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 and single-stranded RNA. [Background technology]

[0002] The synthesis of messenger RNA (mRNA) for gene therapy applications generates preparations containing, in addition to the desired single-stranded (ss) RNA, an undesired subpopulation of double-stranded (ds) RNA. These dsRNA species form post-synthesis through intrastrand interactions of complementary sequences within the base strand. The formation of dsRNA sequences can also occur through pairing with complementary sequences between adjacent ssRNA molecules, thereby creating nonspecific interstrand dimers and higher-order multimers that may also contain intrastrand ds sequences. Removal of double-stranded RNA is a particular purification goal because it induces unwanted and potentially lethal immune responses when injected into a subject.

[0003] Methods for reducing the dsRNA content from mRNA preparations are known. The level of dsRNA contamination can be reduced by affinity adsorption chromatography using cellulose-based chromatography media [1-3]. While the exact mechanism of adsorption is unclear, dsRNA binds under certain conditions, while ssRNA passes through. While this method is simple and effective at the laboratory scale, it is burdensome due to its low capacity. At the manufacturing scale, this low capacity is associated with large columns requiring large amounts of buffer, large manufacturing area, and extended processing time, which reduces the productivity of the manufacturing facility. This method also results in dilution of the processed ssRNA, which is associated with increased product volume, which burdens subsequent purification steps.

[0004] Alternatively, dsRNA levels can be reduced by reversed-phase chromatography (RPC) using a styrene-divinyl-benzene (SDVB) solid phase [4-7]. RPC uses toxic and flammable organic solvents, requiring specialized equipment that is prohibitively expensive on an industrial scale to mitigate the risk of fire and explosion. RPC also imposes additional safety concerns related to organic solvent toxicity and hazardous waste disposal issues in the work environment. In addition to solvent issues, RPC separations often impose the additional burden of requiring high temperatures for best results.

[0005] Anion exchange chromatography has shown utility in the purification of small mRNAs (<1000 bases) [8]. Anion exchange media evaluated to date include so-called strong anion exchangers, which refer to quaternary amine (QA) anion exchangers. So-called weak anion exchangers, particularly diethylaminoethyl (DEAE) anion exchangers that use tertiary amine ligands, have also been evaluated.

[0006] Anion exchange chromatography has shown limited utility in removing DNA and protein contaminants from large mRNAs (1,000–10,000 bases) and only at elevated operating temperatures. [9] Large mRNAs can be eluted in a sodium chloride gradient at temperatures up to 65°C. However, high-temperature operation imposes complex logistical burdens, as buffers, samples, and columns must all be pre-equilibrated and precisely maintained at specific operating temperatures throughout the process, and reproducibly maintained across all batches for the product's manufacturing lifespan, potentially several years.

[0007] Proteins become more electropositive and less electronegative with decreasing pH, and It is known that proteins can be eluted from anion exchangers by a descending pH gradient, which has the effect of stopping their association with the anion exchanger. This approach does not work with RNA, as the charge characteristics of RNA remain constant from about pH 2.6 to pH 13.0. It is known that increasing the pH causes RNA to bind more strongly, so elution of biomolecules from anion exchangers by this method does not work. Proteins Rise pH gradient To distribute Thus, although it can be eluted from the cation exchanger, the method is not useful for separating dsRNA from ssRNA because ssRNA and dsRNA do not bind to the cation exchanger. Summary of the Invention [Problem to be solved by the invention]

[0008] A novel method for removing dsRNA from mixed preparations with ssRNA has been developed, which represents an improvement over known methods. This is relevant for mRNAs of all sizes, but particularly for large or giant mRNAs, such as those in the size range of 1,000 to 25,000 bases. This method allows for the separation of ssRNA and dsRNA bound to a primary amino solid phase using an ascending 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 single-stranded RNA purification is claimed, said method comprising: 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; Includes:

[0010] In some embodiments of the method of the present invention, at least one step may be provided, after applying the sample and before eluting the single-stranded RNA, of washing the solid phase with a wash buffer having a higher ionic strength than the elution buffer used to elute the single-stranded RNA.

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

[0012] In a further embodiment of the method of the invention, after applying the sample and before eluting the single-stranded RNA, at least one washing step may be provided using a wash buffer with an elevated pH, which keeps the single-stranded RNA adsorbed to the solid phase and desorbs 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 wash buffer can be selected within 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 wash buffer can be 2.5 M. In another example, if the ionic strength at which ssRNA elutes is 0.5 M, the ionic strength of the wash buffer can be 1.0 M.

[0015] Typically, the molarity of the wash buffer ranges from 0.51 M to 12.0 M, while the molarity at which ssRNA is eluted ranges from 0.01 M to 0.5 M.

[0016] In yet another embodiment of the method of the present invention, the ionic strength of the wash buffer may be adjusted by the concentration of a chaotropic salt, in particular 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 single-stranded RNA can be eluted from the surface of the solid phase 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.

[0018] In yet a further embodiment of the method of the present invention, applying said sample to said solid phase may occur at a pH value below about pH 8.5.

[0019] In still further embodiments of the method of the invention, a chelating agent may be present in the essentially aqueous mixture, in the environment of the surface of the solid phase prior to said contact with the aqueous mixture, 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.

[0020] In another embodiment of the present invention, the chelating agents 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 has a size ranging from 1,000 bases to 25,000 bases.

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

[0023] The surprising nature of this method is underscored by the fact that it is contrary to the teachings of the prior art, which teach that it is not possible 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 thought impossible to elute any biomolecular species from any anion exchanger by increasing the pH highlights a second surprising feature of the present invention. This contradicts a fundamental principle known in the art for decades: that acidic solutes (net negatively charged) bind stronger to strong anion exchangers with increasing 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 subjected to elution exclusively with a descending pH gradient.

[0025] The present invention embodies a third surprising feature that is unprecedented in the art: Preliminary data indicate that elution of ssRNA from a primary amino solid phase by a pH gradient separates ssRNA species according to size, with higher pH values ​​required to elute larger ssRNA species. Notably, size discrimination between dsRNA species is relatively poor.

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

[0027] No theory has yet been developed to explain the paradoxical behavior of primary amino solid phases eluted with an ascending pH gradient, why it contradicts predictions based on traditional anion exchangers, how dsRNA is separated from ssRNA, or how size fractionation of ssRNA is achieved.

[0028] Rather than simply displacing dsRNA prior to elution of ssRNA, the high-salt wash offers a unique opportunity to dissociate otherwise stable complexes between ssRNA and contaminants such as proteins and DNA. This provides a solution to a major problem only beginning to be recognized in the field. Nucleic acids often exist in stable complexes with contaminants. Some of these complexes elute under the same or nearly the same conditions as pure nucleic acids. These complexes are therefore Trojan horses, introducing contaminants into what would otherwise be pure ssRNA elution fractions, even though their independent properties would preclude this possibility. The dissociation high-salt wash offers the potential to stop this contamination pathway. 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 presence of a chelating agent can further enhance complex dissociation.

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

[0030] It will be recognized by those skilled in the art that this simplification of the workflow is a slight process variation that uses the same principle as that used in the basic configuration of the present invention, which is that dsRNA will not bind to a primary amino solid phase in the presence of high salt concentrations.

[0031] A final surprising feature of the present invention is that the elution behavior of contaminants that differ in 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. Most of them are also removed by salt applied to the solid phase before separating their trace residues from ssRNA with an increasing pH gradient.

[0032] General description of the invention In one general aspect, the present invention is a solid-phase extraction method for removing double-stranded messenger RNA (dsRNA) from a preparation containing a mixture of 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 invention relates to the separation of dsRNA from ssRNA and / or the separation of ssRNA species according to their size, where the dsRNA and ssRNA are bound by non-covalent interactions to a primary amino solid phase.

[0034] In another specific embodiment, the 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, with smaller ssRNA species eluting at a lower pH value than larger ssRNA species.

[0035] In more specific embodiments, the present invention relates to pH gradients spanning pH 3.5 to 11.5, or pH 4.5 to 11.5, or pH 5.5 to 11.5, or 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 higher, lower, or intermediate pH ranges. The pH ranges specified are due to the fact that the values ​​required to separate dsRNA from a particular ssRNA or to fractionate different sized ssRNA species depend on their respective sizes.

[0036] In certain embodiments, the pH value can be increased continuously over a certain range, forming a so-called linear gradient. In related embodiments, the pH can be increased discontinuously, in discrete steps, forming a so-called step gradient. In other related embodiments, the gradient can consist of a single step.

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

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

[0039] In certain embodiments, the chelating agent can be ethylenediaminetetraacetic acid (EDTA) at a concentration 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, intermediate, or higher ranges up to full saturation. In closely related embodiments, the chelating agent can 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 spanning the same ranges as described for EDTA.

[0040] In certain embodiments of the invention, treatment with a chelating agent can include any of the following for performing the steps of the method, including during pH elution: adding a chelating agent to the sample, buffer exchanging the sample into a metal ion-free buffer, buffer exchanging the sample into a chelating buffer, and using a metal ion-free or chelating buffer. In some such embodiments, the type of chelating agent and / or their concentration can be different for each step of the method.

[0041] In one aspect, the invention relates to the separation of dsRNA and ssRNA in which a primary amino solid phase is washed with salt to remove a subset of dsRNAs, before the final separation of dsRNA and ssRNA is carried out by increasing the pH. In all such embodiments, the concentration of salt during the wash is higher than the concentration of salt present during elution.

[0042] In certain embodiments, the salt used to wash the solid phase prior to elution by increasing the pH can be any type of salt at any concentration, including up to full saturation. In one such embodiment, where the salt is sodium chloride at approximately 5.0 M saturation, the salt concentration can range from 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 any intermediate range, but preferably ranges from 1 M to 5 M. In a closely related embodiment, the salt can be potassium chloride. It will be appreciated that RNA can be precipitated by other high concentrations of neutral salts, such as lithium chloride, as well as by sodium chloride or potassium chloride. It will further be appreciated that precipitation of RNA during sample application or elution is highly undesirable, as it can interfere with buffer flow through the solid-phase chromatography device. Strictly speaking, this is not an issue if a salt wash is performed after sample loading to remove excess salt before elution, but it is often preferable to avoid the problem altogether by using a non-RNA precipitating salt instead.

[0043] In some embodiments where the RNA precipitating salt is replaced with a non-RNA precipitating salt, the salt is a chaotropic salt such as guanidinium hydrochloride that is saturated at about 6M, and the concentration can be in the range of 10mM to 6M, or 50mM to 6M, or 100mM to 6M, or 500mM to 6M, or 1M to 6M, or 2M to 6M, or 3M to 6M, or 4M to 6M, or 5M to 6M, or any intermediate range, preferably in the range of 3M to 4M. In a closely related embodiment, where the salt is a chaotropic salt such as guanidine thiocyanate, which saturates at about 12 M, the concentration can 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 an intermediate range, preferably a value in the range of 1.5 M to 3.0 M. From these two salt examples, it will be apparent that other salts can be similarly used up to but not beyond the point of saturation.

[0044] In certain embodiments, a chelating agent can be used in conjunction with a salt. In various embodiments of the present invention, treatment with a salt and / or chelating agent can include any of the following: adding salt and a chelating agent to the sample, exchanging the sample's buffer into a salt- and / or chelating agent-containing buffer, exchanging the sample's buffer into a salt- and / or chelating agent-containing buffer, and using a salt- and / or chelating agent-containing buffer to perform the method steps, including eluting ssRNA. In some such embodiments, the type of salt, chelating agent, and their respective concentrations can be different for each step of the method.

[0045] In some embodiments, dsRNA can be separated from ssRNA by equilibrating the sample with a high concentration of a non-RNA precipitating 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. During the application of the sample to the solid phase, most of the dsRNA does not bind to the solid phase. At the end of the washing step using the same concentrations of guanidine thiocyanate and EDTA, the dsRNA has been reduced to trace levels. The guanidine thiocyanate and EDTA are then washed out of the system with a buffer that does not contain them. The remaining dsRNA is separated from the ssRNA using an increasing pH gradient.

[0046] In preparative embodiments, where the goal is to isolate a quantity 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 ​​approaching pH 9 or higher. Experimental data indicate that ssRNA exposed to alkaline conditions for only the period required to perform the methods of the present invention retains its native composition and remains stable indefinitely. Essentially instantaneous neutralization can be achieved by collecting fractions in a neutralizing solution. Alternatively, rapid neutralization can be achieved by adding a neutralizing solution immediately after fraction collection. Neutralization can also be achieved after fraction collection 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 methods of the invention can be carried out using any apparatus conventional in the art, for example, the primary amino solid phase can be placed in a chromatography device. Typically, the solid surface can be in the form of a monolith, a packed particle column, a packed nanofiber column, a membrane sorber, or a hydrogel, among other chromatography formats.

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

[0049] The methods of the invention may be preceded, followed, or preceded and followed by one or more additional processing methods to purify ssRNA to a greater extent than can be achieved by any single processing method alone. [Brief explanation of the drawings]

[0050] [Figure 1] Separation of dsRNA from ssRNA is shown using a primary amino solid phase, and the dsRNA is removed by a sodium chloride step before eluting the ssRNA using a pH gradient at ambient temperature. [Figure 2] Separation of dsRNA from ssRNA using a primary amino solid phase is shown, with the dsRNA being removed by a 6 M guanidine step prior to eluting the ssRNA using a pH gradient at ambient temperature. [Figure 3] Figure 1 shows the failure of eluting ssRNA with a pH gradient at ambient temperature from strong and weak anion exchangers. [Figure 4] Separation of plasmid DNA from ssRNA by pH gradient at ambient temperature using a primary amino solid phase is demonstrated. [Figure 5] Separation of plasmid DNA from ssRNA using a primary amino solid phase is demonstrated, with DNA being removed by a salt step prior to elution of the ssRNA using a pH gradient at ambient temperature. [Figure 6]1 shows the effect of combining salt and pH gradient elution at ambient temperature on the separation of DNA and ssRNA. [Figure 7] We demonstrate the separation of large DNA and giant dsRNA from small ssRNA on a primary amino solid phase by a pH gradient at ambient temperature. [Figure 8] Separation of dsRNA from ssRNA using a primary amino solid phase is shown, and DNA is removed by a chelator-chaotrope combination step prior to eluting the ssRNA using a pH gradient at ambient temperature. [Figure 9] 1 shows the decrease in ssRNA elution pH in a pH gradient due to the inclusion of chelating salts. [Figure 10] FIG. 1 shows monitoring of an in vitro transcription reaction at different time points using a primary amine solid phase eluted with a pH gradient in the presence of salt. DETAILED DESCRIPTION OF THE INVENTION

[0051] The term "primary amino solid phase" refers to a solid phase suitable for performing chromatography that predominantly or exclusively carries 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 minority on the surface of the solid phase. Tertiary and quaternary amines should be absent or present in minority. 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 linked by a single covalent bond to each of two hydrogen atoms and also linked by a single covalent bond to a carbon atom. A primary amino group may be directly covalently attached to a solid phase through its carbon atom. Alternatively, a primary amino group may be indirectly attached to a solid phase by covalent attachment of its carbon atom to a so-called spacer arm that is covalently attached to the solid phase. A primary amino group may also be part of a polymer structure covalently attached to a solid phase that contains primary amino groups, where the repeating subunits of the polymer contain primary amino groups.

[0053] "Solid phase" may 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-bearing polymer phase synthesized on a monolith initially synthesized as a macroscaffold. Any of these solid phase materials can be provided within a housing to facilitate the performance of chromatography. A chromatographic solid phase within a housing is generally referred to as a chromatographic device and is often referred to as a chromatographic column, or simply a column.

[0054] Chromatographic solid phases bearing primary amino groups are known and commercially available. One example is a product manufactured by Tosoh Biosciences and sold under the name Toyopearl NH2-750F, where "NH2" refers to the primary amino group [www.separations.eu.tosohbioscience.com / solutions / process-media-products / by-mode / ion-exchange / anion-exchange / toyopearl-nh2-750f]. According to the product literature, the primary amino groups are in the form of a polyamine, meaning that it is a polymer with repeating primary amine subunits covalently anchored to 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 residues from primary to secondary amino groups, thereby producing 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 primary amine [www.sartorius.com / shop / ww / en / usd / sartobind-stic (R) -pa / c / M_Sartobind_STIC_PA]. The sales literature indicates that the primary amino groups are in the form of a polymer, specifically polyallylamine, with repeating primary amino subunits covalently attached to the solid phase. Similar to the above-mentioned products, 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 residues from primary to secondary amino groups, thereby producing 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 routine or experimental basis.

[0057] Considering that chromatographic solid phases bearing primary amino groups may not be named in a manner 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 the appropriate properties for practicing 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 so-called ssRNA ladders, each containing a subset of RNA molecules with a different size. Such RNA ladders generally cover size ranges of 50-500 bases, 100-1000 bases, 200-6000 bases, or some other range, and are commercially available from common suppliers such as Thermo Scientific and New England BioLabs. After sample injection and a brief wash with the equilibration buffer to remove unbound sample components, the primary amino chromatographic device is subjected to elution with a linear pH gradient from pH 7.5 to approximately pH 11. A primary amino solid phase elutes ssRNA in order of increasing size as shown in Figure 1. Failure to elute ssRNA or elution of only the smallest ssRNA species may indicate that the solid phase contains an excessive proportion of non-primary amino groups, including any one or combination of secondary, tertiary, and quaternary amino groups.

[0058] The term "RNA size" or "size of RNA" refers to the number of bases in a nucleotide chain. A base is commonly referred to as "b." Thus, 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, which are both the same size in terms of number of bases because they are both derived from the same DNA plasmid. In some cases, ssRNA strands may be clipped or truncated during processing, either through shear stress or enzymatic lysis, resulting in the formation of a fragment subpopulation of the previous complete strand. In other cases, truncated forms may hypothetically arise from incomplete transcription. Whatever the source, the ability of the methods of the present invention to fractionate ssRNA according to size provides a tool for removing undesired fragment forms.

[0059] The terms "equilibrated" or "equilibration" refer to a chemical conditioning step performed on a solid phase and / or a sample to create a specific chemical environment. Solid phases are usually conditioned by exposing them to a buffer embodying the desired pH and salt composition. Samples are usually conditioned by titration of pH, sometimes by dilution to reduce salt concentration, sometimes by buffer exchange techniques including chromatography, or by dialysis, or by diafiltration 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 contacting an equilibrated sample with an equilibrated solid phase bearing predominantly positively charged primary amino groups. This is typically accomplished using a chromatographic device by forcing the sample through the device by gravity or an external force such as pumping.

[0061] The term "adsorption" refers to the process of binding a biological product to a chemically complementary surface. Adsorption is similar to the uptake of water by a sponge through the physical action of capillary action, but differs from "absorption," which does not involve chemical interaction. Complementarity in this case is understood to include electrostatic charge. Negative electrostatic charges on the surface of the RNA mediate its adsorption to the surface of a solid phase made electropositive by primary amino groups on its surface. 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 prevent the binding of most dsRNA while allowing the binding of ssRNA.

[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, desorption of ssRNA typically requires an alkaline pH value of at least pH 9.0, or pH 9.5, pH 10.0, or higher. Salts can 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, no salt or salt combination achieves significant elution of ssRNA, regardless of concentration.

[0064] The term "selective desorption" refers to a situation in which one or more adsorbed species are released from a solid surface by a change in conditions that leaves one or more other species still adsorbed. A different set of conditions can then be applied to release a different subset of species from the solid 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 be selectively desorbed. The ssRNA is then selectively desorbed in a subsequent step 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 wash buffer with the purpose of repelling unbound species from the pores or channels within the device. The term "rinsed" has the same meaning in this context. In the most basic case, the wash buffer has the same composition as the equilibration buffer. In more complex configurations, the wash buffer may have the additional role of chemically releasing a subset of weakly bound contaminants so that they are chemically removed prior to elution of the desired product. Alternatively, there may be two or more wash steps, the first using conditions identical to the equilibration buffer, but the second using conditions that repel the subset of weakly bound contaminants from the solid phase, allowing their removal prior to elution. Washing may 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 that repels contaminants, it may be desirable to perform a salt-free wash to establish conditions for eluting ssRNA with a salt-free pH salt gradient. Without that wash, elution would start at high salt by default, creating a gradient of decreasing salt concentration while increasing pH.

[0066] The term "elution" represents a special case of the term "desorption" as it relates to the field of chromatography. It refers to a process that changes the chemical environment in which the solid phase resides, causing dissociation of interactions between the primary amino solid phase and species that remain bound after the loading and washing steps. Once dsRNA and proteins have been removed from the solid phase, ssRNA can be eluted by simply increasing the pH, or by increasing the pH in conjunction with salts.

[0067] Elution can be carried out in one or a series of steps, each step reducing the strength of the interaction between the solid phase and ssRNA.The change in conditions can also be made in a continuous or linear manner, with weakly bound species being desorbed in the early stages of the series, while strongly bound species are eluted in the later stages of the series.Whether in a stepwise or linear manner, the change in operating conditions is generally referred to as a gradient, and specifically as an elution gradient.A stepwise gradient is often considered more convenient, but a linear gradient typically supports better reproducibility.

[0068] The term "ambient temperature" is generally considered to be analogous to the expressions "room temperature" or "normal temperature." It typically corresponds to a temperature in the range of about 20-22°C, but may include a broader range, such as about 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 ranking on the Hofmeister series of lyotropic and chaotropic ions. Lyotropic ions are at one end of the Hofmeister series; chaotropic ions are at the opposite end of the series. Chaotropic ions are often described as preferentially bound by biomolecules. Chaotropic salts have the effect of relaxing noncovalent interactions within and between biomolecules, sometimes to the point of destabilizing and dissociating interactions between components in multicomponent noncovalent mixtures. They usually have the effect of increasing solubility. Examples of chaotropic salts include guanidinium salts, thiocyanates, and perchlorates, among others. In some cases, as in the case of guanidinium 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 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, promoting nonspecific association between individual components of a mixture. Strong lyotropic ions typically reduce the solubility of large biomolecules. Examples of lyotropic salts include ammonium sulfate, potassium phosphate, and sodium citrate, among others. Salts that represent intermediates in the Hofmeister series tend to have only a moderate or minimal effect on stability, association-dissociation, or solubility for 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, their effect is primarily mediated through Coulombic (electrostatic) forces.

[0070] The term "polyvalent metal cation" refers to a positively charged ionic form of a metal, where the ionic charge is two or greater. Polyvalent metal cations include calcium, magnesium, and zinc (all of which have a net charge of 2+), and iron (iron), which has a net charge of 3+, among other species with similar or different valencies. All of these ions have an affinity for nucleic acids, binding via coordinate bonds. Coordinate bonds are 15 to 60 times stronger than ionic bonds, meaning that binding of polyvalent metal cations to nucleic acids or other biomolecules persists even at saturating salt 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 complexes (associations).

[0071] The term "chelating agent," in the context of the methods of the present invention, refers to a molecule capable of forming a strong coordinate bond with a polyvalent metal cation such that it can competitively remove the metal ion from a pre-existing complex between the polyvalent metal cation and a biomolecule, including a nucleic acid, including mRNA.

[0072] The term "nuclease" or "nuclease enzyme" refers to a protein capable of cleaving a strand of nucleic acid, ideally into individual nucleotides, doublets, or triplets. They can be divided into two major classes: DNAse enzymes, which lyse DNA, and RNAase enzymes, which lyse RNA. RNAses should be strictly avoided because they destroy ssRNA products. DNAses are often used to simplify purification by destroying the DNA plasmid template used to produce mRNA. DNAse enzymes often require the use of a polyvalent metal cation cofactor to function properly. Polyvalent metal cations can interfere with RNA purification, as discussed above.

[0073] The terms "protease" or "proteinase" or "proteolytic enzyme" refer to proteins that have the ability to cleave other proteins into fragments. This is sometimes used as a method to reduce protein contamination prior to chromatography steps that may be burdened by such contamination. Many proteases, such as trypsin, require a polyvalent metal cation cofactor to function properly. Polyvalent metal cations can interfere with RNA purification, as discussed above. Proteases commonly used for this purpose include proteinase K, which provides good results even in the absence of a polyvalent metal cation cofactor.

[0074] If desired or necessary, the decision to include salt during the implementation of the method may depend in part on the selection of subsequent analytical methods or purification steps. For example, if the subsequent method is intolerant of salt, it may be advantageous to elute the ssRNA from a positively charged solid phase containing predominantly primary amino groups in the absence of salt or at a sufficiently low salt concentration to avoid interference. If the subsequent method is highly tolerant of salt, elution of the device can use substantial concentrations of salt species that are tolerant to the subsequent step.

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

[0076] In certain embodiments, pH gradient elution from a primary amino solid phase is performed to separate ssRNA species according to their size, with smaller species eluting earlier in the gradient than larger species.

[0077] The following general, non-limiting descriptions of a series of basic method options illustrate variations in how the method may be performed and provide a platform for a more detailed discussion of the operating variables. The buffer conditions mentioned in each of these scenarios are intended to provide a general idea of ​​how the method may be performed, with the understanding that optimization of buffer composition will be required given the different sizes of ssRNA species and different contaminant loads involved.

[0078] In one embodiment, a primary amino solid phase in the form of a chromatographic device, such as a monolith, is equilibrated to a near-neutral pH, such as 20 mM Tris, 20 mM Bis-Tris-propane, pH 7.5±0.5. A sample containing a mixture of dsRNA and ssRNA is equilibrated to 20 mM Tris, 20 mM Bis-Tris-propane, 20 mM glycine, pH 7.5±0.5 by buffer exchange. The chromatographic device is then eluted with a linear pH gradient of more than 20, 50, or 100 device volumes from the equilibration buffer to an endpoint buffer of 20 mM Tris, 20 mM Bis-Tris-propane, 20 mM glycine, 100 mM NaCl, pH 11±0.5, where the number of device volumes is used as a means of controlling the rate of pH change during the gradient, i.e., the slope of the gradient. This approach can be particularly useful for analytical applications because dsRNA, ssRNA, DNA fragments, and proteins all bind to the primary amine-containing solid phase and elute within the gradient, thus providing an indication 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 preparative separations.

[0079] In a different embodiment, a primary amine solid phase can be loaded under the same conditions and then washed with a 3M NaCl solution to remove most of the dsRNA, DNA, and protein contamination. The NaCl itself is then removed by a subsequent wash without excess salt, allowing the pH gradient elution of ssRNA to begin without excess salt. Comparison of the results with the previous embodiment allows for evaluation of the extent to which the NaCl wash improves ssRNA purity. Determining the most preferable concentration of NaCl is a routine activity well known in the art of chromatography, with the goal being to achieve the best balance between product purity and product yield.

[0080] In a closely related embodiment, either or both of the foregoing embodiments may include 50 mM sodium chloride in the pH gradient buffer. In another closely related embodiment, 100 mM sodium chloride may be included in the pH gradient buffer. In another closely related embodiment, any other salt may be substituted for sodium chloride.

[0081] In another related embodiment, a primary amino solid phase can be loaded under the same conditions and then washed with a solution of 3 M guanidine-HCl to remove the majority of the dsRNA before eluting the desired ssRNA product using a pH gradient. The guanidine wash is followed by a wash without guanidine salt, allowing the pH gradient elution of ssRNA to begin in the absence of excess salt. In a closely related embodiment, 100 mM sodium chloride can be included in the pH gradient buffer. In another closely related embodiment, guanidine-HCl can be replaced by guanidine isothiocyanate. Comparison of the results with the previous two embodiments allows assessment of the extent to which the guanidine wash improves ssRNA purity.

[0082] In a related embodiment extending the above sequence, 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 metal-stabilized complexes and repel non-ssRNA species from the solid phase prior to elution of the ssRNA. Comparison of the results from the three previous embodiments allows for an assessment of the extent, if any, that the chaotrope-chelator wash improves ssRNA purity.

[0083] In other related embodiments, washes with other salt species or combinations of salt species at various pH values ​​can be evaluated prior to elution of ssRNA to maximize clearance of dsRNA. The underlying concept is generally to apply the most dissociating salt concentration at the highest pH that does not cause elution of ssRNA, with the idea that such conditions are likely to remove the largest subset of undesired contaminants prior to elution of the desired ssRNA. Adjustments can then be made to determine the minimum salt concentration required to achieve the desired effect.

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

[0085] In some embodiments, the pH increase to elute the desired ssRNA and separate it from the dsRNA can be performed in a stepwise manner. If the dsRNA content has been sufficiently reduced in the previous washing step, the ssRNA can be eluted in a single step to simplify the method and obtain eluted ssRNA at the highest possible concentration and in the lowest possible volume. Alternatively, stepwise elution can be performed in a series of pH-increasing steps. Each step can be relatively mild or large, depending on the needs of a particular preparation. In some embodiments, the pH increase to elute 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 the majority of dsRNA. In some such embodiments, depending on the size of the mRNA, the pH during sample loading can be pH 8.0, or pH 8.5, or pH 9.0, or a higher pH that does not prevent binding of ssRNA.

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

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

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

[0090] In a related embodiment, the presence of a non-ionic chaotrope, which competes for hydrogen bonding between the ssRNA and the primary amino groups of the solid phase, is expected to enhance separation of the dsRNA from the dsRNA by increasing the pH, resulting in elution of the 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 can range from 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 values ​​within higher, lower, or intermediate ranges. In another such embodiment, the chaotrope is dimethyl sulfoxide. In some such embodiments, the concentration of dimethyl sulfoxide is up to 99%. In other related embodiments, a non-ionic chaotrope can be applied during a wash step prior to eluting the desired ssRNA by pH gradient.

[0091] In another embodiment, the presence of an alkaline amino acid is expected to enhance separation of dsRNA from dsRNA by increasing the pH, resulting in 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, histamine, lysine, arginine, another alkaline amino acid, or a mixture of alkaline amino acids. In one such embodiment, the histidine concentration can 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 any higher, lower, or intermediate value. In another such embodiment, the lysine concentration can 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 any higher, lower, or intermediate value. In other such embodiments, the concentration of arginine can 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 any higher, lower, or intermediate value within the range.

[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 ions and the negatively charged phosphatidic acid residues along the nucleic acid backbone. Calcium and magnesium are both known to participate in such interactions; both are divalent metal cations with a 2+ charge. Iron, a trivalent cation with a 3+ charge, interacts more aggressively with nucleic acids. Each time any one of these ions interacts with a nucleic acid, it neutralizes an equivalent number of negative charges. This creates the superficial expectation that the negative charge on a given mRNA molecule will be reduced, weakening the mRNA's interaction with the anion exchanger. Instead, the addition of polyvalent metal cations typically results in the formation of nonspecific crosslinks, which cause ssRNA to form large aggregates that do not elute from the chromatography device.

[0093] Because polyvalent cations are commonly added to mRNA preparations, it is recommended in all embodiments to reduce their content, preferably completely eliminate them, before loading the sample onto a solid phase bearing primarily positively charged primary amino groups; or at least to perform a step to remove them before the device is subjected to elution. There are at least two additional reasons for removing polyvalent metal cations in advance. First, polyvalent metal cations may tend to promote the formation or stabilization of intrastrand and interstrand dsRNA sequences. Second, metal ions stabilize nonspecific associations between nucleic acids and proteins, essentially forming stable crosslinks between them. Because coordinate bonds are 15 to 60 times stronger than ionic bonds, coordination complexes readily tolerate exposure to saturating levels of NaCl and nonmetallic salts, including guanidinium salts. This makes it essential to extract as much of the polyvalent metal cations as possible to obtain the highest ssRNA purity and recovery from the methods 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 lesser extent with high concentrations of non-chaotropic salts such as NaCl.

[0094] In some embodiments where chelator or chelator-high salt dissociation of nucleic acid-metal-contaminant complexes is performed during sample preparation and / or a chelating wash or a chelating-chaotrope wash is performed, a subsequent wash step can be performed that does not contain salt beyond the agent used to provide pH control, allowing the ssRNA to be eluted in a low salt environment.

[0095] In other embodiments, the chelator concentration and high salt concentration can be maintained during elution of the ssRNA by increasing the pH. In other embodiments, the chelator can be eliminated while the high salt is maintained. In other embodiments, the high salt can be removed while the chelator is maintained.

[0096] A particular benefit of the present invention is that its ability to remove DNA plasmids from ssRNA preparations eliminates the need to perform nuclease digestion of in vitro transcription mixtures or partially purified in vitro transcription mixtures. This is disproportionately valuable given that nuclease digestion requires the addition of magnesium ions to activate the enzymes. These magnesium ions potentially contribute to crosslinking of the desired ssRNA with itself and other sample components, with the practical consequence of reducing the recovery of the desired ssRNA product. By eliminating the need for nuclease digestion, the addition of magnesium ions is not required, and ssRNA yield is not compromised.

[0097] Regardless of the ability of the methods of the present invention to separate plasmid DNA from ssRNA, nuclease digestion can be carried out to the desired extent, and the positively charged solid phase, which contains predominantly primary amino groups, can then be washed with excess chelating agent to remove residual magnesium ions. In such an embodiment, nuclease digestion of plasmid DNA in the presence of 5 mM nuclease enzyme can be followed by a chelating wash containing EDTA in the range of 10-50 mM or more.

[0098] Starting with an in vitro transcription mixture, in one embodiment, EDTA is added to a final concentration of 10-50 mM 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, if necessary, and then loaded onto a primary amino acid chromatography device equilibrated in 50 mM Tris, 100 mM NaCl, 10 mM EDTA, pH 8.0. After loading the sample, the device is washed with 10-20 device volumes of 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 flush the guanidine and EDTA from the device and prepare it for elution of ssRNA. The ssRNA is then eluted with a linear gradient over 50 device volumes to 20 mM Tris, 20 mM Bis-Tris-propane, 20 mM glycine, 250 mM arginine, pH 11.0. Immediately after fraction collection, the vessel containing the ssRNA peak is neutralized by adding 1 M acetic acid.

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

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

[0101] Starting with the in vitro transcription mixture, in one embodiment, the mixture is precipitated by adding lithium chloride (LiCl) to a final concentration of 2.0M-2.5M. The supernatant is discarded, and the precipitate is resuspended in 50 mM Tris, 100 mM NaCl, 20 mM EDTA, pH 8.0, and filtered, if necessary, to remove turbidity. 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 device is washed with 10-20 device volumes of 50 mM Tris, 1.5 M guanidine isothiocyanate, 20 mM EDTA, pH 8.0. The column is then washed with 20 mM Tris, 20 mM Bis-Tris-propane, 20 mM glycine, 100 mM NaCl, pH 8.0 to flush guanidine and EDTA from the device and prepare it for elution of the ssRNA. The ssRNA is then eluted with a 50-device-volume linear gradient to 20 mM Tris, 20 mM Bis-Tris-propane, 20 mM glycine, 100 mM NaCl, pH 11.0. Immediately after fraction collection, the vessel containing the ssRNA peak is neutralized by adding 1 M acetic acid to a final concentration of 5% of the fraction volume. In a closely related embodiment, mRNA is precipitated with NaCl or another salt instead of LiCl. In another closely related embodiment, mRNA is precipitated by adding ethanol to a final concentration of about 2.5%.

[0102] Industrial process developers often prefer to avoid the use of enzymes because they increase the cost of the process and because any additions must later be removed and testing must be performed to document their removal. However, using enzymes early in a development program can be a convenient shortcut that allows companies to enter clinical trials sooner, with more advanced versions of the process that do not require the use of enzymes being developed later. As noted above, in vitro transcription mixtures are typically treated with a DNAase enzyme to eliminate the DNA plasmid used as a template for mRNA production. RNA purification can also use proteolytic enzymes to reduce the protein contaminant load in a given in vitro transcription mixture. In some embodiments, the in vitro transcription mixture can first be treated with DNAse to remove the plasmid, and then with a proteolytic enzyme, such as proteinase K, to remove the DNAse and large amounts of other protein contaminants before performing the methods of the invention. In other embodiments, the methods of the invention obviate the need for DNAse, and the in vitro transcription mixture can be treated with proteinase K or another proteolytic enzyme alone.

[0103] In one embodiment, the in vitro transcription mixture is treated with proteinase K to reduce protein load. The sample is filtered through a membrane filter to remove particulates 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 device is washed with 10-20 device volumes of 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, 50 mM NaCl, pH 8.0 to flush the guanidine and EDTA from the device and prepare it for elution of the ssRNA. The ssRNA is then eluted with a 50 device volume linear gradient to 20 mM Tris, 20 mM bis-Tris-propane, 20 mM glycine, 50 mM NaCl, pH 11.0. Immediately after fraction collection, the vessel containing the ssRNA peak is neutralized by adding 1 M acetic acid.

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

[0105] The present invention offers the advantage of postponing the need to perform separation of dsRNA and ssRNA at high temperatures, but does not preclude the possibility of doing so.

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

[0107] In some embodiments, the methods of the invention can be carried out at elevated temperatures, such as 37° C., 45° C., 56° C., 60° C., 70° C., or intermediate, higher, or lower temperatures. In such embodiments, the ssRNA elutes at a lower pH than it elutes at ambient temperature.

[0108] In some embodiments, the sample is equilibrated to an elevated temperature and the method may be carried out at the elevated temperature.

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

[0110] In certain embodiments, the methods of the present invention can be combined with a method of hydrophobic interaction chromatography (HIC). The two methods can be combined in any order desired. In one such embodiment, the hydrophobic ligands on the HIC solid phase can comprise phenyl groups. In another such embodiment, the hydrophobic ligands on the HIC solid phase can comprise butyl groups. In another such embodiment, the hydrophobic ligands on the HIC solid phase can comprise hexyl groups. In other such embodiments, the hydrophobic ligands on the HIC solid phase can comprise different aliphatic or aromatic groups, or groups embodying both aliphatic and aromatic properties.

[0111] In certain embodiments, the methods of the present invention can be combined with a method of reversed-phase chromatography (RPC). The two methods can be combined in any order desired. In one such embodiment, the hydrophobicity of the solid phase surface can be imparted by the inherent 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 imparted by the hydrophobicity of a ligand immobilized on the surface of the solid phase, where the ligand represents an aliphatic hydrocarbon, or 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 the method of hydroxyapatite chromatography. The two methods can be combined in any order desired.

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

[0114] In certain embodiments, the methods of the present invention can be used to remove ssRNA from DNA plasmid preparations. In a closely related embodiment, the methods of the present invention can be used to remove ssRNA from protein preparations. In such embodiments, the methods can be used to remove contaminating ssRNA from enzyme preparations used in mRNA synthesis. In any of these embodiments, samples can be loaded under acidic to neutral pH conditions and low salt concentrations, where DNA and proteins bind to a positively charged solid phase bearing 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 to remove ssRNA, sample and solid phase conditions can include high salt concentrations at acidic to neutral pH, where DNA and proteins flow through the solid phase while RNA remains bound.

[0115] In certain embodiments, the methods of the present invention can be used as an analytical tool to quantify the amount of ssRNA in a sample. In such embodiments, a sample at 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 eluted using an increasing pH gradient to sort the bound ssRNA by increasing size. In a closely related embodiment, the primary amine solid phase can be eluted in a single step to a pH sufficiently alkaline to elute all ssRNA in a single peak, in order to maximize assay sensitivity. In a related embodiment, the primary amine solid phase can be eluted with a linear gradient toward 25 mM NaOH, or 50 mM NaOH, or 100 mM NaOH, or higher, lower, or intermediate concentrations. In another such embodiment, the primary amino solid phase can be eluted in increasing steps to 25 mM NaOH, or 50 mM NaOH, or 100 mM NaOH, or higher, lower, or intermediate concentrations. In another such embodiment, the primary amino solid phase can be eluted in a single step with 25 mM NaOH, or 50 mM NaOH, or 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 an embodiment, the dye can be RiboGreen.

[0116] In certain embodiments, the methods of the present invention can be provided in the form of a kit to facilitate their implementation. The kit can include two or more solid phases, at least one of which is a positively charged solid phase bearing predominantly primary amino groups, and the kit can also include instructions describing the methods of the present invention. In such embodiments, the second solid phase is an oligo-dT chromatography device. In another such embodiment, the second solid phase is a hydrophobic interaction chromatography device. In another such embodiment, the second solid phase is an oligo-dT chromatography device and the third solid phase is a hydrophobic interaction chromatography device.

[0117] In a typical protocol for the purification of mRNA using a solid phase used in accordance with the present invention, a 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, protein, and endotoxin while fractionating ssRNA in ascending size order (Figure 1). Primary amine monoliths can be used for single-step purification of research-grade ssRNA or as a high-resolution capture step in multistep purification processes. They also enable rapid, high-resolution analytical characterization of in vitro transcription mixtures, partially purified samples, chromatographic fractions, and formulated drug substances.

[0119] The primary amine monolith uses a unique combination of anion exchange and hydrogen bonding to purify ssRNA in an ascending pH gradient. DNA, protein, and dsRNA elute before ssRNA. Purification performance is enhanced by a high-salt wash, which removes the majority of dsRNA, DNA, and protein before elution. Remarkably, ssRNA remains bound even at saturating concentrations of chaotropic salts. The inclusion of a chelating agent during the high-salt wash further enhances contaminant removal, 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, if desired, by affinity chromatography using CIMmultus Oligo dT, hydrophobic interaction chromatography using CIMmultus C4 HLD, or reversed-phase chromatography using CIMmultus SDVB (contact BIA Separations for more detailed information regarding any of these columns). Primary amine monoliths can also be used as a polishing method, especially after high salt steps like precipitation or hydrophobic interaction chromatography, since high salt samples can be loaded without further sample preparation.

[0121] The primary amine monolith is a radial flow chromatography device. It is designed to distribute 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, which improves separation performance. Before performing an experiment, be sure to connect the device to the chromatograph so that the flow direction is aligned with the markings on the device. Note that some chromatographs have a default reverse flow function built into the software, which can reverse the flow direction without warning. Ensure that this function is disabled before performing an experiment.

[0122] The primary amine monolith is delivered in 20% ethanol. It is recommended to disinfect and regenerate it as described below before use. It is also recommended to perform a run without a sample to provide a baseline for comparing experimental results. Some buffer components absorb UV, and some transfer between buffers can produce refractive index artifacts that can confound the interpretation of experimental results.

[0123] Sample 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 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) prior to injection. The pH of the sample should be between 6.0 and 8.0. Salt content is not considered.

[0124] Buffer A: Equilibration buffer / gradient start 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: Renaturation buffer. 3.0 M ammonium acetate.

[0125] Equilibrate the column with Buffer A: Pump 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. Monitor the operating pressure while applying large volumes of sample, especially crude samples such as in vitro transcription mixtures. Reduce the flow rate as necessary to maintain the operating pressure within an acceptable range.

[0127] Wash 1:10 with buffer A. Equilibration buffer for 20 CV. There is no need to wait for the UV signal to completely return to baseline, as contaminants at the rear end will be removed by the subsequent high-salt wash step.

[0128] Wash 2 with Buffer B: 10-20 CV of equilibration buffer. Note that guanidine absorbs UV, resulting in an immediate peak. DNA and dsRNA typically elute as a fairly sharp peak approaching the apex of the peak. Continue washing until the guanidine peak reaches a level plateau.

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

[0130] Elution gradient towards Buffer C: 50-100 CV linear gradient towards 100% gradient end buffer, then hold at 100% for 10 CV. Neutralize fractions immediately after elution.

[0131] Wash / sanitize with Buffer D. A 10-20 CV treatment with sanitizing buffer is recommended after each run to determine whether significant amounts of material remain bound to the column at the end of the pH gradient. The contents of the sanitizing step may be collected during elution and neutralized for further analysis. If the column is loaded with an in vitro transcription mixture, the duration of the sanitizing step may need to be extended to 1 hour. In cases of extreme fouling, the sanitizing period may need to be extended to 16-24 hours. Maintaining a minimum flow rate during sanitizing tends to yield better results, as it continuously replenishes OH ions and flushes contaminants from the column rather than simply hydrolyzing them in situ.

[0132] Regenerate the column: Wash the NaOH out of the column with 20 CV of Buffer A or water, then wash with 20 CV of Buffer E. This is to remove the hydroxide counterions from the surface of the monolith, which would otherwise slow column equilibration and create pH artifacts during elution.

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

[0134] A representative chromatogram is shown in FIG.

[0135] Variations, optimizations, and troubleshooting

[0136] Use the chromatogram as a guide to optimize the times of the individual steps.

[0137] For example, the high salt wash can be significantly modified using different concentrations of chaotropic salts up to full saturation, and different concentrations of chelating agents. The concentrations given in the protocol are intended as a starting point. If a lower concentration provides equivalent purity, using such a smaller amount reduces material costs and simplifies buffer preparation.

[0138] Chaotropic salts can be replaced with nonchaotropic salts, but care must be taken when applying high concentrations of salt that precipitate RNA. These include sodium chloride, potassium chloride, and lithium chloride, among others. Preliminary results indicate that washing with 1 M sodium chloride removes the majority of dsRNA and DNA, but prolonged washing may be required to restore UV absorbance to baseline (Figure 1).

[0139] A simplified workflow can be implemented by adding guanidine and EDTA directly to the sample and equilibrating the column to a high-salt wash buffer. Sample load is followed by a first wash with a high-salt buffer, then a second wash with Buffer A from above, followed by elution. Note that this approach may also support higher ssRNA binding capacity because it prevents dsRNA and DNA from competing for binding surface area. Column fouling may also be reduced with this approach.

[0140] The high-salt wash step can be omitted entirely, which may be preferable for analytical characterization of samples aimed at determining the relative amounts of dsRNA and ssRNA, or DNA and ssRNA. The 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 yield and also increases the pH at which elution occurs. The use of salts that precipitate RNA is acceptable as long as their concentrations are kept well below precipitation levels (Figure 6). Salts that promote RNA solubility, such as chaotropes, can also be used during pH elution, but consider that they may interfere with subsequent purification methods.

[0142] The operating temperature may increase during the pH gradient, with the effect of causing solutes to elute faster. Uncontrolled operating temperature can impair reproducibility.

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

[0144] Signs of inadequate cleaning may include a gradual increase in operating pressure over a series of runs, selectivity shifts where a given species elutes earlier or later than in a previous run, and / or the appearance of ghost peaks, which are peaks that appear during elution even though no sample was injected.

[0145] The present invention is further illustrated by the following non-limiting examples. [Example]

[0146] Example 1 Separation of dsRNA from ssRNA using a primary amino solid phase with a pH gradient at ambient temperature, with a salt step to remove dsRNA before eluting the ssRNA Samples containing a dsRNA ladder containing dsRNA molecules ranging from 21 b to 500 b and a ssRNA ladder containing ssRNA molecules ranging from 200 b to 6000 b were applied to a primary amine solid phase in the form of a monolith chromatography device. 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 wash was applied to remove unbound material from the channels within the monolith. A wash step using 20 mM Bis-Tris-propane, 20 mM glycine, 1 M NaCl, pH 8.0 was then applied, followed by another wash 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, pH 11.0. As shown in Figure 1, all dsRNA species eluted at the NaCl step, while ssRNA remained bound and eluted later in the pH gradient. No obvious size separation is observed between species within the dsRNA ladder, but a clear separation is observed between species within the ssRNA ladder.

[0147] Example 2. Separation of dsRNA from ssRNA using a primary amino solid phase with a pH gradient at ambient temperature involves removal of dsRNA by a wash step containing 6 M guanidine-HCl and 20 mM EDTA before elution of the ssRNA. A sample containing a dsRNA ladder containing dsRNA molecules ranging from 21 b to 500 b and a 5000 b ssRNA was applied to a primary amine solid phase in the form of a monolith chromatography device. 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 wash solution was applied to remove unbound material from the channels within the monolith. A wash step using 20 mM Bis-Tris-propane, 20 mM glycine, 6 M guanidine, 20 mM EDTA, pH 8.0 was then applied, followed by another wash step to remove the 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 at the salt step, while ssRNA remained bound and eluted later in the pH gradient.

[0148] Example 3 pH gradients fail to elute ssRNA from strong and weak anion exchangers The behavior of ssRNA in a pH gradient was characterized by comparing the performance of pH gradient elution on a primary amino solid phase with a strong anion exchanger (quaternary amine, QA) and a weak anion exchanger (tertiary amine, DEAE) as experimental controls. The physical form of both anion exchangers was a monolithic device with a volume of 1 mL. The column was equilibrated with 20 mM Tris, 20 mM bis-tris-propane, 20 mM glycine, pH 8.0. A sample containing approximately 5000 nucleotide bases (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, pH 11.0. FIG. 3 shows that ssRNA did not elute within the pH gradient on either the strong or weak anion exchanger, but was only removed by washing the column with 1 M NaOH.

[0149] Example 4. Separating plasmid DNA from ssRNA using a primary amino solid phase using a pH gradient at ambient temperature A sample containing a supercoiled dsDNA plasmid approximately 6,000 base pairs in size and a ssRNA approximately 5,000 bases in size was applied to a primary amine solid phase in the form of a monolith chromatography device. 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 wash solution was applied to remove unbound material from the channels within 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 eluted first and was well separated from the ssRNA, which eluted later.

[0150] Example 5. Separation of plasmid DNA from ssRNA using a primary amino solid phase with a pH gradient at ambient temperature, with a salt step to remove DNA before eluting the ssRNA A sample containing a supercoiled dsDNA plasmid with a size of approximately 6,000 base pairs and a ssRNA with a size of approximately 5,000 bases was applied to a primary amine solid phase in the form of a monolith chromatography device. 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 wash solution was applied to remove unbound material from the channels within the monolith. A wash step using 20 mM Bis-Tris-propane, 20 mM glycine, 1 M NaCl, pH 8.0 was then applied, followed by another wash 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, pH 11.0. As shown in Figure 5, the DNA plasmid eluted during the salt step, while the ssRNA remained bound and eluted later in the pH gradient.

[0151] Example 6 Effect of combined salt and pH gradient elution at ambient temperature on the separation of DNA and ssRNA A series of separations were performed comparing simple pH gradient elution, pH gradient elution with an end buffer containing 50 mM NaCl, and pH gradient elution with an end buffer containing 1000 mM NaCl. In each experiment, a sample containing a supercoiled dsDNA plasmid approximately 6000 base pairs in size and a ssRNA approximately 5000 bases in size was applied to a primary amine solid phase in the form of a monolith chromatography device. The primary amine monolith was equilibrated with 100 mM Tris, pH 8.0. The sample was applied, and a wash solution was applied to remove unbound material from the channels within the monolith. In the first experiment, the monolith was eluted with a linear gradient toward 125 mM glycine, pH 10.5. In the second experiment, the monolith was eluted with a linear gradient toward 125 mM glycine, 50 mM NaCl, pH 10.5. In the third experiment, the monolith was subjected to elution with a linear gradient toward 125 mM glycine, 100 mM NaCl, pH 10.5. As shown in Figure 6, without sodium chloride, the center of the ssRNA peak occurred at pH 10.2. With 50 mM sodium chloride in the gradient end buffer, ssRNA eluted at pH 10.0. With 100 mM sodium chloride in the gradient end buffer, ssRNA eluted at pH 9.9. When sodium chloride was maintained at a concentration level of 50 mM throughout the pH gradient, ssRNA eluted at pH 9.8 (not shown). When sodium chloride was maintained at a concentration level of 100 mM throughout the pH gradient, ssRNA eluted at pH 9.6 (not shown). The inclusion of sodium chloride caused other interesting effects. The separation between DNA and ssRNA was greatest 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 Uniformity of reaction with dsRNA and DNA As evidenced by the preceding experiments, dsRNA and DNA behave similarly, both being eliminated by salt washes and both suggesting that they should be separated from ssRNA in a pH gradient, suggesting that DNA can be used as a model for dsRNA behavior to characterize the method of the present invention. Experiments were conducted to test the performance of the method of the present invention in an extreme worst-case scenario that never occurs during the purification of ssRNA from a typical transcription mixture. This test involved separating a relatively small ssRNA of approximately 1700 bases from a dsRNA of approximately 13,000 bases in size, and comparing the elution behavior of a 13 kb dsRNA with a DNA plasmid of approximately 6000 base pairs. A primary amine monolith was equilibrated with 100 mM Tris, pH 8.0. In each experiment, sample was applied, and a wash solution was applied to remove unbound material from the channels within the monolith. The monolith was then subjected to elution with a linear gradient toward 125 mM glycine, pH 10.5. As shown in Figure 7, consistent with expectations, DNA and large dsRNA largely co-eluted, with both eluting before the much smaller ssRNA. While the degree of resolution between ssRNA and the two larger contaminants was lower compared to other examples, clear separation was nonetheless evident. In fact, the dsRNA in a given in vitro transcription mixture contains the same number of bases as the ssRNA. Compared to Example 4, which shows that ssRNA elutes later in the pH gradient according to its size, this suggests that the present invention provides good separation between dsRNA and ssRNA regardless of their size. While this point may seem insignificant given that dsRNA can be easily removed by a salt wash prior to eluting the ssRNA in the pH gradient, it demonstrates that the present invention can achieve effective separation even in the absence of a salt wash. The results in Figure 7 are also notable in showing that the present invention should work well with ssRNAs of 15,000 bases or larger, possibly 25,000 bases or larger.

[0153] Example 8 Separation of dsRNA from ssRNA using a primary amino solid phase with a pH gradient at ambient temperature, with DNA removed by a chelator-chaotrope combination step before eluting the ssRNA A sample containing a 6000 bp supercoiled dsDNA plasmid and a 5000 bp ssRNA was applied to a primary amine solid phase in the form of a monolith chromatography device. 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 wash solution was applied to remove unbound material from the channels within the monolith. A wash step using 20 mM Bis-Tris-propane, 20 mM glycine, 1 M guanidine-HCl, 20 mM EDTA, pH 8.0 was then applied, followed by another wash 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 the guanidine-EDTA (chaotrope-chelator) wash. The remaining bound DNA eluted ahead of the ssRNA in a pH gradient.

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

[0155] Example 9. The inclusion of polyvalent anions reduces the pH of ssRNA elution A series of experiments was performed in which a mixture of DNA and ssRNA was fractionated by 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 of 125 device volumes was applied 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 decreased the pH at which both DNA and ssRNA eluted, with EDTA producing the strongest observed pH decrease. The magnitude of the pH decrease in both cases was also greater than that achieved with 100 mM sodium chloride (Figure 6). Consistent with the sodium chloride results, the presence of salt increased the yield of DNA and ssRNA in the elution gradient. Another similarity was that elution with either sodium chloride, citrate, or EDTA did not elute ssRNA at pH values ​​below pH 8.0.

[0156] Example 10. Purification of ssRNA using a high-salt wash followed by elution using a pH gradient in the presence of salt A primary amine solid phase in the form of a chromatographic device, such as a monolith, is equilibrated to 50 mM Tris, 20 mM EDTA, 2 M guanidine isothiocyanate, pH 8.0 ± 0.5. The UV monitor is zeroed. EDTA and guanidine isothiocyanate are added to the sample, and the sample pH is adjusted to pH 8.0 ± 0.5. If necessary, the sample is filtered to remove turbidity. The sample is applied to the primary amine monolith, and the monolith is washed with equilibration buffer until the baseline returns to zero. The sample application and washing in a strong chelating-chaotrope solution is intended to prevent binding and / or remove trace levels of dsRNA, DNA, and protein. The monolith is then washed with at least 10 device volumes with 50 mM Tris, 5 mM EDTA, 100 mM guanidine isothiocyanate, pH 8.0 ± 0.5. The ssRNA is then eluted with a linear gradient toward 65 mM glycine, 5 mM EDTA, 100 mM guanidine isothiocyanate, pH 10.5±0.5. The presence of a chelating agent is maintained throughout the method to prevent adverse effects from the potential presence of polyvalent metal cations. To avoid reducing the solubility of the ssRNA, 100 mM guanidine isothiocyanate is used instead of 100 mM sodium chloride as described in Example 6 or 100 mM citric acid or 100 mM EDTA as described in Example 9. If desired, in parallel experiments, the concentration of guanidine isothiocyanate is increased to further lower the pH at which the ssRNA elutes. If desired, in parallel experiments, the chaotropic salt guanidine thiocyanate is replaced with a different salt. If desired, the species and concentration of the chelating agent are varied in parallel experiments.

[0157] Example 11 Eluting the in vitro transcription mixture from the 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, 200 mM sodium pyrophosphate, pH 8.5; Buffer C containing 100 mM sodium hydroxide, 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 taken 30 seconds into the reaction was injected. The column was washed with 20 CV of Buffer A, followed by a 40 CV linear gradient to 20% Buffer B, followed by a 10 CV gradient hold at 20% Buffer B. This segment eluted nucleotides and double-stranded species, including DNA plasmids. The mRNA was eluted by applying a 10 CV linear gradient to 50% Buffer B, followed by a gradient hold at 50% B. An additional 10 CV linear gradient to 100% B was applied, followed by a 10 CV gradient hold. The column was washed with Buffer C and then returned to pH 7 with Buffer D. The column was re-equilibrated, and a sample collected 4 h after the start of in vitro transcription was injected. Elution was performed similarly to the previous sample. After completion of the run, the column was washed with 20% ethanol and stored in that solution. All chromatographic steps were performed at ambient temperature. The elution profile is shown in Figure 10. The profile corresponding to the sample collected at 30 s is shown as a long-dashed line. The profile corresponding to the sample collected at 4 h is shown as a solid line. The short-dashed line indicates conductivity. As expected, the levels of constitutive nucleotides such as CTP, UTP, ATP, and GTP decreased to lower levels over the course of in vitro transcription, reflecting their incorporation into newly synthesized mRNA.

[0158] References All references cited herein are incorporated herein by reference, unless such incorporation would contradict the explicit teachings of this specification. [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 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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