Method for separating single-stranded RNA molecules from other nucleic acid species, use of solid phases therefor, and method for separating double-stranded RNA molecules by size - Patents.com

Abstract

The present invention relates to a method for separating single-stranded RNA (ssRNA) molecules from other nucleic acid species, and the use of solid phases to separate ssRNA molecules from other nucleic acid species. The present invention claims that ssRNA can be eluted in an ascending pH gradient from a solid phase medium containing a multimodal ligand containing a nitrogen-containing heterocycle, 1) with high product recovery at room temperature, 2) at near-neutral pH, and 3) without harmful additives.

Description

[Technical field]

[0001] The present invention relates to a method for separating single-stranded RNA (ssRNA) molecules from other nucleic acid species, in particular double-stranded RNA (dsRNA) species, and to the use of solid phases and associated methods for separating single-stranded RNA from other nucleic acid species.The present invention further relates to a method for separating double-stranded RNA molecules by size. [Background technology]

[0002] RNA and DNA-related processes for drug substance manufacturing in the biopharmaceutical industry are driving the need for more diverse separation methods. As an example, in the production of messenger RNA (mRNA), the rapid development and mass production of mRNA-based vaccines is becoming increasingly important, as seen in the recent COVID-19 pandemic. Synthesis of mRNA for gene therapy and vaccination applications by in vitro transcription (IVT) results in preparations that contain undesired subpopulations of various nucleic acid species, including but not limited to double-stranded (ds) RNA, in addition to the desired single-stranded (ss) RNA. Removal of dsRNA is a particular purification goal, as dsRNA can trigger undesired immune responses when injected into a subject. mRNA preparations also contain residual dsDNA from the plasmid template (pDNA) used to synthesize the mRNA. Although dsDNA levels can be reduced by post-transcriptional nuclease enzyme treatment, dsDNA fragments may still be present in the preparation. mRNA preparations also contain proteins, which must be removed to reduce the chance that patients will have a non-specific immune response to the proteins. These proteins include enzymes such as RNA polymerase, pyrophosphatase, and nucleases that are used to degrade the dsDNA template after transcription.

[0003] Standard laboratory purification of RNA employs DNase enzymatic treatment to degrade DNA, followed by LiCl precipitation to purify and concentrate the RNA. This approach offers cheap and rapid purification of RNA, but is chemically harsh, difficult to scale up, and requires subsequent removal of nucleases.

[0004] Methods are known to reduce the content of dsRNA from mRNA preparations. For example, affinity adsorption chromatography on cellulose-based chromatography media can reduce the level of dsRNA contamination. Such methods are simple and effective on laboratory scale, but have very low binding capacities, making scale-up a burden. On the other hand, chromatography offers an alternative and easily scalable approach to producing high-purity preparations. Various approaches have been developed for RNA purification, as described below.

[0005] The levels of dsRNA and dsDNA in mRNA preparations can be reduced by affinity chromatography using oligodeoxythymidine (dT)-functionalized solid phases. This approach utilizes specific oligo-dT ligands that hybridize to polyadenine (A) regions of nucleic acids, allowing the isolation of mRNAs due to their poly-A tails. However, poly-A regions are also present in the template dsDNA and the generated dsRNA populations, all of which at least partially share the sequence of the target mRNA and do not completely separate from the mRNA under some conditions. Furthermore, the production of oligo-dT ligands may become a global bottleneck due to the growing need for mRNA therapeutics. Another drawback of this approach is that it cannot be used to isolate ssRNAs that lack poly-A tails, a notable subgroup.

[0006] Primary and secondary amine-based chromatography can separate mRNA from oligonucleotides, enzymes and DNA at room temperature. Such methods employ a pH gradient to elute ssRNA in the pH range above 9.5, while mRNA is eluted at high concentrations of OH. -It has the disadvantage of being easily hydrolyzed by ions.

[0007] Furthermore, pyrophosphate anion gradients can be used for primary and secondary amine-based chromatography, allowing elution of ssRNA at neutral pH at ambient temperature. Pyrophosphate is not widely recognized as a common anion in chromatography, which is a burden, at least for preparative applications. In addition, it is not hydrolytically stable in aqueous solutions, making it unlikely to be widely used.

[0008] Hydrophobic interaction chromatography (HIC) uses high salt concentrations in the mobile phase, which can lead to precipitation of mRNA in solution or on the column. High salt buffers also increase the pressure on the chromatographic system, limiting the flow rate to lower values. On the other hand, reversed-phase chromatography employs organic solvents that are best avoided for GMP manufacturing, and uses high temperatures to successfully bind and elute ribonucleic acid, which are major obstacles in the production of biopharmaceuticals for human use.

[0009] Standard ion exchangers, specifically quaternary and tertiary amine ion exchangers, have been used for dsRNA separation in ascending NaCl gradients, but the recovery of ssRNA was low. Successful elution of ssRNA longer than 1000 bases from standard ion exchangers requires the use of high temperatures (e.g., 65° C.) or high concentrations of toxic organic denaturants (e.g., acetonitrile).

[0010] The current state of the art further teaches that solid phase media derivatized with nitrogen-containing heterocycles, such as histamine-modified chromatographic columns, have been successfully applied to the separation of dsDNA (pDNA) from fragmented RNA, such as that present in E. coli lysates. In one example, the HIC principle was used with histamine- and histidine-modified chromatographic columns, alone or in combination with a descending pH gradient. The same principle cannot be directly applied, especially for ssRNAs longer than 1000 bases, because of the precipitation of mRNA in buffers containing ammonium sulfate in the applied concentration range. In another application with histamine-modified chromatographic columns, ion exchange conditions with an ascending salt gradient were applied to the separation of pDNA. In the case of mRNA, the same principle cannot be applied because of the low mRNA recovery from the column, similar to the separation efficiency with standard anion exchangers.

[0011] Surprisingly, in contrast to the teachings of the prior art, the present inventors have found that solid phases containing nitrogen-containing heterocycles can solve the above-mentioned problems and have been able to show that such solid phases behave differently from the above-mentioned expectations. The nitrogen-containing heterocycles are present on the solid phase surface and act as multimodal ligands. In particular, the present inventors have found that solid phase media derivatized with multimodal ligands containing nitrogen-containing heterocycles allow the development of methods that can overcome some practical limitations associated with existing methods for separating ssRNA from other nucleic acids. Summary of the Invention [Problem to be solved by the invention]

[0012] In view of the above, the object of the present invention is to provide a simple, low-risk and inexpensive method for separating ssRNA from other nucleic acids and biomolecules, which achieves high separation resolution and high recovery of ssRNA under mild operating conditions, i.e., at an ambient temperature of 15° C. to 30° C., at a near-neutral pH, and without the use of chemicals that are to be avoided in GMP manufacturing, as summarized, for example, in guidelines such as the European Medicines Agency's (EMA) EMA / CHMP / ICH / 82260 / 2006-ICH Guideline on Impurities Q3C(R8): Guideline on Residual Solvents. [Means for solving the problem]

[0013] According to the present invention, the object is to provide a method for separating single-stranded RNA from other nucleic acid species, comprising the steps of: a) providing a solid phase comprising a multimodal ligand having a pKa value between 4.0 and 8.5, the multimodal ligand comprising at least a nitrogen-containing heterocycle; b) loading a sample onto a solid phase, the sample containing the target single-stranded RNA molecules to be separated; c) separating and eluting the target single-stranded RNA molecules by applying an ascending pH gradient approaching or exceeding the pKa value of the multimodal ligand; This is achieved by providing a method comprising:

[0014] The surprising advantages of the present invention include the ability to elute ssRNA from solid phases containing multimodal ligands containing nitrogen-containing heterocycles at 1) high product recovery at room temperature, 2) near-neutral pH, and 3) without harmful additives, a combination of all three advantages not known in the prior art.

[0015] The present invention, including preferred embodiments thereof, will now be described in more detail in conjunction with the accompanying drawings. [Brief description of the drawings]

[0016] [Figure 1]FIG. 1 shows separation of dsRNA and plasmid dsDNA from mRNA using an ascending pH gradient at ambient temperature using a histamine-modified monolith (Example 2). [Diagram 2] FIG. 1 shows size separation of dsRNA and NTPs by ascending pH gradient at ambient temperature using histamine modified monolith (Example 3). [Diagram 3] FIG. 1 shows separation of plasmid dsDNA from mRNA using a 4-mercaptopyridine monolith with an ascending pH gradient at ambient temperature (Example 5). [Figure 4] FIG. 13. Application of a salt wash at ambient temperature to separate plasmid dsDNA from mRNA using a CIM PrimaH monolith (Example 6). [Diagram 5] FIG. 1 shows separation of plasmid DNA and mRNA using a combined pH and salt gradient at ambient temperature using a CIM PrimaH monolith (Example 7). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The separation method of the present invention employs a solid phase comprising a multimodal ligand. The multimodal ligand comprises at least one nitrogen-containing heterocycle as described above. In a preferred embodiment, the solid phase is a support matrix to which the multimodal ligand is attached. The type of solid phase is not particularly limited as long as it is suitable for use in liquid chromatography and can comprise a multimodal ligand.

[0018] Preferably, the solid phase has chemical functional groups on its surface to which the multimodal ligand is attached or to which the multimodal ligand can be attached. These groups can be already present on the surface of the solid phase or can be introduced by a suitable method. Thus, according to the invention, it is possible to use chromatographic solid phases that inherently contain functional groups (such as glycidyl-containing polymethacrylates) or to use chromatographic supports on which suitable functional groups can be introduced by surface modifications known to those skilled in the art. In this context, examples of known surface modifications include substitution and addition reactions, reaction with functional epoxides, activated acids or active esters, plasma treatment, e-beam (electron beam) treatment, gamma irradiation, coating, hydrolysis, aminolysis, oxidation, reduction, activation by reaction with functional carbenes and / or nitrenes, etc.

[0019] According to one embodiment of the present invention, the solid phase comprises at least one material selected from the group consisting of natural or synthetic fibers, (polymeric) membranes, natural or synthetic porous particles, natural or synthetic non-porous particles, natural or synthetic perfusion-based particles, natural or synthetic monolithic solid supports, polymer gels, films, non-woven fabrics and woven fabrics. The solid phase may be an organic or inorganic material. In a preferred embodiment, the solid phase is an organic-based monolithic solid support or a membrane.

[0020] Examples of natural or synthetic fibers that can be used as the material of the solid phase include electrospun cellulose fibers (such as "Fibro" from Cytiva), polyester fibers (such as "Winged Fibers" from Allasso Industries, which are composed of polyethylene terephthalate (PET), or "4DG™ Fibers" from Fiber Innovation Technology, which are composed of polyethylene terephthalate), as well as fibers that contain cellulose derivatives, nylon, polyethylene (PE), polyamide (PA), sulfone (PES), polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), polypropylene (PP) and polysulfone as structuring components, which can be used individually or in the corresponding combinations.

[0021] Examples of (polymer) membranes that can be used as the material of the solid phase include membranes that contain as structuring components cellulose, cellulose derivatives, nylon, polyester, polyethylene (PE), polyamide (PA), sulfone (PES), polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), polypropylene (PP) and polysulfone, which can be used alone or in corresponding combinations. Preferably, membranes based on cellulose and cellulose derivatives, in particular cellulose hydrate membranes, or polyethylene membranes are used.

[0022] Examples of polymer gels that can be used as the solid phase material include agarose, dextran, cellulose, polymethacrylate, polyvinyl ether, polyacrylamide, polystyrene-divinylbenzene copolymer, silica dextran, agarose acrylamide, and dextran acrylamide.

[0023] Examples of films and woven fabrics that can be used as the material of the solid phase include films and woven fabrics made of the above-mentioned polymeric materials that can be used for the (polymer) membrane. Examples of non-woven fabrics that can be used as the material of the solid phase include polyester / polypropylene / polyamide non-woven fabrics (such as "Pluratexx 2317 S" manufactured by Freudenberg) and the above-mentioned polymeric materials that can be used for the (polymer) membrane.

[0024] Examples of monolithic solid supports include molded monoliths synthesized from a single polymer mixture composed of, for example, poly(glycidyl methacrylate-co-ethylene dimethacrylate) or a single polymer mixture composed of polystyrene-divinylbenzene. A particular example is the CIM monolithic chromatography column manufactured by Sartorius BIA Separations. Another example is the so-called hydrogels, which are first synthesized as a monolith as a macroscaffold onto which a secondary ligand-bearing polymer phase is synthesized. The solid support may be provided within a housing to facilitate carrying out the chromatography.

[0025] The above materials may inherently contain the multimodal ligand or may be functionalized with the multimodal ligand. The binding of the multimodal ligand may be achieved via non-covalent or covalent bonds, preferably covalent bonds are used. According to the present invention, the multimodal ligand comprising at least one nitrogen-containing heterocycle is bound to the surface of the material of the solid phase directly or via a polymeric spacer element to form the solid phase.

[0026] In the solid phase used in the method of the present invention, the multimodal ligand is preferably bound via a polymeric spacer element between the surface of the material of the solid phase and the multimodal ligand to form the solid phase. The binding between the surface of the material of the solid phase and the spacer element preferably occurs or has occurred via originally existing functional groups or via functional groups generated by surface modification of the solid phase. The polymeric spacer element that can serve as a binding unit between the surface of the material of the solid phase and the multimodal ligand in the solid phase can advantageously obtain a high ligand density that allows a high binding capacity for target substances.

[0027] In the sense of the present invention, the term "multimodal" is understood to mean that the ligand contains two or more different functional groups, and therefore undergoes interaction with the target single-stranded RNA molecule based on different chemical and / or physical mechanisms, resulting in the binding of the target single-stranded RNA molecule to the solid phase. Examples of these interactions include hydrophobic interactions, electrostatic interactions, ionic interactions, hydrogen bonds, and π-π interactions.

[0028] pKa is the acid dissociation constant (also called acidity constant or acid ionization constant) and is a quantitative measure of the strength of an acid in solution. In the scope of this application, pKa is defined as the pH value at standard conditions where half of the functional groups that are part of the nitrogen-containing heterocycle are positively charged and half are uncharged. The pKa values ​​of multimodal ligands can be determined, for example, by potentiometric titration at ambient temperature.

[0029] According to the present invention, the multimodal ligand has a pKa value of 4.0 to 8.5 and comprises at least one nitrogen-containing heterocycle. According to the present invention, a multimodal ligand having a pKa value of 4.0 to 8.5 means that at least one nitrogen-containing heterocycle has at least one nitrogen atom whose conjugate acid has a pKa value in the range of 4.0 to 8.5.

[0030] Multimodal ligands containing nitrogen-containing heterocycles are capable of multimodal interactions that allow fine-tuning the separation of chemically similar RNA / DNA species for three important reasons:

[0031] First, multimodal ligands containing nitrogen-containing heterocycles can be neutral or positively charged at a certain pH. Their pKa values ​​depend on the number and position of the nitrogen atoms in the ring, as well as the size of the ring. According to the present invention, the ring structure is such that the pKa of the multimodal ligand when bound to a solid phase is between 4.0 and 8.5. In a preferred embodiment, the pKa value is between 5.0 and 8.0. The charge of the multimodal ligand varies with pH. When the pH is below the pKa, the multimodal ligand is positively charged ("binding mode" to nucleic acid). When the pH is two units higher than the pKa value of the multimodal ligand, the charge is reduced to practically zero ("elution mode"). The positive charge helps the nitrogen-containing heterocycles contained in the multimodal ligand on the solid phase to interact with the backbone of the nucleic acid, in particular the negatively charged phosphate backbone. Since single-stranded RNA molecules become unstable when exposed to high pH conditions for long periods of time, a charge switch point close to or slightly below neutral pH is an important feature for optimal elution conditions. Elution of bound nucleic acids is achieved by an increasing pH gradient that approaches or exceeds the pKa value of the multimodal ligand, while keeping the conductivity of the running buffer constant. In another embodiment, the increasing pH gradient is combined with an increasing or decreasing salt gradient. Thus, elution in the neutral pH range is possible.

[0032] According to the present invention, a pH gradient close to the pKa value of the multimodal ligand is a pH gradient that starts at a pH value below the pKa value of the multimodal ligand and ends at a pH value close to but not reaching the pKa value of the multimodal ligand.Similarly, a pH gradient above the pKa value of the multimodal ligand is a pH gradient that starts at a pH value below the pKa value of the multimodal ligand and ends at a pH value equal to or higher than the pKa value of the multimodal ligand.

[0033] Second, nitrogen-containing heterocycles can form hydrogen bonds with nucleic acid residues. This mode of interaction is useful for fine-tuning the strength of the bond and separating otherwise chemically very similar species such as mRNA, linear DNA (linDNA), dsRNA, and pDNA, because each species has a different profile and potential for hydrogen bonding. The relative strength of hydrogen bonds is higher at lower pH, allowing an ascending pH gradient as a preferential elution mechanism. However, this type of interaction is not possible with quaternary ammonium groups.

[0034] Third, nitrogen-containing heterocycles can be aromatic and therefore can exhibit hydrophobic interactions with aromatic bases of nucleic acids under salt exclusion conditions. The probability of salt-exclusion-based interactions is strongly influenced by "strand flexibility" and is higher for ssRNA (mRNA) than for its double-stranded counterparts dsRNA and linDNA, which are structurally much more rigid and limit the potential for hydrophobic interactions to the groove.

[0035] The relative strength of interactions based on salt exclusion is higher at higher pH due to deprotonation of the nitrogen-containing heterocycles. Thus, pH gradients allow the separation of nucleic acid species on the basis of their relative hydrophobicity.

[0036] Thus, the methods of the present invention are capable of selectively separating nucleic acid molecules based at least on their charge and ability to form hydrogen bonds. The methods of the present invention are further capable of separating nucleic acids by hydrophobic interactions.

[0037] In a preferred embodiment, the nitrogen-containing heterocycle is an aromatic nitrogen-containing heterocycle, in which case the multimodal ligand is more sensitive to the aromatic character of the nucleic acid molecule.

[0038] In particular, an important aspect of aromatic nitrogen-containing heterocycles is their ability to delocalize charge, altering their interactive properties compared to other amine ligands, including primary, secondary, tertiary and quaternary ammonium ligands, which do not have this ability.

[0039] According to the invention, an ascending pH gradient is used that approaches or exceeds the pKa value of the multimodal ligand. Conventionally, pH gradients used in protein biochromatography are applied within the pH range where the stationary phase is charged and stable, but without exceeding the pKa of the multimodal ligand. Thus, for anion exchangers, it is desirable to start at a high pH where electronegative proteins bind to the positively charged stationary phase, and then lower the pH until the proteins become more electropositive and are eluted by electrostatic repulsion. Both the initial and final pH values ​​are below the pKa of the multimodal ligand. The separation mechanism follows the change in charge of the analytes according to the respective isoelectric points (pI) of the molecules. However, this principle does not work for nucleic acids, since the pI of nucleic acids is very low (2-3) and similar for RNA and DNA molecules.

[0040] According to the present invention, the multimodal ligand has a pKa value between 4.0 and 8.5. Upon application of an ascending pH gradient approaching or exceeding the pKa of the multimodal ligand, the multimodal ligand loses charge, thereby reducing the interaction between the multimodal ligand and the nucleic acid. For strong anion exchangers, this mechanism is not possible, since they do not lose charge. On the other hand, for weak anion exchangers, such as simple amine ligands (e.g., diethylaminoethyl, diethylaminopropyl, etc.), with a pKa above 8.5, the desorption of nucleic acid occurs at high pH (above 10). Prolonged exposure to such alkaline pH is undesirable, since it reduces the chemical stability of single-stranded RNA. According to the present invention, the multimodal ligand has a pKa value between 4.0 and 8.5, and therefore transitions from a positive charge to a neutral charge under neutral or weakly acidic conditions. This allows the elution of nucleic acid at weakly acidic / neutral to weakly alkaline pH (below pH 9), where nucleic acid, especially mRNA, is relatively stable. Moreover, under certain conditions, mRNA and other nucleic acids can also bind to solid phases at pH values ​​above the pKa, since at one pH unit above the pKa, the material still retains 10% of the positively charged groups. Thus, it is technically possible to separate nucleic acids in a pH gradient that starts above the pKa value, but does not exceed or is close to the pKa value. However, a practical limitation of this approach is the low binding capacity of the solid phase, since there are fewer ligand groups available to bind nucleic acids.

[0041] It is important to emphasize here that while charge transfer elutes nucleic acids, it is not sufficient by itself to achieve substantial separation between different groups of DNA or RNA molecules, so additional multimodal interactions are required to fine-tune and improve the separation of these very similar species.

[0042] Thus, the present invention provides nucleic acid separations based on the combination of specific multimodal ligands and an ascending pH gradient. The use of an ascending gradient over the pH range where charge loss of the ligand occurs influences hydrogen bonding, salt exclusion and hydrophobic based interactions as well as other interactions mentioned above and allows for distinct modes of operation of "bind" and "elute", resulting in fine separation of similar species (RNA and DNA).

[0043] According to the present invention, the separation of ssRNA from other nucleic acids is based, at least in part, on hydrogen bonds between lone pairs or acidic hydrogens of the conjugated acid of the nitrogen-containing heterocycle contained in the multimodal ligand and various functional groups on the nucleic acid. These interactions are stronger at lower pH, so an upward gradient allows the detachment of the bound molecules. dsRNA / DNA molecules are base-paired and thus hidden in the core of the double helix. Furthermore, the double-stranded species are more rigid and cannot easily fit onto the solid phase surface to which the multimodal ligand is bound, resulting in a weaker interaction with the solid phase. Also, the base-paired bases are not fully available on the solid phase for hydrogen-bonding interactions with the multimodal ligand, so dsRNA / DNA interacts more with the solid phase through electrostatic interactions. This is in contrast to ssRNA and ssDNA, where the contribution of hydrogen bonds between the solid phase and the analyte is relatively greater due to the exposure of more nucleic acid bases, which retards the detachment of ssRNA and ssDNA from the support.

[0044] According to the present invention, the nitrogen-containing heterocycle may further contain other heteroatoms, such as, but not limited to, oxygen and / or sulfur. The nitrogen-containing heterocycle is preferably selected from the group consisting of purine, pyrimidine, pyridine, diazole, triazole, morpholine and tetrazole, and derivatives thereof. Examples of such compounds include imidazole, benzimidazole, quinoline, isoquinoline, and indoline.

[0045] In a further preferred embodiment, the nitrogen-containing heterocycle is selected from the group consisting of imidazole, imidazole derivatives, pyridine and pyridine derivatives. Preferred imidazole derivatives include histidine and histamine. A preferred pyridine derivative is 4-mercaptopyridine.

[0046] Preferably, the solid phase according to the present invention contains only one type of multimodal ligand, which means that only identical ligands are contained in the solid phase. This facilitates the functionalization of the solid phase and allows higher resolution when eluting the target single-stranded RNA molecules, since the target substance binds to each multimodal ligand in the same way. Therefore, the target substance is desorbed from all multimodal ligands under similar conditions.

[0047] According to the method of the invention, as step b), a sample containing a target ssRNA is loaded onto the solid phase, the target ssRNA being the ssRNA that is intended to be separated from other nucleic acid species by the method of the invention.

[0048] The size and conformation of the target ssRNA are not particularly limited. Typically, the target ssRNA has a length of 1000 bases to 25000 bases. Similarly, the conformation of the target ssRNA is not particularly limited, and the target ssRNA may exist in various conformations such as linear and cyclic. In a preferred embodiment of the present invention, the size and conformation of the target ssRNA are the same throughout one sample. This allows the target ssRNA to be clearly separated and eluted in one elution peak.

[0049] The sample containing the target single-stranded RNA molecule may be, for example, the result of an in vitro transcription process or the result of alkaline lysis of a cell suspension. Furthermore, the sample containing the target single-stranded RNA molecule may be the result of chemical synthesis or may be derived from a biological sample. The sample may be used as obtained or may be subjected to a pre-purification process to remove proteins and other non-nucleic acid molecules.

[0050] In a further preferred embodiment, the target single-stranded RNA molecule is an mRNA molecule.

[0051] In addition to ssRNA, the sample loaded onto the solid phase may contain one or more of the following other nucleic acid species: single-stranded DNA molecules, double-stranded DNA molecules, double-stranded RNA molecules, and plasmids. The sample may also contain other biological molecules (carbohydrates, lipids, proteins, etc.) and / or small organic compounds (nucleotides, sugars, etc.).

[0052] Before loading the sample onto the solid phase, the sample may be equilibrated to a suitable pH value, where the pH value chosen depends on the solid phase used and the type of multimodal ligand contained in the solid phase. Preferably, the sample is equilibrated to a pH value that is 2 units lower than the theoretical or determined pKa of the multimodal ligand contained in the solid phase.

[0053] The type of buffer used is not particularly limited, and any conventional buffer used in liquid chromatography can be used as long as it satisfies the above pH conditions. Buffers used include, among others, TRIS, bis-TRIS-propane, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), phosphate, and acetate. Furthermore, the equilibration buffer and loading buffer may contain additives as described herein below, if necessary.

[0054] Furthermore, before loading the sample onto the solid phase, the solid phase can be equilibrated to an appropriate pH value, preferably 2 units lower than the theoretical or determined pKa value of the multimodal ligand, to maximize the charge and capacity of the multimodal ligand. In a preferred embodiment, the solid phase is adjusted to the same pH value as the sample to be loaded onto the solid phase. In this way, acid-base reactions between the sample and the solid phase can be avoided.

[0055] Additionally or alternatively, it may be preferable to include various salts in the equilibration and / or loading buffers, for example 50 mM sodium phosphate or sodium chloride may be included in the buffers.

[0056] After loading the sample, unbound species can be washed away by rinsing the solid phase with a clean equilibration buffer, preferably having the same composition as the buffer used to equilibrate the solid phase and the sample solution.

[0057] After a first washing step to wash away unbound species, a second washing step may be applied at a pH value higher than the pH value to which the solid phase was equilibrated, but below the elution pH of the target ssRNA. This washing step can remove contaminants such as other nucleic acids or proteins that bind more loosely to the solid phase than the target ssRNA.

[0058] Additionally or alternatively, a second washing step may be performed with a buffer having a high salt concentration to selectively elute proteins and / or other contaminating species but not the target ssRNA. For example, washing with a high NaCl concentration (>0.2M) can elute some loosely bound contaminants, such as dsRNA and dsDNA molecules, from the surface. The concentration of salt in the washing buffer can have a high or low ionic strength compared to the ionic strength of the elution buffer.

[0059] In the final step of the method of the invention, the target single-stranded RNA molecules are selectively separated and eluted by applying an ascending pH gradient that approaches or exceeds the pKa of the multimodal ligand, which may be applied in a continuous manner, a stepwise manner, or a manner that includes both continuous and stepwise portions.

[0060] The elution buffer may have a similar composition to the buffer used to equilibrate the sample and the solid phase, but may differ at least in its pH value, and may differ in salt concentration and / or type of salt contained in the buffer, conductivity and / or additives added.

[0061] As outlined above, increasing the pH value of the elution buffer to approach or exceed the pKa of the multimodal ligand causes the multimodal ligand to gradually lose positive charge, thereby reducing its ion exchange binding ability to the nucleic acid species. However, this charge loss does not occur simultaneously and coordinately for all multimodal ligands as the pKa value is exceeded, but rather represents a statistical transition of the multimodal ligand from positive to neutral around the pKa value of the multimodal ligand. The pH range for charge loss is approximately 2 pH units below the pKa value of the ligand at which 99% of the multimodal ligand molecules are positively charged to approximately 2 pH units above the pKa value of the ligand at which 99% of the multimodal ligand molecules are uncharged. The optimal pH range for the ligand to lose charge must be determined experimentally, since pKa is not a fixed value but depends on temperature, buffer composition, salt concentration, density of immobilized multimodal ligand, etc. In view of the above, the starting and ending values ​​of the pH gradient are not particularly limited, as long as the pH gradient increases, i.e., starts at a lower pH than the initial pH and ends at a higher pH, however, this does not necessarily mean that the pH gradient crosses the middle of the range of pKa values.

[0062] The cumulative effect of electrostatic, hydrogen bonding, and hydrophobic interactions between the solid phase and the nucleic acid allows the target single-stranded RNA molecules to be separated from other nucleic acid species.

[0063] In a preferred embodiment, the pH gradient starts below the pKa value of the multimodal ligand and ends above the pKa value, and the pH gradient is achieved over approximately 4 pH units, however, this does not necessarily mean that the pH gradient crosses the middle of the pKa value.

[0064] In one embodiment of the present invention, the separation and elution of the target single-stranded RNA molecules is performed by applying an additional increasing salt gradient in combination with the increasing pH gradient. This change can further increase the separation efficiency of the method of the present invention, since the increase in salt concentration reduces the electrostatic interaction between the multimodal ligand and the nucleic acid species, thus lowering the pH required for elution of the target single-stranded RNA.

[0065] In one embodiment of the present invention, the separation and elution of the target single-stranded RNA molecules is performed by applying an ascending pH gradient while maintaining a salt concentration at an initial pH at which the target ssRNA does not elute. For example, NaCl or other types of salts up to 2 M salt concentration may be employed. This change may decrease the elution pH of the target ssRNA molecules, since the presence of salt weakens the electrostatic interactions between the biomolecule and the multimodal ligand.

[0066] The salt used is not particularly limited, and any salt conventionally used in chromatography can be adopted, including any ion from the Hofmeister series. Preferably, the salt is a monovalent or divalent salt. The cation of the salt is preferably selected from alkali metal cations, alkaline earth metal cations, transition metal cations and nitrogen-based cations. The anion of the salt is preferably selected from halogen anions (e.g., chloride, bromide, iodide, etc.), or inorganic acid anions (e.g., phosphate, phosphite, sulfate, sulfite, nitrate, nitrite, chlorate, carbonate, etc.), or organic acid anions (acetate, citrate, lactate, etc.). In a specific embodiment, the salt is NaH2PO4 or NaCl.

[0067] In the method according to the present invention, Mg is used to enhance the resolution between eluting species and improve the separation selectivity. 2+ , Zn 2+ , Ca 2+ Multivalent cations such as may be added to the loading and elution buffers.

[0068] Furthermore, in the method according to the present invention, a metal chelator such as ethylenediaminetetraacetic acid (EDTA), citric acid, ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), or tris(2-aminoethyl)amine (TREN) may be added to the loading buffer and elution buffer in order to increase the resolution between eluting species and change the separation selectivity.

[0069] Furthermore, in the method according to the invention, additives that change the interactions between the biomolecules themselves and / or between the solid phase and the biomolecules may be added to the loading buffer and / or the elution buffer. Examples of such additives include amino acids (e.g. arginine), carbohydrates (e.g. sorbitol), denaturants (e.g. urea, guanidine), organic acids, nitrogen-containing heterocycles (e.g. imidazole) and organic solvents (e.g. isopropanol).

[0070] As outlined above, the described method is also capable of separating double-stranded RNA molecules by size. Thus, the present invention provides a method for separating double-stranded RNA molecules by size, comprising: a) providing a solid phase comprising a multimodal ligand having a pKa value between 4.0 and 8.5, the multimodal ligand comprising a nitrogen-containing heterocycle; b) loading a sample onto a solid phase, the sample comprising two or more double-stranded RNA molecules of different lengths; c) size-separating and eluting the double-stranded RNA molecules by applying an ascending pH gradient approaching or exceeding the pKa value of the multimodal ligand; The method further includes the steps of:

[0071] Further variations of this method include the same as those described above for the method of separating single-stranded RNA molecules from other nucleic acid species.

[0072] In summary, the method of the present invention can be employed to perform analytical or preparative applications. The specific chromatographic conditions and the degree of separation between various DNA / RNA species may vary depending on the conformation and length of the nucleic acids and the different ligands used. This can be exploited for more efficient separations to achieve higher resolution (analytical) and / or higher purity of biological molecules (preparative scale).

[0073] The present invention is further illustrated in the following non-limiting examples. EXAMPLES

[0074] Example 1: Preparation of histamine-modified monolith A histamine modified monolith was prepared using the following exemplary procedure. Briefly, a 0.1 mL CIMmic-CDI monolith (Sartorius BIA Separations doo, Cat. No. 103.8000-2) was used to immobilize histamine on the monolith via carboxyimidazole (CDI) activation chemistry. The immobilization procedure consisted of pumping a histamine solution (10 g of histamine dihydrochloride dissolved in 100 ml of 1.0 M NaOH) through the monolith column, followed by thermostating the column at 25° C. for 48 hours. After modification, the monolith surface was exposed to 0.5 M NaOH to inactivate the remaining CDI groups.

[0075] Example 2: Separation of dsRNA and plasmid dsDNA from mRNA using histamine-modified monoliths with an ascending pH gradient at ambient temperature A histamine-modified solid phase in the form of a 0.1 mL monolithic chromatography device was used in combination with a PATfix HPLC system (Sartorius BIA Separations doo). The system was operated at ambient temperature. The histamine monolith was equilibrated with 10 mM Tris, 10 mM BTP, pH 6.0. Samples containing dsRNA ladder molecules ranging from 21 bp to 500 bp, 7300 bp plasmid dsDNA, 1000 bp mRNA ssRNA were prepared by dilution in 10 mM Tris, 10 mM BTP, pH 6.0 and applied to the histamine-modified monolith. A 5 CV wash with 10 mM Tris, 10 mM BTP, pH 6.0 was then applied to remove unbound material from the channels in the monolith. The monolith was then eluted with a linear gradient to 10 mM Tris, 10 mM BTP, pH 9.5 for 100 CV at ambient temperature with a flow rate of 10 CV / min.

[0076] As shown in Figure 1, all dsRNA molecules and plasmid dsDNA were eluted at the pH at which mRNA ssRNA remained bound. mRNA ssRNA was eluted at higher pH values ​​than dsRNA molecules and plasmid dsDNA. By further separating the nucleotides present in the sample, some size separation was observed between dsRNA molecules, which, like other nucleic acid molecules, were eluted at a lower pH due to their much weaker interactions compared to nucleic acid polymers. The elution recovery rates of all nucleic acids used for separation were higher than 85%.

[0077] Example 3: Size separation of dsRNA and NTPs using histamine-modified monoliths with an ascending pH gradient at ambient temperature A histamine-modified solid phase in the form of a 0.1 mL monolithic chromatographic device was used in combination with a PATfix HPLC system (Sartorius BIA Separations doo). The system was operated at ambient temperature. The histamine monolith was equilibrated with 10 mM Tris, 10 mM BTP, 20 mM guanidinium chloride, pH 6.0. Samples containing dsRNA ladder molecules (NEB, N0363S) ranging from 21 bp to 500 bp, or the individual nucleotide triphosphates (NTPs) adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP) and uridine triphosphate (UTP) were prepared by dilution in 10 mM Tris, 10 mM BTP, 20 mM guanidinium chloride, pH 6.0, and applied to the histamine-modified monolith. A wash with 10 mM Tris, 10 mM BTP, 20 mM guanidinium chloride, pH 6.0 was then applied to remove unbound material from the channels within the monolith, and the monolith was then eluted with a linear gradient to 10 mM Tris, 10 mM BTP, 20 mM guanidinium chloride, pH 9.5 over 100 CV at ambient temperature with a flow rate of 10 CV / min.

[0078] As shown in Figure 2, chaotropic guanidinium was included throughout the separation procedure to improve size separation of dsRNA molecules. Purine NTPs clearly separate from pyrimidine NTPs, which elute at higher pH values. Furthermore, NTPs separate from minor nucleotide diphosphates (NDPs), which elute at lower pH values ​​than NTPs.

[0079] From the above, the method of the present invention can be applied to monitor ssRNA production by IVT reactions, since the final product ssRNA is separated from the template molecule dsDNA and the nucleotides used as reaction substrates.

[0080] Example 4: Preparation of 4-mercaptopyridine modified monolith 4-mercaptopyridine modified monoliths were prepared by covalently attaching 4-mercaptopyridine to epoxide-loaded CIM monoliths (Sartorius BIA Separations doo). Briefly, 0.1 mL CIMmic epoxy monoliths (Sartorius BIA Separations doo) were used to immobilize 4-mercaptopyridine on the monoliths via epoxy activation chemistry. The immobilization procedure consisted of pumping 4-mercaptopyridine solution (10% (v / v) 4-mercaptopyridine solution in 96% EtOH) through the monolith column, followed by thermostating the column at 50°C for 48 hours. After modification, the monolith surface was exposed to 0.5 M H2SO4 at 65°C for 5 hours to deactivate the residual epoxy groups.

[0081] Example 5: Separation of plasmid dsDNA from mRNA using 4-mercaptopyridine monolith with ascending pH gradient at ambient temperature A 4-mercaptopyridine modified solid phase in the form of a 0.1 mL monolithic chromatography device was used in combination with a PATfix HPLC system (Sartorius BIA Separations doo). The system was operated at ambient temperature and the results are shown in Figure 3. The 4-mercaptopyridine monolith was equilibrated with 10 mM MES, 10 mM HEPES, 7% (v / v) isopropanol, pH 5.5. Samples containing plasmid dsDNA and mRNA were prepared by dilution in 10 mM MES, 10 mM HEPES, 7% (v / v) isopropanol, pH 5.5 and applied to the 4-mercaptopyridine modified monolith. A wash with 10 mM MES, 10 mM HEPES, 7% (v / v) isopropanol, pH 5.5 was then applied to remove unbound material from the channels in the monolith. The monolith was then eluted with a linear gradient to 10 mM MES, 10 mM HEPES, 7% (v / v) isopropanol, pH 8.0 over 100 CV at ambient temperature with a flow rate of 10 CV / min.

[0082] Due to the relatively high hydrophobicity of the ligand, isopropanol was included in the separation procedure to improve the elution profile of the nucleic acid. This example demonstrates that the method of the present invention is also applicable to pyridine-based multimodal ligands, but is not limited thereto. Furthermore, this example demonstrates that the separation conditions, including buffer composition and pH, can be adjusted to suit the properties of different ligands.

[0083] Example 6: Separation of plasmid dsDNA from mRNA using CIM PrimaH monolith with salt wash at ambient temperature A CIM PrimaH monolith in the form of a 0.1 mL chromatographic device was used in combination with a PATfix HPLC system (Sartorius BIA Separations doo). The system was operated at a flow rate of 1 mL / min and at ambient temperature, and the results are shown in Figure 4. The monolith was equilibrated with 20 mM citrate, 20 mM phosphate, pH 5.0. Samples containing plasmid dsDNA or mRNA were prepared by dilution in 20 mM citrate, 20 mM phosphate, pH 5.0 and applied to the CIM PrimaH monolith. A wash with 20 mM citrate, 20 mM phosphate, pH 5.0 was then applied to remove unbound material from the channels in the monolith. Elution was performed in two ways. First, the plasmid dsDNA was eluted by a salt wash with 20 CV of 20 mM citrate, 20 mM phosphate, 1 M NaCl, pH 5.0. The column was then equilibrated with 25 CV of 20 mM citrate, 20 mM phosphate, pH 5.0 Finally, the mRNA was eluted with a 50 CV step pH gradient of 20 mM citrate, 20 mM phosphate, pH 7.5.

[0084] Example 7: Separation of plasmid dsDNA and mRNA using CIM PrimaH monoliths by combining pH and salt gradients at ambient temperature A CIM PrimaH monolith in the form of a 0.1 mL chromatographic device was used in combination with a PATfix HPLC system (Sartorius BIA Separations doo). The system was operated at a flow rate of 1 mL / min and ambient temperature, and the results are shown in Figure 5. The monolith was equilibrated with 20 mM citrate, 20 mM phosphate, pH 5.0. Samples containing plasmid dsDNA or mRNA were prepared by dilution in 20 mM citrate, 20 mM phosphate, pH 5.0 and applied to the CIM PrimaH monolith. A wash with 20 mM citrate, 20 mM phosphate, pH 5.0 was then applied to remove unbound material from the channels in the monolith. The monolith was then eluted with a linear gradient to 20 mM citrate, 20 mM phosphate, 150 mM NaCl, pH 7.5 over 50 CV at ambient temperature and a flow rate of 10 CV / min.

Claims

1. 1. A method for separating single stranded RNA molecules from other nucleic acid species, comprising the steps of: a) providing a solid phase comprising a multimodal ligand having a pKa value between 4.0 and 8.5, said multimodal ligand comprising a nitrogen-containing heterocycle; b) loading a sample onto the solid phase, the sample containing the target single-stranded RNA molecules to be separated; c) separating and eluting the target single-stranded RNA molecules by applying an increasing pH gradient approaching or exceeding the pKa value of the multimodal ligand; A method comprising:

2. The method of claim 1 , wherein the multimodal ligand has a pKa value of 5.0 to 8.

0.

3. The method according to claim 1 or 2, wherein the nitrogen-containing heterocycle is an aromatic nitrogen-containing heterocycle.

4. The method according to any one of claims 1 to 3, wherein the nitrogen-containing heterocycle is selected from the group consisting of purine, pyrimidine, pyridine, diazole, triazole, morpholine, and tetrazole.

5. The method of claim 4, wherein the nitrogen-containing heterocycle is selected from the group consisting of imidazole, an imidazole derivative, pyridine or a pyridine derivative.

6. The method according to any one of claims 1 to 5, wherein the solid phase is a chromatography column comprising a solid stationary phase.

7. The method of any one of claims 1 to 6, wherein the sample is loaded onto the solid phase at a pH below the pKa of the multimodal ligand.

8. The method according to any one of claims 1 to 7, wherein the target single-stranded RNA molecule is an mRNA molecule or a derivative of a single-stranded RNA or mRNA molecule.

9. 9. The method according to claim 1, wherein the sample loaded onto the solid phase comprises as the other nucleic acid species one or more of single-stranded DNA molecules, double-stranded DNA molecules, double-stranded RNA molecules and plasmids.

10. The method according to any one of claims 1 to 9, further comprising the step b1) of washing away unbound species after step b) and before step c).

11. The method according to claim 10, further comprising, after step b1) and before step c), a step b2) of washing with a solution having a higher pH value than the solution used for loading the sample onto the solid phase in step b), but lower than the elution pH of the target single-stranded RNA molecules.

12. 12. The method of any one of claims 1 to 11, wherein the increasing pH gradient starts at a pH value at least 1 pH unit below the pKa value of the multimodal ligand and ends at a pH value at least 1 pH unit above the pKa value of the multimodal ligand.

13. The method according to any one of claims 1 to 12, wherein separation and elution of the target single-stranded RNA molecules is performed by applying an increasing salt gradient in combination with said increasing pH gradient.

14. 1. Use of a solid phase comprising a multimodal ligand having a pKa value between 4.0 and 8.5 for separating single-stranded RNA molecules from other nucleic acid species, said multimodal ligand comprising a nitrogen-containing heterocycle.

15. 15. The use according to claim 14, wherein the multimodal ligand is an imidazole or an imidazole derivative.

16. 1. A method for separating double-stranded RNA molecules by size, comprising: a) providing a solid phase comprising a multimodal ligand having a pKa value between 4.0 and 8.5, said multimodal ligand comprising a nitrogen-containing heterocycle; b) loading a sample onto the solid phase, the sample comprising two or more double-stranded RNA molecules of different lengths; c) size-separating and eluting the double-stranded RNA molecules by applying an ascending pH gradient approaching or exceeding the pKa value of the multimodal ligand; A method comprising:

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