Method for purifying single-stranded RNA

Abstract

Disclosed is a method for purifying single-stranded RNA (ssRNA). More specifically disclosed is a method of purifying an ssRNA-containing sample containing double-stranded RNA (dsRNA) as an impurity using two different chromatography steps. The method includes steps of: (a) subjecting a sample containing ssRNA and containing double-stranded RNA (dsRNA) as an impurity to primary purification using affinity chromatography; and (b) subjecting the sample to secondary purification using at least one chromatography selected from the group consisting of size-exclusion chromatography (SEC), anion-exchange chromatography (AEX), and hydrophobic interaction chromatography (HIC). The method has a high mRNA recovery and a dsRNA removal rate of 95% or more, and is useful because it can minimize or appropriately control the dsRNA content in an mRNA sample, so that the mRNA can be developed into a drug regardless of the type of sequence, and can also maximize patient safety and drug efficacy.

Description

METHOD FOR PURIFYING SINGLE-STRANDED RNA

[0001] The present invention relates to a method for purifying single-stranded RNA (ssRNA), and more specifically, to a method for purifying an ssRNA-containing sample containing double-stranded RNA (dsRNA) as an impurity using two different chromatography steps.

[0002]

[0003] It has been reported that, during synthesis of mRNA byin vitrotranscription (IVT) using T7 RNA polymerase (Yin et al., Cell, Vol. 116, pp. 393-404, 2004), significant amounts of aberrant products, including double-stranded RNA (dsRNA), are produced due to unconventional activity of the enzyme (Gong et al., JBC, Vol. 281, pp.23533-23544, 2006). It is known that, when dsRNA enters the human body, it activates antiviral and inflammatory signaling pathways (TLR3-, RIG-I-, and MDA-5-mediated), resulting in multiple immune responses and toxic responses, including cell growth inhibition and apoptosis (Poynter SJ. et al., Front Immunol. Vol. 9(829), 2018; Wu MZ. et al., RNA, Vol. 26(3), pp. 345-360, 2020).

[0004] Representative pattern recognition receptors (PRRs) that recognize dsRNA include TLR3, RIG-I, and MDA-5. TLR3 is localized in the endosome and recognizes dsRNA of 40-50 bp in length, RIG-I is localized in the cytosol and recognizes dsRNA of 500 bp or less in length, and MDA-5 is one of the RIG-I-like receptors (RLR) that recognizes long dsRNA of 2,000 bp or more in length. When dsRNA is recognized by TLR3, RIG-I, and MDA-5, type I interferons (IFNs, IFN-σ and IFN-β) and proinflammatory cytokines are secreted. At this time, chemokines, interferon gamma-induced protein 10 (IP-10, CXCL10) and monocyte chemoattractant protein 1 (MCP-1, CCL2), secreted under the influence of type I IFNs, are known to contribute to the recruitment of immune cells such as T cells, monocytes, and dendritic cells (Crowl JT. et al., Annu Rev Immunol. Vol. 35, pp. 313-336, 2017; Dousis, A. et al., Nat Biotechnol, Vol. 41, pp. 560-568, 2023; Verbeke R. et al., Immunity, Vol. 55(11), pp. 1993-2005, 2022; Chen, Y.G. et al., Nat Rev Mol Cell Biol, Vol. 23, pp. 286-301, 2022).

[0005] Therefore, it is very important to remove dsRNA from IVT mRNA to be used as a therapeutic agent.

[0006] Various methods for removing dsRNA from mRNA preparations are known. For example, dsRNA can be removed by affinity chromatography on cellulose-based chromatography media (Baiersdorfer M, et al., Mol. Ther. Nucleic Acids. Vol.15(15), pp.26-35, 2019; Urayama S, et al., Microbes Environ. Vol. 30(2), pp.199-203; Korean Patent No. 10-2565881). This mechanism of adsorption has not been defined but dsRNA binds under certain conditions while ssRNA flows through. The method is effective at laboratory scale but it is burdened by low capacity. Low capacity corresponds to large column volumes at manufacturing scale, which require large buffer volumes, large manufacturing areas, and extended process time that depresses productivity of the manufacturing facility. The method also causes dilution of the processed ssRNA, corresponding to an increase in product volume that complicates follow-on purification steps.

[0007] dsRNA may also be removed by ion-pair reverse phase chromatography (RPC) using a styrene-divinyl-benzene (SDVB) solid phase (Nwokeoji AO, et al., J. Chromatogr B Analyt Technol Biomed Life Sci. Vol.1104, pp.212-219, 2019). RPC employs toxic flammable organic solvents that require extremely expensive specialized equipment at industrial scale to mitigate the risk of fire and explosion. RPC also imposes the further burden of safety issues as they pertain to organic solvent toxicity in the work environment and to hazardous waste disposal issues. In addition to the solvent issues, RPC separations often bear the further burden of requiring elevated temperature to obtain the best results.

[0008] dsRNA may also be removed using size exclusion chromatography (Kim I, et al., RNA, Vol. 13(2), pp. 289-94, 2007). However, size exclusion chromatography has disadvantage that the resulting IVT product, ssRNA, is difficult to separate from dsRNA, which is similar in size to ssRNA, and requires high-performance columns and sophisticated conditions to achieve sufficient separation.

[0009] dsRNA may also be removed using anion-exchange chromatography (A Romanovskaya, et al., J. Chromatography A, Vol.1278, pp.54-60, 2013). However, anion exchange chromatography has shown limited utility for removal of DNA and protein contaminants from large mRNA (1,000-10,000 bases) and shown utility only at elevated operating temperatures. Elevating the temperature to 65°C enables elution of large mRNA in a sodium chloride gradient. However, high temperature operation imposes a compound logistical burden because the buffers, samples, and column should all be pre-equilibrated to and maintained precisely at the specified operating temperature for the entire duration of the process, and reproducibly across all batches for the manufacturing life of the product, potentially for years.

[0010] dsRNA may also be removed using hydrophobic interaction chromatography (Pete Gagnon et al., Cell and Gene therapy insights. Vol.6(7), pp.1035-1046, 2020). However, hydrophobic interaction chromatography has disadvantages in that the purity of the purified product is low and the purification time is long.

[0011] dsRNA may also be removed usingE. coliRNaseIII that specifically hydrolyzes dsRNA but not ssRNA (WO 2013 / 102 203 A1). However, RNaseIII can induce undesired reactions (such as an undesired immune reaction) in the patient to be treated with the RNA. Thus, before administering the RNA to the patient, it is necessary to remove the enzyme, which increases the complexity and cost of the method. Moreover, the use of RNaseIII often leads to a partial degradation of ssRNA, especially long ssRNA, during incubation.

[0012] Accordingly, the present inventors have made extensive efforts to solve the above-described problems and develop a purification method having a high recovery rate of ssRNA and an increased removal rate of dsRNA, and as a result, have found that, when primary purification is performed using affinity chromatography and then secondary purification is further performed using size exclusion chromatography, anion exchange chromatography or hydrophobic interaction chromatography, ssRNA from which 95% or more of dsRNA has been removed while maintaining a yield of about 80% or more can be obtained, thereby completing the present invention.

[0013]

[0014] SUMMARY OF THE INVENTION

[0015] An object of the present invention is to provide a method of purifying single-stranded RNA (ssRNA) by specifically removing dsRNA as an impurity.

[0016] To achieve the above object, the present invention provides a method for purifying single-stranded RNA (ssRNA), including steps of: (a) subjecting a sample containing ssRNA and containing double-stranded RNA (dsRNA) as an impurity to primary purification using affinity chromatography; and (b) subjecting the sample to secondary purification using at least one chromatography selected from the group consisting of size-exclusion chromatography (SEC), anion-exchange chromatography (AEX), and hydrophobic interaction chromatography (HIC).

[0017]

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 is a schematic diagram showing an ssRNA purification process according to the present invention.

[0020] FIG. 2 shows the results of Superdex 200 PG resin-based size-exclusion chromatography performed according to one example of the present invention.

[0021] FIG. 3 shows the results of Superose 6 PG resin-based size-exclusion chromatography performed according to one example of the present invention.

[0022] FIG. 4 shows the results of Sepharose 6 FF resin-based size-exclusion chromatography performed according to one example of the present invention.

[0023] FIG. 5 shows the results of Sephacryl S-300 HR resin-based size-exclusion chromatography performed according to one example of the present invention.

[0024] FIG. 6 shows the results of comparison between Superose 6 PG resin-based size-exclusion chromatography and Sepharose 6 FF resin-based size-exclusion chromatography performed under the same conditions according to one example of the present invention.

[0025] FIG. 7 shows the results of stepwise elution in C4-HLD resin-based hydrophobic interaction chromatography performed according to one example of the present invention.

[0026] FIG. 8 shows the results of gradient elution of Benzyl Ultra resin-based hydrophobic interaction chromatography performed according to one example of the present invention.

[0027] FIG. 9 shows the results of gradient elution in Capto Phenyl (High Sub) resin-based hydrophobic interaction chromatography performed according to one example of the present invention.

[0028] FIG. 10 is a schematic diagram showing an ssRNA purification process performed according to one example of the present invention.

[0029] FIG. 11 shows the results of measuring the level of IFN-σ, a factor related to the innate immune response of mice, depending on the concentration of dsRNA by ELISA according to one example of the present invention.

[0030] FIG. 12 shows the results of measuring factors related to the innate immune response of mice depending on the concentration of dsRNA concentration according to one example of the present invention. Specifically, (A), (B), (C), and (D) show the results of measuring the levels of IFN-α, IFN-β, MCP-1, and IP-10, respectively, by flow cytometry.

[0031] FIG. 13 shows the results of measuring the level of IFN-β, a factor related to the innate immune response of mice, depending on the concentration of dsRNA by ELISA according to one example of the present invention.

[0032] FIG. 14 shows the results of measuring factors related to the innate immune response of mice depending on dsRNA concentration according to one example of the present invention. Specifically, (A), (B), (C), and (D) show the results of measuring the levels of IFN-α, IFN-β, MCP-1, and IP-10, respectively.

[0033] FIG. 15 shows the results of evaluating thein vivotoxicity of dsRNA in mice depending on the concentration of dsRNA according to one example of the present invention. Specifically, (A) and (B) show the results of measuring the secretion levels of ALT and AST, respectively.

[0034]

[0035] DETAILED DESCRIPTION OF THE INVENTION

[0036] Unless otherwise defined, all technical and scientific terms used in the present specification have the same meanings as commonly understood by those skilled in the art to which the present disclosure pertains. In general, the nomenclature used in the present specification and the experimental methods described below are well known and commonly used in the art.

[0037] In the present invention, it has been found that, when a product obtained through IVT is subjected to primary purification by affinity chromatography and then subjected to secondary purification using the differences in size, charge, and hydrophobicity that distinguish the polyA tail-containing dsRNA, which is an impurity, from ssRNA, the removal rate of the dsRNA is increased.

[0038] That is, in one example of the present invention, it was confirmed that, when an mRNA product obtained through IVT was subjected to primary purification using oligo-dT-based affinity chromatography and then subjected to secondary purification using size-exclusion chromatography, anion exchange chromatography, or hydrophobic interaction chromatography, the removal rate of dsRNA was increased while a high recovery of ssRNA was maintained (FIG. 1).

[0039] Therefore, in one aspect, the present invention is directed to a method for purifying single-stranded RNA (ssRNA), including steps of:

[0040] (a) subjecting a sample containing ssRNA and containing double-stranded RNA (dsRNA) as an impurity to primary purification using affinity chromatography; and

[0041] (b) subjecting the sample to secondary purification using at least one chromatography selected from the group consisting of size-exclusion chromatography (SEC), anion-exchange chromatography (AEX), and hydrophobic interaction chromatography (HIC).

[0042] In the present invention, the RNA may be, without limitation, any type of RNA, and is preferably mRNA, without being limited thereto.

[0043] In the present invention, the sample may be obtained throughin vitrotranscription (IVT), without being limited thereto.

[0044] In the present invention, the dsRNA may be any double-stranded RNA produced as a result of IVT, and is preferably a dsRNA containing a poly(A) tail, without being limited thereto.

[0045] In the present invention, step (a) of subjecting the sample to primary purification using affinity chromatography may include steps of:

[0046] (a-i) mixing the sample containing ssRNA with a sample preparation buffer and loading the mixture into a column containing a resin that complementarily binds to the poly(A) tail;

[0047] (a-ii) washing the column using a wash buffer; and

[0048] (a-iii) recovering bound ssRNA by elution with an elution buffer.

[0049] In the present invention, the resin that complementarily binds to the poly(A) tail may be a polythymidine resin (oligo dT resin), without being limited thereto.

[0050] In the present invention, the sample preparation buffer may be a solution (pH 6.0 to 7.0) containing: 10 to 150 mM, preferably 10 to 100 mM sodium phosphate, tris(hydroxymethyl)aminomethane, tris), sodium citrate, or sodium acetate; 1 to 20 mM, preferably 1 to 10 mM EDTA (ethylene-diamine-tetraacetic acid); and 100 to 1000 mM, preferably 100 to 900 mM, more preferably 100 to 800 mM sodium chloride. More preferably, the sample preparation buffer may be a solution (pH 6.3 to 6.8) containing: 20 to 100 mM sodium phosphate; 5 to 10 mM EDTA; and 250 to 700 mM sodium chloride. Most preferably, the sample preparation buffer may be a solution (pH 6.5) containing: 100 mM sodium phosphate; 10 mM EDTA; and 700 mM sodium chloride or a solution (pH 6.6) containing: 20 mM sodium phosphate; 10 mM EDTA; and 500 mM sodium chloride or a solution (pH 6.5) containing: 20 mM sodium phosphate; 10 mM EDTA; and 700 mM sodium chloride.

[0051] In the present invention, the term “sample preparation buffer” refers to a buffer that adjusts the ionic strength, etc. of the sample so that the sample may be loaded onto a column containing a chromatography resin, and the term is used with the same meaning as an n-fold concentrated equilibration buffer. For example, a 2X equilibration buffer is a buffer prepared by adding twice as many reagents as the equilibration buffer, and may have the same meaning as the sample preparation buffer, and in the case of buffers containing salts, it will be apparent to those skilled in the art that the pH may vary as the concentration of the salt varies.

[0052] In the present invention, step (a) may further include, before step (a-ii), a step of additionally loading an equilibration buffer.

[0053] In the present invention, the equilibration buffer may be a solution (pH 6.0 to 7.0) containing: 10 to 100 mM sodium phosphate, tris(hydroxymethyl)aminomethane, tris), sodium citrate, or sodium acetate; 1 to 10 mM EDTA (ethylene-diamine-tetraacetic acid); and 100 to 500 mM sodium chloride. More preferably, the equilibration buffer may be a solution (pH 6.5 to 7.0) containing 20 to 80 mM sodium phosphate; 1 to 8 mM EDTA; and 100 to 400 mM sodium chloride. Most preferably, the equilibration buffer may be a solution (pH 6.7) containing: 50 mM sodium phosphate; 5 mM EDTA; and 350 mM sodium chloride or a solution (pH 6.8) containing: 10 mM sodium phosphate; 5 mM EDTA; and 250 mM sodium chloride or a solution (pH 6.8) containing: 10 mM sodium phosphate; 5 mM EDTA; and 350 mM sodium chloride.

[0054] In the present invention, the wash buffer may be a solution (pH 6.0 to 7.0) containing: 10 to 100 mM sodium phosphate, Tris, sodium citrate or sodium acetate; and 1 to 10 mM EDTA. More preferably, the wash buffer may be a solution (pH 6.7) containing 50 mM sodium phosphate and 5 mM EDTA.

[0055] In the present invention, the elution buffer may be a solution (pH 6.0 to 7.0) containing 0.01 to 10 mM sodium phosphate, Tris, sodium citrate or sodium acetate. More preferably, the elution buffer may be a solution (pH 7.0) containing 5 mM Tris or a solution (pH 6.0 to 6.5) containing 5mM sodium citrate.

[0056] In the present invention, it is obvious that, if the pH of the buffers and the concentrations of the components are lower or higher than the values described above, the chromatography performance is reduced and thus a purified product cannot be obtained at a high recovery rate.

[0057] In the present invention, step (b) of subjecting the sample to secondary purification using size-exclusion chromatography may include steps of:

[0058] (b-1-i) mixing the primarily purified sample, eluted in step (a), with a 2X equilibration buffer, and loading the mixture into a column containing a size exclusion resin;

[0059] (b-1-ii) eluting bound RNA using the equilibration buffer; and

[0060] (b-1-iii) obtaining the last eluted RNA as a fraction containing ssRNA.

[0061] In the present invention, the size-exclusion chromatography is also known as molecular sieve chromatography, and is a chromatography method that separates molecules according to size and molecular weight. In the present invention, size-exclusion chromatography may be performed using various size-exclusion chromatography resins known in the art or columns containing the same. For example, the size-exclusion chromatography resin may typically include a polymer with fine porous beads, and substances are separated by the pore size of the beads. In the present invention, the polymer may be, for example, dextran, agarose or polyacrylamide, preferably SuperdexTM 200, SuperoseTM 6, SepharoseTM 6, SepharcylTM S-300, most preferably SuperoseTM 6, without being limited thereto.

[0062] In the present invention, the size exclusion resin can separate molecules of 10 to 7,000 kDa, more preferably molecules of 1,000 to 6,000 kDa, most preferably molecules of 1,500 to 5,000 kDa, according to size.

[0063] In the present invention, the equilibration buffer may be, but is not limited to, a solution (pH 3.0 to 7.0) containing: 1 to 100 mM sodium citrate, sodium phosphate, Tris, or sodium acetate; and 0.01 to 1,000 mM sodium chloride, preferably a solution (pH 4.0 to 6.5) containing: 10 to 80 mM sodium citrate, sodium phosphate, Tris, or sodium acetate; and 0.1 to 700 mM sodium chloride, more preferably a solution (pH 4.5 to 5.5) containing: 30 to 70 mM sodium citrate, sodium phosphate, tris, or sodium acetate; and 100 to 500 mM sodium chloride, and most preferably a solution (pH 5.0) containing 50 mM sodium citrate and 200 mM sodium chloride.

[0064] In the present invention, it is obvious that, if the pH of the buffers and the concentrations of the components are lower or higher than the values described above, the chromatography performance is reduced and thus a purified product cannot be obtained at a high recovery rate.

[0065] In the present invention, step (b) of subjecting the sample to secondary purification using anion-exchange chromatography may include steps of:

[0066] (b-2-i) mixing the primarily purified sample, eluted in step (a), with a sample preparation buffer and loading the mixture into a column containing an anion exchange resin; and

[0067] (b-2-ii) collecting the eluted flow-through (FT) fraction as a fraction containing ssRNA.

[0068] In the present invention, the ssRNA may be collected using a flow-through mode in which the flow-through (FT) eluted from chromatography is collected as a fraction containing the ssRNA.

[0069] The terms "flow-through (FT)", "flow-through mode", and "flow-through purification", as used interchangeably herein, refer to a separation technique in which at least one target molecule (e.g., ssRNA) contained in a biopharmaceutical preparation along with one or more impurities passes through a material, which usually binds to the one or more impurities, where the target molecule usually does not bind (i.e., flows through).

[0070] In the present invention, the term “anion-exchange chromatography” refers to a process of separating substances based on charge by an ion-exchange resin containing a positively charged group such as diethylaminoethyl (DEAE).

[0071] In the present invention, various commercially available anion-exchange chromatography resins may be used. Examples of the anion-exchange chromatography resins include, but are not limited to, those substituted with diethylaminoethyl (DEAE), trimethylaminoethyl (TAME), triethylaminoethyl (TEAE), aminoethyl (AE), diethylaminopropyl (ANX), or quaternary ammonium (Q) groups. Preferably, the anion-exchange chromatography resin is any one selected from among anion-exchange resins having a strongly basic quaternary ammonium group or anion-exchange resins having a weakly basic diethylaminoethyl (DEAE) group. More preferably, the anion-exchange chromatography resin may be any one selected from anion exchange resins having a strongly basic quaternary ammonium (Q) group. Most preferably, the anion-exchange chromatography resin may be Q Sepharose, without being limited thereto.

[0072] In the present invention, the sample preparation buffer may be, but is not limited to, a solution (pH 7.0 to 8.0) containing: 10 to 50 mM Tris, sodium phosphate, sodium citrate or sodium acetate; 1 to 10 mM EDTA; and 500 to 1000 mM sodium chloride, and more preferably a solution (pH 7.3) containing: 20 mM Tris; 5 mM EDTA; and 700 mM sodium chloride.

[0073] In the present invention, it is obvious that, if the pH of the buffers and the concentrations of the components are lower or higher than the values described above, the chromatography performance is reduced and thus a purified product cannot be obtained at a high recovery rate.

[0074] In the present invention, step (b) of subjecting the sample to secondary purification using the hydrophobic interaction chromatography may include steps of:

[0075] (b-3-i) mixing the primarily purified sample, eluted in step (a), with a sample preparation buffer, and loading the mixture into a column containing a hydrophobic interaction resin;

[0076] (b-3-ii) washing the column using an equilibration buffer; and

[0077] (b-3-iii) recovering bound ssRNA by elution with an elution buffer.

[0078] In the present invention, the hydrophobic interaction chromatography is a method of separating target molecules based on their degree of hydrophobicity. For example, hydrophobic groups such as phenyl, octyl, and butyl may be attached to the HIC resin (stationary phase) through hydrophobic interaction with the target molecule. In the present invention, various commercially available hydrophobic interaction chromatography resins may be used. For example, the hydrophobic interaction chromatography resin may include a hydrophobic moiety selected from among alkyl groups, aromatic groups, and ether. More specifically, the alkyl groups include lower alkyl groups such as n-propyl, isopropyl, n-butyl, iso-butyl and n-octyl, and the aromatic groups include a substituted or unsubstituted phenyl, without being limited thereto. In addition, the hydrophobic interaction chromatography resin may include a matrix selected from among agarose, Sepharose (GE Healthcare), polystyrene, divinylbenzene, and combinations thereof. More preferably, the hydrophobic interaction chromatography resin may be C4-HLD, Benzyl Ultra, or Capto Phenyl, most preferably a C4-HLD resin, without being limited thereto.

[0079] In the present invention, the sample preparation buffer or the equilibration buffer may be, but is not limited to, a solution (pH 6.5 to 7.5) containing: 10 to 100 mM sodium phosphate, tris, sodium citrate, or sodium acetate; 1 to 20 mM EDTA; and 100 to 2,800 mM sodium chloride, sodium sulfate, ammonium sulfate, potassium sulfate, disodium phosphate, lithium chloride or potassium thiocyanate, and more preferably a solution (pH 7.0) containing: 50 mM sodium phosphate; 10 mM EDTA; and 1,600 mM sodium chloride, or a solution (pH 7.0) containing: 25mM Tris; 10 mM EDTA; and 2500mM ammonium sulfate.

[0080] In the present invention, step (b) may further include, before step (b-3-ii), a step of additionally loading an equilibration buffer.

[0081] In the present invention, the elution buffer may be, but is not limited to, a solution (pH 6.5 to 7.5) containing: 10 to 100 mM sodium phosphate, tris, sodium citrate, or sodium acetate; 1 to 20 mM EDTA; and 100 to 1,000 mM sodium chloride, and more preferably a solution (pH 7.0) containing: 50 mM sodium phosphate; 10 mM EDTA; and 500 mM to 600 mM sodium chloride.

[0082] In the present invention, it is obvious that, if the pH of the buffers and the concentrations of the components are lower or higher than the values described above, the chromatography performance is reduced and thus a purified product cannot be obtained at a high recovery rate.

[0083] In the present invention, the purification method may further include, before step (a), after step (a), or after step (b), an ultrafiltration / diafiltration (UF / DF) step.

[0084] In the present invention, "UF diafiltration" refers to a technique that removes or collects any component (e.g., particles) from a target substance (solution) using a permeable filter capable of achieving separation according to the molecular weight (molecular size) of the component, thereby increasing the purity of the target substance. Ultrafiltration / diafiltration (UF / DF) may be performed using a conventional UF / DF system, and may include a change to a constant osmotic pressure, exchange of a buffer, and adjustment of the concentration.

[0085] In the present invention, the ultrafiltration step may be performed by a tangential flow filtration (TFF) method. The “tangential flow filtration (TFF)” is also known as “cross-flow filtration” and refers to a filtration method that passes water and a sample tangentially across a membrane.

[0086] In the present invention, the ssRNA obtained by the secondary purification may have a dsRNA removal rate of 80% or more, more preferably 90% or more, most preferably 95% or more.

[0087] Examples

[0088] Hereinafter, the present invention will be described in more detail through examples. These examples are intended only to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

[0089] Experimental Method 1. Determination of mRNA Concentration

[0090] In the following examples, the concentration of mRNA was determined by measuring the absorbance at 260 / 280 nm using UV spectrophotometry (NanoDrop, Thermo Fisher Scientific, USA).

[0091] Experimental Method 2. Measurement of dsRNA Concentration

[0092] In the examples below, the concentration of dsRNA concentration was measured in the following manner.

[0093] First, capture antibody (SCICONS, Netherlands) was diluted in 1x PBS to 3 μg / mL, and 100 μL of the dilution was loaded into each well of a 96-well microplate and incubated overnight at 4°C. The plate was then inverted to discard the solution and any remaining solution was removed. Thereafter, the process of adding 300 ㎕ of washing buffer (0.5% tween in PBS) to each well and then discarding the solution was repeated three times, and then the remaining solution was completely removed by patting the plate on several layers of paper towels. After adding 200 ㎕ of blocking buffer (ThermoFisher, USA) to each well, the plate was covered with a plate sealer and incubated at 37°C for 2 hours. Next, the plate was inverted to discard the solution and then any remaining solution was removed. The process of adding 300 ㎕ of washing buffer to each well and then discarding the solution was repeated three times, and then the remaining solution was completely removed by patting the plate on several layers of paper towels. 100 ㎕ of each of the standard solution (Abnova, Taiwan) and test solution diluted in STE buffer (100 mM NaCl, 50 mM Tris, 1 mM EDTA, pH 7.0) to an analyzable concentration was loaded in duplicate into wells and incubated at 37°C for 1 hour. After completion of the incubation, the plate was inverted to discard the solution, and then any remaining solution was removed. The process of adding 300 ㎕ of washing buffer to each well and discarding the solution was repeated three times, and then the remaining solution was completely removed by patting the plate on several layers of paper towels.

[0094] Detection antibody (SCICONS, Netherlands) was diluted 1:1 with 1xPBS, and 100 ㎕ of the dilution was loaded in duplicate into wells. The plate was covered with a sealer and incubated at 37°C for 1 hour. The plate was then inverted to discard the solution and then any remaining solution was removed. The process of adding 300 ㎕ of washing buffer to each well and then discarding the solution was repeated three times, and then the remaining solution was completely removed by patting the plate on several layers of paper towels. HRP-conjugated anti-IgM antibody (EMD Millipore, USA) was diluted 1 / 10,000 in blocking buffer, and 100 ㎕ of the dilution was loaded into each well. The plate was covered with a sealer and incubated at 37°C for 1 hour. The plate was then inverted to discard the solution, and then any remaining solution was removed. The process of adding 300 ㎕ of washing buffer to each well and discarding the solution was repeated three times, and then the remaining solution was completely removed by patting the plate on several layers of paper towels.

[0095] 100 ㎕ of 1-step ultra TMB-ELISA (ThermoFisher, USA) was added to each well, and the plate was covered with a plate sealer and incubated at room temperature for 15 minutes. Then, 100 ㎕ of stop buffer (1M H2SO4) was added to each well to stop the reaction, and the absorbance was measured at 450 nm.

[0096] After subtracting the blank absorbance from the absorbance of the standard solution and test solution, the regression equation was obtained using a 4-parameter logistic model with the concentration of the standard solution as the X-axis and the response value as the Y-axis, thereby establishing a standard curve. The dsRNA concentration was calculated by substitution with the response value of the test solution.

[0097]

[0098] Example 1. Preparation of Nucleic Acid for Synthesis of mRNA Containing dsRNA as Impurity

[0099] 1-1. Linearization for Template DNA Preparation

[0100] Linearization was performed using a restriction enzyme that cuts the portion downstream of the poly A site in the plasmid. The linearized DNA was isolated using AMICON, and then whether or not the plasmid DNA was cut was identified on a 1% agarose gel.

[0101] 1-2.In VitroTranscription

[0102] In vitrotranscription is the process of synthesizing mRNA.

[0103] The prepared template DNA was reacted with T7 RNA polymerase, buffer, NTPs (including natural and chemically modified NTPs) and other necessary elements of IVT at 37°C for 4 hours as shown in Table 1 below.

[0104]

[0105] After the reaction was completed, 1 μg of DNA was treated with 1U of DNase I, followed by reaction at 37°C for 15 to 30 minutes, and template DNA was removed and used in a subsequent purification process.

[0106] Example 2. Primary purification Using Affinity Chromatography

[0107] The IVT product obtained in Example 1 was subjected to primary purification using a column containing oligo dT that binds to the poly(A) tail.

[0108] The specific method is summarized in Table 2 below.

[0109]

[0110] That is, a CIMmultus Oligo dT (C12 linker) 8 mL (2 ㎛) (Sartorius, USA) column was mounted on an AKTA Avant 150 (Cytiva, USA), and 2.5 mg / mL of the IVT product of Example 1 mixed with 3 mL of sample preparation buffer(100mM sodium phosphate + 700mM NaCl + 10mM EDTA, pH 6.5) was loaded into the column at the flow rate shown in Table 2 above. Then, the column was re-equilibrated with 5 CV of equilibration buffer, and the column to which the product bound was washed with 40 CV or less of wash buffer, and then 5 CV of elution buffer was loaded to recover the poly(A) tail-containing IVT product bound to the column. Thereafter, the column was washed with CIP buffer, and then the concentrations of mRNA and dsRNA were measured as described in Experimental Methods 1 and 2 above.

[0111] Example 3. Secondary Purification Using Size-Exclusion Chromatography (SEC) and Evaluation of Effect

[0112] A wide variety of types of dsRNA can be generated in the IVT described in Example 1, but can be broadly classified into three types. First, if the abortive transcript generated during the mRNA synthesis process has a complementary sequence, it can bind to mRNA to generate dsRNA. In this case, the dsRNA formed has a poly(A) tail.

[0113] In addition, if a T7 promoter-like sequence is present in non-template DNA, a reverse transcript may be generated. The possibility of dsRNA being generated by antisense transcription by recognizing this non-template DNA is low if a poly(A) tail is present, but it is possible that dsRNA will be generated because promoter-independent reverse transcripts can be generated.

[0114] In addition, IVT terminates in a run-off manner, which can form an mRNA that is longer than the original mRNA. The longer mRNA is a loop back dsRNA generated by self-primed extension through the formation of a hairpin structure at the 3' end, and has a poly(A) tail.

[0115] Since most of the above dsRNAs have a poly(A) tail, they cannot be removed by the oligo dT process. Also, since dsRNA is formed by attaching a complementary sequence to mRNA, dsRNA is expected to be larger than mRNA. Considering this, the present inventors sought to examine the dsRNA removal rate using a size exclusion resin.

[0116] The characteristics of the size exclusion resin used in the examples below are as shown in Table 3 below.

[0117]

[0118] 3-1. Secondary purification Using Superdex 200 PG Resin and Evaluation of Effect

[0119] The secondary purification process using Superdex 200 PG resin is summarized in Table 4 below.

[0120]

[0121] That is, the primary purification product obtained in Example 2 was subjected to UFDF (at 3,000 g for 20 minutes) using Amicon (Merck, Germany), and then mixed with the 2X equilibration buffer shown in Table 4, thus preparing an injection sample. A Hiload 26 / 600 Superdex 200 pg (Cytiva, USA) column was mounted to an AKTA Avant 150 (Cytiva, USA), and the sample having a concentration of 1,011 μg / mL was injected into the column at a flow rate of 2.6 mL / min under the conditions shown in Table 4. Then, 2 CV (column volume) of equilibration buffer was flowed, and the eluted flow-through (FT) factions were collected. The total mRNA concentration and dsRNA concentration in each fraction were measured by the methods of Experimental Methods 1 and 2.

[0122] As a result, as shown in Table 5 below and FIG. 2, it was confirmed that, although the peak on the chromatogram was not separated, dsRNA was distributed in the front fraction as expected, and when the fraction was collected based on an mRNA recovery of 79%, the dsRNA removal rate was about 72%.

[0123] In the examples below, the mRNA recovery and the dsRNA removal rate were calculated as follows.

[0124] Collected fraction = Fraction taken after excluding the front fraction containing a lot of dsRNA.

[0125] mRNA recovery (%) = (mRNA content of collected fraction / mRNA content of load) * 100

[0126] dsRNA removal rate (%) = (1 - (dsRNA content of collected fraction / dsRNA content of load)) * 100

[0127] For example, the recovery in Table 5 below was calculated as follows.

[0128] mRNA recovery (%) = (Fr. 11 - Fr. 45 mRNA content / mRNA content of load) * 100

[0129] dsRNA removal rate (%) = (1 - (Fr. 11 - Fr. 45 mRNA content / mRNA content of load)) * 100

[0130]

[0131] 3-2. Secondary Purification Using Superose 6 PG Resin and Evaluation of Effect

[0132] It was confirmed in Example 3-1 that dsRNA was larger in size than ssRNA and that the SEC column had a higher dsRNA removal rate than other columns. However, in order to further increase the dsRNA removal rate, secondary purification was performed using Superose 6 PG resin, which has a wider working range, and the pH and salt concentration were adjusted to further increase the dsRNA removal rate. The method is summarized in Table 6 below.

[0133]

[0134] That is, the primary purification product obtained in Example 2 was subjected to UFDF (at 3000 g for 20 min) with Amicon (Merck, Germany), and then mixed with the 2X equilibration buffer shown in Table 6, thus preparing an injection sample. A HiLoad 16 / 600 Superose 6 pg (Cytiva, USA) column was mounted to an AKTA Avant 150 (Cytiva, USA), and the sample having a concentration of 760 μg / mL was injected into the column at 1 mL / min under the conditions shown in Table 6. Then, 2 CV (column volume) of equilibration buffer was flowed, and the eluted flow-through (FT) fractions were collected. The total mRNA concentration and dsRNA concentration in each fraction were measured by the methods of Experimental Methods 1 and 2.

[0135] As a result, as shown in Table 7 below and FIG. 3, it was confirmed that the dsRNA peak was concentrated in the front, similar to the case of Superdex 200 pg, and that the higher the pH and the higher the salt concentration, the larger the size of the front peak. In addition, it was confirmed that as the front peak on the chromatogram became smaller, it was possible to maximize the mRNA recovery and dsRNA removal rate when collecting the rear peak (Run 1 in Table 7).

[0136] That is, as shown in Table 7 below, the results of Run 1, which had the smallest front peak, indicated a dsRNA removal rate of 97% at an mRNA recovery of 79%.

[0137]

[0138] 3-3. Secondary Purification Using Sepharose 6 FF Resin and Sephacryl S-300 HR Resin and Evaluation of Effect

[0139] Secondary purification was performed using Sepharose 6 FF, which has a working range similar to Superose 6 PG used in Example 3-2 but a larger resin bead size, and Sephacryl S-300 HR, which has a narrower working range but a similar bead size. The method is summarized in Table 8 below.

[0140]

[0141] That is, the primary purification product obtained in Example 2 was subjected to UFDF (at 3000 g for 20 minutes) using Amicon (Merck, Germany), and then mixed with the 2X equilibrium buffer shown in Table 6, thus preparing an injection sample.

[0142] In the case of Sepharose 6 FF, a Sepharose 6 Fast Flow (Cytiva, USA) column was mounted on AKTA Avant 150 (Cytiva, USA), and the sample having a concentration of 725 μg / mL was injected into the column at a flow rate of 1 mL / min under the conditions shown in Table 8. Then, 2 CV (column volume) of equilibration buffer was flowed, and the eluted flow-through (FT) fractions were collected. The total mRNA concentration and dsRNA concentration in each fraction were measured by the methods of Experimental Methods 1 and 2. In the case of Sephacryl S-300 HR, a Sephacryl S-300 HR (Cytiva, USA) column was mounted on AKTA Avant 150 (Cytiva, USA), and the sample having a concentration of 725 μg / mL was injected into the column at a flow rate of 1 mL / min under the conditions shown in Table 8. Then, 2 CV (column volume) of equilibration buffer was flowed, and the eluted flow-through (FT) fractions were collected. The total mRNA concentration and dsRNA concentration in each fraction were measured by the methods of Experimental Methods 1 and 2.

[0143] As a result, as shown in FIG. 4, it was confirmed that, similar to Example 3-2, in the case of Sepharose 6 FF resin, the front peak increased as the pH increased and the salt concentration increased. As shown in FIG. 5, it was confirmed that, in the case of Sephacryl S-300 HR resin, the peak was not separated even though purification was performed under the conditions where the dsRNA removal rate in Example 3-2 was the highest.

[0144] 3-4. Secondary Purification Using Superose 6 PG Resin and Sepharose 6 FF Resin under the Same Conditions and Evaluation of Effect

[0145] As a result of repeating the same process under the conditions of run 1 in Example 3-2 and Sepharose 6 FF resin run 1 in Example 3-3, as shown in FIG. 6, it was confirmed that the front peak of Sepharose 6 FF resin was high, indicating that Superose 6 PG resin was the most effective in ensuring the mRNA recovery and the dsRNA removal rate.

[0146] The results of Examples 3-1 to 3-3 are summarized in Table 9 below.

[0147]

[0148] That is, since dsRNA removal rate and mRNA yield are inversely proportional, it was confirmed that, under the conditions where the secondary purification using the SEC process was performed using Superose 6 PG resin and the equilibration and elution buffer composition was 50 mM citrate + 200 mM NaCl, pH 5.0, a dsRNA removal rate of 95% or higher was ensured while an mRNA recovery of about 80% or higher was maintained.

[0149] Example 4. Secondary purification Using Anion-Exchange Chromatography (AEX) and Evaluation of Effect

[0150] The AEX process for removing dsRNA from IVT products is known, but this process is performed by binding ssRNA to a column and removing dsRNA by washing. However, based on the principle of AEX, it is expected that, when the type and concentration of salt are controlled, the efficiency of the process can be increased by binding dsRNA to the column and collect ssRNA through FT. Therefore, the AEX process was performed in FT mode and the dsRNA removal rate was measured.

[0151] The use of FT mode has an advantage over the use of elution mode in that a significantly increased capacity may be used. For example, when the load sample is assumed to contain 0.5% dsRNA, about 200 times the capacity may be used. In addition, there is an advantage in that since the elution step can be omitted, process time and reagents can be saved.

[0152] Specific AEX conditions are shown in Table 10 below.

[0153]

[0154] That is, the primary purification product obtained in Example 2 was mixed with the 2X equilibrium buffer shown in Tables 11 to 18 below, thus preparing an injection sample.

[0155] A HiScreen Q FF (Cytiva, USA) column was mounted on AKTA Avant 150 (Cytiva, USA), and AEX was performed under the conditions shown in Tables 11 to 18. FT was collected, and the presence or absence of dsRNA in FT and the recovery rate were measured using the methods of Experimental Methods 1 and 2.

[0156]

[0157] First, as a result of performing AEX under the conditions shown in Table 11, it was confirmed that 161 ㎕ was obtained in flow-through, no mRNA was measured in elution, and a peak of 2,000 mAU or higher appeared in CIP. This was believed to be because mRNA was strongly bound to the resin and thus did not appear in the FT. Thus, AEX was performed under the conditions shown in Table 12 below, in which the binding force between mRNA and resin was relatively weakened by increasing the ionic strength of the EQ buffer.

[0158]

[0159] As a result, it was confirmed that mRNA was still not measured in the flow-through and eluate, and a peak of 3,000 mAU or higher appeared in CIP. In other words, it was confirmed that mRNA was still strongly bound to the resin and thus did not appear in the FT. Thus, AEX was performed under the conditions shown in Table 13 below using sodium chloride, which has a lower ionic strength than ammonium sulfate.

[0160]

[0161] As a result, it was confirmed that 35 ㎍ of mRNA was measured in the elution, and the rest corresponded to a peak appearing in the CIP. That is, in 200 mM sodium chloride, mRNA was strongly bound to the resin and thus did not appear in the FT. Thus, the ionic strength of the EQ buffer was increased and AEX was performed under the conditions shown in Table 14 below.

[0162]

[0163] As a result, it was confirmed that 123 ㎍ was measured in the elution, 322 ㎍ was measured in the elution (pH 10), and the rest corresponded to a peak in the CIP. The combined recovery in the elution was 44.5% (445 ㎍), indicating that mRNA was strongly bound to the resin and thus was not sufficiently recovered. Thus, AEX was performed under the conditions shown in Table 15 below, in which the ionic strength was further increased.

[0164]

[0165] As a result, it was confirmed that 661 ㎍ was measured in the elution and the rest corresponded a peak appearing in the CIP. The combined recovery in the elution was 70% (661 ㎍), indicating that mRNA was strongly bound to the resin and thus sufficient recovery was not possible. Thus, AEX was performed under the conditions of Table 16 below, in which the ionic strength was further increased.

[0166]

[0167] As a result, it was confirmed that 719 ㎍ was measured in the flow-through and the rest corresponded to a peak appearing in the CIP. As described in Table 17 below, it was confirmed that the mRNA recovery was 80%, which was an appropriate level, and the dsRNA removal rate was also 55%, which was equal to or higher than that when the elution mode was used.

[0168]

[0169] To examine mRNA recovery rate and dsRNA removal rate under higher salt conditions, AEX was performed under the conditions shown in Table 18 below.

[0170]

[0171] As a result, it was confirmed that 672 ㎍ was measured in the flow-through and the rest correspond to a peak appearing in the CIP. This corresponded to a recovery rate of 75%, which was not significantly different from that when 700 mM sodium chloride was used. Accordingly, in order to confirm the results at a higher ionic strength, AEX was performed under the conditions shown in Table 19 below.

[0172]

[0173] As a result, it was confirmed that 1 mg of mRNA was measured in the flow-through. This corresponded to a recovery rate of 85%, which was not significantly different from that when 700 mM or 800 mM sodium chloride was used.

[0174] Example 5. Secondary Purification Using Hydrophobic Interaction Chromatography and Evaluation of Effect

[0175] Since mRNA has a different structure from dsRNA, it is expected that there will also be a difference in hydrophobicity therebetween. Therefore, the present inventors sought to confirm the possibility of removing dsRNA using HIC resin.

[0176] The characteristics of the hydrophobic interaction resin used in the examples below are shown in Table 20 below.

[0177]

[0178] 5-1. Secondary Purification Using C4-HLD Resin and Evaluation of Effect

[0179] The secondary purification process using C4-HLD resin is summarized in Table 21 below.

[0180]

[0181]

[0182] That is, the primary purification product obtained in Example 2 was mixed with the 2X equilibrium buffer shown in Table 21, thus preparing an injection sample.

[0183] A CIMmultus™ C4 HLD - 4 mL (2 μm) (Sartorius, USA) column was mounted on an AKTA Avant 150 (Cytiva, USA), and 48 mL of the sample having 184.8 μg / mL was injected into the column at a flow rate of 4 mL / min under the conditions shown in Table 21, and the column was washed with the equilibration buffer until the UV value dropped to the baseline. Then, the column was washed with the elution buffer under each of gradient conditions and stepwise conditions, and the eluted fractions (volume of each fraction: e.g., 1 mL) were collected. The total mRNA concentration and dsRNA concentration in each fraction were measured by the methods of Experimental Methods 1 and 2.

[0184] As a result, as shown in Tables 22 and Table 23 below and FIG. 7, it was confirmed that, in the gradient elution in Table 22, the dsRNA / mRNA ratio in the earlier elution fractions was low compared to that in the load, but the dsRNA / mRNA ratio in the later elution fractions was more similar to that in the load, and overall, the distribution of mRNA and the distribution of dsRNA were generally similar in the later elution fractions. It was confirmed that the mRNA recovery was very low if only the earlier fractions were taken, and that a certain amount of dsRNA had to be taken to obtain a certain level of mRNA recovery. As shown in Table 23, as a result of performing a step elution test to achieve an mRNA recovery of 80% or more, it was confirmed that an mRNA recovery of 83% and a dsRNA removal rate of 47% were achieved at a NaCl concentration of 500 mM.

[0185]

[0186]

[0187] The above results were obtained when the dsRNA / mRNA ratio of the load was 0.03%, and a spiking test was performed to check whether a similar removal rate could be ensured when the dsRNA / mRNA ratio of the load was higher. In the spiking test, the dsRNA / mRNA ratio of the load was set to 0.241%, which is about 8 times higher than 0.03% in the above test, and the salt concentration of the first elution buffer was changed because it was expected that the elution pattern would change as the amount of dsRNA increased.

[0188] As a result, as described in Table 24 below, it was confirmed that an mRNA recovery of 70% and a dsRNA removal rate of 40% were achieved at a NaCl concentration of 600 mM. It was confirmed that the dsRNA removal rate tended to decrease as the mRNA recovery increased, indicating that the dsRNA removal rate decreased as the dsRNA / mRNA ratio of the load increased.

[0189]

[0190] 5-2. Secondary Purification Using Benzyl Ultra Resin and Evaluation of Effect

[0191] The secondary purification process using Benzyl Ultra resin is summarized in Table 25 below.

[0192]

[0193] That is, the primary purification product obtained in Example 2 was mixed with the 2X equilibrium buffer shown in Table 25, thus preparing an injection sample.

[0194] A GoPure Benzyl Ultra (Thermo Fisher, USA) column was mounted on an AKTA Avant 150 (Cytiva, USA), and 20 mL of the sample having a concentration of 249 μg / mL was injected into the column at a flow rate of 1.67 mL / min (Run 1) or 1.1 mL / min (Run 2) under the conditions shown in Table 25. Then, the column was washed with the equilibration buffer until the UV value dropped to the baseline. Then, the elution buffer was injected under gradient conditions, and the eluted fractions (volume of each fraction: e.g., 1 mL) were collected. The total mRNA concentration and dsRNA concentration in each fraction were measured using the methods of Experimental Methods 1 and 2.

[0195] As a result, as shown in Tables 26 and 27 below and FIG. 8, it was confirmed that, in the case of Run 1 described in Table 26, unlike Example 5-1, the dsRNA / mRNA ratio of latter fractions was higher than that of the load, indicating that that there was a certain degree of separation between dsRNA and mRNA. However, since the mRNA recovery of the fraction to ensure a dsRNA removal rate similar to that of C4-HLD (43%) was confirmed to be 59%, Run 2 was performed to check whether additional separation between dsRNA and mRNA was possible by decreasing the elution gradient and increasing the elution time.

[0196] From the results of Run2 shown in Table 27 below, it was confirmed that, when an mRNA recovery (61%) similar to that in Run 1 was ensured, the dsRNA removal rate was 56%, indicating that additional dsRNA removal was possible by changing the elution method.

[0197]

[0198]

[0199] 5-3. Secondary Purification Using Capto Phenyl Resin and Evaluation of Effect

[0200] The secondary purification process using Capto Phenyl resin is summarized in Table 28 below.

[0201]

[0202] That is, the primary purification product obtained in Example 2 was mixed with the 2X equilibrium buffer shown in Table 28 above, thus preparing an injection sample.

[0203] A Hiscreen Capto Phenyl (High Sub) (Cytiva, USA) column was mounted on AKTA Avant 150 (Cytiva, USA), and 21 mL of the sample having a concentration of 249.2 μg / mL was injected into the column at a flow rate of 1.67 mL / min under the conditions shown in Table 28 above. Then, the column was washed with the equilibration buffer until the measured UV value dropped to the baseline. Then, the elution buffer was injected under gradient conditions, and the eluted fractions (volume of each fraction: e.g., 1 mL each). The total mRNA concentration and dsRNA concentration in each fraction were measured by the methods of Experimental Methods 1 and 2.

[0204] As a result, as shown in Table 29 below and FIG. 9, it was confirmed that the dsRNA removal rate was 50% at an mRNA yield of 81%. In other words, it was confirmed that the Capto Phenyl resin showed the best performance compared to C4-HLD and Benzyl Ultra.

[0205]

[0206] The results of Examples 5-1 to 5-3 are summarized in Table 30 below.

[0207]

[0208] That is, it was confirmed that, although there was a difference in the dsRNA removal rate between the HIC resins, the mRNA recovery was about 80% and the dsRNA removal rate was about 50%, and that the process time was the shortest (shorter by about 3 times) for the monolith used in Example 5-1.

[0209] Example 6. mRNA Production, Purification, and Sample Preparation for Evaluation of Innate Immune Response and Toxicity at Varying dsRNA Concentrations Depending on Purification Process of IVT Product

[0210] Five different samples with different dsRNA concentrations were prepared, andin vivoinnate immune response and toxicity evaluations were performed on each sample. In order to evaluate the influence of dsRNA at varying concentrations, mRNA synthesis and purification were performed through the process shown in FIG. 10.

[0211] 6-1.In VitroTranscription

[0212] In vitrotranscription is the process of synthesizing mRNA.

[0213] The prepared template DNA was reacted with T7 RNA polymerase, buffer, NTPs (including natural and chemically modified NTPs) and other necessary elements of IVT at 37°C for 4 hours as shown in Table 31 below.

[0214]

[0215] * All reagents were added based on each total IVT reaction volume, and water was added to reach the final volume.

[0216] After the reaction was completed, DNA was treated with 50 U / Ml of DNase I (03539121103, Roche, USA), followed by reaction at 37°C for 30 minutes, and template DNA was removed, treated with 70 mM EDTA, and incubated for 3 minutes. A 5X volume of RNase-free water was added thereto and centrifuged, and the resulting IVT product was used in a subsequent purification process.

[0217] 6-2. Primary Purification Using Affinity Chromatography

[0218] The IVT product obtained in Example 6-1 was subjected to primary purification by the method described in Example 2 using a column containing oligo dT that binds to the poly(A) tail.

[0219] A specific method is summarized in Table 32 below.

[0220]

[0221] That is, a CIMmultus Oligo dT (C12 linker) 8 mL (2 ㎛) (Sartorius, USA) column was mounted on an AKTA Avant 150 (Cytiva, USA), and 2.0 mg / mL of the IVT product of Example 6-1 mixed with 3 mL of sample preparation buffer(20mM sodium phosphate + 500mM NaCl + 10mM EDTA, pH 6.6) was loaded into the column. Then, the column was re-equilibrated with 10 CV of equilibration buffer, and the column to which the product bound was washed with 40 CV or less of wash buffer, and then 6 CV of elution buffer was loaded to recover the poly(A) tail-containing IVT product bound to the column. Thereafter, the column was washed with CIP buffer.

[0222] The obtained mRNA purified solution was concentrated to 2.5 mg / mL using Amicon Ultra-15 30 KD (UFC903024, MILLIPORE, USA) at 3,000 g and 6°C, and then subjected to buffer exchange 1 mM sodium citrate + 200 mM NaCl (pH 5.7).

[0223] 6-3. Secondary Purification Using Size-Exclusion Chromatography

[0224] The purified solution obtained in Example 6-2 was subjected to secondary purification using the method described in Example 3-1. As described in Example 3, it was confirmed that, when the SEC process was performed, the dsRNA concentration was high in the front part of the peak. Therefore, after performing the SEC process using the method described in Table 33 below, the flow-through peaks were collected. The front peak with a high dsRNA concentration was named high dsRNA and the rear peak with a low dsRNA concentration was named low dsRNA.

[0225] For example, for high dsRNA, flow-through fraction 1-5 in Example 3 was pooled and used, and as for low dsRNA, fraction 18-27 in Example 3 was used.

[0226] A specific method is summarized in Table 33 below.

[0227]

[0228] The obtained mRNA purified solution (each collected sample) was concentrated to 0.7 mg / mL or more using Amicon Ultra-15 30 KD (UFC903024, MILLIPORE, USA) at 3,000 g and 6°C, and then subjected to buffer exchange with 1 mM sodium citrate (pH 6.4).

[0229] 6-4. LNP Encapsulation and Sample Preparation

[0230] Encapsulation with ALC-0315 LNP was performed on each of the obtained high dsRNA and low dsRNA. The final concentration was set to 0.2 mg / mL. After dispensing and freezing, the sample was thawed and used on the day of thein vivoinnate immune response evaluation and toxicity evaluation test.

[0231] The dsRNA concentrations of the obtained high dsRNA and low dsRNA samples were analyzed using the method described in Experimental Method 2. Then, preliminary experimental samples for innate immune response were prepared at the concentrations described in Table 34 below, and main experimental samples for innate immune response and toxicity test samples were prepared at the concentrations described in Table 35 below.

[0232]

[0233]

[0234] Example 7. Experimental Method for Evaluating Innate Immune Response and Toxicity at Varying dsRNA Concentrations Depending on Purification Process of IVT Product

[0235] 7-1. Animal Experiment

[0236] 7-1-1. Innate Immune Response

[0237] Five-week-old female Balb / C and C57BL / 6 mice (Orient Bio, female) were purchased, acclimatized for one week, and used at 6 weeks of age in the experiment. Samples were prepared according to the experimental plan and taken with an insulin syringe (33 gauge, 0.5 mL), and 50 to 200 ㎕ per mouse were administered intramuscularly into the right thigh. Then, 500 ㎕ or more of blood was collected per mouse by whole blood collection.

[0238] To facilitate serum separation, the collected blood was allowed to stand at room temperature for 30 minutes or more to coagulate, and then centrifuged (at 10,000 rpm and 4°C for 10 minutes), and the supernatant was transferred to a fresh 1.5 mL tube. To separate pure serum, the blood was centrifuged once more (at 10,000 rpm and 4°C for 10 minutes), and the supernatant was transferred to a fresh 1.5 mL tube and stored in a freezer at -20°C until analysis.

[0239] 7-1-2. Toxicity

[0240] Six-week-old female Balb / C mice (Orient Bio, female) were purchased, acclimatized for one week, and used at 7 weeks of age in the experiment. Samples were prepared according to the experimental plan and taken with an insulin syringe (33 gauge, 0.5 mL), and 200 ㎕ per mouse was administered intravenously through the mouse tail vein. Then, 500 ㎕ or more of blood was collected per mouse by whole blood collection.

[0241] To facilitate serum separation, the collected blood was allowed to stand at room temperature for 30 minutes or more to coagulate, and then centrifuged (at 10,000 rpm and 4°C for 10 minutes), and the supernatant was transferred to a fresh 1.5 mL tube. To separate pure serum, the blood was centrifuged once more (at 10,000 rpm and 4°C for 10 minutes), and the supernatant was transferred to a fresh 1.5 mL tube and stored in a freezer at -20°C until analysis.

[0242] 7-2. Measurement of IFN-α, IFN-β, IP-10 and MCP-1 by Multi-Analyte Flow Assay for Evaluation of Innate Immune Response

[0243] To measure IFN-α, IP-10, and MCP-1, LEGENDplex Mouse Anti-Virus Response Panel (13-plex) with Filter Plate (Biolegend, 740621), a kit, was used. The assay was performed as follows according to the kit’s manual.

[0244] First, one vial of lyophilized standard in the LEGENDplex kit was taken out and reconstituted in 250 μL of LEGENDplex assay buffer at room temperature for 10 minutes. Then, a 4-fold serial dilution was performed starting from the top standard, and a total of 8 standards including buffer only were prepared in 1.7-mL tubes. In addition, 25 μL of serum obtained in Example 7-1 was diluted 2-fold with 25 μL of LEGENDplex assay buffer, thus preparing a sample.

[0245] Considering the number of standards and samples to be analyzed, pre-mixed beads were prepared. 25 μL of pre-mixed beads were used per analysis sample. The pre-mixed beads included in the kit were vortexed for 1 minute or more. 25 mL of 20X LEGENDplex wash buffer was diluted with 475 mL of distilled water, stored at 4°C, and used. 5 mL of LEGENDplex assay buffer was added to a bottle containing lyophilized matrix A and reconstituted for 15 minutes.

[0246] The filter plate included in the kit was used. 100 μL of LEGENDplex wash buffer was added to each well of the filter plate and left at room temperature for 1 minute. The LEGENDplex wash buffer present in the plate wells was removed using a vacuum manifold, and then 25 μL of matrix A and 25 μL of standard were added to each plate well, and then 25 μL of the analysis sample and 25 μL of the assay buffer included in the kit were added to each plate well. Next, the pre-mixed beads were vortexed for 30 seconds, and then 25 μL of the beads were added to each well. To prevent the beads from settling, the beads were mixed intermittently. For reaction, the plate was shaken using a plate shaker at 500 rpm for 2 hours at room temperature.

[0247] After the reaction was completed, the plate was placed on a vacuum manifold, and the process of draining the plate by aspiration under vacuum and washing all wells with 200 μL of LEGENDplex wash buffer was repeated twice. Then, 25 μL of the detection antibody included in the kit was added to each well. Then, for reaction, the plate was shaken using a plate shaker at 500 rpm for 1 hour at room temperature. During the shaking, the plate was wrapped in aluminum foil to block light.

[0248] After completion of the reaction, the reaction solution in the wells of the plate was removed using the vacuum manifold, and then all wells were twice washed with 200 μL of LEGENDplex wash buffer. Then, 150 μL of LEGENDplex wash buffer was added to each well to resuspend the beads, and the suspension was transferred to 1.1-mL tubes.

[0249] Sample analysis was performed using a flow cytometer (BD, FACSymphony A3), and the levels of IFN-α, IFN-β, IP-10, and MCP-1 in the samples were quantitatively analyzed using the LEGENDplex data analysis program (Biolegend).

[0250] Statistical analysis was performed using Graphpad Prism 10. Normality test was performed using D'Agostino & Pearson test. Ordinary one-way ANOVA test was used if normal distribution was followed, and Kruskal-Wallis test was used if normal distribution was not followed. Turkey's test was used for post hoc analysis (p value < 0.05, *; p value < 0.005, **; p value < 0.0005, ***; p value < 0.0001, ****).

[0251] 7-3. Measurement of IFN-α by ELISA to Evaluate Innate Immune Response

[0252] To measure serum IFN-α, the mouse IFN-alpha ELISA kit (BMS6027, Invitrogen, USA) was used. The assay was performed as follows according to the kit’s manual.

[0253] If there was a crystal of wash buffer, the wash buffer was completely dissolved at room temperature, and then 50 mL of wash buffer concentrate is diluted with 950 mL of distilled water. If there was a crystal of assay buffer, the assay buffer was completely dissolved at room temperature, and then 10 mL of assay buffer concentrate was diluted with 190 mL of distilled water. One vial of mouse IFN-α standard was completely dissolved by adding the indicated amount of distilled water, and then 4,000 pg / mL standard stock was serially diluted 1 / 2 with a calibrator diluent seven times (2,000, 1,000, 500, 250, 125, 62.5, and 31.3 pg / mL). The serum obtained in Example 7-1 was thawed at room temperature and then diluted 1 / 1.5 with assay buffer (16.7 μL sample diluent + 33.3 μL serum), the biotin conjugate was diluted 1 / 100 in assay buffer (120 μL biotin conjugate + 11.88 mL assay buffer (1x)), and streptavidin-HRP was diluted 1 / 300 in assay buffer (40 μL streptavidin-HRP + 11.96 mL assay buffer (1x)).

[0254] The plate provided in the kit was washed twice with 400 μl of wash buffer, and then 50 μL of assay buffer, 50 μL of serum diluted 1 / 1.5, and 50 μL of prepared standard were added into each well, and 50 μL of calibrator diluent was added into the blank well. The plate was covered with a plate sealer and incubated at 650 rpm and room temperature for 1 hour. Then, the plate was inverted to discard the solution, and each well was washed four times with 300 μL of wash buffer each time, and the remaining solution was completely removed by patting the plate on several layers of paper towels. Then, 100 μL of diluted streptavidin-HRP was added to each well and the plate was incubated at 450 rpm and room temperature for 1 hour. The plate was then inverted to discard the solution, and each well was washed 4 times with 400 μL of wash buffer each time, and the remaining solution was completely removed by patting the plate on several layers of paper towels. Then, 100 μL of TMB solution was added to each well and the plate was incubated at room temperature for 30 minutes. At this time, the plate was wrapped in foil to block light. 100 μL of stop solution was added to each well to stop the reaction, and the absorbance was measured at 450 nm and 620 nm using a microplate reader within 5 minutes. Then, correction was performed by O.D. at 450 nm - O.D. at 620 nm.

[0255] The analysis of the experimental results was performed using the SoftMax Pro program. That is, the standard curve was drawn using 5-parameter, and then the IFN-α concentration in the sample was calculated and multiplied by the dilution factor to calculate the IFN-α concentration in the serum.

[0256] Normality test was performed using D'Agostino & Pearson test. Ordinary one-way ANOVA test was used if normal distribution was followed, and Kruskal-Wallis test was used if normal distribution was not followed. Turkey's test was used for post hoc analysis (p value < 0.05, *; p value < 0.005, **; p value < 0.0005, ***; p value < 0.0001, ****).

[0257] 7-4. Measurement of IFN-β by ELISA to Evaluate Innate Immune Response

[0258] To measure serum IFN-β, the Mouse IFN-Beta ELISA Kit (42410-1, PBL, USA), a high sensitivity kit, was used. The assay was performed as follows according to the kit’s manual.

[0259] 60 μL of 1,000 pg / mL standard working stock was taken and diluted in 940 μL sample diluent (60 pg / mL). Serial dilution of 1 / 2 was performed to prepare 7 dilutions (60, 30, 15, 7.5, 3.75, 1.87, and 0.94 pg / mL). The serum obtained in Example 7-1 was thawed at room temperature and then diluted 1 / 3 with sample diluent (sample diluent 40 μL + serum 20 μL). The antibody solution was prepared by diluting 1:60 in antibody diluent 15 minutes before use, and the HRP solution was prepared by diluting 1:70 in HRP diluent 15 minutes before use. The wash buffer was prepared by adding 50 mL of wash solution concentrate with completely dissolved crystals to a bottle containing 450 mL of distilled water and mixing.

[0260] 50 μL of serum buffer, 50 μL of diluted serum, and 50 μL of prepared standard were added into each well, and 50 μL of sample diluent was added into the blank well. Then, the plate was covered with a plate sealer and incubated at 650 rpm and room temperature for 1 hour. The plate was then inverted to discard the solution, and each well was washed 4 times with 300 μL of wash buffer each time, and the remaining solution was completely removed by patting the plate on several layers of paper towels. Then, 50 μL of the prepared antibody solution was added to each well and the plate was incubated at 650 rpm and room temperature for 30 minutes. The plate was then inverted to discard the solution, and each well was washed four times with 300 μL of wash buffer, and the remaining solution was completely removed by patting the plate on several layers of paper towels. Then, 50 μL of the prepared HRP solution was added to each well and the plate was incubated at 650 rpm and room temperature for 10 minutes. The plate was then inverted to discard the solution, each well was washed four times with 300 μL of wash buffer each time, and finally, the remaining solution was completely removed by patting plate on several layers of paper towels. Then, 100 μL of TMB solution was added to each well and the plate was incubated at room temperature for 10 minutes. At this time, the plate was wrapped in foil to block light. Then, 100 μL of stop solution was added to each well to stop the reaction, and the absorbance was measured at 450 nm using a microplate reader (Molecular Devices, Spectramax M3) within 5 minutes.

[0261] The analysis of the experimental results was performed using the SoftMax Pro program. That is, the standard curve was drawn using 4-parameter, and then the IFN-β concentration in the sample was calculated and multiplied by the dilution factor to calculate the IFN-β concentration in the serum.

[0262] Statistical analysis was performed using GraphPad Prism 10. Normality test was performed using D'Agostino & Pearson test. Ordinary one-way ANOVA test was used if normal distribution was followed, and Kruskal-Wallis test was used if normal distribution was not followed. Turkey's test was used for post hoc analysis (p value < 0.05, *; p value < 0.005, **; p value < 0.0005, ***; p value < 0.0001, ****).

[0263] 7-5. Measurement of ALT and AST for Cytotoxicity Evaluation

[0264] Serum ALT and AST levels were measured by Chaon Co., Ltd. (Korea) using the AU680 clinical chemistry analyzer (Beckman Coulter, USA).

[0265] Example 8. Preliminary Experiment for Evaluating Innate Immune Response at Varying dsRNA Concentrations Depending on Purification Process of IVT Product

[0266] Since the innate immune response depending on the dsRNA concentration has not been previously evaluated, a preliminary experiment was conducted as shown in Table 36 below to select the dsRNA concentration, blood collection time point, and mouse strain suitable for comparing the secretion levels of type I IFN and chemokines.

[0267]

[0268] Blood collection was performed at 1 hour, 3 hours, and 6 hours after administration. Blood was collected from 4 animals at each time point, for a total of 12 animals, and serum was obtained using the method of Example 7-1. Analyses for IFN-α, IFN-β, IP-10, and MCP-1 were performed using the methods of Examples 7-2 to 7-4.

[0269] As a result, as shown in FIG. 11, when the concentration of IFN-α was analyzed by the ELISA method, IFN-α was not detected regardless of the dsRNA concentration at 1 and 3 hours after administration, and IFN-α could be detected only at 6 hours. In addition, it was confirmed that the secretion level of IFN-α tended to increase as the dsRNA concentration increased, and that the secretion level of IFN-α was higher in the C57BL / 6 strain than in the Balb / C strain in the medium dsRNA group (dsRNA / mRNA (%) = 0.03).

[0270] In addition, in order to simultaneously confirm the detection of IFN-α, IFN-β, IP-10, and MCP-1, multi-analyte flow assay was performed using the method of Example 7-2. Since serum samples were limited, the analysis was performed using the serum from the Balb / C strain (6 hours after immunization), not the C57BL / 6 strain, which secretes a high level of IFN-α.

[0271] As a result, as shown in FIG. 12, it was confirmed that the secretion level of IFN-α increased with increasing dsRNA concentration, and that IFN-α, IP-10, and MCP-1 were detected at all dsRNA concentrations except IFN-β.

[0272] In addition, in order to check whether IFN-β is secreted even from samples with lower dsRNA concentrations than high dsRNA samples, the serum from the C57BL / 6 strain (6 hours after immunization) was analyzed by the method of Example 7-3 using an IFN-β ELISA kit with a lower detection limit.

[0273] As a result, as shown in FIG. 13, it was confirmed that IFN-β, which was not detected in the multi-analyte flow assay, was detectable by the IFN-β ELISA kit with a low detection limit, and that, similar to the correlation between the dsRNA concentration and the IFN-α secretion level shown in FIGS. 11 and 12, the secretion level of IFN-β increased as the dsRNA concentration increased.

[0274] Based on the above results, the blood collection time for this experiment was selected as 6 hours after immunization when IFN-α is detected, and the mouse strain was selected as the C57BL / 6 strain in which higher IFN-α is detected at low dsRNA concentrations. In addition, since the minimum dsRNA concentration at which the secretion of IFN-α and IFN-β could be identified was 0.03% (dsRNA / mRNA (%)) corresponding to the medium dsRNA group, the dsRNA concentration in the high dsRNA group in this experiment was set to 0.04% (dsRNA / mRNA (%)).

[0275] Example 9. Evaluation of Innate Immune Response at Varying dsRNA Concentrations Depending on Purification Process of IVT Product

[0276] This experiment was conducted as shown in Table 37 below using the mouse strains and blood collection time points determined in Example 8. Serum was obtained by the method of Example 7-1, and the secretion levels of IFN-a, IFN-b, IP-10, and MCP-1 depending on dsRNA concentration in the obtained serum were analyzed by the methods of Examples 7-2 and 7-4. That is, the secretion levels of IFN-α, IP-10, and MCP-1 were analyzed by the method of Example 7-2, and the secretion level of IFN-β was analyzed by the method of Example 7-4 using an ELISA kit with a low detection limit.

[0277]

[0278] As a result, as shown in FIG. 14, it was confirmed that the secretion levels of IFN-α, IFN-β, IP-10, and MCP-1 all tended to increase as the dsRNA concentration increased, and that the low dsRNA group (dsRNA / mRNA (%) = 0.002) and the medium 3 dsRNA group (dsRNA / mRNA (%) = 0.01) did not have a statistically significant difference from the PBS group, a control group, in all evaluation items.

[0279] Therefore, it was confirmed that a dsRNA concentration of 0.02% (dsRNA / mRNA (%)) or higher, corresponding to the medium 2 dsRNA group, was a dsRNA concentration capable of inducing an innate immune response.

[0280] That is, it was confirmed that mRNA purified through the primary affinity chromatography and secondary SEC process according to the present invention had a low dsRNA concentration and did not induce an innate immune response or was significantly reduced.

[0281] Example 9.In VivoToxicity Evaluation at Varying dsRNA Concentrations Depending on Purification Process of IVT Product

[0282] Considering the biodistribution after systemic administration of mRNA / LNP, the organ in which mRNA / LNP is mainly distributed is the liver. Thus, aspartate aminotransferase (AST) and alanine aminotransferase (ALT), which are liver damage markers, were selected as toxicity evaluation factors (Hou, X. et al., Nat. Rev. Mater. Vol. 6, pp. 1078-1094, 2023; Musunuru, K. et al., Nature, Vol. 593, pp. 429-434, 2021), and the experiment was conducted at the dsRNA concentrations shown in Table 35 above.

[0283] As shown in Table 38 below, experiments were conducted on a total of 6 groups including a control group, and the time of blood collection was set to 24 hours after administration, the time point at which acute hepatotoxicity was observed. The analysis according to the method of Example 7-5 was conducted on AST and ALT depending on dsRNA concentration in the serum obtained by the method of Example 7-1.

[0284]

[0285] The results of toxicity evaluation depending on the dsRNA concentration were statistically analyzed using GraphPad Prism 10. One-way ANOVA test was used, and post-hoc analysis was performed using Turkey’s test.

[0286] As a result, as shown in FIG. 15, it was confirmed that the secretion levels of ALT and AST all tended to increase as the dsRNA concentration increased. In addition, in the statistical analysis, it was confirmed that only the high dsRNA group (dsRNA / mRNA (%) = 0.04) showed significant ALT and AST levels compared to the other groups, and that the other groups, excluding the high dsRNA group, did not show significant differences from the vehicle (empty LNP) group, which is the control group.

[0287] Therefore, it was confirmed that, although there may be differences in toxicity depending on the GOI, mouse strain and other species and the LNP, dosage and method used, toxicity due to dsRNA did not appear up to 0.03% (dsRNA / mRNA (%)), which is the medium 1 dsRNA group.

[0288] That is, it was confirmed that the mRNA purified through the first affinity chromatography and second SEC process according to the present invention had a low dsRNA concentration and did not exhibit or significantly reducein vivotoxicity.

[0289] Example 10. Optimization of salt concentration in sample preparation buffer and equilibration buffer for primary purification using affinity chromatography

[0290] The binding of Oligo dT resin to mRNA occurs by hydrogen bonding as NaCl masks the repulsive force between the (-) charged phosphate group of the resin ligand and the (-) charged phosphate group of the mRNA. Therefore, the DBC (Dynamic Binding Capacity) of the resin can be increased by increasing the salt concentration of the equilibration buffer. However, exposure of mRNA to high concentrations of NaCl can cause aggregation, so it is necessary to select the appropriate NaCl concentration of the EQ buffer before checking the DBC. Therefore, we checked the changes in mRNA productivity and quality according to NaCl concentration for each construct to secure the appropriate process range.

[0291] IVT was performed as in Example 1 to obtain Gene 1, Gene 2, and Gene 3 mRNAs, equilibration buffers were prepared with the conditions in Table 39, the turbidity of the Oligo dT load was checked under each condition, and affinity chromatography was performed twice on each of the obtained products as in Table 40, followed by analysis of the eluates.

[0292] The turbidity was checked by measuring UV at 350 nm using a UV spectrophotometer instrument (Libra S50), and the amount and yield of eluate were analyzed as in experimental method 1.

[0293]

[0294]

[0295] That is, a CIMmultus Oligo dT (C12 linker) 0.2 mL 96-well plate (Sartorius, USA) column was mounted on an AKTA Avant 150 (Cytiva, USA), and 1.0 mg / mL of the IVT products are mixed with sample preparation buffer(20mM sodium phosphate + 2X concentration of NaCl in table 39 + 10mM EDTA, pH 6.5) was loaded into the column. Then, the column was re-equilibrated with 10 CV of equilibration buffer, and the column to which the product bound was washed with 40 CV or less of wash buffer, and then 6 CV of elution buffer was loaded to recover the poly(A) tail-containing IVT product bound to the column. The column was then washed with 30 CV injections of CIP buffer and stored in a 20% ethanol solution.

[0296] As a result, as shown in Tables 41 through 43, turbidity was increased with increasing NaCl concentration in all constructs, although there were differences in the degree of increase in turbidity between constructs.

[0297]

[0298]

[0299]

[0300] In addition, for GENE 1, as shown in Tables 41, 44, and 45, precipitation of the Oligo dT Load occurred from 600 mM and the process could not proceed, but at 300 - 500 mM, where the process was possible, the %CV between yields was within 2%, the %CV between purity was within 1%, and the %CV between dsRNA content was 10.20%, confirming that there was no difference in QA / PA within the processable range. In other words, all QA / PA acceptance criteria were met with Yield (%) ≥ 80.0, dsRNA content (ng / mg) ≤ 1000, and Purity (%) ≥ 80.0 at 300 - 500 mM.

[0301]

[0302]

[0303] In the case of Gene 2, as shown in Tables 42, 46, and 47, precipitation of Oligo dT Load occurred from 700 mM, and high visual turbidity was observed at 500 mM and 600 mM, and the column clogged when the process was actually run. At 300 - 400 mM, where the process was possible, the %CV between yields was within 2%, the %CV between purity was within 1%, and the %CV between dsRNA content was 7.17%, confirming that there was no difference in QA / PA within the processable range. That is, all QA / PA acceptance criteria were met with Yield (%) ≥ 80.0, dsRNA content (ng / mg) ≤ 1000, and Purity (%) ≥ 80.0 at 300 - 400 mM.

[0304]

[0305]

[0306]

[0307]

[0308] In conclusion, it was confirmed that there is a difference in the NaCl concentration that can be used in the EQ buffer for each construct: 300 - 500 mM for Gene 1, 300 - 400 mM for Gene 2, and 300 - 800 mM for Gene 3. Also, it was confirmed that the NaCl concentration of 300 - 400 mM in the EQ buffer is the concentration that all constructs can be applied.

[0309] Example 11. Evaluation of composition and pH of elution buffer in primary purification using affinity chromatography

[0310] Since the ligand and mRNA of the oligo dT resin are hydrogen bonded at high salt, elution is performed using a buffer with low salt in the elution step. Therefore, based on the above examples and existing literature, the range of elution buffers was selected as shown in Table 50, and the mRNA productivity and quality changes according to the elution buffer composition and pH for each construct were checked to secure an appropriate process range.

[0311] That is, the eluent was analyzed by the method of Example 10 after the chromatography (Oligo dT) process by the method of Table 51 with 2 runs in each condition, and since the holding step of the eluent may be necessary before entering the next process, the mRNA UF / DF process, the purity was analyzed at room temperature for 4 days and at refrigeration for 7 days to select the buffer composition and pH that can secure a certain level of stability.

[0312]

[0313]

[0314] That is, a CIMmultus Oligo dT (C12 linker) 0.2 mL 96-well plate (Sartorius, USA) column was mounted on an AKTA Avant 150 (Cytiva, USA), and the IVT products are mixed with sample preparation buffer(20mM sodium phosphate + 700 mM NaCl + 10mM EDTA, pH 6.5) was loaded into the column. Then, the column was re-equilibrated with 10 CV of equilibration buffer, and the column to which the product bound was washed with 40 CV or less of wash buffer, and then 6 CV of elution buffer was loaded to recover the poly(A) tail-containing IVT product bound to the column. The column was then washed with 30 CV injections of CIP buffer and stored in a 20% ethanol solution.

[0315] As a result, for Gene 1, as shown in Tables 52 and 53, the %CV between yields was within 2% and %CV between purity was within 1%, regardless of elution buffer composition and pH, confirming that there was no difference in QA / PA within the processable range, and the dsRNA content of the eluent was checked ant confirmed that the %CV between the dsRNA content was 30.07%, which varied depending on the elution buffer composition and pH. Except for the citrate buffer (pH 5.0), it was confirmed that the dsRNA content was lower in the citrate buffer compared to other buffers. This is due to the difference in conductivity of the buffers, and determined that the conductivity of the citrate buffer except citrate buffer (pH 5.0) was more than 900 mS / cm, which was about twice as high as other buffers, so could not elute dsRNA that was strongly bound to Oligo dT resin.

[0316] Also, as shown in Table 53, it was confirmed that the storage stability of the eluent was stable for 4 days at room temperature and 7 days at room temperature in all elution buffers, with a maximum purity %CV of 1.06% between day 0 and 4 days at room temperature and 0 and 7 days at room temperature.

[0317] That is, regardless of the elution buffer, all QA / PA met the acceptance criteria and were stable for 4 days at room temperature and 7 days in refrigeration, but to reduce the dsRNA content, it was determined that citrate buffer pH 5.5 - 7.5 was suitable as an elution buffer to reduce the dsRNA content.

[0318]

[0319]

[0320]

[0321]

[0322] On the other hand, for Gene 2, as shown in Tables 55 and 56, it was confirmed that there was no difference in QA / PA within the processable range with %CV between yields within 2% and %CV between purity within 1% regardless of elution buffer composition and pH, and the dsRNA content of eluent was checked and confirmed that the %CV between dsRNA content was 21. 89%, confirming that there is a difference depending on the elution buffer composition and pH, and that the dsRNA content is lower than 10 ng / mg in citrate buffer (pH 6.0 - 7.5) and acetate buffer (pH 6.5) compared to other buffers.

[0323] Also, as shown in Table 57, it was confirmed that the storage stability of Eluent is stable for 4 days at room temperature and 7 days at room temperature in all elution buffers with a maximum purity %CV of 0.66% between day 0 and 4 days at room temperature and 0 and 7 days at room temperature.

[0324] That is, regardless of the elution buffer, all QA / PA met the acceptance criteria and were stable for 4 days at room temperature and 7 days in refrigeration, but to reduce the dsRNA content, it was determined that citrate buffer (pH 6.0 - 7.5) and acetate buffer (pH 6.5) were suitable as elution buffers.

[0325]

[0326]

[0327]

[0328]

[0329] In addition, for Gene 3, as shown in Tables 58 and 59, it was confirmed that there was no difference in QA / PA within the processable range with %CV between yields within 2% and %CV between purity within 1%, regardless of elution buffer composition and pH, and the dsRNA content of the eluent was checked and confirmed that the %CV between dsRNA contents was 17.72%, which is within the level of method variation (criterion: %CV ≤ 20), so it was determined that there was no difference in dsRNA content depending on the elution buffer.

[0330] Also, as shown in Table 60, it was confirmed that the storage stability of Eluent was stable for 4 days at room temperature and 4 days at room temperature and 7 days at room temperature with a purity %CV within 2%, except for citrate buffer (pH 7.0).

[0331] That is, all buffers met the acceptance criteria without any difference in QA / PA depending on the elution buffer, but all buffers except citrate buffer (pH 7.0) were judged to be suitable because the room temperature stability of citrate buffer (pH 7.0) decreased.

[0332]

[0333]

[0334]

[0335]

[0336] In conclusion, it was confirmed that citrate buffer pH 5.5 - 7.5 is applicable for Gene 1, citrate buffer (pH 6.0 - 7.5) and acetate buffer (pH 6.5) are applicable for Gene 2, and all buffers are suitable as elution buffers except citrate buffer (pH 7.0) for Gene 3. Therefore, considering all three constructs, it was determined that citrate buffer (pH 6.0 - 6.5) and citrate buffer (pH 7.5) are suitable for all constructs.

[0337] Although the present invention has been described in detail with reference to specific features, it will be apparent to those skilled in the art that this description is only of a preferred embodiment thereof, and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereto.

[0338]

[0339] The ssRNA purification method according to the present invention has a high mRNA recovery and a dsRNA removal rate of 95% or more, which are higher than those in existing methods for dsRNA removal (IP / RP, etc.), and thus may produce an mRNA sample having a dsRNA content similar to that of a mutant T7 RNA polymerase that drastically reduces the amount of dsRNA produced. In addition, the amount of dsRNA generated during mRNA synthesis varies depending on the sequence, and for a certain sequence, excessive dsRNA may be generated, making it difficult to use the certain sequence as a drug. However, the ssRNA purification method according to the present invention is useful because it can minimize or appropriately control the dsRNA content contained in an mRNA sample, so that the mRNA can be developed into a drug regardless of the type of sequence, and can also maximize patient safety and drug efficacy.

Claims

1.A method for purifying single-stranded RNA (ssRNA), comprising steps of:(a) subjecting a sample containing ssRNA and containing double-stranded RNA (dsRNA) as an impurity to primary purification using affinity chromatography; and(b) subjecting the sample to secondary purification using at least one chromatography selected from the group consisting of size-exclusion chromatography (SEC), anion-exchange chromatography (AEX), and hydrophobic interaction chromatography (HIC).2.The method of claim 1, wherein the sample is obtained through in vitro transcription (IVT).3.The method of claim 1, wherein step (a) of subjecting the sample to the primary purification using the affinity chromatography comprises:(a-i) mixing the sample containing ssRNA with a sample preparation buffer and loading the mixture into a column containing a resin that complementarily binds to a poly(A) tail;(a-ii) washing the column with a wash buffer; and(a-iii) recovering bound ssRNA by elution with an elution buffer.4.The method of claim 3, wherein the sample preparation buffer is a solution (pH 6.0 to 7.0) containing:10 to 150 mM sodium phosphate, tris(hydroxymethyl)aminomethane, tris), sodium citrate, or sodium acetate;1 to 20 mM EDTA (ethylene-diamine-tetraacetic acid); and100 to 1,000 mM sodium chloride.5.The method of claim 3, wherein step (a) further comprises, before step (a-ii), a step of additionally loading an equilibration buffer.6.The method of claim 5, wherein the equilibration buffer is a solution (pH 6.0 to 7.0) containing:10 to 100 mM sodium phosphate, tris(hydroxymethyl)aminomethane, tris), sodium citrate, or sodium acetate;1 to 10 mM EDTA (ethylene-diamine-tetraacetic acid); and100 to 500 mM sodium chloride.7.The method of claim 3, wherein the wash buffer is a solution (pH 6.0 to 7.0) containing:10 to 100 mM sodium phosphate, Tris, sodium citrate or sodium acetate; and1 to 10 mM EDTA.8.The method of claim 3, wherein the elution buffer is a solution (pH 6.0 to 7.0) containing 0.01 to 10 mM sodium phosphate, Tris, sodium citrate or sodium acetate.9.The method of claim 1, wherein step (b) of subjecting the sample to the secondary purification using the size-exclusion chromatography comprises steps of:(b-1-i) mixing the primarily purified sample, eluted in step (a), with a 2X equilibration buffer and loading the mixture into a column containing a size exclusion resin;(b-1-ii) eluting bound RNA using the equilibration buffer; and(b-1-iii) obtaining the last eluted RNA as a fraction containing ssRNA.10.The method of claim 9, wherein the size exclusion resin separates molecules of 1,500 to 5,000 kDa according to size.11.The method of claim 9, wherein the equilibration buffer is a solution (pH 3.0 to 7.0) containing:1 to 100 mM sodium citrate, sodium phosphate, Tris, or sodium acetate; and0.01 to 1,000 mM sodium chloride.12.The method of claim 1, wherein step (b) of subjecting the sample to the secondary purification using the anion-exchange chromatography comprises steps of:(b-2-i) mixing the primarily purified sample, eluted in step (a), with a sample preparation buffer and loading the mixture into a column containing an anion exchange resin; and(b-2-ii) obtaining an eluted flow-through (FT) as a fraction containing ssRNA.13.The method of claim 12, wherein the sample preparation buffer is a solution (pH 7.0 to 8.0) containing:10 to 50 mM Tris, sodium phosphate, sodium citrate or sodium acetate;1 to 10 mM EDTA; and500 to 1,000 mM sodium chloride.14.The method of claim 1, wherein step (b) of subjecting the sample to the secondary purification using the hydrophobic interaction chromatography comprises steps of:(b-3-i) mixing the primarily purified sample, eluted in step (a), with a sample preparation buffer, and loading the mixture into a column containing a hydrophobic interaction resin;(b-3-ii) washing the column using the sample preparation buffer; and(b-3-iii) recovering bound ssRNA by elution with an elution buffer.15.The method of claim 14, wherein the sample preparation buffer is a solution (pH 6.5 to 7.5) containing:10 to 100 mM sodium phosphate, tris, sodium citrate, or sodium acetate;1 to 20 mM EDTA; and100 to 2,800 mM sodium chloride, sodium sulfate, ammonium sulfate, potassium sulfate, disodium phosphate, lithium chloride or potassium thiocyanate.16.The method of claim 14, wherein step (b) further comprises, before step (b-3-ii), a step of additionally loading the sample preparation buffer.17.The method of claim 14, wherein the elution buffer is a solution (pH 6.5 to 7.5) containing:10 to 100 mM sodium phosphate, Tris, sodium citrate, or sodium acetate;1 to 20 mM EDTA; and100 to 1,000 mM sodium chloride.18.The method of claim 1, further comprising, before step (a), after step (a), or after step (b), an ultrafiltration step.19.The method of claim 1, wherein ssRNA obtained by the secondary purification has a dsRNA removal rate of 80% or more.

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