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

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 subjecting the RNA preparation to purification to remove fragments generated by dissociation of double-stranded and / or multi-stranded nucleic acid impurities.

Description

[Technical field]

[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 subjecting the RNA preparation to purification to remove fragments generated by dissociation of double-stranded and / or multi-stranded nucleic acid impurities. [Background technology]

[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 using an RNA polymerase enzyme in an in vitro transcription (IVT) reaction. RNAs used for therapeutic applications include messenger RNA (mRNA), self-amplifying RNA (saRNA) and circular RNA. Impurities resulting from the IVT reaction include a range of double-stranded and multi-stranded nucleic acid species, with the major impurity being double-stranded RNA (dsRNA). dsRNA is an important impurity in RNA therapeutics due to the immunogenic response that may result from the host immune system recognizing dsRNA as an invading virus. Cellular innate immune responses to dsRNA by-products can lead to undesirable outcomes including suppression of protein synthesis and cell death, thereby detrimentally impacting the efficacy of RNA therapeutics.

[0004] Two main types of dsRNA by-products have been identified: one is formed by 3' extension of the run-off product and anneals to a complementary sequence in the body of the run-off transcript either in cis (folding back on the same RNA molecule) or in trans (annealing with a second RNA molecule) to form an extended duplex. The second type of dsRNA is formed by hybridization of an antisense RNA molecule with the run-off transcript. Antisense RNA molecules have been reported to be formed independently of the promoter and the run-off transcript. RNA polymerase can switch to the non-template strand and generate an RNA molecule complementary to the run-off product.

[0005] Thus, dsRNA is not a well-defined impurity, but rather forms a heterogeneous population of RNA sequences of different lengths.

[0006] Removal of dsRNA is typically accomplished by one of two chromatographic techniques, both of which exploit the difference in the relative hydrophobicity of single-stranded (ssRNA) versus dsRNA. The first of these techniques is reversed-phase (RP) HPLC, and the second is chromatography using chromatographic cellulose media. However, these current techniques are nonspecific and poorly scalable. In particular, RP chromatography utilizes both high temperatures and organic solvents that are undesirable in the manufacture of biological therapeutics. Furthermore, while 3' extension products are removed because they are longer and therefore more hydrophobic than ssRNA, antisense transcripts that are generated as run-off transcripts are not. Furthermore, cellulose as a chromatographic medium is poorly defined and not easily scalable. Furthermore, the forces driving the separation are not understood and therefore not well controlled.

[0007] The third method for removing dsRNA is enzymatic digestion using RNase III. However, RNase III is non-specific, i.e., it digests not only dsRNA but also ssRNA. Moreover, the use of enzymes in the manufacturing process is undesirable, so this method is not feasible for commercial-scale manufacturing of RNA.

[0008] A second important class of impurities 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 gene of interest, the 5' and 3' UTR regions, the polymerase binding site, and the selection marker. The presence of residual plasmid template in the RNA preparation can cause an immunogenic response, so the residual plasmid must be removed from the final RNA preparation. Typical purification techniques include a post-IVT deoxyribonuclease treatment combined with a purification step (precipitation, tangential flow filtration (TFF) or chromatography). This technique is highly selective and efficient, but introduces additional contaminants (e.g. enzymes) that need to be removed in the purification process. In the absence of DNase treatment, affinity purification of RNA (e.g., oligo dT purification of polyadenylated mRNA) achieves high but not complete levels of DNA removal, presumably because residual plasmids after affinity purification interact with the RNA molecules via specific or non-specific base pairing interactions (forming what are defined herein as double-stranded and / or multi-stranded nucleic acid impurities). Summary of the Invention [Problem to be solved by the invention]

[0009] The technical problem underlying the present invention is therefore the provision of an improved means for removing double-stranded and / or multi-stranded nucleic acid impurities from RNA preparations, which should exhibit high specificity, good scalability and therefore be suitable for the production of RNA-based therapeutics on a commercially viable scale. [Means for solving the problem]

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

[0011] In particular, the present invention provides a method for removing double-stranded and / or multi-stranded nucleic acid impurities from an RNA preparation, comprising the steps of: (a) incubating the RNA preparation at a pH in the range of pH 1 to pH 5; (b) subjecting the RNA preparation to purification to remove fragments generated by dissociation of double-stranded and / or multi-stranded nucleic acid impurities; The present invention relates to a method comprising the steps of:

[0012] Double-stranded and / or multi-stranded nucleic acid impurities that may be associated with the preparation of RNA and that may 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 heteroduplex structures, and multi-stranded homo- or heteromeric nucleic acid structures (e.g., formed by non-specific (i.e., non-Watson-Crick-based) association of RNA strands and / or DNA strands (e.g., RNA / DNA triplexes)). In certain embodiments, double-stranded and / or multi-stranded nucleic acid impurities that may be associated with the preparation of RNA and that may be removed according to the present invention include dsRNA, RNA-DNA heteroduplex structures, and multi-stranded homo- or heteromeric nucleic acid structures, with dsRNA as an impurity being particularly preferred.

[0013] In this context, the term "removal of double-stranded and / or multi-stranded nucleic acid impurities" explicitly includes the situation where a contaminating RNA or DNA single strand, or a DNA double strand, anneals to a target RNA (i.e., the RNA prepared in an RNA preparation) and the resulting dsRNA structure, or RNA-DNA heteroduplex, or RNA-DNA triplex, or other multi-stranded structure, is subsequently removed by causing dissociation of the contaminating RNA or DNA strand(s) from the target RNA.

[0014] According to the 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 dissociate the individual nucleic acid strands from one another.

[0015] 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 ranging from pH 1 to pH 5 includes any means of providing the target RNA with the respective pH environment, for example by acidifying the buffer in which the target RNA is present (for example by combining the buffer with an acidic buffer) or by transferring the target RNA to a new (acidic) buffer.

[0016] According to the invention, the RNA preparation is incubated at a pH in the range of pH 1 to pH 5, the range explicitly including the boundaries pH 1 and pH 5. In a preferred embodiment, the RNA preparation is incubated at a pH in the range of pH, the lower limit of which is selected from the group consisting of 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 the upper limit of which is selected from the group consisting of 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 certain embodiments, the RNA preparation is incubated at a pH ranging from pH 2 to pH 4, from pH 2 to pH 3.5, or from pH 2 to pH 3. For example, the RNA preparation can be incubated at about pH 3.

[0017] The incubation in step (a) of the method of the present invention can be carried out for a time sufficient to dissociate most of the nucleic acid strands from each other. The respective durations depend on various factors including temperature, pH, and the nature of the impurities (e.g., with respect to the length of the double-stranded and / or multi-stranded regions) and can be appropriately selected by the skilled artisan. The respective durations range from a few seconds (including, for example, 1 second) to a few hours, and exemplary duration ranges include incubation for 25 seconds to 1 hour, 1 minute to 1 hour, 10 minutes to 40 minutes, 20 minutes to 40 minutes, 25 minutes to 35 minutes, for example, about 30 minutes.

[0018] In certain embodiments, step (a) of the method of the 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 the skilled artisan. Exemplary buffers in this regard include those with a pK in the range of 2 to 5. a Specific exemplary buffer solutions include citrate buffer, acetate buffer, glycine buffer, glycylglycine buffer, malic acid buffer, maleic acid buffer, phosphate buffer, succinic acid buffer, formic acid buffer, and combinations thereof. Each buffer solution may contain a denaturant and / or a chelating agent, and the denaturant is preferably a chaotropic agent, an organic solvent, and combinations thereof. Each chaotropic agent, organic solvent, and chelating agent 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 (ethylenediaminetetraacetic acid).

[0019] Step (a) of the method of the invention is preferably carried out at a temperature between 4°C and 50°C, e.g. between 8°C and 40°C, 15°C and 30°C, or between 20°C and 25°C, e.g. at about 20°C or 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, e.g. between 25°C and 50°C, 25°C and 40°C, or 30°C and 40°C. In any case, step (a) of the method of the invention is not carried out at a temperature which itself causes dissociation of double-stranded and / or multi-stranded nucleic acid impurities, e.g. the method is not carried out at a temperature higher than 50°C.

[0020] In a particular embodiment, step (a) of the method of the invention is carried out as part of an affinity chromatography step, in which the RNA preparation is loaded onto the 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, thus carrying out step (a) of the method of the invention. Suitable affinity ligands in this regard are not particularly limited and are known in the art. They include nucleic acids complementary to the target RNA, for example oligo dT.

[0021] In step (b) of the method of the invention, the RNA preparation is subjected to purification to remove fragments generated by dissociation of double-stranded and / or multi-stranded nucleic acid impurities.

[0022] The fragments generated by dissociation of double-stranded and / or multi-stranded nucleic acid impurities removed in this step (b), i.e., the fragments generated by dissociation of the strands of double-stranded and / or multi-stranded nucleic acid impurities from each other, are contaminating single-stranded RNAs different from the target RNA, or contaminating single-stranded DNAs. The means for carrying out the purification of each of the RNA preparations are not particularly limited and are known in the art. However, such means are preferably as defined below.

[0023] In certain embodiments, the RNA preparation is subjected to purification at the pH used in step (a), i.e. the pH is not adjusted prior to purification. In other particular embodiments, the pH of the RNA preparation is raised to a higher pH than that used in step (a), but the pH is below pH 5 (including pH 5) prior to purification. These embodiments do not apply, for obvious reasons, if step (a) has already been carried out at pH 5. In yet other particular embodiments, the pH of the RNA preparation is raised to a pH in the range of above pH 5 to pH 7 (including pH 7) prior to purification.

[0024] In a particular embodiment, step (a) of the method of the invention is carried out at a pH in the range of pH 2 to pH 4, e.g. at about pH 3, and then the pH is increased to a pH in the range of pH 4.5 to pH 5, e.g. to pH 5, before purification in step (b).

[0025] As described hereinbefore, steps (a) and (b) are separate steps performed sequentially in the order of step (a) first and step (b) second. Thus, in certain embodiments, steps (a) and (b) are separate steps performed sequentially in the order shown. As noted above, in such embodiments, step (b) can be performed at the same pH used in step (a), or the pH can be increased prior to step (b).

[0026] However, in certain other embodiments, steps (a) and (b) can be performed simultaneously, i.e. incubation of the RNA preparation at a pH in the range of pH 1 to pH 5 according to step (a) can be part of a purification process for removing fragments generated by dissociation of double-stranded and / or multi-stranded nucleic acid impurities in step (b). By way of example, in case of purification by chromatography-based techniques as described herein, step (a) of the method of the invention can be performed while the RNA preparation is bound to a chromatography medium, and step (b) of the method of the invention is performed using the chromatography medium. In such an embodiment, pH denaturation according to the invention is carried out by binding the RNA preparation to a chromatography medium under conditions favouring the 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, e.g. at a pH of about 7), then nucleic acid strand dissociation is carried out on the chromatography medium by lowering the pH to a pH range of pH 1 to pH 5 according to step (a) of the method of the invention, and subsequent or simultaneous elution of the dissociated ssRNA and nucleic acid impurities is carried out, e.g. by a pH gradient, a salt gradient or a combination thereof.

[0027] As mentioned above, the means for carrying out each purification of RNA preparation is preferably as defined herein. In particular, purification can be carried out 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. The means for carrying out such techniques are not particularly limited and are known in the art.

[0028] For chromatographic techniques such as those described above, the chromatographic material may be in any suitable form, such as porous particles, membranes, nanofibers, filters, and monoliths known in the art.

[0029] In a particular embodiment, the purification is carried out by anion exchange chromatography, using an anion exchange ligand, carried out at a pH in the range of pH 1 to pH 5. The respective anion exchange ligands are not particularly limited and are known in the art. Exemplary anion exchange ligands that can be used in this regard include quaternary amines (QA), as well as tertiary, secondary and primary amines, with diethylaminoethyl (DEAE) and dimethylaminoethyl (DMAE) being particularly preferred. As mentioned above, the anion exchange chromatography is carried out at a pH in the range of pH 1 to pH 5 (with the bounds pH 1 and pH 5 expressly included) or in the preferred range of pH as defined above for step (a) of the method of the invention. In a related preferred embodiment, the anion exchange chromatography is carried out at the same pH as step (a) of the method of the invention.

[0030] In another particular embodiment, the purification is carried out by cation exchange chromatography carried out at a pH in the range of pH 1 to pH 5 using a cation exchange ligand. The respective cation exchange ligand is not particularly limited and is known in the art. Exemplary cation exchange ligands that can be used in this regard include cation exchange ligands containing sulfonic acid groups, cation exchange ligands containing sulfate groups, and cation exchange ligands containing carboxyl groups. As mentioned above, the cation exchange chromatography is carried out at a pH in the range of pH 1 to pH 5 (this range expressly including the boundaries pH 1 and pH 5) or in the preferred range of pH as defined above for step (a) of the method of the invention. In a related preferred embodiment, the cation exchange chromatography is carried out at the same pH as step (a) of the method of the invention.

[0031] In further particular embodiments, the purification is carried out by multimodal chromatography carried out at a pH ranging from pH 1 to pH 5 using multimodal ligands, such as ligands with combined ion exchange / hydrogen bonding properties or ligands with combined ion exchange / aromatic moieties, or combinations thereof. The respective multimodal ligands are not particularly limited and are known in the art. Exemplary multimodal ligands that can be used in this regard include CIM PrimaS, CIM PrimaH, CIM H-bond, Capto MMC, Sartobind STIC, and Toyopearl NH2. In general, any type of multimodal ligand comprising multiple types of chemical moieties selected from the group consisting of affinity moieties, hydrophobic interaction moieties, ion exchange moieties, hydrogen bonding moieties, metal chelating moieties, and aromatic moieties, and any combination thereof, may be suitable for the method of the invention. As mentioned above, the multimodal chromatography is carried out at a pH ranging from pH 1 to pH 5 (this range expressly including the boundaries pH 1 and pH 5) or at a pH in the preferred range defined above for step (a) of the method of the invention. In a related preferred embodiment, the multimodal chromatography is carried out at the same pH as step (a) of the method of the invention.

[0032] In yet further particular embodiments, purification is performed by affinity chromatography using an affinity ligand, such as oligo dT or other sequence specific nucleic acid ligand complementary to the target RNA, performed at a pH in the range pH 1 to pH 5. As mentioned above, affinity chromatography is performed at a pH in the range pH 1 to pH 5 (this range expressly including the boundaries pH 1 and pH 5) or in the preferred range of pH as defined above for step (a) of the method of the invention. In related preferred embodiments, affinity chromatography is performed at the same pH as step (a) of the method of the invention.

[0033] In alternative particular embodiments, purification comprises: (i) size exclusion chromatography; (ii) reverse phase chromatography; (iii) hydrophobic interaction chromatography; (iv) IMAC (immobilized metal affinity chromatography); (iv) molecular weight cut-off filtration, or (v) precipitation / extraction techniques; and are carried out at a pH ranging from pH 1 to pH 5 or at a pH ranging from above pH 5 to pH 7. As mentioned above, these purification techniques can be carried out at a pH ranging from pH 1 to pH 5 (this range expressly including the limit values ​​pH 1 and pH 5) or in the preferred range of pH defined above for step (a) of the method of the invention. In related preferred embodiments, these purification techniques are carried out at the same pH as step (a) of the method of the invention. Alternatively, the above mentioned purification techniques can be carried out at a pH ranging from above pH 5 to pH 7 (this range expressly including the limit value pH 7), i.e. these techniques can be carried out after a step of adjusting the pH of the RNA preparation to the respective pH after step (a) and before step (b) of the method of the invention.

[0034] The target RNA, i.e. the RNA prepared in the RNA preparation used in the method of the present invention, can be selected from the group consisting of messenger RNA (mRNA), self-amplifying RNA (saRNA) and circular RNA (circRNA), with mRNA being particularly preferred.

[0035] As used herein, the term "comprising" expressly includes the terms "consisting essentially of" and "consisting only of," i.e., all of these terms can be used interchangeably herein.

[0036] Furthermore, as used herein, the term "about" preferably refers to a modifier of ±10%, more preferably ±8%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%, or ±0.5% of the specified value. Thus, by way of example, the term "about 100" can include ranges of 90-110, 92-108, 94-106, 95-105, 96-104, 97-103, 98-102, 99-101, or 99.5-100.5.

[0037] The present invention describes a method for removing double-stranded and / or multi-stranded nucleic acid impurities from RNA preparations. The method applies the use of pH adjustment to remove impurities from RNA-based therapeutics, such as mRNA, saRNA, or circRNA. The pH adjustment is combined with a purification step to remove single-stranded fragments generated upon denaturation of the double-stranded and / or multi-stranded starting material.

[0038] The formation of double-stranded and / or multi-stranded nucleic acid structures, as well as the stability of the structures, depend on the basic principle of nucleic acid base pairing. The formation is driven by standard hydrogen bond interactions between CG and AU base pairs (Figure 1). The formation of accurate base pairs, and therefore the formation of dsRNA, is driven by the formation of standard bond interactions, i.e., CG and AU. To form standard interactions, guanosine requires protonation of N1 (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).

[0039] In the pH range typically used for RNA production, purification, and storage (i.e., pH 5-9), all four nucleosides are therefore favorably protonated for base pairing, resulting in duplex formation. The phosphate backbone is deprotonated at pH>1, and thus RNA contains a negatively charged molecular surface (the phosphate backbone) at the working pH, a property that is used in many purification techniques.

[0040] In the pH range typically used for RNA production, purification, and storage (i.e., pH 5-9), all four nucleosides are therefore suitably protonated for base pairing with an affinity ligand, e.g., poly-deoxythymidinic acid (oligo dT), immobilized on a chromatographic support. Lowering the pH according to the invention disrupts the base pairing with the affinity ligand, thus releasing the target RNA molecule and simultaneously denaturing the multi-stranded and / or double-stranded interactions (RNA-RNA or RNA-DNA).

[0041] For short RNA duplexes (less than 20-mers), it has been demonstrated that protonation of hydrogen bond acceptors disrupts base pairing and leads to denaturation of the RNA duplex, measured as a decrease in the melting temperature of the RNA duplex with decreasing pH. As a more general point, it has previously been observed that low pH leads to denaturation (or at least destabilization) of nucleic acids due to protonation of GC base pairs and the resulting base pairing. Furthermore, protonation has been demonstrated to stabilize non-canonical interactions in DNA duplexes, such as AC and CC. Denaturation of double-stranded DNA has been shown by incubation in acidic conditions. However, this concept has not been demonstrated in the context of purification of biological products, where short RNA duplexes do not represent therapeutically relevant RNA such as mRNA, and dsDNA does not represent ssRNA contaminated with dsRNA, ssRNA, ssDNA or other double-stranded and / or multi-stranded nucleic acid impurities.

[0042] Thus, the present invention relates to denaturing double-stranded and / or multi-stranded nucleic acid impurities present in an RNA preparation to single-stranded form by low pH-driven strand separation, causing disruption of canonical base pairing. Double-stranded and / or multi-stranded nucleic acid impurities are dissociated from the RNA by incubation of the RNA preparation at ambient to slightly elevated temperatures in the pH range of pH 1 to pH 5, preferably pH 2 to pH 4. This pH range is sufficient to protonate the bases involved in base pairing, making it unable to support canonical base pairing. The phosphate backbone remains unprotonated, and thus the RNA retains an overall negative charge. Citrate, acetate, glycine, or other acidic buffers can be used. Additives that promote denaturation, such as chaotropes, 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 assist denaturation. Chelators, such as EDTA, which facilitate removal of structural divalent cations, can be used to facilitate double- and / or multi-stranded denaturation under acidic conditions. The temperatures required for denaturation at low pH are ambient to slightly elevated temperatures (e.g., 4° C. to 50° C., preferably ambient).

[0043] According to the invention, 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 species, and then the pH is raised to above pH 5 to pH 7. This increase in pH confers long-term stability to the RNA, but because the impurities are not reformed, or are not reformed completely, potential immune responses triggered by such impurities are not activated upon administration of the RNA in vivo.

[0044] Additionally, 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 species, then the pH is raised to pH 5-pH 7 (pH 5 if the low pH is below pH 5), and the preparation is purified to remove fragments resulting from double-stranded and / or multi-stranded dissociation. Alternatively, RNA containing the respective impurity can be incubated at low pH, then purified to remove fragments resulting from double-stranded and / or multi-stranded dissociation, with purification being performed in the pH range used for denaturation (e.g., pH 2-pH 4).

[0045] The present invention can utilize unusual pH ranges used for disrupting dsRNA and RNA-DNA double-stranded and / or multi-stranded structures, and binding of prepared RNA to chromatographic media (porous particles, nanofibers, membranes, monoliths) used for ion exchange (cation and anion exchange), size exclusion, reversed phase, hydrophobic interaction, or multimodal chromatographic supports. Additionally, molecular weight cut-off devices, such as tangential flow filtration devices, can be used.

[0046] In this context, anion exchange ligands can be used in unusual pH ranges. Due to the strong negative charge of 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 (e.g. DEAE), secondary amines (e.g. ethylenediamine), primary amines, or multimodal ligands containing at least one anion exchange modality, are positively charged in the pH range of pH 1 to pH 11 or pH 1 to pH 13 (weak and strong anion exchangers, respectively). Thus, RNA can bind to positively charged ligands in the low pH range of pH 1 to pH 2. This is not a common practice and is actually counterintuitive, since the use of anion exchange ligands in the art is in neutral to basic conditions and the lowest pH value reported for the use of anion exchangers for the purification of mRNA is pH 5. The present invention takes advantage of the charge complementarity of RNA and anion exchange ligands in the unconventional pH range of pH 1 to pH 5, specifically pH 2 to pH 4. The RNA fragments annealed to the target RNA forming dsRNA structures and the residual DNA fragments forming RNA-DNA structures are separated from the main ssRNA strand using a salt or pH gradient in a selected buffer (e.g., citrate, acetate, glycine, glycylglycine, malate, maleate, phosphate, succinate, or formate). In a further embodiment, any undissociated double-stranded and / or multi-stranded structures can bind to the anion exchange ligand differently than the ssRNA structures, thus providing an additional layer of purification control. Additives known in the art that promote denaturation, such as chaotropes, e.g., urea or guanidine, or chelating agents, e.g., EDTA or citrate, or organic solvents, e.g., acetonitrile or ethanol, can be used to promote double-stranded and / or multi-stranded denaturation in the mobile phase, thereby further enhancing the selectivity of the method. The separation temperature can be optimized to provide an optimal balance of chromatographic separation and double- and / or multi-stranded denaturation.

[0047] Alternatively, cation exchange ligands can be used to purify the 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, whereas cation exchange chromatography supports bind positively charged species.

[0048] Additionally, size exclusion chromatography can be used to separate single-stranded fragments resulting from pH denaturation of double-stranded and / or multi-stranded impurities from the desired RNA target molecule. Size exclusion chromatography can be performed in the pH range used for denaturation (e.g., pH 2-pH 4) or at neutral / slightly acidic pH (e.g., pH 5-pH 7, or pH 5 if the pH range used for denaturation is less than pH 5). Small fragments that formed double-stranded and / or multi-stranded species by hybridization with the parent RNA, and 3'-extended dsRNA species are separated from the target RNA by size.

[0049] Additionally, reversed-phase or hydrophobic interaction chromatography can be used to separate single-stranded fragments resulting from pH denaturation of double-stranded and / or multistranded impurities from the desired RNA target molecule based on the different hydrophobicity of the fragments compared to the target RNA. Reversed-phase and hydrophobic interaction chromatography can be performed in the pH range used for denaturation (e.g., pH 2-pH 4) or at neutral / slightly acidic pH (e.g., pH 5-pH 7, or pH 5 if the pH range used for denaturation is less than pH 5).

[0050] In all embodiments that use chromatographic purification, the chromatographic purification is not limited to a particular chromatographic medium, but can use any format, such as a monolith, membrane, filter, nanofiber, or porous particle, that contains an appropriately charged polymer that facilitates binding at the target pH range.

[0051] Additionally, tangential flow filtration operated in the pH range used for denaturation (e.g., pH 2-pH 4) or in an elevated pH range (e.g., pH 5-pH 7, or pH 5 if the pH range used for denaturation is below pH 5) can be used to separate fragments / impurities resulting from pH denaturation of double-stranded and / or multi-stranded nucleic acids from the target RNA molecules. [Brief description of the drawings]

[0052] [Figure 1] FIG. 1 shows (A) the chemical structure of dsRNA and (B) the protonation equilibrium of nucleosides, with boxes representing the protonation states achieved by low pH treatment. [Diagram 2] Figure 1: pH treatment of mRNA reduces dsRNA content without affecting mRNA stability. Re-neutralization to pH 6 does not cause reformation of dsRNA. Left: dot blot (J2 antibody). Right: agarose gel electrophoresis. [Diagram 3] A. Denaturation of dsRNA by low pH is temperature dependent. eGFP (995 nt) was incubated at different pH values ​​ranging from 2.9 to 4.1 for 30 min at 4°C, room temperature (RT) and 40°C. Dot blots with J2 antibody were performed to determine the amount of dsRNA. B. Denaturation of long dsRNA strands requires high temperature along with low pH for denaturation. Magi2 dsRNA (1000 bp full complement dsRNA) was incubated at pH 3 (sample 1) or pH 6 (sample 3) for 20 min at 50°C and then buffer exchanged to pH 3 (sample 2 and sample 4, respectively). Denaturation was monitored by A) PAGE and B) J2 dot blot. [Figure 4]Figure 1: Exposure of affinity purified mRNA to low pH for up to 60 min does not cause mRNA degradation. A. eGFP (995 nt) was incubated at pH 3 for i) 15 min, ii) 30 min or iii) 60 min, then the pH was raised to pH 6. A fragment analyzer (BioAnalyzer, Agilent) was used to determine the extent of fragmentation immediately after neutralization to pH 6 (i-iii) or after storage of the same samples at 4°C-8°C for 1 day (iv-vi). B. Agarose gel electrophoresis of samples i-vi. Lane 1: Riboruler HR, lane 2: pH 3, 15 min, lane 3: pH 3, 30 min, lane 4: pH 3, 60 min, lane 5-7: samples in lanes 2-4 analyzed after storage at 4°C-8°C for 1 day. [Diagram 5] Figure 1 shows that exposure of IVT mixtures containing mRNA to low pH causes a decrease in dsRNA content as measured by J2 dot blot: i) IVT mixtures containing mRNA were incubated at pH 3 for 30 min at T (25°C). ii) IVT mixtures containing mRNA were analyzed in IVT reaction buffer (pH 7.9). [Figure 6]Figure 1 shows an example of low pH treatment of mRNA combined with removal of dsRNA structures by chromatography. eGFP (995 nt) was incubated in 100 mM glycine at pH 3.0 for 25 min at ambient temperature. Afterwards, the pH was increased to pH 6 with 100 mM glycine pH 10. The sample was then loaded onto a CIM PrimaH column (a prototype multimodal column combining weak anion exchanger, aromaticity, and hydrogen bonding elements) at pH 5 and eluted with a pH gradient (pH 5 to pH 7.5) made with a 20 mM citrate, 20 mM Na phosphate, 10 mM EDTA buffer system. A: Elution chromatogram of eGFP mRNA incubated at pH 3 in CIM PrimaH. Red: 260 nm, blue: 280 nm, green: linear gradient, grey: pH trace, brown: conductivity. B: PAGE gel of starting material, CIM PrimaH elution fraction 1 and elution fraction 2, and in situ wash (high pH wash, CIP). C: J2 dot blot of eGFP(L) and CIM PrimaH elution fractions 1 and 2 and in situ wash (high pH wash, CIP).D: AGE of gels of starting material, CIM PrimaH elution fractions 1 and 2 and in situ wash (high pH wash, CIP). [Figure 7] Figure 1 shows an example of low pH treated mRNA for removal of dsRNA structures by cation exchange chromatography. eGFP (995 nt) was incubated in 100 mM glycine at pH 3.0 for 20 min at ambient temperature or directly prepared for cation exchange chromatography. Samples were diluted in mobile phase A, 50 mM glycine pH 3, loaded onto a CIM SO3 column at pH 3 and eluted with a pH gradient from pH 3 to pH 10. A: Elution chromatogram of eGFP mRNA with CIM SO3. Green: untreated mRNA, Red: pH 3 treated mRNA, B: polyacrylamide gel electrophoresis (PAGE) of starting material (pretreated at pH 3 or not), flow-through (FT) fraction and eluted fractions E1, E2 and CIP. C: J2 dot blot of the corresponding fractions. [Figure 8]Figure 1 shows that low pH treatment in combination with chromatographic separation results in the removal of residual DNA template. eGFP (995 nt) was incubated in 100 mM glycine at pH 3.0 for 25 min at ambient temperature or was not treated with low pH. Both samples were then purified with CIM PrimaH using a pH gradient. The main elution fraction was analyzed for the presence of residual DNA plasmid template by RNase treatment and AGE. Lane 1: GeneRuler, lane 2: 50 ng eGFP DNA template, lane 3: 30 ng eGFP DNA template, lane 4: 10 ng eGFP DNA template, lane 5: 5 ng eGFP DNA template, lane 6: 2 ng eGFP DNA template, lane 7: 1 ng eGFP DNA template, lane 8: oligo dT purified mRNA (starting material), lane 9: PrimaH elution fraction of pH 3 treated mRNA, lane 10: PrimaH elution fraction of untreated mRNA. [Figure 9] mRNA (eGFP, 995 nt) was incubated for 15 min at ambient temperature in different concentrations of glycine (5 mM to 200 mM, as indicated) at pH 3.0 or was not treated (lane "mRNA"). The resulting material was analyzed by J2 dot blot to detect the presence of dsRNA and agarose gel electrophoresis (2% gel) to assess the stability of mRNA under denaturing conditions. A minimum of 25 mM glycine pH 3 was required for dsRNA denaturation. [Figure 10] 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 concentrations above 250 mM prevented denaturation of dsRNA at low pH. This means that NaCl concentrations above 250 mM cannot be used with pH denaturation. [Figure 11]Figure 1 shows mRNA (eGFP, 995 nt) incubated in 75 mM glycine pH 3 for 20 min. The resulting product was loaded onto a CIM C4 HLD column and the column was washed with 10 mM glycine pH 3 at 1 mL / min. The mRNA was eluted with a step gradient into mobile phase B (10 mM citric acid pH 5.1). The resulting elution fractions were analyzed by J2 dot blot to confirm removal of dsRNA and to assess mRNA integrity by agarose gel electrophoresis (2% gel). i) HPLC chromatogram of CIM C4 HLD purification (number "1" represents elution fraction with retention time 4.1 min - 4.3 min). ii) J2 dot blot: 1: mRNA eGFP (untreated), 2: CIM C4 HLD elution fraction (retention time 4.1 min - 4.3 min). iii) AGE: 1: Riboruler HR, 2: mRNA eGFP (untreated), 3: CIM C4 HLD elution fraction. [Figure 12] Figure 1 shows an in vitro transcription (IVT) reaction mixture containing mRNA (Cas9, 4000 nt) incubated in 25 mM glycine at pH 3 for 20 min. The resulting product was loaded onto a CIM C4 HLD column and the column was washed with 10 mM glycine pH 3 at 1 mL / min. The mRNA was eluted with a step gradient into mobile phase B (10 mM citric acid pH 5.1). The resulting elution fractions were analyzed by J2 dot blot to confirm removal of dsRNA. i) HPLC chromatogram of CIM C4 HLD purified elution fractions, number 1 represents the elution fraction (retention time 4.1 min - 4.4 min). ii) J2 dot blot: 1: IVT mRNA Cas9 (untreated), 2: CIM C4 HLD flow-through (FT) fraction, 3: CIM C4 HLD elution fraction (retention time 4.1 min - 4.4 min). [Figure 13]mRNA (eGFP, 995 nt) was diluted in mobile phase A (50 mM Na phosphate, 0.5 M NaCl, pH 7.5) and loaded onto a CIM oligo dT column (1 mL) at 1 mL / min. The column was washed with 10 mL of mobile phase B (50 mM Na phosphate, pH 7.5) and then eluted with a step gradient to mobile phase C (100 mM glycine, pH 3). Elution fractions were analyzed by J2 dot blot to confirm dsRNA denaturation. Elution fractions 1 and 2 (retention time 22.8 min-24.2 min) showed partial denaturation, and elution fraction 3 (retention time 24.2 min-26 min) showed complete denaturation. The stability of elution fraction 3 was confirmed by agarose gel electrophoresis. i) HPL chromatogram of CIM oligo dT purification. Numbers represent elution fractions (1: retention time 22.8 min - 23.8 min, 2: retention time 23.8 min - 24.2 min, 3: retention time 24.2 min - 26 min). ii) J2 dot blot: 1 - mRNA eGFP (untreated), 2: CIM oligo dT elution fraction 1, 3: CIM oligo dT elution fraction 2, 4: CIM oligo dT elution fraction 3. iii) AGE: 1: Riboruler HR, 2: mRNA eGFP (untreated), 3: CIM oligo dT elution fraction 3. [Figure 14]mRNA (eGFP, 995 nt) was diluted in mobile phase A (50 mM Na phosphate, 0.5 M NaCl, pH 7.5) and loaded onto a Poros Oligo(dT)25 column (1 mL) at 1 mL / min. The column was washed with 9 mL of mobile phase B (50 mM Na phosphate, pH 7.5) and then eluted with a step gradient to mobile phase C (100 mM glycine, pH 3). Elution fractions were analyzed by J2 dot blot to confirm denaturation of dsRNA. Partial denaturation was shown in elution fraction 1 (18.4-18.9) and complete denaturation was shown in elution fraction 2 (retention time 18.9 min-20.3 min). 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: retention time 18.4 min to 18.9 min, 2: retention time 18.9 min to 20.3 min. ii) J2 dot blot: 1-mRNA eGFP (untreated), 2-Poros oligo(dT)25 elution fraction 1, 3-Poros oligo(dT)25 elution fraction 2. iii) AGE: 1:mRNA eGFP (untreated), 2:Poros oligo(dT)25 elution fraction 1, 3-Poros oligo(dT)25 elution fraction 2. [Figure 15]FIG. 1 shows mRNA (eGFP, 995 nt) was diluted with mobile phase A (50 mM Na phosphate, 0.5 M NaCl, pH 7.5) and loaded onto a Poros oligo dT column (1 mL) at 1 mL / min. The column was washed with 9 mL of mobile phase B (50 mM Na phosphate, pH 7.5) and then eluted with a step gradient to mobile phase C (100 mM glycine, pH 3). The resulting pH 3 eluate was then diluted with mobile phase A (10 mM glycine, pH 3) and loaded onto a CIM C4 HLD column at 1 mL / min. The mRNA was eluted with a step gradient to mobile phase B (50 mM citrate, pH 5.1). The resulting elution fractions were analyzed by J2 dot blot to confirm removal of dsRNA. A) HPLC chromatogram of Poros oligo(dT)25 purification. Number 1 represents the elution fraction with retention time 18.4-18.9 min, number 2 represents the elution fraction with retention time 18.9-20.3 min. B) HPLC chromatogram of CIM C4 HLD purification of oligo dT eluate. Number 1 represents the elution fraction with retention time 7.9-8.3 min. C) J2 dot blot: 1: mRNA eGFP (untreated), 2: CIM C4HLD elution fraction 1. [Figure 16] RNA (eGFP, 995 nt) incubated in 25 mM glycine at pH 3 for 15 min. The resulting product was diluted with mobile phase A (50 mM citric acid, pH 5) and loaded onto a CIM PrimaH column at 1 mL / min. mRNA was eluted with a linear gradient to mobile phase B (100 mM phosphate, pH 7.5). The resulting elution fractions were analyzed by J2 dot blot to confirm removal of dsRNA. i) HPLC chromatogram of CIM PrimaH separation of low pH denatured mRNA. Number 1 represents the elution fraction with retention time 9.6-9.9 min, number 2 represents the elution fraction with retention time 9.9-10.5 min. ii) J2 dot blot: 1-mRNA eGFP (untreated), 2: CIM PrimaH elution fraction 1, 3: CIM PrimaH elution fraction 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] The present invention is further illustrated by the following non-limiting examples. EXAMPLES

[0054] Example 1: mRNA encoding eGFP (enhanced green fluorescent protein, 995 nucleotides) was generated in an IVT (in vitro transcription) reaction and purified by oligo dT affinity chromatography, which does not remove dsRNA (double-stranded RNA). The presence of dsRNA impurities in the samples was confirmed by dot blot analysis using the J2 antibody, which is specific for dsRNA sequences of at least 40 base pairs.

[0055] The pH range required for denaturation of dsRNA was tested by incubation of mRNA in a wide pH range (2.9-4.1, glycine buffer was used at the indicated pH values) at room temperature. Samples were incubated for 30 min at room temperature before loading into AGE (agarose gel electrophoresis, 100 ng) or dot blot assays (800 ng). Incubation of mRNA at pH 2.9-4.1 did not reduce the intensity of the main mRNA AGE band, but the intensity of the dot blot signal decreased with decreasing pH. The signal was strongest at pH 6, and a strong signal was still observed at pH 3.9-4.1. The signal was significantly reduced at pH 3.5 and was not visible below pH 3.2, suggesting that the dsRNA structure was dissociated but the mRNA was intact.

[0056] The mRNA was then incubated at pH 3 for 20 min and then increased to pH 6. AGE results showed the appearance of a low molecular weight band upon increasing the pH to 6, but the dot blot showed no signal, suggesting that the dsRNA structure was not reformed (Figure 2).

[0057] The temperature dependence of dsRNA denaturation was demonstrated by incubating eGFP (995 nt) at 4 °C, room temperature (RT), and 40 °C for 30 min at different pH values ​​ranging from 2.9 to 4.1. Dot blots with J2 antibody were performed to determine the amount of dsRNA and demonstrate the temperature- and pH-dependent decrease of dsRNA signal. At 4 °C, dsRNA signal was still detected at pH 3.2, whereas at 40 °C, dsRNA signal was only detected at pH 3.9 and above (Figure 3).

[0058] To demonstrate that pH rather than temperature is necessary for duplex denaturation, the perfectly complementary dsRNA sequence (1000 base pairs) Magi2 was incubated at 50°C for 20 min at either pH 3 or pH 6. At pH 6, high temperature (50°C) was not sufficient to denature the duplex as shown by the J2 dot blot (Figure 3B), whereas at pH 3 the dsRNA signal disappeared. Buffer exchange to pH 3 eliminated the dsRNA signal (Figure 3B, sample 4).

[0059] 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 (Figures 4A and 4B, respectively). No significant degradation was observed by either method, indicating that exposure of mRNA to low pH for the duration required for denaturation of dsRNA does not cause mRNA degradation.

[0060] To demonstrate the generality of the approach, we next incubated unpurified IVT reaction mixtures containing eGFP mRNA at pH 3 or without treatment, and the dsRNA signal was measured by J2 dot blot in the absence of RNA purification. The dot blot demonstrated disappearance of the dsRNA signal at pH 3 (Figure 5i), but not in the absence of pH treatment (Figure 5ii).

[0061] To demonstrate purification of low-pH-treated mRNA, mRNA encoding eGFP was incubated in 100 mM glycine at pH 3.0 for 20 min at ambient temperature. The pH of the sample was then raised to pH 6 with glycine buffer (pH 10), and the sample was then loaded onto a prototype multimodal column (PrimaH) combining weak anion exchanger, aromaticity, and hydrogen bonding elements at pH 5 and eluted with a pH gradient (pH 5 to pH 7.5) created with a 20 mM citric acid, 20 mM Na phosphate, 10 mM EDTA buffer system (Figure 6A). Two elution fractions (1 and 2) were analyzed by dot blot using the J2 antibody.

[0062] The results demonstrated strong binding of mRNA to PrimaH when incubated at pH 3 (Figure 6). pH treatment of the mRNA resulted in a significant decrease in dsRNA content in the eluted fractions as demonstrated by J2 dot blots, fraction 1 and fraction 2 (Figure 6).

[0063] To demonstrate purification of low pH treated mRNA by cation exchanger, mRNA encoding eGFP was incubated in 100 mM glycine at pH 3.0 for 20 min at ambient temperature or left untreated. Both samples were diluted separately in mobile phase A (MPA, 50 mM glycine pH 3), loaded onto a Convective Interaction Media (CIM) SO3 column and eluted with a pH gradient from 3 to 10 (Figure 7A). The starting material (mRNA treated or untreated at low pH), the flow-through fraction and the two elution fractions (1 and 2) were analyzed by PAGE and dot blot with J2 antibody (Figure 7B, Figure 7C). The results demonstrated that both pretreatment at pH 3 or dilution of the sample with MPA (50 mM glycine pH 3) resulted in denaturation of dsRNA, as demonstrated by a decrease in the dot blot signal. ssRNA elutes in the flow-through and the dot blot shows the absence of dsRNA (Figure 7C).

[0064] To demonstrate that low pH treatment in combination with chromatographic separation results in removal of residual DNA template, eGFP (995 nt) was incubated for 25 min at ambient temperature in 100 mM glycine at pH 3.0 before neutralization, and a control was not treated with low pH. Both samples were then purified with CIM PrimaH using a pH gradient as described above. The main elution fraction was analyzed for the presence of residual DNA plasmid template by treating 10 μg of mRNA with ribonuclease A (to enzymatically digest mRNA) and loading the product of ribonuclease A digestion of 10 μg of mRNA onto AGE. Ribonuclease A selectively digests RNA but not DNA, comparing the residual DNA signal to a standard dilution (range 50 ng to 1 ng) of the parent linearized plasmid. The PrimaH elution of pH 3 treated mRNA was analyzed by AGE using our method, and no signal for residual plasmid was detected. PrimaH elution of pH-untreated mRNA showed the presence of 2 ng to 5 ng of DNA plasmid per 10 µg of mRNA (Figure 8).

[0065] Example 2: mRNA encoding eGFP (enhanced green fluorescent protein, 995 nucleotides) was generated in an IVT (in vitro transcription) reaction and purified by oligo dT affinity chromatography, which does not remove dsRNA (double-stranded RNA). The presence of dsRNA impurities in the samples was confirmed by dot blot analysis using the J2 antibody, which is specific for dsRNA sequences of at least 40 base pairs.

[0066] The buffer molarity at pH 3 required for denaturation of dsRNA was tested by incubation of mRNA in a wide range of buffer concentrations (5 mM to 200 mM glycine buffer at pH 3) at room temperature. Samples were incubated for 15 min at room temperature before loading on AGE (2% agarose gel electrophoresis, 500 ng) or dot blot assay (1000 ng). Incubation of mRNA in a range of concentrations from 5 mM to 200 mM glycine buffer pH 3 resulted in a decrease in dot blot intensity. The J2 signal was strongest in the untreated sample (untreated control), and a strong signal was still observed in the sample incubated with 5 mM buffer at pH 3. The signal was significantly reduced in 10 mM buffer and was not visible in buffers above 25 mM, suggesting that the dsRNA structure was dissociated, whereas the agarose gel showed that the mRNA was intact in all concentrations of glycine buffer tested (Figure 9).

[0067] Example 3: The salt dependence of dsRNA denaturation was demonstrated by incubating eGFP (995 nt) in 25 mM glycine pH 3 in the presence of different concentrations of NaCl ranging from 250 mM to 2 M for 15 min at room temperature (RT). Dot blots with J2 antibody were performed to determine the amount of dsRNA signal, which was not detectable in the presence of 50 mM NaCl, but at salt concentrations of at least 250 mM, the J2 dot blot signal increased, suggesting that NaCl prevents dsRNA denaturation at pH 3 (Figure 10).

[0068] Example 4: To demonstrate purification of low pH treated mRNA by hydrophobic interaction chromatography, mRNA encoding eGFP was incubated in 75 mM glycine at pH 3.0 for 20 min at ambient temperature. The sample was loaded onto a CIM C4 HLD column and eluted with a step gradient to 10 mM citric acid pH 5.1 (Figure 11, i). The starting material (untreated mRNA) and eluted fractions were analyzed by AGE and dot blot with J2 antibody (Figure 11, iii). The results demonstrated that purification by hydrophobic chromatography column following low pH incubation resulted in denaturation of dsRNA and purification of ssRNA, as demonstrated by the reduction of dot blot signal in the eluted fractions (Figure 11, ii).

[0069] Example 5: To demonstrate the generality of the methodology, crude IVT reaction mixtures containing Cas9 mRNA (4000nt) were incubated in 25 mM glycine pH 3 for 20 min at ambient temperature. The samples were loaded onto a CIM C4 HLD column and eluted with a step gradient to 10 mM citric acid pH 5.1 (Figure 12, i). The dsRNA signal in the resulting elution fractions was measured by J2 dot blot, demonstrating a significant decrease in dsRNA signal 3 (Figure 12, ii), confirming that the dsRNA was denatured and the ssRNA was purified.

[0070] Example 6: The possibility of mRNA denaturation by low pH treatment on the column was demonstrated by affinity chromatography. mRNA encoding eGFP was diluted in mobile phase A (50 mM Na phosphate, 0.5 M NaCl, pH 7.5), loaded onto a Convective Interaction Media (CIM) oligo dT column and then washed with mobile phase B (50 mM Na phosphate pH 7.5). The mRNA was eluted with a step gradient to 100 mM glycine pH 3 (Figure 13, i). The starting material (mRNA) and the eluted fractions were analyzed by AGE and dot blot with J2 antibody (Figure 13, ii and iii). No significant degradation was observed with AGE, indicating that exposure of the mRNA to low pH during the purification process does not affect the RNA integrity (Figure 13, iii). Elution of the mRNA by oligo dT at pH 3 resulted in denaturation of the dsRNA, confirmed by a decrease in the dot blot signal in the eluted fractions (Figure 13, ii).

[0071] Example 7: To demonstrate the generality of the method, mRNA encoding eGFP was diluted in mobile phase A (50 mM Na phosphate, 0.5 M NaCl, pH 7.5), loaded onto a Poros oligo(dT)25 column, and then washed with mobile phase B (50 mM Na phosphate, pH 7.5). The mRNA was eluted with a step gradient to 100 mM glycine pH 3 (Figure 14, i). The starting material (mRNA) and the eluted fractions were analyzed by AGE and dot blot with J2 antibody (Figure 14, ii and iii). No significant degradation was observed, indicating that exposure of the mRNA to low pH during the purification process does not affect the RNA integrity (Figure 14, iii). Elution of the mRNA with oligo dT at pH 3 resulted in denaturation of the dsRNA, confirmed by a decrease in the dot blot signal in the eluted fractions (Figure 14, ii).

[0072] Example 8: To remove dsRNA fragments resulting from denaturation at low pH of the elution from the oligo dT column, chromatographic separation of the oligo dT eluate by CIM C4 HLD was performed. The mRNA coding for eGFP (995 nt) was diluted in mobile phase A (50 mM Na phosphate, 0.5 M NaCl, pH 7.5), loaded onto a Poros oligo(dT)25 column and then washed with mobile phase B (50 mM Na phosphate, pH 7.5) (Figure 15, A). The resulting pH 3 eluate was then diluted with 10 mM glycine pH 3 and loaded onto a CIM C4 HLD column. The mRNA elution was performed by a step gradient to 50 mM citrate at pH 5.1 (Figure 15, B). The starting material (mRNA) and the C4 HLD elution fractions were analyzed by dot blot with the J2 antibody (Figure 15, C). Dot blots demonstrated a significant reduction in dsRNA signal in the C4 HLD elution, suggesting that the dsRNA structure was dissociated and removed, while the mRNA was intact.

[0073] Example 9: Purification of ssRNA from low-pH treated mRNA samples was further demonstrated by low-pH treatment combined with multimodal chromatography, where eGFP (995 nt) was incubated in 25 mM glycine at pH 3.0 for 15 min at ambient temperature. The resulting product was diluted with mobile phase A (50 mM citric acid, pH 5) and purified with CIM PrimaH using a pH gradient (pH 5 to pH 7.5) made with 50 mM citrate and 100 mM Na phosphate buffer system (Figure 16, i). The resulting elution fractions were analyzed by dot blot with J2 antibody, showing a significant reduction in dsRNA content in elution fraction 1 (Figure 16, ii).

Claims

1. A method for removing double-stranded and / or multi-stranded nucleic acid impurities from an RNA preparation, (a) A step of incubating the RNA preparation at a pH in the range of pH 1 to pH 5, (b) A step of purifying the RNA preparation to remove fragments resulting from the dissociation of the double-stranded and / or multi-stranded nucleic acid impurities, Methods that include...

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

3. The method according to claim 1 or 2, wherein step (a) is carried out in a buffer containing a denaturing agent and / or a chelating agent.

4. The method according to claim 1 or 2, wherein step (a) is performed at a temperature of 4°C to 50°C.

5. The method according to claim 1 or 2, wherein step (a) is performed as part of an affinity chromatography step, the RNA preparation is loaded onto an affinity chromatography medium at a pH in the range of pH 6 to pH 8, and then the pH is reduced to a pH in the range of pH 1 to pH 5.

6. (i) The RNA preparation is subjected to the purification at the pH used in step (a), or (ii) If step (a) has not already been performed at pH 5, the pH of the RNA preparation is raised to a pH in the range of pH to pH 5 that is higher than the pH used in step (a) before purification, or (iii) The pH of the RNA preparation is raised to a pH in the range of pH greater than 5 to pH 7 before the purification. The method according to claim 1 or 2.

7. The method according to claim 1 or 2, wherein the purification is carried out by a technique selected from the group consisting of cation exchange chromatography, anion exchange chromatography, size exclusion chromatography, reversed-phase chromatography, hydrophobic interaction chromatography, multimodal chromatography, affinity chromatography, IMAC (immobilized metal affinity chromatography), molecular weight cutoff filtration, and precipitation / extraction techniques.

8. (i) The purification is carried out by a technique selected from the group consisting of cation exchange chromatography, anion exchange chromatography, size exclusion chromatography, reversed-phase chromatography, hydrophobic interaction chromatography, multimodal chromatography, affinity chromatography, and IMAC (immobilized metal affinity chromatography). (ii) Steps (a) and (b) are performed simultaneously while the RNA preparation is bound to the chromatography medium. The method according to claim 1 or 2.

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

10. The method according to claim 9, wherein the anion exchange ligand is diethylaminoethyl (DEAE) or dimethylaminoethyl (DMAA).

11. The method according to claim 7, wherein the technique is a cation exchange chromatography performed at a pH in the range of pH 1 to pH 5 using a cation exchange ligand selected from the group consisting of 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 according to claim 7, wherein the technique is multimodal chromatography performed at a pH in the range of pH 1 to pH 5 using a multimodal ligand comprising a plurality of types of chemical residues selected from the group consisting of affinity residues, hydrophobic interaction residues, ion exchange residues, hydrogen bonding residues, metal chelate residues, aromatic residues, and combinations thereof.

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

14. The method according to claim 1 or 2, wherein the RNA prepared in the RNA preparation is selected from the group consisting of messenger RNA (mRNA), self-amplified RNA (saRNA), and circular RNA (circRNA).

15. The method according to claim 1 or 2, wherein the double-stranded and / or multi-stranded nucleic acid impurity is a non-single-stranded nucleic acid structure selected from the group consisting of double-stranded RNA (dsRNA), double-stranded DNA (dsDNA), RNA-DNA heteroduplex structures, and multi-stranded homo or heteromeric nucleic acid structures.

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