nitrogen atom that is linked by a single covalent bond to each of two hydrogen atoms, and also by a single covalent bond to a carbon atom. The primary amino group may be directly covalently bonded to the solid phase through its carbon atom. Alternatively, the primary amino group may be indirectly bonded to the solid phase by the covalent bond of its carbon atom to so-called spacer arms that are covalently bonded to the solid phase. The primary amino group may also be part of a polymer structure covalently bonded to a solid phase containing the primary amino group, where repeating subunits of the polymer contain the primary amino group.

[0053] The term "solid phase" can refer to a chromatographic solid phase in the form of one or more porous membranes, one or more fibers, one or more porous or non-porous particles, a monolithic solid phase (including monoliths synthesized from a single polymer mixture), or a so-called hydrogel having a secondary ligand-supported polymer phase synthesized on top of a monolith initially synthesized as a macroframework. Any of these solid phase materials can be placed within a housing to facilitate the performance of chromatography. The chromatographic solid phase within the housing is generally referred to as a chromatography apparatus, and is often called a chromatography column, or simply a column.

[0054] Chromatographic solid phases containing primary amino groups are known and commercially available. One example is Toyopearl NH2-750F, manufactured by Tosoh Bioscience, where "NH2" refers to a primary amino group [www.separations.eu.tosohbioscience.com / solutions / process-media-products / by-mode / ion-exchange / anion-exchange / toyopearl-nh2-750f]. According to the sales materials, the primary amino group is in the form of a polyamine, meaning it is a polymer having repeating primary amine subunits covalently immobilized on the solid phase. Such polymers are typically linked to the surface of the solid phase via one or more of their primary amino groups. This linkage has the effect of converting the linked amino residue from a primary amino group to a secondary amino group, thereby creating a mixture of primary and secondary amino groups on the surface of the solid phase.

[0055] Another example is manufactured by Sartorius under the name Sartobind STIC PA, where "PA" refers to a primary amine [www.sartorius.com / shop / ww / en / usd / sartobind-stic (R) -pa / c / M_Sartobind_STIC_PA]. According to the sales materials, the primary amino group is in the form of a polymer, specifically a polyallylamine, which has repeating primary amino subunits covalently bonded to the solid phase. Similar to the products described above, such polymers are typically linked to the surface of the solid phase via one or more of their primary amino groups. This bonding has the effect of converting the linked amino residue from a primary amino group to a secondary amino group, thereby creating a mixture of primary and secondary amino groups on the surface of the solid phase.

[0056] All major commercial manufacturers of chromatographic solid phases produce products (including anion exchangers) with amino derivatives on their surfaces, demonstrating the knowledge and resources necessary to produce primary amine-containing solid phases on a regular or experimental basis.

[0057] Considering that chromatographic solid phases containing primary amino groups may not be named in a way that clearly or completely reveals their composition, it is useful to have a simple analytical method for determining whether a given chromatographic solid phase has suitable properties for carrying out the present invention. One simple method for making this determination is to equilibrate the chromatographic solid phase in question with a buffer such as 50 mM Tris, pH 7.5, and then inject a sample consisting of a so-called ssRNA ladder containing subsets of RNA molecules of different sizes. Such RNA ladders generally cover sizes of 50–500 nucleotides, or 100–1000 nucleotides, or 200–6000 nucleotides, or several other ranges, and are commercially available from common suppliers such as Thermo Scientific and New England BioLabs. After removing unbound sample components with a simple wash in the sample injection and equilibration buffer, a primary amino chromatography apparatus is subjected to elution in a linear pH gradient from pH 7.5 to approximately pH 11. As shown in Figure 1, the primary amino solid phase elutes ssRNAs in ascending order of size. If no ssRNAs are eluted, or only the smallest ssRNA species are eluted, it may suggest that the solid phase contains an excessive proportion of non-primary amino groups, including one or any combination of secondary, tertiary, or quaternary amino groups.

[0058] The term "RNA size" refers to the number of bases in a nucleotide chain. A base is commonly denoted as "b". Therefore, the designation 100b refers to an RNA strand of 100 bases. RNA size is independent of RNA conformation, which refers to single-stranded (ssRNA) and double-stranded (dsRNA). A given in vitro transcription mixture refers to a mixture of reagents and products and by-products generated during mRNA synthesis, and may contain both ssRNA and dsRNA, but since both originate from the same DNA plasmid, they are both the same size in terms of the number of bases. In some cases, ssRNA strands may be clipped or truncated during processing, either through shear stress or enzymatic lysis, resulting in the formation of fragmentary subpopulations of the former complete strand. In other cases, truncated forms may hypothetically arise from incomplete transcription. Regardless of the source, the ability of the present invention's method to fractionate ssRNA according to size provides a tool for removing undesirable fragment morphologies.

[0059] The term "equilibriumized" or "equilibriumization" refers to chemical conditioning steps performed on a solid phase and / or sample to create a specific chemical environment. Solid phases are typically prepared by exposing them to buffers that embody a desired pH and salt composition. Samples are typically prepared by pH titration, sometimes by dilution to reduce salt concentration, sometimes by buffer exchange techniques including chromatography, or by dialysis, or by dialysis using tangential flow filtration membranes. All of these methods and the criteria for selecting one or another have been known in the art for decades.

[0060] The term "loading" or "sample application" refers to the process of bringing an equilibrated sample into contact with a solid phase that primarily supports equilibrated, positively charged primary amino groups. This is typically done using a chromatography apparatus, by passing the sample through the apparatus using gravity or other external forces such as pumping.

[0061] The term "adsorption" refers to the process of binding biological products to a chemically complementary surface. Adsorption is similar to the absorption of water by a sponge through the physical action of capillary action, but it differs from "absorption," which does not involve chemical interaction. In this case, complementarity is understood to include static charge. The negative static charge on the surface of RNA mediates its adsorption to the surface of a solid phase that has been electrically positively charged by primary amino groups on its surface. The adsorption of biological products is often referred to by the more general term "binding."

[0062] The term "selective adsorption" refers to conditions that allow the adsorption of at least one species while preventing the adsorption of one or more other species. In this case, the operating conditions can be adjusted to allow the binding of ssRNA while preventing the binding of most dsRNAs.

[0063] The term "desorption" refers to the process of releasing a biological product from a chemically complementary surface to which it was previously adsorbed. In the methods of the present invention, ssRNA desorption typically requires an alkaline pH value of at least pH 9.0, or pH 9.5, or pH 10.0 or higher. Salts may be used to enhance desorption, typically with the effect of achieving elution of a given ssRNA species at a lower pH than in the absence of salt. However, at acidic or neutral pH, salts or combinations of salts, at any concentration, will not achieve large ssRNA elution.

[0064] The term "selective desorption" refers to a situation in which one or more adsorbed species are released from a solid phase surface due to a change in conditions in which one or more other species remain adsorbed. A further set of different conditions can then be applied to release a subset of different species from the solid phase surface. In one such example, by applying a high concentration of salt at a neutral or near-neutral pH, most of the dsRNA can be removed from the primary amino solid phase while the ssRNA remains bound. The dsRNA is said to have been selectively desorbed. ssRNA is selectively desorbed in subsequent steps at an alkaline pH, possibly in the presence of salt.

[0065] The term "washed" refers to the process of exposing a loaded column to a washing buffer for the purpose of removing unbound species from pores or channels within the instrument. The term "rinsed" has the same meaning in this context. In its most basic form, the washing buffer has the same composition as the equilibrium buffer. In more complex configurations, the washing buffer may have the additional role of chemically releasing a subset of weakly bound contaminants so that they are chemically removed before the desired product is eluted. Alternatively, there may be two or more washing steps, with the first using the same conditions as the equilibrium buffer, but the second using conditions that remove the subset of weakly bound contaminants from the solid phase so that they can be removed before elution. Washing can also be used to transition to a buffer that allows elution under a different or more controlled set of conditions. For example, after a high-salt wash to remove contaminants, it may be desirable to perform a salt-free wash to set conditions for eluting ssRNA under a salt-free pH gradient. Without this washing process, elution begins with a high salt concentration by default, creating a gradient where the salt concentration decreases while the pH rises.

[0066] The term "elution" is a special case of the term "desorption" in the field of chromatography. This refers to the process of altering the chemical environment in which the solid phase exists, causing the dissociation of the interaction between the primary amino solid phase and the species that remains bound after the loading and washing steps. Once the dsRNA and proteins have been removed from the solid phase, the ssRNA can be eluted by simply increasing the pH, or by increasing the pH with salts.

[0067] Elution can be performed in one or a series of steps, each of which reduces the strength of the interaction between the solid phase and the ssRNA. The changes in conditions can also be sequential or linear, with weakly bound species being desorbed early in the sequential steps, while strongly bound species are eluted later in the sequential steps. Whether in a stepwise or linear form, the changes in operating conditions are generally referred to as a gradient, and more specifically, an elution gradient. Stepwise gradients are often considered more convenient, but linear gradients typically support better reproducibility.

[0068] The term "ambient temperature" is generally considered similar to the expressions "room temperature" or "normal temperature." It typically corresponds to temperatures in the range of approximately 20-22°C, but may also include a broader range such as approximately 18-25°C.

[0069] The term "chaotropic salt" refers to a salt species in which at least one of its constituent ions has a high chaotropic rank in the Hofmeister series of lyotropic and chaotropic ions. Lyotropic ions are located at one end of the Hofmeister series, while chaotropic ions are located at the opposite end. Chaotropic ions are often described as preferentially binding to biomolecules. Chaotropic salts have the effect of relaxing non-covalent interactions within and between biomolecules, sometimes to the extent that they destabilize and dissociate the interactions between individual elements in a multi-component non-covalent mixture. They usually have the effect of increasing solubility. Examples of chaotropic salts include, among others, guanidinium salts, thiocyanates, and perchlorates. In some cases, as in the case of guanidine thiocyanate, both the anion and cation of a particular salt are strongly chaotropic. Such salts have a higher chaotropic potential than salts containing only a chaotropic anion or chaotropic cation. Lyotropic ions are located at the opposite end of the Hofmeister series. They are often described as being preferentially excluded by biomolecules. Lyotropic ions have a stabilizing effect on biomolecules and promote nonspecific association between individual elements of a mixture. Strong lyotropic ions typically reduce the solubility of large biomolecules. Examples of lyotropic salts include, among others, ammonium sulfate, potassium phosphate, and sodium citrate. Salts representing intermediates in the Hofmeister series tend to have a moderate or little effect on stability, association-dissociation, or solubility to biomolecules. Examples include so-called neutral salts such as sodium chloride and potassium chloride. To the extent that such salts can affect stability, association-dissociation, or solubility, these effects are mediated primarily through Coulomb (electrostatic) forces.

[0070] The term "polyvalent metal cation" refers to a positively charged ionic form of a metal with a net charge of 2 or more. Examples of polyvalent metal cations include calcium, magnesium, and zinc (all with a net charge of 2+), and secondary iron (with a net charge of 3+), among others with similar or different valencies. All of these ions have an affinity for nucleic acids, which bind via coordinate bonds. Coordinate bonds are 15 to 60 times stronger than ionic bonds, meaning that the binding of polyvalent metal cations to nucleic acids or other biomolecules persists even at salt saturation concentrations. Polyvalent metal cations can be problematic in RNA purification because they can promote the formation of dsRNA sequences or stabilize existing dsRNA sequences. They can also promote the formation of complexes between multiple RNA molecules, between RNA and DNA molecules, and between RNA, DNA, and contaminating proteins, or stabilize existing such complexes (aggregates).

[0071] In the context of the method of the present invention, the term "chelating agent" refers to a molecule that has the ability to form a strong coordination bond with a polyvalent metal cation, enabling the competitive removal of metal ions from a previously existing aggregate of a polyvalent metal cation and a biomolecule containing nucleic acid, including mRNA.

[0072] The term "nuclease" or "nuclease enzyme" refers to a protein that has the ability to cleave a chain of nucleic acids, ideally into individual nucleotides, doublets, or triplets. They can be classified into two main classes: DNAse enzymes, which lyse DNA, and RNAase enzymes, which lyse RNA. RNAse should be strictly avoided because it destroys ssRNA products. DNAse is often used to simplify purification by destroying the DNA plasmid template used to produce mRNA. DNAse enzymes often require the use of polyvalent metal cation cofactors to function properly. Polyvalent metal cations can interfere with RNA purification, as mentioned above.

[0073] The terms "protease," "proteinase," or "protein-degrading enzyme" refer to proteins that have the ability to cleave and fragment other proteins. This can be used as a method to reduce such contamination before chromatographic processes that may be burdened by protein contamination. Many proteases, such as trypsin, require polyvalent metal cation cofactors to function properly. Polyvalent metal cations can interfere with RNA purification, as described above. A protease commonly used for this purpose is proteinase K, which provides good results even in the absence of polyvalent metal cation cofactors.

[0074] If desired or necessary, a decision may be made to include salt during the implementation of this method, partly in the selection of subsequent analytical methods or purification steps. For example, if the subsequent method is not salt-tolerant, it is advantageous to elute ssRNA from a positively charged solid phase mainly consisting of primary amino groups in the absence of salt, or at a sufficiently low salt concentration, to avoid interference. If the subsequent method is highly salt-tolerant, the elution apparatus can use a salt species tolerant to the subsequent step at a substantial concentration.

[0075] In one embodiment, the presence of 50 mM NaCl in the pH gradient endpoint buffer leads to elution of ssRNA at a lower pH than when NaCl is absent from the endpoint buffer. The presence of 50 mM NaCl only in the gradient endpoint buffer suggests that ssRNA is eluted not by the pH gradient alone, but by a simultaneous gradient of increasing NaCl and rising pH. In a closely related embodiment, the presence of 100 mM NaCl in the pH gradient endpoint buffer leads to RNA elution at a lower pH than when the gradient endpoint buffer contains 50 mM NaCl. In both cases, the presence of NaCl in the gradient endpoint buffer significantly increases the recovery rate of ssRNA. Separation between dsDNA and ssRNA is widest with 0 mM NaCl. This decreases when the endpoint buffer contains 50 mM NaCl and further decreases when the endpoint buffer contains 100 mM NaCl, but separation remains good even at 100 mM. Other salts, such as chaotropic and chelated salts, can be substituted at similar concentrations.

[0076] In one embodiment, pH gradient elution from a primary amino solid phase is performed to separate ssRNA species according to their size, where smaller species elute faster on the gradient than larger species.

[0077] The following general and non-restrictive description of a series of basic method options illustrates variations in how this method may be performed and provides a platform for a more detailed discussion of the instrumental variables. The buffer conditions mentioned in each of these scenarios are intended to provide a general concept of how this method may be performed, and it should be understood that optimization of the buffer composition will be necessary due to the presence of different ssRNA species of different sizes and different contaminant loads.

[0078] In one embodiment, a primary amino solid phase in the form of a chromatographic apparatus such as a monolith is equilibrated to a near-neutral pH value such as 20 mM Tris, 20 mM bis-tris-propane, and pH 7.5 ± 0.5. A sample containing a mixture of dsRNA and ssRNA is equilibrated by buffer exchange to 20 mM Tris, 20 mM bis-tris-propane, 20 mM glycine, and pH 7.5 ± 0.5. The chromatographic apparatus is then eluted from the equilibration buffer toward an endpoint buffer of 20 mM Tris, 20 mM bis-tris-propane, 20 mM glycine, 100 mM NaCl, and pH 11 ± 0.5, with a linear pH gradient of over 20 apparatus volumes, over 50 apparatus volumes, or over 100 apparatus volumes, where the apparatus volume value is used as a means to adjust the rate at which the pH changes during the gradient, in other words, the slope of the gradient. This approach may be particularly useful for analytical applications because dsRNA, ssRNA, DNA fragments, and proteins all bind to a primary amine-containing solid phase and elute within the gradient, thus providing an indicator of the total content of the sample (i.e., product and contaminants). This approach can also be used as a starting point for developing conditions for preparatory separation.

[0079] In a different embodiment, the dsRNA, DNA, and protein contaminants can be removed by loading them onto a primary amine solid phase under the same conditions and then washing them with a 3M NaCl solution. The NaCl itself is then removed by a subsequent wash in the absence of excess salt, allowing the pH gradient elution of ssRNA to begin in the absence of excess salt. By comparing the results with those of the previously described embodiments, it is possible to evaluate the extent to which NaCl washing improves ssRNA purity. Determining the most preferred concentration of NaCl is a well-known and routine activity in the field of chromatography, with the aim of achieving the best balance between product purity and product yield.

[0080] In closely related embodiments, one or both of the embodiments described above may contain 50 mM sodium chloride in the pH gradient buffer. In other closely related embodiments, 100 mM sodium chloride may be included in the pH gradient buffer. In other closely related embodiments, any other salt may be used instead of sodium chloride.

[0081] In another related embodiment, the dsRNA can be loaded onto a primary amino solid phase under the same conditions and then washed with a 3M guanidine-HCl solution to remove most of the dsRNA before eluting the desired ssRNA product using a pH gradient. Following the guanidine wash, a wash without guanidine salt is performed to allow pH gradient elution of ssRNA to begin in the absence of excess salt. In a closely related embodiment, 100 mM sodium chloride can be added to the pH gradient buffer. In another closely related embodiment, guanidine-HCl may be replaced with guanidine isothiocyanate. By comparing the results with the two embodiments described above, it is possible to evaluate the extent to which guanidine washing improves the purity of ssRNA.

[0082] In related embodiments that extend the above series of processes, a chelating agent, such as but not limited to ethylenediaminetetraacetic acid (EDTA) at a concentration of 20 mM, can be combined with guanidine to dissociate the metal-stabilizing complex and remove non-ssRNA species from the solid phase before eluting ssRNA. By comparing the results with the three aforementioned embodiments, it is possible to evaluate the extent to which chaotrope-chelating agent washing improves (if any) the purity of ssRNA.

[0083] In other relevant embodiments, to maximize dsRNA clearance, washing with other salts or combinations of salts at various pH values ​​can be evaluated prior to ssRNA elution. The underlying concept is generally that the concentration of the most dissociative salt at the highest pH that does not cause ssRNA elution is likely to remove the largest subset of undesirable contaminants prior to the elution of the desired ssRNA, and such conditions are applied based on this idea. Subsequently, adjustments can be made to determine the minimum salt concentration required to achieve the desired effect.

[0084] In some embodiments, the linear pH gradient may be modified to alter the degree of separation between species eluting at the gradient, or in particular to improve fractionation between ssRNA molecules of different sizes. If elution at a pH gradient of 10 instrument volumes does not produce the desired degree of separation, the duration may be extended to 20 instrument volumes, 50 instrument volumes, 100 instrument volumes, or more.

[0085] In some embodiments, the pH increase for eluting the desired ssRNA and separating it from dsRNA can be performed in a stepwise manner. If the dsRNA content has been sufficiently reduced in the previous washing step, the method can be simplified to obtain eluted ssRNA in a single step to obtain the highest possible concentration and lowest possible volume of eluted ssRNA. Alternatively, stepwise elution may be performed in a sequence of pH-increasing steps. Each step may be relatively mild or large depending on the needs of the particular preparation. In some embodiments, the pH increase for eluting the desired ssRNA can be performed in a single step.

[0086] In some embodiments, a sample containing dsRNA and ssRNA can be loaded onto a primary amino solid phase at a pH that prevents binding by most of the dsRNA. In some such embodiments, depending on the size of the mRNA, the pH during sample loading can be pH 8.0, pH 8.5, pH 9.0, or a higher pH that does not hinder ssRNA binding.

[0087] In a representative embodiment for a particular case, ssRNA can be eluted by a salt gradient at a constant pH. This requires first raising the pH to a value just below the value at which ssRNA elutes in the absence of salt. Then, the salt gradient is applied at that pH. While this embodiment has a superficial similarity to the standard anion exchange elution method of salt gradients at a fixed pH, it remains distinctive in the field of separating dsRNA from ssRNA because salt gradients for large mRNAs are only successful otherwise at elevated operating temperatures.

[0088] Many methods for equilibrating a sample to the conditions for loading it onto a chromatography column are known to those skilled in the art. Any of these methods can be used without altering the true nature of the method. These methods include laboratory-scale dialysis, dialysis filtration using tangential flow filtration membranes, and buffer exchange chromatography. In some cases, proper sample equilibration can be achieved by titrating the sample to a target pH and, if necessary, diluting the sample with water or a low-salt or non-salt buffer.

[0089] In some embodiments, the presence of a sugar competing for hydrogen bonding between ssRNA and the primary amino groups of the solid phase is expected to enhance the separation of dsRNA from dsRNA by increasing the pH, resulting in elution of ssRNA at a lower pH than in the absence of sugar. In one such embodiment, the sugar is sorbitol, or xylitol, or mannitol, trehalose, or sucrose, or another sugar, or a combination of sugars. In some such embodiments, the sugar concentration may be in the range of 0.1% to 20%, or 1% to 20%, or 5% to 20%, or 10% to 20%, or higher, lower, or intermediate values.

[0090] In relevant embodiments, the presence of a nonionic chaotrope competing for hydrogen bonds between ssRNA and primary amino groups of the solid phase is expected to enhance the separation of dsRNA from dsRNA by increasing the pH, resulting in elution of ssRNA at a lower pH than in the absence of the chaotrope. In one such embodiment, the chaotrope is urea. In some such embodiments, the concentration of urea may be in the range of 0.1 M to 10 M, or 1 M to 9 M, or 2 M to 8 M, or 4 M to 6 M, or higher, lower, or intermediate values. In another such embodiment, the chaotrope is dimethyl sulfoxide. In some such embodiments, the concentration of dimethyl sulfoxide is up to 99%. In other relevant embodiments, the nonionic chaotrope may be applied during the washing step before eluting the desired ssRNA by the pH gradient.

[0091] In another embodiment, the presence of an alkaline amino acid is expected to enhance the separation of dsRNA from dsRNA by increasing the pH, resulting in the elution of ssRNA at a lower pH than in the absence of the alkaline amino acid. In one such embodiment, the alkaline amino acid is histidine, or histamine, or lysine, or arginine, or another alkaline amino acid, or a mixture of alkaline amino acids. In one such embodiment, the concentration of histidine may be in the range of 1 mM to 250 mM, or 10 mM to 250 mM, or 20 mM to 250 mM, or 50 mM to 250 mM, or 100 mM to 250 mM, or higher, lower, or intermediate. In another such embodiment, the concentration of lysine may be in the range of 1 mM to 10 M, or 10 mM to 10 M, or 100 mM to 10 M, or 1 M to 10 M, or higher, lower, or intermediate. In another such embodiment, the arginine concentration may be in the range of 1 mM to 850 mM, or 10 mM to 850 mM, or 100 mM to 850 mM, or 425 mM to 850 mM, or higher, lower, or intermediate values.

[0092] Nucleic acids, including mRNA, are known to have a high affinity for polyvalent metal cations. They form strong associations with each other, primarily through coordination bonds between the metal ion and negatively charged phosphatidic acid residues along the nucleic acid backbone. Both calcium and magnesium are known to be involved in such interactions and are both divalent metal cations with a 2+ charge. Secondary iron, a trivalent cation with a 3+ charge, interacts more actively with nucleic acids. At each point in time that one of these ions interacts with a nucleic acid, it neutralizes an equivalent number of negative charges. This leads to the superficial expectation that the negative charge on a given mRNA molecule decreases, weakening the mRNA's interaction with the anion exchanger. Rather, the addition of polyvalent metal cations typically results in the formation of nonspecific crosslinks, which causes ssRNA to form large aggregates that do not elute from the chromatography apparatus.

[0093] Since polyvalent cations are generally added to mRNA preparation, in all embodiments it is recommended to reduce their content, preferably completely remove polyvalent metal cations from the sample, before loading the sample onto a solid phase mainly supporting positively charged primary amino groups; or at least perform a step to remove polyvalent metal cations before the apparatus is subjected to elution. There are at least two further reasons for pre-removing polyvalent metal cations. The first reason is that polyvalent metal cations may have a tendency to promote the formation or stabilization of intra-chain and inter-chain dsRNA sequences. The second reason is that metal ions stabilize nonspecific associations between nucleic acids and proteins, essentially forming stable crosslinks between them. Since coordination bonds are 15 to 60 times stronger than ionic bonds, coordination complexes readily withstand exposure to saturated levels of NaCl and non-metallic salts, including guanidinium salts. This makes it essential to extract as many polyvalent metal cations as possible in order to obtain the best ssRNA purity and recovery rate from the method of the present invention. Considering the effective extraction of polyvalent metal cations, effective dissociation of nucleic acid-protein complexes can be achieved with high concentrations of chaotropic salts such as guanidinium salts, and to a lower degree, with high concentrations of non-chaotropic salts such as NaCl.

[0094] In some embodiments in which chelating or chelating-high-salt dissociation of nucleic acid-metal-contaminant complexes is performed during sample preparation, and / or chelation washing or chelation-chaotrope washing is performed, a salt-free washing step beyond the agent used to provide pH control may then be performed. This allows ssRNA to be eluted in a low-salt environment.

[0095] In other embodiments, the chelating agent concentration and high salt concentration may be maintained during ssRNA elution by increasing the pH. In other embodiments, the chelating agent may be removed while the high salt concentration is maintained. In other embodiments, the high salt concentration may be removed while the chelating agent is maintained.

[0096] A particularly advantageous aspect of the present invention is that its ability to remove DNA plasmids from ssRNA preparations eliminates the need for nuclease digestion of in vitro transcription mixtures or partially purified in vitro transcription mixtures. This is of disproportionate value, given that nuclease digestion requires the addition of magnesium ions to activate the enzyme. These magnesium ions potentially contribute to the crosslinking of the desired ssRNA with itself and other sample components, and as a practical consequence, reduce the recovery of the desired ssRNA product. By eliminating the need for nuclease digestion, the addition of magnesium ions is eliminated, and the yield of ssRNA is not impaired.

[0097] Regardless of the ability of the present invention to separate plasmid DNA from ssRNA, nuclease digestion may be carried out to the desired extent, and then the positively charged solid phase, mainly having primary amino groups, may be washed with an excess chelating agent to remove residual magnesium ions. In such embodiments, plasmid DNA may be digested with a nuclease in the presence of a 5 mM nuclease enzyme, followed by chelation washing with EDTA in the range of 10 to 50 mM or more.

[0098] Starting with an in vitro transcription mixture, in one embodiment, EDTA is added to a final concentration of 10–50 ml for the purpose of capturing polyvalent metal cations. Assuming the pH is in the range of pH 7–8.0, the sample can be filtered as needed and then loaded into a primary aminochromatograph equilibrated with 50 mM Tris, 100 mM NaCl, 10 mM EDTA, pH 8.0. After loading the sample, the apparatus is washed 10–20 times by volume with 50 mM Tris, 3 M guanidine-HCl, 20 mM EDTA, pH 8.0. The column is then washed with 20 mM Tris, 20 mM bis-tris-propane, 20 mM glycine, pH 8.0 to wash away guanidine and EDTA from the apparatus and prepare for ssRNA elution. Next, the ssRNA is eluted at a volume of 50 using a linear gradient toward pH 11.0 with 20 mM Tris, 20 mM bis-tris-propane, 20 mM glycine, and 250 mM arginine. Immediately after fraction collection, the container containing the ssRNA peak is neutralized by adding 1 M acetic acid.

[0099] In one extended embodiment of the above embodiment, the neutralized sample after the method of the present invention is applied to an oligo-dT affinity chromatography apparatus for final purification.

[0100] In an alternative embodiment that extends the above embodiment, the neutralized sample is applied to a hydrophobic interaction chromatography apparatus for final purification.

[0101] In one embodiment, the process begins with an in vitro transfer mixture, and the mixture is precipitated by adding lithium chloride (LiCl) to a final concentration of 2.0 M to 2.5 M. The supernatant is discarded, and the precipitate is resuspended in 50 mM Tris, 100 mM NaCl, 20 mM EDTA, pH 8.0, and filtered to remove turbidity as necessary. The sample is then loaded onto a primary amino solid phase equilibrated to 50 mM Tris, 100 mM NaCl, 10 mM EDTA, pH 8.0. After loading the sample, the apparatus is washed with 50 mM Tris, 1.5 M guanidine isothiocyanate, 20 mM EDTA, pH 8.0 at a volume of 10 to 20 units. Next, the column is washed with 20 mM Tris, 20 mM bis-tris-propane, 20 mM glycine, 100 mM NaCl, pH 8.0 to wash away guanidine and EDTA from the instrument and prepare for ssRNA elution. Then, the ssRNA is eluted with a linear gradient of 50 instrument volume toward 20 mM Tris, 20 mM bis-tris-propane, 20 mM glycine, 100 mM NaCl, pH 11.0. Immediately after fraction recovery, the container containing the ssRNA peak is neutralized by adding 1 M acetic acid until the final percentage is 5% of the fraction volume. In closely related embodiments, mRNA is precipitated with NaCl or another salt instead of LiCl. In another closely related embodiment, mRNA is precipitated by adding ethanol until the final percentage is about 2.5%.

[0102] Industrial process developers often prefer to avoid the use of enzymes. This is because the use of enzymes increases the cost of the process, and because the added substances must be removed later, and testing must be performed to document that they have been removed. However, the use of enzymes in the early stages of a development program can be a convenient shortcut that allows companies to participate in clinical trials sooner, after which more advanced versions of the process that do not require the use of enzymes are developed. As described above, in vitro transcription mixtures are generally treated with DNAase enzymes to remove DNA plasmids used as templates for mRNA production. In RNA purification, proteases may also be used to reduce the protein contaminant load in a given in vitro transcription mixture. In some embodiments, the in vitro transcription mixture can be first treated with DNAse to remove plasmids, and then treated with a protease such as proteinase K to remove DNAse and large amounts of other protein contaminants before carrying out the method of the present invention. In other embodiments, the in vitro transcription mixture can be treated with proteinase K or another protease alone, as the method of the present invention makes the use of DNAse unnecessary.

[0103] In one embodiment, the in vitro transcription mixture is treated with proteinase K to reduce the protein load. The sample is filtered through a membrane filter to remove particles and then loaded onto a primary amino solid phase equilibrated to 50 mM Tris, 10 mM EDTA, pH 8.0. After loading the sample, the apparatus is washed with 50 mM Tris, 3 M guanidine-HCl, 20 mM EDTA, pH 8.0 at a volume of 10-20. The column is then washed with 20 mM Tris, 20 mM bis-tris-propane, 20 mM glycine, 50 mM NaCl, pH 8.0 to wash away guanidine and EDTA from the apparatus and prepare for ssRNA elution. The ssRNA is then eluted with a linear gradient of 50 units of apparatus volume toward 20 mM Tris, 20 mM bis-tris-propane, 20 mM glycine, 50 mM NaCl, pH 11.0. Immediately after fraction collection, the container containing the ssRNA peak is neutralized by adding 1M acetic acid.

[0104] In one embodiment, the in vitro transfer mixture is treated with 2M guanidine isothiocyanate and 20mM EDTA. The pH is adjusted to 7.5 ± 0.5 if necessary, and the sample is filtered to remove solids if necessary. The primary amino solid phase is equilibrated with 2M guanidine isothiocyanate and 20mM EDTA in 20mM Tris, 20mM bis-tris-propane, 20mM glycine, pH 8.0. The sample is loaded onto the solid phase and then tracked with equilibration buffer until the UV absorbance approaches zero. The solid phase is then washed with 20mM Tris, 20mM bis-tris-propane, 20mM glycine, 50mM NaCl, pH 8.0, and then eluted with a pH gradient toward 20mM Tris, 20mM bis-tris-propane, 20mM glycine, 20mM EDTA, pH 11.0. The gradient can be performed as a single step, a series of steps, or in a continuous (linear) format. Immediately after fraction recovery, the container containing the ssRNA peak is neutralized by adding 1 M acetic acid.

[0105] This invention offers the advantage of being able to postpone the need to separate dsRNA and ssRNA at high temperatures, but it does not rule out the possibility of doing so.

[0106] In some embodiments, the sample may be equilibrated to a high temperature, such as 37°C, 45°C, 56°C, 60°C, 70°C, or an intermediate, higher, or lower temperature, before the method is carried out at ambient temperature.

[0107] In some embodiments, the method of the present invention may be carried out at high temperatures such as 37°C, 45°C, 56°C, 60°C, 70°C, or intermediate, higher, or lower temperatures. In such embodiments, ssRNA is eluted at a pH lower than that at ambient temperature.

[0108] In some embodiments, the sample is equilibrated at a high temperature, and the method can be carried out at a high temperature.

[0109] In one embodiment, the method of the present invention can be combined with an affinity chromatography method using an oligo-dT(OdT) ligand. The two methods can be combined in any desired order.

[0110] In some embodiments, the method of the present invention can be combined with a hydrophobic interaction chromatography (HIC) method. The two methods can be combined in any desired order. In one such embodiment, the hydrophobic ligand on the HIC solid phase may consist of a phenyl group. In another such embodiment, the hydrophobic ligand on the HIC solid phase may consist of a butyl group. In yet another such embodiment, the hydrophobic ligand on the HIC solid phase may consist of a hexyl group. In yet another such embodiment, the hydrophobic ligand on the HIC solid phase may consist of different aliphatic groups or aromatic groups, or groups that embody both aliphatic and aromatic properties.

[0111] In some embodiments, the method of the present invention can be combined with a reversed-phase chromatography (RPC) method. The two methods can be combined in any desired order. In one such embodiment, the hydrophobicity of the solid-phase surface can be conferred by the intrinsic hydrophobicity of the styrene-divinylbenzene (SDVB) polymer used to synthesize the solid-phase. In another such embodiment, the hydrophobicity of the solid-phase surface can be conferred by the hydrophobicity of a ligand immobilized on the surface of the solid-phase, where the ligand represents an aliphatic hydrocarbon, an aromatic hydrocarbon, or a ligand having a mixture of aliphatic and aromatic properties.

[0112] In one embodiment, the method of the present invention can be combined with a hydroxyapatite chromatography method. The two methods can be combined in any desired order.

[0113] In one embodiment, the method of the present invention can be combined with affinity chromatography and RPC using oligo-dT ligand. The three methods can be combined in any desired order.

[0114] In one embodiment, the method of the present invention can be used to remove ssRNA from a DNA plasmid preparation. In a closely related embodiment, the method of the present invention may be used to remove ssRNA from a protein preparation. In such an embodiment, the method may be used to remove contaminating ssRNA from an enzyme preparation used for mRNA synthesis. In any of these embodiments, the sample can be loaded under acidic to neutral pH conditions and low salt concentrations, where the DNA and protein are bound to a positively charged solid phase having primarily primary amino groups. They can then be eluted with a salt gradient to increase their purity relative to contaminants while leaving ssRNA bound to the surface of the solid phase. In a closely related embodiment intended solely for ssRNA removal, the sample and solid phase conditions may include high salt concentrations at an acidic to neutral pH, where the DNA and protein flow through the solid phase while the RNA remains bound.

[0115] In some embodiments, the method of the present invention can be used as an analytical tool for quantifying the amount of ssRNA in a sample. In such embodiments, a sample with an acidic to neutral pH can be mixed with a high concentration of salt to prevent most non-ssRNA molecules from binding. The primary amino solid phase is then subjected to elution by increasing the pH gradient to sort the bound ssRNA by size. In closely related embodiments, the primary amine solid phase may be subjected to elution in a single step to an alkaline pH sufficient to elute all ssRNA in a single peak, for the purpose of maximizing the sensitivity of the assay. In related embodiments, the primary amine solid phase may be subjected to elution in a linear gradient toward 25 mM NaOH, 50 mM NaOH, 100 mM NaOH, or higher, lower, or intermediate concentrations. In another such embodiment, the primary amino solid phase may be subjected to elution in gradual steps toward 25 mM NaOH, 50 mM NaOH, 100 mM NaOH, or higher, lower, or intermediate concentrations. In another such embodiment, the primary amino solid phase may be subjected to elution in a single step with 25 mM NaOH, 50 mM NaOH, 100 mM NaOH, or higher, lower, or intermediate concentrations. In any of the above embodiments, after loading the sample onto the primary amino solid phase, a dye that interacts with RNA to produce fluorescence can be injected, and the fluorescent dye-RNA complex can be passed through a fluorescence monitor to amplify the sensitivity of the assay. In such embodiments, the dye may be ribogreen.

[0116] In one embodiment, the method of the present invention may be provided in the form of a kit for ease of implementation. The kit may comprise two or more solid phases, at least one of which is a positively charged solid phase primarily supporting primary amino groups, and the kit may also comprise instructions describing the method of the present invention. In such an embodiment, the second solid phase is an oligo-dT chromatography apparatus. In another such embodiment, the second solid phase is a hydrophobic interaction chromatography apparatus. In yet another such embodiment, the second solid phase is an oligo-dT chromatography apparatus and the third solid phase is a hydrophobic interaction chromatography apparatus.

[0117] In a typical protocol for mRNA purification using a solid phase as used in accordance with the present invention, the primary amine monolith is used as follows:

[0118] Primary amine monoliths purify large single-stranded mRNA (ssRNA) under aqueous conditions at ambient temperature. They remove dsRNA, DNA, proteins, and endotoxins, while fractionating ssRNA in ascending order of size (Figure 1). Primary amine monoliths can be used for one-step purification of research-grade ssRNA or as a high-resolution capture step in multi-step purification processes. They also enable rapid, high-resolution analytical characterization of in vitro transcription mixtures, partially purified samples, chromatographic fractions, and formulated drug substance ingredients.

[0119] Primary amine monoliths utilize a unique combination of anion exchange and hydrogen bonding to purify ssRNA under an ascending pH gradient. DNA, proteins, and dsRNAs elute before ssRNAs. Purification performance is enhanced by high-salt washing, which removes most dsRNAs, DNA, and proteins before elution. Notably, ssRNAs remain bound even at saturation concentrations of chaotropic salts. The inclusion of chelating agents during high-salt washing further enhances the removal of contaminants, allowing residual trace levels of contaminants and aggregates to be removed by the pH gradient.

[0120] The ssRNA fraction from the primary amine monolith can be further purified as desired by affinity chromatography using CIMmultus Oligo dT, hydrophobic interaction chromatography using CIMmultus C4 HLD, or reverse-phase chromatography using CIMmultus SDVB (see BIA Separations for more detailed information on any of these columns). The primary amine monolith can also be used as a polishing method, particularly after high-salt step-like precipitation or hydrophobic interaction chromatography, as it can be loaded with high-salt samples without further sample preparation.

[0121] A primary amine monolith is a radial flow chromatography apparatus. It is designed to distribute the flow from the outside to the inside of the cylinder. This has the effect of stabilizing the physical structure of the cylinder and also has a concentration effect during elution that improves separation performance. Before conducting experiments, ensure that the apparatus is connected to the chromatograph so that the flow direction aligns with the apparatus markings. Note that some chromatographs have a default backflow function built into the software, which may reverse the flow direction without warning. Ensure that this function is disabled before conducting experiments.

[0122] Primary amine monoliths are delivered in 20% ethanol. Disinfection and regeneration as described below are recommended before actual use. It is also recommended to perform a sample-less run to provide a baseline for comparing experimental results. Some buffer components absorb UV light, and some transfer between buffers may produce refractive index artifacts that could confuse the interpretation of experimental results.

[0123] Samples and Preparation: Primary amine monoliths can be used to process in vitro transcription mixtures including those after digestion with DNAse and / or proteinase K, as well as ssRNA resuspended from salt precipitates or organic solvent precipitates, or partially purified ssRNA from other purification methods. Samples containing divalent metal cations should be treated with a chelating agent at approximately 10 times the estimated concentration of the metal ion. Particles must be removed by centrifugation or filtration (0.45 μm) before injection. The pH of the sample should be pH 6.0 to 8.0. Salt content is not considered.

[0124] Buffer A: Equilibrium buffer / gradient initiation buffer. 20 mM Tris, 20 mM bis-tris-propane, 20 mM glycine, 50 mM NaCl, pH 8.0. Buffer B: High salt wash buffer. 50mM Tris, 3.0M Guanidine-HCl, 20mM EDTA, pH 8.0. Buffer C: Gradient endpoint buffer. 20 mM Tris, 20 mM bis-tris-propane, 20 mM glycine, 50 mM NaCl, pH 11.0. Buffer D: Washing / disinfection buffer. 2M NaCl, 1M NaOH. Buffer E: Regenerated buffer. 3.0M ammonium acetate.

[0125] Equilibrate the column using buffer A: Pump the equilibration buffer through the column until the pH and conductivity of the output buffer are the same as those of the input buffer. Flow rate: 10 column volumes (CV) / min.

[0126] Inject the sample. Observe the operating pressure while applying a large amount of sample, especially crude samples such as in vitro transfer mixtures. Reduce the flow rate as needed and maintain the operating pressure within acceptable limits.

[0127] Washing with buffer A at a 1:10-20CV equilibration buffer. Since the contaminants at the trailing end are removed by the subsequent high-salt washing step, there is no need to wait for the UV signal to return completely to the baseline.

[0128] Wash with buffer B: 10-20 CV of equilibrating buffer. Note that guanidine absorbs UV light, so a peak will appear immediately. DNA and dsRNA typically elute as fairly sharp peaks approaching the peak apex. Continue washing until the guanidine peak reaches a level plateau.

[0129] Wash with buffer A: 3:10-20 CV of equilibration buffer, or until UV returns to baseline.

[0130] Elution gradient toward buffer C: A linear gradient of 50–100 CV toward the 100% gradient endpoint buffer, then held at 100% for 10 CV. The fraction is neutralized immediately after elution.

[0131] Wash / disinfect with buffer D. After each run, it is recommended to treat with 10-20 CV of disinfectant buffer to determine if a significant amount of material remains bound to the column at the end of the pH gradient. The contents of the disinfection step may be collected at elution and neutralized for further analysis. If the column is loaded with an in vitro transfer mixture, the duration of the disinfection step may need to be extended to 1 hour. In cases of extreme contamination, it may be necessary to extend the disinfection period to 16-24 hours. Maintaining a minimum flow rate during disinfection tends to yield better results as it continuously replenishes OH ions and washes the contaminants off the column, as well as simply hydrolyzing them in situ.

[0132] Regenerating the column: Wash the column with 20 CV of buffer A or water to remove NaOH, and then wash with 20 CV of buffer E. This removes hydroxide counterions from the monolith surface; otherwise, the hydroxide counterions can slow the column's equilibration and generate pH artifacts during elution.

[0133] Storage: After disinfection, rinse the column with buffer A or water and store it in 20% ethanol.

[0134] A typical chromatogram is shown in Figure 8.

[0135] Variations, optimization, troubleshooting

[0136] Chromatograms are used as a guide to optimize the time for each individual step.

[0137] For example, significant modifications to high-salt washing are possible by using chaotropic salts at different concentrations up to complete saturation, and by using chelating agents at different concentrations. The concentrations shown in the protocol are intended as a starting point. If equivalent purity can be achieved at lower concentrations, using smaller quantities will reduce material costs and simplify buffer preparation.

[0138] Chaotropic salts can be replaced with non-chaotropic salts, but caution is necessary when applying high concentrations of salts that precipitate RNA. These include, among others, sodium chloride, potassium chloride, and lithium chloride. Preliminary experimental results have shown that washing with 1M sodium chloride removes most of the dsRNA and DNA, but may require prolonged washing to restore UV absorbance to baseline (Figure 1).

[0139] A workflow simplification can be implemented by directly adding guanidine and EDTA to the sample and equilibrating the column toward a high-salt wash buffer. Following sample loading, an initial wash is performed using the high-salt buffer, followed by a second wash from above using buffer A, and then elution. It should be noted that this approach may also support higher ssRNA binding capacity because it prevents dsRNA and DNA from competing for binding surface area. This approach may also reduce column fouling.

[0140] The high-salt washing step can be completely omitted. This may be preferable for analytical characterization of samples aimed at determining the relative amounts of dsRNA and ssRNA, or DNA and ssRNA. This method generally does not distinguish between dsRNA and DNA.

[0141] Removing salts from the pH gradient increases the separation of dsRNA and ssRNA, but doing so reduces the yield and raises the pH at which elution occurs. The use of RNA-precipitating salts is permissible as long as their concentration is maintained well below the precipitation level (Figure 6). Salts that promote RNA solubility, such as chaotrope, can also be used during pH elution, but it should be considered that they may interfere with subsequent purification methods.

[0142] The operating temperature may rise in the pH gradient, which has the effect of eluting the solute more quickly. Uncontrolled operating temperatures can impair reproducibility.

[0143] A linear pH gradient can be converted to a stepwise gradient format. In some cases, such as when CIMmultus dsX-β is combined with an orthogonal purification method, it may be practical to elute ssRNA in a single pH step.

[0144] Signs of improper washing may include a gradual increase in operating pressure over a series of runs, a selectivity shift in which a given species elutes earlier or later in previous runs, and / or the appearance of ghost peaks where peaks appear during elution even though no sample has been injected.

[0145] The present invention can be further described by the following non-limiting embodiments. [Examples]

[0146] Example 1. When separating dsRNA from ssRNA using a primary amino solid phase with a pH gradient at ambient temperature, the dsRNA is removed by a salt step before eluting the ssRNA. A sample containing a dsRNA ladder with dsRNA molecules in the range of 21b–500b and an ssRNA ladder with ssRNA molecules in the range of 200b–6000b was applied to a primary amine solid phase in the form of a monolith chromatography apparatus. The primary amine monolith was equilibrated with 20 mM Tris, 20 mM bis-tris-propane, 20 mM glycine, pH 8.0. The sample was applied, and a washing solution was applied to remove unbound material from the channels in the monolith. A washing step with 20 mM bis-tris-propane, 20 mM glycine, 1 M NaCl, pH 8.0 was then applied, followed by another washing step to remove NaCl. The monolith was then subjected to elution at a linear gradient toward 20 mM Tris, 20 mM bis-tris-propane, 20 mM glycine, pH 11.0. As shown in Figure 1, all dsRNA species eluted in the NaCl step, while ssRNA remained bound and eluted later in the pH gradient. No clear size segregation was observed between species within the dsRNA ladder, but clear segregation was observed between species within the ssRNA ladder.

[0147] Example 2. When separating dsRNA from ssRNA using a primary amino solid phase with a pH gradient at ambient temperature, the dsRNA is removed by a washing step containing 6M guanidine-HCl and 20mM EDTA before eluting the ssRNA. A sample containing a dsRNA ladder with dsRNA molecules in the range of 21b to 500b and ssRNA at 5000b was applied to a primary amine solid phase in the form of a monolithic chromatography apparatus. The primary amine monolith was equilibrated with 20 mM Tris, 20 mM bis-tris-propane, 20 mM glycine, pH 8.0. The sample was applied, and a washing solution was applied to remove unbound material from the channels in the monolith. A washing step was then applied with 20 mM bis-tris-propane, 20 mM glycine, 6 M guanidine, 20 mM EDTA, pH 8.0, followed by another washing step to remove guanidine and EDTA. The monolith was then subjected to elution with a linear gradient toward 20 mM Tris, 20 mM bis-tris-propane, 20 mM glycine, pH 11.0. As shown in Figure 2, all dsRNA species eluted in the salt step, while ssRNA remained bound and eluted later in the pH gradient.

[0148] Example 3. The pH gradient for eluting ssRNA from strong and weak anion exchangers failed. The behavior of ssRNA in a pH gradient was characterized by comparing its pH gradient elution performance on a primary amino solid phase with that of a strong anion exchanger (quaternary amine, QA) and a weak anion exchanger (tertiary amine, DEAE) as experimental control groups. Both anion exchangers were physically monolithic devices with a volume of 1 mL. The column was equilibrated with 20 mM Tris, 20 mM bis-tris-propane, 20 mM glycine, and pH 8.0. A sample containing approximately 5000 nucleotides (5000 b) of ssRNA was injected, and the column was washed with equilibration buffer to remove unbound material. The column was then subjected to elution with a linear gradient toward 20 mM Tris, 20 mM bis-tris-propane, 20 mM glycine, and pH 11.0. Figure 3 shows that ssRNA did not elute within the pH gradient in either the strongly or weakly anion-exchanged column, and was removed only by washing the column with 1M NaOH.

[0149] Example 4. Plasmid DNA is separated from ssRNA using a primary amino solid phase with a pH gradient at ambient temperature. A sample containing a supercoiled dsDNA plasmid approximately 6000 base pairs in size and ssRNA approximately 5000 base pairs in size was applied to a primary amine solid phase in the form of a monolith chromatography apparatus. The primary amine monolith was equilibrated with 20 mM Tris, 20 mM bis-tris-propane, 20 mM glycine, pH 8.0. The sample was applied, and a washing solution was applied to remove unbound material from the channels in the monolith. The monolith was then subjected to elution with a linear gradient toward 20 mM Tris, 20 mM bis-tris-propane, 20 mM glycine, pH 11.0. As shown in Figure 4, the DNA plasmid was eluted first and was sufficiently separated from the ssRNA which was eluted later.

[0150] Example 5. When separating plasmid DNA from ssRNA using a primary amino solid phase with a pH gradient at ambient temperature, the DNA is removed by a salt step before eluting the ssRNA. A sample containing a supercoiled dsDNA plasmid with a size of approximately 6000 base pairs and an ssRNA with a size of approximately 5000 base pairs was applied to a primary amine solid phase in the form of a monolithic chromatography apparatus. The primary amine monolith was equilibrated with 20 mM Tris, 20 mM bis-tris-propane, 20 mM glycine, and pH 8.0. The sample was applied, and a washing solution was applied to remove unbound material from the channels in the monolith. Next, a washing step with 20 mM bis-tris-propane, 20 mM glycine, 1 M NaCl, and pH 8.0 was applied, followed by another washing step to remove NaCl. The monolith was then subjected to elution with a linear gradient toward 20 mM Tris, 20 mM bis-tris-propane, 20 mM glycine, and pH 11.0. As shown in Figure 5, the DNA plasmid eluted at the salt stage, while the ssRNA remained bound and eluted later in the pH gradient.

[0151] Example 6. The effect of combining salt and pH gradient elution on the separation of DNA and ssRNA at ambient temperature. A series of separations were performed comparing simple pH gradient elution, pH gradient elution with an endpoint buffer of 50 mM NaCl, and pH gradient elution with an endpoint buffer of 1000 mM NaCl. In each experiment, a sample containing a supercoiled dsDNA plasmid with a size of approximately 6000 base pairs and an ssRNA with a size of approximately 5000 base pairs was applied to a primary amine solid phase in the form of a monolith chromatography apparatus. The primary amine monolith was equilibrated with 100 mM Tris, pH 8.0. The sample was applied, and a washing solution was applied to remove unbound material from the channels in the monolith. In the first experiment, the monolith was subjected to elution with a linear gradient toward 125 mM glycine, pH 10.5. In the second experiment, the monolith was subjected to elution with a linear gradient toward 125 mM glycine, 50 mM NaCl, pH 10.5. In the third experiment, the monolith was subjected to elution using a linear gradient toward 125 mM glycine, 100 mM NaCl, and pH 10.5. As shown in Figure 6, in the absence of sodium chloride, the center of the ssRNA peak occurred at pH 10.2. When 50 mM sodium chloride was included in the gradient endpoint buffer, ssRNA eluted at pH 10.0. When 100 mM sodium chloride was included in the gradient endpoint buffer, ssRNA eluted at pH 9.9. When sodium chloride was maintained at a concentration level of 50 mM across the pH gradient, ssRNA eluted at pH 9.8 (not shown). When sodium chloride was maintained at a concentration level of 100 mM across the pH gradient, ssRNA eluted at pH 9.6 (not shown). The inclusion of sodium chloride caused other interesting effects. Separation between DNA and ssRNA was maximized in the absence of NaCl, and large amounts of NaCl further reduced the separation. However, the yield of ssRNA was clearly improved in the presence of sodium chloride.

[0152] Example 7. Homogeneity of reactions involving dsRNA and DNA As is evident in the experiments described above, dsRNA and DNA behave similarly, both being eliminated by salt washing and both being separated from ssRNA within a pH gradient, suggesting that DNA can be used as a model of dsRNA behavior to characterize the method of the present invention. Experiments were conducted to test the ability of the method of the present invention in the most extreme worst-case scenario, which never occurs during the process of purifying ssRNA from a typical transcription mixture. This test involved separating a relatively small ssRNA of about 1700 bases from a dsRNA of about 13,000 bases in size, and comparing the elution behavior of a 13kb dsRNA with that of a DNA plasmid of about 6000 base pairs. Primary amine monoliths were equilibrated at 100 mM Tris, pH 8.0. In each experiment, the sample was applied, and a washing solution was applied to remove unbound material from the channels in the monolith. The monolith was then subjected to elution in a linear gradient toward 125 mM glycine, pH 10.5. As shown in Figure 7, as expected, DNA and large dsRNAs co-eluted for the most part, with both eluting before the much smaller ssRNAs. Although the degree of separation between the ssRNAs and the two larger contaminants was lower compared to other examples, clear separation was still evident. In fact, the dsRNAs in a given in vitro transcription mixture contain the same number of bases as the ssRNAs. Compared to Example 4, which shows that the ssRNAs elute later in the pH gradient according to their size, this suggests that the present invention provides good separation between dsRNAs and ssRNAs regardless of their size. This point may seem meaningless considering that dsRNAs can be easily removed by salt washing before eluting ssRNAs in the pH gradient, but it shows that the present invention can achieve effective separation even in the absence of salt washing. The results in Figure 7 are also noteworthy in that the present invention should work well even with ssRNAs of 15,000 b or more, possibly 25,000 b or more.

[0153] Example 8. When separating dsRNA from ssRNA using a primary amino solid phase with a pH gradient at ambient temperature, DNA is removed by a chelating agent-chaotrope combination step before eluting the ssRNA. A sample containing a 6000 bp supercoiled dsDNA plasmid and 5000 bp ssRNA was applied to a primary amine solid phase in the form of a monolith chromatography apparatus. The primary amine monolith was equilibrated with 20 mM Tris, 20 mM bis-tris-propane, 20 mM glycine, pH 8.0. The sample was applied, and a washing solution was applied to remove unbound material from the channels in the monolith. A washing step was then applied using 20 mM bis-tris-propane, 20 mM glycine, 1 M guanidine-HCl, 20 mM EDTA, pH 8.0, followed by another washing step to remove guanidine and EDTA. The monolith was then subjected to elution with a linear gradient toward 20 mM Tris, 20 mM bis-tris-propane, 20 mM glycine, pH 11.0. Figure 8 shows that the majority of the DNA plasmid was removed by guanidine-EDTA (chaotrope-chelating agent) washing. The remaining bound DNA elutes before the ssRNA under a pH gradient.

[0154] Comparing Figure 8 (using 1M guanidine) with Figure 5 (using 6M guanidine), the results suggest that 1M guanidine-HCl is insufficient to completely remove DNA before eluting ssRNA. In Figure 5, the dsRNA peak was concentrated at the very front of the 6M guanidine wash peak. In Figure 8, DNA begins to elute towards the front of the guanidine wash, but significantly later than in Figure 5, and exhibits a prominent tail. The relative size of the DNA / ssRNA peak eluting immediately before the ssRNA peak further supports the conclusion that 1M guanidine-HCl does not preemptively remove DNA / dsRNA. However, it should also be noted that while ssRNA elutes as a single peak after pre-washing with 1M guanidine (Figure 8), in Figure 5 (after 6M guanidine), although the ssRNA peak was observed as expected, it was followed by eluted material interpreted as representing aggregates. This comparison suggests that adjusting the chaotrope concentration to provide optimal DNA / sdRNA clearance without altering the desired ssRNA behavior is useful as part of a process optimization routine.

[0155] Example 9. Decreased ssRNA elution pH due to the presence of polyvalent anions. A series of experiments were conducted to fractionate a mixture of DNA and ssRNA using a pH gradient. A primary amine solid phase was equilibrated with 100 mM Tris, pH 8.0. The sample was then loaded and washed with the equilibration buffer. A linear gradient with a 125-volume instrument was applied down to an endpoint buffer of 125 mM glycine, pH 10.5. In the second experiment, the conditions were repeated except that 100 mM citrate was added to the buffer. In the third experiment, the conditions were repeated except that 100 mM ethylenediaminetetraacetic acid (EDTA) was added to the buffer. As shown in Figure 9, both additives strongly reduced both the pH at which DNA and ssRNA eluted, with EDTA exhibiting the strongest pH reduction observed. The degree of pH reduction was also greater for both than that achieved with 100 mM sodium chloride (Figure 6). A common finding with the sodium chloride results was that the presence of the salt increased the yield of DNA and ssRNA in the elution gradient. Another similarity was that elution with sodium chloride, citrate, or EDTA did not elute ssRNA at pH values ​​below 8.0.

[0156] Example 10. Purification of ssRNA by high-salt washing prior to elution using a pH gradient in the presence of salt. Equilibrate the primary amine solid phase in the form of a chromatographic apparatus such as a monolith to 50 mM Tris, 20 mM EDTA, 2 M guanidine isothiocyanate, pH 8.0 ± 0.5. Set the UV monitor to zero. Add EDTA and guanidine isothiocyanate to the sample and adjust the sample pH to pH 8.0 ± 0.5. If necessary, filter the sample to remove turbidity. Apply the sample to the primary amine monolith and wash the monolith with equilibration buffer until the baseline returns to zero. The application and washing of the sample in a strong chelate-chaotrope solution is intended to prevent and / or remove trace levels of dsRNA, DNA, and protein binding. Then, wash the monolith with 50 mM Tris, 5 mM EDTA, 100 mM guanidine isothiocyanate, pH 8.0 ± 0.5 for at least 10 instrument volumes. Next, the ssRNA is eluted in a linear gradient toward 65 mM glycine, 5 mM EDTA, 100 mM guanidine isothiocyanate, and pH 10.5 ± 0.5. The presence of the chelating agent is maintained throughout the method to prevent adverse effects from the potential presence of polyvalent metal cations. To avoid a decrease in ssRNA solubility, 100 mM guanidine isothiocyanate is used instead of 100 mM sodium chloride as described in Example 6 or 100 mM citrate or 100 mM EDTA as described in Example 9. If desired, the concentration of guanidine isothiocyanate is increased in a parallel experiment to further lower the pH at which ssRNA is eluted. If desired, the chaotropic salt guanidine thiocyanate is replaced with a different salt in a parallel experiment. If desired, the type and concentration of the chelating agent are changed in a parallel experiment.

[0157] Example 11. An in vitro transfer mixture is eluted from a primary amine solid phase using a pH-salt gradient combination. The following buffers were prepared: Buffer A containing 50 mM Hepes, pH 7.0; Buffer B containing 50 mM Hepes and 200 mM sodium pyrophosphate, pH 8.5; Buffer C containing 100 mM sodium hydroxide and 2.0 M sodium chloride; and Buffer D containing 1.5 M Hepes, pH 7.0. A primary amine monolith with a 2 μm channel and a bed volume of 100 μL, stored in 20% ethanol, was washed with 50 CV of water at a flow rate of 2 mL / min (20 CV / min). The column was equilibrated with Buffer A, and a 25 μL in vitro transcription mixture sample, collected 30 seconds after the start of the reaction, was injected. The column was washed with 20 CV of Buffer A, then a 40 CV linear gradient toward 20% Buffer B was applied, followed by a 10 CV gradient hold with 20% Buffer B. This segment eluted nucleotides and double-stranded species containing DNA plasmids. The mRNA was eluted by applying a 10 CV linear gradient toward 50% buffer B, followed by holding the gradient at 50% B. An additional 10 CV linear gradient toward 100% B was applied, followed by holding the gradient for 10 CV. The column was washed with buffer C, and then returned to pH 7 with buffer D. The column was re-equilibrated, and a sample taken 4 hours after the start of in vitro transcription was injected. Elution was performed in the same manner as with the previous sample. After the run was complete, the column was washed with 20% ethanol and stored in the solution. All chromatographic steps were performed at ambient temperature. The elution profiles are shown in Figure 10. The profile corresponding to the sample taken at 30 seconds is shown by the long dashed line. The profile corresponding to the sample taken at 4 hours is shown by the solid line. The short dashed line indicates conductivity. As expected, the levels of constituent nucleotides such as CTP, UTP, ATP, and GTP decreased to lower levels throughout the in vitro transcription process, reflecting their incorporation into the newly synthesized mRNA.

[0158] References All references cited herein are incorporated by reference unless their incorporation would contradict the express teachings herein. [1] M Baiersdorfer, G Boros, H Murumatsu, A Mahini, I Vlatkovic, U Sahin, K Kari-ko, A fascile method for the removal of dsRNA contaminant from in vitro-transcribed mRNA, Molecular therapy: nucleic acids, 15 (2019) [2] S. Urayama, Y. Yoshida-Takashima, M. Yoshida, Y. Tomaru, H. Moriyama, K. Takai, T. Nunoura, A New Fractionation and Recovery Method of Viral Genomes Based on Nucleic Acid Composition and Structure Using Tandem Column Chroma-tography. Microbes Environ. 30, (2015) 199-203. [3] R. Franklin, Purification and properties of the replicative intermediate of the RNA bacteriophage R17. Proc. Natl. Acad. Sci. USA 55, (1966) 1504-1511. [4] A Nwokeoji, AW Kung, P Kilby, D Portwood, M Dickman, Purification and char-acterization of dsRNA using ion pair reverse phase chromatography and mass spectrometry, J. Chromatography A 1484 (2017) 14-25. [5] A Nwokeoji, M Earll, P Kilby, D. Portwood, M. Dickman, High resolution finger-printing of double-stranded RNA using ion-pair reversed phase chromatography, J. Chromatography B 1104 (2019) 212-219.

[0159] [6] K Kariko, H Muramatsu, J Ludwig, D. Weismann, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves transla-tion of nucleoside-modified, protein-encoding mRNA, Nucleic acid research, 39 (2011) el42. [7] D. Weismann, N. Pardi, H Murumatsu, K Kariko, HPLC purification of in vitro transcribed long RNA, Methods Molecular Biology, 969 (2013) 43-54. [8] A Romanovskaya, LP Sarin, DH Bramford, MM Poranen, High-throughput puri-fication of double-stranded R.N.A. molecules using convective interaction media monolithic anion exchange columns, J. Chromatography A, 1278 (2013) 54-60. [9] WO 2014 / 144767 A1

Claims

1. applying a sample containing single-stranded RNA to a solid phase carrying predominantly or exclusively primary amino groups on its surface at a pH of less than 8.5 which binds at least predominantly said single-stranded RNA, eluting the adsorbed single-stranded RNA from the surface of the solid phase by exposing the surface of the solid phase to an ascending pH gradient beginning at a pH equal to or greater than the pH used to apply the sample to the solid phase; A method for purifying single-stranded RNA, comprising:

2. 2. The method of claim 1, wherein after applying the sample and before eluting the single-stranded RNA, at least one step is provided of washing the solid phase with a wash buffer having a higher ionic strength than an elution buffer used to elute the single-stranded RNA.

3. 3. The method of claim 2, wherein the at least one washing step is provided to reduce the higher ionic strength.

4. 4. The method of claim 1, wherein after applying the sample and before eluting the single-stranded RNA, at least one washing step is provided with a wash buffer having an elevated pH, which keeps single-stranded RNA adsorbed to the solid phase and desorbs remaining double-stranded RNA.

5. 5. The method of claim 2, wherein the ionic strength of the wash buffer is in the range of 0.5 M to 12 M, or 1.0 M to 10 M, or 2.0 M to 8.0 M, or 4.0 M to 6.0 M higher than the molarity of salt required to elute the single-stranded RNA.

6. The method according to any one of claims 2 to 5, wherein the ionic strength of the washing buffer is adjusted by the concentration of a chaotropic salt.

7. 7. The method of claim 6, wherein the chaotropic salt is selected from the group consisting of guanidinium salts, thiocyanates, perchlorates, and combinations thereof.

8. A method described in any one of claims 1 to 7, wherein the increasing pH gradient comprises a pH range of pH 8.5 to pH 9.

0.

9. 9. The method according to claim 1, wherein the elution of the single-stranded RNA from the surface of the solid phase is carried out with an elution buffer having a pH in the range of pH 7.5 to pH 12.0, or pH 8.0 to pH 11.5, or pH 8.5 to pH 11, or pH 9.0 to pH 10.

5.

10. The method according to claim 1, wherein the elution of the single-stranded RNA from the surface of the solid phase is carried out with an elution buffer having an alkaline pH value of pH 9.0 or higher.

11. 11. The method of any one of claims 1 to 10, wherein a chelating agent is present in the sample, in the environment of the surface of the solid phase prior to the application of the sample, in a buffer for eluting the single-stranded RNA from the surface of the solid phase, and / or in a separate buffer used during the steps of applying the sample to the solid phase and / or eluting the single-stranded RNA from the surface of the solid phase.

12. 12. The method of claim 11, wherein the chelating agents are independently selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), citrate, phosphate, or ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), tris(2-aminoethyl)amine (TREN), and mixtures thereof.

13. The method according to any one of claims 1 to 12, wherein the single-stranded RNA has a size ranging from 1,000 bases to 25,000 bases.

14. 1. Use of a solid phase containing mainly or only primary amino groups on its surface for the purification of single-stranded RNA, wherein the single-stranded RNA is purified from double-stranded RNA by an ascending pH gradient, and elution of the single-stranded RNA from the surface of the solid phase is carried out with an elution buffer having an alkaline pH value of 8.5 or higher.

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