Method for removing double-stranded and / or multi-stranded nucleic acid impurities from RNA preparations by low pH treatment

Abstract

The present invention relates to a method for removing double-stranded and / or multi-stranded nucleic acid impurities from an RNA preparation, comprising the steps of incubating the RNA preparation at a pH in the range of pH 1 to pH 5, and purifying the RNA preparation to remove fragments produced by dissociation of the double-stranded and / or multi-stranded nucleic acid impurities.

Description

[0001] The present invention relates to a method for removing double-stranded and / or multi-stranded nucleic acid impurities from an RNA preparation, comprising the steps of incubating the RNA preparation at a pH in the range of pH 1 to pH 5, and purifying the RNA preparation to remove fragments generated by the dissociation of the double-stranded and / or multi-stranded nucleic acid impurities.

[0002] The recent clinical success of mRNA-based COVID-19 vaccines has triggered unprecedented investment in the development of RNA-based therapeutics as vaccines in multiple therapeutic areas.

[0003] RNA is produced by enzymatic conversion of a DNA template into RNA in an in vitro transcription (IVT) reaction using RNA polymerase. RNA for therapeutic applications includes messenger RNA (mRNA), self-amplifying RNA (saRNA) and circular RNA. The impurities produced by the IVT reaction include a variety of double-stranded and multi-stranded nucleic acids, the main impurity being double-stranded RNA (dsRNA). Due to possible immunogenic responses, dsRNA is a key impurity in RNA therapy, in which the host immune system recognizes dsRNA as an invading virus. Cellular innate immune responses to dsRNA byproducts can lead to undesirable results, including inhibition of protein synthesis and cell death, which in turn can adversely affect the efficacy of RNA therapy.

[0004] Two major types of dsRNA byproducts have been identified: one is formed by 3'-extension of the runoff product annealing to a complementary sequence within the runoff transcript in cis (by folding back on the same RNA molecule) or in trans (annealing to a second RNA molecule), thereby forming an extended duplex. The second type of dsRNA is formed by hybridization of an antisense RNA molecule to the runoff transcript. Antisense RNA molecules have been reported to form in a promoter-independent and runoff transcript-independent manner. RNA polymerase may switch to a non-template strand, resulting in an RNA molecule that is complementary to the runoff product.

[0005] Therefore, dsRNA forms a heterogeneous population of RNA sequences of varying lengths rather than a well-defined impurity.

[0006] dsRNA removal is usually performed by one of two chromatography methods, both of which exploit the difference in relative hydrophobicity of single-stranded (ssRNA) relative to dsRNA. The first of these methods is reverse phase (RP) HPLC, and the second is chromatography using a chromatographic cellulose medium. However, these current methods are non-specific and have poor scalability. Specifically, RP-chromatography utilizes high temperatures and organic solvents, both of which are undesirable in the manufacture of biotherapeutics. In addition, they remove 3'-extended products because they are longer than ssRNA and therefore more hydrophobic, but not antisense transcripts produced as runoff transcripts. In addition, cellulose as a chromatographic medium is difficult to define and is not easily scalable. In addition, the forces driving the separation are not understood and therefore cannot be well controlled.

[0007] The third method of removing dsRNA is to use RNAse III for enzymatic degradation. However, RNAse III is non-specific, i.e. it digests dsRNA as well as ssRNA. In addition, it is undesirable to use enzymes in the production process, so this method is not feasible for commercial-scale production of RNA.

[0008] The second important impurity associated with RNA preparations is the residual DNA template used for in vitro transcription into RNA. The DNA template is usually a linearized form of DNA encoding the target gene, 5' and 3' UTR regions, polymerase binding sites and selection markers. The presence of residual plasmid templates in RNA preparations may lead to immunogenic responses, so that residual plasmids must be removed from the final RNA preparations. Typical purification methods include DNAse treatment after IVT combined with purification steps (precipitation, tangential flow filtration (TFF) or chromatography). Although highly selective and efficient, this method introduces additional contaminants (such as enzymes), which need to be removed during the purification process. In the absence of DNAse treatment, affinity purification of RNA (such as oligo dT purification of polyadenylated mRNA) achieves high but incomplete DNA removal levels, where it is assumed that the residual plasmid after affinity purification interacts with RNA molecules through specific or non-specific base pair interactions (forming substances defined as double-stranded and / or multi-stranded nucleic acid impurities herein).

[0009] The technical problem underlying the present invention is therefore to provide an improved method for removing double-stranded and / or multi-stranded nucleic acid impurities from RNA preparations, which should show high specificity, good scalability and thus be suitable for the preparation of RNA-based therapeutics on a commercially viable scale.

[0010] The solution to the above technical problem is achieved by the embodiments described in the claims.

[0011] Specifically, the present invention relates to a method for removing double-stranded and / or multi-stranded nucleic acid impurities from an RNA preparation, comprising the following steps:

[0012] (a) incubating the RNA preparation at a pH in the range of pH 1 to pH 5; and

[0013] (b) purifying the RNA preparation to remove fragments generated by dissociation of double-stranded and / or multi-stranded nucleic acid impurities.

[0014] Double-stranded and / or multi-stranded nucleic acid impurities that can be associated with the preparation of RNA and that can be removed according to the present invention include any non-single-stranded nucleic acid structure, in particular dsRNA, dsDNA (e.g., template DNA from an IVT reaction), RNA-DNA heteroduplexes, and multi-stranded homologous or heterologous nucleic acid structures, such as those formed by non-specific (non-Watson-Crick) binding of RNA and / or DNA chains (e.g., RNA / DNA triplexes). In a specific embodiment, double-stranded and / or multi-stranded nucleic acid impurities that can be associated with the preparation of RNA and that can be removed according to the present invention include dsRNA, RNA-DNA heteroduplexes, and multi-stranded homologous or heterologous nucleic acid structures, wherein dsRNA as an impurity is particularly preferred.

[0015] Herein, the term "removal of double-stranded and / or multi-stranded nucleic acid impurities" explicitly includes the situation where contaminating RNA or DNA single strands or DNA double strands have been annealed to the target RNA (i.e., the RNA to be prepared in the RNA preparation), and the resulting dsRNA structure or RNA-DNA heteroduplex or RNA-DNA triplex or other multi-stranded structure is subsequently removed from the target RNA by causing dissociation of the contaminating RNA or DNA strands.

[0016] According to the present invention, an RNA preparation containing double-stranded and / or multi-stranded nucleic acid impurities is subjected to low pH conditions in the range of pH 1 to pH 5, which results in the dissociation of the corresponding nucleic acid chains from each other.

[0017] The term "RNA preparation" as used herein relates to any physical solution containing the target RNA in a suitable buffer. Incubation of the RNA preparation at a pH in the range of pH 1 to pH 5 includes any means of providing a corresponding pH environment to the target RNA, for example, by acidifying the buffer in which the target RNA is present (e.g., by combining the buffer with an acidic buffer), or by transferring the target RNA to a new (acidic) buffer.

[0018] According to the invention, the above-mentioned RNA preparation is incubated at a pH in the range from pH 1 to pH 5, which range explicitly includes the boundary values ​​pH 1 and pH 5. In a preferred embodiment, the RNA preparation is incubated at a pH within a range whose lower boundary is selected from pH 1.0, pH 1.1, pH 1.2, pH 1.3, pH 1.4, pH 1.5, pH 1.6, pH 1.7, pH 1.8, pH 1.9 and pH 2.0, and whose upper boundary is selected from pH 5.0, pH 4.9, pH 4.8, pH 4.7, pH 4.6, pH 4.5, pH 4.4, pH 4.3, pH 4.2, pH 4.1, pH 4.0, pH 3.9, pH 3.8, pH 3.7, pH 3.6, pH 3.5, pH 3.4, pH 3.3, pH 3.2, pH 3.1 and pH 3.0. In specific embodiments, the RNA preparation is incubated at a pH in the range of pH 2 to pH 4, in the range of pH 2 to pH 3.5, or in the range of pH 2 to pH 3.5. For example, the RNA preparation can be incubated at about pH 3.

[0019] The incubation in the step (a) of the inventive method can be enough to allow the time of most of nucleic acid chains to dissociate from each other. The respective duration depends on various factors, including the property (such as the length of double-stranded region and / or multi-stranded region) of temperature, pH and impurity, and can be suitably selected by those skilled in the art. Each duration range is from a few seconds (including such as one second) to a few hours, wherein exemplary duration range includes hatching 25 seconds to 1 hour, 1 minute to 1 hour, 10 to 40 minutes, 20 to 40 minutes, 25 to 35 minutes, such as about 30 minutes.

[0020] In a specific embodiment, step (a) of the method of the present invention is carried out in an acidic buffer. Suitable buffers for any selected pH or pH range are known in the art and can be selected or designed by a person skilled in the art. Exemplary buffers in this regard include pK a Buffer in the range of 2 to 5. Specific exemplary buffers include citrate buffer, acetate buffer, glycine buffer, glycylglycine buffer, malate buffer, maleate buffer, phosphate buffer, succinate buffer, formate buffer and combinations thereof. The corresponding buffer may contain denaturants and / or chelating agents, wherein the denaturant is preferably a chaotropic agent, an organic solvent and a combination thereof. Various chaotropic agents, organic solvents and chelating agents are not particularly limited and are known in the art. Such chaotropic agents include, for example, urea, guanidine and combinations thereof. In addition, organic solvents include, for example, acetonitrile. In addition, chelating agents include, for example, EDTA (ethylenediamine-tetraacetic acid).

[0021] Step (a) of the method of the present invention is preferably carried out at a temperature of 4°C to 50°C, for example 8°C to 40°C, 15°C to 30°C or 20°C to 25°C, for example at about 20°C or at about 25°C. In particular, step (a) can be carried out at ambient temperature. Alternatively, step (a) can be carried out at a temperature elevated relative to ambient temperature, for example at a temperature of 25°C to 50°C, 25°C to 40°C or 30°C to 40°C. In any case, step (a) of the method of the present invention is not carried out at a temperature that will itself lead to the dissociation of double-stranded and / or multi-stranded nucleic acid impurities. For example, the method is not carried out at a temperature above 50°C.

[0022] In a specific embodiment, step (a) of the method of the invention is performed as part of an affinity chromatography step, wherein the RNA preparation is loaded onto an affinity chromatography medium at a pH in the range of pH 6 to pH 8, for example at a pH of about 7, and then the pH is lowered to a pH in the range of pH 1 to pH 5, thereby completing step (a) of the method of the invention. In this regard, suitable affinity ligands are not particularly limited and are known in the art. They include nucleic acids complementary to the target RNA, such as oligo-dT.

[0023] In step (b) of the method of the present invention, the RNA preparation is purified to remove fragments generated by the dissociation of double-stranded and / or multi-stranded nucleic acid impurities.

[0024] The fragments produced by the dissociation of double-stranded and / or multi-stranded nucleic acid impurities, i.e., the fragments produced by the dissociation of the chains of double-stranded and / or multi-stranded nucleic acid impurities from each other, which will be removed in this step (b), are contaminated single-stranded RNAs other than the target RNA, or contaminated single-stranded DNAs. The methods for purifying the RNA preparations separately are not particularly limited and are known in the art. However, such methods as defined below are preferred.

[0025] In specific embodiments, the RNA preparation is purified at the pH used in step (a), i.e., the pH is not adjusted prior to purification. In other specific embodiments, the pH of the RNA preparation is raised to a pH higher than the pH used in step (a), but not higher than pH 5, including pH 5, prior to purification. For obvious reasons, these embodiments are not applicable in the case where step (a) is already carried out at pH 5. In other specific embodiments, the pH of the RNA preparation is raised to a pH greater than the pH range of pH 5 to pH 7, including pH 7, prior to purification.

[0026] In a specific embodiment, step (a) of the method of the invention is carried out at a pH in the range of pH 2 to pH 4, for example at a pH of about pH 3, and subsequently, prior to purification in step (b), the pH is raised to a pH in the range of pH 4.5 to pH 5, for example to pH 5.

[0027] As described so far, steps (a) and (b) are separate steps that are performed in the order of first performing step (a) and then performing step (b). Therefore, in specific embodiments, steps (a) and (b) are separate steps that are performed in the order shown. As described above, in such embodiments, step (b) can be performed at the same pH as the pH used in step (a), or the pH can be raised prior to step (b).

[0028] However, in other specific embodiments, steps (a) and (b) can be performed simultaneously, i.e., incubating the RNA preparation at a pH in the range of pH 1 to pH 5 according to step (a) can be part of the purification process to remove fragments generated by the dissociation of double-stranded and / or multi-stranded nucleic acid impurities in step (b). For example, in the case of purification by chromatography-based techniques as described herein, step (a) of the method of the invention can be performed when the RNA preparation is combined with a chromatography medium, and step (b) of the method of the invention is performed using the chromatography medium. In such embodiments, the pH denaturation of the invention is achieved by combining the RNA preparation to the chromatography medium under conditions that are favorable for respective binding (e.g., at a pH in the range of pH 1 to pH 8, or at a pH in the range of pH 6 to pH 8, such as at a pH of about 7), and then step (a) of the method of the invention lowers the pH to a pH range of pH 1 to pH 5 to achieve nucleic acid chain dissociation on the chromatography medium, and then or simultaneously eluting the dissociated ssRNA and nucleic acid impurities, such as by a pH gradient, a salt gradient, or a combination thereof.

[0029] As mentioned above, the methods for purifying the RNA preparations are preferably as defined herein. Specifically, purification can be performed by a technique selected from cation exchange chromatography, anion exchange chromatography, size exclusion chromatography, reverse phase chromatography, hydrophobic interaction chromatography, multimodal chromatography, affinity chromatography, IMAC (immobilized metal affinity chromatography), molecular weight cutoff filtration and precipitation / extraction techniques. The methods for performing such techniques are not particularly limited and are known in the art.

[0030] In the case of chromatography techniques as described above, the chromatography material can be in any suitable form, for example in the form of porous particles, membranes, nanofibers, filters and blocks as known in the art.

[0031] In a specific embodiment, purification is carried out by anion exchange chromatography, and the anion exchange chromatography uses anion exchange ligands to carry out at a pH in the range of pH 1 to pH 5. The corresponding anion exchange ligands are not particularly limited and are known in the art. Exemplary anion exchange ligands that can be used in this respect include quaternary amines (QA), and tertiary amines, secondary amines and primary amines, wherein diethylaminoethyl (DEAE) and dimethylaminoethyl (DMAE) are particularly suitable. As mentioned above, anion exchange chromatography is carried out at a pH in the range of pH 1 to pH 5, and this range clearly includes boundary values ​​pH 1 and pH 5, or at a pH in the preferred range defined as above for the step (a) of the method of the present invention. In a relevant preferred embodiment, anion exchange chromatography is carried out at the pH identical with the step (a) of the method of the present invention.

[0032] In another specific embodiment, purifying is carried out by cation exchange chromatography, and the cation exchange chromatography uses cation exchange ligands to carry out at a pH within the scope of pH 1 to pH 5. The corresponding cation exchange ligands are not particularly limited and are known in the art. The exemplary cation exchange ligands that can be used in this respect include cation exchange ligands containing sulfonic acid groups, cation exchange ligands containing sulfate groups and cation exchange ligands containing carboxyl groups. As mentioned above, cation exchange chromatography is carried out at a pH within the scope of pH 1 to pH 5, and this scope clearly includes boundary values ​​pH 1 and pH 5, or is carried out at a pH within the defined preferred range as above for the step (a) of the method of the present invention. In a relevant preferred embodiment, cation exchange chromatography is carried out at a pH identical with the step (a) of the method of the present invention.

[0033] In another specific embodiment, purification is carried out by using a multimodal ligand, such as a ligand in conjunction with ion exchange / hydrogen bond properties or a ligand in conjunction with ion exchange / aromatic moieties or a combination thereof, at a pH in the range of pH 1 to pH 5 by multimodal chromatography. The corresponding multimodal ligand is not particularly limited and is known in the art. Exemplary multimodal ligands that can be used in this regard include CIM PrimaS, CIM PrimaH, CIM H-bonds, Capto MMC, Sartobind STIC and Toyopearl NH2. Typically, any type of multimodal ligand comprising multiple types of chemical residues selected from affinity residues, hydrophobic interaction residues, ion exchange residues, hydrogen bonding residues, metal chelating residues and aromatic residues and any combination thereof can be applied to the method of the present invention. As described above, multimodal chromatography is carried out at a pH in the range of pH 1 to pH 5, the range explicitly including boundary values ​​pH 1 and pH 5, or at a pH in the preferred range defined as above for step (a) of the method of the present invention. In a related preferred embodiment, the multimodal chromatography is performed at the same pH as step (a) of the method of the invention.

[0034] In another specific embodiment, purification is performed by affinity chromatography, which is performed at a pH in the range of pH 1 to pH 5 using an affinity ligand, such as oligo dT or other sequence-specific nucleic acid ligands complementary to the target RNA. As described above, the affinity chromatography is performed at a pH in the range of pH 1 to pH 5, which range explicitly includes the boundary values ​​of pH 1 and pH 5, or at a pH in the preferred range as defined above for step (a) of the method of the invention. In a related preferred embodiment, the affinity chromatography is performed at the same pH as step (a) of the method of the invention.

[0035] In other embodiments, purification is performed by

[0036] (i) size exclusion chromatography,

[0037] (ii) reverse phase chromatography,

[0038] (iii) hydrophobic interaction chromatography,

[0039] (iv) IMAC (immobilized metal affinity chromatography),

[0040] (iv) molecular weight cut-off filtration,

[0041] (v) precipitation / extraction techniques,

[0042] That is, at a pH in the range of pH 1 to pH 5, or at a pH greater than pH 5 to pH 7. As described above, these purification techniques can be performed at a pH in the range of pH 1 to pH 5, which range explicitly includes the boundary values ​​of pH 1 and pH 5, or at a pH in the preferred range as defined above for step (a) of the method of the present invention. In a related preferred embodiment, these purification techniques are performed at the same pH as step (a) of the method of the present invention. Alternatively, the above-mentioned purification techniques can be performed in a pH range of greater than pH 5 to pH 7, which range explicitly includes the boundary value pH 7, that is, the technique can be performed after the step (a) of the method of the present invention and before step (b) after adjusting the pH of the RNA preparation to the corresponding pH.

[0043] The target RNA, i.e., the RNA to be prepared in the RNA preparation used in the method of the present invention, can be selected from self-amplifying RNA (mRNA), self-amplifying RNA (saRNA) and circular RNA (cirRNA), among which mRNA is particularly preferred.

[0044] As used herein, the term "comprising" expressly includes the terms "consisting essentially of" and "consisting of", ie, all terms are interchangeable with each other herein.

[0045] In addition, as used herein, the term "about" preferably means a variation of ±10%, more preferably ±8%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1% or ±0.5% of a particular value. Thus, for example, the term "about 100" may include a range of 90 to 110, 92 to 108, 94 to 106, 95 to 105, 96 to 104, 97 to 103, 98 to 102, 99 to 101 or 99.5 to 100.5.

[0046] The present invention describes a method for removing double-stranded and / or multi-stranded nucleic acid impurities from RNA preparations. The method uses pH adjustment to remove impurities from RNA-based therapeutics, such as mRNA, saRNA, or circular RNA. pH adjustment is combined with a purification step to remove single-stranded fragments generated when double-stranded and / or multi-stranded starting materials are denatured.

[0047] The formation of double-stranded and / or multi-stranded nucleic acid structures and the stability of the structures depend on the basic principles of nucleic acid base pairing. Canonical hydrogen bonding interactions between CG and AU base pairs drive formation (Figure 1). Correct base pair formation, and thereby dsRNA formation, is driven by the formation of canonical binding interactions, namely CG and AU. To form canonical interactions, guanosine requires N1 protonation (pH>1.6), adenosine requires unprotonated N1 (pH>3.5), cytosine requires unprotonated N3 (pH>4.2), and uridine requires protonated N3 (pH<9.2).

[0048] At the pH range standard for RNA production, purification, and storage (i.e., pH 5 to 9), all four nucleosides are therefore properly protonated for base pairing to form a double strand. The phosphate backbone is deprotonated at pH>1; therefore, RNA contains a molecular surface (phosphate backbone) that is negatively charged at the working pH, a feature used in many purification methods.

[0049] At the pH range standard for RNA production, purification and storage (i.e. pH 5 to 9), all four nucleosides are therefore also appropriately protonated for base pairing with an affinity ligand immobilized on a chromatographic support, e.g., polydeoxythymidylic acid (oligo-dT). The pH reduction according to the invention disrupts base pairing with the affinity ligand, thereby releasing the target RNA molecule and simultaneously denaturing multi-stranded and / or double-stranded interactions (RNA-RNA or RNA-DNA).

[0050] It has been demonstrated on short RNA duplexes (<20mers) that protonation of hydrogen bond acceptors disrupts base pairing and leads to denaturation of RNA duplexes, which is measured as a decrease in the melting temperature of RNA duplexes as pH decreases. As a more general point, it has been previously observed that low pH leads to denaturation (or at least destabilization) of nucleic acids due to the protonation of GC base pairs and the formation of the resulting base pairs. In addition, protonation has been demonstrated to stabilize non-canonical interactions in DNA duplexes, such as AC and CC. Denaturation of double-stranded DNA was shown by incubation under acidic conditions. However, this concept has never been demonstrated in the purification of biologics, where short RNA duplexes are not representative of therapeutically relevant RNAs such as mRNA, and dsDNA is not representative of ssRNA contaminated by dsRNA, ssRNA, ssDNA or other double-stranded and / or multi-stranded nucleic acid impurities.

[0051] Therefore, the present invention relates to the chain separation driven by low pH, and the double-stranded and / or multi-stranded nucleic acid impurities present in the RNA preparation are denatured into single-stranded forms, resulting in the destruction of the base pairing of the specification. By incubating the RNA preparation at ambient temperature to a slightly elevated temperature in the pH range of pH 1 to pH 5, preferably pH 2 to pH 4, the double-stranded and / or multi-stranded nucleic acid impurities are dissociated from the RNA. This pH range is enough to protonate the bases involved in the base pairing so that they cannot support the base pairing of the specification. The phosphate backbone remains unprotonated, so that the RNA maintains an overall negative charge. Citrate, acetate, glycine or other acidic buffers can be used. Additives that promote denaturation, such as chaotropic agents such as urea or guanidine, can be used to promote double-stranded and / or multi-stranded denaturation under acidic conditions. Other denaturants known in the art, such as acetonitrile can be used at low pH to help denaturation. Chelating agents such as EDTA that help to remove structural divalent cations can be used to promote double-stranded and / or multi-stranded denaturation under acidic conditions. The temperature required for denaturation at low pH is ambient to slightly elevated (eg, 4°C to 50°C, preferably ambient).

[0052] According to the present invention, RNA containing double-stranded and / or multi-stranded nucleic acid impurities can be incubated at low pH to dissociate the double-stranded and / or multi-stranded species, and then the pH is raised to greater than pH 5 to pH 7. This increase in pH confers long-term stability to the RNA without the impurities reforming, or reforming completely, thereby activating potential immune responses triggered by such impurities when the RNA is administered in vivo.

[0053] In addition, RNA containing double-stranded and / or multi-stranded nucleic acid impurities can be incubated at low pH to dissociate double-stranded and / or multi-stranded substances, and then the pH is raised to pH 5 to pH 7, pH 5, and the preparation is purified to remove fragments generated by double-stranded and / or multi-stranded dissociation at a low pH of less than pH 5. Alternatively, RNA containing the corresponding impurities can be incubated at low pH and then purified to remove fragments generated by double-stranded and / or multi-stranded dissociation, wherein the purification is performed within the pH range used for denaturation (e.g., pH 2 to pH 4).

[0054] The present invention can utilize unusual pH ranges for disrupting dsRNA and RNA-DNA duplexes and / or multi-stranded structures and for binding the RNA to be prepared to chromatographic media (porous particles, nanofibers, membranes, monoliths) for ion exchange (cation and anion exchange), size exclusion, reverse phase, hydrophobic interaction or multimodal chromatography supports. In addition, molecular weight cutoff devices, such as tangential flow filtration devices, can be used.

[0055] In this article, anion exchange ligands can be used in unusual pH ranges. Due to the strong negative charge carried by the phosphate backbone, RNA molecules such as mRNA are strongly negatively charged at pH 1.6 and above. Anion exchange ligands such as quaternary amines (QA), tertiary amines (such as DEAE), secondary amines (such as ethylenediamine), primary amines or multimodal ligands comprising at least one anion exchange form are positively charged in the pH range of pH 1 to pH 11 or pH 1 to pH 13 (weak anion exchangers and strong anion exchangers, respectively). Therefore, RNA can be combined with positively charged ligands in the pH range of as low as pH 1 to pH 2. This is not common practice, and is actually counterintuitive, because in the art, the use of anion exchange ligands is under neutral to alkaline conditions, and the lowest pH value reported by the use of anion exchangers to purify mRNA is pH 5. The present invention utilizes the complementarity of the charge of RNA and anion exchange ligands in the abnormal pH range of pH 1 to pH 5, particularly pH 2 to pH 4. A salt gradient or pH gradient will be used to separate RNA fragments annealed with the target RNA forming the dsRNA structure and residual DNA fragments forming the RNA-DNA structure from the main ssRNA chain in a selected buffer (e.g., citrate, acetate, glycine, glycylglycine, malate, maleate, phosphate, succinate or formate buffer). As another aspect, any undissociated double-stranded and / or multi-stranded structure can be combined with an anion exchange ligand different from the ssRNA structure to provide an additional purification control layer. Additives that promote denaturation as known in the art, such as chaotropic agents such as urea or guanidine, or chelating agents such as EDTA or citrate, or organic solvents such as acetonitrile or ethanol, can be used to promote double-stranded and / or multi-stranded denaturation in the mobile phase, thereby further improving the selectivity of the method. The temperature of the separation can be optimized to result in an optimal balance between chromatographic separation and double-stranded and / or multi-stranded denaturation.

[0056] Alternatively, cation exchange ligands can be used for purification of target RNA after low pH treatment. This is counterintuitive because, for example, mRNA is strongly negatively charged in the pH range of pH 1.9 to pH 13, while cation exchange chromatography supports bind positively charged species.

[0057] In addition, size exclusion chromatography can be used to separate single-stranded fragments produced by pH denaturation of double-stranded and / or multi-stranded impurities from the desired RNA target molecule. Size exclusion chromatography can be performed at a pH range for denaturation (e.g., pH 2 to pH 4) or at a neutral / weakly acidic pH (e.g., pH 5 to pH 7, or pH 5 if the pH range for denaturation is less than pH 5). Small fragments of double-stranded and / or multi-stranded material that have been formed by hybridization with the parent RNA, as well as 3' extended dsRNA material, are separated from the target RNA by size.

[0058] In addition, based on the different hydrophobicity of the fragments and the target RNA, the single-stranded fragments produced by pH denaturation of double-stranded and / or multi-stranded impurities can be separated from the desired RNA target molecule using reverse phase chromatography or hydrophobic interaction chromatography. Reverse phase chromatography and hydrophobic interaction chromatography can be performed within the pH range for denaturation (e.g., pH 2 to pH 4) or at a neutral / weakly acidic pH (e.g., pH 5 to pH 7, or pH 5 if the pH range for denaturation is less than pH 5).

[0059] In all embodiments using chromatographic purification, the latter is not limited to a particular chromatographic medium, wherein any format, such as a block, membrane, filter, nanofiber or porous particle containing an appropriately charged polymer that promotes binding at the pH range of interest can be used.

[0060] In addition, tangential flow filtration operating in the pH range used for denaturation (e.g., pH 2 to pH 4) or an increased pH range (e.g., pH 5 to pH 7, or pH 5 if the pH range used for denaturation is less than pH 5) can be used to separate fragments / impurities generated by pH denaturation of double-stranded and / or multi-stranded nucleic acids from target RNA molecules.

[0061] The attached figure shows:

[0062] Figure 1:

[0063] (A) Chemical structure of dsRNA and (B) protonation equilibrium of nucleosides. The boxes represent the protonation states obtained by low pH treatment.

[0064] Figure 2 ：

[0065] pH treatment of mRNA reduces dsRNA content without affecting mRNA stability. Re-neutralization to pH 6 does not lead to reformation of dsRNA. Left: Dot blot (J2 antibody). Right: Agarose gel electrophoresis.

[0066] Figure 3:

[0067] A. Denaturation of dsRNA at low pH is temperature dependent. EGFP (995nt) was incubated at 4°C, room temperature (RT) and 40°C for 30 minutes at different pH values ​​ranging from 2.9 to 4.1. Dot blots were performed with J2 antibody to determine the amount of dsRNA. B. Denaturation of long dsRNA chains requires elevated temperatures and low pH for denaturation. Magi2 dsRNA (1000bp fully complementary dsRNA) was incubated at 50°C, pH 3 for 20 minutes (sample 1) or pH 6 (sample 3), and then buffer exchanged to pH 3 (samples 2 and 4, respectively). Denaturation was monitored by A) PAGE and B) J2 dot blots.

[0068] Figure 4:

[0069] Exposure of affinity purified mRNA to low pH for 60 minutes does not cause degradation of the mRNA. A. eGFP (995nt) was incubated at pH 3 for i) 15 minutes, ii) 30 minutes or iii) 60 minutes, and then the pH was raised to pH 6. The extent of fragmentation was determined using a fragment analyzer (BioAnalyzer, Agilent) directly after neutralization to pH 6 (i to iii) or after the same samples were stored at 4 to 8°C for 1 day (iv to vi). B. Agarose gel electrophoresis of samples i to vi. Lane 1: Riboruler HR, Lane 2: pH 3, 15 minutes, Lane 3: pH 3, 30 minutes, Lane 4: pH 3, 60 minutes, Lanes 5 to 7: Samples from lanes 2 to 4 were analyzed after storage at 4 to 8°C for 1 day.

[0070] Figure 5 ：

[0071] Exposure of the mRNA-containing IVT mixture to low pH resulted in a decrease in dsRNA content measured by J2 dot blot.

[0072] i) Incubate the IVT mixture containing mRNA at pH 3 at T (25°C) for 30 min

[0073] ii) The IVT mixture containing mRNA was analyzed in IVT reaction buffer (pH 7.9).

[0074] Figure 6:

[0075] An example of low pH treatment of mRNA bound to dsRNA structure by chromatography. EGFP (995nt) was incubated at ambient temperature for 25 minutes in 100mM glycine, pH 3.0. Afterwards, the pH was raised to pH 6 with 100mM glycine pH 10. The sample was then loaded onto a CIM PrimaH column (prototype multi-mode column, combining weak anion exchangers, aromaticity and hydrogen bond elements) at pH 5 and eluted with a pH gradient (pH 5 to pH 7.5) prepared with a 20mM citrate, 20mM sodium phosphate, 10mM EDTA buffer system. A: Elution chromatogram of eGFP mRNA incubated at pH 3 on CIM PrimaH. Red: 260nm, blue: 280nm, green: linear gradient, gray: pH tracer, brown: conductivity. B: PAGE gel of starting material, CIM PrimaH elution fractions 1 and 2 and in situ cleaning (high pH washing, CIP). C: J2 dot blot of eGFP (L) and CIM PrimaH, CIM PrimaH elution fractions 1 and 2 and wash in place (high pH wash, CIP). D: AGE of gel starting material, CIM PrimaH elution fractions 1 and 2 and wash in place (high pH wash, CIP).

[0076] Figure 7:

[0077] Example of low pH treatment of mRNA to remove dsRNA structure using cation exchange chromatography. EGFP (995nt) was incubated in 100mM glycine, pH 3.0 at ambient temperature for 20 minutes, or directly prepared for cation exchange chromatography. The sample was diluted in mobile phase A 50mM glycine pH 3 and loaded onto a CIM SO3 column at pH 3 and eluted with a pH gradient pH 3 to pH 10. A: Elution chromatogram of eGFP mRNA on CIM SO3. Green: untreated mRNA, red: pH 3 treated mRNA, B: polyacrylamide gel electrophoresis (PAGE) of starting material (pretreated in pH 3, or without pretreatment), flow-through (FT) fractions and elution fractions E1, E2 and CIP. C: J2 spot blot of the corresponding fractions.

[0078] Figure 8 ：

[0079] Low pH treatment combined with chromatographic separation results in the removal of residual DNA template. EGFP (995nt) was incubated in 100mM glycine, pH 3.0 at ambient temperature for 25 minutes or without low pH treatment. Both samples were then purified using a pH gradient on CIM PrimaH. The main elution fractions were analyzed for the presence of residual DNA plasmid template by RNAse treatment and AGE. Lane 1: GeneRuler, Lane 2: 50ng eGFP DNA template, Lane 3: 30ng eGFP DNA template, Lane 4: 10ng eGFP DNA template, Lane 5: 5ng eGFP DNA template, Lane 6: 2ng eGFP DNA template, Lane 7: 1ng eGFP DNA template, Lane 8: mRNA purified with oligo dT (starting material). Lane 9: PrimaH elution fraction of pH 3 treated mRNA, Lane 10: PrimaH elution fraction of untreated mRNA

[0080] Fig. 9 ：

[0081] mRNA (eGFP, 995 nt) was incubated in various concentrations of glycine, pH 3.0 (from 5 mM to 200 mM, as indicated) at ambient temperature for 15 minutes or was left untreated (lane 'mRNA'). The resulting material was analyzed by J2 dot blot to detect the presence of dsRNA and agarose gel electrophoresis (2% gel) to evaluate the stability of mRNA under denaturing conditions. A minimum of 25 mM glycine pH 3 was required for dsRNA denaturation.

[0082] Fig.10 ：

[0083] mRNA (eGFP, 995 nt) was denatured in 35 mM glycine pH 3 in the presence of different concentrations of NaCl (250 mM to 2 M) and analyzed by J2 dot blot. The dot blot showed that NaCl at a concentration of 250 mM or above prevented dsRNA from low pH denaturation. This means that NaCl concentrations of 250 mM and above cannot be used in combination with pH denaturation.

[0084] Fig.11 ：

[0085] mRNA (eGFP, 995nt) was incubated in 75mM glycine, pH 3 for 20 minutes. The resulting product was loaded onto a CIMC4 HLD column and the column was washed with 10mM glycine pH 3 at 1 mL / min. The mRNA was eluted with a step gradient to mobile phase B (10mM citrate pH 5.1). The resulting elution fractions were analyzed by J2 dot blot to confirm the removal of dsRNA and the mRNA integrity was evaluated by agarose gel electrophoresis (2% gel). i) HPLC chromatogram of CIM C4HLD purification (number '1' indicates the elution fraction from retention time 4.1 minutes to 4.3 minutes). ii) J2 dot blot: 1: mRNA eGFP (untreated); 2: CIM C4 HLD elution fraction (from retention time 4.1 minutes to 4.3 minutes). iii) AGE: 1: Riboruler HR, 2: mRNA eGFP (untreated); 3: CIM C4 HLD elution fraction.

[0086] Fig.12 ：

[0087] The in vitro transcription (IVT) reaction mixture containing mRNA (Cas9, 4000nt) was incubated in 25mM glycine, pH 3 for 20 minutes. The resulting product was loaded onto a CIM C4 HLD column and the column was washed with 10mM glycine pH 3 at 1 mL / min. The mRNA was eluted into mobile phase B (10mM citrate pH 5.1) using a step gradient. The resulting elution fractions were analyzed by J2 dot blot to confirm the removal of dsRNA. i) HPLC chromatogram of CIM C4 HLD purified elution fractions, number 1 represents the elution fraction (retention time 4.1 minutes to 4.4 minutes). ii) J2 dot blot: 1: IVT mRNA Cas9 (untreated); 2: CIM C4HLD flow-through (FT) fraction; 3: CIM C4 HLD elution fraction (retention time 4.1 minutes to 4.4 minutes).

[0088] Fig.13 ：

[0089] mRNA (eGFP, 995nt) was diluted in mobile phase A (50mM sodium phosphate, 0.5M NaCl, pH 7.5) and loaded onto a CIM oligo dT column (1mL) at a speed of 1mL / min. The column was washed with 10mL mobile phase B (50mM sodium phosphate, pH 7.5) and then eluted to mobile phase C (100mM glycine, pH 3) with a step gradient. The eluted fractions were analyzed by J2 dot blot to confirm the denaturation of dsRNA. Partial denaturation (RT 22.8 to 24.2 minutes) was shown in elution fractions 1 and 2, and complete denaturation (RT 24.2 to 26 minutes) was shown in elution fraction 3. The stability of elution fraction 3 was confirmed by agarose gel electrophoresis. i) HPL chromatogram of CIM oligo dT purification. Numbers indicate elution fractions (1: Rt 22.8 to 23.8 min; 2: Rt 23.8 to 24.2 min; 3: Rt 24.2 to 26 min). ii) J2 dot blot: 1-mRNA eGFP (untreated); 2: ClM oligo dT elution fraction 1; 3: ClM oligo dT elution fraction 2; 4: ClM oligo dT elution fraction 3. iii) AGE: 1: Riboruler HR, 2: mRNA eGFP (untreated); 3: ClM oligo dT elution fraction 3.

[0090] Fig.14 ：

[0091] mRNA (eGFP, 995nt) was diluted in mobile phase A (50mM sodium phosphate, 0.5M NaCl, pH 7.5) and loaded onto a Poros oligo (dT) 25 column (1mL) at a speed of 1mL / min. The column was washed with 9mL mobile phase B (50mM sodium phosphate, pH 7.5) and then eluted to mobile phase C (100mM glycine, pH 3) with a step gradient elution. The eluted fractions were analyzed by J2 dot blot to confirm the denaturation of dsRNA. Partial denaturation (18.4 to 18.9) was shown in elution fraction 1, and complete denaturation (RT 18.9 to 20.3 minutes) was shown in elution fraction 2. The stability of elution fraction 2 was confirmed by agarose gel electrophoresis. i) HPLC chromatogram of Poros oligo (dT) 25. Elution fractions are indicated by numbers 1: Rt 18.4 to 18.9 minutes; 2: RT 18.9 to 20.3 minutes. ii) J2 dot blot: 1-mRNA eGFP (untreated); 2-Poloxamer oligo(dT)25 elution fraction 1; 3-Poloxamer oligo(dT)25 elution fraction 2. iii) AGE: 1: mRNA eGFP (untreated); 2:Poloxamer oligo(dT)25 elution fraction 1; 3-Poloxamer oligo(dT)25 elution fraction 2.

[0092] Fig.15 ：

[0093] mRNA (eGFP, 995nt) was diluted in mobile phase A (50mM sodium phosphate, 0.5M NaCl, pH 7.5) and loaded onto a Poros oligo dT column (1mL) at a speed of 1mL / min. The column was washed with 9mL mobile phase B (50mM sodium phosphate, pH 7.5), and then mobile phase C (100mM glycine, pH 3) was eluted with a step gradient. The resulting pH 3 eluent was then diluted in mobile phase A (10mM glycine, pH 3) and loaded onto a CIM C4 HLD column at 1mL / min. mRNA was eluted to mobile phase B (50mM citrate, pH 5.1) with a step gradient. The resulting eluted fraction was analyzed by J2 dot blot to confirm the removal of dsRNA. A) HPLC chromatogram of Poros oligo (dT) 25 purification. Number 1 indicates the elution fraction from RT 18.4 to 18.9 minutes; number 2 indicates the elution fraction from RT 18.9 to 20.3 minutes. B) HPLC chromatogram of CIM C4 HLD purification of oligo dT eluate. Number 1 indicates the elution fraction from RT 7.9 to 8.3 minutes. C) J2 dot blot: 1: mRNA eGFP (untreated); 2: CIM C4 HLD elution fraction 1.

[0094] Fig.16 ：

[0095] RNA (eGFP, 995nt) was incubated in 25mM glycine, pH 3 for 15 minutes. The resulting product was diluted in mobile phase A (50mM citric acid, pH 5) and loaded onto a CIM PrimaH column at 1 mL / min. The mRNA was eluted to mobile phase B (100mM phosphate, pH 7.5) using a step gradient. The resulting elution fractions were analyzed by J2 spot blot to confirm the removal of dsRNA. i) HPLC chromatogram of CIM PrimaH separation of low pH denatured mRNA. Number 1 indicates elution fraction RT 9.6 to 9.9 minutes; number 2 indicates elution fraction RT 9.9 to 10.5 minutes. ii) J2 spot blot: -mRNA eGFP (untreated); 2: ClM PrimaH elution fraction 1; 3: ClM PrimaH elution fraction 2.

[0096] The present invention will be further illustrated by the following examples, but is not limited thereto. Example

[0097] Embodiment 1:

[0098] mRNA encoding eGFP (enhanced green fluorescent protein, 995 nucleotides) was produced in an IVT (in vitro transcription) reaction and purified by oligo-dT affinity chromatography, which does not remove dsRNA (double-stranded RNA). Dot blot analysis with the J2 antibody specific for dsRNA sequences of at least 40 base pairs confirmed the presence of dsRNA impurities in the samples.

[0099] The pH range required for dsRNA denaturation was tested by incubating mRNA at room temperature over a wide pH range (2.9 to 4.1; glycine buffer was used at the indicated pH values). Samples were incubated at room temperature for 30 minutes before loading on AGE (agarose gel electrophoresis; 100 ng) or dot blot assay (800 ng). Incubation of mRNA at pH 2.9 to 4.1 did not reduce the intensity of the main mRNA AGE band, while the intensity of the dot blot signal decreased with decreasing pH. The signal was strongest at pH 6, and strong signals were still observed at pH 3.9 to 4.1. The signal was significantly reduced at pH 3.5 and was not visible at pH 3.2 and below, indicating that the dsRNA structure was dissociated while the mRNA was intact.

[0100] The mRNA was then incubated at pH 3 for 20 min and then raised to pH 6; AGE results showed the appearance of a lower molecular weight band upon increasing to pH 6, while dot blots showed no signal, indicating that the dsRNA structure was not reformed ( Figure 2 ).

[0101] The temperature dependence of dsRNA denaturation was demonstrated by incubating eGFP (995nt) at different pH values ​​ranging from 2.9 to 4.1 at 4°C, room temperature (RT) and 40°C for 30 minutes. Dot blotting was performed with J2 antibody to determine the amount of dsRNA and to demonstrate the temperature and pH-dependent decrease in dsRNA signal. The dsRNA signal was still detected at pH 3.2 and 4°C, while at 40°C, the dsRNA signal was only detected at pH 3.9 or higher (Figure 3).

[0102] To demonstrate that pH rather than temperature is required for duplex denaturation, the fully complementary dsRNA sequence (1000 base pairs) Magi2 was incubated at 50°C for 20 min at pH 3 or pH 6. At pH 6, high temperature (50°C) was not sufficient to denature the duplex, as shown by J2 dot blots ( Figure 3B ), while at pH 3, the dsRNA signal disappeared. When the buffer was exchanged to pH 3, the dsRNA signal disappeared ( Figure 3B , sample 4).

[0103] To demonstrate that low pH treatment does not affect mRNA stability, eGFP (995 nt) was incubated at pH 3 for 15, 30, and 60 min at room temperature and then neutralized to pH 6. Fragmentation was then measured by BioAnalyzer and AGE ( Figure 4A and 4B ). No significant degradation was observed by either method, indicating that exposure of the mRNA to low pH for the time required for dsRNA denaturation does not result in mRNA degradation.

[0104] To demonstrate the versatility of this approach, unpurified IVT reaction mixtures containing eGFP mRNA were then incubated at pH 3 or left untreated, and the dsRNA signal was measured by J2 dot blot without RNA purification. Dot blots confirmed the disappearance of the dsRNA signal at pH 3 ( Figure 5 i), but did not disappear without pH treatment ( Figure 5 ii).

[0105] To demonstrate the purification of mRNA treated at low pH, mRNA encoding eGFP was incubated in 100 mM glycine, pH 3.0 at ambient temperature for 20 minutes. The pH of the sample was then raised to pH 6 with glycine buffer (pH 10) and then loaded onto a prototype multimodal column that combines elements of weak anion exchanger, aromaticity, and hydrogen bonding (PrimaH) at pH 5 and eluted with a pH gradient (pH 5 to pH 7.5) prepared from a 20 mM citrate, 20 mM sodium phosphate, 10 mM EDTA buffer system ( Fig. 6A ). Two elution fractions (1 and 2) were analyzed by dot blot using J2 antibody.

[0106] The results demonstrated strong binding of mRNA to PrimaH when incubated at pH 3 (Figure 6). pH treatment of mRNA resulted in a significant reduction in the dsRNA content in the eluted fractions, as shown in J2 dot blot, fractions 1 and 2 (Figure 6).

[0107] To demonstrate the purification of mRNA using low pH treatment with a cation exchanger, mRNA encoding eGFP was incubated in 100 mM glycine, pH 3.0, at ambient temperature for 20 min or left untreated. Both samples were diluted in mobile phase A (MPA; 50 mM glycine pH 3) and loaded onto a CIM (convective interaction media) SO3 column and eluted with a pH gradient from 3 to 10 ( Fig. 7A The starting material (mRNA treated with or without low pH), the flow-through fraction, and the two elution fractions (1 and 2) were analyzed by PAGE and dot blotting with J2 antibody ( Figure 7B,C). The results showed that pretreatment with pH 3, or dilution of the sample with MPA (50 mM glycine pH 3), resulted in denaturation of dsRNA, as evidenced by a decrease in the dot blot signal. ssRNA eluted in the flow-through; the dot blot indicated the absence of dsRNA (Figure 7C).

[0108] To demonstrate that low pH treatment combined with chromatographic separation results in the removal of residual DNA template, eGFP (995nt) was incubated in 100mM glycine, pH 3.0 at ambient temperature for 25 minutes and then neutralized; the control was not treated with low pH. Both samples were then purified on CIM PrimaH using a pH gradient as described above. The presence of residual DNA plasmid template in the main elution fraction was analyzed by treating 10μg mRNA with RNAse A (to enzymatically digest mRNA); the RNAse A digest of 10μg mRNA was loaded onto AGE. RNAse A selectively digests RNA but not DNA, and the residual DNA signal was compared to a standard dilution of the parent linearized plasmid (range 50ng to 1ng). When the PrimaH elution of pH 3 treated mRNA was analyzed by AGE using this method, no signal for residual plasmid was detected. PrimaH elution of pH untreated mRNA showed the presence of 2 to 5ng DNA plasmid / 10μg mRNA ( Figure 8 ).

[0109] Embodiment 2:

[0110] mRNA encoding eGFP (enhanced green fluorescent protein, 995 nucleotides) was produced in an IVT (in vitro transcription) reaction and purified by oligo-dT affinity chromatography, which does not remove dsRNA (double-stranded RNA). Dot blot analysis with the J2 antibody specific for dsRNA sequences of at least 40 base pairs confirmed the presence of dsRNA impurities in the samples.

[0111] The buffer molarity of pH 3 required for dsRNA denaturation was tested by incubating mRNA at room temperature in a wide buffer concentration range (5 to 200 mM glycine buffer at pH 3). The samples were incubated at room temperature for 15 minutes and then loaded into AGE (2% agarose gel electrophoresis; 500 ng) or dot blot assay (1000 ng). Incubation of mRNA in a concentration range of 5 to 200 mM glycine buffer pH 3 resulted in a decrease in the intensity of the dot blot. The J2 signal was the strongest in the untreated sample (untreated control), and a strong signal was still observed in the sample incubated with 5 mM buffer (pH 3). The signal was significantly reduced in 10 mM buffer, and the signal was not visible in 25 mM and higher buffers, indicating that the dsRNA structure was dissociated, while the agarose gel showed that the mRNA was intact in all concentrations of glycine buffer tested ( Fig. 9 ).

[0112] Embodiment 3:

[0113] The salt dependence of dsRNA denaturation was demonstrated by incubating eGFP (995 nt) in 25 mM glycine pH 3 for 15 min at room temperature (RT) in the presence of different concentrations of NaCl ranging from 250 mM to 2 M. Dot blotting was performed with J2 antibody to determine its amount. No dsRNA signal was detected in the presence of 50 mM NaCl, while at salt concentrations of at least 250 mM, the J2 dot blot signal increased, indicating that NaCl prevents dsRNA denaturation at pH 3 ( Fig.10 ).

[0114] Embodiment 4:

[0115] To demonstrate the purification of low pH treated mRNA using hydrophobic interaction chromatography, mRNA encoding eGFP was incubated in 75 mM glycine, pH 3.0 at ambient temperature for 20 min. The sample was loaded onto a CIM C4 HLD column and eluted using a step gradient to 10 mM citrate pH 5.1 ( Fig.11 , i). The starting material (untreated mRNA) and the elution fractions ( Fig.11 , iii). The results indicate that low pH incubation followed by purification by a hydrophobic chromatography column leads to denaturation of dsRNA purification of ssRNA as evidenced by a decrease in the dot blot signal in the elution fractions ( Fig.11 , ii).

[0116] Example 5 :

[0117] To demonstrate the generality of this approach, an unpurified IVT reaction mixture containing Cas9 mRNA (4000 nt) was incubated in 25 mM glycine pH 3 at ambient temperature for 20 min. The sample was loaded onto a CIM C4 HLD column and eluted using a step gradient to 10 mM citrate pH 5.1 ( Fig.12 , i). The dsRNA signal in the resulting elution fractions was measured by J2 dot blot, which confirmed that the dsRNA signal 3 was significantly reduced ( Fig.12 , ii), confirmed the denaturation of dsRNA and purified ssRNA.

[0118] Embodiment 6:

[0119] The possibility of mRNA denaturation on a low pH column was demonstrated using affinity chromatography. The mRNA encoding eGFP was diluted in mobile phase A (50 mM sodium phosphate, 0.5 M NaC, pH 7.5) and loaded onto a CIM (convective interaction medium) oligo dT column, followed by washing with mobile phase B (50 mM sodium phosphate, pH 7.5). The mRNA was eluted using a step gradient to 100 mM glycine pH 3 ( Fig.13 , i). The starting material (mRNA) and the eluted fractions ( Fig.13 , ii and iii). No significant degradation was observed by AGE, indicating that exposure of mRNA to low pH during purification did not affect RNA integrity ( Fig.13 , iii). Elution of mRNA from oligo dT at pH 3 resulted in denaturation of dsRNA, as evidenced by a decrease in dot blot signal in the eluted fractions ( Fig.13 , ii).

[0120] Embodiment 7:

[0121] To demonstrate the generality of the method, mRNA encoding eGFP was diluted in mobile phase A (50 mM sodium phosphate, 0.5 M NaCl, pH 7.5) and loaded onto a Poros oligo(dT)25 column, followed by washing with mobile phase B (50 mM sodium phosphate, pH 7.5). The mRNA was eluted using a step gradient into 100 mM glycine pH 3 ( Fig.14 , i). The starting material (mRNA) and the eluted fractions ( Fig.14 , ii and iii). No significant degradation was observed, indicating that exposure of mRNA to low pH during purification does not affect RNA integrity ( Fig.14, iii). Elution of mRNA from oligo dT at pH 3 resulted in denaturation of dsRNA, as evidenced by a decrease in dot blot signal in the eluted fractions ( Fig.14 , ii).

[0122] Embodiment 8:

[0123] To remove dsRNA fragments generated by denaturation eluted at low pH from the oligo dT column, chromatographic separation of the oligo dT eluate using CIMC4 HLD was performed. mRNA encoding eGFP (995 nt) was diluted in mobile phase A (50 mM sodium phosphate, 0.5 M NaCl, pH 7.5) and loaded onto a Poros oligo (dT) 25 column, followed by washing with mobile phase B (50 mM sodium phosphate, pH 7.5) ( Fig.15 The resulting pH 3 eluate was then diluted in 10 mM glycine pH 3 and loaded onto a CIMC4 HLD column. The mRNA was eluted via a step gradient to 50 mM citrate, pH 5.1 ( Fig.15 , B). Starting material (mRNA) and C4HLD elution fractions ( Fig.15 , C). Dot blot shows a significant decrease in dsRNA signal in the C4 HLD eluate, indicating that the dsRNA structure is dissociated and removed, while the mRNA is intact.

[0124] Embodiment 9:

[0125] Purification of ssRNA from low pH treated mRNA samples was further confirmed by low pH treatment combined with multimodal chromatography. eGFP (995 nt) was incubated in 25 mM glycine, pH 3.0 at ambient temperature for 15 min. The resulting product was diluted in mobile phase A (50 mM citric acid, pH 5) and purified on CIM PrimaH using a pH gradient (pH 5 to pH 7.5) prepared from a 50 mM citrate and 100 mM sodium phosphate buffer system ( Fig.16 , i). The eluted fractions were analyzed by dot blot using J2 antibody ( Fig.16 , ii), indicating that the dsRNA content in elution fraction 1 was significantly reduced.

Claims

1. A method for removing double-stranded and / or multi-stranded nucleic acid impurities from RNA preparations, wherein The following steps are involved: (a) incubating the RNA preparation at a pH in the range of pH 1 to pH 5, and (b) purifying the RNA preparation to remove fragments generated by dissociation of double-stranded and / or multi-stranded nucleic acid impurities.

2. The method of claim 1, wherein the RNA preparation is incubated in step (a) for 25 seconds to 1 hour.

3. The method of claim 1 or 2, wherein step (a) is performed in a buffer comprising a denaturant and / or a chelating agent.

4. The method according to any one of claims 1 to 3, wherein step (a) is carried out at a temperature of 4°C to 50°C.

5. The method of any one of claims 1 to 4, wherein step (a) is performed as part of an affinity chromatography step, wherein the RNA preparation is loaded onto an affinity chromatography medium at a pH in the range of pH 6 to pH 8 and the pH is subsequently lowered to a pH in the range of pH 1 to pH 5.

6. The method according to any one of claims 1 to 5, wherein (i) purifying the RNA preparation at the pH used in step (a), or (ii) if step (a) was not carried out at pH 5 prior to said purification, raising the pH of said RNA preparation to a pH greater than the range of pH to pH 5 used in step (a), or (iii) prior to said purification, raising the pH of said RNA preparation to a pH greater than in the range of pH 5 to pH 7.

7. The method of any one of claims 1 to 6, wherein the purification is performed by a technique selected from the group consisting of cation exchange chromatography, anion exchange chromatography, size exclusion chromatography, reverse phase chromatography, hydrophobic interaction chromatography, multimodal chromatography, affinity chromatography, IMAC (immobilized metal affinity chromatography), molecular weight cutoff filtration and precipitation / extraction techniques.

8. The method according to any one of claims 1 to 6, wherein (i) the purification is performed by a technique selected from the group consisting of cation exchange chromatography, anion exchange chromatography, size exclusion chromatography, reverse phase chromatography, hydrophobic interaction chromatography, multimodal chromatography, affinity chromatography and IMAC (immobilized metal affinity chromatography), and (ii) while the RNA preparation is being bound to the chromatography medium, steps (a) and (b) are performed simultaneously.

9. The method of claim 7 or 8, wherein the technique is anion exchange chromatography performed at a pH in the range of pH 1 to pH 5 using anion exchange ligands selected from quaternary amines (QA), tertiary amines, secondary amines and primary amines.

10. The method of claim 9, wherein the anion exchange ligand is diethylaminoethyl (DEAE) or dimethylaminoethyl (DMEA).

11. The method of claim 7 or 8, wherein the technique is cation exchange chromatography performed at a pH in the range of pH 1 to pH 5 using a cation exchange ligand selected from a cation exchange ligand containing a sulfonic acid group, a cation exchange ligand containing a sulfate group, and a cation exchange ligand containing a carboxyl group.

12. The method of claim 7 or 8, wherein the technique is multimodal chromatography performed at a pH ranging from pH 1 to pH 5 using a multimodal ligand comprising multiple types of chemical residues selected from affinity residues, hydrophobic interaction residues, ion exchange residues, hydrogen bonding residues, metal chelating residues, aromatic residues and combinations thereof.

13. The method of claim 7 or 8, wherein the technique is affinity chromatography using an affinity ligand at a pH in the range of pH 1 to pH 5.

14. The method according to any one of claims 1 to 13, wherein the RNA to be prepared in the RNA preparation is selected from the group consisting of mRNA, self-amplifying RNA (SaRNA) and circular RNA (cirRNA).

15. The method of any one of claims 1 to 14, wherein the double-stranded and / or multi-stranded nucleic acid impurities are non-single-stranded nucleic acid structures selected from double-stranded RNA (dsRNA), double-stranded DNA (dsDNA), RNA-DNA heteroduplexes, and multi-stranded homologous or heterologous nucleic acid structures.

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